Search

PNS Bacteria: Denitrification Powerhouses

Every aquarist possesses a baseline knowledge of nitrification. This is the oxidization of ammonia into nitrate by certain aerobic bacteria and archaea. Those metabolic processes are summarized here:


NH3 + 1.5 O2 NO2- + H+ + H20

NO2- + ½ O2 NO3


Elevated nitrate concentrations are the inevitable result of nitrification. Chronically-high nitrate levels can induce unpleasant algae blooms as well as stress fish and invertebrates that are adapted to oligotrophic (i.e. nutrient-poor) ecosystems such as shallow tropical coral reefs. Conventional methods for removing nitrate include water changes and chemical treatments, both of which are generally costly and laborious.


Natural methods of nitrate removal are comparatively inexpensive and simple. One particularly effective tool is denitrification. Denitrification is an anaerobic process which involves the reduction of nitrite, nitrate, nitric oxide and nitrous oxide into dinitrogen (N2) gas. This requires a series of interdependent reactions which are enzymatically specific to individual species/strains. The generalized equation for the denitrification of nitrate is:


2 NO3− + 10 e− + 12 H+ → N2 + 6 H2O


There is mounting evidence that denitrification was one of the earliest metabolic strategies that has emerged on this planet and that it has been the subject of numerous vertical and horizontal genetic adaptations/refinements throughout evolutionary history. As a result, an assortment of marine and freshwater microbes species conduct denitrification in order to derive energy in areas devoid of oxygen and in excess of nitrogenous compounds. Through this respiratory strategy, denitrifying species conquer and cleanse anoxic sediments and the inner crevices of rockwork. Through their metabolic activity they play a silent, but invaluable, role in driving the world’s carbon, nitrogen, sulfur and phosphorus cycles.

The enzymes behind these reactions have been classically generalized as “nitrite reductase,” “nitrate reductase” and “nitrous oxide reductase,” but in reality, there is a huge swath of enzymatic diversity associated with the molecular infrastructure behind denitrification. To overgeneralize, nitrate reductases act to facilitate the conversion of nitrate to nitrite, whereas nitrite reductases act to convert nitrite to nitric oxide, nitrous oxide or directly to dinitrogen gas. The reduction of nitrous oxide to dinitrogen is coupled with ATP production, allowing the organisms to derive metabolic energy by using nitrogenous compounds as final electron acceptors.


The efficiency of each of these enzymes are contextualized by environmental and nutritional factors such as carbon availability and illumination. Denitrification can be conducted either heterotrophically (where an organic carbon source is required) or autotrophically (where inorganic carbon may be utilized). The molecular infrastructure used by denitrifiers to process nitrogenous compounds also play a role in their ability to utilize sulfides and orthophosphates. But as a whole, enzymes associated with denitrification are repressed by free oxygen (O2). As these enzymes cease to function even under seemingly minute oxygen concentrations, denitrification is largely an anaerobic process.


Through denitrification, nitrogenous “waste” is converted into nitrogen gas (N2). This N2 gas is harmless and escapes into the air. This reduces the need for water changes (at least those carried out specifically for the purpose of reducing nitrate concentration). Thus, it is essential for conserving water use and reducing the overall environmental impact of nutrient accumulation. Whether it’s stabilizing low-nutrient levels in a reef tank or managing the copious waste of a commercial shrimp pond, denitrification is an often overlooked albeit valuable biochemical tool.


Limitations of a conventional denitrifying biofilter

Denitrifying bioreactors are an attractive solution to persistent issues in aquaculture and domestic wastewater management. Industrial denitrifying bioreactors have been designed along with specialized strains of denitrifying bacteria. However, these conventional species and designs share collective attributes which limit both their scale and their application in the home aquarium. The most predominant of these limitations is the abject vulnerability of most conventional denitrifying microbes to oxygen. Many obligately anaerobic non-photosynthetic denitrifying species will perish when exposed to O2. This means that such denitrifiers must exist in zones where limited water exchange occurs and thus participate little in the “active water” of an aquarium.

Packed-bed reactors have also been traditionally deployed to promote denitrification in recirculation aquaculture systems. But these require constant input of organic carbon and are still vitally sensitive to oxygen contamination and are prone to systemic biofouling issues. If O2 is exposed to these conventional reactors, it could induce complete culture collapse and require long re-establishment periods. Because of these reasons, conventional anaerobic denitrifying bioreactors traditionally are enclosed, single pass and are designed to process wastewater that is not part of an aerobic production/display system.


Advancements in denitrifying filter designs include the development of hybrid-systems, such as fluidized bed reactors, which cater to the metabolism of aerobic and anaerobic, photosynthetic and nonphotosynthetic microorganisms. In such reactors, both aerobic and anaerobic microzones are created and rapidly ‘shuffled’ in between areas of low to mild illumination. Such hybridized reactors are more applicable for use in the home aquaria and have demonstrated potentially greater overall nutrient-consumption capability than conventional anaerobic designs. But more importantly, they are more resistant to oxygen contamination. However, conventional denitrifying species are ill-suited for such reactors--rather biofilter species of multi-faceted metabolism is required. Species which will not be shocked by the depletion of oxygen, but instead, use it as an opportunity to consume nitrate and convert it back to harmless N2 gas.


Purple nonsulfur bacteria (PNSB) such as Rhodopseudomonas palustris are ideal denitrifying organisms. They may be used in many types of denitrifying filters, but can also survive and indeed perform denitrification in rather unspecialized environments such as live rock, sand beds, loose filter sponges, etc.

Rhodopseudomonas palustris: An ideal denitrifier

Live PNSB products (such PNS ProBio) are applicable for all forms of aquaculture, from reef tanks to tilapia ponds, because they are so proficient at denitrification. Rhodopseudomonas palustris has a Swiss army knife metabolism, allowing it to conduct aerobic nitrification alongside conventional biofilter species. But whereas those species will promptly die in areas devoid of oxygen, PNSB will still thrive. In anaerobic conditions, R. palustris activates genes responsible for denitrification and subsequently metabolizes nitrates. R. palustris can conduct denitrification autotrophically, utilizing light and inorganic carbon, but can also utilize organic carbon and denitrify in the dark as well. In the absence of carbon, PNSB can utilize excess sulfides, hydrogen, manganese and/or iron as electron donors. This metabolic versatility allows them to colonize deep inside the rockwork of reefs or the bottom of sediments and essentially convert those places into areas of efficient anaerobic biofiltration.

R. palustris is extraordinarily versatile in its ability to utilize a diverse array of organic and inorganic carbon sources. Kim et al 2000 demonstrated greater conversion of waste carbon to fatty acids (stearic & oleic acid) by R. palustris than yeast or Chlorella grown under the same conditions. Its powerful metabolic abilities allow it to grow exponentially on waste carbon and nitrate and to convert these back into microbial protein and fats. Because of their extreme metabolic versatility, PNSB are an ideal denitrifying seed species for hybridized biofiltration designs and for colonizing the many diverse microhabitats of a reef aquarium.


Things that can be done to encourage denitrification


1. Seed with known denitrifying bacterial strains

For sure, various types of denitrifying microbes inadvertently get introduced into most aquarium systems. Nevertheless, directly seeding with highly desirable species can greatly promote denitrification. Because they hungrily strip waste products out of the aquarium, PNSB deprive opportunistic bacteria and pest algae the fuel they need to spread. Therefore, the earlier PNSB are inoculated into a system, the greatest effect they can have preventing such issues. Unlike obligately anaerobic denitrifying species, PNSB may be seeded into fluidized bed reactors and other hybridized semi-aerated biofiltration systems.


2. Have complex biomedia with anaerobic zones

Complex biomedia are paramount to facilitating effective denitrification. “Healthy” anaerobic zones occur in the underlayers of sediments and (to a lesser degree) deep in the pores and crevices of live rock. Such areas have the key combination of seclusion so as to limit oxygenation (which is consumed by aerobes associated with the upper/outer layer), but at the same time, receive nutrients via a constant, minute flow from the surrounding water. These anaerobic zones are rich in CO2 and have a lower pH compared to the water column. Kim et al 1999 reported maximum denitrification activity in R. palustris at a pH of 5.3!


3. Illuminate the anaerobic zones

R. palustris is a powerful denitrifier because it can photosynthesize in the absence of oxygen. Kim et al 1999 demonstrated that this species exhibits greatest denitrifying potential while illuminated. This particular experiment reported maximum denitrification activity at 88ppfd/umol/s/m2; however, some ambiguity remains as to which specific spectrum and photoperiod of light best facilitates the denitrifying potential of the species. But with this in mind, illumination of secluded anaerobic zones may be the inspiration of future novel biofilter designs.

Conclusion

Denitrification is an integral part of Nature’s nitrogen, phosphorus and sulfur cycles. Robust denitrifying microbial communities are likely responsible for the stability of more than one well-aged aquarium. The denitrifying power of PNSB is now recognized for their potential to reduce nitrate, reduce waste carbon, modulate phosphates/sulfides and overall reduce the need for water changes. Because of their multifaceted metabolism, PNSB can utilize an enormous array of carbon/electron sources and can adapt to changing levels of oxygen and illumination. This makes them more robust and more adaptable than conventional non-photosynthetic denitrifying species. R. palustris is a powerhouse of denitrification--aggressively modulating wastes on anaerobic and aerobic fronts, all while benefiting the overall stability, appearance and nutrition of the aquatic system it occupies.

Literature Consulted

Debarbadillo, C., Rectanus, R., Canham, R., & Schauer, P. (2006). Tertiary denitrification and very low phosphorus limits: a practical look at phosphorus limitations on denitrification filters. Proceedings of the Water Environment Federation, 2006(9), 3454-3465.

Ferguson, S. J. (1994). Denitrification and its control. Antonie van Leeuwenhoek, 66(1), 89-110.

Gibson, J., Dispensa, M., Fogg, G. C., Evans, D. T., & Harwood, C. S. (1994). 4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation. Journal of Bacteriology, 176(3), 634-641.

Khanna, P., Rajkumar, B., & Jothikumar, N. (1992). Anoxygenic degradation of aromatic substances by Rhodopseudomonas palustris. Current microbiology, 25(2), 63-67.

Kim, J. K., Lee, B. K., Kim, S. H., & Moon, J. H. (1999). Characterization of denitrifying photosynthetic bacteria isolated from photosynthetic sludge. Aquacultural engineering, 19(3), 179-193.

Kim, J. K., & Lee, B. K. (2000). Mass production of Rhodopseudomonas palustris as diet for aquaculture. Aquacultural engineering, 23(4), 281-293.

Kundu, B., & Nicholas, D. J. D. (1985). Denitrification in Rhodopseudomonas sphaeroides f. denitrificans. Archives of Microbiology, 141(1), 57-62.

Hamlin, H. J., Michaels, J. T., Beaulaton, C. M., Graham, W. F., Dutt, W., Steinbach, P., ... & Main, K. L. (2008). Comparing denitrification rates and carbon sources in commercial scale upflow denitrification biological filters in aquaculture. Aquacultural engineering, 38(2), 79-92.

Han, D. W., Yun, H. J., & Kim, D. J. (2001). Autotrophic nitrification and denitrification characteristics of an upflow biological aerated filter. Journal of Chemical Technology & Biotechnology: International Research in Process, Environmental & Clean Technology, 76(11), 1112-1116.

Harwood, C. S., & Gibson, J. (1988). Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris. Applied and Environmental Microbiology, 54(3), 712-717.

Hochstein, L. I., & Tomlinson, G. A. (1988). The enzymes associated with denitrification. Annual Reviews in Microbiology, 42(1), 231-261.

Jones, C. M., Stres, B., Rosenquist, M., & Hallin, S. (2008). Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification. Molecular biology and evolution, 25(9), 1955-1966.

Song, B., & Ward, B. B. (2005). Genetic diversity of benzoyl coenzyme A reductase genes detected in denitrifying isolates and estuarine sediment communities. Applied and environmental microbiology, 71(4), 2036-2045.

Van Rijn, J., Tal, Y., & Schreier, H. J. (2006). Denitrification in recirculating systems: theory and applications. Aquacultural engineering, 34(3), 364-376.

Zhao, Y., Zhao, C. G., Chen, Y., & Yang, S. P. (2012). Denitrifying characterizations from inorganic nitrogen-polluted wastewater by Rhodopseudomonas palustris CQV97. Journal of Shanxi University (Natural Science Edition), 3.