Treatment Plant Operator’s Bug of the Month is an ongoing series that spotlights the organisms present in wastewater microbiology. Each month a new organism is featured, giving readers a profile of the species and how it functions in a wastewater treatment setting. 

In this peek under the microscope, take an up-close look at Nitrobacter. 

Nitrobacter is a nitrite oxidizing bacteria (NOB) that oxidizes nitrite to nitrate and one of two genus of nitrifying bacteria that can be readily identified at phase contrast microscopy 1000x magnification. The other genus of bacteria visible with microscopy that have a predominant role in nitrification are Nitrosomonas, which convert ammonia to nitrite (AOBs). Currently the MIDAS field guide recognizes 20 individual species within the Nitrosomonas genus. Nitrobacter is typically observed on the edges of the flocs where there is the most dissolved oxygen present and appears as colonies of small rod-shaped microbes. 

While it was once believed that Nitrobacter and Nitrosomonas were the only two nitrifying genus found in activated sludge, recent advancements in genetic testing have revealed the genus Nitrospira (currently six recognized species) to be the predominant NOB microbe in most activated sludge systems. Unlike Nitrobacter, Nitrospira cannot be viewed with traditional phase-contrast microscopy. 

Growth conditions

While the growth conditions that determine which nitrifying bacteria types compete is relatively unknown, not seeing Nitrobacter in a sample is not cause for panic. If nitrite concentrations are low and Nitrobacter is not viewed, chances are there are a different group of NOB (likely Nitrospira) completing this function. Drawing from personal experience, I would estimate that Nitrobacter is visible in approximately 25% of nitrifying activated sludge samples with no specific trend to any particular type of wastewater. 

Overall, Nitrobacter appears to play a small role in activated sludge processes. Nitrobacter can grow lithoautotrophically under oxic conditions where nitrite is the preferred energy source and carbon dioxide is the main source of carbon, or it may also grow chemoorganotrophically under anoxic conditions by anaerobic respiration with nitrate.

Conditions needed to maintain a stable population of nitrifying bacteria include adequate dissolved oxygen concentrations (typically greater than 1.5-2 mg/L), sufficient alkalinity ( greater than 75 mg/L), pH values in desired ranges (typically 7-8), reduction of the majority of carbonaceous BOD, adequate sludge age (typically more than five days), and lack of any inhibiting compounds. Note that the NOB bacteria are typically more sensitive than the AOB in regards to stressful conditions. When only partial nitrification occurs nitrite concentrations will increase (often referred to as “nitrite lock”). It is unusual to see high concentrations of nitrite in wastewater effluents and concentrations of nitrite greater than 0.1-0.2 mg/L may potentially indicate issues. Where nitrite can be problematic is disinfection (high chlorine demand), and if there is a total nitrogen limit that must be met in the effluent discharge. 

Building a database

Midwest Contract Operations Inc. is working with a genetic sequencing lab called Aster Bio to create a comprehensive database comparing microscopy findings to DNA analysis to allow for trending and “translation” for further understanding of the genetic diversity amongst filamentous bacteria morphotypes and other microbes observed in different types of samples. 

For this database, we are seeking plants that would be interested in sending in samples in which microscopy and DNA would be tested. We would grant anyone willing to submit a sample access to the database and its use as a reference.

For more information or to volunteer to send samples to help build the database, email rhennessy@mco-us.com.

---

About the author: Ryan Hennessy is the microbiology and operations specialist at Midwest Contract Operations Inc. He 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 rhennessy@mco-us.com.