H2O systems introduced the Waveline DC pumps and RLSS protein skimmers in 2012.  These products are just now coming to market, and Advanced Aquarist has the first review of the new RLSS line.

The Design

The RLSS R10-U is a conventional high-end in-sump needle-wheel design but with an unconventional pump.   A single, submersed Waveline DC-5000 pump performs both air fractionation and water delivery for the skimmer.  Unlike the single speed AC pumps employed by nearly every other skimmer, the Waveline is a speed-controllable direct current pump.  Users can select between six preset RPMs via the included DC pump controller to tune their skimmer's performance.  Six green LEDs indicate the speed.  Users can also engage a 10 minute feed/service shutoff timer;  The pump restarts automatically after 10 minutes or restarts immediately by pressing the timer button for a second time.  The Waveline DC-5000 is a soft-start pump, meaning the impeller will gradually ramp up to full speed on start-up.

H2O Systems includes an air silencer, custom venturi intake, and all the silicone tubing required for operation.  The entire skimmer system (including the Waveline pump) is shipped in a single box.  Some assembly is required, and H2O Systems will soon provide instructions on their website.  Unboxing and assembly took us less than 10 minutes, and no tools were required.

The R10-U measures 10" diameter at the base of the reaction chamber and collection cup (hence the R10 model number).  The entire skimmer (sans the grey PVC plumbing) is manufactured from sturdy acrylic. Fit and finish is top notch.  Read more specifications at the end of this review.

RLSS skimmer users can remove the entire bottom plate (above, left), which is held to the main skimmer body with four plastic thumbscrews.  The bubble plate diffuser is also removable, allowing unobstructed access inside the skimmer body.  Pictured (above, right) is the underside of RLSS' bubble plate diffuser.

The custom RLSS needle-wheel is a generous 2" diameter and spun on an impressively large diameter but short ceramic shaft, reducing potential for imbalances that can generate both noise/vibration as well as excessive wear.  All friction points are ceramic to ceramic, including the bushings (no rubber whatsoever).  This is as robust a needle-wheel assembly as I've seen.

The needle-wheel features dual diameter pins and perforations on the back plate.  H2O Systems claims the stepped pins create finer bubbles and the perforations help reduce heat buildup at the motor.

As an energy-efficient DC pump, the Waveline runs very cool.  In theory, this should also improve reliability and reduce calcium buildup.

The RLSS collection cup is connected to the skimmer body via a slip joint fitted with an o-ring. The connection is watertight and solid.  In fact, if I was to levy my first critical comment, it's that the fitting was too tight.  Removing the collection cup proved awkward.  In theory, a slip fitting should be easier to remove than a screw-on or union collection cup, but I found this was not the case with the review unit.  But I'm nitpicking.

Performance

Let's start with the skimmer's noise level.  The RLSS R10-U is one quiet skimmer, on par with the best Askoll-based (e.g. Red Dragon) protein skimmers such as Bubble Kings.  The Waveline pump generates virtually no noise or vibrations; Whatever vibration that exists is absorbed by the silicone and rubber feet on the skimmer and pump respectively.  The silicone tubing also prevents transference of vibration from pump to skimmer.  The only audible noise comes from the air bubbles and a very low level rotor hum.

How does the R10-U skim?  To find out, I seeded the 20 gallon test reservoir with one cup of tea-colored skimmate (produced with another protein skimmer).  The RLSS R10-U developed a thick head of stable foam nearly instantaneously.  Since there are no standard metrics to quantify skimming performance, I'll let the photos and video speak for themselves.

The noise you hear in the video is the bubbles popping with the lid removed.

The Waveline pump generates a wall of fine bubbles with virtually no microbubbles returned back to the sump, evidenced by the photo above. I found the RLSS R10-U requires 8 inches of water to operate effectively.  Anything less than 7 inches and the pump (at full speed)  will suck air from the water's surface.

Despite the curved cone design and bubble diffuser plate, the internal water column was more turbulent than some of its competitors.  This did not appear to hinder the skimmer's performance as the photos/video show. Unfortunately there is no way to modify the water to air ratio to reduce turbulence; Reducing the pump's speed results in a linear reduction of both water and air.  Again, the skimmer is  clearly capable of producing very thick and stable foam, so the turbulence "problem" may be academic.  And in all fairness, the skimmer may require more time to break in; I spent one week with the review unit before putting the skimmer through its paces.

Conclusion

At $799.99, the RLSS R10-U is not a cheap protein skimmer, but it represents a terrific value compared to its competitors (some costing more than twice the R10-U).  We know this time-tested skimmer body (based on the JNS design) works well, but the heart of any needle-wheel protein skimmer is its pump.  The Waveline DC pump proves it is an effective protein skimmer pump capable of producing ample fine bubbles with minimal noise, heat, and electrical consumption.

Specifications & Pricing

• 10" diameter in-sump protein skimmer
• 18 x 18 x 21.5" (455 x 455 x 550mm)
• 1 x Waveline DC-5000 pump (40 watts)
• Air draw: 900-1800lph
• Rated for 396-660 gallons (1500-2500 liters)
• $799.99 USD

The manufacturer provided this product to Advanced Aquarist for review.

Phosphate is an ion of great concern to reef aquarists. In fact, aside from calcium and alkalinity, it is probably the chemistry topic on which reef aquarists focus the most. Much of this concern is warranted, with phosphate potentially contributing to algae problems, poor coloration of corals and other invertebrates, and growth of most photosynthetic organisms. For these reasons, every reef should have a plan for export of phosphate in one or more ways, and the choices abound. Topics relating to desired target levels and the many export methods have been covered in detail by me and other authors in the past, and I won't dwell on them here.

What I will focus on relates to the various sources of phosphate in reef aquaria, and how important each one actually is to an operating aquarium. There have long been numerous misunderstandings of these source issues, but in the last two years it seems that these misunderstandings have become more common and are sometimes driving aquarists to unwarranted actions.

In part, I attribute this recent uptick in confusion to the newly available phosphate checkers from Hanna. Without getting into any discussion of their relative accuracy or the merits of using them, I think many aquarists are being lured into inappropriate actions by finding phosphate in various additives that they were not aware of previously. To paraphrase Field of Dreams: "Build it and they will find phosphate". In order for aquarists to interpret what these found values mean, they need to have an understanding of the overall phosphate balance in coral reef aquaria. This article strives to provide the understanding necessary to put phosphate issues into proper perspective.

So just for starters, let's see how many of you would be concerned by each of these scenarios:

1. My purified fresh water I use for top off has 0.05 ppm phosphate in it. Since I'm trying to keep my tank at 0.02 ppm, that's obviously too much. What should I do?
2. I put a cube of my frozen fish food in a half cup of water and did a phosphate test on the water. I got a whopping value of 1.0 ppm!!! That's a huge problem, right?
3. I put a teaspoon of my GAC (granular activated carbon) in a glass of fresh water, and then tested the water for phosphate. It was dark blue and I could not even get a reading there was so much. Time to look for another brand, right?
4. I know I feed my fish and corals, but I carefully rinse my foods, use only frozen foods, and only feed enough that all is eaten. So I have no idea where my phosphate is coming from. Must be my RO/DI isn't working as there is no other possible source.

Experience tells me that 95% or more of reef aquarists agree that the first three are problems to be solved, and at least half have the same feelings as expressed in number four. In reality, the first three are really not substantial issues at all and reefers need to understand why. Scenario four sums up the main point reefers need to understand: foods are the primarily source of phosphate in almost any aquarium that is being fed, regardless of the choice of foods and rinsing etc. In most aquaria, this source dominates all other sources by a factor of ten to a hundred or more, even if scenarios 1-3 are also true!

Food Sources of Phosphate

In order to begin to understand phosphate sources in aquaria, let's first look at foods. Contrary to what many suppose, phosphate is not something that can be avoided in a nutritious fish food. Phosphorus is present in many of the biomolecules of life, so every natural tissue that goes into a fish food will have substantial phosphorus in it. Phospholipids make up a substantial part of cell membranes. The genetic code of every organism is made of DNA, and that DNA contains a phosphate bridge between each base pair (Figure 1 shows how much phosphate is really in DNA). The way that all cells get energy is through conversion of ATP to ADP, and these molecules contain 3 and 2 phosphate moieties respectively (Figure 2).

Most importantly for nutritional aspects of foods and phosphate, proteins contain phosphate. Many budding chemists understand that proteins are made of amino acids, and none of the standard amino acids contain phosphate, so where does the phosphate come from? It turns out that organisms attach phosphate to the hydroxyl group of the amino acids threonine and tyrosine in proteins to turn on and off many types of protein functions. Consequently, proteins often contain a lot of phosphate, making it very hard to make nutritionally complete foods without substantial phosphate. The relationship between phosphate and protein is so tight that people who suffer from excess phosphate (typically those with kidney disease, and especially those on dialysis) are unable to get enough protein without getting too much phosphate. These patients take oral phosphate binders to bind dietary phosphate before it is absorbed from their small intestines because restricting dietary phosphate just is not effective enough.

Quantitation of Phosphate in Foods

How much phosphate is in foods? A lot, but it does depend on the food. Some aquarium foods state on the label a minimum specified amount of phosphorus (phosphorus is the atom, P, at the center of a phosphate ion, PO₄^---). Analyses of foods often quote an amount of phosphorus instead of phosphate as the unit of measure. Like feet and inches, these are just different units of measure of the same thing, and values of phosphorus are multiplied by 3.1 to get values to phosphate.

For foods that are intended for human consumption (such as shrimp or clams), we can look up known data on phosphorus content. Unfortunately, many manufacturers of aquarium foods do not supply phosphorus values and they are complex mixtures of many ingredients which preclude us from looking them up. Perhaps some manufacturers are concerned about scaring aquarists about the phosphate content. Fortunately, Ron Shimek analyzed a variety of foods several years ago and this data set can also be used to understand how much phosphorus is in various commercial aquarium foods. Tables 1-3 contain data on the phosphorus and protein content of many aquarium foods, with Table 1 containing dry foods, Table 2 containing frozen foods, and Table 3 containing grocery store foods that are sometimes used in aquaria.

These raw phosphorus values alone allow us to know how much phosphorus is entering the aquarium by feeding a certain food, and we will use these numbers in several analyses later in this article. One must be wary of relying too much on such numbers for comparative purposes, however, since some foods have substantially more moisture or fillers in the food, which serve to drop the percent phosphorus, but are not necessarily better choices from a phosphorus standpoint. One simply needs to feed more of a wetter food than a drier one to attain the same total nutrition, offsetting the lower claimed phosphorus value.

One useful way to compare foods to each other is by comparing the phosphorus to protein ratio. In this crude way, we can eliminate effects due to moisture and fillers. Obviously this method has its own flaws since the nutritional value of foods is far more complex than a protein value alone provides, but it will allow us to understand among similar types of foods, which ones seem to provide more or less phosphorus. In order to help guide the reader, I have chosen to show the higher phosphorus to protein foods in red and the lower ones in green in Tables 1-3. The cutoff values I used are completely arbitrary, and one should definitely not make a choice of food based on this criterion alone. Nevertheless, several points are apparent:

• Dry and frozen foods vary substantially in phosphorus to protein content. There are high and low phosphorus foods in both categories. On balance, one could not say from this data that typical dry foods are any worse in this regard than are typical frozen foods.
• With the exception of seaweeds, grocery store foods do generally seem to have a lower phosphorus to protein ratio than many other choices, although several Ocean Nutrition frozen foods are similarly low. This presumes that one is using grocery store foods that are not treated with phosphate to preserve freshness in the processing plant, but that is a concern for many of these materials.
• Shrimp seem to be a standout in terms of a low protein to phosphorus ratio for grocery store foods.
• Fish with bones have a high phosphorus content, since bones are modified calcium phosphate, but whether these bones are fully digested or not probably depends on what eats them and how long one otherwise waits for them to decompose.

Table 1. Phosphorus and protein content of some common dry aquarium foods.

Food	Protein (mg/g)	Phosphorus (mg/g)	Phosphorus/Protein(w/w)	Source of Data
Brine Shrimp Direct Golden Pearls	550	15	0.027	Shimek
Brine Shrimp Direct Plankton Gold Flakes	490	8.3	0.017	Shimek
Cobalt Aquatics Brine Shrimp Flakes	440	10	0.023	Label
Cobalt Aquatics Spirulina Flakes	450	10	0.022	Label
IO Marine Chips Herbivore	460	9	0.020	Label
IO Marine Chips Omnivore	460	11	0.024	Label
IO Marine Grazing Block	40	0.5	0.013	Label
IO Marine Pellets Herbivore	440	10	0.023	Label
IO Marine Pellets Omnivore	470	12	0.026	Label
IO Seaweed Grazing Blocks	25	0.3	0.012	Label
Nutrafin Max Marine Angel Sinking Pellets	440	8	0.018	Label
OSI Marine Flake	470	6	0.013	Label
TetraAlgae Vegetable Enhanced Crisps	460	10	0.022	Label
TetraMarine Flakes	460	12	0.026	Label
TetraMarine Granules	440	14	0.032	Label
Vibragro Saltwater Staple	300	15	0.050	Shimek

Table 2. Phosphorus and protein content of some common frozen aquarium foods.

Food	Protein (mg/g)	Phosphorus (mg/g)	Phosphorus/Protein(w/w)	Source of Data
Direct Tahitian Blend Cryopaste	44	1.4	0.032	Shimek
Frozen Plankton/Krill Brine Shrimp	88	1.6	0.018	Shimek
Gamma Foods Lancefish	180	4.4	0.024	Shimek
Hikari Bio-Pure Clam On A Half Shell	32.9	3	0.090	Label
Hikari Bio-Pure Clam On A Half Shell (meat only)	128.4	4.1	0.032	Label
Ocean Nutrition Frozen Formula 1	160	1.1	0.007	Shimek
Ocean Nutrition Frozen Formula 2	62	1.2	0.019	Shimek
Ocean Nutrition Frozen mysis	52	0.1	0.002	Label
Ocean Nutrition Frozen Prime Reef	130	0.9	0.007	Shimek
Oregon Desert Brine Shrimp Company Silversides	42	4	0.095	Shimek
San Francisco Brand Frozen Brine Shrimp	31	0.72	0.023	Shimek

Table 3. Phosphorus and protein content of some common grocery store foods used in reef aquaria.

Food	Protein (mg/g)	Phosphorus (mg/g)	Phosphorus/Protein(w/w)	Source of Data
Broccoli	30	0.66	0.022	analysis
Clams (no shell)	128	1.69	0.013	analysis
Clams (no shell)	128	1.69	0.013	analysis
Cod	179	1.74	0.010	analysis
Cod	229	2.23	0.010	analysis
Eel	184	2.16	0.012	analysis
Eel	186	2.16	0.012	analysis
Kelp	17	0.42	0.025	analysis
Mackerel	186	2.17	0.012	analysis
Mussel	119	1.97	0.017	analysis
Nori	290	6.4	0.022	Shimek
Octopus	149	1.86	0.013	analysis
Oyster (no shell)	70	1.35	0.019	analysis
Oyster (no shell)	71	1.35	0.019	analysis
Salmon	204	2.4	0.012	analysis
Sardine	209	3.66	0.018	analysis
Scallops	168	2.19	0.013	analysis
Shrimp	209	1.36	0.007	analysis
Shrimp	210	0.7	0.004	analysis
Spirulina	59	1.1	0.019	analysis
Squid	156	2.2	0.014	analysis
Wakame (seaweed)	30	0.8	0.027	analysis

Impact of Foods on the Aquarium Phosphate Balance

Now we come to the heart of the issue. The actual amount of phosphorus present in foods and what it means. In order to understand the effects of foods, we need to understand what happens to them when added to an aquarium. Some aquarists are under the misconception that eaten foods do not contribute to the free phosphate in the water. Many aquarists are told the mantra of feeding only as much as is eaten, and they confound this idea with the assumption that when doing so, one minimizes the phosphate release. That idea is simply untrue.

A fish or other organism that eats foods takes in substantial phosphate, as shown above. But what happens to it? If the organism is not actually expanding in size (such as an adult green chromis, or a person), the phosphate that is taken in is almost entirely excreted back into the water. The only exception to that process is the very small amount of phosphorus that goes into eggs or sperm, and since in most aquaria those items are rapidly consumed by other organisms, the phosphorus will ultimately get into the water.

Growing organisms do take up a small amount of phosphorus from the diet and retain it in their growing tissues, but the emphasis is on small. A study of a fish farm with rapidly growing rainbow trout in the ocean showed that 78-82% of the phosphorus feed to the fish was lost to the environment. A second aquaculture study using normal fish foods showed that 62% of the fed phosphate was released to the environment, with 35% being released as soluble phosphate available directly to algae, and 27% as phosphorus in fecal pellets (which if not removed, will break down in an aquarium releasing the phosphate again). Another study showed that 81.5% of commercial diet phosphate was released to the environment, but that with a "special" diet with low phosphate and low fish meal this could be reduced to 64% lost. A fourth study showed that growing fish fed slightly less phosphate than they need (to optimize theoretical uptake) take up and retain different phosphate sources differently. Using a purified protein diet, they observed retention of 72% of the phosphorus, 51% retention of phosphorus from added fish bone meal, and higher levels of uptake and retention for inorganic phosphate supplements (such as sodium phosphate).

This sort of study is of concern in aquaculture settings due to environmental contamination due to the released phosphorus and nitrogen. To my knowledge, however, it has never been done in a reef aquarium. Such phosphorus balance studies have also been performed in people for many years. In adults it is clear that nearly all phosphate taken up is excreted, mostly in the urine and some in the feces. Even in young growing children, the amount of phosphorus retained from the diet is only 5-20% of that consumed, with 80-95% excreted in the urine and feces. While such studies are fairly far removed from reef aquaria, they do supporting the idea that organisms take in a lot more phosphorus than they retain, even when growing.

Consequently, reef aquarists should expect that much of the phosphorus added to a reef aquarium in the form of foods ultimately ends up in the water as phosphate. Whether that portion getting into the water is 95% or 35% won't substantially impact the conclusions below that foods add a very large amount of phosphate.

Quantitation of the Food Impact Aquarium Phosphate

Using the assumption that most of the phosphorus present in foods ultimately ends up as phosphate in the aquarium, we can calculate roughly what that effect is. Even if the actual number is a half or a quarter of that added, getting the ballpark information is very useful to gauge the importance of this phosphate source. Obviously the calculated value depends on how much of what is fed to what size aquarium.

Table 4 shows a variety of possible feeding schemes that an aquarist might use on a hypothetical 100 gallon (379 L; actual water volume) aquarium. Aquarists can decide for themselves how these regimens compare to their own feed schedules.

Table 4. Phosphate additions to a 100 gallon actual water volume aquarium with different daily feeding regimens.

Foods Fed	Phosphorus Added Daily (mg)	Equivalent Phosphate Added Daily (mg)	Equivalent Phosphate Concentration Added Daily (ppm)
1 Prime Reef Cube	2.7	8.4	0.022
1 Prime Reef Cube1 Formula 1 Cube	6.0	18.6	0.049
1 Formula 2 Cube1 Mysis Cube	3.9	12.1	0.032
IO Marine Omnivore Chips (2 g)	22	68	0.18
IO Marine Omnivore Chips (1 g)Silversides (1/2 teaspoon)Nori (2.5 g = large sheet)	37	115	0.30

Obviously there is a big range depending on how much is fed. What is surprising to many folks, however, is how large that number is relative to typical target levels of phosphate in reef aquaria, which might be something like 0.03 ppm or less. Even the light feeding of a single cube of a relatively low phosphate frozen food to this aquarium supplied most of that target amount in a single feeding. Heavy feeding added ten times that amount in a single day. In short, this high daily addition rate is why phosphate control is often very difficult or reef aquaria.

Rinsing Foods and the Effect on Phosphate

Now that we have some information on the phosphate in foods, we can critically examine the concern that many aquarists have about foods, and specifically their rinsing of frozen foods before use. A typical test you see is someone taking a cube of fish food, thawing it, and putting it into a half cup of water. They then test that water for phosphate and find it "off the charts". Let's assume that means 1 ppm phosphate, which would give a very dark blue color in many phosphate tests. Bear in mind this is a thought problem, not an actual measured value, but it is typical of what people think the answer is.

Is that a lot of phosphate? Well, there are two ways to think of the answer.

The first way is as a portion of the total phosphate in that food. A half cup of water at 1 ppm (1 mg/L) phosphate contains a total of 0.12 mg of phosphate. A cube of Formula 2 contains about 11.2 mg of phosphate. So the hypothetical rinsing step has removed about 1 percent of the phosphate in that food. Not really worthwhile, in my opinion, but that decision is one every aquarist can make for themselves.

The second way to look at this rinsing is with respect to how much it reduces the boost to the aquarium phosphate concentration. Using the same calculation as above of 0.12 mg of phosphate, and adding that to 100 gallons total water volume, we find that phosphate that was rinsed away would have boosted the "in tank" phosphate concentration by 0.12 mg/379 L = 0.0003 ppm. That amount washed away does not seem significant with respect to the "in tank" target level of about 50-100 times that level (say, 0.015 to 0.03 ppm), nor does it seem significant relative to the total amount of phosphate actually added each day in foods (which is perhaps 50-1000 times as much, based on input rates from Table 4. Again, the conclusion I make is that rinsing is not really worthwhile, in my opinion.

Comparison of Food Sources of Phosphate to Other Sources

What about other sources of phosphate, like the "crappy" RO/DI water containing 0.05 ppm phosphate? A similar analysis will show it equally unimportant relative to foods.

Let's assume that the aquarist in question adds 1% of the total tank volume each day with RO/DI to replace evaporation. Simple math shows that the 0.05 ppm in the RO/DI becomes 0.0005 ppm added each day to the phosphate concentration in the aquarium. That dilution step is critical, taking a scary number like 0.05 ppm down to an almost meaningless 0.0005 ppm daily addition. Since that 0.0005 ppm is 40-600 times lower than the amount added each day in foods (Table 4), it does not seem worthy of the angst many aquarists put on such measurements. That said, tap water could have as much as 5 ppm phosphate, and that value could then become a dominating source of phosphate and would be quite problematic. Purifying tap water is important for this and many other reasons.

The same sort of calculation applies to analyzing other phosphate issues, such as the GAC in scenario three. The issue of finding "high" phosphate in GAC soaked in fresh water was frequently quoted as a reason to use one or the other brand of GAC, and probably still is. But simple analysis as shown above for the food rinsing puts the lie to this being a big problem.

One needs to consider how much GAC one will really use in the aquarium and how often it is added in order to interpret how important the added phosphate is. A typical recommendation might be 1 cup of GAC per 100 gallons of aquarium water, and to change it in 4-6 weeks. Let's assume we detect 0.5 ppm phosphate when a teaspoon is placed in a cup of water, and we get scared by the dark blue color during the test. Is this reasonable? That 0.5 ppm from a teaspoon in a cup of water translates to 0.015 ppm phosphate when a cup is used in 100 gallons.

That 0.015 ppm may be significant, being a typical target concentration level for reef aquaria and amounting to about half to a twentieth of the amount added daily in foods, but remember, it is used for 4-6 weeks. During those 4-6 weeks before the next replacement, foods add 50-700 times as much phosphate. So while it is not unreasonable to look for another brand of GAC, to blame phosphate or algae issues in the aquarium on its use would stretch credibility because it is a very tiny portion of the total phosphate being added.

Conclusion

Foods are by far the most important source of phosphate in most aquariums. While there are big variations between foods, it does not appear in this analysis that dry foods are the nasties they are often made out to be, relative to frozen foods. There are better and poorer choices (with respect to phosphate) to be made within each food type. Avoiding foods with bones, however, might be worthwhile if delivering less phosphate is a goal. Additionally, fresh grocery store shrimp seems to be one of the best foods from this standpoint.

In considering whether sources of phosphate other than foods are important, one must carefully look to the actual amounts involved to determine whether other sources are even worth trying to minimize. It can be scary to learn that your purified fresh water has phosphate in it, or that your salt mix has detectable phosphate, or that your supplements or whatever have some phosphate. But just because you detect something, and maybe you even detect a concentration far higher than in your aquarium, that does not by any means imply that those sources are significant enough to warrant some sort of corrective action. Our analytical tools have become fairly sensitive, allowing us to detect things which might sound like trouble, but really aren't. We need to understand the various dilution issues involved as well as the overall phosphate balance in a reef aquarium to evaluate the importance of different measurements.

Just use some math and put it all into perspective, before using some dollars or time to chase a trivial "problem".

Happy Reefing

When I first started work in a laboratory, determination of various aqueous constituents was a laborious task, beginning with mixing standards, manually graphing their absorbances, and then making comparisons with samples' absorbance tests results. We manually recorded this information and charted it on graph paper. It was state-of-the-art for the 1970's. We would be (and are) amazed with today's analytical instruments. Compact, battery-operated colorimeters (a type of spectrometer) use conveniently packaged chemicals to quickly determine concentrations of various aqueous substances.

Of particular interest to me in this new world of laboratory instruments are devices used to analyze for alkalinity and phosphorus. Hanna Instruments offers relatively inexpensive colorimeters called 'Checkers' priced at about $50-$60 each (including a small supply of reagents, 2 test tubes, and carrying case). These instruments are small and highly portable. But how accurate are they, and are they worthy of your consideration? This article will attempt to answer these questions.

Hanna Instruments

Hanna Instruments (Woonsocket, Rhode Island, USA) has been in business since 1978, and today offers over 3,000 products to its customers worldwide. Many of their products are of interest to aquarist and Hanna has in fact targeted the aquarium market. For more details, see 'Contact Information' near the close of this article. Hanna imports the Checkers and their reagents from Europe (Romania).

Why Test?

Since you're reading an article about testing for phosphorus and alkalinity, I'll make an assumption you understand the role they play in aquaria. If you'd like to learn more about these, see:

1. Phosphates: http://reefkeeping.com/issues/2006-09/rhf/index.php
2. Alkalinity: http://www.advancedaquarist.com/2002/11/chemistry

Glossary

The following terms are used in this article:

Cuvette

A vessel for holding water, especially a transparent laboratory vessel (such as a test tube)

Colorimeter

A device for determining the concentration of a substance dissolved in liquid by comparing the intensity of its color with that of standard solution(s) of known concentration(s)

mg/L

milligrams per liter, essentially the same thing as ppm

ppm

parts per million

Reagent

A substance for use in a chemical reaction, especially for analysis

Spectrometer

For our purposes, an optical instrument capable of measuring intensity of transmitted light at a specific (but varying) wavelength, especially for determining the concentration of a dissolved substance

Titrant

The liquid reagent used in titrations

Titration

A method for determining the concentration of an aqueous substance by adding a liquid reagent of known concentration and measuring the volume necessary to convert the substance from one form to another

Methods and Materials

First of all, the results shown here are simply comparisons of those gathered by different analytical means. No standards were tested, and I am operating on the assumption that results of Hach's EPA-approved methods and a 'laboratory-grade' spectrometer are most accurate. In addition, only one Hanna instrument each was used in these comparisons.

Water, gathered from a functioning marine fish-only aquarium, was used for the testing.

Samples tested for phosphate were gathered in acid-washed glassware and analyzed within a few hours' time. Initial analyses indicated the phosphate content was at the upper detection limits of both instruments used (a Hach DR2800 spectrometer and the Hanna HI-713 colorimeter). Simple dilution with deionized water brought the phosphate to concentrations spanning the full range of both instruments and to levels realistically found in many reef aquaria. The Hanna and Hach devices both use the ascorbic acid chemistry method for analyses.

Comparing alkalinity results is not as straight-forward. The Hanna device estimates alkalinity through colorimetric analysis. Apparently Hanna has made correlation of chromatic shifts (indicating pH) and the impact of an acidic titrant on alkalinity. While making for a quick and convenient test, there are caveats. First, the amount of alkalinity determines the titration endpoint. In addition, the presence or absence of phosphate and/or silica also affects testing protocol (See Table 1 for titration endpoints).

Table 1. Titration end-point pH Values.

Test Condition		End-Point pH Values
Alkalinity Concentration (as ppm CaCO3):	30	4.9
	150	4.6
	500	4.3
Silicates, Phosphates present or suspected		4.5

For comparative purposes, alkalinity values were determining using the Hanna's colorimetric method and an end-point pH titration method. Hach reagents (sulfuric acid, either 0.160 or 1.600N) were used to titrate a magnetically-stirred sample. A calibrated meter monitored pH.

End-points depended upon alkalinity values shown in Table 1 while Figure 1 shows various endpoints based on alkalinity concentrations. Figure 2 is a photo of equipment used in measuring alkalinity according to EPA guidelines.

With background information out of the way, we can now turn our attention to Hanna's HI-755 and HI-713 devices.

Alkalinity: Hanna Instrument HI-755

Hanna advertises these specifications for their Alkalinity Checker:

Hana Specifications for Alkalinity Checker

Item	Specification
Range:	0 to 300 ppm (mg/L) (some retailer ads incorrectly say 250 ppm)
Resolution:	1 ppm (or mg/L, if you prefer)
Accuracy :	±5 ppm (mg/L) ±5% of reading @ 25°C (77°F)
Battery Type:	One 1.5V AAA (included)
Light Source:	LED @ 610 nm
Light Detector:	Silicon photocell
Environment:	0 to 50°C (32 to 122°F); 95% Relative Humidity maximum, non-condensing
Auto-off:	Yes, after ten minutes of non-use
Dimensions:	81.5 x 61 x 37.5 mm (3.2 x 2.4 x 1.5")
Weight:	64 grams (2.25 oz.) - actually a little heavier (~75 g - yes, I checked!)
	Colorimetric method, using a liquid pH indicator (bromcresol green)

As discussed in detail in the Methods and Materials section, the results from the Hanna Checker were compared to analyses performed by the acid titration method using endpoints dictated by alkalinity concentrations and the presence of phosphates and/or silica. The results are shown in Figure 3.

Next, we'll look at the Hanna Phosphorus Checker. Hanna advertises these specifications:

Phosphorus (Low Range), Hanna Instrument HI-713

Suitable for freshwater, brackish and seawater. Model Tested: HI-713 Phosphorus, Low Range

Hana Specifications for Low Range Phosphorus

Item	Specification
Range:	0.00 - 2.50 ppm (mg/l) as Phosphate (PO43-)
Resolution:	0.01 ppm (mg/l)
Accuracy :	±4 % of reading @ 25°C (77°F)
Battery Type:	One 1.5V AAA (included)
Light Source:	LED @ 610 nm
Light Detector:	Silicon photocell
Environment:	0 to 50°C (32 to 122°F); Relative humidity: 95% maximum, non-condensing
Auto-off:	After two minutes of non-use, and ten seconds after reading
Dimensions:	81.5 x 61 x 37.5 mm (3.2 x 2.4 x 1.5")
Weight:	64 grams (2.25 oz.)
Analytical Chemistry:	Ascorbic Acid Method: Ammonium molybdate and potassium antimonyl tartrate react in an acid medium with ortho-phosphate to form phosphomolybdic acid that is reduced to molybdenum blue by ascorbic acid.
Interferences:	Silicates in excess of 10 mg/l; highly colored water (such as visibly yellow water)

As discussed in detail in the Methods and Materials section, the results from the Hanna Checker were compared to analyses reported by a Hach 2800 spectrometer and ascorbic acid reagent. The results are shown in Figure 4.

Comments and Recommendations

In many cases, results from colorimeters (and other photometric devices such as spectrometers) are superior to visually judging (comparing) colored samples. In fact, Standard method states: Visual comparisons of treated samples in Nessler tubes is not better than 5% (more often 10%) whereas photometric analyses can be reliable to within 3%, depending upon many factors. Naturally, the photometric device can eliminate personal biases of the user as well as accurately and precisely differentiate between small increments of sample absorbance/transmission.

If photometric analyses are potentially superior, the question of device engineering and quality becomes the issue (that is, will an inexpensive device deliver results comparable to an expensive one?). Under the conditions stated in the Methods and Materials section (above), these observations and recommendations are made:

Hanna Alkalinity Checker

These observations apply to the Hanna Checker HI-755 (Note the instrument is marked 'Checker Marine' suggesting it is intended for seawater analyses only):

Likes:

• Easy to use
• Results are generated in seconds
• The procedure is simple - no drip titration is required (just add reagent, shake, and read)
• Does not require any manual conversions (results are expressed as parts per million calcium carbonate)
• Replacement reagent is inexpensive
• Auto-shutoff
• Reagents marked with expiration date
• Small sample size (10 milliliters)
• Spare cuvette included
• Directions and MSDS available online

Dislikes:

• Alkalinity measurements were consistently higher than those generated by the titration/pH end-point method
• Multiple measurements are not possible without zeroing the instrument each time
• No Material Safety Data Sheet included (see below for link)
• Reagent will stain skin and clothing (all procedures using organic dyes carry the same risk)
• Battery replacement requires a small screwdriver and removal of a tiny screw

Recommendation (Alkalinity):

Though my quick comparisons showed the Hanna Alkalinity Checker delivered results consistently higher than those obtained with an EPA-approved method, I am impressed with the speed and ease of measurement. If I were to establish a correction (and comfort) factor to apply to the Hanna Checker's results, this would be my method of choice for establishing alkalinity concentrations.

I recently received a Hach catalog advertising their version of a colorimetric alkalinity test. Hach's chemistry, just as Hanna's, apparently uses bromocresol green sodium salt in the analysis method. It seems this method is becoming widely accepted, if only for estimations (this is not an EPA-accepted procedure).

Recommendation: Yes, with some reservations

Footnote:

• 1 ppm Alkalinity as CaCO₃ = 0.02 milliequivalent per liter (meq/L)
• 1 ppm Alkalinity as CaCO₃ = 0.056 degrees Carbonate Hardness (dKH, or German Hardness)

Hanna Phosphate Checker

These observations apply to the Hanna Checker HI-713 (Low Range Phosphate):

Likes:

• Results compare very favorably with those generated by a much more expensive device
• Much less expensive than a full-blown spectrometer or photometer
• Easy to use and portable
• Results are generated in several minutes (including a 3-minute reaction time)
• The Checker incorporates a timer
• The procedure is simple - Add reagent, shake and bake for several minutes, and read results in parts per million as phosphate
• Replacement reagent is competitively priced
• Auto-shutoff
• Reagents work in fresh, brackish, and saltwater
• Reagents marked with expiration date
• Low Battery, Dead Battery, Under Range, Over Range, Inverted Cuvettes, High Light and Low Light errors are displayed when appropriate
• Small sample size (10 milliliters)
• Spare cuvette included
• Directions (but not MSDS) available online

Dislikes:

• Test results for phosphate are displayed for only 10 seconds after they are reported
• Multiple measurements are not possible without zeroing the instrument each time
• No Material Safety Data Sheet available
• Battery replacement requires a small screwdriver and removal of a tiny screw

Recommendation (Phosphate): Yes!

Footnote: To convert PO₄³ to P, divide by 3.066

Reagent Cost Comparisons

The following approximate prices are for replacement reagents only, and do not include instruments (unless noted), taxes, insurance, or shipping costs.

Phosphate:

• Hanna Phosphate Reagents: $27.99 U.S. per 100 tests
• Hach PhosVer3: $28.19 U.S. per 100 tests (10 milliliter sample)
• Footnote: The Hanna HI-714 phosphate colorimeter can use Hach reagents (ascorbic acid powder pillows).

Alkalinity:

• Hanna Alkalinity: $9.99 U.S. for 25 tests (~40 cents per test)
• Hach: Alkalinity for digital Titrator, $59.17 - includes sulfuric titration cartridges, phenolphthalein and bromcresol green/methyl red reagents. $219 includes reagents and Digital Titrator. To comply with EPA guidelines, a pH meter is also required, and a magnetic stirrer is useful.
• Hach's new colorimetric Total Alkalinity test (25-4,000 mg/l as CaCO₃): 25 tests for $31.45 ($1.26 U.S. per test) using only their spectrometer models DR2800, 3900 and 5000 (Note: These spectrometers begin at about $3,500 U.S.)

Material Safety Data Sheets (MSDS)

The two kits I obtained did not include Material Safety Data Sheets. Here is a link to the Alkalinity reagent: http://www.hannainst.com/sds/SDS_HI%20755S_2010-12-14.pdf I could not find a link to the Low Range Phosphate reagents on Hanna's website at the time of this writing.

Sampling Procedures

"Garbage in; Garbage out" is a saying applicable to results generated when improper sampling procedures are in place. Use reasonable care when gathering samples for analyses. It is recommended that the vials (cuvettes) supplied by Hanna are used when sampling to avoid contamination.

Alkalinity: Analyze immediately or fix sample by refrigerating at 4°C (39.2°F) for up to 24 hours. Bring to room temperature before analysis. If the sample is gathered in a container other than Hanna's cuvette, either clean plastic or glass is OK, and it should be completely filled to avoid prolonged exposure to any air trapped in the bottle. Filter if the sample contains excessive suspended particles.

Phosphate: Analyze immediately. Use the Hanna 10 milliliter cuvette to draw the sample if possible. If not possible, use clean plastic or glass containers - they should be scrupulously clean - preferable acid-washed with 1:1 hydrochloric acid and rinsed with de-ionized water. To fix the sample, exclude particulate matter via filtration and refrigerate at 4°C (or 39.2°F). Maximum holding time is 48 hours.

Treat the cuvettes with respect, and keep them clean and free of scratches. Don't allow the treated samples to remain in the cuvettes any longer than necessary as the chemicals might stain the glass. If the vials do become stained, gently clean them with paper used for sensitive applications (such as that used to clean camera lenses). In extreme cases, a dilute bleach solution may be required to remove the stains. Some hobbyists keep the vials filled with deionized water between uses to prevent spotting within them.

Other Instruments from Hanna

Many of Hanna's other instruments may be of interest to aquarist, including:

• HI-727 Color of Water (0-500 Platinum-Cobalt Units; absorbance @470nm)
• HI-736 Phosphate Ultra-low Range (0-200ppb; Ascorbic Acid Method)
• HI-718 Iodine (0-12.5 mg/l; DPD Method)
• HI-706 Phosphate High Range (0-15mg/l; Ascorbic Acid Method)
• HI-717 Phosphate High Range (0-30mg/l; Ascorbic Acid Method)
• HI-764 Nitrite Ultra-low Range (0-200 ppb)

Contact Information

Hanna Instruments has targeted the aquarium market and has devoted a webpage to hobbyists. See: aquariums@hannainst.com

Specific questions concerning Hanna instruments and aquaculture can be addressed to Jessica Hoagland, Email: jhoagland@hannainst.com

Or write to:

Hanna Instruments, Inc.
584 Park East Drive
Woonsocket, RI 02895

Additional Comments

These products were obtained through normal retail channels, and descriptions, specifications and other information were current at the time of writing.

Questions? Comments? I am best reached at: RiddleLabs@aol.com or sound off in the comments below.

References

1. Holmes-Farley, R., 2006. Phosphate and the reef aquarium. http://reefkeeping.com/issues/2006-09/rhf/index.php
2. Holmes-Farley, R., 2002. Chemistry and the aquarium: Solving calcium and alkalinity problems. http://www.advancedaquarist.com/2002/11/chemistry

Nuisance algae are perhaps the number one challenge facing aquarists and various strategies exist to prevent their growths. Pretreatment of makeup water (including reverse osmosis filtration, deionization), additions of high pH solutions of water and calcium hydroxide (kalkwasser), protein skimming, algal scrubbing with macro-algae, use of fish foods with low phosphorus content, etc. all can help control nutrients responsible for algal outbreaks. These methods have one thing in common - they target the element phosphorus.

Phosphorus is an element essential for all life forms including plants and algae. Indeed, bags of lawn fertilizer are marked with numbers (such as 10-5-5) indicating the amount of nitrogen, phosphorus, and potassium ('macro-nutrients'), respectively. If we were to remove or limit one of these macro-nutrients, we would be able to control the growth of plants and algae. This concept is known as Liebig's Law of the Minimum, which states plant growth is not encouraged by the total amount of nutrients available but instead by the one least available. Hence, phosphorus is often the element targeted for removal. Biological removal is possible, but requires a certain sequence of biochemical events. More often, phosphorus is removed by chemical means and is often done on industrial levels in wastewater treatment plants. It can be done in aquaria as well.

Recommended phosphate levels for reef aquaria vary somewhat although most are generally low (~0.03 ppm, or mg/l - 3 parts in 100,000,000 parts - quite low indeed).

Silica (SiO₂) is of concern to hobbyists because it is used by diatoms to create their skeletons. Outbreaks of diatoms are characterized by brown coatings or filaments. While diatoms in themselves are not harmful (useless they smother sessile invertebrates) they can be unsightly. Some artificial saltwater manufacturers will not include silica in their mix. However, drinking water can often contain significant amounts of silica (~14mg/l). Since diatoms are photosynthetic, limiting phosphorus along with silica can control their population within aquaria. Silica can also 'plate out' on hot surfaces (such as heaters or pumps allowed to run dry) and can be difficult to remove.

Definitions

ppm

Parts per million, essentially the same thing as milligrams per liter (mg/l)

Phosphorus

A non-metal element considered essential for all life forms. It has an atomic weight of 30.9738 g/mol; its symbol is P.

Phosphate

Phosphorus bound with oxygen, and almost always the form of phosphorus found in aquatic environments.

Ortho-phosphate or reactive phosphate

This form is readily 'bio-available' for plant and algal growths and is the type measured by hobbyist test kits. It may be suspended or dissolved in the water column. Often reported as PO₄^-3, the recommended amount in a successful reef aquarium's water should be low (less than 0.05 ppm, or mg/l, if you prefer). When we consider that the phosphorus (P) portion of phosphate (PO₄^-3) is only about 1/3 of its weight, we can easily see that the 'reactive' part is only about 0.017 ppm. However, hobbyist test kits report ortho-phosphate as PO₄^-3; hence the recommended value is 0.05 ppm or less. To convert PO₄^-3 to P, divide by 3.066. To convert P to PO₄^-3, multiply by 3.066.

Total Phosphate

the sum of ortho-phosphate (easily measured) and that phosphate converted to ortho-phosphate by a severe digestion process employing potassium persulfate and sulfuric acid. Total phosphate can be suspended, dissolved, or incorporated in animal and plant tissues and is usually less bio-available for encouraging algal growths.

 

Two Little Fishies

Two Little Fishies, Inc. (or TLF, Miami Gardens, Florida USA) has been providing products to the pet industry since 1991. They offer a variety of products ranging from fish and invertebrate foods to aquarium supplements. Two of their offerings were selected for this month's Product Review - the PhosBan Reactor (model 150) and PhosBan phosphate/silica adsorbent.

The PhosBan reactor is a 'fluidized bed' (meaning the water to be treated flows upwards through the media and gently suspends at least some of the media particles). Treated water exits the reactor and returns to the aquarium. If all goes according to plan, the reactor's effluent will be depleted of phosphate and silica. How well do these products perform and are they worthy of your consideration? These are the recommendations made by TLF:

• For use on freshwater or marine aquaria, up to 150 gallon (568 liter) capacity.
• Flow rate through reactor should be 20 - 30 gallons (76 - 114 liters) per hour - this will require a pump capacity of 100 - 200 gallons (379 - 757 liters) per hour. A Maxi-Jet 500 (a misprint in the directions - a Maxi-Jet 400 or similar pump will work just fine) is recommended, but throttling will be required using the supplied ball valve. Excessive flow rates may cause the media to crumble - the directions use the synonym 'friable').
• Reactor capacity depends upon the media. The 150 reactor can hold up to a maximum 200 grams of PhosBan media (5 inches or 12.7cm height within the column, with a recommended minimum of 2 inches or 5cm). The column can be filled to capacity when using granular activated carbon.
• If an amount of PhosBan required is below the minimum depth within the reactor, TLF recommends mixing PhosBan with activated carbon.

Phosphorus Removal by Granular Ferric Oxide (GFO)

Iron particles possess a natural positive charge (these ions can have a valence of 2 or 3) while phosphorus possesses a negative charge. Since opposites attract, the phosphorus can be bound to iron (in this case, the ferric state, or Fe ⁺³) thus removing it from solution and preventing it from fueling algal growths.

It is uncommon (and difficult) for hobbyists to distinguish between 'bio-available' phosphate (called 'reactive' or 'ortho-phosphate') and total phosphate (organically-bound) since the latter requires specific laboratory testing procedures and equipment. I wondered what impact PhosBan would have on organic phosphate concentrations and decided to test for it (see the 'Methods and Materials' section below for details). Figure 1 shows the results of testing for Total and Reactive ('ortho-') Phosphate over a period of 12 hours. Figure 2 demonstrates that, when ortho-phosphate content is subtracted from the amount of total phosphate, the level remains relatively stable, indicating removal of reactive ortho-phosphate only.

Silica Removal

TLF also claims that silica is removed by PhosBan as well. Figure 3 shows the results of testing 3 samples gathered over a 21-hour period after initiating treatment with PhosBan.

Effects on pH

A Hach HQ40d data logger equipped with a calibrated pH probe recorded pH values every five minutes over the course of 36 hours. pH fell slightly, but some of this drop is attributable to rising temperature (temp data not shown, and due to powerheads' operation within the aquarium). See Figure 4 and the Discussion section below for further comments.

The effects of PhosBan on alkalinity would be a better metric of its impact. See below.

Effects on Alkalinity

Directions for use of the PhosBan Reactor state that alkalinity will drop when 5x the recommended amount of PhosBan is used. Under the conditions of this experiment, alkalinity dropped by about 19% when PhosBan was applied at about twice the recommended amount. See Figure 5. At face value, this may seem negative until we consider that the presence of both phosphorus and silica lend to alkalinity (Standard Methods, 1998). Therefore, a portion of alkalinity drop could be attributable to their removal, and results generated by alkalinity testing in this situation could be partially attributed to removal of 'false' alkalinity and not a drop in carbonate content.

Water Color

Color of water can be estimated using a comparator or a colorimeter/spectrometer. Color is due to dissolved organic substances (called gelbstoff, German for 'yellow matter') in the water column. Generally, color due to these substances is yellowish and is reported in Platinum/Cobalt units. A few milligrams of dry PhosBan was placed in a beaker containing 100ml deionized water and allowed to sit overnight. A sample was analyzed for color (at 455nm). PhosBan did not impart color to the water.

On the other hand, GFO has the potential to remove organic substances causing yellow water. This was not tested.

Discussion and General Impressions

The PhosBan Reactor 150 is constructed entirely of plastic and acrylic materials and is safe for use in freshwater and saltwater applications. The clear acrylic material of the reactor itself appears to be about 1/16" (~1.6mm) thick and is rugged enough to withstand some abuse. The 90°elbows fitting the barbed fittings atop the reactor are made of a soft rubber-like material and are friction fits. Use cable ties to affix these 90's to the reactor if desired - avoid stainless steel worm clamps that might compress and break the barbs.

This reactor holds about 36 fluid ounces (1,075ml) and has a useable capacity of about 26.5 fluid ounces (~785 milliliters). Assuming PhosBan has a consistent density, the 150 reactor will hold approximately 7.3 ounces (207 grams) of this material. Using the manufacturer's recommendations for loading, the reactor will hold enough PhosBan to treat a maximum marine aquarium size of 200 gallons (757 liters; using 50 grams for 50 liters). It should be sufficient to treat a 400-gallon (~1,500 liter) freshwater aquarium. Note that these ratings are higher than TLF's recommendation of a maximum aquarium size of 150 gallons.

Under the conditions of these tests, TLF's 150 Reactor containing PhosBan quickly removed both phosphates and silica to levels below the detection limits of the spectrometers (for our purposes, the concentration of both silica and ortho-phosphate fell to 'zero').

GFO can potentially remove other chemicals including cobalt, manganese, zinc and nickel (Holmes-Farley, 2008). All (save the latter) are important micronutrients for algal growth. The effectiveness and rapidity of GFO in sequestering these is unknown to me.

Likes:

• Reactor is well made yet relatively inexpensive
• Media is relatively inexpensive and lives up to manufacturer's claims
• Ortho-phosphate and silica are effectively and quickly removed to levels approaching zero
• Recommended pump (Maxi-Jet, not included) is readily available, reliable, inexpensive to purchase and operate
• Device (ball valve) to regulate water flow through the reactor is included
• Directions are appropriate and easy to follow
• The reactor can be used with other media, such as activated carbon

Dislikes:

• Effectiveness of any GFO product in removing some 'trace elements' (Mn, Zn, Co, for example) from artificial seawater is unknown
• Instructions not available on TLF's website (I routinely misplace directions and did so for a while during the course of writing this article)

Conclusion

TLF's 150 Reactor and PhosBan media performed as advertised. Under the conditions of this testing, both phosphate and silica concentrations fell to levels below the detection limits of 'laboratory grade' instruments.

There is some concern about depleting reactive phosphate levels too quickly and/or for extended periods of time (remember Liebig's Law of the Minimum discussed earlier). Proceed slowly with the application of any product in a reef aquarium and closely watch corals for negative reactions.

While reactive phosphate is effectively removed, the same cannot be said about the form of phosphorus (acid hydrolysable) that requires vigorous laboratory digestion processes before analysis. Information suggests that almost all of the acid-hydrolysable phosphorus is suspended - not dissolved - in the water column and this form should not be of much concern to hobbyists.

The media reactor is well made and should provide years of service. The adsorption material is relatively dust-free (although it should be flushed with water per the manufacturer's recommendations) and has not crumbled and added any apparent particulates or color to the aquarium water. In addition, there were apparently no significant negative impacts on water chemistry even when used at twice the recommended amount (however I would strongly advise hobbyists to follow the manufacturer's directives).

The successful application of these products in aquaria around the world is a testament to their functions. If you're battling algae outbreaks and need a quick solution, give these products a try.

Materials and Methods

A 55-gallon (208 liters) was filled with 48 gallons (182 liters) of well-aged artificial seawater from a fish-only aquarium. A PhosBan Reactor 150 was installed according to manufacturer's recommendations. Since the suggested amount of PhosBan for 48 gallons of seawater did fill the reactor to the minimum recommended level, the dosage was doubled to 100 grams of PhosBan phosphorus adsorbent to fill the reactor to the recommended depth of 2 inches (~50mm). A Maxi-Jet 400 provided flow, which was regulated to 19.6 gallons per hour (~1,240 milliliters per minute) using the supplied in-line ball.

Baseline water samples from the aquarium were gathered in PO₄^-3-free bottles, and again at 0.5, 1, 2, 3, 11.5, 21, and 36 hours after starting the reactor's pump. These were analyzed for Total Phosphorus and Reactive Phosphorus using a Hach 2800 Spectrometer, DRB 200 Dry Thermostat Reactor, and reagents from Hach (potassium persulfate and sulfuric acid for digestion, and ascorbic acid method for analyses). Hach advertises the range of the ortho-phosphate analysis to be 0.02-2.50 ppm and total phosphate as 0.06-3.50 ppm (both expressed as PO₄^-3). Alkalinity (as calcium carbonate; CaCO_3;) was determined using a Hach Digital Titrator and alkalinity reagents, using a calibrated pH meter and probe to determine titration endpoints (which varied from 4.5 to 4.8, depending upon phosphate concentrations and the presence or absence of silica) . A Hach HQ40d data logger equipped with a pH probe measured and recorded pH and water temperature every 5 minutes. Silica was determined by a LaMotte Smart2 colorimeter using LaMotte silica reagents (high range - 0-75 ppm, using ammonium molybdate in an acidic state, with oxalic acid added to prevent a reaction with phosphate and molybdate).

References

1. American Public Health Association, 1998. Standard Methods for the Examination of Water and Wastewater. Washington, DC.
2. Holmes-Farley, R., 2008. Reef Alchemy: Iron oxide hydroxide (GFO) phosphate binders. Reef Keeping Magazine Online.

The nitrogen cycle plays a highly important role in a closed environment like that of an aquarium. Due to its presence, it is possible keep the fish and invertebrates alive, in a small viable space, therefore it is fundamental to learn to know it, mainly in respect of the life forms that we nurture.

Until a few years ago, it was thought that the nitrogen cycle in its complexity, was a complete linear process. However, most recent scientific discoveries have greatly revolutionized our well-established knowledge on the nitrogen cycle and on the micro-organisms involved in such processes. As a matter of fact, the global cycle of nitrogen in the environment, particularly in that of marine, has been integrated with at least three new links which include:

1. the oxidation of ammonium by a particular group of micro-organisms, the archaeabacteria (AOA);
2. the anaerobic reduction of nitrates into ammonium ion (DNRA);
3. the anaerobic oxidation processes of ammonium (ANAMMOX).

In the first part of this article, I will try to review the essential and more predominant aspects of the nitrogen cycle: the transformation processes of the main components (atmospheric nitrogen, ammonium ion, nitrite, nitrate) and the role played by the bacterial species involved.

In the second part, new ways will be explored with particular reference on the role of bacteria, focusing on the implications that these new discoveries have brought in the global cycle of nitrogen.

The Canonical Nitrogen Cycle

Nitrogen (N) is an essential nutrient for all organisms, and it is a critical element of protein, vitamins and DNA, and is important in biochemical structures and process that define life.

Nitrogen exists in different states of oxidation and in many chemical forms and is quickly converted by the microorganisms both on earth and the sea.

In the marine environment, nitrogen is present in 5 forms:

1. Gaseous nitrogen (N₂), stable molecules that require specialized enzyme systems (present in some types of bacteria) for fixation and later use;
2. Ammonium ion (NH₄⁺), the most reduced natural specie of nitrogen, and the most biologically available in an oxygen-less environment;
3. Nitrate ion (NO₃^-), the most oxidized form of nitrogen and mostly usable in an aerobic environment;
4. Particulate organic nitrogen (PON), organic form of nitrogen predominant in sediments;
5. Dissolved organic nitrogen (DON), a rich mixture of molecules with a wide range of composition.

A complex network of reactions links these nitrogen forms in processes that as a whole, is called the nitrogen cycle (Figure 1). The greatest source of nitrogen comes in the form of inert gas N₂ (N ≡ N), representing 78% of the atmosphere. A small part of the atmospheric N₂ is fixed by particular bacteria called nitrogen-fixing (nitrogen fixation) and is reduced to ammonium ion (NH₄⁺) which can be easily usable for other organisms. In a marine environment which inhabited by particular bacteria, ammonium is quickly oxidized to nitrate in aerobic conditions (nitrification). Nitrate is then reduced again to an N₂ gas in anaerobic conditions (denitrification), thereby completing the cycle (Figure 1).

Nitrogen Fixation and Ammonification

Nitrogen fixation

The biological fixation of nitrogen can be synthetically represented by the following global formula:

N₂ + 8H⁺ + 6e^- → 2NH₄⁺

Which means that for each molecule of atmospheric nitrogen, 2 ammonium ions are formed with the absorption of 6 electrons and 6H+, this last process tends to increase the pH.

It is interesting to note that, ultimately, the ammonium ion in the water is in balance with ammonia (NH3) based on the following stoichiometry:

NH₃ + H₂O ↔ NH₄⁺ + OH^-

The concentration of the two chemical species relies largely on the pH, in short, the higher the alkalinity, the larger will be the quantity of ammonia; or proportion-wise, the lower the pH is (more acid), the larger will be the quantity of ammonium ion (less toxic than ammonia). As can be seen in Figure 2, in an average range of pH in the seawater, the percentage of NH4+ is higher (82-97%) compared to that of NH3 (3-18%).

As we have previously stated, the atmospheric nitrogen N₂, before being incorporated into the biological molecules, has to be reduced to NH₄⁺, through a series of reactions called biological fixation of nitrogen. Such reactions are catalyzed by a particular enzyme, nitrogenase, which is present in some nitrogen-fixing bacteria belonging mainly to Cyanobacteria phylum. One of the peculiar characteristics of this enzyme is that it comes irreversibly inhibited by the molecular oxygen (O₂); and since fixation is a process that happens in an aerobic environment, it creates an apparent paradox. In reality, cyanobacteria are able to negotiate the activities of nitrogenase, an enzyme which is essentially anaerobic, with the inevitable presence of oxygen (resulting from photosynthetic processes), through not yet well-known mechanisms. In the marine environment, the nitrogen-fixing bacteria (some of which also belong to Clostridium and Azobacter genera) can be found both in free form and in symbiosis with other organisms (ex. Sponge).

But what is the source of nitrogen in an aquarium? Certainly, the biological fixation of nitrogen is an extremely important process in the ocean, but it has a limited role in the tank.

The main source of nitrogen is obtained from the nourishment both of the fish and invertebrates, particularly in the form of protein and single amino acids, assuming they are directly administered into the tank. Even minor vitamins and other molecules like the DNA, contain nitrogen but the quantity is decisively less than that of protein's. In proteins, nitrogen forms a part of the framework and of some single amino-acids' lateral chains, as the tryptophan, asparagin, glutamine, lysin, arginine, histidine.

The oxidative degradation of amino acids leads to the release of ammonia nitrogen into the tank. In what way? On one hand, the protein ingested by the fish or by the other organisms are broken down into single amino acids. In turn, amino acids can be used to build new proteins within the organism or be oxidized to supply energy. The degradation of amino acids by the animals leads to the elimination of varied by-products. For example, the fish release nitrogen as ammonia, while the majority of organisms may release it in the form of uric acid (fowls, reptiles), or urea (humans). On the other hand, in the presence of a strong organic charge, protein and amino acids in waste products, in sediments and in organic decay are decomposed in a process called ammonification, carried out by particular decomposer bacteria which release ammonium into the water by degrading the aminoacidic nitrogen.

Nitrification

Nitrification occurs in two distinct stages:

1. oxidation of ammonium to nitrite (nitrosation) and
2. oxidation of nitrite to nitrate (nitration).

1) Nitrosation: in the first stage, ammonium ion is oxidized to nitrite in two steps:

1. The first step is catalyzed by the enzyme, monooxygenase which forms the hydroxylamine by using O₂ as oxidant:
   2NH₄⁺ + O₂ → 2NH₂OH + 2H⁺
2. In the second step, hydroxylamine is oxidized to nitrite by the enzyme hydroxylamine-dehydrogenase:
   2NH₂OH + 2O₂ → 2H⁺ + 2H₂O + 2NO₂^-

2)   Nitration:  the oxidation of nitrite to nitrate, which occurs through the activity of the nitrite oxidase enzyme, completes the process of nitrification:

2NO₂^- + O₂ → 2NO₃^-

The conventional view of nitrification occurs in the presence of oxygen and anticipates the oxidation of ammonium to nitrate based on the following global synthetic formula (see Figure 1):

2NH₄⁺ + 4O₂ → 4H⁺ + 2H₂O + 2NO₃^-

But who directs the music? The metabolic work of nitrification is entrusted to two groups of nitrifying bacteria:

1. bacteria which oxidize ammonium (ammonia-oxidizing bacteria or AOB), also called nitrous bacteria. They belong chiefly to the Nitrosococcus and Nitrosomonas species;
2. bacteria which oxidize nitrite (Nitrite-oxidizing bacteria or NOB) also called nitric bacteria. They form a part of the Nitrobacter, Nitrococcus and Nitrospina species.

The nitrifying bacteria are generally obliged aerobes and obliged chemoautorophs because they directly use CO₂ as a source of carbon, while organic substances can be toxic.

Denitrification

Here I describe the four stages of denitrification process in detail. The oxidation state of nitrogen is indicated by the enclosing parentheses, after the names of chemical species.

1. Reduction of nitrate (+5) to nitrite (+3). This reaction is catalyzed by nitrate reductase (NAR) which exists in the (internal) cytoplasmic part of bacterial membrane. Nitrate is carried within the bacterial cell by a specialized carrier (AP in Figure 3), defined as antiport because it exchange ion nitrate upon entry with the nitrite which is produced in the reaction and must be carried to the (external) periplasmatic space for the subsequent reaction.
   2NO₃^- + 4H⁺ + 4e^- → 2NO₂^- + 2H₂O
2. Reduction of nitrite (+3) to nitric oxide (+2). The nitrite which is now at the periplasmatic space is reduced by nitrite reductase (NIR), releasing nitric oxide (NO). NO is a remarkably important molecule, from the bacteria to humans (but this is another story).
   2NO₂^- + 4H⁺ + 2e^- → 2NO + 2H₂O
3. Reduction of nitric oxide (+2) to nitrous oxide (+1). NO is reduced by nitric oxide reductase (NOR) to nitrous oxide (also called nitrogen protoxide otherwise known as the laughing gas). Both oxides represent a strong stimulus to the reductase synthesis in the presence of nitrates and under anaerobic conditions.
   2NO + 2H⁺ + 2e^- → N₂O + H₂O
4. Reduction of nitrous oxide (+1) to gaseous nitrogen (0). The last reaction in the denitrification process is the reduction of nitrous oxide to molecular nitrogen in gaseous form by the nitrous oxide reductase. This reaction should complete the denitrification process and conclude the nitrogen cycle.
   N₂O + 2H⁺ + 2e^- → N₂ + H₂O

Denitrification is one of the key processes within the nitrogen cycle and anticipates the reduction of nitrates to gaseous nitrogen, passing through nitrite, nitric oxide (nitrogen monoxide) and nitrous oxide (nitrogen protoxide).

The global reaction of denitrification (without considering the organic molecular degradation eventually associated) can be synthesized with the following formula (for details see Figure 1):

2NO₃^- + 12H⁺ + 10e^- → N₂ + 6H₂O

Denitrification is mainly a heterotrophic option and occurs in anaerobic conditions. A wide range of bacteria called precisely denitrifying bacteria are able to carry out the entire sequence of reactions, being equipped with a complete enzyme apparatus.

The denitrifying bacteria are able to accomplish the anaerobic respiration of nitrates by using the nitrate in place of oxygen, as acceptor of the electrons released during the respiratory process. These bacteria possess special enzymes (Figure 3), as the nitrate reductase (NAR) and nitrite reductase (NIR), which allows the electrons to flow towards nitrate or nitrite, in the absence of oxygen. They are flexible enzymes which form in the cellular membrane only under anaerobic conditions: as a matter of fact, a part of NAR, the reductase synthesis is inhibited in the presence of oxygen.

Some bacterial species of the Pseudomonas, Thiobacillus, Paracoccus and Naisseria classes, are considered denitrifying.

New Developments and New Prospects

The previous description represents a well-known scenario for a long time. In the course of the recent years however, our references concerning the nitrogen cycle have drastically changed to the extent that the principle of closed linear cycle itself is being questioned. This is because new reactions have been discovered and consequently new microorganisms that make the entire nitrogen cycle even more complex and twisted (Figure 4). In the second part of this article, I will try to clarify some important ways which will be inserted within the canonical nitrogen cycle:

1. Ammonium oxidation by a particular group of microorganisms, the archaeabacteria (AOA)
2. The anaerobic reduction of nitrates to ammonium (DNRA):
3. The anaerobic oxidation processes of ammonium (ANAMMOX)

AOA: Ammonium Oxidizing Archaeabacteria

Recently, new important components of nitrogen cycle, which form the part of the richer and diffused group of micro-organisms in the planet, the archaeabacteria, have been identified. In spite of the group's evolutive line being unclear, the archaeabacteria (Archaea or Archeobacteria) combined with the eukaryotes and with eubacteria, are some of the fundamental domains of the living beings.

The archaeabacteria, like the bacteria, consist of single cells without nucleus and in the past they were classified as prokaryotes together with the bacteria. Based on the DNA analysis, the archaeabacteria were re-grouped into three phyla: Crenarchaeota, Euryarchaeota and Korarchaeota. The Euryarchaeota bacteria are the most prominent and they include methane producers and holophiles. The Crenarchaeota bacteria include thermophilic microorganisms, while the Korarchaeota bacteria are less known because only their DNA is recognized but no microorganism has so far been isolated. Originally, it was thought that the archaeabacteria were just inhabitants of a harsh and most hostile environment on the face of the earth. The thermophiles can grow at a temperature higher than 100°C, the psychrophiles are those which grow at temperatures lower than -10°C, while the acidophilus and the alkaliphiles grow in extremely acidic or alkaline environments, respectively. Finally, the halophiles prefer the highly saline environment. Today, we know that archaeabacteria are present in all habitats: for example the Crenarchaeota bacteria are considered ubiquitous components of zooplankton.

In 2004, a particular gene called the ammonium mono-oxygenase (amoA) was discovered in marine Crenarchaeota, indicating the capacity to oxidize ammonium. The definite and convincing link between this new gene and the ammonium oxidation in archaeabacteria has been recently established in Crenarchaeota, the Nitrosopumilus maritimus, which was isolated from the water of aquarium. N. maritimus is chemoautrophic: as a matter of fact it grows with bicarbonate as the only source of carbon (organic carbon inhibits its growth) and converts NH₄⁺ in NO₂^- (green line in Figure 4 and Figure 7). Other archaeabacteria have been successively identified with this property and have been named as Ammonium oxidizing archaeabacteria (AOA). An accurate analysis of the AmoA gene in many archaeabacteria has revealed diverse isoforms of this gene, each one is associated to a microorganism which is present in different habitats (with little overlapping, for example, between sediment and water column). Symbiont archaeabacteria have also been identified, like for example the Cenarchaeum symbosiosum, a symbiont Crenarchaeota with a sponge. Surprisingly, it was observed that this archaeabacteria is not able to produce hydroxylamine as intermediate reaction (see the Nitrosation process in the BOX 2) indicating that ammonium oxidization occurs with a mechanism which is different from that of the classic nitrification. Finally, the most recent studies conclude that the majority of Crenarchaeota are AOA's and AOA's are microorganisms whose presence is numerically predominant in the ocean.

DNRA

During the recent years, the anaerobic reduction of nitrates/nitrite or, DNRA (acronym for Dissimilatory Nitrate/nitrite Reduction to Ammonium) has stirred up a certain interest as a relevant reaction both in the terrestrial and marine eco-systems. The reaction has been described in anoxic sediments and in the presence of bacteria of the Thioploca and Thiomargarita species. Both types of bacteria are able to concentrate nitrates within their own cells for the subsequent oxidation of sulphur-containing compounds in reduced form. In this way, they are able to reduce nitrate to ammonium passing through nitrite as an intermediary compound (blue line in Figure 4 and Figure 7). This reaction, although still needing clarification, would potentially supply nitrite and ammonium to the ANAMMOX reaction (see subsequent paragraph) in anoxic sediments.

ANAMMOX: anaerobic ammonium oxidation

As we have previously seen in the description of AOA and nitrification stages, ammonium oxidation is a strictly aerobic process. In reality, we have also seen that ammonium can be generated in hypoxic and anoxic environment (for example in sediments) through the re-mineralization of organic nitrogen process (ammonification), and/or the anaerobic nitrite reduction (DNRA). For many years, it has been thought that ammonium is inert in anaerobic conditions, which is to say, useless for living things. The problem, however, is that no bacteria that are able to metabolize ammonium without oxygen have been identified, especially due to the technical difficulty of cultivating in the laboratory bacterial strains with these characteristics. In 2008, many of these difficulties have been overcome and some laboratories were able to identify, cultivate and characterize some types of ANAMMOX bacteria (Acronym for ANaerobic AMMonium OXidation) which are capable of oxidizing ammonium to gaseous nitrogen (N₂) (red line in Figure 4 and Figure 7) by using nitrite as electron acceptor, instead of oxygen.

The first bacteria that are isolated in a marine environment belongs to the Scalindua species (Sc. sorokinii) although a probe regarding the presence of other species like Brocadia and Kuenenia, is being conducted. The common characteristic of these bacteria, unique in its class, is the presence of a specialized organelle called anammoxosoma which is surrounded by a particular lipid (fat) that contains hydrazine oxide reductase, an exclusive enzyme which is able to combine nitrite and ammonium is a single step (Figure 6). These bacteria use a rather complex mechanism that involves hydrazine as an intermediary. However, the following reaction, which is incomplete and stoichiometrically unprecise, can suggest the idea of an ANAMMOX process.

NH₄⁺ + NO₂^- → N₂ + 2H₂O

This reaction has been described for the first time in sediment samples taken from particular marine ecosystems (the Black Sea, for example). It has been observed that in those experimental conditions, the ANAMMOX process was responsible for the loss of 30-50% of inorganic nitrogen from the sea (in the form of N₂), significantly making it of the same level as the classic denitrification. But to analyze it closely, if we associate the above-mentioned DNRA process with ANAMMOX, we have a real and actual anaerobic denitrification, clearly not canonical. Indeed, DNRA supplies nitrite (as a reaction intermediary beginning from nitrate) and ammonium (the latter having been obtained also through organic nitrogen ammonification), and ANAMMOX transforms everything into gaseous nitrogen. A lovely and good denitrification, clearly with different mechanisms and bacterial strains, but nevertheless, a denitrification.

The question now is: under analogous conditions, can ANAMMOX occur in an aquarium? Obviously we are not able to establish that but we can make some considerations. A driven DSB can reproduce optimum conditions for this process. In fact, nitrogen bubbles are visible in the depths of the sediment. In line with this, it has been experimentally observed (but not in an aquarium) that the higher the layer of sediment, the more pushed the anoxic condition (oxygen inhibits the reaction) and the faster is the reaction. Therefore the high efficiency of a DSB in removing nitrates can be also due to a non-canonical denitrification, besides the classic anaerobic denitrification; obviously, the eventual presence of qualified bacterial strains has yet to be defined. Figure 7 illustrates the schematic diagram of integrated nitrogen cycle with the new reactions and the areas in which they occur.

In conclusion, these constant discoveries clarify many obscure points of the nitrogen cycle but at the same time they open up new horizons and paradigms that make these processes even more complex and fascinating. On the other hand, I hope not to have further complicated the well-established ideas on nitrogen cycle, with this article. I also wish that over time, these new discoveries can be applied and integrated with the aquarium, in spite of the scarce scientific researches in this area. However, even if just theoretical, this knowledge allows us to better understand that which can occur in our tanks and nurture our passion for aquarium.

References

1. Arrigo K. R. (2005) Marine microorganisms and global nutrient cycles. Nature 437: 349-355.
2. Berman-Frank I., Lundgren P., & Falkowski P. (2003) Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res.Microbiol. 154: 157-164.
3. Brandes J. A., Devol A. H., & Deutsch C. (2007) New developments in the marine nitrogen cycle. Chem.Rev. 107: 577-589.
4. Francis C. A., Beman J. M., & Kuypers M. M. (2007) New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. ISME.J. 1: 19-27.
5. Jetten M. S. (2008) The microbial nitrogen cycle. Environ.Microbiol. 10: 2903-2909.

Acknowledgements

Marco Colasanti aka marcola62 (moderator of Reefitalia forum). A special thanks is owed to the staff of ReefItalia community for their support.

About the Author

MARCO COLASANTI, born in Rome, Italy, September 15, 1962, has a degree in biology and holds a Ph.D. in Neuroscience. He is currently a Full Professor in Cell Biology at the Department of Biology, Faculty of Sciences of the University of Rome, ITALY. Entrusted with holding courses of Cellular Biology and Laboratory of Cellular Biotechnology for the University Degree of Biology. He entered the aquarium hobby with freshwater tanks (1982) and set up his first saltwater tank in 1995. He is currently a Staff Member as a Moderator of ReefItalia, an Italian reef community. Over the last fifteen years, active in the scientific research on Nitric Oxide (NO) pathway in different models and systems, including fish and invertebrates. Co-author of more than 70 publications in international peer-reviewed ISI journals or books.

Departments of Chemistry (Ken S. Feldman, Allison A. Place) and Industrial and Manufacturing Engineering (Sanjay Joshi), The Pennsylvania State University, University Park, Pennsylvania 16802, and Route 66 Marine, Gardena, California (Gary White)

1. Introduction

Bacteria are ubiquitous in the marine environment and they play absolutely decisive roles in every conceivable ecological niche. Numerous studies have documented the impact of bacterial action on life processes and energy transduction in natural reefs, as detailed below. However, a corresponding influx of information about bacteria biology in our captive marine aquaria has been lacking. For example, the inextricable connection between Total Organic Carbon (TOC) in the natural marine environment and one it its major consumers, bacterioplankton, constitutes the most fundamental level of the marine food web (Johannes, 1967; Ducklow, 1979; Eppley, 1980; Ducklow, 1983; Gottfried, 1983; Moriarity, 1985). Thus, bacterial grazing on this carbon-rich food source is an absolutely obligatory first step in the incorporation of this central nutrient into the food chain. In addition to a carbon source, bacteria require nitrogen and phosphorus compounds in significant quantities along with trace amounts of many other elements, perhaps the most critical of which is iron. Deficiencies of any of these macro- or micronutrients can, in principle, serve as a limiter of growth (details below).

On the other hand, much less is known about bacterial nutrient needs in the captive environment of a reef aquarium, and in fact there have not been any studies that document bacterial growth responses to any specific nutrient in a reef tank. Nevertheless, one of the more recent aquarium nutrient export methodologies is based upon the hypothesis that bacteria growth in reef aquaria is carbon limited. This methodology has been dubbed "Carbon Dosing" (Walton, 2008; Michael, 2008), and it has three basic premises:

1. Bacterial growth in reef aquaria is carbon source limited.
2. Adding a digestible carbon source therefore will spur bacteria population growth, and with that growth, other necessary nutrients, like nitrogen- and phosphorus-containing compounds, will be scavenged from the water column.
3. Mechanical filtration via protein skimming (and possibly Granular Activated Carbon (GAC)) will remove bacteria from the aquarium water column, and with them their C/N/P nutrient load.

Thus, according to this approach, the export of undesired nutrients (nitrogen and phosphorus compounds) will be achieved upon carbon dosing. Much variation in carbon sources, including vodka (EtOH), sucrose (table sugar), vinegar (acetic acid), and solid biopellets (biodegradable polyesters), have been promoted. In addition, media that are alleged to facilitate bacterial biofilm formation, like Zeolites, have been incorporated into this emerging technology. Of course, bacteria export by skimming requires that the bacteria actually reside in the water column and not as a film on a solid support, and so mechanical mechanisms to dislodge the biofilms from these media have been engineered into the process, such as agitation of the (Zeolitic) media on a frequent basis. Finally, several commercial proponents of this methodology offer pre-packaged bacterial mixes and bacteria foods as starter kits. However, despite the proselytizing of its advocates, this approach to nutrient export rests entirely on the untested hypotheses that are listed above. Does it really work as advertised?

Our earlier research on the topic of carbon nutrient levels in marine aquaria (Feldman, 2008; Feldman, 2009; Feldman, 2010) has provided experimental documentation for four conclusions that impact on TOC management in our reef tanks:

1. Reef aquaria utilizing active filtration (GAC, skimming) maintain equilibrium TOC levels within the range found on healthy tropical reefs.
2. Protein skimming (i.e., bubbles) is not very effective at removing TOC from aquarium water, depleting typical reef tank water of only ~ 20 - 35% of the post-feeding TOC present.
3. GAC filtration is quite effective at stripping reef tank water of its TOC load, removing 60 - 85% of the TOC present.
4. And, quite intriguingly, the natural biological filtration, which starts with bacteria and other microbes, is remarkable in its capacity to remediate reef tank water of TOC, easily removing 50% or more of the post-feeding TOC increase in tank water.

Conclusions (2) and (3) describe the consequences of mechanical filtration on TOC levels, but the 4th conclusion emphasizes the importance of the "hidden" part of the remediation equation, bacterial predation, for gaining an understanding of the dynamics of carbon commerce in our aquaria. In fact, this observation, coupled with the advent of Carbon Dosing strategies for nutrient export, led to a new series of questions regarding the perhaps pivotal role of bacteria, or at least skimmable water column bacteria, in successful reef aquarium husbandry.

1.1 The goal of our study - testing the validity of the Carbon Dosing hypothesis

With this information as preamble, we set out to explore the following questions:

1. What are the bacteria populations in the water column of reef tanks, and how does that value compare with bacterial counts in authentic reef water?
2. Does carbon dosing indeed increase water column bacteria populations (i.e., is growth carbon limited)?
3. Does mechanical filtration (protein skimming and/or GAC filtration) actually remove bacteria from the water column, and if so, how much?

1.2 Bacteria: A general introduction

Bacteria are found everywhere on the Earth's surface where water is at least temporarily available. Their ubiquity, along with their sheer numbers and fast growth rates, means that they are extremely important geochemical agents. By facilitating aqueous oxidation-reduction processes, concentrating metal cations and anions, creating stored chemical energy, decomposing organic material, and mediating mineral precipitation and dissolution, they drive the Earth's biogeochemical cycles. Similarly, bacteria are essential agents for the biogeochemical processes that take place in marine aquaria. These biogeochemical processes include decomposition of organic matter, nitrification, denitrification, nutrient and element assimilation, sequestration and release ("cycling"), and chelation and the formation of complexes with a wide variety of ions and compounds.

As recently as the mid-1960's, the scientific community was not entirely convinced that "marine bacteria" existed as a distinct group. It turned out that marine bacteria have special requirements for inorganic ions to supply their needs for growth and metabolism, as well as for maintaining the integrity of their cells. They have a highly specific need for sodium, a need for higher concentrations of potassium, calcium and magnesium than are typically available in either freshwater or terrestrial environments, and exhibit a tolerance to halide ions that many other groups of bacteria lack. Many strains of marine bacteria have a preference for amino acids as sources of carbon, nitrogen, and energy, and require vitamins and other growth factors (MacLeod 1965). It is because of differences in cell wall composition, ionic requirements, and tolerances of inorganic ions that the majority of bacteria associated with freshwater aquaria cannot survive a direct transition to marine aquaria.

Bacterial Physiology

Bacteria are the most physiologically diverse group of organisms in a marine aquarium. They are unicellular (they do not differentiate into multicellular forms) prokaryotic (they have no nuclear membrane, and their DNA is not organized into chromosomes) and they reproduce by cell division. A typical bacterial cell is about 1 micrometer (uM) in diameter or width, compared with most eukaryotic cells that vary from 10 to 100 uM in diameter. Bacteria demonstrate three general shapes: rods ("bacilli"), spheres ("cocci"), and helical shapes ("spirilla" or "vibrio"). When individual cells grow end on end, they form what are known as filaments. They have five characteristic structural components: Unbound DNA ("nucleoid"), ribosomes (protein-making "factories"), a plasma membrane, a cell wall, and a surface layer which may or may not be an inherent part of the cell wall. These structures are arranged into three architectural regions: (1) a cytoplasmic region that is typically over 70% water and near pH neutrality. This region contains the cell's genetic material, ribosomes, gas vacuoles that confer buoyancy to the cell, and various "inclusions" and granules that serve as specific storage sites. The soluble portion ("cytosol") contains a variety of small organic molecules and dissolved inorganic ions; (2) a rigidifying cell wall on top of a plasma membrane that provides support for the cell, protection from external physical stresses, and governs the cell's shape ("morphogenesis"). This region also acts as a filter by controlling the passage of dissolved molecules into the cell. The cell wall also may display attached appendages including flagella (filamentous protein structures to generate movement in motile strains), and fimbriae & pilli (hair-like proteins, which exhibit a wide diversity of form and arrangement, involved in the attachment of cells to surfaces, substrates, and other cells); and (3) a surface layer. The three major types of surface layers are extracellular polymers ("capsules"), sheaths, and S-layers. This region's functions include (a) protection from environmental and pH extremes, (b) attachment & adherence to surfaces, (c) stabilizing biofilm communities, (d) storage of carbon and energy reserves, (e) binding of metals, (f) formation of minerals that can serve as chemical buffers at the cell's periphery, (g) alteration of light dispersal patterns, and (h) the masking of the electrochemical charge characteristics of the cell wall, thereby mediating physiochemical reactions between the cell and the ions & solids of the external environment (Konhauser, 2007).

Bacterial Surface Charge and Protein Skimming

Extending from the cell wall and surface layer regions are organic "functional groups". Functional groups are specific collections of atoms bound together into well-defined structures; these structures typically confer one distinct chemical reactivity pattern to the collection of atoms. Many of these organic functional groups can either bind hydrogen ions ("protonation") or release hydrogen ions ("deprotonation"), depending upon the pH of the bacteria cell's immediate environment. The combined protonation and deprotonation states of the functional groups on the cell wall largely determine the hydrophobic and hydrophilic characteristics of a bacterial cell at any given pH. Functional groups may also act as ligands that can bind with other ions or compounds to form larger molecular complexes. For example, it has been estimated that as much as 99% of the dissolved iron in ocean surface waters is bound to organic ligands (Granger, 1999). Such complexes also may impart a charge to the cell wall, thereby affecting the cell's hydrophobic-hydrophilic characteristics. Functional groups may be specialized to act as chelating agents for the capture and transport of specific ions ("siderophores"). The resultant complex may also affect surface charge.

A bacterial cell's surface charge affects its hydrophobic/hydrophilic characteristics and therefore that cell's potential to be removed from solution by foam fractionation ("protein skimming"). Bubble-based removal of particles like bacteria, as well as dissolved organic carbon (DOC), is largely dependent upon the hydrophobic character of the particle/molecule; the more hydrophobic patches, the greater the binding energy to the bubble's surface, and hence the greater likelihood for bubble-based removal (Feldman, 2010, and references cited therein). The addition of inorganic salts to a solution containing bacteria has been demonstrated to increase the rate of removal of bacterial cells from that solution (Gaudin, 1960 & 1962). Magnesium chloride and calcium chloride have been demonstrated to increase the concentration of bacterial cells in the foam generated by protein skimming (Bretz, 1966). Differences between the types of functional groups present on the bacterial cell walls, bacterial cell surface charge, response to ambient pH, and the concentration of inorganic salts all influence the degree to which a bacterial cell is available for export via protein skimming. This influence occurs independently of the physical operating characteristics of a given protein skimmer.

1.3 Bacterial life processes

Bacterial Metabolism

Bacterial metabolism describes the set of chemical processes that allows bacteria to grow, reproduce, maintain structures, and interact with their environment. The physical and genetic characteristics of bacteria profoundly influence their metabolism. The small size of bacteria allows them to acquire external energy and nutrients at the cell's periphery and to optimize transport processes into/out of the cell. These transport processes include passive & facilitated diffusion, active transport, and engulfing materials with their cell membrane (endocytosis). Bacteria synthesize proteins to regulate every phase of their metabolic behavior. The types of proteins that a bacterium can synthesize are determined by its genetic characteristics.

Bacterial Growth

Bacterial populations in marine aquaria may complete their life cycles entirely as isolated, or semi-isolated, strains within the system's water column ("free-living"), or attached to surfaces in a colony consisting of multiple strains ("biofilms" or "mats"). Bacteria populations demonstrate a growth cycle with four stages: (1) a "lag" phase, which is the initial period of time within which cells adjust to their new surroundings and synthesize proteins in response to that environment; (2) an "exponential growth" phase during which the population demonstrates a rapid increase in biomass; (3) a "stationary" phase during which there is no net increase or decrease in overall cell number, and groups within the population may be either metabolically active or inactive; (4) a "death" phase during which the number of viable cells decreases. As a population enters death phase, members of the population may compensate by budding and by the formation of protective structures such as exospores, endospores and cysts. Such protective structures have proven to be an extraordinarily successful evolutionary adaptation; for example, Bacillus spores have been resuscitated after preservation in amber for 25 million years (Cano, 1995). Factors characteristic to a bacteria's environment can also regulate bacterial growth patterns. These factors include nutrient availability, temperature, substrate supply, predation, and viral mortality.

Bacterial Nutrients

Marine bacteria utilize a vast group of elements and compounds to meet their energy and nutrient requirements. Marine bacteria have a special need for sodium. Other major elements required for marine bacteria growth and reproduction include carbon, nitrogen, phosphorous, oxygen, hydrogen, sulfur, potassium, magnesium, calcium, and iron. Common trace element requirements consist of manganese, cobalt, zinc, copper, and molybdenum. Chemical groups fulfilling energy and nutrient needs span an even broader range; inorganic carbon compounds, organic carbon compounds (generally, but not limited to, sugars and amino acids), inorganic & organic nitrogen compounds, organic sulfur compounds, inorganic phosphate compounds, and potassium, magnesium, calcium, and iron salts. Marine bacteria can sequester elements, such as potassium, within their cells at concentrations far in excess of the concentration of the element in their environment (Thompson & MacLeod, 1974). Such sequestration presents the possibility of some degree of elemental depletion in a marine aquarium by bacterial export via protein skimming.

Manipulating Bacterial Growth

A "limiting nutrient" is a nutrient that has the ability, though its presence or absence, to restrict the utilization of other nutrients. Bacterial growth rates, bacterial carbon production, and bacterial growth efficiency all increase with the addition of organic carbon supplements in certain groups of marine bacteria (Carlson, 1996). The presence of a readily assimilated carbon source has been demonstrated to increase the uptake of ammonium in certain groups of marine bacteria (Goldman, 2000). The availability of a particular nutrient can not only affect a bacterial population's growth rate, but also the metabolic functioning of the population. The availability of organic carbon has been shown to not only limit the growth rate of denitrifying bacteria, but also to limit the rate at which denitrification takes place (Brettar, 1992). Chemical entities other than organic carbon, such as inorganic phosphate, also can function as limiting nutrients (Rivkin, 1997). Indeed, given the rapid and dynamic shifts in marine bacteria metabolic behavior over time in response to changing nutrient availability, it may be inappropriate to describe marine bacteria as being limited by a single nutrient.

The frequency with which nutrient enrichments are supplemented has the potential to exert a significant effect on bacteria populations. Nutrient regimes of different periodicities have been shown to result in mixed heterotrophic bacterial communities with distinct physiological properties. It also was observed that the interplay between various bacterial strains and other participants within the microbial community may be equally as important as the selective forces of the environment in structuring microbial communities. Perhaps most interestingly, when replicate bacterial cultures were subjected to identical nutrient supplementation, the microbial community functionality was conserved despite the fact that community compositions were significantly different (Carrero-Colon, 2006). Thus, there appears to be a great deal of bacteria community plasticity in response to environmental changes, one of the hallmarks of bacteria as a successful species over evolutionary time.

The Coral Holobiont

A coral's holobiont is comprised of close associations between the coral animal itself, its symbiotic zooxanthellae, and a diversity of associated microbes including bacteria, archaea, algae, and fungi. These associations can take place in the coral's immediate environment, on its surface, within its tissues, and within its skeleton (if present). This paradigm emphasizes the potential contributions of each component to the overall function and health of the coral (Rypien, 2010). The dynamic nature of these relationships can be seen in a comparison between freshly collected corals from the Red Sea region that were then placed into marine aquaria. A microbial community shift in the bacteria inhabiting the surface mucus layer was documented for collected corals when placed into the captive marine aquarium. The differences that emerged between corals from natural and captive environments suggested an adaptation of the mucus bacterial communities to the different conditions (Kooperman, 2007).

Disruptions within a coral's holobiont have the potential to negatively impact the coral's health. Altered bacterial community structures have been linked to both coral disease and bleaching (Kvennefors, 2010). Coral bleaching occurs if the endosymbiosis between corals and their symbiots disintegrates during stress (Ainsworth, 2008). Even so, shifts in the holobiont's bacterial community component may not be a direct cause of coral bleaching. While bacterial communities play important roles in coral stasis and health, environmental stressors appear to be the primary triggers for coral bleaching, and bacterial involvement in patterns of bleaching appear to be the result of opportunistic colonization (Ainsworth, 2008).

"Probiotic" Application of Bacteria

A "probiotic" can be defined as a live microbial adjunct that offers a benefit to animals, algae, plants, corals, or the ambient microbial community. This benefit can be assessed in terms of an improved use of food (i.e., enhanced nutritional value), enhanced disease resistance, or by improving the quality of the ambient environment (Verschuere et al., 2000). The introduction of live bacteria cultures into a marine aquarium may be viewed as a "probiotic" husbandry technique.

Deterministic (non-random) factors influencing the survivability of bacteria that are deliberately introduced into a marine aquarium include salinity, temperature, oxygen concentration, and the quality and quantity of available nutrients. The development of a microbial community within a marine aquarium is also influenced by stochastic (random) phenomena: chance favors organisms that happen to be in the right place at the right time to enter the system and to proliferate if suitable conditions exist. It is this stochastic aspect that explains why different marine aquaria may evolve distinctly different microbial communities despite the appearance of nearly identical conditions. The idea that both environmental conditions and random chance influence the emergence of microbial communities provides the context for the concept of probiotics as biological conditioning and control agents. During a time period in which a stable microbial community in a marine aquarium is still emerging, a single addition of a live bacterial culture may suffice to achieve colonization and persistence in the ambient environment, provided that the strains being added are well adapted to the prevailing environmental conditions. When the environment already carries a relatively stable microbial community, it is more likely that a live bacterial culture will have to be added on a regular basis in order to achieve an artificially stable presence in the microbial community (Verschuere et al 2000).

1.4 Counting bacteria in the water column

Probing all of the issues of bacterial commerce in the reef aquarium as listed in 1.1 above requires access to one specific experimental methodology; the capability to count bacteria in marine water. Enumeration (counting) bacteria in marine water samples proved to be a pivotal early technology in developing an understanding of the ecological significance of bacterial populations in various environmental niches (Robertson, 1989; Button, 1989; Troussellier, 1995; Marie, 1997). Early (and later) attempts to quantify marine bacteria by first culturing them in growth media mimicked the similar successful approaches of medical microbiologists in growing pathogenic bacteria with specific growth media that crudely reproduced the body's environment. Unfortunately, a lack of appreciation of appropriate growth media for "environmental" marine bacteria, as opposed to "human" bacteria, has resulted in culture efficiencies of no better than the 1-10% range (Porter, 2004). This limitation still holds true today. Consequently, alternative counting methodologies, which did not require independent culturing, were explored.

One early and quite successful approach utilized direct counting via epifluorescence microscopy (Hobbie, 1977). This technique involves nothing more conceptually complicated than "manually" counting bacteria made visible via a microscope's optics. To implement this counting methodology, a water sample is filtered through a special polycarbonate filter with 0.2 um pores, and the bacteria laden filter pad is then examined under a special type of microscope. The bacteria that do not pass through the filter then can be counted using any one of several image analysis software packages. The bacteria must be stained in order to visualize them. It is a "direct" technique that provides an absolute count of bacteria present for a given volume of sample. It has the advantages of being relatively fast and technically easy, and it can account for bacterial "clumping" as is sometimes seen. In practice, it suffers from some limitations that can compromise its effectiveness. For example, one problem that arises is the limited lifetime of the "stain" - the light emitted from the microscope will photobleach the staining compound, rendering the bacteria invisible, on a time scale similar to that needed to complete the measurement. In addition, the entire surface of the membrane filter is too large to be examined, and so only selected areas are chosen and counted. Then, various assumptions are made about homogeneity of the sample, and mathematical algorithms are applied to the raw data set to permit extrapolation from the counted regions to the entire filter disk in order to estimate an overall count. Thus, there is a significant level of subjectivity that attends epifluorescence microscopy measurements ("Is that dim spot a bacterium whose stain has faded, or is it junk?" "Did the filtration process lead to a homogeneous distribution of bacteria on the filter, or was there wicking towards the outer rim of the circular disk?"). Nevertheless, an experienced operator can generate a self-consistent bacterial count rather rapidly. As an example, Fig. 1 illustrates the results of a bacteria staining/counting experiment with water from the author's 175-gallon reef aquarium. The bacteria are stained with a dye called SYBR1-Green (more on this dye below), and this photograph represents one "field" of the microscope's view. The entire 25 mm-diameter filter disk is composed of approximately 10,000 such fields. Note that there is significant variation in the size, and in the color intensity, of the green "dots"; which are bacteria?

A second independent technique for marine water bacteria enumeration has emerged recently, once again from the medical microbiology community, as an alternative to epifluorescence microscopy; this newer methodology is called flow cytometry (Robertson, 1989). Flow cytometry, like epifluorescence microscopy, is a direct counting technique that requires initial bacteria staining. SYBR1-Green has emerged as the dye (stain) of choice for a variety of reasons, not the least of which are (1) its relative resistance to photobleaching, and (2) the fact that it turns "on", or glows green, only when it is embedded ("intercalated" is the correct term) into double-stranded DNA, see Fig. 2 (Marie, 1997; Noble, 1998; Lebaron, 1998). Thus, the stain used for these experiments only registers at the detector if it is intercalated in double-stranded DNA as would be found in biological specimens like bacteria, but not in random small particles of junk.

One major advantage of flow cytometry over epifluorescence microscopy stems from the fact that crude water samples can be used without filtration, thus removing the possibility of inadvertent sample sorting and loss of some bacteria. In addition, incorporation of size-specific calibration beads in the sample allows for a crude determination of size, and so it is possible to discriminate between "objects" and exclude those outside of a given (bacterial) size range. Flow cytometry uses a sophisticated fluid transfer system that exploits hydrodynamic focusing in order to pass a bacteria-containing water sample that includes a known quantity of the added calibration beads through an aperture so that particles (bacteria, calibration beads, detritus, etc.) pass through a laser beam single file, Fig. 2. High-end optics collect and then focus the scattered light from each particle. Sensitive photodetectors then discriminate between the scatter and fluorescence from individual particles. The residence time of the stained bacterium in the laser beam is short enough that photobleaching is not a problem. Perhaps more importantly, all of the bacteria in a given sample volume can be counted both rapidly (~ 5 min for a 1 mL sample) and objectively by applying standard counting parameters that do not vary from sample-to-sample, operator-to-operator, or from day-to-day.

A cytometer operator does have to decide which particle-based fluorescence intensities to include or exclude. Bacteria that have lost DNA possibly due to environmental conditions would exhibit very weak intensities and therefore might be missed. However, once the decision is made about what constitutes a baseline for intensities to include in the count, that baseline value remains constant for subsequent samples and experiments. Thus, this consistency, along with the size discrimination capability, adds another level of confidence that the objects counted as bacteria are actually bacteria. Flow cytometry does have some liabilities that are not shared by epifluorescence microscopy; the complicated fluidics can get clogged by detritus in the water column, and bacterial clumps, which are characteristic of some types of bacteria, can be missed. Finally, since cytometry is both a light scattering technique and a fluorescence measuring technique, the minimum size of the particle that can be detected is dependent on the wavelength of the light used and the aperture of the collection optics; in our experiments, wavelengths in the 0.4 - 0.6 um range impose a practical lower limit for distinguishing particle sizes of about 0.5 um, which is towards the lower end of bacteria sizes. Epifluorescence microscopy suffers from this drawback as well; detection limits in microscopy also are limited by wavelength and the quality of the microscope optics. Overall, the speed, convenience, and objective and unbiased counting protocol recommend flow cytometry as a useful technique for marine water bacteria enumeration. More recently, a third direct approach for estimating (not counting) bacteria in marine water samples has been described (Wegley, 2006).

Several reports that compare the results of epifluorescence microscopy-based bacteria counting to flow cytometry-based bacteria counting have confirmed that there are only negligible differences between the measured populations of bacteria in both marine and non-marine water samples (Robertson, 1989; Monfort, 1992). In the experiments that follow, the flow cytometry technique is used exclusively to count bacteria in aquarium (and other) water samples.

2. Experimental Approach

We adapted for our studies procedures reported to be successful for bacteria enumeration in marine water samples (Marie, 1999; Grégori, 2001). The protocol involves collecting a water sample in a sterile vessel, preserving it with formaldehyde solution, adding a precise quantity of 6 um diameter counting beads and SYBR1-Green dye to a measured portion of the sample, and running it on a Coulter XL cytometer (see Fig. 3). Dedicated data analysis software then provides a direct count of both the beads and the "bacteria" in a given volume of water sample. In these experiments, the counted "bacteria" include any particle that meets the following criteria: (1) it is between ~0.5 and 6 um in diameter, (2) it contains double-stranded DNA, and (3) it does not fall into a region of the data plot where control experiments indicate that only debris and/or chlorophyll-containing organisms (i.e., phytoplankton), and not bacteria, show up (more details below). The "countable" size range of ~ 0.5 - 6 um was chosen to overlap with the typical size ranges of bacteria from marine environments; some phytoplankton likely will "leak through" and be counted as well. We did not view including some phytoplankton in the overall bacteria count as a problem, since both of these water column microorganisms have TOC removing capabilities, and also both are likely to serve as food for filter feeders and other predators as well. Larger organisms (diatoms, ciliated protozoa, copepods, etc.) will not be counted by this method, nor will smaller organisms (viruses).

2.1 General experimental

A 25.0 mL water sample was collected in a sterile VWR brand centrifuge tube and immediately 0.5 mL of a 37% aqueous formaldehyde solution as a preservative was added. The tube was capped, shaken vigorously, and placed in a -4^oC freezer pending analysis. If this sample was drawn from an aquarium, an effort was made to avoid surface water. The tube was immersed upside down about 4" - 6" below the water surface, rapidly inverted, and then removed from the aquarium and drained until the volume was 25.0 mL. Other water samples either were collected on site (Florida Keys mangrove estuary) by this procedure, or the sample tube was simply filled to the 25.0 mL mark with the appropriate water specimen (Aquafina, RO/DI/0.2 um filtered, tap water, etc.) prior to formaldehyde addition. The samples were rapidly thawed by immersion in warm water just prior to analysis. After vigorous agitation via a Vortex mixer, three independent 960 uL samples were removed from each tube and placed in sterile 2 mL centrifuge tubes. Into each tube was placed 20.0 uL of a diluted (see below) SYBR1 Green solution and then 20.0 - 60.0 uL of a 3.3 x10⁷ bead/mL solution in 3 mM aqueous NaN₃ (as a bactericidal agent) (volume depended upon the anticipated bacteria count). The SYBR1 Green stock solution was freshly prepared just prior to each run as follows: 20 uL of a commercially available solution of SYBR1 Green in DMSO (Invitrogen; the exact concentration is proprietary information, but the dilution that we used follows their suggested cytometry protocols) was added to 380 uL of an aqueous DMSO solution that was made from 40 uL of DMSO + 360 uL of RO/DI/0.2 um filtered water. This solution was vigorously mixed with a Vortex mixer, passed through two successive 0.2 um filters, and used immediately. The bead solution was prepared by adding 10 uL of a 3.3 x 10⁸ bead/mL stock solution (Polysciences) to 90 uL of 3 mM aqueous NaN₃. This solution was vigorously mixed with a Vortex mixer and used immediately. The 1000 - 1040 uL sample (water sample + beads + SYBR1 Green) was inserted into the Coulter XL inlet and the enumeration run was initiated and monitored by the dedicated software. Note that each water sample was run in triplicate.

2.2 Control experiments and bacterial contamination

Several of the procedures described above were tested for the possibility that contaminating bacteria were introduced, by running appropriate controls. For example, the formaldehyde solution was pre-filtered through a 0.2 um filter prior to use, although we observed that ± this filtration treatment did not lead to any significant difference in bacteria counts for the same water sample. Testing for bacterial introduction via contaminated reagents, or through contamination in the XL cytometer itself, was conducted along with every run. A "blank" composed of pure RO/DI/0.2 um filtered water, either treated with formaldehyde or not, was used for this test. In all cases, the blank bacteria counts were < 10000/mL, and in most experiments < 2000/mL. In comparison, actual bacteria counts in marine water were on the order of 10⁵ - 10⁶ bacteria/mL. The effect of freezing the samples on bacteria counts was tested by splitting a sample between fresh and frozen, and running both samples on different days. No significant differences between fresh and frozen bacteria counts were observed. On the other hand, leaving a formaldehyde-treated sample at room temperature for several days before analysis led to an approximately 10-20% decrease in the bacteria count compared to a freshly run sample.

2.3 Data workup

The Coulter XL Flow Cytometer has sophisticated internal electronics that aid in one of the primary functions of the instrument in addition to strictly counting; "gating", or discriminating among the particles in the sample in order to analyze the specific species of interest, in this case bacteria and other microbes of related size. Because flow cytometry counts particles irrespective of sample volume, beads with known concentrations are counted in the same volume as the bacteria, thus permitting the determination of the sample volume holding a certain count of bacteria (=> bacteria "concentration"). The use of the fluorescent indicator SYBR1-Green provides the means to identify and then count "objects" that correspond to bacteria. In addition, the added 6.0 um diameter calibration beads, which are chosen to have physical characteristics that allow them to be easily distinguished from bacteria, are identified and counted. The emission spectrum of SYBR1-Green bound to double stranded DNA is illustrated in Fig. 4 below. In order to discriminate between actual bacteria and similarly sized detritus and phytoplankton (to a degree), we measure and plot the intensity of the green fluorescence (530 nm, the top of the large peak of the green line) vs. the red fluorescence (the signal at 610 nm). Note that for SYBR1-Green, the green/red signal strength ratio is a constant, ~ 7:1. Thus, any increase in green fluorescence intensity (530 nm), such as might accompany, for example, multiple and independent binding of several SYBR1-Green molecules to the double stranded DNA of a single bacterium, should be matched by a proportional increase in the red (610 nm) signal; that is, plotting green vs. red fluorescence intensity should produce a straight line. In actuality, it produces an elliptical region on the red/green plot illustrated in the representative cytogram shown in Fig. 5c below. Note that damaged or stressed bacteria might have less highly coiled DNA, which could alter the green/red ratio as well.

The drawn perimeter in this Green/Red plot (Fig. 5c) contains the counted responses. All other detector responses outside of this region are ignored. What is the basis of ignoring these other responses? In actuality, there are two factors that contribute to the choice of the region to count. (1) Plankton-derived contaminants, in particular chlorophyll-containing detritus, have a natural red autofluorescence (Marie, 1999). Thus, signals that have "too much red", i.e., are off the counted ellipse toward the higher red end, are likely to reflect contamination by phytoplankton/chlorophyll (or possibly other red autofluorescing detritus). These particles are less likely to be examples of the bacteria of interest, which lack chlorophyll, and so they are omitted. (2) Pure water blanks that lack any substantial bacteria are run to establish a baseline detritus region on the red/green plot. These bacteria-independent signals tend to congregate at low green fluorescence intensity and towards the higher red end (lower right of the Fig. 5b red/green plot). They can be easily avoided when determining the bacteria count (= the L-gate) by excluding them from the counted region, as illustrated with Fig. 5c. As a practical matter, we can count these "excluded" signals as well by using data within the FS/SS graph (= entire population of counted signals; FS = forward scatter, SS = side scatter), and typically the excluded signals ( bacteria) in the red/green graph constitute about 10 - 40 % of the entire population of counted signals. So, most of the particles that meet the "bacteria" size criteria are counted in any event. For example, in Fig. 5c, 10000 out of a possible 13647 signals are counted as bacteria.

By this analysis, it is apparent that, much like with epifluorescence microscope visual inspection, the instrument operator must make a decision about which data points correspond to valid bacteria counts, and which do not. Both techniques use a dye (SYBR1-Green) that only lights up when attached to objects containing double-stranded DNA, so that requirement eliminates most similar-sized but non-bacterial detritus. However, with cytometry, there is one additional and valuable input criterion that can be used to distinguish bacteria; red/green intensity plots. Thus, a second check on non-bacterial detritus/phytoplankton can be accomplished.

The counts from two gates illustrated on the cytogram in Fig. 5c are relevant to the bacteria enumeration question; the L gate, which displays the count of bacteria, and the E gate, which displays the bead count. The instrument is set up to stop counting when the bacteria count reaches 10000 or after 300 seconds have elapsed, whichever occurs first. The former criterion is almost always reached in authentic marine water, reflecting the relatively large (compared to blank controls) number of bacteria present. In water blanks, considerably less bacteria are counted and the 300-second limit is usually reached. The real number of value, then, is the bead count from the E gate. Since we know the concentration of beads in the sample, and the cytogram gives us the ratio of beads counted to bacteria counted for a given volume of sample (the amount that passes until the bacteria count reaches 10000), we can use a simple ratio to get the bacteria population in a known volume of the sample. An illustration of the calculation for the data in Fig. 5c is given below:

Bead population: 3.29 x 10⁷ beads/mL starting stock solution concentration x 0.060 mL = 2.0 x 10⁵ beads added to the sample. The sample volume = 0.960 mL + 0.060 mL (bead solution) + 0.020 mL (SYBR solution) = 1.040 mL total volume, so the final bead concentration = 2.0 x 10⁵ beads/1.040 mL = 1.9 x 10⁵ beads/mL.

So, bacteria/mL = bacteria (L-gate)/beads (E-gate) x 1.9 x 10⁵ beads/mL

However, we have not yet accounted for either (a) the initial dilution of the 25.0 mL sample by addition of 0.5 mL of formaldehyde, or (b) the dilution of this water/formaldehyde solution upon addition of the bead solution and the SYBR1-Green solution. So, we have to multiply the above quantity by both (a) 25.5 mL total volume/25 mL sample volume (= 1.02) and (b) 1.040 mL total volume/0.960 mL sample volume (= 1.08):

(1) Bacteria/mL = L-gate(bacteria #)/E-gate(bead #) x 1.9 x 10⁵ beads/mL x 1.02 x 1.08

Eq. (1) is the formula that allows us to arrive at a bacteria population in a water sample from the raw cytometer data. In the specific case illustrated in Fig. 5c,

bacteria/mL = 10000/2049 x 1.9x10⁵ x 1.02 x 1.08 ~ 1,021,149 bacteria/mL. Significant figure restrictions ("0.060 mL" of bead solution is only reported to 2 significant figures) would require that this value be reported as 1,000,000 bacteria/mL.

Each water sample was run in triplicate, and the averaged values with standard deviation (although not technically applicable to a three sample data set) are reported in the Results section below.

3. Results and Discussion

3.1 Baseline bacteria counts

The initial experimental foray in reef tank water bacteria enumeration was focused on establishing baselines for bacteria populations under different husbandry regimes. Later, these baselines will be used as comparison points in experiments where the system is perturbed, either by carbon dosing or by mechanical filtration (skimming, GAC). Much prior effort in the area of bacteria counting from authentic reef water has led to an expansive body of work. A few representative examples are documented in Table 1, where bacterial counts from reef water in Hawaii, Micronesia, Key Largo, and the Northern Line Islands all converge on a span of bacteria populations in the 500K - 1500K/mL range or so. The counts from the Northern Line Islands, Ponape Island, and Key Largo were derived from epifluorescence microscopy measurements, whereas the Kaneohe Bay data were acquired via flow cytometry. In addition, we sampled water from a mangrove estuary in the Florida Keys, and not surprisingly, this highly sedimented shoreline water exhibited a bacterial population (3300-4400K/mL) much higher than those observed in the more pristine reef waters. These values serve as standards for comparison to aquarium water, and they will help address the question, "Are our reef tanks similar to, or different than, an authentic reef with respect to water column bacterial populations?"

A second series of bacterial counts focused on control water samples of various origins. These data represent low-end standards that delineate the limits of our counting technique. For example, every experimental run was accompanied by an RO/DI/0.2 um filtered water blank to ensure that the flow cytometry instrument itself was not a source of bacterial contamination. It did not seem to matter whether these samples were treated with formaldehyde preservative or not; similar counts were obtained in both cases. Overall, these "sterile" water blanks typically displayed counts in the 1000 - 5000 bacteria/mL range; occasionally, values as high as 10000 bacteria/mL were observed. Since bacteria counts of authentic aquarium or marine water samples were 1-2 orders-of-magnitude greater than these blank values, we concluded that instrument contamination was not likely to increase the measured bacteria counts in any significant way. Both Aquafina bottled water and the RO/DI water produced in the 175-gallon aquarium make-up water system were almost sterile: < 1500 bacteria/mL. State College Pennsylvania tap water, on the other hand, did carry a modest bacterial load; 27K/mL.

The surveyed reef aquariums divided into two distinct sets of husbandry protocols; aggressive and passive (see Fig. 6 for pictures of these aquariums). The aggressive husbandry practices included protein skimming, GAC filtration, and regular water changes in an active effort to scrub the water of nutrients. The passive approach did not involve any of these procedures. Interestingly, the aquaria subjected to passive husbandry exhibited bacterial counts that fell within the range seen on authentic reefs; 200 - 1000K/mL. On the other hand, the tanks that "benefited" from careful attention to nutrient removal protocols displayed bacteria/mL counts that fell far short of these numbers; only 90-140K/mL. In addition to monitoring water column bacteria counts, the TOC (Total Organic Carbon, see Feldman, 2008) levels were examined as well. Not surprisingly, the tanks with "unpurified" water exhibited TOC levels greater than those seen with the skimmed/GAC-filtered tanks. The "purified" aquaria's TOC levels fall within the typical TOC range seen on authentic, healthy reefs (Feldman, 2008); the passively husbandry tanks were 2-3x higher.

The observation that, at least among this small set of aquaria examined, the water within the skimmed/filtered tanks had only ~ 1/10th of the population of bacteria that the unskimmed/unfiltered tanks had was a real surprise. It speaks to one aspect of aquarium husbandry in which a perhaps important parameter (?), water column bacteria counts from authentic and healthy reefs, is not reproduced at all effectively in these home aquaria. Sensitive corals, like Acropora, do not thrive in the high-bacteria-count/high-TOC-level tanks examined, although soft corals do well (see pictures). On the other hand, SPS corals do well in the low-bacteria-count/low-TOC-level tanks (Fig. 6). These observations raise a number of questions, chief among them perhaps are, (1) "Do water column bacteria counts have any relevance to the short-term or long-term prospects for maintaining SPS in captive aquaria?", and (2) "What is the relationship between TOC and water column bacteria population?" The former question, whereas perhaps more interesting, remains unanswered. The latter question (TOC vs. bacteria population), which bears on the topic of carbon dosing, will be addressed below.

Table 1. Bacterial counts from authentic marine water, various control samples, and several reef tanks.

Sample	Water volume (gal)	skimmer	GAC	GFO	Water change	TOC ppm	Sandbed	Bacteria/mL
aMonger, 1993 bDinsdale, 2008 cYoshinaga, 1991 dPaul, 1993 eThis work
Kaneohe Bay, Hawaiia								800-1500K
Northern Line Islandsb								50 - 800K
Ponape Island, Micronesiac								800-1600K
Key Largo, Floridad								300-1000K
Mangrove estuary, Fl. Keyse								3300-4400K
RO/DI/0.2 uM filtered H2O								1-10K
KSF RO/DI								1.1±0.3K
Aquafina								1.5±0.5K
State College PA tap water								27±6K
Sanjay 55	55	no	no	no	rarely	2.72	no	590±70K
Sanjay 29	29	no	no	no	rarely	1.69	no	1000±80K
Sanjay 28	28	no	no	no	rarely	2.14	yes	220±10K
KSF 175	168	yes	yes	yes	17%/wk	0.50	yes	105±10K
PSU HUB 500	500	yes	yes	yes	10%/wk	1.0	yes	91±13K
Sanjay 500 Zeovit	500	yes	yes	yes	14%/2-3 wks	0.90	yes	140±10K

The reef aquariums that were monitored for their bacterial populations:

The bacteria/mL counts for the aquaria described in Table 1 reflect single-time-point measurements, and it is possible that hourly, daily, or other fluctuations in bacterial populations have been missed. These modulations in bacterial populations might result from the light on/off cycle, food additions, pH swings, etc. In order to probe this point, a few week-in-the-life longitudinal studies were conducted on both the 175-gallon reef tank and the 55-gallon reef tank. The first experiment covered 5 days of typical aquarium life, with the skimmer deliberately off for the first three days, the UV sterilizer on, and the GAC and GFO filters on, Fig. 7. Over the course of this experiment, the water column bacteria population fluctuated between ~ 95K and 115K bacteria/mL, a ~ 20% spread in values. The skimmer was turned off at t = 0, and the count rose steeply over the next 12 hours, and then declined to the starting point at the 2-day mark. At that point, a gradual rise to the maximum recorded value, 115K/mL occurred. Turning the skimmer back on at the 3.5-day mark may have slowed the rate of this increase slightly, but the data are not compelling on this point.

It is temping to attribute the initial steep rise to a presumed corresponding increase in the carbon source TOC, since the TOC-removing skimmer was turned off at t = 0. During this growth phase, nitrogen and phosphorus nutrients as well as C would be stripped from the water column also. In this interpretation, the subsequent decline in bacteria population at the 12-hour mark might reflect a depletion of these N and P nutrients required for bacteria population growth; that is, perhaps the initial growth spurt might reflect an increase in C concentration (skimmer off) in a C-limiting bacterial growth regime, and the subsequent decline at 12 hours might indicate a switch over to a N and/or P limited growth regime. This hypothesis dovetails nicely with the Carbon Dosing ideas described earlier. However, it is important to appreciate that simply observing the expected result of a hypothesis does not validate that hypothesis - the strongest conclusion that legitimately can be offered is simply that the data is consistent with the predictions of the hypothesis. Other interpretations of the bacteria population data in Fig. 7 cannot be excluded at this point. Furthermore, the "rebound" in bacterial growth after 2 days is more difficult to interpret without some further knowledge about commensurate C, N, and P levels in the tank. A more controlled experimental plan to monitor bacteria population change contemporaneously with C and N concentration measurements might settle this issue; this experiment will be discussed shortly.

One concerning point in the experiment described in Fig. 7 involves the role that the UV sterilizer might play in influencing bacterial levels; Are we killing significant numbers of bacteria by UV treatment, thus suppressing population growth? The UV sterilizer in use is a 57W flow-through model from Aqua Ultraviolet. In order to probe this question, we re-ran the "week-in-the-life" experiment with the UV sterilizer off, but the skimmer on continuously, Fig. 8. The observed bacteria/mL values over the course of 5 days fluctuated between 60K and 90K (~ 50% change) for this particular time period. Thus, there did not appear to be any significant bacteria population increase in the water column when the UV sterilizer was off, and it is probably safe to conclude that the UV sterilizer does not have a significant effect on the bacteria population levels in the tank's water column.

A final "week-in-the-life" experiment was conducted on the 55-gallon tank lacking any active filtration, Fig. 9. In addition, this tank had been treated daily with vodka as a carbon source for 6 months prior to water removal. This vodka addition started after the original bacteria population reading reported in Table 1 was taken. Since there was no active bacteria removal mechanism (i.e., skimming), it was not clear, a priori, how the bacteria population might change compared to the pre-vodka value of 590±70 K/mL. In fact, the bacteria population clearly had risen to a significantly higher level than observed in the pre-vodka time period, and now it hovered around 1500-2500K/mL. Once again, significant (~ 60% !) fluctuations in bacteria/mL counts over time appear to be the norm, at least for the two aquaria examined.

Overall, the major conclusions from these preliminary studies on baseline bacteria counts are

1. Actively purified aquaria have water column bacteria populations that are approximately 1/10^th those of authentic healthy reefs.
2. Unpurified aquaria have water column bacteria populations that are approximately the same as those of authentic healthy reefs.
3. UV sterilization does not significantly influence aquarium water column bacteria populations.
4. There is substantial fluctuation (20-50%) in the measured water column bacteria populations over a several-day time scale in aquaria.

3.2 Carbon dosing (planned and inadvertent) - How does it affect water column bacteria levels?

The data from section 3.1 established that skimmed/GAC filtered aquaria exhibited baseline bacteria/mL counts in the 70-140K/mL range, whereas unskimmed/GAC-less tanks had approximately 10x that amount. Furthermore, the bacteria level fluctuated on a daily basis as much as 20 - 50%. Now it was time to examine how these baseline bacteria/mL levels responded to deliberate carbon addition. If bacteria population growth is carbon-limited, we might anticipate a significant increase. If it is not, the bacteria population numbers shouldn't scale with carbon dose. In the first trial, we removed 30 gallons of water from the KSF aquarium approximately 17 hours after the last feeding, and about 1 hour after the lights came on. This water sample was placed in an uncovered Rubbermaid tub with continuous powerhead mixing. The fact that the tub was left uncovered might lead to some bacterial introduction via airborne sources; we felt that this configuration best approximated how an aquarist might actually run their aquarium/water change apparatus. Ethanol (EtOH) was dosed as a rate of 0.29 mL/day (= 1 ppm of C/day). Water samples were removed just before each EtOH dose over the course of 5 days. In addition, TOC levels were measured over this time period, and beginning and ending [NO₃] values were recoded with a Salifert kit. The [PO₄] level was unmeasurable via the Merck kit (< 0.024 ppm). The results are displayed in Fig. 10.

The initial bacteria/mL population increased from 80K/mL prior to the first EtOH dose up to about 1200K/mL (~ 15x increase) over the course of a day, and maximized at 3900K/mL (~ 50x increase) after 2 days. The bacteria level then receded to about 1500K/mL (~ 19x increase) at the 5-day mark. The TOC level changes started predictably; the initial level = 0.61 ppm, and just after addition (3.5 h mark) of 0.29 mL of EtOH (~ 1 ppm of C), the recorded TOC level elevated to 1.58 ppm. Thus, the addition of EtOH did, in fact, raise the TOC level the expected amount- an internal check on the measurement methodology. The curious and potentially revealing observation came after the 1-day mark; continued additions of EtOH did not lead to increases in the TOC level at day 2 and day 3, and only slight increases (~ 33%) at day 5. The [NO₃] value decreased from 2.5-5 ppm at the beginning of the experiment to 1-2.5 ppm at day 5. The uncertainty in reading the results of the Salifert kit make drawing any definitive conclusions about nitrate reduction over this experimental time course difficult.

These observations can be interpreted within the context of the carbon dosing hypothesis described earlier, specifically the initial premise that addition of a metabolizable carbon source will lead to an increase in the bacteria population (i.e., bacteria growth is C-limited in aquarium water). Over the first 2 days, bacteria growth does respond as predicted by this hypothesis. In addition, the TOC behavior can be rationalized in light of this hypothesis as well; the initial and subsequent day 2 additions of EtOH (= TOC) are consumed by bacteria, both fueling growth and depleting, or at least stabilizing, the TOC pool. Although the TOC load within intact bacteria will be picked up by the TOC analyzer, it is likely that normal bacterial metabolism will convert a lot of this TOC into CO₂, which will not be detected by the TOC analysis. However, at the approximately 2-day mark, the dynamic situation in this water volume changes; the bacteria population declines, and the TOC level begins to rise. Perhaps at this time point, the growth in bacteria population is no longer carbon limited, and so further carbon addition doesn't fuel growth. Continuing on in this speculative vein, perhaps in the presence of now surplus carbon, bacterial growth is now limited by the availability of some other nutrient (N or P). In this interpretation, the growth in bacteria population over days 1 and 2 would have depleted enough N and/or P from the water that one (or both) of these nutrients is now deficient from the bacterium's perspective. Of course, from the aquarist's perspective, this depletion of N and/or P in the water column is desirable and in fact is the whole point of the carbon dosing procedure.

We sought to repeat this experiment, given our caveat from above ("observing the expected result of a hypothesis does not validate that hypothesis - the strongest conclusion that legitimately can be offered is simply that the data is consistent with the predictions of the hypothesis"). However, in this second trial, we included a control experiment for testing the importance of the EtOH addition itself to bacteria growth. Specifically, we ran the 30-gallon water sample experiment exactly as described for Trial 1, and the data are reported as the aqua (bacteria/mL) and blue (TOC) lines of Fig. 11. These data follow closely those from Trial 1; an initial rapid bacteria population rise (~ 30x the initial level) over the first day, followed by a slight decline over the next few days. The TOC level's response was consistent with Trial 1 as well; an initial ~ 1 ppm (0.64 ppm to 1.89 ppm) jump upon the first EtOH addition followed by a decline as bacterial growth peaked, and then a slight rise as (perhaps) the bacterial population entered a non-C-limited growth phase.

The real surprise is the red line of Fig. 11; it documents the substantial increase in bacteria/mL for a control water sample that was not treated with EtOH. We simply removed about ½ gallon of water from the tub at the beginning of the experiment, and placed it in an open Tupperware container under fluorescent room lighting. This control portion of tank water was sampled for bacteria content daily just like the EtOH-treated larger tub water sample. Remarkably enough, the bacteria population in this control sample experienced a slower but still significant rise, increasing to a maximum of ~ 27x the original level after 3 days. What is fueling this bacterial growth? Since we have already determined that the bacteria population in this aquarium water sample is carbon limited, it seems reasonable to hypothesize that the Tupperware vessel itself is providing a metabolizable source of carbon! This vessel had been used in various water transfers previously, and it was rinsed with tap water and dried between uses. Could organic residue or bacterial films coat the plastic? Is the plastic itself "digestible" to the water column bacteria? These questions are not answerable at present, but the overall observation that the plastic container itself (or residue on its surface), and not necessarily the EtOH addition, can contribute to a significant increase in water column bacteria population raises some concerns about the interpretation of the results in Fig 10 (remember the caveat).

These observations prompted a more direct examination of the actual Rubbermaid tub's capacity to contribute to bacterial growth - maybe the Tupperware vessel was unusually contaminated with organics. In this experiment, we removed a 30-gallon water sample from the KSF aquarium and transferred it to the uncovered Rubbermaid tub, as described previously. Water circulation was provided by powerheads but otherwise the system remained undisturbed. Water samples were withdrawn daily for 5 days, and the bacteria/mL counts are displayed in Fig. 12. Clearly, the tub itself (or organic material coating it) is providing a suitable carbon source for bacterial growth. The bacterial population rises some 26x over the initial value over 2.3 days, before declining slightly; a rationale for the burst in bacteria population at day 5 cannot be offered at present.

Do these control experiments negate the carbon dosing hypothesis? No. These results merely suggest that ANY metabolizable carbon source, whether it is unknown organic "stuff" from the surface of a Rubbermaid tub or deliberately added EtOH, will fuel bacterial growth in a C-limited environment such as, apparently, aquarium water. The Rubbermaid tub was cleaned out with tap water and then distilled water between uses. Any solid buildup was removed by rinsing with 1 M HCl followed by distilled water. So, this protocol, which might reflect the typical procedures used by an aquarist for their make-up water or saltwater holding containers, clearly contributes to an organic-rich environment that promotes bacterial growth. This observation may hold some interest to aquarists who believe that they need to seed their tanks with bacteria ("probiotics") as part of a carbon dosing scheme to lower nutrients, and therefore are contemplating purchasing a bacterial "elixir" to accomplish this seeding.

The observation that a plastic container, which is re-used in the course of normal aquarium husbandry, can contribute carbon nutrients to fuel bacterial growth opened up a new line of inquiry; what role might standard water changes that use such containers play in modulating (increasing) bacterial levels in reef tank water? Specifically, can water-change water introduce significant bacteria populations into the aquarium? This question was probed by simply monitoring the bacteria/mL count within a 30-gallon saltwater sample made to 35 ppt salinity by mixing Instant Ocean Reef Crystals salt mix with 30 gallons of distilled water in a loosely sealed Rubbermaid garbage can (different than the tub used for the previous experiments). This container had been used for weekly water changes for the preceding 6 years. Following each water transfer, the container was washed with distilled water and wiped dry prior to mixing the next batch of saltwater. Furthermore, it was periodically cleaned with 1M HCl to remove solid deposits, but in general it carried with it some solid residue on the plastic surface. The data (Fig. 13) unequivocally illustrate that indeed this freshly mixed saltwater is a major source of bacteria introduction into the KSF reef tank. The initial distilled water exhibited negligible bacteria population (~ 10K/mL), but that value increased dramatically to over 900K/mL in just one day after IORC addition. The bacteria/mL value decreased and leveled off at ~ 500K - 600K/mL by day 2. Thus, preparing IORC saltwater under typical aquarium husbandry conditions does lead to a remarkable bacteria load, fueled by some as yet unidentified carbon source, although it should be noted that IORC does include "extra vitamins" that could serve as an organic carbon fuel source. The bacteria population peaked in this experiment at about the 1-day mark, suggesting that addition of this saltwater to an aquarium after one day of mixing, as part of a routine water change, could serve as a inexpensive, simple and functionally useful way to introduce bacteria. This premise was tested next.

Does adding bacteria-rich saltwater to a reef aquarium increase its bacterial load? This question was probed by the experiment described in Fig. 14. As part of routine weekly tank maintenance, 30 gallons of water was removed from the KSF aquarium, and immediately 30 gallons of IORC saltwater (bacteria count = 280K/mL in this instance) was added. The KSF tank bacteria/mL count was monitored daily over the course of a week; midway through this experiment, a second smaller water change with Instant Ocean saltwater (bacteria count not measured) was conducted. The active filtration (skimmer/GAC/UV) was on throughout the week-long time course. As illustrated in Fig. 14, the tank's bacteria population did experience a sharp increase upon addition of the bacteria-rich water-change saltwater; after 2 hours post IORC saltwater addition, the count increased by 1.7x. Likewise, by 1 day post IO saltwater addition, the count had enlarged again, also by 1.7x. So, addition of bacteria-rich saltwater does indeed lead to an observable and significant increase in the tank's bacteria population. In fact the dilution math works out perfectly and serves as an internal check of the counting methodology, much like it did with the TOC analysis of Figs. 10 and 11. Specifically, the entire tank water volume is 168 gallons. Adding 30 gallons of 280K bacteria/mL to 138 gallons of tank water (total volume - amount remove during water change) of 61K bacteria/mL should lead to a predicted final 168 gallon volume at 100K bacteria/mL - at 2 hours after the water change, the bacteria/mL was measured at 101±5K/mL.

The more interesting observation is what happens after the bolus of bacteria is added with the water change. After both water changes, the ~ 1.7x increase in bacteria count diminished back to a "baseline" value for the KSF aquarium of ~ 60-70K/mL within one day. The bacterial population at any instant will be determined by the interplay of population-increasing events (direct addition of bacteria and growth, if appropriate and sufficient nutrients are present) and population-decreasing events, such as death and predation by higher organisms in the food chain. The fact that a rapid increase in bacteria is equally rapidly "absorbed" so that an equilibrium level is maintained speaks to the dynamic nature of these events. The larger microfauna and sessile filter feeders seem to be likely candidates for bacteria removal culprits that in concert balance out any induced spike in population. The level of bacteria addition that might overwhelm this balance is not known, but the fact that a biological "mechanism" appears to be in place in order to return population swings to an equilibrium level poses an interesting problem for the carbon dosing hypothesis; perhaps higher bacteria levels cannot be sustained in an authentic reef tank even upon carbon dosing, if the bacteria consumption mechanisms increase in kind. In this scenario, nutrient "export" would occur, at least in part, via bacteria consumption by endogenous organisms and not just by physical removal from the water via protein skimming; that is, some of the nutrients would stay in the system, albeit in new trophic levels. This issue of water column bacteria population increase via carbon dosing in an authentic, thriving reef tank can be probed directly, and the results of that experiment are described below. First, however, there is one more question on the topic of (inadvertent) bacteria addition via water changes that should be addressed; do the bacteria come from the salt mix itself, or are they introduced via the (non-sterile) mixing vessel?

This question was examined by removing the container as a source of bacterial contamination and/or food. Specifically, saltwater mixes (to 35 ppt salinity) were prepared from RO/DI/0.2 uM-filtered water in sterile plastic Erlenmeyer flasks (Nalgene 500 mL sterile disposable PETG flask). Six salt mixes were examined: Instant Ocean Reef Crystals (IORC), Instant Ocean (IO), Tropic Marin, Oceanic, Red Sea, and Kent, and the bacteria population in each flask was monitored over 5 days. The flasks were all sealed and covered with aluminum foil to avoid exposure to standard fluorescent room lighting. A control flask containing only RO/DI/0.2 uM-filtered water was included, and the results are depicted in Fig. 15.

Five of the six salt mixes (Red Sea excepted) displayed little bacterial contamination beyond the pure water control, and the bacteria populations fluctuated but did not consistently rise over time as might be expected if sufficient nutrients were available (cf. Fig. 13). Thus, there is no reason to suspect that any of these five salt mixes themselves contribute to the high levels of bacteria in the make-up saltwater of Fig. 13. Red Sea salt, on the other hand, does appear to bring with it a not insignificant bacterial load. Once again, there was little increase over 5 days, indicating that sufficient nutrients for growth were not available under these "sterile" conditions. What is so special about Red Sea salt? This salt, uniquely among the six mixes tested, is made, at least partially, by drying authentic seawater. Thus, it appears to retain some viable bacteria from the drying process. The other five salt mixes are prepared from mixing strictly chemical sources of the components. Overall, it appears justified to conclude that the significant populations of bacteria in mixed saltwater are a result of container contamination and not salt mix introduction per se.

The final question of merit in testing the "carbon addition" part of the carbon dosing hypothesis was alluded to earlier; what happens in authentic reef tank water over time upon deliberate addition of a carbon source? Will the natural bacterial population control mechanisms handle the induced population growth so that the normal equilibrium level of bacteria is maintained? Or, will these mechanisms be overwhelmed and therefore will the bacteria population rise? To probe this key point, we adopted the carbon dosing protocol described by Walton and Bjornson (Walton, 2008). Specifically, we added pure EtOH to the KSF 175 gallon reef tank at the following schedule:

Day 1-4: 0.28 mL (~ 0.l7 ppm of C in the tank volume of the KSF aquarium)

Day 5-8: 0.58 mL (~ 0.35 ppm of C)

Day 9-15: 0.78 mL (~ 0.47 ppm of C)

Day 16-21: 0.98 mL (~ 0.60 ppm of C)

Day 22-27: 1.18 mL (~ 0.71 ppm of C)

Day 28-30: 1.38 mL (~ 0.84 ppm of C)

The EtOH was adding in the early morning, prior to lights on, and approximately 17 hours after the last tank feeding. Water samples for bacteria count analysis were removed just prior to the EtOH addition. Periodic measurements of [NO₃] via the Salifert kit were conducted also. During this month-long experiment, the tank was maintained under its normal husbandry conditions; a weekly 30 gallon (17%) water change with IORC saltwater (standing for 1 week prior to use), biweekly change of GAC and GFO, continuous protein skimming (H&S 200-1260), UV sterilization, light cycle of 8 hours on/16 hours off. The results of this experiment are illustrated in Fig. 16.

The interpretation of this experiment is clouded somewhat by a complicated backstory. This experiment as described above was planned to proceed over 30 days. Unfortunately, at the 20-day time point, the Coulter XL cytometer broke, and it took several weeks for the instrument to be repaired. The data for the three weeks of samples were compromised, and the entire experiment had to be re-run. The KSF 175 gallon aquarium was maintained as described above for 2 more months without EtOH addition before the dosing experiment was restarted, following the protocol described above, and the data displayed in Fig. 16 came from the second complete run.

However, between approximately the 2^nd week of the initial EtOH addition experiment and the start of the second EtOH experiment about 9 weeks later, two significant changes in the KSF aquarium were observed. Cyanobacteria made an unwelcome appearance after vanishing from the KSF aquarium as a result of a Chemiclean treatment 6 months prior. The cyanobacteria built up to a significant coating on the sand bed and rockwork by the time the second EtOH addition experiment ended at the 30-day mark. In addition, four larger and relatively old Acropora coral colonies developed white "dead" spots near their bases that gradually spread upward, eventually consuming the corals. It is not possible to establish whether causality or coincidence is at work here. However, the bacteria level at the t = 0 starting point for the second EtOH dosing regimen, ~ 190K bacteria/mL, is significantly higher that the steady-state bacteria populations in the KSF tank measured over the preceding 9 months (~ 70 - 100k bacteria/mL). Water blank controls and examination of a water sample from the PSU HUB tank (see Table 1) ascertained that the now repaired Coulter XL cytometer was still giving reliable readings. It is possible that the now higher bacteria/mL starting point reflects the presence of lots of cyanobacteria in the water column, absent in all of the earlier readings. After the 30-day EtOH dosing experiment of Fig. 16 ended, the KSF tank was treated with Chemiclean as per label directions to remove the cyanobacteria, and then a 90% water replacement (five 60-gallon (34%) water changes conducted over 48 hrs) was performed. Ten days after the completion of this water replacement, the bacteria count of the KSF tank registered at 97000 ± 3000 bacteria/mL, just about where it was prior to the EtOH dosing campaign. The affected corals, unfortunately, did not recover, but frags clipped from the living branches subsequently did thrive.

Nevertheless, even given the elevated starting bacteria count, it is clear that the measurable bacteria population in the water column of the KSF 175 gallon aquarium did not undergo the rapid increase upon EtOH dosing that was observed with the removed 30-gallon water samples, Figs. 10 and 11. In addition, there was no detectable trend in [NO₃] levels; the value hovered around the 2-5 ppm level throughput. The bacteria population observation is a reminder that the detected bacteria/mL count really reflects a dynamic equilibrium between bacteria introduction (primarily via growth) and bacteria removal by death/predation. In the case of the 30-gallon water samples removed from the tank and placed in a Rubbermaid tub, it seems reasonable to posit that since the sedentary filter feeders (= direct bacteria consumers, or consumers of organisms that consume bacteria; i.e., the "predators") were no longer present, the observed bacteria population increased dramatically upon addition of a fuel source. With the 175 gallon reef tank of Fig. 16, however, the bacteria consumers are still present, and in fact may respond to an EtOH-fueled bacteria population increase by increasing their own numbers as well. The most immediate bacteria consumers, the larger microfauna in the water column, may therefore experience an increase in population also, although our water examination method would not have picked up this increase. Eventually, this carbon fuel (= EtOH) may work its way up the food chain until sedentary filter feeders in the tank (corals, sponges, fan worms, etc) consume it, thereby removing it from the water column (but not from the tank!). Thus, we speculate that the bacteria population may not show evidence of an increase upon EtOH dosing, as any increase is counterbalanced by a corresponding increase in removal via predation. Some of these larger water column microfauna will be removed via skimming*, and so the overall plan of nutrient export will be realized. However, some of the introduced carbon nutrient is likely to be retained within the sedentary filter feeding livestock, and hence it will remain within the overall tank ecosystem. Presumably, as long as these end-consumers live and grow as a result of the added carbon nutrients, they pose no immediate risk for contributing to the waste nutrient level of the aquarium.

* In unpublished results, examination of skimmate from the KSF 175 gallon aquarium, removed by the H&S 200 skimmer, via high-resolution optical microscopy revealed the presence of many examples of microfauna of the diatom and foraminifera families. It is likely that members of these families, at least within the foraminifera, do account for some bacterial predation. We thank Jamie Kunkle of the Pennsylvania State University Materials Research Lab for providing these photographs.

Overall, the major conclusions from these carbon dosing experiments are

1. Addition of a carbon source (EtOH or uncharacterized organic "gunk") to reef tank water removed from a reef environment leads to dramatic increases in the water column bacteria load.
2. Addition of a carbon source (EtOH) to an active reef tank via a recommended schedule does not lead to any measurable increase in water column bacteria load.
3. Fresh 1-day old saltwater prepared in a non-sterile environment has a bacteria load approximately 10x that of skimmed reef tank water.

3.3 Bacteria removal via mechanical filtration - how effective?

The third and final research topic under the general heading of modulating bacteria populations in aquarium water focuses on the question of mechanical bacteria export, a necessary requirement for the overarching goal of carbon dosing-based nutrient depletion. We have already seen circumstantial evidence for bacteria removal from the water column by predation, but that process does not remove the bacterium's nutrient content from the closed aquarium system. For that function, some type of mechanical filtration that specifically targets bacteria for removal is required. The carbon dosing hypothesis cites protein skimming as the mechanism for physical removal of bacteria, with their nutrient load, from the aquarium water. Note that skimming, or any type of mechanical filtration, can only remove bacteria that are in the water column; bacterial biofilms on surfaces can only be cleared if they are dislodged and enter the water column. Therefore, we set out to examine the capability of a protein skimmer, in our case the Bubble King mini 160, to scrub authentic aquarium water of its bacterial load. There are reports in the literature (Brambilla, 2008; Suzuki, 2008) that document the ability of foam fractionation (= protein skimming) to clear bacteria from both coastal seawater and also a closed circulation fish rearing system. In the former case (Suzuki, 2008), a coastal seawater sample from a fishing port near Miyazaki Japan was continuously skimmed with a DYI protein skimmer, and the removal of different types of water column bacteria, reported as efficiency of removal (100% = complete removal of bacteria) follows: Enterococci, Vibrio and Salmonella, 55-60%; Fecal coliform, 5%. Thus, it appears that not all types of bacteria respond identically to bubble-based extraction. The latter experiment (Brambilla, 2008) documented that in a closed, recirculating, fish-only system, water column bacteria populations could be reduced from 32-88% by skimming, depending upon the experimental details. We interpreted these two independent sets of results as providing strong precedent for the expectation that we would observe similar behavior when skimming aquarium water. In addition to protein skimming, many aquarists utilize Granular Activated Carbon (GAC) as an active filtration medium to clear TOC from aquarium water, and so we wondered if GAC might also contribute to the mechanical removal of bacteria from aquarium water as well.

The question of GAC-based bacterial water remediation was studied by removing 5 gallons of KSF tank water that had been prepared as follows; the KSF tank's H&S 200 skimmer was turned off for 42 hours before water removal, a 17% (30 gallon) water change with IORC saltwater was conducted 24 hours before water removal, and the tank was fed 3.5 hours before water removal. This 5 gallon water sample was placed in a plastic container and, mindful of the rapid growth of the bacteria population in non-sterile plastic containers, this sample was immediately recirculated through a Phosban reactor containing 150 gm of HC2 GAC. Prior to starting the Phosban reactor pump, five 25 mL water samples were removed from the 5-gallon bucket and placed in sterile centrifuge tubes, capped, and set aside. These five tubes serve as a control for bacteria growth; at 30-minute time intervals during the GAC run, 0.5 mL of 37% formaldehyde solution was added to each tube in succession, and these control tubes were immediately frozen pending later analysis. These tubes are not a perfect control, because they do not account for plastic-container-induced bacteria growth, but they do serve as a check against overall contamination. Samples (25 mL, worked up with formaldehyde as described in the General Experimental) were removed periodically from the 5 gallon volume during the course of the GAC run. An entire experimental time period of 120 minutes was chosen to minimize the opportunity for plastic container-fueled bacteria growth, which might mask detection of any GAC-induced depletion.

The data are displayed in Fig. 17. Clearly, there is no evidence that supports the notion that GAC filtration can serve as an effective method for bacteria removal from the water column. Rather than going down, the bacteria levels in the water sample rise slightly (~ 1.4x compared to the starting point) and then decline. This behavior is no different from what might be expected if the container's surface organic load fueled some bacteria growth, at least until the carbon source was consumed. The control samples also rose a little (1.3x) over the same 2-hour time period, a reminder that some small bacterial growth is almost unavoidable in standing water samples. Thus, GAC does not seem to be an effect method for removing bacteria from the water column of reef tanks.

We next turned our focus to the larger question of skimmer-based bacteria depletion of reef tank water. As in the GAC experiment, we first "prepared" the KSF tank water sample by (a) turning off the tank skimmer and GAC filter for 42 hours prior to water removal, and (b) feeding the tank 3.5 hours prior to water removal. In this instance, the usual 30 gallon water sample was removed from the KSF tank and placed in the Rubbermaid tub containing the Bubble King mini 160 protein skimmer; water circulation was provided by two powerheads. Immediately, five 25 mL control water samples were removed and set aside; these control tubes were dosed in sequence with 0.5 mL of 37% formaldehyde solution at 30-minute time intervals during the skimmer run and frozen for later analysis, as described above. The skimmer was turned on, and the riser tube was adjusted in order to keep the foam height within the boundaries recommended by the manufacturer. 25 mL water samples were removed periodically over 120 minutes and worked up as described above. The data resulting from this study are displayed in Fig. 18.

Inspection of these data reveal that, like the GAC experiment, there is no evidence which suggests that this skimmer was able to remove bacteria from this water sample over the 2-hour time period of the experiment. Both the skimmed water samples (pink) and the control samples (aqua) fluctuate in their bacteria/mL values over a fairly narrow range (75 - 90K/mL) without any meaningful trend emerging. Since the skimmed water sample exhibited the same behavior as the unskimmed control water samples, it is not possible to draw any further conclusions from this experiment. These results were quite unsettling; why wasn't the skimmer stripping the aquarium water of at least some of its bacterial load? Is the problem with the skimmer, or is the problem with the bacteria?

One approach to probing this puzzling point is to use aquarium water that had been "artificially" spiked with bacteria; perhaps with a much greater bacterial load, the skimmer will function as desired. Towards this end, we took advantage of the previous observation that allowing aquarium water to sit in a plastic container of several days leads to a dramatic increase in the water's bacteria population. We removed a 30 gallon sample of KSF tank water, prepared as described previously, and placed it in the uncovered Rubbermaid tub with the skimmer present but off, and the two powerheads on for circulation. After 5 days, the tub was covered with foil, the skimmer was turned on, and the skimmer experiment was run for 120 minutes. In addition, the usual control samples were removed at the beginning of the skimmer run. In this instance, the water's bacteria population had built up to approximately 7000K/mL, much higher than the 75K/mL starting point of the experiment described in Fig. 18. The results of this "doped" water run are given in Fig. 19.

This experiment does demonstrate that the Bubble King mini 160 skimmer is effective at removing bacteria from the modified aquarium water sample, at least to the extent of approximately 39% over 2 hours. The control water samples remained fairly constant in comparison. Thus, for the first time, we can conclude that skimming does clear some bacteria from the water column of a reef tank. However, the artificial nature of this experiment, with its induced high bacteria population, raises the important question of its relevance to bacteria removal from an untreated reef tank water sample. Nevertheless, the bubble-based mechanism of protein skimming itself is demonstrated to be capable of bacteria export, and so that component of the Fig. 18 puzzle is exonerated.

How can we design an experiment that probes the question of skimmer-based bacterial export in a more realistic reef tank-like setting? One possibility is to rely on our EtOH dosing regimen to provide the necessary "bacteria-rich" but otherwise realistic aquarium water sample. This idea was pursued by removing a 30 gallon water sample from the KSF tank, prepared as described above, and placing it in the Rubbermaid tub with circulation provided by two powerheads. The Bubble King skimmer was in the water, but not running. EtOH (0.29 mL, ~ 1 ppm of C) was added daily for three days. At the 3-day mark, the skimmer was activated, and water samples were removed and worked up as described previously. In addition, the TOC levels of this water sample were monitored during the 120-minute skimming run. The results are presented in Fig. 20.

The starting bacteria population in this instance, ~ 2300K/mL, was lower than that generated by simply using the tub itself (or its organic surface coating) as a source of bacterial growth nutrients (cf. Fig. 19). The reason for this difference is not clear. Nevertheless, the bacteria depletion profile generated in this experiment (pink line in Fig. 20) is quite similar to that one observed in the experiment of Fig. 19, although the overall % decrease in bacteria population is a little less (28%, vs. 39% in Fig. 19). The non-skimmed control water sample remained relatively constant in its bacteria population over the 2-hour course of this experiment. The TOC level in the water sample is depleted by skimming as well, dropping from about 2.5 ppm down to about 2.0 ppm (~ 25% decrease) over the 2-hour skimmer run. Both the bacteria population and the TOC level appear to have reached their "floors"; no further removal of each was detected over the last third of the run. Once again, we have confirmed that if the aquarium water sample is "spiked" with bacteria, skimmer-based removal is observed. However, the results of both of these experiments (Figs. 19 and 20) indicate that only a component of the entire water column bacteria cohort appears to be susceptible to skimmer-based removal.

One last attempt to detect skimmer-based removal of bacteria from KSF aquarium water that is not too perturbed was pursued, Fig. 21. The observation (Fig. 14) that IORC water-change water introduces some bacteria into the aquarium served as the catalyst for this last skimmer run. In this instance, the H&S skimmer and GAC filter were turned off 48 hours prior to water removal. Then, two 30-gallon water changes were conducted on two successive days just before the experimental water sample was removed. Thus, the aquarium water's bacteria population should have been elevated relative to its equilibrium (and unskimmable) levels, but not to an artificially high value like those that attended either the EtOH dosing run (Fig. 20) or the tub contamination run (Fig. 19). This water sample was skimmed immediately upon transfer into the Rubbermaid tub. As it turned out, the initial bacteria population level was not particularly high, around 90K/mL. Over the first 10 minutes of skimmer operation, this value increased to about 100K/mL, presumably as a consequence of added bacteria from the tub/skimmer/powerhead setup. Despite this initial surprising data point, the remaining data set taken over the next 110 minutes followed the expected course, and the bacteria level depleted overall by about 29%. The unskimmed control water sample did not show any notable fluctuations in its bacteria content over the course of the experiment. For the first time, we have obtained evidence that protein skimming will deplete some bacteria from authentic aquarium water that has bacteria/mL levels not so far out of line from an unperturbed system (cf. Table 1). Nevertheless, one message comes through clearly from all of these experiments, regardless of the starting bacteria/mL level; only a minor fraction (~ 28 - 39%) of the bacteria present are susceptible to extraction by bubbles.

Finally, we wondered how the skimming results with these "captured" water samples, i.e., samples removed from an authentic reef setting and thus lacking all of the dynamic bacteria growth/removal mechanisms present in a reef tank, might compare to the results obtained upon skimming an actual functioning reef tank. Of course, such a tank cannot be pre-skimmed or we will be back to where we started with Fig. 18. The SJ 55 gallon tank fits this requirement (see Fig. 6); it has a healthy, thriving population of fish, soft corals, invertebrates, etc., but this tank has not been skimmed or treated with GAC filtration for approximately six months. It has, however, been subjected to daily vodka additions as described in the discussion of Table 1 and Fig. 9. The Bubble King mini 160 protein skimmer was placed in the sump of this aquarium system, turned on, and adjusted as described above. Water samples (25 mL) were removed periodically over 24 hours, dosed with 0.5 mL of 37% formaldehyde, and frozen for later analysis. No control samples were utilized in this experiment. The results are depicted in Fig 22.

Clearly, the Bubble King skimmer has performed effectively in dropping the bacteria load of this naturally bacteria-rich tank water by approximately 37%. In fact, this decrease was recorded by the 8-hour mark, and even after 2 hours (the time period of the KSF tank water skimming experiments), the bacteria population decrease was significant; ~ 22%. It should be noted that no late rise in the bacteria population was seen; the skimmer was effective at keeping the level at some baseline value (~ 1500K/mL) for this tank. Interestingly, even thought the bacteria population starting points in the KSF tank water skimming experiments (Figs. 19, 20, and 21) and the SJ 55 skimming experiment were very different, in all cases, only about 28-39% of the original bacteria were removed before the data "flatlined".

It is likely a significant observation that there is a floor in aquarium water bacteria populations that skimming will not breach. Perhaps in both water sources, the SJ 55 naïve tank water and the KSF modified (or not) tank water, there appears to be two functionally distinct populations of bacteria; one that is susceptible to bubble-based removal, and one that is not. What is this functional difference, as far as the skimmer is concerned? An earlier publication describes the limitations of bubble-based mechanisms in scrubbing TOC from aquarium water (Feldman, 2009). The argument forwarded in that case may very well apply here as well, Fig. 23. It is plausible that the requirement for hydrophobic patches on particles (i.e., bacteria, TOC molecules or clusters; refer to Bacterial Surface Charge and Protein Skimming, Section 1.2 above) that must be met for successful bubble-based extraction may only apply to some but not all of the aquarium water column bacteria (approximately 28-39%, by our studies). Thus, there may be some discrimination by the skimmer based upon bacteria surface properties. In addition, some but not all bacteria form multicellular clumps (flocs) that may be susceptible to foam-based extraction based upon simple buoyancy and not bubble-surface chemistry; once again, this physical process constitutes a basis for selecting between different bacteria types. So, the bottom line appears to be that some but not all bacteria can be removed by protein skimming.

Overall, the major conclusions from these water column bacteria removal experiments are

1. GAC (Granular Activated Carbon) filtration does not remove bacteria from the water column.
2. Protein skimming (bubbles) removes approximately 30 - 40% of the bacteria in the water column of carbon-treated or organic rich water, but the remainder is not susceptible to bubble-based removal.
3. Steady state bacteria populations in skimmed reef tank water are not subject to further skimmer-based bacteria removal - there is a baseline value that the skimmer will not go below.

4. Conclusions

The preliminary studies described herein document, for the first time, the modulation of water column bacteria population in reef tank water as a consequence of either (a) carbon source addition or (b) mechanical filtration (GAC, skimming). This information bears on the Carbon Dosing hypothesis for nutrient removal in marine aquaria.

Aquaria subjected to active filtration via skimming present water column bacteria populations that are approximately 1/10 of those observed on natural reefs. The consequences of this disparity on the long-term health of the tank's livestock are not known. How do reef tank organisms adapt to such a bacteria-deficient environment? Is the whole food web in an aquarium perturbed, or are there compensatory mechanisms that maintain an appropriate energy transduction through all of the trophic levels? Is "old tank syndrome" related to possible nutritional deficiencies stemming from this bacteria "gap"? Alternatively, could "old tank syndrome" be symptomatic of a gradual decrease of bacterial diversity as a consequence of selective skimmer-based removal of only bubble-susceptible bacteria? At present, it is not possible to go beyond speculation on these points - further research is needed.

On the other hand, our studies have shown that bacterial growth appears to be carbon limited in reef aquarium water. However, there is a demonstrable difference between reef tank water in an active reef tank, and reef tank water removed from the tank. In the latter case, bacteria consumers are largely absent, and so fueling bacteria growth via carbon addition translates to rapid and large increases in bacteria population. In an active reef tank, however, this population increase is not manifest, presumably because active predation keeps the overall level in check. Thus, the highly dynamic nature of bacteria populations in the water column of reef aquaria is highlighted by these studies. From a different perspective, the bacteria population in a reef tank seems to act as a buffer to help dissipate the otherwise potentially serious negative consequences of (inadvertent?) tank pollution via rapid carbon addition, at least perhaps up to a saturation point.

Finally, mechanical filtration in the form of skimming but not GAC does provide an effective means of bacteria export, at least up to a point. It appears likely that some types of bacteria are indeed "skimmable", but others are not. Thus, skimming inadvertently provides severe (?) evolutionary pressure to skew the tank's resident water column bacteria population to favor the "non-skimmable" cohort.

The bottom line with respect to the carbon dosing hypothesis is clear; the basic tenets of this theory appear to hold up to experimental scrutiny; carbon dosing does increase water column bacteria populations, and skimming does remove some bacteria with their attendant nutrient loads. Thus, the underlying science behind this approach to nutrient export appears valid.

5. Acknowledgments

We thank the Eberly College of Science at the Pennsylvania State University and E. I DuPont de Nemours and Co. for financial support. The expertise and assistance of Dr. Elaine Kunze, Susan Magaree, and Nicole Zembower of the Flow Cytometry laboratory within the Huck Institutes of the Life Sciences at Penn State is gratefully acknowledged. Furthermore, we thank Mr. David Jones of the Pennsylvania State University Department of Civil and Environmental Engineering for assistance with the Shimadzu 5000 TOC Analyzer.

6. References

1. Ainsworth, T.D.; Fine, M.; Roff, G.; Hoegh-Guldberg, O. 2008. "Bacteria are Not the Primary Cause of Bleaching in the Mediterranean Coral Oculina patagonia." ISME J. (Multidisciplinary Journal of Microbial Ecology), 2, 67-73.
2. Azam, F.; Malfatti, F. 2007. "Microbial Structuring of Marine Ecosystems." Nature Rev. Microbiol., 5, 782-791.
3. Bergey, D.H. 1994. Bergey's Manual of Determinative Bacteriology, 9th ed. (Edited by John G. Holt et al.) Baltimore: Williams & Wilkins.
4. Brambilla, F.; Antonini, M.; Ceccuzzi, P.; Terova, G.; Saroglia, M. 2008. "Foam Fractionation Efficiency in Particulate Matter and Heterotrophic Bacteria Removal from a Recirculating Seabass (Dicentrarchus labrax) System." Aquacultural Engineering, 39, 37-42.
5. Brettar, I.; Rheinheimer, G. 1992. "Influence of Carbon Availability on Denitrification in the Central Baltic Sea." Limnol. Oceanogr., 37, 1146-1163.
6. Bretz, H.W.; Wang S.L.; Grieves, R.B. 1966. "Variables Affecting the Foam Separation of Escherichia coli." Appl. Microbiol., 14, 778-783.
7. Button, D. K.; Robertson, B. R. 1989. "Kinetics of Bacterial Process in Natural Aquatic Systems Based on Biomass as Determined by High-Resolution Flow Cytometry." Cytometry, 10, 558-563.
8. Cano, R.J.; Borucki, M., 1995. "Revival and Identification of Bacterial Spores in 25 to 40 million year old Dominican Amber." Science, 268, 1060-1064.
9. Carlson, C.A.; Ducklow, H.W. 1996. "Growth of Bacterioplankton and Consumption of Dissolved Organic Carbon in the Sargasso Sea." Aq. Microbial Ecol., 10, 69-85.
10. Carrero-Colon, M.; Nakatsu, C.H.; Konopka, A. 2006. "Effect of Nutrient Periodicity on Microbial Community Dynamics." Appl. Environ. Microbiol. 72, 3175-3185.
11. Dinsdale, E. A.; Pantos, O.; Smriga, S.; Edwards, R. A.; Angly, F.; Wegley, L.; Hatay, M.; Hall, D.; Brown, E.; Haynes, M.; Krause, L.; Sala, E.; Sandin, S. A.; Thurber, R. V.; Willis, B. L.; Azam, F.; Knowlton, N.; Rohwer, F. 2008. "Microbial Ecology of Four Coral Atolls in the Northern Line Islands." PLoS One, 3, e1584, 1-17.
12. Ducklow, H. W.; Mitchell, R. 1979. "Bacterial Populations and Adaptations in the Mucus Layers on Living Corals." Limnol. Oceanogr., 24, 715-725.
13. Ducklow, H. W. 1983. "Production and Fate of Bacteria in the Oceans." BioScience, 33, 494-501.
14. Eppley, R. W. 1980. "Estimating Phytoplankton Growth Rates in the Central Oligotrophic Oceans." Brookhaven Symp. Biol., 31, 231-242.
15. Feldman, K. S.; Maers, K. M. 2008. "Total Organic Carbon (TOC) and the Reef Aquarium: an Initial Survey. Part 2." Advanced Aquarist, http://www.advancedaquarist.com/2008/9/aafeature2/
16. Feldman, K. S.; Maers, K. M. 2008. "Total Organic Carbon (TOC) and the Reef Aquarium: an Initial Survey. Part 1." Advanced Aquarist, http://www.advancedaquarist.com/2008/8/aafeature3/
17. Feldman, K. S.; Maers, K. M.; Vernese, L. F.; Huber, E. A.; Test, M. R. 2009. "The Development of a Method for the Quantitative Evaluation of Protein Skimmer Performance." Advanced Aquarist. http://www.advancedaquarist.com/2009/1/aafeature2/
18. Feldman, K. S.; Maers, K. M. 2010. Further Studies on Protein Skimmer Performance." Advanced Aquarist. http://www.advancedaquarist.com/2010/1/aafeature
19. Gaudin, A.M.; Mular, A.L.; O'Connor R.F. 1960. "Separation of Microorganisms by Flotation. I. Development and Evaluation of Assay Procedures." Appl. Microbiol. 8, 84-90.
20. Gaudin, A.M.; Mular, A.L.; O'Connor R.F. 1960. "Separation of Microorganisms by Flotation. II. Flotation of Spores of Bacillus subtilis var. niger." Appl. Microbiol. 8, 91-97.
21. Gaudin, A.M.; Davis, N.S.; Bangs, S.E. 1962. "Flotation of Escherichia coli with Sodium Chloride." Biotech. Bioeng. 4, 211-222.
22. Goldman, J.C.; Dennett, M.R. 2000. "Growth of Marine Bacteria in Batch and Continuous Culture under Carbon and Nitrogen Limitation." Limnol. Oceanogr., 45, 789 - 800.
23. Gottfried, M.; Roman, M. R. 1983. "Ingestion and Incorporation of Coral Mucus Detritus by Reef Zooplankton." Mar. Biol., 72, 211-218.
24. Granger, J.; Price, N.M. 1999. "The Importance of Siderophores in Iron Nutrition of Heterotrophic Marine Bacteria." Limnol. Oceanogr., 44, 541-555.
25. Grégori, G.; Citterio, S,; Ghiani, A.; Labra, M.; Sgorbati, S.; Brown, S.; Denis, M. 2001. "Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining." Appl. Environ. Microbiol., 67, 4662-4670.
26. Gruber, D.F.; Simjouw, J.-P.; Seitzinger, S. P.; Taghon, G.L., 2006. "Dynamics and Characterization of Refractory Dissolved Organic Matter Produced by a Pure Bacterial Culture in an Experimental Predator-Prey System." Appl. Environ. Microbiol. 72, 4184-4191.
27. Hobbie, J.E.; Daley, R. J.; Jasper, S. 1977. "Use of Nucleopore Filters for Counting Bacteria by Epifluorescence Microscopy. Appl. Environ. Microbiol. 33, 1225-1228.
28. Johannes, R. E. 1967. "Ecology of Organic Aggregates in the Vicinity of a Coral Reef." Limnol. Oceanogr., 12, 189-195.
29. Lebaron, P.; Parthuisot, N.; Catala, P. 1998.
    "Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems." Appl. Environ. Microbiol., 64, 1725-1730.
30. Kirchman, D.L. 1994. "The Uptake of Inorganic Nutrients by Heterotrophic Bacteria." Microbial Ecology, 28, 255-271.
31. Konhauser, K. 2007. Introduction to Geomicrobiology. Massachusetts: Blackwell Publishing.
32. Kooperman, N.; Ben-Dov, E.; Kramarsky-Winter, E.; Barak, Z.; Kushmaro, A. 2007. "Coral mucus-associated bacterial communities from natural and aquarium environments." FEMS Microbiol Lett, 276, 106-113.
33. Kvennefors, E.C.E.; Sampayo, E.; Ridgway, T.; Barnes, A.C.; Hoegh-Guldberg, O. 2010. "Bacterial Communities of Two Ubiquitous Great Barrier Reef Corals Reveals both Site- and Species-Specificity of Common Bacterial Associates." PLoS One, 5, e10401.
34. MacLeod, R.A. 1965. "The Question of the Existence of Specific Marine Bacteria." Bacteriol. Rev., 29, 9-23.
35. Marie, D.; Partensky, F.; Jacquet, S.; Vaulot, D. 1997. "Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green 1." Appl. Environ. Microbiol., 63, 186-193.
36. Marie, D.; Partensky, F.; Vaulot, D.; Brussaard, C. 1999. "Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples." Curr. Prot. Cytometry, 11.11.1-11.11.15.
37. Michael, E. 2008. http://glassbox-design.com/2008/achieved-through-observation-and-experimentation/.
38. Monfort, P.; Baleux, B. 1992. "Comparison of Flow Cytometry and Epifluorescence Microscopy for Counting Bacteria in Aquatic Ecosystems." Cytometry, 13, 188-192.
39. Monger, B. C.; Landry, M. R. 1993. "Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342." Appl. Environ. Microbiol., 59, 905-911.
40. Moriarty, D. J. W.; Pollard, P. C.; Hunt, W. G. 1985. "Temporal and Spatial Variation in Bacterial Production in the Water Column over a Coral Reef." Mar. Biol., 85, 285-292.
41. Noble, R. T.; Fuhrman, J. A. 1998. Use of SYBR Green 1 for Rapid Epifluorescence Counts of Marine Viruses and Bacteria." Aquat. Microb. Ecol., 14, 113-118.
42. Paul, J. H.; Rose, J. B.; Jiang, S. C.; Kellogg, C. A.; Dickson, L. 1993. "Distribution of Viral Abundance in the Reef Environment of Key Largo, Florida." Environ. Microbiol., 59, 718-724.
43. Porter, J. 2004. "Flow Cytometry and Environmental Microbiology." Curr. Protocols Cytometry, 11.2.1-11.2.13.
44. Rivkin, R.B.; Anderson, M.R. 1997. "Inorganic Nutrient Limitation of Oceanic Bacterioplankton." Limnol. Oceanogr., 42, 730-740.
45. Robertson, B. R.; Button, D. K. 1989. "Characterizing Aquatic Bacteria According to Population, Cell Size, and Apparent DNA Content by Flow Cytometry." Cytometry, 10, 70-76.
46. Rypien, K.L.; Ward, J.R.; Azam, F., 2010. "Antagonistic Interactions Among Coral-Associated Bacteria." Environ Microbial., 12, 28-39.
47. Suzuki, Y.; Hanagasaki, N.; Furukawa, T.; Yoshida, T. 2008. "Removal of Bacteria from Coastal Seawater by Foam Separation Using Dispersed Bubbles and Surface-Active Substances." J. Biosci. Bioeng., 105, 383-388.
48. Thompson, J.; MacLeod, R.A. 1974. "Potassium Transport and the Relationship between Intracellular Potassium Concentration and Amino Acid Uptake by Cells of a Marine Pseudomonad." J. Bacteriology, 120, 598-603.
49. Todar, K. 2008. Todar's Online Textbook of Bacteriology. http://www.textbookofbacteriology.net/index.html .
50. Troussellier, M.; Courties, C.; Vaquer, A. 1993. "Recent Applications of Flow Cytometry in Aquatic Microbial Ecology." Biol. Cell, 78, 111-121.
51. Troussellier, M.; Courties, C.; Zettelmaier, S. 1995. "Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picoplankton: Fixation and Storage Effects." Estuar. Coast. Shelf Sci., 40, 621-633.
52. Verschuere, L.; Rombaut, G.; Sorgeloos, P.; Verstraete, W. 2000. "Probiotic Bacteria as Biological Control Agents in Aquaculture." Microbiol. Molec. Biol. Rev., 64, 655-671.
53. Walton, N. A.; Bjornson, M. 2008. "Vodka Dosing…Distilled! A Powerful Method for the Reduction of Nitrates and Phosphates within the Reef Aquaria." ReefKeeping, 8. http://reefkeeping.com/issues/2008-08/nftt/index.php
54. Wegley, L.; Mosier-Boss, P.; Lieberman, S.; Andrews, J.; Graff-Baker, A.; Rohwer, F. 2006. Environ. Microbiol., 8, 1775-1782.
55. Yoshinaga, I.; Fukami, K.; Ishida, Y. 1991. "Comparison of DNA and Protein Synthesis Rates of Bacterial Assemblages Between Coral Reef Waters and Pelagic Waters in Tropical Ocean." Marine Ecol. Prog. Ser., 76, 167-174.

The international pet product trades show in Nuremberg, Germany this past May was a smorgasbord of new marine and reef aquarium products. Some of these new products for aquariums were unique, some creative, some were great and others were really bad. All of these qualities are not mutually exclusive; many of these aquarium products were innovative and many of them deserve further attention. Obviously the question of whether a product is innovative or useful is totally subjective but I hope you'll just take this article as a mini review of products which are interesting enough to write about. If nothing else, take these products as harbingers of concepts which have wider implications for what kind of concepts can help us all to run better marine and reef aquariums.

The Evo3 from Genesis is an interesting device which is kind of like a steampunk DialySeas machine. Instead of using a thin film membrane and water to constantly remove wastes that are dissolved in the water, the Evo3 Titanium uses a roll of filter material and a water wheel to dole out the paper filter as needed based on the amount of waste clogging up the filter paper. The way the Evo3 works is by having the filter paper wrap around a large cylindrical titanium basket. The large basket offers up a large surface area for water to flow through and be mechanically filtered. The filter basket is contained within a box of a modest size and about ten inches deep. As the filter paper around the basket becomes clogged and passes less water, the level of water in the box rises and eventually overflows via a dedicated pipe. This overflow pipe feeds water to a waterwheel which is connected to a waste filter paper collection roll. As the filter paper around the basket becomes clogged, the water level in the Evo3 box rises, overflows to a waterwheel, when it turns so does the waste collection roll which also pulls in fresh paper over the filter basket, causing the water level to drop in the box and the cycle begins all over again.

The Evo3 looks and sounds like a Rube Goldberg machine and I had my doubts when I first came across it. However, watching the Evo3 do its thing over the better part of a week gave me a sense of confidence that the design was robust and in fact, the Evo3 has been cleaning up ponds for the better part of five years. What is new is the Evo3 Titanium which features all custom made bearings, filter cage and bolts that are machined from titanium to absolutely eliminate the risk of corrosion when used in a marine water environment. My enthusiasm for the Evo3 is less about what it can do for marine fish tanks now, than what the concept of constant mechanical nutrient export can do for managing water quality of aquarium water. At $2000-2500 a piece the Evo3 is far from affordable but with future expansions on the concept of automated mechanical filtration perhaps future low nutrient SPS tanks will rely more on steam punk mechanical filtration ingenuity than starving Acros and Montis like bulimic super model corals.

The manual protein skimmer neck cleaner from Deltec is not a standalone product as much as it is a feature. Beginning very soon, selected models of Deltec skimmers will come with the option of having this manual neck cleaner which is basically a double bladed squeegee that rides the inside of the protein skimmer neck. The manual neck cleaner is rotated by twisting the lid of the skimmer cup. The first thing that struck me about the manual neck cleaner for Deltec protein skimmers is why the motorized version came before the manual one. Perhaps the motorized skimmer neck cleaner was first introduced because of the perception that a self cleaning head needed to be a very constant operation to prevent big globs of gunk from falling back into the skimmer body. Depending on how the skimmer is performing, it stands to reason that twisting the lip and neck cleaner every few days would remove a small amount of accumulated proteins which could then be easily foamed out the skimmer neck. My guess is that the increased overall performance of the protein skimmer with a cleaner neck would outweigh some of the protein skimmate which might return to the system. Also keep in mind that this Deltec feature is only the archetype of the manual protein skimmer neck cleaner and the concept is screaming to be elaborated on.

You can picture how my eyes rolled when I was walking past the Aquatic Nature booth and a representative said he had a special fish net he wanted to show me. I could not fathom how in the world a fishnet could ever be worthy of writing about but then he pressed the net against the aquarium glass and the epiphany of this device's usefulness was immediately apparent. You see, the Aquatic nature fish net is not only clear and light colored to make less visible underwater but the fish net incorporates a thick piece of silicone between the head of the net and the end of the handle so that the net can easily be pressed against the aquarium glass to catch the desired aquarium fish and to keep it from escaping. Like Deltec's manual neck cleaner I already wrote about, the flexible fishnet from Aquatic Nature is an idea so simple it's a wonder why no one has ever thought of it. Sure the Flexi net is not going to keep aquarists up at night waiting in anxiety to get their fins on state of the art fish capture technology but, for busy aquarium shops who sell a lot of livestock the flexible fishnet is sure to be a welcome addition to the tools of the trade.

The last really simple and novel product that I came across at InterZoo is actually a whole category of products. In recent years certain aquarium manufacturers began taking notice of the clip on propeller fans which are often used to increase evaporative cooling of aquarium water. These fans are clipped on the side of the aquarium and they are in your face, really taking away from the aesthetic of the aquarium. Vortex fans use a rotor which is more like an impeller in that they "pump" air through a narrow channel and direct this airflow to the aquarium's water surface. What sets vortex aquarium fans apart from regular clip on fans is that the main operating parts can hang on the side of the aquarium and their slim profile makes them easy to hide behind the aquarium. The only part of the vortex aquarium fan which is plainly visible is the small channel that is integrated into the holder on the edge of the aquarium. One model we saw even had a adjustable outlet and when angled straight down it did a fine job of rippling the surface and increasing glimmer lines. Vortex aquarium fans with the hang-on back of the aquarium design were represented by models from Dymax of Singapore, Kotobuki of Japan and several other OEM manufacturers from China.

Speaking of glimmer lines, if someone had told me that two of the more memorable devices at InterZoo would be return/draining devices I would have told them to get outta dodge. First up is the Xinout combination return and draining device from the Italian company Xaqua. The Xinout kit comes with a decent draining component that is both an ideal sized strainer and a silencing kind of drain fitting, a high quality silicone hose for the return line and a vacuum hose for the drain. Sounds pretty mundane but the real fun is where the return device comes in. With no moving parts, the return component of the Xinout pulses the water in a rhythmic motion that is based on the flow rate. There are no diaphragms and no moving parts in this thing and it's just bizarre to watch it pulse with water flowing through it and nothing but fluid dynamics producing the pulsed effect. I took a good look at the inner chamber of the Xinout and all I could gather is that there is an egg shaped cavity at the point where the water is directed at a ninety degree angle. At the moment the pulsing motion is really good at producing extra large ripples which are more aesthetic than anything, helping to increase the amount of glimmer lines coming from point source lighting. With some trial and error it might be possible to adjust an Xinout return device to pulse at a frequency that resonates with the dimensions of an aquarium to get more harmonic wave motion a la Tunze Wavebox or Vortech pump. There is virtually no other information available on the Xinout from Xaqua but I hope that it gets puts through wider aquarium use in the future so we can all learn what this thing is really capable of.

The second noteworthy aquarium water plumbing device I spotted at InterZoo is the Mame Nano Overflow, an elegant and ingeniously designed overflow and return device. If you try to imagine the path that water takes when flowing through a conventional overflow box, and simplify that image to a single continuous tube you get the Nano Overflow. The Nano Overflow is made of three pieces of handmade glass connected together with tightly fitting vinyl tubing. The center piece of glass is actually the middle of the drain line and the only piece of the return line which are fused together by thick sections of solid glass. The return tubing has a built in venturi which is set up to draw in the air that normally accumulates on the last bend of the drain tube, same as you would on the U-tube of a classic overflow box. The venturi of the Nano Overflow can automatically restart the siphon in case of a power interruption or other siphon break. When I looked closely at the Mame Nano Overflow I couldn't help but be reminded of a classic freshwater device, the Lily Pipe made by Aqua Design Amano.

Mame only makes four products so it's such a surprise that another one of their most recent products is also quite memorable from the ocean of aquarium product from InterZoo. I saw well over 100 different LED lights, strips, tubes, lamps and just about every other form factor you can conjure up. There was a number of aquarium lights using RGB LEDs that offered a degree of custom color control but the Mame Eco-Light was a real standout effort at doing RGB reef lighting. The 49 watt Eco-Light is actually the fifth version of this particular product and the build and finish of it really made it stand out from lots of hastily completed prototypes that were the norm at InterZoo. Furthering the cause is Mame's inclusion of yellow LEDs which makes the Mame Eco-Light the first RGBY LED aquarium light and with 49 watts of diodes in less than a square foot, the power ought to be well enough for a medium sized nano reef. If you think that RGB lighting is corny, well that's because you haven't seen what RGB LEDs can do because you haven't seen Mame's RGBY Eco-Light. Furthermore, custom control of color and intensity of LEDs will become the standard for the solid state lighting in the future so we might as well figure out how to make it work for aquariums now.

If you've been jaded by a slew of new LED fixtures that are hard to justify in the reefing budget, then get a hold of the LED tubes from Econlux that are the same T5 and PC shaped lamps you're already using. Sure you've seen fluorescent shaped t00bs with LEDs in them but these are uniquely designed to be powered by the ballast that is driving your existing HO T5 lamps and power compacts. Econlux was showing these plug and play LED replacement tubes for conventional t00b technology in a wide number of sizes and power ratings. Econlux didn't seem too interested in bringing these to market themselves as much as making them for regional lighting markets that would be OEM'd for other companies. Presumably we'll one day be able to walk into a fish store and pretend to buy a replacement in a shape we are familiar with but instead it will be loaded with the light emitting diodes as opposed to the much shorter lived fluorescents. Imagine if a single lamp could be built with a wide range of LED colors with blue and white LEDs that would accomplish similar effects as the dual colored power compact lamps. This hoop-dream of plug and play LED lamps that replace fluorescent lights could take a while to become widely available and it's viability really depends on how cost and performance of full blown LED lights change progresses in the future.

The last really memorable product from InterZoo that I want to tell you about is the HD touchscreen aquarium controller called the Vertex Cerebra. The Cerebra is interesting not only for it's ultra modern touch screen interface and iPad-esque form factor but also for it's applications. Much like the Apple Music store of the App store the Cerebra will have its own app market where developers will be able to sell or give away their aquarium apps. Theoretically the apps could control anything that the Cerebra interfaces with which for now is limited to the Vertex line of products which includes lots and lots of very new products. If you think about it, the Vertex Cerebra is copying Apple's iEcosystem of products and services which in itself is far from innovative. However, if you consider that it might one day be possible for an inexperienced aquarist to buy an equipment set, download an app to control it all and have it tuned to run as a Chalice coral tank or a high energy SPS tank, well that's bold new ground my friend. The concept that the Vertex Cerebra is trying to bring to the aquarium hobby will have an uphill battle of price (the Cerebra will almost surely cost more than an iPad), applications that run on popular smartphones and open software and hardware controllers that are currently under development for the aquarium hobby.

It's easy to criticize new products, products for which a market category may not yet exist. I am sometimes accused of being overly optimistic about the potential of new products for the aquarium hobby. I think it's way too easy to pick at everything that can go wrong with a certain device or invention and it's all too easy to kick the legs out from a concept before it's really been given a chance. InterZoo 2010 certainly delivered on a load of new products for use on small and large aquariums and many of them may not be revolutionary in themselves. However, the ideas these products represent can shed insight on new ways to perform common tasks like lighting a reef tank, catching a fish with a net or getting water in and out of an aquarium. I hope readers will consider the ideas that I tried to illustrate in the new products from this ginormous trade show because it'll be another two years before we get another massive wave of innovation in the aquarium hobby until the next InterZoo conference.

As promised to our freshwater readers, I will report on my 500-gallon freshwater planted aquarium's progress. First of all let me say that I'm enjoying this aquarium immensely. I particularly enjoy the size perspective between the plants, rock, and driftwood with that of the schools of mostly small fish like rummy-nose tetras (Hemigrammus rhodostomus) and cardinal tetras (Paracheirodon axelrodi) that I have introduced to the tank.

As I pointed out before I intended to go low tech, but heeding the device I gleaned from several books on freshwater planted tanks, from readers, and Jake Adams I had to add more light and a CO₂ controller-injection system. The CO₂ system keeps the pH at about 6.6. Currently, I have 12 4-foot T5 54 HO watt bulbs. This gives me a total of 648-watts. However, according to many experts this is not nearly enough for certain plants, especially since my tank is 30-inches high. Nevertheless, most of the plants are doing quite well. The photo period is 12-hours. Half of these bulbs have a color temperature of 10K, and the other half are so-called freshwater bulbs - not sure of their color temperature. This brings me to a question: Should I use all 10K bulbs or continue with half that supposedly have a color temperature specifically designed for freshwater plants?

So far I have not had a problem with algae. I have four algae eating cat fish, two bristlenose plecostomuses (Ancistrus temminckii) and one large common pleco (Plecostomus punctatus), and one whiptail Loricaria sp. I also have two very active red tail sharks (Epalzeorhynchos bicolor). I'm aware that many experts say that large Plecos eat plants, but I have not witnessed that yet. I also have a large Leporinus fasciatus, which I was told would eat my plants, but so far it hasn't done so. It is now about 6-inches long. I also have a lot of fish, which is also a no no, but still no problem.

I suppose it's now time to try some of the more delicate plants, like a Madagascar lace (Aponogeton madagascariensis). I would appreciate any suggestion or advice regarding this plant.

Here is another picture of another section of my freshwater aquarium. The corkscrew Vallisneria (Vallisneria Americana) is now sending out runners in all directions, after not doing anything for several months. I would like to try one or two discus fish, but suspect that the temperature I keep my tank at this time of the year is too low for them -- any advice about this?

The rather counterintuitive observation that protein skimmers remove only 20 - 35% of the measurable Total Organic Carbon (TOC) in reef aquarium water (Feldman, 2009; Feldman, 2010) begs the question, "what is all that "stuff" that collects in our skimmer cups?" Is it really TOC, or at least a labile, or "skimmable", fraction of TOC? Attempts to identify TOC components from authentic ocean water are still in their infancy, and to date this material has resisted detailed chemical analysis. Recent efforts primarily by Hatcher and colleagues (Mopper, 2007; De la Rosa, 2008) using sophisticated mass spectrometry and nuclear magnetic resonance spectroscopy techniques have revealed that authentic ocean TOC is comprised of tens of thousands of discrete compounds that include chemical representatives from all of the major biochemical groups; lipids, peptides, carbohydrates, heterocycles, aromatics, etc. The relationship between ocean TOC and aquarium TOC still remains to be established, but it seems likely that the TOC in our aquaria is equally diverse and rich in its chemical complexity. Thus, it is equally unlikely that a chemical breakdown of aquarium TOC will be forthcoming in the near future. Nevertheless, there are analytical methods that can reveal and quantify most of the elemental components of TOC, and with a little chemical intuition, allow for the assignment of some of these components to chemical categories. These analytical methods are called Elemental (or Combustion) Analysis and Inductively Coupled Plasma Atomic Emission Spectroscopy. Both methods are available from many commercial operations; we used Columbia Analytical Services in Tucson AZ for our skimmate samples (http://www.caslab.com/).

Experimental Results

All skimmate samples were obtained from the collection cup of an H&S 200-1260 skimmer running on a 175-gallon reef tank under the author's care. During the time of these collections, the tank contained 10 fish (pair of Pterapogon kauderni (Banggai cardinals), pair of Liopropoma carmabi (candy bass), Centropyge loriculus (flame angel), Centropyge interrupta (Japanese pygmy angel), Oxycirrhites typus (longnose hawkfish), Zebrasoma flavescens (yellow tang), Amblygobius bynoensis (byno goby) and Synchiropus splendidus (mandarin)), approximately 40 coral colonies from the SPS, LPS and chalice categories, and a few dozen snails and hermit crabs. No soft corals or clams were present. Typical daily feedings included one cube of Hikari mysis shrimp, one cube of PE mysis shrimp, a pinch of flake food, and a pinch of pellet food. Thrice weekly, the Reef Nutrition products Phytofeast, Rotifeast, Oysterfeast and Arctipods were used, and a sheet of nori was added once per week. The skimmer cup was cleaned weekly, and Granular Activated Carbon (GAC), Granular Ferric Oxide (GFO), a calcium reactor, and a UV sterilizer all were used continuously. Seventeen percent of the water volume was changed weekly, and tank parameters were measured on a weekly basis as well; [TOC] = 1.4 ppm (1 hr after feeding) - 0.5 ppm (24 hrs after feeding), [Ca] = 390 - 410 ppm, [Mg] = 1230 - 1260 ppm, [alk] = 3.5 - 4 meq/L, salinity = 34.5 - 36 ppt, pH = 7.8 (lights on) - 8.1 (lights off), [NO₃] < 0.5 ppm, no measurable NH₄, NO₂, or PO₄. Illumination was provided by two 400W 14K Geissmann metal halide bulbs and one 175W 15K Iwasaki metal halide bulb on an 8-hr on, 16-hr off cycle. No additives except CaCl₂•2H₂O were used.

Our initial experiment was designed to probe the composition of the water-insoluble solid material removed by a protein skimmer. Skimmate was collected over 4 days without any food addition to the aquarium, Fig. 1. The liquid and solid contents of the H&S 200-1260 skimmer cup were carefully removed after this time period and concentrated to dryness through initial liquid evaporation under reduced pressure and then vacuum drying at 110 ^oC/0.2 mm. This procedure effectively removes almost all of the water (see below), and of course any volatile components of the skimmate. Seventeen grams of gray-brown solid resulted, see Fig. 1.

Four grams of this crude skimmate were suspending in 100 mL of distilled water and vigorously stirring for several hours. The mixture was then separated by centrifugation at 6000 rpm/10 min, and the supernatant was poured off and discarded. This procedure was repeated 3 times, and then the remaining material was vacuum dried at 110 ^oC/0.2 mm for 48 hours to afford 0.47 gm of gray-green solid. Note that CaCO₃ must be heated to > 900 ^oC to burn off CO₂. This solid was subjected to elemental analysis as described above at Columbia Analytical Services:

• C: 21.08 %
• H: 2.39 %
• N: 2.22 %
• Ca:17.43 %
• Mg: 1.35 %
• Si: 4.76 %
• P: 0.16 %

These data can be interpreted with some application of chemical intuition and some assumptions.

1) Calcium analysis

17.43 % by weight Ca implies that the total amount of Ca in the 470 mg sample is 82 mg. Assuming that all of this Ca is in the form of calcium carbonate (CaCO₃, MW = 100), then the 470 mg of dried skimmate contains 205 mg (44 %) of CaCO₃. Since carbon is 12 % (by weight) of CaCO₃, then the 470 mg of dried skimmate contains ~ 25 mg (~5.2 %) of (inorganic) carbon contributed from the calcium carbonate.

2) Magnesium analysis

1.35 % by weight Mg implies that the total amount of Mg in the 470 mg sample is 6.3 mg. Assuming that all of this Mg is in the form of magnesium carbonate (MgCO₃, MW = 84), then the 470 mg of dried skimmate contains 22 mg (~ 4.7 %) of MgCO₃. Since carbon is 14 % (by weight) of MgCO₃, then the 470 mg of dried skimmate contains ~ 3 mg (~0.7 %) of (inorganic) carbon contributed from the magnesium carbonate.

3) Nitrogen analysis

Living organisms are ~ 5 - 9 % by dry weight nitrogen (we'll use 7% for simplicity), (Sterner, 2002) and so, if we neglect inorganic sources of nitrogen (NH₄, NO₃, and NO₂, which are immeasurably low in the tank water), the 2.22 % by weight of nitrogen implies that there are 10.4 mgs of nitrogen in the 470 mgs of skimmate, which calculates to 149 mgs (~32 %) of organic material present.

4) Hydrogen analysis

Living organisms are ~ 7 % by dry weight hydrogen. (Sterner, 2002) The 2.39 % by weight of hydrogen implies that there are 11.2 mgs of hydrogen in the 470 mgs of skimmate, which calculates to 160 mgs (~34%) of organic material present. Compare this value to the nitrogen analysis-based prediction of organics from (3); 32% - very close agreement!

5) Carbon analysis

21.08 % by weight C implies that the total amount of C present in the 470 mg skimmate sample is 99 mgs. Subtracting the amount of C from the CaCO₃ contribution (25 mgs of C) and the MgCO₃ contribution (3 mgs of C) leaves 71 mgs of C remaining. What is the source of this carbon? Two possibilities seem likely; ejected particulate carbon from the GAC filter, or TOC originating from organic sources. Living organisms are 40 - 50% by dry weight carbon (we'll use 45% for simplicity), (Sterner, 2002). If all of the 71 mg of carbon came from organic sources (= TOC), then there would be ~ 158 mgs (~34 %) of organic material present. Compare this value to both the nitrogen analysis-based prediction of organics from (3); 149 mgs (~ 32%), and the hydrogen analysis-based prediction from (4); 160 mgs of TOC (34%). The concordance between the TOC-carbon-based calculation and the independent hydrogen- and nitrogen-based calculations cannot be ignored. Thus, there is no evidence to contraindicate the conclusion that the remaining 71 mgs of carbon can be attributed to organic sources as TOC; there is no reason to invoke GAC filter ejecta as a source for this carbon.

6) Silicon analysis

The 4.76% by weight silicon present in the 470 mgs of skimmate suggests that there are 22.4 mgs in total of Si present. If we assume that the Si is contributed by biogenic opal from the skeleta of diatoms, (Brzezinski, 1985; Mortlock, 1989) then the Si is in a hydrated polymer of SiO₂ (approx. molecular formula for opal is SiO₂•0.4H₂O, 42% Si by mass). Therefore, we can approximate the amount of biogenic opal present as 53 mgs (~ 11%).

7) Phosphorus analysis

The 0.16% by weight of P present in the 470 mgs of dry skimmate implies that there is 0.75 mgs of P present. Assuming that all of the P is present as phosphate, PO₄^3- (MW = 95, unknown counterion), then there are ~ 2.3 mgs (~ 0.5%) of PO₄^3- present in the 470 mgs of dry skimmate. This amount equals ~ 4900 ppm of phosphate, which is vastly more than the < 0.02 ppm of phosphate in the tank water. Thus, skimming does concentrate phosphate.

Elemental Analysis Summary

In summary, the skimmer is pulling out a solid, water-insoluble mixture of compounds that consist by weight of (approximately):

• 44 % of CaCO₃
• 5% of MgCO₃
• 11% of biogenic opal
• 34% of organic material
• 0.5% of phosphate

Therefore, a total ~ 95% of the dry water-insoluble skimmate is accounted for! What are the sources of these chemical compounds in the skimmate? The biogenic opal is likely from the shells of diatoms, small members of the phytoplankton family of marine microbes. The CaCO₃ (and MgCO₃) might have both biogenic and abiological sources. A calcium reactor was operating throughout the experimental skimmate collection period, and so some of the CaCO₃ might just be microparticulates emitted from this device. Alternatively, the CaCO₃ might arise from the shells of planktonic microbes from the coccolithophore (Mitchell-Innes, 1987; Stanley, 2005) and foraminifera families. These plankton components are prevalent under certain conditions in seawater, but there presence in aquarium water has not been established. It is not possible to distinguish between these biological and abiological sources of CaCO₃ at present. Future experiments in which skimmate is collected without a running calcium reactor might shed some light on this point. The phosphate present in the skimmate could not come from inorganic phosphate in the water column; that ion would have been removed by the thorough washing with water. It is possible that some of this phosphate is in the form of insoluble calcium phosphate, but that occurrence would be unlikely as Ca₃(PO₄)₂ is formed at rather high pH, which is not characteristic of the skimmate liquid (pH = 7.67, see below). By default, then, it is most likely derived from organic phosphate; that is, many biochemicals within diatoms and all other living organisms (coccolithophores, foraminifera, bacteria, humans, etc) have attached phosphate groups. Aquarium organisms recruit these phosphate molecules from the inorganic phosphate in the water column and then attach them to the organic biochemicals. Thus, they effectively concentrate phosphate from the water, and that phosphate is then removed (within the intact organism) upon skimming. From this perspective, skimming does contribute to the removal of inorganic phosphate from aquarium water.

An interesting and perhaps unanticipated observation is that only 34% of this solid skimmate material can be assigned to "organic carbon", TOC. Thus, 2/3 of the solid, water-insoluble part of the skimmate is not TOC, but rather inorganic material that may (or may not) have biogenic origins. If a substantial amount of this inorganic material does come from the shells of plankton, then it stands to reason that a large part of the detected organic material (TOC) probably constitutes the "guts" of these organisms. Thus, perhaps not that much of the TOC removed by skimming is actually free-floating organic molecules. One caveat on this interpretation, of course, is the fact that ~ 90% of the crude original skimmate was washed away with water. Perhaps that water-soluble fraction contained significant quantities of dissolved organic carbon, which would be undetected by the above analysis.

A second, more comprehensive skimmate chemical analysis was pursued to address this concern. In this experiment, the tank was fed daily with a mixture of PE and Hikari mysis shrimp, Ocean Nutrition Formula 1 flakes, Omega One Veggie Flakes, and Aqueon Marine Granules as described above. This daily feeding amounted to a dry weight (110 ^oC/0.2 mm for 48 hrs) of 0.87 gms/day. No Reef Nutrition products were used during this experiment. After 7 days of this feeding regime, the solid and liquid skimmate collected by the H&S 200-1260 skimmer was carefully removed from the skimmer cup and separated by centrifugation (6000 rpm, 40 min). The light brown clear supernatant was poured off and its volume measured; 125 mL. The solid residue was dried in vacuo at 110 ^oC/0.2 mm for 24 hours => 5.18 gm brown solid. 110 mL of the liquid was concentrated under reduced pressure and then vacuum dried (110 ^oC/0.2 mm/24 hr) to yield 2.91 gm of brown solid ( => 3.31 gms of solid from the original 125 mL of liquid recovered). The 15 mL of remaining liquid skimmate was assayed with a Salifert test kit for alkalinity: [alk] = 8.0 meq/L. In addition, the refractive index of 1.023 indicated 31 ppt salinity, and pH = 7.67. An endpoint could not be detected with the Ca or Mg Salifert kits, the Merck phosphate kit, or the Salifert NO₃ kit due to the interfering light brown color of the skimmate liquid. Note that the exceedingly high [alk] measurement does not necessarily suggest that the concentrations of HCO₃^- or CO₃^2- are high; there may be organic acid carboxylates from the TOC pool that are being detected by this alkalinity assay (see below).

The solid derived from evaporation of the liquid portion of the skimmate as well as the solid obtained after centrifugation were both submitted to Columbia Analytical Services for elemental analysis. The results are tabulated in Table1. In addition, the dried food was analyzed for select elements. Natural seawater element content is included for comparison.

Table 1. Results of the elemental analyses of skimmate and food samples.

Element	Solid skimmate (weight %)	Liquid skimmate(weight %)	Natural sea watersolids (weight %)	Food(weight %)
C	22.50	4.50	0.08
N	2.72	0.68	0.04
H	2.37	1.33
S	1.18	2.47	2.6
Ca	10.52	0.60	1.1
Mg	1.99	3.21	3.7
Si	8.94	1.40	< 0.01
Na	3.45	27.25	30.9
Cl	0.40	43.2	55.4
K	0.38	1.17	1.1
Fe	0.93	<0.02	< 0.01
P	0.46	0.08	< 0.01	1.57
I			< 0.01	< 0.1
Cu			< 0.01	< 0.006
Sum	55.84	85.89	95

Food Analysis

The desiccated food was assayed for phosphorus, copper, and iodine content. Neither copper nor iodine registered in these analyses; there can be no more than 100 ppb of either in the food. The phosphorus content, however, was detectable, and 1.57% by weight of P corresponds to approximately 14 mg pf phosphorus in the 0.87 gm of dried food fed to the tank daily. Assuming that all of the P is present as phosphate, PO₄^3- (MW = 95), then there are ~ 42 mgs (~ 5%) of PO₄^3- present in the 0.87 gm of dried food. Note that the frozen mysis shrimp cubes were washed thoroughly with tap water until thawed, and so phosphate content in the water for freezing can be discounted. The daily 42 mg phosphate addition to the 168 gallons of the aquarium water volume represents a nominal addition of approximately 0.06 ppm of phosphate per day. Since Merck phosphate test kit analysis indicates a phosphate level of < 0.02 ppm (test kit limit), the added phosphate appears to be readily removed from the water column.

Skimmate Liquid Analysis

1) Sulfur analysis

The 2.47% by weight sulfur present in the 3.31 gm of solid derived from the skimmate liquid equates to approximately 82 mg of S. This sulfur most likely comes from sulfate, SO₄^2- (MW = 96, 33% S by weight). There is certainly a small amount of "organic" sulfur in the DOC, but that is not likely to add much to the total sulfur %, since sulfur is only ~ 0.1% of the dry weight of living matter. (Sterner, 2002) So, 82 mg of S in the dried liquid skimmate corresponds to 248 mg (7.5%) of sulfate in the dried skimmate liquid.

2) Nitrogen analysis

The 0.68% by dry weight of nitrogen in the 3.31 gm of dried skimmate liquid corresponds to 23 mg of N. Sources of the nitrogen include organic matter (DOC), and of course, inorganic ions; ammonium (NH₄⁺), nitrite (NO₂^-), and nitrate (NO₃^-). There is essentially no measurable (i.e., < 1 ppm) NH₄, NO₂ or NO₃ in the aquarium water, so to a first approximation, the nitrogen in the skimmate can be attributed to "organic" nitrogen. Since organic material derived from living sources is approximately 7% by dry weight nitrogen (see above), the 23 mg of N present in the skimmate liquid suggests that, overall, there is approximately 329 mg (~ 10%) of organic material present.

3) Carbon analysis

The 4.50 % by weight of carbon present in the 3.31 gm of dried skimmate liquid corresponds to 149 mg of C present. Sources of carbon in the skimmate liquid include inorganic carbon as part of the carbonate equilibria, organic carbon (DOC), and carbon particles ejected from the GAC filter. Based upon the argument advanced in (5) above, it seems unlikely that the GAC filter is a source of this carbon. It is not possible to distinguish between the remaining two sources based upon the elemental analysis measurement or the independent [alk] measurement, since that latter assay will detect (organic) carboxylates as well as the inorganic forms, bicarbonate HCO₃^-, and carbonate CO₃^2-. However, it is possible to set an upper limit on the inorganic (bicarbonate and carbonate) content of the skimmate liquid from the Salifert alkalinity measurement. The measured alkalinity via a Salifert test kit was 8 meq/L. If we assume for the purposes of setting this upper limit that all of that alkalinity was due to the carbonate system, then 8.0 meq/L corresponds to 1.0 mmol of alkalinity in the 125 mL of skimmate liquid collected from the centrifugation run. Further, if we assume that all of that alkalinity is in the form of bicarbonate, HCO₃^- (actually, at pH = 7.67, [HCO₃­] is about 96% of the carbonate present), then we would have 1.0 mmol, or 61 mg, of HCO₃^- present in the 3.31 gm of dried skimmate liquid. Thus, at a maximum, 61 mgs of HCO₃^- (= 20% C by weight) would only account for 12 mgs of the total 149 mgs of carbon present in the dried skimmate liquid. In this scenario, 137 mgs of the measured carbon then would be derived from organic sources. Using the estimate that organic material derived from living sources is 45% carbon, then the amount of DOC in the dried skimmate liquid would be 304 mg (~ 9%); not too far off from the nitrogen analysis figure of ~ 10% organic material. If, at the other extreme, all of the measured alkalinity could be attributed to organic carboxylates (assume C18 species on average, so C = 76% of carboxylate mass), then the 1.0 mmol of alkalinity would correspond to 283 mgs of organic carboxylates, 76% of which (= 215 mg) would be carbon. Since the total carbon measured was only 149 mgs, this latter scenario is of course impossible. Most likely, ~ 3 or 4 meq/L of the alkalinity can be assigned to HCO₃^-, so the remaining organic carbon is around 143 mgs => 318 mgs (~ 10%) of organic material - the same value derived from the nitrogen calculation.

4) Hydrogen analysis

The 1.33% by weight hydrogen in the 3.31 gm sample of dried skimmate liquid implies that there are 44 mg of H present. This hydrogen can be contributed from biologically derived organic sources, inorganic sources (HCO₃^- and HSO₄^-), and possibly from water left over from incomplete drying. If all of the measured hydrogen were contributed only from biologically derived organic sources (at ~ 7% by dry weight hydrogen), then we would predict that the dried skimmate liquid contained approximately 629 mg (~ 19%) of organic material. Clearly that value is too large compared to the nitrogen and carbon analyses values, so at least some of the hydrogen must come from either inorganic ions or from water. The amounts contributed from bicarbonate HCO₃^- and carbonic acid H₂CO₃ are negligible, given their relatively small concentrations (see Carbon analysis, above). In addition, at pH = 7.67, there is a vanishingly small amount of bisulfate, HSO₄^-; its pKa = 1.9. So, it is likely that the liquid skimmate sample was not completely dried, and the remaining hydrogen likely comes from that source. Given from the nitrogen analysis that the solid derived from the skimmate liquid contains about 329 mg of organic material, and organic material is about 7% hydrogen, then the hydrogen contributed from this organic material is about 23 mg of the solid residue. If the remaining measured hydrogen (44 - 23 = 21 mg) is from H₂O, then there is 189 mg (~ 6 %) of water present.

5) Silicon analysis

The 1.40% of silicon by weight in the 3.31 gm of solid recovered from the skimmate liquid works out to 46 mg of silicon. This silicon may be derived from either water-soluble orthosilicic acid (Si(OH)₄, 29% Si, 4% H) or from biogenic opal constituting the shell of diatoms as described above (approx. molecular formula for opal is SiO₂•0.4H₂O, 42% Si, 1% H). Note that in either case, the amount of hydrogen contributed by either source of Si is minuscule (~ 0.04% by weight of the original skimmate liquid's derived solid) and scarcely influences the hydrogen analysis's conclusions above. It is not possible to determine how much silicon derives from inorganic orthosilicic acid, and how much can be attributed the shells of diatoms, but the % silicon in each is not that different, and so we will use an average value (36%) to calculate the amount of "SiOxHy" in the crude skimmate liquid's derived solid; approximately 128 mg (~4%) of the skimmate liquid's solid is some form of silicate, SiOxHy.

So, overall, the solid derived from concentration of the skimmate liquid can be partitioned into:

• Inorganic ions (Na, Cl, K, Ca, Mg, SO₄^2-, HCO₃^-, SiO_xH_y) 87%
• Dissolved organic carbon 10%
• Water 6%
• Total 103%

So, we have overshot the theoretical maximum content of 100% by 3%; not too bad, given the many approximations and assumptions that went into the acquisition of these percentages. The bottom line, however, is that the skimmate liquid contains mostly the common inorganic ions that constitute the major ions in seawater. Only a small amount of this material can be arguably assigned to dissolved organic carbon, DOC.

The chemical analysis of the 5.18 gm of skimmate solid follows a similar approach as that described for the heavily washed skimmate solid discussed above. However, in this case, the solid was not washed repeatedly, and so some water-soluble compounds persist, although most of the water was likely removed through vacuum drying. These water soluble species consist of 3.45 wt % sodium, 0.40 wt % chloride, 0.38 wt % potassium, and 1.18 wt % sulfur (= 3.6 wt % sulfate). Furthermore, it is likely that at least some of the measured Ca, Mg, C (as HCO₃) and P might come from water soluble compounds in addition to compounds within the insoluble solid, but the overall amounts of the water soluble fraction of these particular inorganic ions are likely to be small, since the most abundant ion, sodium, is only 3.45 % by weight of the isolated solid (Na/Ca = 28 in seawater). So, to a first approximation, we will neglect their contribution to the water-soluble inorganic part of the solid skimmate. From this perspective, the dried skimmate solid contains about 8 % by weight of normally water-soluble inorganic ions.

1) Calcium analysis

10.52 % by weight Ca implies that the total amount of Ca in the 5.18 mg sample is 545 mg. Assuming that essentially all of this Ca is in the form of water-insoluble calcium carbonate (CaCO₃, MW = 100), then the 5.18 mg of dried skimmate contains 1.36 gm (26 %) of CaCO₃. Since carbon is 12 % (by weight) of CaCO₃, then the 5.18 gm of dried skimmate contains ~ 163 mg (~3.2 %) of (inorganic) carbon contributed from the calcium carbonate.

2) Magnesium analysis

1.99 % by weight Mg implies that the total amount of Mg in the 5.18 gm sample is 103 mg. Assuming that all of this Mg is in the form of magnesium carbonate (MgCO₃, MW = 84), then the 5.18 gm of dried skimmate contains 361 mg (~ 7.0 %) of MgCO₃. Since carbon is 14 % (by weight) of MgCO₃, then the 5.18 gm of dried skimmate contains ~ 51 mg (~ 1 %) of (inorganic) carbon contributed from the magnesium carbonate.

3) Nitrogen analysis

Living organisms are ~ 5 - 9 % by dry weight nitrogen (we'll use 7% for simplicity), (Sterner, 2002) and so, if we neglect inorganic sources of nitrogen (NH₄, NO₃, and NO₂, which are immeasurably low in the tank water), the 2.72 % by weight of nitrogen implies that there are 141 mgs of nitrogen in the 5.18 gm of skimmate solid, which calculates to 2.01 gms (~39 %) of organic material present.

4) Hydrogen analysis

Living organisms are ~ 7 % by dry weight hydrogen. (Sterner, 2002) The 2.37 % by weight of hydrogen implies that there are 123 mgs of hydrogen in the 5.18 gm of skimmate solid, which calculates to 1.75 gms (~34%) of organic material present. Compare this value to the nitrogen analysis-based prediction of organics from (3); 39% organic carbon. In this instance, the hydrogen results are not as close to the nitrogen-based results as they were in the preceding two analyses, but they are not that far off. This hydrogen analysis assumes that there is no water present, or some of the H would be attributable to water and not organics, and the H-based organic calculation would be even smaller.

5) Carbon analysis

22.50 % by weight C implies that the total amount of C present in the 5.18 gm skimmate sample is 1.17 gm. Subtracting the amount of C from the CaCO₃ contribution (163 mgs of C), and the MgCO₃ contribution (51 mgs of C) leaves 952 mgs of C remaining. If we again discount the GAC filter as a source of this carbon, then most (all?) of this carbon comes from "organic" sources. Since living organisms are 40 - 50% by dry weight carbon (we'll use 45% for simplicity), (Sterner, 2002) then 952 mgs of organic C implies that there are ~ 2.12 gms (~ 41 %) of organic material present. Comparison to the nitrogen- (39% organics) and hydrogen- (34% organics) derived values provides a consistent picture of the organic content.

6) Silicon analysis

The 8.94 % by weight silicon present in the 5.18 gm of skimmate solid suggests that there are 463 mgs in total of Si present. If we assume that the Si is contributed by biogenic opal from the skeleta of diatoms, (Brzezinski, 1985; Mortlock, 1989) then the Si is in a hydrated polymer of SiO₂ (approx. molecular formula for opal is SiO₂•0.4H₂O, 42% Si by mass). Therefore, we can approximate the amount of biogenic opal present as 1.10 gm (~ 21%).

7) Phosphorus analysis

The 0.46% by weight of P present in the 5.18 gms of dry skimmate solid implies that there is 24 mgs of P present. Assuming all of the P is present as phosphate, PO₄^3- (MW = 95, unknown counterion), then there are ~ 74 mgs (~ 1.4 %) of PO₄^3- present in the 5.18 gm of dry skimmate solid. This amount equals ~ 14300 ppm of phosphate, which again is vastly more than the < 0.02 ppm of phosphate in the tank water.

8) Iron analysis

The 0.93 % by weight of Fe in the 5.18 gms of dried skimmate solid amounts to 48 mg of Fe present. Inorganic iron salts are quite insoluble in water, and so it is likely that almost all of this iron is either "organic" iron that resided within the bodies of microbes like bacteria, etc. or it is from colloidal iron particles expelled from the GFO reactor. The skimmate's carbon-to-iron weight-percent-ratio of 24 can be compared to the dry weight C:Fe ratios of several planktonic organisms: heterotrophic bacteria: 28500:1, cyanobacteria: 11250:1, eukaryotic phytoplankton: 71250:1. (Tortell, 1996). Since the detected Fe:C ratio is 10000x that of planktonic species, it is highly unlikely that much of the skimmed iron is "organic" in origin. A more likely scenario is that particulate ferric oxide is expelled from the GFO reactor, and that material then constitutes the majority of the iron removed by the skimmer. Ferric oxide has a nominal chemical formula of Fe₂O₃, and it is approximately 70% iron by weight. So, the 48 mg of iron in the skimmate solid corresponds to about 69 mg of Fe₂O₃ (~ 1.3% by weight).

In summary, the skimmer is pulling out a solid mixture of compounds that consist by weight of (approximately):

• 8% inorganic ions
• 26 % of CaCO₃
• 7% of MgCO₃
• 21% of biogenic opal (SiO₂)
• 38% of organic material
• 1.5% of phosphate
• 1.3 % of ferric oxide

These materials sum up to ~ 103%, which is pretty close to the theoretical maximum of 100%. Any discrepancies can be easily explained by the numerical uncertainty introduced through all of the assumptions. That is, even with all of the assumptions and approximations cited in this analysis, the sum total of the mass works out to within 3% of "perfect". Once again, the organic material removed in the skimmate solid is a minor component, although at an average of 38% (C vs. N vs. H analysis), it is a little higher than the 34% value derived from the heavily washed skimmate solid sample and much higher that the amount of DOC in the liquid fraction (~ 10%). In total, the 8.49 gm of total solids removed during the week of skimming contain approximately 318 mg of water-soluble organics (~ 4%) and approximately 2.12 gms of water-insoluble organics (~ 25%). Thus, by a large margin, the bulk of the organics removed by skimming are not DOC (dissolved organic carbon). The inorganic compounds CaCO₃ and SiO₂ constitute the majority of the skimmate solid mass, much as they did in the heavily washed skimmate sample analyzed first. As discussed in that analysis, the source of these compounds is not assignable from these data, but a biological source for the SiO₂ (biogenic opal), diatom shells, is likely. The CaCO₃ might arise from both inorganic sources (i.e., calcium reactor CaCO₃ particle ejection) and organic sources (the shells of foraminifera and/or coccolithophores).

One of the surprising observations to emerge from the original skimmer performance studies is that only approximately 20 - 35% of the measurable TOC in aquarium water is removed by skimming. That observation might now seem a little less surprising when viewed in the context of the skimmate component analysis. Thus, only ~ 29 % (25% from the solid + 4% from the liquid) of the skimmate removed by the H&S 200 skimmer from authentic reef tank water over the course of a week can be assigned to organic material. So, skimming does not remove all that much of the TOC present in aquarium water, and the skimmate does not contain all that much TOC.

So what, exactly, does skimming do? On the subject of water remediation; the most conservative, permissible (but not compelling!) answer is that skimming removes lots of (living or dead? unknown) microorganisms that populate the aquarium water, and in so doing removes the (organic) carbon, phosphorus, and nitrogen that comprise their biochemical makeup. In addition, dissolved organic compounds may also be removed, but the data do not support the proposition that these dissolved organic species constitute a major amount of the total organics removed. In addition to these water purification functions, skimmers serve to oxygenate the water and facilitate gas exchange in general, which are useful activities independent of organic waste removal.

Conclusions

The chemical/elemental composition of skimmate generated by an H&S 200-1260 skimmer on a 175-gallon reef tank over the course of several days or a week had some surprises. Only a minor amount of the skimmate (solid + liquid) could be attributed to organic carbon (TOC); about 29%, and most of that material was not water soluble, i.e., was not dissolved organic carbon. The majority of the recovered skimmate solid, apart from the commons ions of seawater, was CaCO₃, MgCO₃, and SiO₂ - inorganic compounds! The origin of these species is not known with certainity, but a good case can be made that the SiO₂ stems from the shells of diatoms. The CaCO₃ might be derived from other planktonic microbes bearing calcium carbonate shells, or might come from calcium reactor effluent. To the extent that the solid skimmate consists of microflora, then some proportion of the insoluble organic material removed by skimming would then simply be the organic components (the "guts") of these microflora. These microflora do concentrate P, N, and C nutrients from the water column, and so their removal via skimming does constitute a means of nutrient export.

Acknowledgments

We thank the Eberly College of Science at the Pennsylvania State University and E. I DuPont de Nemours and Co. for financial support, and Drs. Sanjay Joshi (Penn State) and Craig Bingman (U. Wisconsin) for many helpful discussions.

References

1. Brzezinski, M. A. 1985. "The Si:C:N Ratio of Marine Diatoms: Interspecific Variability and the Effect of Some Environmental Variables." J. Physiol., 21, 347-357.
2. De la Rosa, J. M.; González-Pérez, J. A.; Hatcher, P. G.; Knicker, H.; González-Vila, F. J. 2008. "Determination of Refractory Organic Matter in Marine Sediments by Chemical Oxidation, Analytical Pyrolysis and Solid-State ¹³C Nuclear Magnetic Resonance Spectroscopy." Eur. J. Soil Sci., 59, 430-438.
3. Feldman, K. S.; Maers, K. M.; Vernese, L. F.; Huber, E. A.; Test, M. R. 2009. "The Development of a Method for the Quantitative Evaluation of Protein Skimmer Performance." Advanced Aquarist http://www.advancedaquarist.com/2009/1/aafeature2/
4. Feldman, K. S.; Maers, K. M. 2010. "Further Studies on Protein Skimmer Performance." Advanced Aquarist
5. Mitchell-Innes, B. A.; Winter, A. 1987. "Coccolithophores: a Major Phytoplankton Component in Mature Upwelling Waters Off the Cape Peninsula, South Africa in March, 1983." Marine Biol., 95, 25030.
6. Mopper, K.; Stubbins, A.; Ritchie, J. D.; Bialk, H. M.; Hatcher, P. G. "Advanced Instrumental Approaches for Characterization of Marine Dissolved Organic Matter: Extraction Techniques, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy." Chem. Rev., 107, 419-442.
7. Mortlock, R. A.; Froelich, P. N. "A Simple Method for the Rapid Determination of Biogenic Opal in Pelagic marine Sediments." 1989. Deep-Sea Res., 36, 1415-1426.
8. Stanley, S. M.; Ries, J. B.; Hardie, L. A. 2005, "Seawater Chemistry, Coccolithophore Population Growth, and the Origin of Cretaceous Chalk." Geology, 33, 593-596.
9. Sterner, R. W.; Elser, J. J. 2002. Ecological Stoichiometry. Princeton University Press, Princeton.
10. Tortell, P. D.; Maldonado, M. T.; Price, N. M. "The Role of Heterotrophic Bacteria in Iron-Limited Ocean Ecosystems." 1996. Nature, 383, 330-332.

We published a paper on skimmer performance in the January 2009 issue of Advanced Aquarist magazine that detailed, for the first time, an experimental methodology to provide meaningful metrics for both the rate at which skimmers removed organics and the extent of the removal of these organics from aquarium water (Feldman, 2009). Highlights of these earlier studies included:

1. Development of a mathematical representation of the skimming process based upon a "continuously stirred reactor" model for both the skimmer and the reservoir (tank) with a given water flow rate between them.
2. Application of that mathematical formalism to both (a) a model system featuring removal of Bovine Serum Albumin (BSA) as a test case protein in freshly prepared saltwater, and (b) authentic TOC (Total Organic Carbon) removal in reef tank water. Key experimental parameters extracted from this mathematical modeling included the rate constant, k, for organic (BSA or TOC) removal, which is a singular metric reflecting the intrinsic capacity of a given skimmer to remove the organics in question, and the total % of the available organics (BSA or TOC) that were removed before the skimmer "flatlined".
3. Analysis of these data for four representative skimmers; a EuroReef CS80 needlewheel skimmer, a Precision Marine ES100 venturi skimmer, a Precision Marine AP624 airstone skimmer, and an ETSS Evolution 500 downdraft skimmer.
4. Conclusions about relative skimmer performance based upon these measurements:
   • All four skimmers removed both BSA and TOC with similar rate constants; in short, "bubbles is bubbles", and there was no significant difference between these four skimmers in their intrinsic abilities to strip organics from saltwater.
   • Only about 20 - 30% of the measurable TOC in reef tank water was removed by skimming, whereas almost all of the BSA was removed from saltwater by skimming.

Over the intervening year, we have continued and expanded these studies of skimmer performance in several directions. In this article, we report the results of these efforts. Specifically, we have:

1. Modified our mathematical model to take into account the observation that there is a (significant) component of TOC that is not skimmable. We have applied this new model to the old skimmer data as well as to new data with new skimmers.
2. Examined the performance of three new skimmers, all of which have bubble plates; the Bubble King Mini 160 needlewheel skimmer, the Royal Exclusiv 170 Cone needlewheel skimmer, and the Reef Octopus 150 recirculating pinwheel skimmer.

The Modified Mathematical Model

The mathematical model derived in the January 2009 Advanced Aquarist article was based upon four assumptions:

1. The water reservoir can be treated as a continuously stirred reactor.
2. The skimmer mixing volume can be treated as a continuously stirred reactor.
3. The reservoir volume is much larger than the skimmer mixing volume.
4. The rate of TOC removal in the skimmer is proportion to the amount of TOC present in the water.

There is no reason to doubt the validity of assumptions 1 - 3. However, assumption #4 does not take into account the experimental observation that only some of the TOC in reef tank water is susceptible to removal by skimming. Thus, a more appropriate and general starting point would involve explicitly breaking down TOC into two functionally distinct components:

1. [TOC_l], or labile TOC that the skimmer will remove
2. [TOC_r], or refractory TOC that the skimmer won't remove

With this distinction made, the prior math can be adapted to arrive at a slightly modified expression that once again allows extraction of the two quantities of interest from the raw data: the mass transfer rate constant, k (units = per minute), for TOC_l removal, and the total amount of TOC remaining, TOC_r, when the skimmer is not pulling out any more (labile) organics from the reservoir water. The recirculating reservoir/skimmer system (Fig. 1) maps quite closely onto a fundamental textbook problem in mass transfer/fluid flow encountered in introductory chemical engineering courses called the "continuously stirred" or "well-stirred" reactor problem. (Felder, 2005) In this instance, both the skimmer and also the reservoir can be treated as "well-stirred reactors" with a given flow Q between them. A component of the water is depleted in the skimmer by bubble-mediated removal.

Where:

1. [TOC_T]_r = the total concentration of TOC in the reservoir, in ppm. Note that:
• [TOC_T]_r = [TOC_l]_r + [TOC_r]_r, where [TOC_l]_r = labile TOC that the skimmer will remove, and [TOC_r]_r = refractory TOC, which the skimmer will not remove
2. [TOC_T]_s = the total concentration of TOC in the skimmer, in ppm. Note that:
• [TOC_T]_s = [TOC_l]_s + [TOC_r]_s, where [TOC_l]_s = labile TOC that the skimmer will remove, and [TOC_r]_s = refractory TOC, which the skimmer will not remove
3. Q = the volumetric water flow rate, in gal/min
4. V_r = the volume of the reservoir, in gal (30 gal)
5. V_s = the mixing volume of the skimmer, in gal

It is important to note that not all of the TOC present in reef tank water is susceptible to skimmer-mediated removal - some types of TOC are not picked up by bubbles. To account for this observation, we have divided the TOC into a labile, or skimmer-removable component [TOC_l], and a refractory, or skimmer-inert component [TOC_r]. Therefore, the total TOC that we measure experimentally is the sum of these two types of TOC: [TOC_T] = [TOC_l] + [TOC_r]. However, only the labile TOC's concentration [TOC_l] is changing upon skimmer action; the refractory TOC's concentration, [TOC_r] does not vary. Thus, we can confine our mathematical derivation to this labile TOC, TOC_l, and at the end take into account the fact that experimentally, we can only measure the total TOC, TOC_T.

Since both "reactors" are interconnected, the level of the labile TOC component will drop in the reservoir as well, and our task will be to develop a mathematical model that relates the removal in the skimmer with the measured depletion in the reservoir. In our experimental setup, a liquid volume V_r (reef tank water in a Rubbermaid tub) has an input stream and an output stream, and TOC in the water becomes depleted over time via bubble-mediated removal in a skimmer with mixing water volume V_s (see Fig. 1). Inspection of the skimmers in action permits measurement of this "active" skimmer mixing volume, which is the value that we will use for V_s. For the purposes of this analysis, we will assume that all of the active volume is water; that is, we will ignore the void volume of the bubbles, as we cannot independently assess the relative contributions of bubbles and water. This assumption will introduce an error into the calculations, but that error should be systematic for all skimmers, and since we are interested in relative and not absolute skimmer performance, this error should not affect the conclusions. Knowledge of the precise mechanism by which the skimmer's bubbles removes the water component(s) is not required; all that we need to know is that the concentration of the measured water component (TOC_T in this case) is diminishing with time in the reservoir.

It is essential for solving this problem that both the reservoir and the skimmer water volumes are well mixed to avoid concentration gradients. The reservoir water mixing in the experiments described below is provided by the skimmer return flow and by two powerheads in the reservoir. We independently tested the "well mixed" assumption in the reservoir by sampling TOC levels at a given time point at different locations (i.e., top, bottom, left side, right side) during a skimmer run. We observed that the site-to-site variation in TOC levels at different locations was no greater than the sample-to-sample variation at one location (both ~ 10%), suggesting that there is no reason to suspect that the "well mixed" assumption is not applicable. The mixing in the skimmer reaction chamber is provided by both rapid water movement and the agitation caused by the motion of the bubble stream. We have no independent experimental measurement/confirmation of mixing behavior in the skimmer.

The application of this mathematical approach to the protein skimmer problem leads ultimately to two important equations, labeled 22 and 24 below. The complete mathematical derivation (i.e., Eqs. (1) - (21)) can be found in the next section. This derivation that is largely taken from the 2009 Advanced Aquarist article. This new version of the derivation takes into account the presence of refractory TOC, TOC_r.

(22) [TOC_T]_r = ([TOC_T]_o - [TOC_r]_o)•e^{-[k•Q/(Vr•(k + Q/Vs))]•t} + [TOC_r]_r

Eq. (22) provides the means to extract the desired quantity, the rate constant k, from the experimental data. Experimentally, the quantities [TOC_T], [TOC_T]_o, time t, Q, V_s, and V_r can all be measured. In addition to this expression which determines the amount of TOC present at a given time t, we can also manipulate the mathematical formula to arrive at an equation that defines the rate of TOC removal, a metric that takes into account the rate constant k as well as the flow Q, and the skimmer and reservoir volumes, V_s and V_r, respectively.

(24) rate = d[TOC_l]_r/dt = -[Q/(V_r•(1 + Q/k•V_s))]• [TOC_l]_r

This version of the rate expression has the useful property of revealing that when Q/k•Vs >>1,

(25) rate = -(V_s•k/V_r)• [TOC_l]_r

That is, under the conditions where Q/k•V_s >> 1, the rate only depends on the rate constant k, the skimmer volume V_s, and the tank volume V_r; it does not depend upon the flow rate Q.

Conversely, if Q//k•V_s << 1, then the rate expression reduces to

(26) rate = - (Q/V_r)• [TOC_l]_r

That is, the rate only depends on the flow rate Q and the tank volume; it does not depend upon either the skimmer size of the rate constant for TOC removal.

For the Royal Exclusiv 170 cone, the Bubble King mini 160, the ETSS evolution 500 and the Reef Octopus 150, Q/k•Vs > 5 (see below), so invoking the simplification of Eq. (25) will introduce less than a 20% error. For the Precision Marine ES100, the Precision Marine AP624 airstone, and the EuroReef CS80, Q/k•Vs is between 1 and 5, and the simplification of Eq. (25) cannot be used. In no case is Q/k•Vs << 1.

Finally, we can easily calculate the amount (%) of TOC removed in a given experiment by simply subtracting [TOC_r]_r, a value available from Eq. (22), from the beginning TOC concentration, [TOT_T]₀, and dividing by [TOC_T]₀.

(27) % of TOC removed = 100 x ([TOC_T]₀ - [TOC_r]_r)/[TOC_T]₀

The Math Behind It

(skip ahead to the next heading if you wish to skip the math behind the modeling)

The fundamental physical property of the system that we will rely on to develop a mathematical model for skimmer performance is called mass balance; conservation of mass dictates that mass (matter, in this case TOC) cannot be created or destroyed, and so the amount of TOC depleted from the reservoir must be equal to the amount of TOC that is removed by the skimmer's bubbles. The fundamental mass balance equation is given in Eq. (1). Details can be found in the Wikipedia entry for "Mass Balance" and "Continuously Stirred-Tank Reactor".

(1) input + generation = output + accumulation

For the problem at hand, there is no generation of TOC; rather, TOC is removed by the skimmer's bubbles, so we will replace the "generation" term with "removal" (actually, just the negative of generation). In addition, since there is no TOC generation, there will be no TOC accumulation; rather, TOC is depleted in the system over time, so we will replace the "accumulation" term with "depletion" (just the negative of accumulation). So, the new mass balance expression applicable to both the reservoir, and independently, the skimmer is:

(2) input + removal = output + depletion

We will focus on the labile TOC in the reservoir first. There is no explicit TOC removal in the reservoir, so "removal" in Eq. (2) = 0. The input is equivalent to the mass of TOC added over time, say milligrams-per-minute (mg/min). In fact, since one term of Eq. (2) has units of mg/min (or more generally, mass/time), then all of the terms in this equation must be expressed in these units. The mass (amount) of TOC added in the input stream is the concentration of TOC, [TOC_l]_s (in mg/gal) times the volumetric flow rate of the input stream, Q (Q in gal/min). That is,

(3) mass of TOC added over time in input stream = Q•[TOC_l]_s

Note how the units of these terms are consistent: Q (in gal/min) • [TOC_l]_s (in mg/gal) = mg/min units, which is just what the "mass of TOC added over time" requires. The output stream of the reservoir can be treated in a similar manner:

(4) mass of TOC removed over time in output stream = Q•[TOC_l]_r

So, referring back to Eq. (2), with removal = 0, we have

(5) Q•[TOC_l]_s = Q•[TOC_l]_r + depletion

What mathematical expression can we use for "depletion"? The amount of TOC in the reservoir volume V_r is simply the product of the TOC concentration, [TOC_l]_r, and the volume:

(6) amount of TOC in the reservoir = V_r•[TOC_l]_r

We can simply define "depletion" to mean the rate of change (decrease) in the TOC amount in the reservoir over time. Note that once again, the units of this term are in mass/time ("mg/min").

(7) depletion = V_r•d[TOC_l]_r/dt

Now, returning to Eq. (5), we can insert the depletion term and generate the full mass balance expression for the reservoir:

(8) V_r•d[TOC_l]_r/dt = Q•([TOC_l]_s - [TOC_l]_r), where:

• V_r = the total volume of the reservoir water, in gal
• [TOC_l]_r = the concentration of labile TOC in the reservoir at any time t and also the concentration of labile TOC in the stream leaving the reservoir and entering the skimmer
• Q = the volumetric flow through the system, in gpm
• [TOC_l]_s = the concentration of labile TOC in the stream leaving the skimmer and entering the reservoir

Eq. (8) says that the change in the amount of TOC in the reservoir (the left hand side) is equal to the difference between the reservoir input and output TOC concentrations ([TOC_l]_s - [TOC_l]_r) times the flow rate (the right hand side). Note that this expression includes information about TOC concentrations in both the reservoir and the skimmer.

A similar expression can be developed for the fate of the TOC concentration just in the skimmer. However, in this case, "removal" does not equal 0, as the bubbles in the skimmer actively remove the organic impurity. So, from Eq. (2),

(9) V_s•d[TOC_l]_s/dt = Q•([TOC_l]_r - [TOC_l]_s) + removal

We must include another term (removal) in Eq. (9) that explicitly accounts for this TOC removal in order to maintain the required mass balance. This TOC removal term must take into account the function of the bubbles. The fundamental chemical equation for TOC removal by the bubbles is:

(10) TOC + bubbles → TOC•bubbles

This simple equation undergirds the assumption that allows the mathematical analysis to proceed: the key assumption is that the rate of labile TOC removed by the skimmer's bubbles is proportional to the amount of labile TOC present in solution. This assumption permits us to connect the [TOC] changes that happen in the skimmer with the [TOC] changes that happen in the reservoir. Since the bubbles are continually being introduced in large excess compared to the TOC concentration in solution, the "concentration" of the bubbles remains for all intents and purposes constant, and so we do not have to worry about how changes in bubble concentration might influence the rate of TOC removal. This model (and its underlying assumptions) greatly simplifies the mathematical analysis. In terms of the discipline of chemical kinetics, this approach is called the "pseudo-first order" approximation. So, the amount of labile TOC in the active skimmer reaction chamber volume V_s is given by the concentration times the volume:

(11) amount of labile TOC in the active water volume of the skimmer = V_s•[TOC_l]_s

By the assumption discussed above, the rate of labile TOC removal by the bubbles is proportional to the amount of labile TOC present:

(12) rate of labile TOC removal by the bubbles µ -V_s•[TOC_l] _s

Note that we must include a "-" sign in front of V_s•[TOC_l]_s because the amount is decreasing with time. We can introduce a proportionality constant, k, to convert Eq. (12) into an equality.

(13) rate of TOC removal by the bubbles = - k•V_s•[TOC_l]_s

The term k•V_s•[TOC_l]_s must have units of mass/time (i.e., mg/min) in order to "fit" into Eq. (9). Since Vs•[TOC_l]_s has units of mass (mg), then k must have units of /time (i.e., per min). As such, k is often referred to as a rate constant; it expresses how something changes over time ("per min"). This proportionality constant is not just a mathematical convenience. It will report on a fundamentally important property of a skimmer. The constant k can be viewed as a measure of how efficiently the skimmer/bubbles remove TOC. The efficiency of TOC removal is a complex function of many factors (skimmer geometry, bubble size/density, bubble residence time, solution diffusion, mass transfer to the bubble, foam coalescence, binding to the bubble surface, etc.). In fact, it is just this value k that we are after; k is actually a singular measure of skimmer efficiency in removing TOC. The larger that k is, the faster that a given skimmer will remove TOC. Thus, k is an intrinsic measure for TOC removal, and it reflects, in the aggregate, all of the parameters that contribute to the efficiency of TOC removal for a given skimmer. However, k does not offer any insight into which parameters, in particular, are more or less important in determining skimmer performance. We can measure experimentally k for different skimmers, and compare these values. Skimmers with larger k values will be more efficient (i.e., faster) at removing TOC from aquarium water. So, by including the information in Eq. (13) in Eq. (9), we have:

(14) V_s•d[TOC_l]_s/dt = Q•([TOC_l]_r - [TOC_l]_s) ­ V_s•k•[TOC_l]_s

This rather complex equation is important because, for the first time, we have mathematically linked the experimentally (indirectly) measurable quantity, the concentration of labile TOC_l in the reservoir, [TOC_l]_r (measured via [TOC_T]_r - [TOC_r]_r), with quantities specific to the skimmer (V_s, [TOC_l]_s). However, Eq. (14) as written is difficult to manipulate, so we will define a new mathematical quantity that will help us simplify the math and arrive at a very useful expression. We define a dimensionless time t = Q•t/V_r. Applying this definition to Eq. (14), we can generate the following expression:

(15) (V_s/V_r)•d[TOC_l]_s/dt = [TOC_l]_r - [TOC_l]_s - V_s•k•[TOC_l]_s/Q

The purpose for this seemingly arbitrary definition/substitution exercise now can be revealed. The use of t allows us to isolate the dimensionless quantity V_s/V_r, which is the ratio of the active skimmer volume to the reservoir volume. We can inject a dose of physical reality into the mathematics at this point. The reservoir volume (30 gallons) is much larger than the skimmers' volumes (0.62 - 1.3 gallons, see below) and so the ratio V_s/V_r hovers in the 0.02 - 0.04 range. If the second term in the left-hand part of Eq. (15), d[TOC_l]_s/dt, is not too large, then the V_s/V_r term will dominate, and we can neglect the entire left-hand term of Eq. (15); that is, we can, to a first approximation, set (V_s/V_r)•d[TOC_l]_s/dt = 0. We have obtained experimental justification for the claim that d[TOC_l]_s/dt is not large. By applying this approximation, we can relate the labile TOC concentration in the skimmer, [TOC_l]_s, to the TOC concentration in the reservoir, [TOC_l]_r:

(16) 0 = [TOC_l]_t - [TOC_l]_s - k•[TOC_l]_s•V_s/Q, or

(17) [TOC_l]_s = [TOC_l]_r/(1 + k•V_s/Q)

We now can plug this [TOC_l]_s value back into Eq. (8) and divide both sides by V_r:

(18) d[TOC_l]_r/dt = (Q/V_r)•([TOC_l]_r/(1 + k•V_s/Q) - [TOC_l]_r)

Rearranging the various terms yields a differential equation:

(19) d[TOC_l]_r/[TOC_l] _r = -[k•Q/(V_r•(k + Q/V_s))]•dt,

and solving the differential equation yields

(20) Ln([TOC_l]_r/[TOC_l] ₀) = -[k•Q/(V_r•(k + Q/V_s))]•t

If we could measure just the labile component of TOC, [TOC_l]_r, then Eq. (20) would suffice for our goals; we would then experimentally measure [TOC_l]_r at different times t, and upon graphing -Ln([TOC_l]_r/[TOC_l] ₀) vs. t, we would be able to extract out the desired quantity, k. However, we cannot measure just the labile component of TOC; we can only measure the total TOC, [TOC_T]_r, and the refractory TOC, [TOC_r]_r. Thus, we have to substitute [TOC_l] = [TOC_T] - [TOC_r] into Eq. (20).

(21) Ln{([TOC_T]_r - [TOC_r]_r)/([TOC_T)_o - [TOC_r]_o)} = -[k•Q/(V_r•(k + Q/V_s))]•t

This expression can be rearranged for convenience into the form:

(22) [TOC_T]_r = ([TOC_T]_o - [TOC_r]_o)•e^{-[k•Q/(Vr•(k + Q/Vs))]•t} + [TOC_r]_r

Eq. (22) provides the means to extract the desired quantity, the rate constant k, from the experimental data. Experimentally, the quantities [TOC_T], [TOC_T]_o, time t, Q, V_s, and V_r can all be measured. Plotting the experimentally measured [TOC_T] vs. time t will give an exponential decay-type curve, and that curve can be fit to the general equation

(23) Y = A•e^-Bx + C

where:

• A = [TOC_T]_o - [TOC_r]_o
• B = k•Q/(V_r•(k + Q/V_s)), and
• C = [TOC_r]_r

Note that [TOC_T]₀ is the concentration of TOC at t = 0, the beginning of the experiment. Remember that the rate constant k for TOC removal has the units min^-1; that is, k is expressed as "per minute". Finally, [TOC_r]_r can be found from this graphing procedure, and then [TOC_l]_r can be calculated via [TOC_T]_r - [TOC_r]_r.

Equation (18) expresses a subtle but important aspect of this modeling; the difference between a rate and a rate constant. Our interest in comparing different skimmers focuses our attention on the rate constant k. As discussed above, this single value is an aggregate measure of the efficiency of a given skimmer in removing TOC; it reflects all of the intimate structural and molecular details that impact on organics removal (see above). However, the overall rate of TOC removal (different than the rate constant!) is expressed by Eq. (18) and rewritten in a more convenient form below as Eq. 24. That is, the rate is defined as the depletion of TOC over time, or d[TOC_l]/dt (the left hand part of Eq. (18)). Eq. (18) indicates that the rate of TOC_l removal is a complex function of k, the flow rate Q, and the system volumes V_s and V_r. Thus, increasing k, or increasing Q, or decreasing V_r will all increase the overall rate of TOC_l removal. There is nothing in the mathematical derivation to indicate whether k itself is a function of Q or not. This point was tested through experiment with the Reef Octopus 150 skimmer, and there was very little change in k as the flow rate increased from 162 gph up to 390 gph (see below).

It is possible to algebraically manipulate the complex "k" term of Eq. (18) to arrive at the following form:

(24) rate = d[TOC_l]_r/dt = -[Q/(V_r•(1 + Q/k•V_s))]• [TOC_l]_r

The Experimental Design

We examined how TOC levels varied over time in reef aquaria prior to initiating the skimmer studies. These preliminary experiments measuring TOC levels in reef aquaria were conducted by removing 20 - 30 mL water samples at the indicated times using VWR brand TraceClean vials equipped with Teflon septa. The vials were dipped upside down under the tank water surface a few inches and then inverted to fill. The vial contents were frozen immediately by placing them in a - 23^oC freezer, and at a later date quickly thawed by placing them in a warm water bath prior to analysis using a Shimadzu 5000 TOC Analyzer calibrated with potassium phthalate as per instrument specifications. Blank samples (Shimadzu pure water - guaranteed < 0.1 ppm of C) were run periodically throughout an experimental run, and blank subtraction preceded final data analysis.

The actual skimmer trials were run as described in the earlier Advanced Aquarist article. All skimmers were cleaned prior to their initial use by running distilled water through them for 24 hr (3 times) and then freshly prepared saltwater for 24 hr (2 times). In the actual experiments, 30 gallons of reef tank water was removed 45 - 60 min after feeding the tank with Reef Nutrition products (Rotifeast, Phytofeast, Oysterfeast, and arctipods), some meaty food (mysis shrimp, chunks of shrimp/clams), and some flake food. The tank's skimmer and granular activated carbon (GAC) filter were off during this feeding period. At the water removal time, no visible evidence of food or food residue remained. The 30 gallons of tank water were transferred to a Rubbermaid tub equipped with two Maxijet 1200 powerheads for circulation and a heater set at 77 ^oF. The skimmer, which was thoroughly cleaned between each run, was placed at the appropriate height in (or above) the water (manufacturer's recommendation) and the pump (again, manufacturer supplied or recommended) was turned on. Any output valve adjustments were quickly made to ensure that the water/foam level was within the manufacturer's recommendations. Approximately 20 - 30 mL water samples were removed at T = 0, 10, 20, 30, 40, 50 60, 90, and 120 min; each water sample was placed in a VWR brand TraceClean vial with a Teflon septum in the cap. The water samples were removed by dunking the vial upside down with tongs about 6" below the water surface, inverted to fill, and then immediately placed in a -23^oC freezer pending analysis. The water samples were all thawed quickly and the TOC concentrations were measured using the Shimadzu 5000 TOC analyzer as described above.

Results

We ran two experiments designed to probe the question, "Is a skimmer really necessary to keep TOC levels low in a reef tank?" prior to examining specific skimmers. The answer to this question is obviously "Yes!" according to skimmer manufacturers, but there are alternative aquarium husbandry techniques (i.e., algae filtration) whose practitioners do claim success, thus raising the larger question of skimmer necessity. Of course, tank water skimming has other substantial benefits in terms of oxygenation and gas exchange in general, but the claim of TOC removal is one that we wished to test.

The first experiment was quite simple; we turned the skimmer off and the GAC canister feed pump off and fed the reef tank as described in the Experimental section. We removed water samples periodically over a 24-hour period and assayed them for TOC content. The results of a typical experiment are shown in Fig. 2a. This experiment was run in triplicate on successive days, and the data normalized to % TOC remaining for ease of comparison, Fig. 2b. These data sets can be fit to an exponential decay curve that in turn can be used to calculate a rate constant k for TOC depletion. This rate constant k bears the same general meaning as the rate constant of Eq. (22); that is, it reports on the intrinsic ability of "something" in the tank to consume TOC and thus remove it from the water column. In the skimmer experiments to follow, that "something" is skimmer bubbles, but in the current experiment, there is no running skimmer or GAC, so whatever removes TOC must be intrinsic to the biota of the tank. The "r²" value is a statistical measure of how closely the actual experimental data (the squares) matches the theoretical mathematical model (the exponential decay lines) from which k is extracted. Any r² value > 0.9 (the maximum is 1.00) indicates a very good fit between data and model. Thus, when no external TOC removal device (GAC or skimmer) is in operation, the TOC levels of the aquarium water elevate initially upon food addition, but then return to approximately (but not necessarily exactly) baseline over 24 hours with rate constants in the 0.21 - 0.26 hr^-1 range. What is responsible for this drop in TOC? The most likely candidates, based upon much extant literature, (Kirchman, 1990; Zweifel, 1993; Covert, 2001; Wild, 2004; de Goeij, 2007; Allers, 2008; Sharon, 2008) are the microbes, mainly bacteria, which inhabit every niche in the tank. So, it appears that this apparently healthy, mature reef tank houses enough TOC consuming organisms to effectively keep TOC levels at some acceptably low equilibrium level without the intervention of a protein skimmer.

Now, if we were to repeat the above experiment, but this time employ an H&S 200-1260 recirculating needlewheel skimmer running throughout the 24-hr experimental period, what might we expect? If the skimmer also aggressively contributes to the removal of TOC from the tank water, we might anticipate that the drop-off in TOC level over time might be precipitous, or at least more rapid than that observed during the skimmerless experiments. This type of increase in rate of TOC removal would be expressed in the k term; a larger k value would signify a faster removal of TOC from the reef tank water. The results, again in triplicate, are illustrated in Fig. 3.

It is apparent that the similarity in k values for the skimmed and the unskimmed tank trials do not support the notion that the skimmer is contributing in any material way to the removal of TOC from the reef tank water. That is, the natural TOC consumers (bacteria and other organisms) are completely adequate for returning the post-feeding TOC levels to approximately baseline values after ~ 24 hrs - the skimmer isn't required in this process. These observations therefore do not support the conventional wisdom that a skimmer is obligate for lowering and/or maintaining low TOC levels in a reef tank.

However, these data may not tell the whole story. If the issue is gradual accumulation of TOC over time, then perhaps a 24-hr experimental period is not sufficient to detect this type of a possible long-term trend. So, we ran a second experiment that was designed to probe for gradual accumulation of TOC over a 30-day time period. Is this instance, two separate trials were pursued, each with a different skimmer, which itself along with GAC was running continuously during the 30-day period. What might we expect? If naturally occurring TOC consumers like bacteria clear up most, but not quite all of the TOC introduced by feeding (its impossible to tell over the 24-hr time period of the above experiments), then perhaps there will be some "remainder" that either accumulates over time, or with skimming, perhaps is removed by that method. These experiments were not run on the author's reef tank like the remainder of the experiments described in this article; rather, they were run on Sanjay Joshi's 500-gallon reef tank. The two skimmers tested in these trials were both homemade; one is a modified ETSS-style downdraft with a Beckett head, and the other is a much more elaborate copy of a Bubble King skimmer complete with Red Dragon pump, bubble plate, etc (see Fig. 4). The experiment began in each case after a large water change, and the water was not changed, nor were there any significant changes in livestock, during the month-long trials. The skimmer was run continuously with weekly cleaning, and GAC was run continuously for the month without recharging. Water samples were removed as previously described and stored frozen pending TOC analysis. The water samples were removed 12 hours after feeding each day. The data are presented in Fig. 4.

Two meaningful conclusions can be drawn upon examination of these data. First and foremost, the TOC level does increase over time. Thus, neither microbial action nor skimming removes all of the accumulating TOC. Second, the less presumptuous downdraft skimmer appears to do a better job at holding TOC levels lower over the course of a month compared to the Bubble King clone. In more quantitative terms, the aggregate TOC values averaged for both skimmers increase from about 0.53 ppm of C at T = 0 to about 0.95 ppm of C at T = 30 days; a 79% increase! If Sanjay would have performed water changes of just 10% at 7, 14, 21, and 28 days during this experimental time course, the TOC level would have increased to only 0.66 ppm of C - a 25% increase. Thus, this experiment illustrates the importance of conducting regular water changes as a means to keep organic nutrients in check.

Skimmer Comparison Studies

Our original studies described in the January 2009 Advanced Aquarist article featured exploration of two experimental systems; BSA in freshly prepared saltwater and TOC in authentic reef tank water. Subsequent to these studies, we embarked on a project designed to provide elemental and in many cases chemical information about the constituents of crude skimmate, as discussed in a companion article to be published in Advanced Aquarist. One unassailable conclusion from those studies was that skimmate contains little, if any, protein, and so the use of a protein (BSA) to measure skimmer performance no longer seemed like a relevant model system. Consequently, the performance tests run on the new trio of skimmers introduced below used only TOC removal from authentic reef tank water as the significant metric; the BSA studies were dropped. Three figures-of-merit were extracted from the mathematical analysis described earlier:

1. the rate constant, k, for TOC removal
2. a quantity (k•Q/(V_r(k + Q/V_s)) proportional to the overall rate, d[TOC]/dt, for TOC removal, and
3. the % of the initial TOC that the skimmer removed

The rate constant k was derived from plotting Eq. (22) with experimental data for [TOC_T]₀, Q, V_r and V_s. The % TOC removed was found through Eq. (27), and the quantity (k•Q/(V_r(k + Q/V_s)), which is proportional to the rate of TOC removal, is derived from Eq. (22) - it is the exponent found through curve fitting. Note that this number is a function of several experimental quantities, including the rate constant k; some simplification is possible under the conditions specified with the discussion of Eq. (25). Pictures of the seven skimmers examined in this and the earlier study are provided in Fig. 5. The reservoir volume, V_r, was always 30 gallons.

Examples of raw data collected in these experiments are illustrated in Fig. 6 for two representative skimmers, the EuroReef CS80 and the Bubble King mini 160. These data sets describe how the measured TOC values in the 30 gallon reservoir drop over the 2-hr time period of the experiment. The squares and triangles are the actual physical measurements; the lines are the mathematical curve fit from Eq. (22). For both of the skimmer runs depicted, r² > 0.9, which indicates that the match between data and model is quite good. It is essential to demonstrate through a control experiment that the depletion in TOC is actually due to the skimmer's action and not, for example, due to either absorption onto the plastic of the reservoir/pump/skimmer or consumption by water borne bacteria (→ CO₂, lost upon analysis). The black line of Fig. 6 is just this control; the reservoir was filled with tank water, but the skimmer was not run. Samples were removed periodically over 2 hours and assayed for TOC content. The data show that at least over the time course of the experiment, the TOC level remains constant - there is no loss that can be attributable to non-skimmer actions. Visual inspection of the EuroReef and Bubble King data draws out some obvious conclusions; the EuroReef seems to drop the TOC level a little faster than the Bubble King, but the Bubble King seems to drop the TOC level to a lower value than the EuroReef at the 2-hr mark. Whereas these observations can provide a qualitative sense of one skimmer's performance vs. another skimmer, a far more valuable outcome of these experiments would be to quantify these differences, and that is where the mathematical modeling/analysis discussed earlier comes in. As detailed with the discussion of Eq. (22), numerical values of both the rate of TOC depletion and the amount of TOC in total removed can be extracted from these data sets.

A tabular survey of the experimentally derived values for the three metrics detailed above is given in Table 1. We have recalculated these quantities with the new mathematical model for the four skimmers reported in the January 2009 Advanced Aquarist article based upon the original raw data, and these new values are reported in Table 1 as well. As an aside, the flow rates for all but the Bubble King and Royal Exclusiv skimmers were measured by simply timing the filling of a vessel of predetermined volume. The two remaining skimmers had flow exit ports below the level of the reservoir water, and so a direct measurement as above could not be performed. A workaround was developed by constructing the plumbing adaptation illustrated in Fig. 5b; the output pipe plumbing was brought exactly to the reservoir water level, thus minimizing any issues with head pressure loss. We then timed the filling of the blue vessel to a predetermined volume. Note that the Red Dragon Bubble King 1000 pump in the Royal Exclusiv 170 cone pushes water about twice as fast through the skimmer body as does the smaller Red Dragon mini Bubble King 600 pump in the Bubble King mini 160.

Table 1. Experimentally derived figures-of-merit for the seven skimmers examined in this study.

Skimmer	Flow Q, g/min	k, min-1	r2 for the data set	ave k, min-1	(k•Q/(Vr(k+Q/Vs))µ rate, min-1	ave ∝ rate, min-1	% drop in TOC	ave % drop in TOC
EuroReef CS80 needlewheel	2.80	0.67	0.88	2.2 ± 1.5	0.012	0.032 ± 0.01	17	20 ± 6
	2.96	0.79	0.89		0.014		32
	2.90	4.61	0.96		0.048		17
	2.90	3.30	0.80		0.040		16
	2.87	1.82	0.90		0.027		26
Precision Marine ES100 venturi	3.59	3.22	0.50	2.3 ± 0.8	0.060	0.048 ± 0.01	17	24 ± 4
	3.74	2.31	0.89		0.051		27
	3.58	1.27	0.65		0.034		24
Precision Marine AP624 airstone	3.41	0.48	0.89	0.84 ± 0.4	0.017	0.027 ± 0.008	26	31 ± 4
	3.49	0.72	0.94		0.024		35
	3.60	1.43	0.87		0.040		32
	3.55	0.75	0.86		0.025		32
ETSS Evolution 500 downdraft	6.63	0.97	0.87	1.3 ± 0.5	0.025	0.031 ± 0.008	41	30 ± 12
	5.83	1.90	0.92		0.043		31
	6.12	0.98	0.85		0.025		18
Reef Octopus 500 recirc pinwheel	2.69	0.19	0.95	0.17 ± 0.03	0.0076	0.0071 ± 0.001	39	35 ± 8
	4.15	0.21	0.96		0.0087		34
	4.73	0.16	0.92		0.0066		25
	6.50	0.13	0.97		0.0055		45
Bubble King Mini 160 needlewheel	2.00	0.77		0.55 ± 0.1	0.014	0.011 ± 0.002	32	37 ± 6
	2.00	0.42			0.0084		30
	2.00	0.51			0.010		45
	2.00	0.50			0.0098		42
Royal Exclusiv 170 Cone needlewheel	4.06	0.64	0.92	0.50 ± 0.10	0.017	0.014 ± 0.002	12	24 ± 12
	4.06	0.43	0.97		0.012		39
	4.06	0.43	0.98		0.012		23

The analysis of Table 1's data begins with a caveat; for some skimmers, the error bars (= "spread" of the measurements) in k are rather large, about 50% of the average value (cf. the EuroReef CS80 and the Precision Marine AP624). Therefore, it would not be appropriate to "over interpret" the significance of the differences in k values for the different skimmers. However, it is possible to use statistical techniques to arrive at an unbiased answer to the question, "Is skimmer A's rate constant significantly different than skimmer B's rate constant?", where "significantly different" is defined as the chance that purely random sampling would result in the averages of two skimmer's k values being as far apart as they are observed to be by experiment. This statistical technique is called an unpaired t-test, and its conclusions are really robust only for data sets containing far more entries than we have in Table 1. In actuality, there is no statistical test designed for data sets having as few as 3-5 entries, and so we will have to bear this caveat in mind when interpreting the t-test data. T-test statistical analysis of the rate constant's k for the seven skimmers examined in this study suggest that:

1. the k value for the Reef Octopus 150 skimmer is different than (smaller than) all of the other skimmers, and
2. the rate constant k for the Precision Marine ES100 skimmer is different than (bigger than) the Bubble King and Royal Exclusive entries.

On the other hand, there are no statistically significant differences between the rate constants k for the EuroReef CS80, the Precision Marine ES100, the Precision Marine AP624 and the ETSS Evolution 500 skimmers. The rate constants k for the Bubble King and the Royal Exclusiv Cone are indistinguishable. Nevertheless, even the statistically significant differences in rate constants for TOC removal are not large, only spanning an approximately 10x range from the smallest to the largest. Since the rate constant k is an intrinsic measure of the skimmer's ability to remove TOC, and it takes into account all of the physical factors that contribute to that removal (i.e., skimmer geometry, bubble size, bubble flow rate, foam coalescence characteristics, water characteristics, etc., etc.), there does not appear to be any compelling reason to favor one type of skimmer design/bubble generation mechanism over any other amongst the seven skimmers examined (Reef Octopus 150 excepted). That is, the inclusion or omission of a bubble plate does not seem to have any decisive effect on the rate constant for TOC removal, nor does a change in skimmer geometry from cylindrical to cone-shaped. Likewise, all methods of bubble generation examined appear adequate.

One question that impacts on the mathematical model derivation, especially the simplification discussed with Eq. (25), involves the relationship between the flow rate Q and the intrinsic rate constant k. There is no provision in the math that links these two quantities; however, if k does depend upon Q, then the simplification of Eq. (25) will not hold. This point was examined experimentally with the Reef Octopus 150 skimmer, as this skimmer has a recirculating pump for bubble introduction and hence the water flow rate Q should not influence the rate of bubble generation. Flow rates from 2.69 gal/min to 6.50 gal/min (= 162 gal/hr to 390 gal/hr) were examined. Within this flow regime, there was no significant variation in the derived rate constant k. So, at least for the Reef Octopus 150 skimmer, and by extension to all of the skimmers, we proceed with the analysis as if k is not dependent on water flow rate Q. Note that in the original January 2009 Advanced Aquarist publication, we attempted to examine the same point using the airstone skimmer (Precision Marine AP624). In those trials using BSA removal as the experimental parameter, we did observe a non-linear response between Q and k; Q = 156 gal/hr, k = 3.1 min^-1; Q = 318 gal/hr, k = 7.6 min^-1; Q = 540 gal/hr, k = 2.5 min^-1. The basis for the discrepancy between the BSA/airstone skimmer results and the TOC/Reef Octopus results is not clear.

The overall rate of TOC removal, as modeled by the exponential term of Eq. (22), is a function of the rate constant k, the skimmer mixing volume V_s, and possibly the flow rate Q (see Eq. (25) and the accompanying discussion). Therefore it is perhaps a more relevant metric for answering overall questions about skimmer performance, since it takes into account the distinct operational parameters (flow, size) of each skimmer. For this metric, large error bars on the order of 10-40% of the average value once again suggest caution in (over) interpreting the data. There are t-test-based statistically significant differences in the values for this rate measurement amongst many the skimmers. Once again, the Reef Octopus 150 displays a rate that is significantly less than all of the other skimmers tested. The Bubble King and Royal Exclusiv skimmers do not display statistically significant differences in their rates of TOC removal, but both of these skimmers do operate at an appreciably slower rate of TOC removal than the Precision Marine ES100, the Precision Marine AP624, and the ETSS Evolution 500.

The skimmers all have different mixing volumes V_s, ranging from a maximum of 1.3 gallons for the Reef Octopus 150 down to a minimum of 0.69 gallons for the Bubble King and 0.62 gallons for the EuroReef. These differences in skimmer sizes become influential in determining the overall rate of TOC removal, whereas the flow rate Q has much less significance (see the discussion along with Eq. (25)). For example, the smallish Bubble King mini 160 skimmer has a relatively small overall rate of TOC removal even though its intrinsic rate constant k is in the middle range; that is, its smaller size really limits its ability to remove TOC rapidly. The similarly sized EuroReef CS80 has a much larger rate of TOC removal since its intrinsic rate constant k is 4x the Bubble King's k value. Conversely, the large volume of the Reef Octopus 150 does not overcome an intrinsic rate constant k at the lower edge of the calculated values and so it exhibits the smallest rate of TOC removal amongst all of the skimmers tested. Of course, all of these skimmers arrive with different price tags, and so a relevant question might focus on the price/performance trade-off. These data are illustrated in Fig. 7. The prices listed are the standard retail price that was paid at the time that these skimmers were purchased. It should not escape notice that the least expensive skimmer (the Precision Marine ES100) offers the greatest rate of TOC removal.

In a practical sense, it is important to resist over interpreting these rate-of-TOC removal data. Most aquarists run their skimmer 24/7, and under that husbandry regime, the major impact of differing rates of TOC removal will be on how often the skimmer cup needs to be cleaned. If the skimmer removes TOC with a faster rate, then the cup will be filled faster and hence have to be cleaned sooner.

One of the more surprising and important observations to emerge from the earlier skimmer studies was that the four original skimmers tested removed only 20 - 30% of the measurable TOC in the reef tank water examined; the remaining 70 - 80% of the TOC was not removed by skimming. Extension of these measurements to the three new skimmers tested in this study did not add much to the argument. The Reef Octopus' removal amount fell within this range, whereas the Bubble King and Royal Exclusiv skimmers appeared to remove incrementally more of the extant TOC, perhaps up to the mid-30% range. An explanation for this observation was offered in the January 2009 Advanced Aquarist article; in summary, skimmers can only remove what bubbles trap, and bubbles only trap molecules and/or particles (i.e., bacteria, diatoms, etc.) with some compelling thermodynamic reason to adhere to the bubble's surface. On the molecular level, this surface association is typically driven by the molecule/particle having a hydrophobic (= water hating) patch that can be buried in the bubble surface/interior. This arrangement avoids the energetically penalizing juxtaposition of hydrophobic surfaces with (hydrophilic) water, and so overall the system energy is lowered (a favorable occurrence). Some of the molecules/particles in aquarium water will meet this hydrophobic region criterion, and some will not. The ones that do not have a sufficiently large hydrophobic patch will not interact with bubbles, and hence will not be removed by skimming. From, the results of the experiments described here, it appears that only 20 - 35 % of the measurable TOC meets this hydrophobicity criterion (= [TOC_l] defined earlier) whereas the remaining 65 - 80 % does not (= [TOC_r] defined earlier). In essence, bubbles are a rather poor media for removal of organic nutrients from aquarium water compared to, for example, GAC. However, they do have the distinct benefit of being cheap.

Conclusions

Many factors contribute to the "value" of a skimmer to an aquarist, including quality of construction, size, footprint, noise level, ease of cleaning, energy efficiency of the pump, and of course, the ability to remove organic waste from aquarium water. Our data show that there are not compelling or remarkably large differences in measurable skimmer TOC removal metrics among the seven skimmers tested, although the Reef Octopus 150 consistently underperformed compared to the other skimmers. However, in the larger picture, it is equally apparent that if an aquarist runs a skimmer continuously (24/7), then any of the skimmers tested would perform adequately in terms of rate of TOC removal; the only practical differences might involve the frequency of skimmer cup cleaning. A perhaps more interesting observation to emerge from these skimmer studies involves not the rate of TOC removal, but rather the amount of TOC removed. None of the skimmers tested removed more than 35% of the extant TOC, leading to the conclusion that bubbles are really not a very effective medium for organic nutrient removal. If fact, the presence of refractory, or unskimmable, TOC, coupled with the likelihood that endogenous TOC consumers (bacteria, among others) also do not remove all of the TOC present (cf. Fig. 4), suggest that in an operational sense, TOC can be categorized as follows:

1. TOC that a skimmer removes
2. TOC that a skimmer does not remove
3. TOC that is consumed by microbes
4. TOC that is not consumed by microbes
5. TOC that is (indirectly or directly) harmful to tank livestock
6. TOC that is not harmful to tank livestock

The last two categories must be included as a result of recent work of Forest Rohwer (See the January 2009 Advanced Aquarist article for a discussion), and they really highlight why an aquarist might be concerned with rising tank TOC levels. Of course, there will be much overlap between these categories. Ultimately, the crucial question for sustaining aquarium livestock health over the long term is, "How much of the harmful TOC (#5) is removed by either biological consumption or by skimming?" That question remains unanswered at present.

The results to date on protein skimming as a means of aquarium water remediation form a consistent picture that is at odds with some of the cherished dogma in the marine husbandry area. According to the data presented in this and the earlier paper (Advanced Aquarist, January 2009), protein skimmers appear to have a much larger variation in their prices than they do in their ability to remove TOC from aquarium water. Recent design innovations like bubble plates, conical sides, or pinwheel impellers do not seem to impact significantly on either rate of TOC removal or amount of TOC removed, at least for the skimmers tested. Thus, skimmer manufacturer claims about enhanced organic removal capabilities should be met with skepticism in the absence of compelling and quantitative TOC removal data.

Acknowledgments

We thank the Eberly College of Science at the Pennsylvania State University and E. I DuPont de Nemours and Co. for financial support, Dr. Bruce Logan and Mr. David Jones of the Pennsylvania State University Department of Civil and Environmental Engineering for use of the Shimadzu 5000 TOC Analyzer, Dr. James Vrentas of the Pennsylvania State University Department of Chemical Engineering for assistance in developing the mathematical model described in this article, and Dr. Sanjay Joshi for the use of his reef tank and for many helpful discussions.

References

1. Allers, E.; Niesner, C.; Wild, C.; Pernthaler, J. 2008. "Microbes Enriched in Seawater after Addition of Coral Mucus." Appl. Environ. Microbiol., 74, 3274-3278.
2. Covert, J. S.; Moran, M. A. 2001. "Molecular Characterization of Estuarine Bacterial Communities that Use High- and Low-Molecular Weight Fractions of Dissolved Organic Carbon." Aquat. Microb. Ecol., 25, 127-139.
3. de Goeij, J. M.; van Duyl, F. C. 2007. "Coral Cavities are Sinks of Dissolved Organic Carbon." Limnol. Oceanogr., 56, 2608-2617.
4. Felder, R. M.; Rousseau, R. W. 2005. Elementary Principles of Chemical Processes, 3^rd Ed., John Wiley and Sons, New York.
5. Feldman, K. S.; Maers, K. M.; Vernese, L. F.; Huber, E. A.; Test, M. R. 2009. "The Development of a Method for the Quantitative Evaluation of Protein Skimmer Performance." Advanced Aquarist http://www.advancedaquarist.com/2009/1/aafeature2/
6. Kirchman, D. L. 1990. "Limitation of Bacterial Growth by Dissolved Organic Matter in the Subartic Pacific." Mar. Ecol. Prog. Ser., 62, 47-54.
7. Mopper, K.; Stubbins, A.; Ritchie, J. D.; Bialk, H. M.; Hatcher, P. G. "Advanced Instrumental Approaches for Characterization of Marine Dissolved Organic Matter: Extraction Techniques, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy." Chem. Rev., 107, 419-442.
8. Sharon, G.; Rosenberg, E. 2008. "Bacterial Growth on Coral Mucus." Curr. Microbiol., 56, 481-488.
9. Wild, C.; Rasheed, M.; Werner, U.; Franke, U.; Johnstone, R.; Huettel, M. 2004. "Degradation and Mineralization of Coral Mucus in Reef Environments." Mar. Ecol. Prog. Ser., 267, 159-171.
10. Zweifel, U. L.; Norrman, B.; Hagström, A. 1993. "Consumption of Dissolved Organic Carbon by Marine Bacteria and Demand for Inorganic Nutrients." Mar. Ecol. Prog. Ser., 101, 23-32.

Once you keep a high energy planted aquarium tank for a while, you are bound to eventually keep hitting such high densities of plant biomass that several difficulties tend to arise: your plants may grow so thick that it is hard to keep the tank clean, the aquascape may have gone out of balance or it becomes difficult to keep up with growing demands for nutrient and carbon dioxide. In another scenario you may have left town for a few weekends in a row and the tank becomes so overgrown that only a complete overhaul can restore aesthetic and biological order to your freshwater aquatic garden. In any case, a planted tank occasionally needs to be bushwhacked and replanted and there a few things the aquarist can do to make the rebirth of their aquarium go as smoothly and efficiently as possible.

Before I even do the actual overhaul of a planted tank, I try to take in all of the required aquarium maintenance that needs to be performed before I start the actual bushwhacking. I find it is a much better practice to do all of these servicing chores before trimming all the plants because you are much more likely to put off more aquarium work after the planting is done. You should clean all filters, top off your dosing, clean the Carbon Dioxide injection mechanism, whether this is a CO₂ reactor or diffuser and by golly, make sure that have plenty of carbon dioxide to last you for at least a couple of weeks after a major replanting. Don't forget to spruce up your lighting system by either replacing your lamps, wiping off the bulbs, reflectors or splashguards and make sure that any fan guard Is free of dust. Also, make sure that all of your replacement water is at a suitably close temperature and pH to add to the tank as soon as it is ready. If you need to spread out duration of the plant tank overhaul, I suggest doing the chores on the first day and then you'll be able to really take your time in redoing the aquascape on a following day.

Now before you start the actual weed wacking, make sure that you have all the proper tools handy: you'll likely need some long handled scissors, some large sturdy tweezers, and a few containers to hold your clean plants and trimmings. If you keep plants that look very different from each other it's very easy to place all your "re-use" plants in the same container but if your aquarium specializes in different varieties of mosses, Cryptocorynes, Rotalas or Eriocaulaceae, you might want to take some precautions to keep you from having to differentiate between closely related plant varieties; even if you're an expert it's a pain to tell the difference between the collapsed state of emerged grown plants, especially when you are trying to tell apart a some of the Syngonanthus or Eriocaulon species. For some plants you can use large shallow trays that you can fill with water or cover with clingwrap and for other more robust species like Anubias and Ferns I like to either throw them into a ziplock bag or tupperware container. Since I often have many similar looking plants, I will label some ziplock bags to hold just one species or type of plant before I even start trimming. Not only do labeled bags help me keep track of what plants are what, when I am done removing plants what I am left with is a clear layout of all the plants I am going to re-use in the layout, as well as the height and quantity of the different plant groups. With such a clear depiction of my plant mass it is much easier to begin planning the replanting of the new aquascape.

Make sure you turn off all your filters and CO₂ dosing before you start messing with the tank because water motion will only blow around all the detritus you will undoubtedly disturb. While you are trimming your plants you will have to decide whether to groom unwanted leaves and ratty stems now or later. You can either trim off the tops of the plants you want and keep trimming the plants until you've removed all that you want to keep. The remaining stem-bottoms may or may not be reusable and the choice to regrow or discard these trimmings will be up to you. If you like the size and placement of this small patch of lower stems you may be able to regrow the plant you desire from this stumpy base and sell or trade off the choice top stems but the capacity for regrowth will also depend on the species of plant. Stem plants from the Ludwigia, Limnophyla, Hygrophyla, Syngonanthus and Rotala genera will likely already have a ton of new buds already appearing at nearly every internode that was left behind. Regrowth from this kind of patch can come back in very quickly and densely and if this is the kind of result you are seeking then by all means leave the patch of stems to regrow.

However, certain stem plants do not respond well to regrowing in this manner and you will not get the desired result with Pogostemon, Ludwigia inclinata var verticillata or Ammania 'Bonsai'. These plants may regrow some buds from the bottom stems but they are more likely to stunt or rot. If you decide to replant from the stem tops, try to remove as much of the bottom stems and roots as possible. At this point you'll want to cut the roots below the surface of the substrate and use your sturdy tweezers to pull out unwanted stems individually. If you try to pull out more than one stem base at a time you risk disturbing a large area of substrate and making a real big mess. For the real heavy, root balling, substrate feeding plants such as Eriocaulon, Echinodorus and Cryptocoryne, you will most certainly need to reach a pair of long scissors with a curved cutting edge deep beneath these plants to cut the roots away from the rootball. Even then you'll want to pull out the plant in stages of uprooting and letting the substrate fall away from the roots a few times until enough of the substrate has been freed from the the substantial roots of these substrate feeding plants. After having uprooted all the plants that need to be refreshed, you'll probably want to take a break to allow the plant matter and detritus to sink or float for a few minutes so that it can be more easily siphoned off of the substrate and hardscape, and then skimmed from the surface with a regular fish net.

Although some of the new-school aquatic gardeners have developed a habit of planting an aquarium mostly without water in the tank, I like to plant my water plants in water, the old fashioned way. If your plant tank aquarium substrate had any areas which were particularly well planted with heavy root forming plants for four to six months, that area of the substrate may have become locally depleted of certain key nutrients. There's no better time to infuse some root tabs into your substrate than before replanting a fresh grouping of heavy root feeders. When it comes to replanting your old or refreshed aquascaping theme, try to anticipate what your pants look like in 4-6 weeks, and plan their growth rates and pattern to coincide with your desired layout and focal points. You'll still need to groom your plants into submission to get the look you desire but proper placement of your plants will still make it easier to reach your aesthetic goals. Once you've got your tank completely replanted filled up and flowing, you'll want to readjust your tank dosing regimen to reflect the reduced biomass of your new layout. If you were dosing a certain amount of NPK, Iron and CO₂ before, you will likely need a much smaller amount and you should reduce your dosing amount to prevent unwanted nutrient or carbon dioxide buildup. Once your plants continue growing again, you'll have to gradually increase your CO2 bubble rate and other dosing to keep pace with your once again flourishing water garden.

The time has finally come to prepare your new reef biosphere for life. The first question is what you want your reef tank to look like. In the early days of reef keeping most aquarists essentially built a “live rock” wall along the back section of their tank, eventually attaching small corals or frags to that wall.

Rock Wall

An example of that technique can seen in the following photograph; it is of a beautiful reef tank setup by the late Greg Schiemer.

This picture is only of a section of the left side of a 500-gallon reef tank. However, it is easy to see how the tank was created.

Low Profile

Another technique that developed some years later I will call the low profile setup. It is the technique I preferred back in those days—around the year 2000. I came upon this technique naturally, because as many of you know, when I moved 3 reef tanks from NY into one 10-foot tank, I didn’t get any new rock and simply placed the corals that had grown large along the bottom of the tank. My hope was that the corals would have plenty of room to grow towards the light. I also hoped that eventually my reef would have a more natural look. From the next photo you can draw your own conclusions.

From the absence of coralline you can tell that the tank was newly setup. I might add that it didn’t take long before corals like Acropora sp. reached the surface of the tank.

Coral Head

Sometime later Julian Sprung setup a 40-gallon cube, within which he created in the center of that tank a coral head. This made this small reef visible from all sides, and allowed the fish to swim and graze around the head. This is clear from the photo coming next.

Julian also wanted this small reef to be representive only of a Caribbean reef, so all of the fauna and flora came from the Caribbean.

Sanjay’s Reef

When he setup his new 500-gallon reef tank Sanjay Joshi perfected what Julian started in his Caribbean reef. Following is a group of photos that demonstrate how to work this technique to perfection. In my opinion, whether in a small reef or a large reef tank this is the ideal way to setup a reef tank. Once populated it gives the fish plenty of swimming room and looks the most natural.

From these 3 photos notice that Sanjay got a vertical pillar of live rock by using a concrete block as a base to which he attached a PVC pole and then used Thorite to attach the rock to the pole and to one another.

NOTE: I have heard that Thorite is no longer manufactured. I suspect that most of the underwater two part epoxies will work as well. Whatever material used it must be thoroughly rinsed before use, and must not leech toxic chemicals into the water after a period of time.

As can be seen from this series of photos, Sanjay was able to create a very functional and beautiful, authentic looking captive reef.

This last photo is of the middle section of Sanjay’s 500-gallon reef a few years later.

There are essentially three types of filtration that the marine aquarist need be concerned with: particle, chemical, and biological. For the marine fish only or the reef tank, biological filtration is essential. Without the establishment of nitrifying bacteria toxic metabolites will reach deadly levels quickly in a marine fish or reef tank. Waste products from the fish's respiration, excrement, etc., will rapidly convert to toxic ammonia. The same process occurs in freshwater aquariums, but in marine aquariums which have a much higher pH - at least 10 times higher - a given amount of ammonia is at least 10 times more toxic. Keep in mind that the higher the pH the more available unionized ammonia, which is the toxic form of ammonia. Therefore, it is essential that the marine aquarist establish a sufficient colony of nitrifying bacteria to convert ammonia to nitrites and finally to nitrates. Nitrates are relatively nontoxic. This must be done before introducing fish into a newly setup marine aquarium. There are any number of ways to accomplish this. One way is to take filter material from an established marine aquarium and place it in the filter system of the new aquarium. Once there are nitrifying bacteria present increasing their number to handle the arrival of fish can easily be done by injecting ammonia into the tank. Even human urine will do nicely. It is then necessary for the aquarist to measure the change from ammonia to nitrites and then to nitrates, a process that usually takes several weeks. Keep in mind that these bacteria are aerobic and should be placed in filter material that is exposed to good water circulation. This is important for fish only marine aquariums. With a new reef tank the presence of conditioned live rock will have already acquired nitrifying bacteria. If the rock is dead and sterile then it will have to be made biologically alive using the same methods as described for new fish only aquariums.

Producers and Consumers of Waste

In successful reef tanks the production of waste, mostly by fish metabolism and uneaten food, is in balance with the capacity of the consumers. The filter feeders and the photosynthetic, symbiotic dinoflagelates (ZOOXANTHELLAE) that live in the hermatypic (reef building corals) corals will consume some waste, but unless there are very few fish in a large thriving reef tank waste will build up. It is therefore necessary for the reef keeper to use water changes, mechanical, and chemical means to maintain this vital balance. The buildup of waste, usually measured by aquarists as nitrates and organic phosphates, if allowed to concentrate will interfere with the ability of many invertebrates to survive, let alone grow. Phosphate (PO₄) at concentrations greater than 0.1 PPM will interfere with coral growth.

Water Changes

One way to dilute unwanted waste in the form called total organic carbon or TOC is through water changes. Many successful reef keepers change 25% of the water monthly. However, for this to be effective the new water must be pure, free of phosphates and other waste products. The new saltwater should be made up from water that has been treated by reverse osmosis and deionization. Otherwise, whatever impurities exist in one's tap water will concentrate with the addition of water to replace seawater from evaporation and during water changes. Note, when making a significant water change it is important to make sure that the new water is at the same salinity, temperature, and pH as the reef tank's water.

Protein Skimming

For a long time marine aquarists believed that protein skimmers (also called foam fractionators) were the be all and end all of waste removal. Furthermore, that they were so efficient that they could remove too much, and thereby starve certain corals. They are effective at removing phytoplankton. However, resent studies by Professor Feldman has shown, regardless of the type of skimmer used, skimmers will only remove about 20% of the unwanted TOC. I strongly suggest that you read Feldman's study published here.

http://www.advancedaquarist.com/2009/1/aafeature2/view?searchterm=feldman

I certainly do not want to give the impression that skimming is useless, only that it is but one of the useful devices available to the aquarist to maintain a balance between the producers and consumers of waste. Also, it greatly helps with gas exchange, especially at night when the production of oxygen stops with the end of photosynthesis after the lights are turned off.

Chemical Filtration

There are two forms of chemical filtration commonly used by marine aquarists, activated carbon and chemical phosphate absorbers. Activated carbon is very useful due to its ability to absorb TOC; however, it requires frequent changing - weekly --as it becomes saturated easily. There are a number of PO₄ absorbers on the market - Phosban and ROWAphos, are the two most popular - which are very effective at removing PO₄ to almost immeasurable levels. To know when to change the PO₄ media requires a PO₄ test kit that will measure to very low levels. Salifert, Hack, and ROWA Phosphate Test Kit, make kits that I know will indicate low enough to be useful; whereas, most other hobby kits are not. There very well maybe others, but these I have used successfully.

I strongly suggest that readers also read Professor Feldman's two articles on TOC published here also.

1. http://www.advancedaquarist.com/2008/8/aafeature3
2. http://www.advancedaquarist.com/2008/9/aafeature2

If there is a heavy bioload (fish) it is difficult to keep the nitrate level below 10-PPM. Still, many of the more delicate invertebrates do better in water that has not accumulated a concentration of nitrate. One way to keep nitrates low is to do regular water changes; how much and how often will depend on the bioload of fish, and how often and how much they are fed. The way to monitor this is with a nitrate test kit. The aquarist should recognize that there is a difference in what some nitrate test kits indicate - nitrate- nitrogen or nitrate (NO₃). If your kit measures nitrate-nitrogen to convert this to the Nitrate ion multiply your reading by 4.4. Some advanced aquarists have experimented with what is called the vodka method to reduce the nitrate and phosphate levels in their aquaria. Vodka is a carbon source, and can stimulate the growth of bacteria that will use nitrates and phosphates as food. To try this method it is vital to have a well functioning skimmer, as the bacteria can reduced the oxygen levels too much. For more information about this method check chapter 6 in The Reef Aquarium: Science, Art, and Technology vol.3 by Delbeek and Sprung. ISBN 1-883693-14-4 In my opinion, if there's one book that the beginning reef keeper should regard as the essential source it is this book.

Algal Turf Scrubbers (ATS)

It is also possible to keep the TOC levels acceptably low by a method known as Algal Turf Scrubbers (ATS). In this method sufficient alga is grown and harvested. The alga uses nitrates and phosphates as food, which is then removed from the aquarium with the harvesting of the algae. It is of course necessary to grow a lot of alga, and to also be concerned with what trace elements are removed from the system with the harvested algae. Another concern with this method is that it yellows the water; however, this can be overcome with the regular use of activated carbon. This method was initially promulgated by Walter Adey.

Check out the following link for more discussion on this and other methods for reducing TOC in reef tanks:

• http://www.advancedaquarist.com/2007/9/diy

Success

A successful reef tank is a thing of beauty and well worth the effort. A picture of my reef tank

I simply cannot recall seeing so many new products hit the scene, each with so much potential to change the way we run a reef tank and shifting paradigms about how certain pieces of equipment should work. Many of these products were revealed for the first time at the Marine Aquarium Expo in Spring but others have just started to gain exposure in the marine aquarium industry.

Twin Arc Metal Halide Lamps from Advanced Lighting Solutions

The Twin Arc metal halide lamp is one of those game-changing products that no one could have seen coming. The Twin Arc bulbs are a standard metal halide bulb which contains two separate inner envelopes. When the Twin Arc bulb is fired, a component within the bulb remembers which envelope was last fired and it directs the current towards the other envelope. The bulb fires the inner envelopes in alternation and in it's most basic application the Twin Arc uses two envelopes of a single color either in 10K, 14K or 20K. In it's most exciting form, the Twin Arc comes with in a dual colored bulb with one envelope pushing 10K colored lighting and the other envelope pushing 20K colored lighting. So each time the dual colored Twin Arc is fired it alternates between pushing "full spectrum" 10K colroed light which is most conducive for growth and 20K colored light which is most conducive to showing off and encouraging coral colors.

Coral farmers have long tossed around the idea of growing corals under full spectrum light and then coloring them up under bluer light for producing more colorful and desirable corals. The concept of growing corals under lights of multiple colors is not a new one, reefers have been using separate lamps of different colors as long as they've been available. Now the multi colored approach togrowing corals is easier than ever; if an aquarist is using a dual colored Twin Arc bulb they can easily switch between the best of both worlds using a simple timer to turn the lights on and off twice a day. We'll have to see how these bulbs hold up in real world applications but we'll be happy even if we just get a single colored Twin Arc lamp which lasts twice as long.

Mag Lights, LED striplights from Advanced Lighting Solutions

The new aquarium lighting company Advanced Lighting Solutions was not content to have simply one revolutionary product on the market so they also released a full line of LED striplights. The line is segmented into the Mini-Mag for pico tanks, the regular Mag Lights and the full length Mag Pro striplights which come in white, blue and mixed light configurations. Unlike the complete stand alone LED light fixtures which are very expensive or no longer warrantied, the Mag LED striplights are modular in nature and they install exactly like one would expect for a striplight. If we were betting folks we'd wager that the ALS LED lights will be the product line which introduces LEDs to the reefing mainstream because there are two very special aces to the ALS LEDs.

First of all, all of the LED striplights are available in a actinic only coloration. LED lights have long been most efficient in the blue spectrum and it is natural that we would now have actinic LED striplights to replace regular actinic 03 and super blue flavors. Now I can already hear all the die-hard VHO actinic fans crying bloody objections to defaming their beloved fluorescent actinics but the quantity and intensity of fluorescence exciting spectrum emanating from actinic LEDs will just about make you forget all about the blue tubes. With fluorescent actinics the 420 and 470nm spectrums are packaged separately meaning that you have to have some actinic 03 bulb and a "super blue" lamp to cover the fluorescent spectrum. With the ALS actinic LED striplight, the proprietary blend of their diodes actually have a broad light emission curve which ranges from 420-460nm meaning that a single striplight can excite more fluorescence than any single fluorescent lamp. Furthermore, ALS has released an Actinic LED striplight which is designed to attach to existing metal halide pendants so that the LEDs can function as the sole source of supplemental blue light.

What is most unique about the ALS LED strips is that they are engineered for long term performance. When comparing different types of light sources many aquarists tend to use watt as the metric of comparing how much power a light source can deliver. However, unlike halides and fluorescents which are designed to operate at a certain power level, the silicon based LEDs are more like a computer CPU which can operate at a range of power levels. AS you might expect, the luminous output of LEDs is not the same whether they are operated at 50% or 100% of their nominal rating. On the one hand a 1 watt LED can bu run at 1.5 watts and it will put out more light but the efficiency and life of the LED will be greatly reduced. However, if you take a 3 watt LED and run it at 1 watt, the luminous efficiency is at it's peak and the life of the bulb is greatly increased. The ALS Mag Pro LED Striplights use exactly this latter scenario with 3 watt LEDs that are operated at 1 watt. In this configuration the lights put out about 100-110 lumens per watt and they are expected to still be at 70-80% intensity after 5 years of operation.

Plasma Arc lighting from Aqua Illumination

Plasma arc lighting is such a new technology that we are surprised to already see a prototype plasma arc light running on a reef tank. Although the plasma arc light from Luxim only hit the mainstream press in the spring of 08, the pioneering techies over at Aqua Illumination couldn't wait to get their hands on the first round of developing kits for the plasma arc light so they could start pushing the envelope. Plasma Arc Lighting was developed by Luxim Corportation, the same manufacturer of many of the better high-ouput LEDs. The Plasma Arc lamps are fully dimmable and the 250 watt pill can put out up to 140 lumens per watt which is twice the efficiency of compact fluorescent lamps and still a good deal better than the 90-110 lumens per watt of high performance LEDs.

The plasma arc light is similar to a metal halide lamp in that the bulb is made up of a small, gas and metal halide filled inner envelope but the similarities end there. The plasma arc lamp is nothing but this little glass envelope and it has no contacts or additional components like a metal halide lamp. Instead, the plasma arc light sits in a small driver which acts produces electromagnetic and radio frequency (RF) waves. The RF waves are directed towards the center of the plasma arc lamp where they vaporize the argon and metal halides and voila, light spills forth. One of the few drawbacks of plasma arc light is that the efficiency of light output and the color spectrum emitted is variable across it's power range and they seem to shift color as they are dimmed. We don't expect to see any production models announced any time soon but as it stands, we are just excited to know that the Plasma Arc light is already logging some time growing aquarium corals.

Apex Aquacontroller from Neptune System

Electronic aquarium controllers have been around for a long, long time but their price tag and complexity have mostly relegated their use to the uber techie reefer. In the last few years, advancements have been made to simplify the set up and operation of aquarium controllers and the increasingly lower price of electronics has been reflected in the prices of consumer aqua-computers. The Apex Aquacontroller from Neptune Systems is one of those breakthrough products which takes a 15 year pedigree of manufacturing aquarium electronics, combines that with what people really want for their reef tanks and delivers an all-in-one package which is affordable and easy to set up. For the base price of $500, the Apex Aquacontroller System includes everything you need to get started monitoring and controlling the most common aquarium applications.

One look at the Apex kit with all of it's neatly labeled components and you get the impression that you may be able to set up the entire system without even reading the directions, let alone having to call the IT guy in your local reef club. One of the distinguishing features of the Apex is a complete separation between the base station and the controller display. The base station can be placed in a convenient place near the sump, where most of the monitoring equipment will reside and the controller display is tethered by a thin cable which can extend the controller between 20 and 100 feet away. The controller feels very much like a remote and it uses descriptive icons and symbols to quickly convey the current status of the aquarium system. The Apex is a broadly expandable, affordable and easy to set up aquarium controller and we expect this product to become the breakthrough controller which makes aquarium controlling and monitoring attractive for the wider marine aquarium community.

EcoTech Marine- Vortech mp10

Ever since the introduction of the first VorTech magnetic water pump, EcoTech's most frequently asked question has been whether the wonder pump would come in a smaller size. After five years of waiting, Ecotech has released the Vortech MP10, a miniaturized version of the VorTech MP40 which above all cuts down the price and the size of the magnetically coupled technology. For an msrp of $195, any aquarist can afford to invest in the efficiency of 1600+ gallons per hour with a maximum 18 watt power draw. The diminutive 2 inch by 1.5" wetside absolutely disappears in the aquarium but yet it still pushes a nice broad, plume of water movement which is the favored method for encouraging mass water movement and gyres in reef aquaria.

The Vortech MP10 ships with the same microprocessor controlled driver board with all of the adjustable, pre-programmed Vortech modes which produce constant flow, random flow, short pulse modes for producing waves and most importantly, a long pulse mode for producing gyre flow. The Vortech MP10 is more expensive than many other similarly rated water pumps but the included controller, diminutive size and the aesthetic of not having any wires in the aquarium makes this propeller pump more than worth the premium. Now that Ecotech has a balanced full line of water pumps, we're calling them out to put some resources behind accessorizing the VorTech pumps to increase their performance and accommodate some custom applications. Propellers and outlets with different flow patterns and larger common strainers to accommodate multiple pumps would really help to diversify the functionality of Vortech pumps.

MCU Research - Silent Air

The Silent Air is a proportional temperature controller for 12 volt DC fans. The small controller box has a temperature sensor which can be used wet or dry and DC current output for up to 8 DC fans. The temperature probe can be placed in the sump or canopy and adjusted to allow for a low temperature range, sump mode, or a high temperature range, canopy mode. The beauty of the Silent Air is that unlike a temperature controller which turns on a chiller or cooling fans full blast, the Silent Air runs the fans at low speeds when there is less heat and high speed when there is more heat. Since the fans spend a lot less time running at 100% capacity, they will put out much less noise than even a chiller or fans.

Since the beginning of reef-keeping aquarists have been using air circulation to help cool their aquariums. Air circulation helps to vent off the heat produced by lights, ballasts and external water pumps and it speeds up evaporation, further helping to cool the aquarium. The most traditional implementation of the air cooled approach involves the use of fans which are controlled by a timer or temperature sensor. In this scenario the fans are operated at a single speed which usually ends up being the fastest (and loudest) setting. This all-or-nothing type of operation produces an inelegant stepwise performance curve. By contrast, the Silent Air operates the fans proportionally to the temperature of the sump or canopy region and it ramps up the fan's RPM as needed to counteract the temperature of the sump or canopy. By delivering an a cooling effect which is proportional to the amount of heat in the canopy or sump, the Silent Air will not only combat maximum temperatures of the aquarium but it will also stabilize temperatures overall.

Euro Reef Nano Skimmers

With the emergence of nano-reef aquariums from the reef hobby as permanent specialty segment of keeping corals in captivity, there has recently been an explosion of new products designed and built exclusively for use with nano reefs. One of of our favorite projects has been baking for over two years and it has very recently been released from the oven. Before they are turned on, Euro Reef Nano skimmers look more or less like any other skimmer: narrow cylindrical body, skimmer cup and a water pump set up in the needle wheel position. However, once you see a Euro Reef Nano skimmer in action, it is clear that the tiny little feed pump is producing tiny bubbles in quantities that seem to well exceed what you'd expect for the pump's size.

The key to the water pump's excellent bubble production lies primarily in the unique metal-alloy mesh wheel which is made from a material that Euro Reef has been sourcing/co-developing for a long time. The new metal-alloy material looks like a pad of fine strings which has been dipped in metallic paint. In the beginning we had needle wheels with two to three rows of pins sticking out from a circular impeller. This design produced only a few layers of chopping air into fine bubbles. The new mesh wheel improves on this design by providing many more layers of air chopping making for a very consistent and dense froth. Currently the Euro Reef Nano skimmer models are custom tailored for 11 different pre-fabbed nano reef set ups and they are mostly under between $125-150. If your aquarium came with a plastic lid, chances are good that Euro Reef has already designed a nano protein skimmer specifically for your tank. Euro Reef could do well to re-manufacture the impellers of other needle wheel skimmers using their mash-alloy material.

Water circulation is undoubtedly the most important parameter for the successful fish only or reef tank. Without water circulation life will not exist; it provides oxygenated water, removes toxic gases like CO₂, and transports nutrients throughout the man made biotope. Without water circulation, whether due to a power outage or pump failure, animals that need oxygen begin to die quite quickly. And, there are biochemical reasons for this.

Respiration

The vast majority of organisms that we keep in our marine tanks respire. They consume oxygen from their environment where it is transported via their circulatory system into their cells. This permits vital metabolism to occur within the cells. Also, as part of this cellular metabolism carbon dioxide (CO₂) is produced. In human beings this is called breathing. We, however, extract oxygen from the air and release carbon dioxide into the air. This is significant because the animals we keep in our marine aquariums need to extract oxygen from water which contains far less oxygen then that of air. The result of that is that our marine fish for example must take in 10 to 30 times the volume of water than terrestrial animals take in from the air to respire successfully. It is therefore vitally important that we supply our critters with water that is constantly well oxygenated through aeration and or circulation.

Gas Exchange

Essentially our goal in creating a successful marine tank is to maintain as close as possible water that is saturated with oxygen and that successfully dispels carbon dioxide. Although aeration from an air stone, or better within the chamber of a protein skimmer contributes somewhat, most of the gas exchange takes place through the water’s surface. The air-water interface is where this gas exchange takes place, and anything that interferes with this vital exchange is dangerous to the health of our organisms. However, between the air and the water is a thin film called by oceanographers a laminar layer, which interferes with said gas exchange. The thinner this film the more efficient the gas exchange.

Laminar layer

In the ocean the thickness of the laminar layer ranges from 0.002 to 0.020 centimeters, mostly determined by surface agitation, like wave action.

How do we then thin or even get close to eliminating this laminar layer or skin, so that efficient gas exchange can take place? This layer is the result of pressure between the atmosphere and the water. Diffusion of gases through this layer increases in efficiency the thinner the laminar layer. The most direct way to promote gas exchange threw this layer is to agitate the water’s surface. On the simplest level stirring the water helps significantly. The can be done with a mechanical paddle or air bubbles breaking the surface. However, there are much better ways.

To complicate matters organic molecules made up of nitrogenous organic material and simple detritus collects on the surface, further impeding necessary gas exchange. When there is no or little surface agitation this organic slick is easily visible to the naked eye, especially when looked at from below the surface. Removal of surface water also aids in the transmission of light to the corals if you are plumbing a reef tank.

Surface Agitation – Plumbing Techniques

When it comes to plumbing a marine aquarium the first goal is to move water from all sections of the tank, especially the lower section to the surface, and at the same time breaking the surface tension. The best way to achieve this is with an overflow, which is the way that most reef keepers go. Following are some pictures, and a video of overflow boxes from two of our sponsors. The one from Marine Depot, if you scroll down shows a video of one at work.

Marine Depot: http://www.marinedepot.com/ps_ViewItem.aspx?idProduct=CR1511&child=CR1515

Champion Lighting: http://www.championlighting.com/product.php?productid=22072&cat=404&page=1

http://www.youtube.com/watch?v=-zVGwdRVHU4

Whether you use a movable overflow box, or a built in inside box overflow the primary issue is capacity. In other words, can the siphon capacity handle the volume of water returned to the tank from the sump pump or pumps, whether said pump or pumps are in the sump or plumbed outside the sump? And, if you’re having the tank drilled be sure to plan for the future in terms of capacity. In a reef tank coral growth often requires greater water movement as growth becomes denser.  Another virtue of surface skimming and overflow boxes is that in the event of a power outage the water level will not drop lower than the top of the overflow.

Caution: In the event of a power failure make sure that there is enough room in the sump for water that will flow into the sump from the display tank. Also, make sure that when the power returns an external overflow box is able to restart siphoning; otherwise the water from the sump will overflow the display tank. This is generally caused by air bubbles getting into the siphons. In fact, this can happen without a power outage. Many external overflow boxes have a nipple for sucking air out of the siphon chamber, and by attaching a small power head to that nipple air bubbles can be kept out of the siphon area. If the outside box uses siphon tubes be sure to check them regularly for air pockets. Remember Murphy’s law: whatever can go wrong will, and at the worst time.

Note: If you have a choice, choose an external pump, because it will transfer less heat into the tank water, and choose one that is energy efficient. On my 700-gallon reef tank I used in the past a Jacuzzi pump, which drew 10-amps, and when I switched to a Reeflo HammerHead pump the water temperature dropped 2 degrees-F, and my electric bill dropped almost $50.00 monthly. The HammerHead moved as much water, but drew only 3-amps – about 350-watts.

The Sump

What kind of sump you utilize is dependent on a number of factors: available space and size. Sumps can be purchased ready-made, or for the more adventurous they can be made. Because I have a pump room located behind the display tank I like to use Rubbermaid stock tanks. They are made of fiberglass, are quite strong, and easy to drill. They also come in sizes ranging from 50 to 300-gallons. See following picture:

In this setup I have two 150-gallon Rubbermaid stock tanks plumbed together with a 2-inch PVC pipe. Using a valve I can isolate the back sump, when I want to use it as a quarantine tank. As you can also see, you can place skimmers, heaters, carbon filters, and calcium reactors in these stock tanks.

Returns to Tank

It should be obvious by now that water circulation is essential; without it, after a relatively short time period – hours – life in your tank will begin to die. How water is returned to the display tank from the sump has two goals: one is to simply get water from the sump back into the display tank, the second is to supply circulation to all areas of the display tank. This can be done simply, or in a more complex way. My way is to utilize devices made by Ocean Motions which have return lines that alternate and heads that rotate creating random water circulation patterns in the display tank. See the following picture:

Caution: If you look carefully at the above picture you will notice that the 4 Ocean Motion returns are lower in the water than the top of the built in overflow box. In the event of a power failure or the failure of the return pump the returns will become siphons with the result that the water will return to the sump from the depth of the returns, which can overflow a sump that doesn’t have enough holding capacity. Furthermore, some aquarists think by using a check valve it will prevent water from siphoning back into the sump, which is what it is designed to do. Check valves will do this quite well when new; however, they tend to clog up after some months of use in seawater and leak. If you need to depend on one than plumb it surrounded by true union valves so that it can be removed for cleaning in a weak acid bath periodically. Incidentally, this is true of all valves; in seawater valves tend to freeze up with deposits of calcium. I make it a practice of turning them off and on vigorously at least once a month.

Defeating Murphy

Backup systems are important, even essential. I always have a second backup pump; in this case a second HammerHead already plumbed and ready to go if necessary. A pair of Haywood true union valves allow me to change pumps in minutes. Also, I never want to rely on one pump, and in this system the main circulation pump. I also use two internal water pumps made by Vortech. And, in the event of a power outage I have 6 battery operated air pumps manufactured by Pennplax that are programmed to turn on if the electrical power goes out.

Notice how the return HammerHead pump is surrounded by true union valves, which allows me to remove the said pump in a matter of minutes. I keep a spare pump already plumbed ready to replace a failed pump. Using true union valves like these shown can also be used to remove and clean a check valve.

Vortech Pumps

I like Vortech pumps because they are unobtrusive and move a lot of water. They can be placed low in the display tank, without worry about siphoning water out of the tank, and because their motor is outside of the tank they will not heat the water. They also come with a battery backup which is useful in the event of a power failure. Advanced Aquarist has reviewed this product twice, see:

1. http://www.advancedaquarist.com/2008/5/review
2. http://www.advancedaquarist.com/2008/11/review2

Tip

You can notice from the plumbing setup in my reef that I use flexible PVC. This allows me to avoid elbows whenever possible. Elbows cause back pressure, reducing flow. Any questions, just post them following this column!