Quantifying viruses and bacteria in wastewater - results, quality control, and interpretation methods

Authors: FRANCY, DONNA - Us Geological Survey (USGS); STELZER, ERIN - Us Geological Survey (USGS); BUSHON, REBECCA - Us Geological Survey (USGS); BRADY, AMIE - Us Geological Survey (USGS); MAILOT, BRIAN - Us Geological Survey (USGS); Spencer, Susan; Borchardt, Mark; ELBER, ASHLEY - Non ARS Employee; RIDDELL, KIMBERLY - Non ARS Employee; GELLNER, TERRY - Non ARS Employee

Submitted to: United States Geological Survey Technical Report
Publication Type: Government Publication
Publication Acceptance Date: 8/31/2011
Publication Date: 10/20/2011
Citation: Francy, D.S., Stelzer, E.A., Bushon, R.N., Brady, A.M., Mailot, B.E., Spencer, S.K., Borchardt, M.A., Elber, A.G., Riddell, K.R., Gellner, T.M. 2011. Quantifying viruses and bacteria in wastewater - results, quality control, and interpretation methods. United States Geological Survey Technical Report. 5150, 44p.

Interpretive Summary:

Technical Abstract: Membrane bioreactors (MBR), used for wastewater treatment in Ohio and elsewhere in the United States, have pore sizes large enough to theoretically reduce concentrations of protozoa and bacteria, but not viruses. Sampling for viruses in wastewater is seldom done and not required. Instead, the bacterial indicators Escherichia coli (E. coli) and fecal coliforms are the required microbial measures of effluents for wastewater discharge permits. Information is needed on the efficiency of MBRs in removing human enteric viruses from wastewaters, particularly as compared to conventional wastewater treatment before and after disinfection. A total of 73 regular and 28 quality-control (QC) samples were collected at three MBR and two conventional wastewater plants in Ohio. Samples were collected at various stages in the treatment processes and analyzed for bacterial indicators E. coli, fecal coliforms, and enterococci by membrane filtration; somatic and F-specific coliphage by the single-agar layer (SAL) method; adenovirus, enterovirus, norovirus GI and GII, rotavirus, and hepatitis-A virus by molecular methods; and viruses by cell culture. While addressing the main objective of the study—comparing removal of viruses and bacterial indicators in MBR and conventional plants—it was realized that work was needed to identify data analysis and quantification methods for interpreting enteric virus and QC data. Therefore, methods for quantifying viruses, qualifying results, and applying QC data to interpretations are described in this report; companion reports address the main objective of the project. During each sampling trip, samples were collected (1) before conventional or MBR treatment (post-preliminary), (2) after secondary or MBR treatment (post-secondary or post-MBR), (3) after tertiary treatment (one conventional plant only), and (4) after disinfection (post-disinfection). Glass-wool fiber filtration was used to concentrate enteric viruses from large volumes and small volume grab samples were collected for direct-plating analyses for bacterial indicators and coliphage. After filtration, the viruses were eluted from the filter and further concentrated. The final concentrated sample volume (FCSV) was used for enteric virus analysis by use of two methods—cell culture and a molecular method, polymerase chain reaction (PCR). Quantitative PCR (qPCR) for DNA viruses and quantitative reverse-transcriptase PCR (qRT-PCR) for RNA viruses were used in this study. To support data interpretations, the assay limit of detection (ALOD) was set for each virus assay and used to determine sample reporting limits (SRLs). The SRLs were different for each sample because effective sample volumes (the volume of the original sample that was actually used in each analysis) were different for each sample. Effective sample volumes were much less than the original sample volumes because of reductions from processing steps and (or) from when dilutions were made to minimize the effects from PCR-inhibiting substances. Codes were used to further qualify the virus data and indicate the level of uncertainty associated with each measurement. Quality-control samples were used to support data interpretations. Field and laboratory blanks for bacteria, coliphage, and enteric viruses were all below detection, indicating that it was unlikely that samples were contaminated from equipment or processing procedures. The absolute value log differences (AVLDs) between concurrent replicate pairs were calculated to identify the variability associated with each measurement. For bacterial indicators and coliphage, the AVLD results indicated that concentrations <10 CFU or PFU/100 mL can differ between replicates by as much as one log, whereas higher concentrations can differ by as much as 0.3 log. The AVLD results for viruses indicated that differences betw