The efficacy of hydrogen sulfide (H2S) tests for detecting microbial contamination in rooftop-harvested rainwater.

As climate change strains the world's freshwater resources, access to safe and clean water becomes limited. The use of alternative water sources, such as rooftop-harvested rainwater, has become one mechanism to address freshwater scarcity in the American Southwest, particularly when it comes to home gardening. The University of Arizona's Project Harvest, in partnership with the Sonora Environmental Research Institute, Inc., is a multi-year, co-created citizen science project aimed at increasing current understanding of harvested rainwater quality. Citizens in four Arizona, USA, communities (Hayden/Winkelman, Globe/Miami, Dewey-Humboldt, and Tucson) submitted harvested rainwater samples over 3 years. The harvested rainwater samples were then analyzed using IDEXX Colilert® for total coliforms and E. coli and using Hach PathoScreen™ test for sulfate-reducing bacteria (SRB). This study design allows for the validation of a low-cost, at-home alternative methodology for testing rainwater for bacteria that may indicate fecal contamination. In total, 226 samples were tested using both methodologies, revealing a positive correlation (r=0.245; p<0.002) between total coliform MPN and SRB MPN, but no discernable correlation between E. coli MPN and SRB MPN. This work indicates a potential value of SRB testing for harvested rainwater if cost, laboratory access, and fecal contamination are of concern.

Assessing the impact of rainwater harvesting infrastructure and gardening trends on microbial indicator organism presence in harvested rainwater and garden soils.

AIM: To assess the microbial water quality of harvested rainwater infrastructure used to supplement household water uses for homegrown produce. METHODS AND RESULTS: Using a co-created community science methodology, between 2017 and 2020, a total of 587 harvested rainwater samples and 147 garden soil samples irrigated with harvested rainwater were collected from four Arizona communities and analyzed for coliform, Escherichia coli, and/or Salmonella. Participants also completed a home description survey regarding their home and surrounding area, water harvesting infrastructure, and gardening habits. CONCLUSION: Chi-Square tests revealed that the quality of harvested rainwater is affected by proximity to a waste disposal or incineration facility, animal presence, cistern treatment, and cistern age (P < 0.05), while soil samples were associated with community (P < 0.05). Coliform and E. coli concentrations in both sample types were greater in the monsoon season.

Increasing Environmental Health Literacy through Contextual Learning in Communities at Risk.

Environmental health literacy (EHL) has recently been defined as the continuum of environmental health knowledge and awareness, skills and self-efficacy, and community action. In this study, an interdisciplinary team of university scientists, partnering with local organizations, developed and facilitated EHL trainings with special focus on rainwater harvesting and water contamination, in four communities with known environmental health stressors in Arizona, USA. These participatory trainings incorporated participants' prior environmental health risk knowledge and personal experiences to co-create training content. Mixed methods evaluation was conducted via pre-post participant surveys in all four trainings (n = 53). Participants who did not demonstrate baseline environmental science knowledge pre-training demonstrated significant knowledge increase post-training, and participants who demonstrated low self-efficacy (SE) pre-training demonstrated a significant increase in SE post-training. Participants overall demonstrated a significant increase in specific environmental health skills described post-training. The interdisciplinary facilitator-scientist team also reported multiple benefits, including learning local knowledge that informed further research, and building trust relationships with community members for future collaboration. We propose contextual EHL education as a valuable strategy for increasing EHL in environmental health risk communities, and for building academia-community partnerships for environmental health research and action.

Assessing environmental impacts of treated wastewater through monitoring of fecal indicator bacteria and salinity in irrigated soils.

To assess the potential for treated wastewater irrigation to impact levels of fecal indicator bacteria (FIB) and salinity in irrigated soils, levels of Escherichia coli, Enterococcus, and environmental covariates were measured in a treated wastewater holding pond (irrigation source water), water leaving the irrigation system, and in irrigated soils over 2 years in a municipal parkland in Arizona. Higher E. coli levels were measured in the pond in winter (56 CFU 100 mL(-1)) than in summer (17 CFU 100 mL(-1)); however, in the irrigation system, levels of FIB decreased from summer (26 CFU 100 mL(-1)) to winter (4 CFU 100 mL(-1)), possibly related to low winter water use and corresponding death of residual bacteria within the system. For over 2 years, no increase in FIB was found in irrigated soils, though highest E. coli levels (700 CFU g(-1) soil) were measured in deeper (20-25 cm) soils during summer. Measurements of water inputs vs. potential evapotranspiration indicate that irrigation levels may have been sufficient to generate bacterial percolation to deeper soil layers during summer. No overall increase in soil salinity resulting from treated wastewater irrigation was detected, but distinct seasonal peaks as high as 4 ds m(-1) occurred during both summers. The peaks significantly declined in winter when surface ET abated and more favorable water balances could be maintained. Monitoring of seasonal shifts in irrigation water quality and/or factors correlated with increases and decreases in FIB will aid in identification of any public health or environmental risks that could arise from the use of treated wastewater for irrigation.

False-positive identification of Escherichia coli in treated municipal wastewater and wastewater-irrigated soils.

The increasing use of treated wastewater for irrigation heightens the importance of accurate monitoring of water quality. Chromogenic media, because they are easy to use and provide rapid results, are often used for detection of Escherichia coli in environmental samples, but unique levels of organic and inorganic compounds alter the chemistry of treated wastewater, potentially hindering the accurate performance of chromogenic media. We used MI agar and molecular confirmatory methods to assess false-positive identification of E. coli in treated wastewater samples collected from municipal utilities, an irrigation holding pond, irrigated soils, and in samples collected from storm flows destined for groundwater recharge. False-positive rates in storm flows (4.0%) agreed closely with USEPA technical literature but were higher in samples from the pond, soils, and treatment facilities (33.3%, 38.0%, and 48.8%, respectively). Sequencing of false-positive isolates confirmed that most were, like E. coli, of the family Enterobacteriaceae, and many of the false-positive isolates were reported to produce the ß-D-glucuronidase enzyme targeted by MI agar. False-positive identification rates were inversely related to air temperature, suggesting that seasonal variations in water quality influence E. coli identification. Knowledge of factors contributing to failure of chromogenic media will lead to manufacturer enhancements in media quality and performance and will ultimately increase the accuracy of future water quality monitoring programs.

Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces.

Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides-specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.

Seasonal variation in accurate identification of Escherichia coli within a constructed wetland receiving tertiary-treated municipal effluent.

As the reuse of municipal wastewater escalates worldwide as a means to extend increasingly limited water supplies, accurate monitoring of water quality parameters, including Escherichia coli (E. coli), increases in importance. Chromogenic media are often used for detection of E. coli in environmental samples, but the presence of unique levels of organic and inorganic compounds alters reclaimed water chemistry, potentially hindering E. coli detection using enzyme-based chromogenic technology. Over seven months, we monitored E. coli levels using m-Coli Blue 24 broth in a constructed wetland filled with tertiary-treated municipal effluent. No E. coli were isolated in the wetland source waters, but E. coli, total coliforms, and heterotrophic bacteria increased dramatically within the wetland on all sampling dates, most probably due to fecal inputs from resident wildlife populations. Confirmatory testing of isolates presumptive for E. coli revealed a 41% rate of false-positive identification using m-Coli Blue 24 broth over seven months. Seasonal differences were evident, as false-positive rates averaged 35% in summer, but rose sharply to 75% in the late fall and winter. Corrected E. coli levels were significantly correlated with electrical conductivity, indicating that water chemistry may be controlling bacterial survival within the wetland. This is the first study to report that accuracy of chromogenic media for microbial enumeration in reclaimed water may show strong seasonal differences, and highlights the importance of validation of microbiological results from chromogenic media for accurate analysis of reclaimed water quality.

Microbial community activities during establishment, performance, and decline of bench-scale passive treatment systems for mine drainage.

Permeable reactive barrier (PRB) technology, in which sulfate-reducing bacteria (SRB) facilitate precipitation of metal sulfides, is a promising approach for remediation of sulfate- and metal-laden mine drainage. While PRBs are easily established, they often decline for reasons not well understood. SRB depend on or compete with multiple dynamic microbial populations within a PRB; as a result, performance depends on the changing PRB chemical composition and on succession and competition within the microbial community. To investigate these interactions, we constructed and monitored eight bench-scale PRBs to define periods of establishment, performance, and decline. We then conducted short-term batch studies, using substrate-supplemented column materials, on Days 0 (pre-establishment), 27 (establishment), 41 (performance), and 99 (decline) to reveal potential activities of cellulolytic bacteria, fermenters + anaerobic respirers, SRB, and methanogens. PRBs showed active sulfate reduction, with sulfate removal rates (SRR) of approximately 1-3 mol/m3/d, as well as effective removal of Zn2+. Potential activities of fermentative + anaerobic respiratory bacteria were initially high but diminished greatly during establishment and dropped further during performance and decline. In contrast, potential SRB activity rose during establishment, peaked during performance, and diminished as performance declined. Potential methanogen activity was low; in addition, SRB-methanogen substrate competition was shown not to limit SRB activity. Cellulolytic bacteria showed no substrate limitation at any time. However, fermenters experienced substrate limitation by Day 0, SRB by Day 27, and methanogens by Day 41, showing the dependence of each group on upstream populations to provide substrates. All potential activities, except methanogenesis, were ultimately limited by cellulose hydrolysis; in addition, all potential activities except methanogenesis declined substantially by Day 99, showing that long-term substrate deprivation strongly diminished the intrinsic capacity of the PRB community to perform.