What qPCR Can Tell You About Your Wastewater System
Identify Critical Populations and Uncover Root Causes of System Upsets.
by Deborah Lee, MS, Microbiologist
Quantitative PCR (qPCR) is also sometimes called real-time PCR. This is PCR, which is a technique used to amplify a specific DNA sequence. However, it differs from conventional PCR by also being able to simultaneously quantify the DNA sequence amplified. This is often helpful in microbiology to measure DNA (or cDNA from RNA) from specific organisms in real time during each cycle of PCR.
How does qPCR work?
Quantitative PCR works by extracting DNA from a sample. Then, using primers, short DNA sequences are amplified that are specific for the gene or organism of interest. This uses a reaction mixture of primers, DNA polymers, probes, and other components, along with a fluorescent dye or fluorescent probe. The mixture is put into tubes and PCR cycles are performed with fluorescence measured at each cycle. As the target DNA is amplified, fluorescence increases proportionally and is detected by the qPCR machine. When a standard curve is also performed, it can be used to quantify the copy numbers. Otherwise, the relative amounts of resulting fluorescence are measured using the cycle number at which the fluorescence in the tube crosses a threshold (Ct = cycle threshold). When RNA is used with reverse transcription to cDNA you can also measure gene expression of a specific microbe of interest.
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When to use qPCR in wastewater systems?
Using qPCR as a monitoring method in wastewater treatment is valuable when you need rapid, specific, and quantitative information about microbial populations or genes that are important for process performance, stability, or regulatory compliance. Some bacteria and archaea that are important in wastewater treatment are known to have specific genes that are conserved across genera. These genes can be targeted to estimate the population of microbes that perform a specific function in nutrient removal, such as the amoA gene for ammonia oxidation. This could also be used to estimate the relative amounts of sulfate-reducing organisms that generate hydrogen sulfide gas, or the relative amount of methanogenic archaea, which can be predictive of an anaerobic system’s health. If optimized, it could also be used to assist in identifying the cause of sludge bulking or foaming.
Key microbial groups that drive treatment processes.
Why might qPCR be a better means of monitoring anaerobic digesters than other conventional monitoring methods??
There are some advantages that qPCR offers over conventional monitoring methods for anaerobic digesters. Traditional methods include measuring pH, alkalinity, COD, VFAs, or biogas production, and while these are necessary and useful, they only reveal symptoms of underlying biological changes and not the cause. An advantage of qPCR is that it can be used as a tool for an early warning system by detecting the change in abundance of target microbial populations before performance degrades. For example, a decline in hydrogenotrophic methanogens may warn of future VFA spikes. Checking on the levels of specific populations, such as Methanosaeta, Syntrophomonas, or sulfate-reducers, can give a clear picture of who is active or missing from the system. This can diagnose the root cause of upsets. For example, if gas drops or VFAs spike, qPCR can reveal whether it’s due to methanogen washout, sulfate-reducer competition, or an overall microbial imbalance. Knowing the acetoclastic-to-hydrogenotrophic ratio could also guide whether to reduce organic loading or make other operational adjustments.
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What are important target organisms to monitor with qPCR in anaerobic digesters?
Methanogens (Methane Producers)
These are the final step organisms that convert intermediates into methane.
Syntrophic Bacteria (Intermediate Degraders)
These organisms oxidize fatty acids and alcohols in partnership with hydrogenotrophic methanogens.
Hydrolytic and Acidogenic Bacteria
They break down complex organics (e.g., proteins, carbs, lipids) into VFAs.
Problematic or Competing Organisms
May indicate instability or competition with methanogens.
About the Author
Deborah holds a Master’s Degree in Microbiology and has been leading the Aquafix research on microbial communities and their relationship with nitrogen and ammonia in wastewater since 2009.