Genetic Analysis in Treatment Process Monitoring

Interest in the use of genetic and genomic data for biological wastewater treatment process monitoring has risen rapidly, evidenced by the recent Water Research Foundation (WRF) request for proposals on the topic (WRF 5291). This presentation will serve as a mini 101 course on genetics and genomics for wastewater professionals, offering accessible information on relevant background, potential applications, types of tools, limitations, biomarker assay design, and study design considerations. 'Genetic tools' refers to technologies based on DNA and other nucleic acids. There are two main types, known as targeted or untargeted methods. Targeted methods typically use PCR (polymerase chain reaction) to identify a specific gene or organism of interest. Targeted genetic tools commonly used in wastewater process monitoring applications include quantitative or digital PCR (qPCR or dPCR). Nontargeted methods employ DNA sequencing, which refers to laboratory and computational methods which determine the order of the DNA building blocks; essentially spelling out the genetic code. A commonly used nontargeted method is metagenomic sequencing. (The 'metagenome' refers to all of the DNA present in a sample; see Figure 1). Nontargeted methods yield information on the composition of the microbial community and its functional potential but are not absolutely quantitative. Each type of method has its own set of advantages and limitations (Table 1). Targeted methods are highly sensitive and specific, and can provide highly accurate quantitative data, but are limited in the amount of information they can provide — typically only a single gene or organism out of thousands of relevant potential targets. Care must be exercised in choosing an appropriate target for monitoring, as well as in interpreting the results. Nontargeted methods, in contrast, provide a wealth of information; however, advanced expertise and substantial computing power is required to parse hundreds of gigabytes of data into actionable conclusions. Additionally, application of the wrong genetic technique or interpretation of genetic data without proper data context can greatly complicate or confound conclusions. Genetic tools are an attractive option for wastewater treatment process monitoring because they provide a look 'under the hood' at what is essentially a black box when it comes to biological treatment processes, particularly biological nutrient removal (BNR). These tools can offer information on the health and functioning of the microbiome, as well as insights into complex metabolic systems which can in turn inform process control. However, the field is still emerging, and many research questions remain regarding which targets are appropriate and informative; whether it's possible to develop 'universal' targets that could be widely used; and how the data should be interpreted. Clean Water Services is leading one such research effort, focused on developing biomarkers for biological phosphorus removal (BPR) process stability (WRF 5273). This presentation will describe the procedure used to develop and test these biomarkers as a case study in the application of both targeted and untargeted genetic technologies (Figure 2). Briefly, metagenomic and metatranscriptomic sequencing was performed to thoroughly characterize the aeration basin microbiomes. From these data, key genes were identified which are differentially expressed during stable versus unstable BPR conditions. These genes were used to design biomarkers which are measured with reverse transcription droplet digital PCR (RT-ddPCR). The biomarker assays were tested virtually (in silico) and validated in the laboratory prior to pilot deployment. Lastly, we will touch on the importance of robust study design when employing genetic tools. Too often, a utility will sequence a low number of samples (n<10) and draw broad conclusions from those results. Unfortunately, this can lead to spurious conclusions and a fundamental misunderstanding of the system. We will offer guidance on robust study design for genetic analyses, and suggestions for interpretation of results. This presentation will describe the different types of genetic tools available, summarize their advantages and disadvantages for biological nutrient removal applications and give an overview of the process used to develop and test biomarker targets for WRF project 5273. The goal is to improve communication between molecular biologists and process engineers to improve collaboration on the beneficial application of genetic tools to improve BNR understanding and operation.