Evaluation of Commercial Products for the Removal of Nitrogenous Waste Products from Water


David V. Straub and Beverly A. Dixon

Introduction

The beneficial role of nitrifying bacteria within saltwater and freshwater aquaria environments is their function in the nitrogen cycle, which involves the conversion of ammonia to nitrite and the subsequent conversion of nitrite to nitrate (Blasiola, 1991). During the process of nitrification, nitrifying bacteria detoxify ammonia in two steps. First, Nitrosomonas spp. converts toxic ammonia to nitrite, which is also toxic to fish. Then in the second step, nitrite is converted to nitrate by the Nitrobacter spp. (Blasiola, 1991) . Nitrate is not considered toxic unless it accumulates in high concentrations. There are numerous commercial products claiming to contain nitrifying bacteria that will facilitate the conversion of toxic ammonia to non-toxic nitrate within aquaria. This is leading to a general confusion amongst aquarists about nitrifying bacteria, due in part to misinformation presented in the advertisements of many commercial products, and the aquarist’s general lack of knowledge about bacteria.

This study was undertaken to investigate the efficacy of commercially available products used to commence and enhance nitrification. Efficacy was determined by measuring the conversion of ammonia to nitrate within an optimal in vitro environment. Furthermore these products were compared to one another to determine, which contained the most efficient source of Nitrobacter spp. and Nitrosomonas spp. Studies in our laboratory, have indicated that not all commercial sources of nitrifying bacteria perform at the same level of efficacy. It has been shown in comparison tests between different brand products, that Fritz-Zyme #7 and #9 (Fritz Industries Inc., Dallas TX) out performs other commercial products.

Materials and Methods

Numerous freshwater and saltwater commercial sources of nitrifying bacteria were used in the study. Freshwater cultures of nitrifying bacteria were grown on Nitrosomonas medium ATCC 221, while saltwater cultures were grown on Nitrosococcus medium ATCC 982. Both types of cultures were placed in shaker baths and maintained at 25oC. Sterile calcium carbonate chips were added to the media to provide growing surfaces for the nitrifying bacteria. Control and experimental cultures containing labeled specified amounts of the various commercial nitrifying products were tested. The chemical parameters of ammonia, nitrite, nitrate and pH were measured every two days for two weeks, to determine the progression of the nitrogen cycle. Parameters were determined by spectrophotometric analysis using the Hach DR/2000. Ammonia was measured using the Nessler method, nitrite using the ferrous sulfate method, and nitrate using the cadmium reduction method, respectively.

Results

In the comparison studies for commercial freshwater sources of nitrifying bacteria, it was found that the Fritz-Zyme #7 out performed all other products in both the areas of conversion of ammonia to nitrite and the conversion of nitrite to non-toxic nitrate. The Fritz-Zyme #7 in each test run converted ammonia to nitrate at a faster rate, showing nitrite/nitrate production and ammonia depletion 4-5 days earlier than all other products. The remaining freshwater commercial sources other than Fritz-Zyme #7, moved at varying rates through the process of converting ammonia to nitrate. Some of these products failed to show any movement at all while others caused a reversal of the nitrification process causing the nitrate which that had produced to be converted back to ammonia.

The comparison studies for saltwater commercial sources of nitrifying bacteria, showed an even more dramatic difference between products. The only commercial source among the saltwater products to produce a conversion of ammonia to nitrate was Fritz-Zyme #9.

Conclusion

Ammonia is constantly being produced in aquaria environments, small amounts can be traced to the diffusion of blood across the gill membranes of fish, but the majority of ammonia is produced through the process of mineralization which involves the conversion of waste products in the aquarium to ammonia by heterotrophic bacteria. Therefore products that are capable of converting toxic ammonia to non-toxic nitrate are an essential part of an aquaria environment.

In the comparison testing performed in the laboratory it was found that the Fritz-Zyme #7 and #9 products performed faster and at superior levels to all other products. These high levels are due to the use of pure cultures of live Nitrosomonas and Nitrobacter bacteria. The other products used in the experiment used combinations of Nitrosomonas and Nitrobacter bacteria at lower levels in conjunction with heterotrophic bacteria, while some of the products tested contained heterotrophic bacteria alone.

The use of heterotrophic bacteria to supplement or replace true nitrifying bacteria belonging to the family Nitrobacteraceae is not an uncommon practice, because heterotrophic bacteria are able to tolerate a wider range of environmental conditions.

The use of heterotrophic bacteria in place of true nitrifiers leads to a number of basic problems. For example, heterotrophs under certain environmental conditions such as low pH and oxygen levels, can operate in reverse directions, converting nitrates back to nitrite, ammonia, and nitrogen gas. This process of denitrification was seen in the comparison testing performed in the lab, and occurred in both the experimental and control flask for that product.

Many of the heterotrophic bacteria used in these products are also capable of forming spores allowing the product to be dried, packaged and sold as a viable culture. Products containing Nitrosomonas or Nitrobacter spp. cannot undergo this drying process because it would destroy the bacteria, however in liquid formulations the nitrifiers can survive as viable inactive cells which can be reactivated during periods of mineralization (Verhagen, et al, 1992). It was also found in our study that dry products performed at some of the lowest levels in the conversion of ammonia to nitrate when placed in an optimal in vitro environment.

In the products that mix heterotrophic bacteria with bacteria from the Nitrobacteraceae family, there is often seen the problem of competition for ammonia between the heterotrophic bacteria and the true nitrifiers. An example of this was seen when Nitrosomonas europea and Nitrobacter winigraosky were grown in a continuously percolated column with heterotrophic bacteria. The results showed that the nitrifying bacteria were less competitive than the heterotrophs (Verhagen et al., 1992). This fact is further supported by our results which show that the only pure nitrifier culture in the experiment far out performed those products containing a mix culture.

The most dramatic difference in the product testing came in the comparison of saltwater products for the removal of ammonia through nitrification. In this section of our testing only one product produced results, all other products failed to convert the ammonia. Most of the products tested using the saltwater formula claimed to be capable of working in both saltwater and freshwater, however marine species of Nitrobacter and Nitrosomonas differ from those that live in freshwater. Therefore it is believed that these products actually contained only the freshwater species, since they were able to convert ammonia to nitrate in the freshwater testing but failed to produce results when placed in a saltwater environment.


Footnote

David V. Straub and Beverly A. Dixon, Department of Biological Sciences, California State University, Hayward, CA 94542


References

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Verhagen, F. J. M., H. Duyts and H. J. Laanbroek. 1993. Competition for ammonium between nitrifying and heterotrophic bacteria in contiguously percolated soil columns. Appl. Environ. Microbiol. 58: p3303 (9).

Verhagen, F. J. M., H. Duyts and H. J. Laanbroek. 1993. Effects of grazing by flagellates on competition for ammonium between nitrifying and heterotrophic bacteria in soil columns. Appl. Environ. Microbiol. 59: p2099 (8).


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