Quantitative Assessment of Microbial Pathogens and Indicators of Wastewater Treatment Performance for Safe and Sustainable Water Reuse in India

Wastewater treatment plants (WWTPs) have rapidly increased in India during the last decade. Nonetheless, there are only a few labs in India that can perform culture-based screening for microbial quality. ABSTRACT Currently, there is no data on the molecular quantification of microbial indicators of recycled water quality in India. In this study, multiple microbial pathogens and indicators of water quality were evaluated at three wastewater treatment plants located in two Indian cities (New Delhi and Jaipur) to determine the treatment performance and suitability of recycled water for safe and sustainable reuse applications. Real-time polymerase chain reaction (PCR) was used for the rapid evaluation of six human pathogens and six microbial indicators of fecal contamination. Among the microbial indicators, pepper mild mottle virus (PMMoV), F+RNA-GII bacteriophage, Bacteroides thetaiotamicron, and four human pathogens (Norovirus genogroups I & II, Giardia, and Campylobacter coli) were detected in all of the influent samples analyzed. This work suggests that the raw influents contain lower levels of noroviruses and adenoviruses and higher levels of Giardia compared to those reported from other geographic regions. Overall, the efficacy of the removal of microbial targets was over 93% in the final effluent samples, which is consistent with reports from across the world. PMMoV and Giardia were identified as the best microbial targets, from the microbial indicators spanning across bacteria, bacteriophages, DNA/RNA viruses, and protozoan parasites, by which to evaluate treatment performance and recycled water quality in Indian settings, as they were consistently present at high concentrations in untreated wastewater both within and across the sites. Also, they showed a strong correlation with other microbial agents in both the raw influent and in the final effluent. These findings provide valuable insights into the use of culture-independent molecular indicators that can be used to assess the microbial quality of recycled water in Indian settings. IMPORTANCE Wastewater treatment plants (WWTPs) have rapidly increased in India during the last decade. Nonetheless, there are only a few labs in India that can perform culture-based screening for microbial quality. In the last 2 years of the pandemic, India has witnessed a sharp increase in molecular biology labs. Therefore, it is evident that culture-independent real-time PCR will be increasingly used for the assessment of microbial indicators/pathogens in wastewater, especially in resource-limited settings. There is no data available on the molecular quantitation of microbial indicators from India. There is an urgent need to understand and evaluate the performance of widely used microbial indicators via molecular quantitation in Indian WWTPs. Our findings lay the groundwork for the molecular quantitation of microbial indicators in WWTPs in India. We have screened for 12 microbial targets (indicators and human pathogens) and have identified pepper mild mottle virus (PMMoV) and Giardia as the best molecular microbiological indicators in Indian settings.