Scientists have reported the first successes using real-time polymerase chain reaction (PCR) technology to monitor the population dynamics of nitrifying bacteria in a wastewater treatment plant (WWTP) in research recently posted to ES&T’s Research ASAP website. This PCR method, which so far has mainly been used in biological and medical research, could detect and possibly prevent unwanted changes in critical microbial subpopulations in WWTPs before serious problems arise, says Hebe Dionisi, one of the study’s coauthors. Compared to other PCR methods, real-time PCR offers higher throughput, less labor and costs, and a much wider detection range.

The PCR process, in which a specific DNA fragment is amplified by the polymerase enzyme, can be used to detect and quantify specific groups of bacteria after extraction of their DNA. In contrast to conventional quantitative PCR methods, which detect the amount of final amplified product and back-calculate the initial DNA concentration after complex post-PCR processing, real-time PCR follows the DNA production during each PCR cycle and quantifies it through a fluorescent reporter signal. Monitoring the PCR reaction during its early, exponential phase, it is possible to determine the first significant increase in the amount of PCR product, which correlates with the initial amount of target DNA.

Gary Sayler and colleagues at the University of Tennessee–Knoxville have developed and standardized this real-time PCR approach for use in wastewater treatment monitoring. The researchers quantified different groups of nitrifying bacteria from a local WWTP treating mainly municipal wastewater over the course of a year. The activity and abundance of these nitrifyers, which are responsible for the stepwise transformation of toxic ammonia to nitrate, are crucial for controlling effluent toxicity in a WWTP. The next step for the research team will be the analysis of the microbes’ messenger RNA to quantify the relationship between their number and activity, Dionisi says.

Sayler says that, in WWTPs, molecular techniques can complement the more traditional approaches for solving problems such as bulking, in which the necessary settling of activated sludge is prevented by the growth of filamentous bacteria that need to be identified and quantified. The unwanted bacteria lead to the formation of sludge flocs with high volume-to-mass ratios that do not settle properly.

Real-time PCR is indeed a promising method for environmental monitoring, agrees molecular microbiologist Jan-Roelof van der Meer of the Swiss Federal Institute for Environmental Science and Technology (EAWAG). He stresses that because the technique is simpler than conventional quantitative PCR, more samples can be analyzed with less time and effort. However, the method’s accuracy varies with the efficiency of DNA extraction, which can be a problem with complex environmental samples, van der Meer points out.

Real-time PCR is not the only candidate competing for molecular analysis in WWTPs. Fluorescence in situ hybridization (FISH) is being promoted as the method of choice for quantifying nitrifying bacteria in WWTPs by Vermicon AG, a spin-off company from the Technical University of Munich, Germany. In FISH, target DNA or RNA sequences are hybridized with fluorescence-marked nucleotide probes and subsequently detected microscopically. Vermicon microbiologist Tanja Linner says that FISH avoids two main drawbacks of real-time PCR—the amplification can be inhibited by substances present in complex samples, and the method fails to differentiate between dead and live cells.

Nevertheless, Sayler suggests that real-time PCR could play an important future role in other environmental areas, such as detecting pathogens in drinking water, food safety, and bioterrorism monitoring. Newly developed field-portable units can be used by nonexperts and can give a quantitative response to the occurrence of microorganisms, such as the pathogen Bacillus anthracis, within less than 4 hours, he adds. —ANKE SCHAEFER