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DNA uniquely diagnoses major upset at wastewater plant

DNA uniquely diagnoses major upset at wastewater plant

DNA uniquely diagnoses major upset at 10 MGD wastewater treatment plant

A 10 MGD municipal wastewater treatment plant in the southwest USA experienced a major plant upset due to a significant unplanned change in the influent characteristics, and called Microbe Detectives to help diagnose the problem.

Problem #1 – Total Nitrogen (TN) removal plummeted

The first major problem was negative impacts to the biological nitrogen removal process. Prior to the upset, the wastewater treatment plant maintained a daily average total nitrogen (TN) concentration in the effluent of < 6 mg/L, which at the time was below the limit in their discharge permit. After the plant upset, TN persistently increased, up to 18 mg/L, falling outside of permit requirements.

Likely root causes

  1. The % relative abundance of Microbes with nitrogen removal capabilities dropped to less than half the amount observed prior to the upset (~45% down to ~20%). This is illustrated above.
  2. Thaurea, the most well known Nitrate Reducing Bacteria (NRB) in wastewater treatment systems, was nearly wiped out (down from ~10% to ~0.01%). Nitrate reducing bacteria are referred to as denitrifiers. Denitrification is a microbial facilitated process where nitrate (NO3−) is reduced to Nitrite (NO2-) and ultimately molecular nitrogen (N2).
  3. Dechloromonas, also known to have denitrification capabilies was also nearly wiped out (down from ~12% to 0.04%).
  4. Thaurea are also known to have Simultaneous Nitrification-Denitrification (SND) capabilities. 

Problem #2 – Final effluent clarity plummeted

The second major problem was that the daily average turbidity, measured as Nephelometric Turbidity Units (NTUs), of final effluent increased by a factor greater than 10X, up from <0.5 to 5.7 NTUs, falling outside of permit requirements.

Likely root causes

  1. The % relative abundance observed of Zooglea plummeted after the upset. Zooglea is one of the more well known filamentous bacteria in wastewater treatment systems, known to help facilitate settling and the production of clear, low-turbidity effluent.
  2. The relative abundance of Candidatus Microthrix was also nearly eliminated, down from ~1.2% to <0.01%. Microthrix is also well known for its beneficial properties to facilitate settling.

Other problems

Phosphorus Accumulating Organisms (PAOs) plummeted

The % relative abundance observed of two well known PAOs plummeted after the upset. Dechloromonas, as mentioned above, dropped from ~12% to ~0.04%. Rhodocyclus, dropped from ~5% to 0.2%.

At the time, Phosphorus was not regulated in the discharge permit at the wastewater treatment plant. However, the plant was anticipating new limits on Phosphorus when its discharge permit is renewed.

Biological diversity dropped 32%

Biological diversity in wastewater treatment generally is associated with system resiliency. The higher the diversity, the more resilient or resistant to system upsets. Biological diversity observed, measured as Shannon’s Diversity Index, dropped ~32% after the upset, from a value of 3.8 (on a scale of 0 to 5), to 2.6.

Diagnostic clues to potential sources of the upset

The largest increases in % abundance observed after the upset were generally fecal associated bacteria


The largest increase in % relative abundance observed after the plant upset was with Citrobacter, with a ~29% increase. Citrobacter spp. primarily are inhabitants of the intestinal tract of mammals and other vertebrates. Their isolation from environmental sources such as water and soil likely is the result of fecal excretion.(1)


The second largest increase in % relative abundance observed was with Pantoea with a ~27% increase. As a member of the Order Enterobacterales, Pantoea is commonly associated with human gut bacteria.


The third largest increase observed was with Klebsiella, with a ~9% increase. Klebsiella is a type of gram-negative bacteria that can cause different types of healthcare-associated infections. Increasingly, Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems. Klebsiella bacteria are normally found in the human intestines and in the human stool (feces) and are commonly associated with healthcare facilities. (2)

Raoultella and Faecalibacterium

The fourth and fifth largest increases in % relative abundance observed after the upset were with Raoultella (up ~2%) and Faecalibacterium (up ~2%). Both are commonly associated with fecal as well.

The prime suspect

Since the significant majority of microbes observed with the highest increases in % relative abundance after the upset were all fecal associated microbes, a large, unplanned discharge of human waste (feces) is therefore a likely suspect for the root cause of the upset. Industrial dischargers are not likely to have been the source. More specifically, since Klebsiella is commonly associated with healthcare facilities, it may be a worthwhile activity to consider healthcare facilities that discharge to the wastewater treatment plant as possible sources.

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  1. Stella Antonara, Monica I. Ardura, 141 – Citrobacter Species,
    Editor(s): Sarah S. Long, Charles G. Prober, Marc Fischer, Principles and Practice of Pediatric Infectious Diseases (Fifth Edition), Elsevier, 2018, ISBN 9780323401814, (
  2. Centers for Disease Control and Prevention, website (