Bug of the Month: Nitrosomonas and Nitrification in WWTPs

In this wastewater microbiology spotlight, learn about Nitrosomonas and how they function within wastewater treatment plant processes

Bug of the Month: Nitrosomonas and Nitrification in WWTPs

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Typically considered the predominant genus responsible for the first step of nitrification in wastewater treatment plants, the Nitrosomonas genus is recognized by the Microbial Database for Activated Sludge (MiDAS) field guide as having 20 individual species.

These species include Nitrosomonas oligotrophaNitrosomonas europaeaNitrosomonas communisNitrosomonas eutropha, as well as other currently unnamed species. Also, based on 16SrRNA reads, it is suspected that an unclassified read in the same family as Nitrosomonas (Aster Bio Tax ID #206379) is also fairly common in many systems.

In the majority of wastewater treatment plants Nitrosomonas appears to be the predominant genus responsible for the first step of converting ammonia to nitrite. Bacteria capable of oxidizing ammonia to nitrite are often referred to as ammonia oxidizing bacteria (AOB).

Nitrosomonas can be readily viewed at 1000x phase contrast microscopy or 1000x differential interference contrast (DIC) magnification and are recognized by their honeycomb-like appearance. Typically Nitrosomonas are viewed on the edges of flocs where the most dissolved oxygen tends to be present.

Based on 16SrRNA sequencing, in our experience most successful nitrifying municipal plants tend to have Nitrosomonas reads between 0.5-1.5% of the total sequencing reads. In industrial treatment plants — depending on factors such as incoming BOD:N ratio, systems that have a high nitrogen surplus have obtained reads as high as 5-10% Nitrosomonas in certain instances.

Other roles in treatment

A unique feature of Nitrosomonas is that they have other potential roles in wasetwater treatment processes other than strictly nitrification. Nitrosomonas may grow mixotrophically with organic substrates, meaning they can take up organic compounds in addition to inorganic compounds (alkalinity).

Under mixotrophic conditions, members of the genus can also nitrify and denitrify simultaneously. In instances in which Nitrosomonas may denitrify molecular hydrogen or other organic compounds, they can act as electron donors with nitrite as the electron acceptor.

Nitrite lock

Both AOB and nitrate oxidizing bacteria (NOB) have the potential to be inhibited due to a wide range of potential conditions. In general, NOB tend to be more sensitive to various stresses. In instances where NOB are inhibited and AOB function normally, a condition commonly referenced as nitrite lock may apply. During nitrite lock, nitrite concentrations in the filtered mixed liquor or effluent are elevated (under normal circumstances, the presence of any significant nitrite concentrations is rare). Nitrite lock can be detrimental for plants that have an effluent total nitrogen limit and also can cause issues with disinfection due to the high chlorine demand required to oxidize nitrite and obtain a free chlorine residual.

While less sensitive than NOB, AOB can also be inhibited under a wide range of conditions. These conditions include low or rapidly changing temperatures; lack of alkalinity (we typically recommend a 125 mg/L alkalinity residual in the filtered mixed liquor at all times); low pH values (under 7); inadequate sludge retention time, usually less than 4-5 days; and the presence of inhibitory compounds such as peracetic acid, quatenarmy ammonia compounds, and a wide range of others at a threshold in which inhibition or toxicity may occur.

Resolving nitrification issues

Addressing issues in which nitrification is not occurring may be challenging. The first steps are ensuring that the proper environment for nitrifying bacteria exist within the system. Through microscopy, if Nitrosomonas are viewed within the flocs and ammonia concentrations are still high, it is likely that adequate nitrifying populations may be present but inhibited to function as AOB. In these instances, high fluctuations of effluent ammonia concentrations (sometimes as often as on a daily basis) may be encountered.

Based on my experience, it is also somewhat remarkable how long Nitrosomonas populations may remain present in mixed liquor samples with prolonged absence of nitrification. The theory for this phenomenon is likely due to the ability of Nitrosomonas to have the dual ability to behave as a heterotroph using carbonaceous material for growth.

Nitirifcation testing studies can be simulated at bench scale for troubleshooting. Bioaugmentation of nitrifying bacteria have been noted to help speed up recovery of nitrification (generally by a day or two in our experience) in many instances. Note that unless the conditions within the plant exist for AOB and NOB to function and grow, then supplemental nitrifying bacteria will not “catch” and addition of nitifying bacteria will therefore fail. When possible, there may be value in DNA testing (16SrRNA or qPCR) in addition to microscopy to help gauge nitrification bacteria populations to help make more informed decisions for situations such as supplementing nitrifying bacteria via bioaugmentation.

About the author: Ryan Hennessy is the principal scientist at Ryan Hennessy Wastewater MicrobiologyHe was trained and mentored by Dr. Michael Richard for over 10 years in wastewater microbiology, and serves as a microbiology services consultant. Hennessy is a licensed wastewater treatment and municipal waterworks operator in the state of Wisconsin and fills in as needed for operations at several facilities. He can be reached at ryan@rhwastewatermicrobiology.com.


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