This post is the first in a series on using molecular diagnostics in wastewater operations. Our goal is to provide operators with an understanding of the tools and how they are used in day-to-day operations. We will also use real-world case studies to show how molecular testing is being used in working facilities.
What are molecular diagnostics?
Most people know of molecular diagnostics (MD) as medical tests that use the genetic information present in a patient's sample to gain insight into the health (or illness) of a patient. Outside of medical applications, these are just a set of laboratory techniques for analyzing any bit of genetic information - including the genetics of wastewater treatment!
There are many different MD tests available today. Two of the most common are quantitative polymerase chain reaction (qPCR) and microbial community analysis (MCA, among many different names). Both have been used by academic laboratories to analyze the microbes making up the biomass in environmental samples for almost 20 years. But, due to the relatively high costs of the tests, they were not commonly used in operations. As the costs for these tests have dropped, more and more WWTPs are using MD tests as routine tools for monitoring plant operations.
(Quantitative) Polymerase Chain Reaction
The polymerase chain reaction (PCR) is an enzymatic method that amplifies (copies) specific DNA sequences. The target DNA sequence to amplify is determined by the oligonucleotides ("primers", which are short, single-stranded bits of DNA that have been synthesized to encode specific sequences of Gs, Ts, As, and Cs - the genetic alphabet). There's a lot of both science and trial & error that goes into designing PCR primers. Primers determine the specificity and sensitivity of the assay. Depending on how primers are designed, a (q)PCR assay detects most members of a genus of bacteria – or the assay can distinguish between two strains of bacteria that are identical except for a single mutation!
In qPCR, the machine that runs the reaction (a thermocycler) is also able to measure the amount of DNA amplification occurring over time. The resulting amplification curve can then be analyzed to estimate how many copies of the target DNA were present in the original reaction or sample. A qPCR assay can, theoretically, detect a single target molecule in a reaction, although the practical limits are usually around 10 to 50 target molecules in a reaction. The linear range of detection typically covers 5 to 7 logs.
Some of the use cases for qPCR include:
Monitoring the key microbial populations involved in biological nutrient removal (AOBs, NOBs and PAOs) to provide early warning for potential loss of nutrient removal, giving operators a chance to take corrective action ahead of the problem
Checking the nitrifier population in the event of a loss of nitrification (should you bioaugment with expensive nitrifier cultures, adjust operating conditions, or both?)
Tracking the major causes of non-filamentous bulking, usually strains of Thauera and Zoogloea, or filamentous bulking (e.g., Thiothrix, Microthrix, Type 1701, and even Methanothrix-associated problems in anaerobic digesters)
And again, qPCR results can be ready very quickly, even on the day the lab receives a sample if necessary.
Microbial Community Analysis
Microbial Community Analysis (MCA) is a census of the microbial population present in a WWTP: it tells you what types of bacteria are present in your system, and in what relative amounts. MCA is based on high-throughput, next-generation DNA sequencing. It starts with a PCR reaction that can amplify the 16S ribosomal RNA gene from most all bacteria. This "library" of 16S rRNA genes is sequenced, generating 10,000 to 50,000 individual data points, which we usually call sequencing reads. Using bioinformatics, we then compare each individual sequencing read against a database of known bacteria to identify the highest probability matches. These database matches are then converted into a report.
Some of the use cases for MCA include:
Routine monitoring of several microbial populations at one time – if you are interested in nitrifiers, PAOs, and multiple filaments, the MCA test makes more sense than qPCR
Diagnosing the cause(s) of upset operating conditions, including such things as loss of nitrification, fouling/loss of transmembrane pressure in membrane bioreactors (MBRs), or low methane production in anaerobic digesters
