Human mitochondrial DNA and endogenous bacterial surrogates for risk assessment of graywater reuse

Previous graywater risk assessment studies have focused on fecal contamination, yet the low density of fecal indicators may not provide the most useful approach to assess pathogen removal during graywater treatment. In this study, we employed high throughput bacterial sequencing and qPCR to elucidate potential microbial surrogates in wastewater sourced from an industrial laundry. In addition, we explored human mitochondrial DNA (HmtDNA) as a new, potentially more reliable molecular marker, because it can be unambiguously sourced, has a high copy number per cell, and is persistent when released from cells with no self-replication in graywater. Pyrosequencing and qPCR revealed that laundry water microbiota was dominated by the skin-associated bacteria Staphylococcus, Corynebacterium, and Propionibacterium (6.5, 5.7, 5.4 log10 copies/100 mL, respectively). While HmtDNA was less abundant (2.8 log10 copies/100 mL), it showed a strong positive correlation with the opportunistic pathogen Staphylococcus aureus (r=0.54, P=3.2×10(-4)) and closely followed a first-order exponential decay model (R2=0.98), remaining detectable in stored laundry graywater for up to 6 days at 20 °C. Based on abundance and persistence, we propose HmtDNA and total Staphylococcus as future laundry graywater treatment surrogates to potentially assess a wide dynamic range of pathogen removal.