Better Biological Nutrient Removal Through Bioscience

Biological Nutrient Removal (BNR) is critical for the preservation of our lakes and streams. Large amounts of waste products are passed through our sewage treatment systems on a continuous basis. Nutrient loads discharged from these systems must be reduced to avoid eutrophication of the receiving waters. Microbes are particularly useful in biodegrading most of the compounds treated by wastewater treatment plants, in particular carbonaceous, nitrogenous, and phosphoric compounds. They do this in nature on a continuous basis in both high and low nutrient environments.

Bacteria dominate in an ecosystem as primary decomposers because they can utilize dissolved organic substrates at low concentrations, assimilate dissolved inorganic nutrients such as nitrate and phosphorus, and at the same time decompose nutrient-poor plant tissue (Fenchel and Jorgensen, 1977). Many bacteria, particularly Bacillus, produce enzymes that are excreted into the local aqueous environment and hydrolyze the solid substrates so that the carbon-based compounds can be transported into the cell as smaller solubilized molecules for use in assimilation or energy production (Priest, 1977). When the bacteria degrade carbonaceous compounds, they utilize essential nutrients such as nitrogen and phosphorus, and do so at different rates. (This is reflected in the often used ratio of C:N:P as 146:16:3).

There are many different types of bacteria and each is particularly suited for the environmental niche in which it is found. Heterotrophic bacteria are present in high numbers within domestic wastewater and include both gram-positive and gram-negative bacteria. The population densities of heterotrophic bacteria in domestic wastewater have been found to be as high as 1011 CFU/g dry weight (Roth, 1994). Many of these heterotrophic bacteria are capable of degrading polymers, lipids, complex carbohydrates, and proteins, and also convert ammonia and phosphate to useable forms. However, under conditions without In-Pipe microbial treatment (non-IPT), the primary source of these heterotrophs is human fecal matter. The bacteria found in fecal matter have become acclimated to living within the intestine and are primarily anaerobic. These bacteria metabolize organic material but they have already degraded most of the wastewater constituents that they are capable of degrading before entering the collection system. Thus, the fecal bacteria are not very efficient treating wastewater.


Gottschalk (1986) indicates that nitrogen comprises about 10% of the dry weight of bacteria, thus it is required in large quantities for microbial growth. In comparison, the dry weight of a bacterial cell is about 50% carbon, 20% oxygen, 9% hydrogen, and 2% phosphorus (Metcalf and Eddy, 2003). Ammonia is the preferred source of nitrogen for bacteria. Practically all organisms can and do utilize ammonia through assimilatory nitrate reduction. Nitrate is also taken up and utilized by many organisms but not all. Before nitrate can be incorporated into cellular material, it must be reduced to ammonia. Nitrite is the product of nitrate-nitrite respiration and of the metabolic activities of bacteria such as Nitrosomonas. Nitrite is used by a particularly interesting group of bacteria with potential process value, the anaerobic ammonia oxidizing bacteria (Anammox), as the primary electron acceptor (Strous and Jetten, 2004).

Nitrogen metabolism in bacteria is very diverse among the many species. Three mechanisms of particular interest to wastewater treatment are heterotrophic nitrification, aerobic denitrification, and Anammox (Richardson and Watmouth, 1999).

Heterotrophic Nitrification

Nitrification is defined as the oxidation of ammonia to nitrate and is an aerobic process. Typically, nitrification is a two-step process and attention has historically concentrated on two groups of chemoautotrophic bacteria, the Nitrosomonas, which oxidize ammonia to nitrite and the Nitrobacter which oxidize nitrite to nitrate. Both Nitrosomonas and Nitrobacter are chemolithotrophs and use the energy derived from nitrification to assimilate CO2. The rate of nitrification is greatly affected by temperature when Nitrosomonas and Nitrobacter are involved. Autotrophic nitrifying bacteria are notoriously slow growing at the low temperatures typical of domestic wastewater, and often result in performance limitations regarding ammonia and nitrogen in cold water.

Often ignored by those unfamiliar with the metabolic capabilities of bacteria is the fact that many heterotrophic bacteria are able to oxidize nitrogen compounds. Not only do these organisms oxidize ammonium, hydroxylamine, and nitrite but also a number of other organic nitrogenous substances. Identified products of heterotrophic nitrification include nitrate and nitrite, hydroxamic acids, oximes, amine oxides, nitroso and nitro compounds (Verstraete and Alexander, 1972). For example, heterotrophic nitrification by Arthrobacter results in high levels of hydroxylamine and nitrite (Verstraete and Alexander, 1972). The nitrification pathway is significantly different between chemoautotrophic and heterotrophic nitrification. Heterotrophic nitrification has not been found to be inhibited by typical nitrification inhibitors used in conventional BOD testing (Roberson et al, 1989). One of the most active heterotrophic nitrifiers in the literature is an Alcaligenes isolated from soil (Castignetti and Hollocher, 1982). This particular organism was observed to yield large amounts of nitrite when cultured with pyruvic oxime as the sole source of carbon and nitrogen. The bacterium could also oxidize hydroxylamine to nitrite and synthesize the entire complement of denitrifying enzymes under appropriate conditions. As illustrated by these examples, the nitrogen cycle is more complex than indicated in many textbooks. When one considers the large population of heterotrophs present in wastewater, one cannot ignore their impact on the nitrification-denitrification cycle. They might not all contain fully functional nitrification pathways but by working together they can have a significant effect reducing the levels of nitrogen entering a treatment facility.

Aerobic Denitrification

Denitrification is the reduction of nitrate to nitric oxide, nitrous oxide and nitrogen gas. A wide range of bacteria have been shown to be capable of denitrification. Because of the role denitrification plays in generating anaerobic energy it has been assumed that it was only an anaerobic process. Over the last decade it has become clear that significant numbers of diverse bacterial species can denitrify under aerobic conditions (Richardson et al, 1998).

The first step, nitrate reduction, is catalyzed by two enzymes, membrane-bound nitrate reductase (NAR) and periplasmic nitrate reductase (NAP). The membrane-bound NAR is the most important enzyme under anoxic conditions while the periplasmic NAP is more important under oxic conditions (Richardson et al, 1998). Like aerobic respiration, denitrification effects the complete oxidation of the organic substrate to CO2. Heterotrophic bacteria which are capable of at least partial denitrification include the following genera: Achromobacter, Acinetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Bacillus, Chromobacterium, Corynebacterium, Flavobacterium, Hypomicrobium, Moraxella, Neisseria, Paracoccus, Propionibacterium, Pseudomonas, Rhizobium, Rhodopseudomonas, Spirillium, and Vibrio (Tchobanoglous et al., 2003; Drysdale et al., 2001). Most of these bacteria are present in low quantities in raw domestic wastewater. A study performed on an NDBEPR activated sludge system revealed that ordinary heterotrophic organisms interactively contribute to the denitrification occurring in the system (Drysdale et al., 2000). However, there was a large fraction of ordinary heterotrophic organisms that were incapable of any nitrate or nitrite production.


Phosphorus is another compound that can be removed from wastewater through microbiological activity. Municipal wastewaters may contain from 4 to 16 mg/L as P. The usual forms of phosphorus that are found in aqueous solutions include orthophosphate, polyphosphate, and organic phosphate. Orthophosphates (PO4 , HPO4 , H2PO4 , and H3PO4 for example) are readily available for biological metabolism. Polyphosphates undergo hydrolysis in aqueous solutions to orthophosphate forms, but this is a slow process.

Organically bound phosphate is usually of minor importance in most domestic wastewater but it can be an important component of industrial wastewaters and wastewater sludges, due to the high concentrations of phosphorous in chemicals and wasted biomass. The biological removal of phosphorus from wastewater is accomplished by incorporation into the cellular biomass which is subsequently removed from the process through sludge wasting. Some bacteria have the capability to accumulate excess amounts of phosphorus as polyphosphates within their cells. These bacteria are called phosphorus accumulating organisms (PAO). In an EBPR plant, the PAOs are encouraged to grow and incorporate phosphorus through a specific setup involving an anaerobic stage followed by an aerobic stage. Under anaerobic conditions, PAOs will assimilate fermentation electron acceptor (Strous and Jetten, 2004).

Nitrogen metabolism in bacteria is very diverse among the many species. Three mechanisms of particular interest to wastewater treatment are heterotrophic nitrification, aerobic denitrification, and Anammox (Richardson and Watmouth, 1999).


See More

Andrews JH, Harris RF. 1977. r- and K-Selection and Microbial Ecology. Adv Microbial Ecol. 9:99-147.

Atlas, RM and Bartha R. 1987. Microbial Ecology: Fundamentals and Applications.

Benjamin/Cummings Publishing Co. Menlo Park CA USA.

Castignetti, D. and Hollocher T. 1982. Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil. Appl Environ Microbiol. 44(4): 923- 928.

Fenchel TM and Jorgensen BB. 1977. Detritus Food Chains of Aquatic Ecosystems: The Role of Bacteria. Adv Microbiol Ecol. 1:1-58.

Gottschalk, G. 1986. Bacterial Metabolism, 2nd edition. Springer-Verlag, Inc. NY.

Gray TR, Parkinson D (editors). 1968. The Ecology of Soil Bacteria. University of Toronto Press, Canada. 681 pages.

Kuenen JG and Gottschal JC. 1982. Competition among Chemolithotrophs and Methylotrophs and their interactions with Heterotrophic Bacteria in Microbial Interactions and Communities Volume 1, pp 153-186.

La Riviera JWM. 1977. Microbial Ecology of Liquid Waste Treatment. Adv Microbiol Ecol. 1:215-259.

Priest, F. 1977. Extracellular Enzyme Synthesis in the Genus Bacillus. Bacteriol Rev. 41(3): 711-753.

Richardson DJ, Watmough NJ. 1999. Inorganic Nitrogen Metabolism in Bacteria. Curr Opin Chem Biol. 3:207-219.

Richardson DJ, Wehrfritz JM, Keech A, Crossman LC, Roldan MD, Sears HJ, Butler CS, Reilly A, Moir JW, Berks BC. 1998. The diversity of redox proteins involved in bacterial heterotrophic nitrification and aerobic denitrification. Biochem Soc Trans. 26:401-408.

Robertson LA, Cornelisse R, Zeng R, Kuenen JG. 1989. The effect of thiosulfate and other inhibitors of autotrophic nitrification on heterotrophic nitrifiers. Antonie van Leeuwen. 56:301-309.

Roth D. and Lemmer H. 1994. Biofilms in Sewer Systems-

Characterization of the bacterial biocenosis and its metabolic activity. Wat. Sci. Tech. 29(7): 385-388.

Seviour RJ, Mino T, Onuki M. 2003. The Microbiology of Biological Phosphorus Removal in Activated Sludge Systems. FEMS Microbiol Rev. 27: 99-127.

Strous M and Jetten MSM. 2004. Anaerobic Oxidation of Methane and Ammonium. Annu. Rev. Microbiol. 58: 99-117.

Tchobanoglous G, Burton FL, Stensel HD. 2003. Wastewater Engineering: Treatment and Reuse/Metcalf & Eddy 4th edition. Tata McGraw-Hill Publishing Company Limited. New York USA.

Verstraete W and Alexander M. 1972. Heterotrophic Nitrification by Arthrobacter sp. J Bacteriol. 110(3): 955-961.

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