Different strains of bacteria each have unique genetic fingerprints encoded in DNA. qPCR (quantitative polymerase chain reaction) tests count these DNA fingerprints. Depending on how the test is run, a qPCR test can count all the bacteria, or just a specific subset, like the ammonia-oxidizing bacteria, present in a sample.
While qPCR can give absolute values (for example, 1.3 × 10^9 bacteria per milliliter of MLSS), the accuracy of the measurement depends on a lot of factors that are difficult to control. For this reason, we usually report relative numbers, which requires that we always run at least two tests on each sample.
The “Denominator” Test is for Total BacteriaFor every sample, we measure all the bacteria present in a sample using a standardized, reproducible protocol. This test measures a key gene, the 16S ribosomal RNA gene, that is found in all bacteria (and humans, too!). This number ends up being the denominator in all future calculations. Typical measurements of MLSS samples range from 1 × 10^6 to 5 × 10^7 copies per reaction.
The “Numerator” Test Varies
qPCR tests can be incredibly specific, able to detect and measure quantities of bacteria at the subspecies level! The test is also very sensitive: a properly designed test can measure 1 to 10 copies of the DNA fingerprint in a single reaction! And, samples can always be diluted to keep them within the upper limits of detection We routinely test for ammonia-oxidizing bacteria (AOBs), filaments, and zoogloeal bulking.
qPCR results are often reported as a fraction or percentage. For example, in a wastewater system with nitrification, the ammonia-oxidizing bacteria usually range from 0.1% to 1.0% of the total count. Lower numbers may indicate loss of nitrification, while higher numbers may indicate unusually heavy loadings of ammonia. Type 1701 filaments range from 0.002% to as high as 20%!
While the relative numbers are useful, we find the trend in the numbers to be the real value. Each wastewater treatment plant is a little different, so the acceptable range for a particular microorganism differs from plant to plant. But, the trends within a single plant can reveal a slow increase in problematic filaments or foaming microorganisms to allow operators to take action before there is a crisis. Likewise, slow drops in nitrifiers can signal chronic toxicity before the entire population is lost.