While visiting a fish store during a recent trip to the mainland, I saw products claiming to hasten “cycling” of marine aquaria. I was intrigued by the possibility, purchased the products, and began formulating a testing protocol. These products are Brightwell Aquatics” MicroBacter7 and Continuum Aquatics” BacterGen.M.

Successful reefkeeping depends upon natural cycles doing what they do, whether it involves cycling of carbon, nitrogen, or other elements. Perhaps the most important is the nitrogen cycle since it involves ammonia, which can be toxic to fishes at high concentrations. Ammonia is excreted by fishes and their waste products contain proteins that eventually degrade to ammonia. Ammonia is converted to nitrite (also toxic) by a certain group of bacteria (usually designated as Nitrosomonas species.) Nitrite is then converted to relatively non-toxic nitrate by another group of bacteria (generally referred to as Nitrobacter species.) Bacteria that convert nitrogenous wastes tend to be rather sensitive to environmental conditions (such as pH) and slow growing, hence it is critical that a newly set up aquarium be given sufficient time for bacteria involved in the aerobic portion of the nitrogen cycle to become established (it has been my experience that this requires about 30 days once an aquarium is inoculated.)

Before beginning, let’s define some of the terms used in this article.

Glossary

Aerobic: Condition where molecular oxygen (O2) is present.

Anaerobic: Condition where no molecular oxygen is present.

Biochemical Oxygen Demand (BOD): A test that determines the amount of oxygen required to stabilize biodegradable wastes. Usually, the test duration is five days at a temperature of 20C (68F.) The formula is:

(a – b) * c Where: a = initial oxygen concentration (mg/l); b = oxygen concentration after the 5-day incubation period; c = dilution factor. This test is sometimes incorrectly called biological oxygen demand . The answer is biochemical oxygen demand in milligrams per liter (or parts per million.)

Carbonaceous: Of, like, or containing the element carbon.

Conductivity : A measure of an aqueous solution’s ability to conduct electricity. Pure water is a very poor conductor, while water containing, say, inorganic compounds is a much better conductor. It is reported in fractions of a Siemen (denoted as “s”, which is the reciprocal of an ohm) – usually micro-S (uS) or milli-S (mS), and the distance between measuring probes is also reported (the standard is 1 centimeter), hence Conductivity might be reported as uS.cm.

Denitrification: Biochemical conversion of nitrate to nitrogen gas, usually under anaerobic conditions.

mg/L : Milligrams per liter, essentially the same thing as parts per million.

Nitirification: Biochemical oxidation of ammonia to nitrate under aerobic conditions.

Nitrogenous : Of, like, or containing the element nitrogen.

Pathogenic: Able to cause ailment or sickness.

ppt: Parts per thousand. Used to describe the degree of salinity in the context of this article.

ppm: Parts per million.

Spore, or Endospore: Walled, single- to many-celled reproductive bodies of an organism, capable of giving rise to a new individual. Bacteria capable of forming endospores number perhaps 100 genera including Bacillus , Sporolactobacillus , Clostridium , Sporosarcina , and others. Interestingly, nitrifying bacteria genera ( Nitrobacter and Nitrosomonas ) do not produce spores.

Products Tested, and Comments

MicroBacter7 ^TM

Marketed by: Brightwell Aquatics
Appearance: Turbid (cloudy) suspension
Odor: Slightly sweet smell

Manufacturer’s Overview: “Complex system of non-pathogenic aerobic and anaerobic microbes…specifically formulated to establish biological filtration in new aquarium setups, and to enhance the rate of nitrification, de-nitrification, and organic waste degradation in marine and freshwater aquaria through complete nutrient remineralization.”

Recommended Dosage (marine and freshwater aquarium a startup): 5 ml for 25 US gallons (4 drops per gallon daily for 2 weeks.)

BacterGen.M ^TM

Marketed by: Continuum Aquatics
Appearance: Turbid (cloudy) suspension
Odor: Slightly sweet smell

Manufacturer’s Overview: “The addition of… BacterGen.M will cause a rapid increase in the population of beneficial microorganisms with a resulting increase in water clarity and a drop in unbeneficial nutrients, particularly ammonia and nitrogen compounds , as well as phosphates. BacterGen.M contains both aerobic and anaerobic microbes which will generate new populations and greatly increase existing bacterial populations to establish and accelerate nitrification and denitrification, lowering ammonia and nitrites, as well as lowering phosphates, nitrates, and organic pollutants significantly.”

Recommended Dosage (marine aquarium startup) : 5 ml for 25 US gallons (4 drops per gallon daily for 2 weeks.)

Comparison of Lab Results for Brightwell and Continuum Products

Preliminary testing of the two products (pH, conductivity, salinity, ammonia, and nitrate) found them to be remarkably similar. See Figures 1-4.

Figure 1. pH of the two products was remarkably similar.

Figure 2. Salinity of the Brightwell and Continuum products.

Figure 3. Conductivity refers to these solutions” ability to conduct an electrical current.

Figure 4. Ammonia content of the two products.

Figure 5. Nitrate content (as NO3-N) of the two products.

Biochemical Oxygen Demand

Biochemical Oxygen Demand (BOD) is an empirical test used to determine oxygen requirements (consumption) of bacteria in the stabilization of wastes. The test is performed under standardized laboratory conditions, and usually takes 5 days to complete, although this time period may be extended. BOD may be due to stabilization of carbonaceous wastes (where it is called CBOD), nitrogenous wastes (such as ammonia and nitrite), or both.

If the products tested contained living aerobic bacteria, oxygen consumption after their introduction into a sterile hospitable environment containing food would confirm their presence. If these tests are conducted under controlled conditions, comparisons of the effectiveness of the two products can be made. In addition, a proper number of bacteria would be required to properly “seed” this sterile environment. To test for the presence of carbonaceous bacteria, the manufacturers” recommended dosages were made to a sterile environment (BOD bottles containing water treated by reverse osmosis and spiked with necessary nutrients such as iron, calcium, phosphate, magnesium, and so on.) This water also contained a small amount solution of known BOD strength. This target BOD would be 198 ppm after 5 days of incubation at 20C. As Figure 6 shows, both products contained enough bacteria to consume the amount of oxygen required over the 5 days to calculate “proper” BOD.

Figure 6. Time – course oxygen demand of the bacteria and calculated biochemical oxygen demand (BOD) of the two products. Since the initial bottles and dilution water were sterile, this demonstrates that bacteria within the products were of sufficient number to exert a proper oxygen demand during the testing period.

Figure 7 shows the time-course oxygen levels in the experiment’s “sterile” controls and bottles containing the glucose-glutamic acid solution of known carbonaceous BOD strength and inoculated by the products” carbonaceous bacteria. Recall that both products contain ammonia (with the Continuum product having more) as well nitrifying bacteria (that also exert an oxygen demand.)

Figure 6. Biochemical Oxygen Demand of the products, when the bottles were spiked with a solution of known carbonaceous BOD.

Figure 7 shows time-course dissolved oxygen concentrations of the “sterile” controls and the samples inoculated with ammonia and the products” nitrifying bacteria.

Figure 7. Oxygen demand (BOD) of solutions spiked with a known amount of ammonia (with some ammonia added by the content of the products.)

Confirmation of the presence of ammonia oxidizing bacteria is confirmed by a reduced concentration of ammonia in the inoculated samples (with the control showing no reduction in the level of ammonia over the course of the incubation. See Figure 8.

Figure 8. Ammonia concentrations of the control (practically unchanged) and those observed in samples (inoculated with Brightwell and Continuum products) after 16 days. Note that the two products added ammonia, hence true final ammonia concentrations would likely have been lower – perhaps much lower – than seen here.

Results

The goal of these experiments was to confirm the presence of living carbonaceous and nitrogenous bacteria. Both products apparently contain sufficient numbers of these bacteria to inoculate a sterile environment and begin carbonaceous and nitrogenous cycling. Other uses could include possible hastening of cycling when using conventional cycling methods (introduction of live rock, live sand, and/or a couple of hardy “starter” fishes, or inoculating an existing tank after some sort of upset has occurred.

Comments on Dosing These Products

Based on the testing performed during the investigation of these products” performance, and using the manufacturers” recommended dosages, these products do seem to have positive benefits in hastening carbon and aerobic nitrogen cycling. However, neither products warns against possible effects of drastic, accidental over-dosing (a possible scenario includes spilled most or all of a bottle’s contents into a nano-reef containing a small volume of water.) Both products are acidic (pH values of slightly below 4.5 standard pH units) and overdosing in such a scenario would have immediate and negative effects on pH, alkalinity, calcium content and so on resulting in stressful (potentially fatal) consequences for fishes and invertebrates. Ammonia content in the products differed by a few parts per million (depending upon differences in the age of the products (?), since ammonia would “feed” bacteria within the bottle over the course of time) but meaningful (perhaps harmful) amounts of ammonia could be added as well.

I would recommend testing for ammonia and nitrite as usual to confirm proper cycling even when using these products.

Test Protocols

In a perfect world, identical parameters could be maintained in aquaria with identical bioloads and meaningful comparisons could be made. Realistically, this is at best impractical (if not impossible) so alternative experimental protocols had to be established.

Hence, testing consisted of time-course monitoring of oxygen consumption under controlled conditions in freshwater containing known amounts of carbonaceous and nitrogenous compounds and spiked with bacteria from the two products. These samples were tested for Biochemical Oxygen Demand (BOD), pH, ammonia, nitrite, nitrate, and total iron. All glassware were sterilized with a solution of sodium hypochlorite and de-chlorinated with sodium thiosulfate. Rinse water was analyzed for Total Chlorine using the DPD method and confirmed no residual chlorine.

Standard 300-ml BOD bottles were filled with deionized water containing commercially available buffers (Hach “BOD pillows” containing essential compounds such as calcium, iron, phosphorus, etc.) For this reason, phosphorus reduction as claimed by a manufacturer could not be performed.

A glucose/glutamic acid (GGA) solution was prepared. This adds a carbon source that biodegrades slowly (through addition of glutamic acid) while consuming oxygen and allows an estimation of carbon cycling. Use of this solution is a standard quality control method used in the BOD test.

Many of the tests performed during product testing were colorimetric in nature. This sort of testing involves adding reagent(s) to a known volume of sample (usually 10 milliliters, sometimes 25 mL.) After a given reaction time, absorption is measured by a colorimeter (or spectrometer.) Absorption of light is proportional to the concentration of the substance tested for. Colorimetry is subject to a number things that can cause false high or low measurements (called interferences.) Many common interferences are listed by the manufacturer of the colorimeter. However, some interferences are uncommon, or unknown. With that said, testing followed the following protocols, and common interferences with the chemistries are listed.

Ammonia

Ammonia was determined by a Hach DR890 colorimeter using the salicylate method. Monochloramine is formed when ammonia combines with chlorine, and monochloramine reacts with salicylate to form 5-aminosalicylate. This, in turn, is oxidized in the presence of sodium nitroprusside to form a blue-colored compound. This blue color is masked by excess reagent to form a final greenish solution. Iron causes interference. Since iron was added to the BOD dilution water, its presence was analyzed to determine its content. Total iron (Hach FerroVer chemistry) found iron at 0.01 ppm (95% confidence level is 0.02 ppm) hence, for practical purposes, no iron was present.

Conductivity

Conductivity was determined through use of a Corning Model 311 conductivity meter.

pH

pH was determined through use of a Denver Instruments Model UB-5 UltraBasic pH meter calibrated to two points (4 and 7, using freshly prepared buffer solutions.)

Iron (Total)

Total iron was determined by a Hach DR2000 spectrometer using their FerroVer (1, 10 phenanthroline) chemistry. Minimum detection limit (MDL) is 0.02 ppm. Iron can interfere with the nitrate test.

Nitrate

Nitrate was determined by a Hach DR890 colorimeter using the cadmium reduction method. Cadmium reduces nitrate within the sample to nitrite, which reacts in an acidic environment with sufanilic acid. An intermediate diazonium salt couples with gentistic acid to form an amber colored solution which is proportional to nitrate content. Chloride levels exceeding 100 ppm will cause low results (chloride was determined to be less than 500 ppm in the Continuum product.) No correction for chloride concentration was attempted. Presence of ferric iron causes high results and therefore must be absent (total iron of the samples was found to be absent in straight samples of the two products. In the control (with buffers known to add ferric iron) total iron was reported as 0.01 ppm. The Minimum Detection Limit (95% confidence level) for this chemistry is 0.02 ppm.

Nitrite

Nitrite was determined by a Hach DR890 colorimeter using the diazotization method. Nitrite reacts with sulfanilic acid to form an intermediate diazonium salt. When coupled to chromotropic acid, a pink complex forms, which is proportional to the nitrite content. Nitrite results were at or below the MDL (minimum detection limit, which for this chemistry is 0.002 ppm.)

Biochemical Oxygen Demand/Dissolved Oxygen Testing

Oxygen content was determined through use of a Hach HQ40d dissolved oxygen meter and self-stirring BOD probe (luminescent technology). This device allows measurements to 1/100 ^th of a milligram dissolved oxygen. Samples were stored in a darkened incubator with a temperature of 20C (+/-1 C) until the oxygen content fell to about 2 ppm (ammonia samples) and 1 ppm (glucose-glutamic acid samples.) Controls : Three controls were set up. One was DI water only (except for added Hach buffers.) A second control contained DI water spiked with 6 ml of the GGA solution. None of the controls were spiked with bacteria.

Chloride

Chloride content was determined to estimate its impact on nitrate analyses (high chloride results in reports of low nitrate.) A 50 milliliter sample of the Continuum product was tested for chloride using the Silver Nitrate titration method. An endpoint could not be established, so 10 milliliters of the product was diluted by 90 milliliters of DI water. A clean endpoint was apparent and it was determined the product contains about 560 ppm chloride. Method Summary: Silver nitrate reacts with chloride to produce insoluble silver chloride. Continued addition of silver reacts with potassium chromate (previously added to the sample) to form a red-brown silver chromate. The appearance of this red-brown coloration marks the endpoint of titration. Adjustment of sample pH was not necessary. It is assumed interferences (such as iodide, bromide, sulfide, and sulfite) were not present. The Brightwell product was not tested for chloride. See Figure 10.

Figure 9. Chloride content of the Continuum product.

Dosing Protocols

Brightwell: One drop of the Brightwell product (diluted in DI water to meet the dosing requirements established by the manufacturer) was added daily to 300 ml BOD bottles, containing a) 6 ml GGA solution, and b) DI water spiked with ammonium hydroxide to a concentration of 0.4 mg/l. A commercially available nutrient buffer (containing calcium chloride, magnesium sulfate, potassium phosphate (monobasic and bibasic), sodium phosphate (dibasic) and ferric chloride) was added at recommended concentrations to the deionized water in order to create a matrix suitable for bacterial growth.

Continuum: One drop of the Continuum product (diluted in DI water to meet the dosing requirements established by the manufacturer) was added to 300 ml BOD bottles, containing a) 6 ml GGA solution, and b) the ammonium hydroxide solution with a NH ₃ ; concentration of 0.4 mg/l. A commercially available nutrient buffer (containing calcium chloride, magnesium sulfate, potassium phosphate (monobasic and bibasic), sodium phosphate (dibasic) and ferric chloride) was added at recommended concentrations to the deionized water in order to create a matrix suitable for bacterial growth.

Since the Brightwell and Continuum products are acidic, pH was determined of the water before and after the addition of the products. The pH was not significantly affected. See Figure 9.

Figure 10. In the recommended dosages, these products, although acidic, will not significantly affect water pH.

Reference

1. Bhaskar, K.V., and P.B.B.N. Charyulu, 2005. Effect of environmental factors on nitrifying bacteria isolated from the rhizosphere of Setaria italica (L.) Beauv. African J. Biotech., 4:1145-1146.