Erik R Coats

Intracellularly stored carbon (ISC) plays a central role in enabling carbon-efficient nitrogen removal, yet its contributions to full denitrification (FdN) and partial denitrification (PdN) remain incompletely understood. This review synthesizes current knowledge of ISC anabolism and catabolism across phosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), PHA-storing organism (PSOs), glycogen-storing organism (GSOs), and oleaginous microorganisms under feast–famine, nutrient limitation, and variable redox regimes. Major ISC storage and utilization pathways are discussed alongside two conceptual models of ISC metabolism (sequential storage–growth versus parallel storage–growth) in the context of mainstream biological nutrient removal. Subsequently, the influence of wastewater characteristics, reactor configuration, feast–famine structure, dissolved oxygen control, sludge age, biomass concentration, and ISC composition on ISC-driven denitrification is analyzed. For ISC-FdN, evidence from bench- to full-scale shows that post-anoxic ISC utilization can achieve low effluent nitrogen while greatly reducing external carbon dosing and mixed-liquor recirculation. For ISC-PdN, slow ISC depolymerization, nitrate residuals, and nitrite sinks via anammox are highlighted as key kinetic drivers of denitritation suppression and/or nitrite detouring to shortcut N removal pathway, linked to the activity of taxa including Candidatus Competibacter, Candidatus Accumulibacter, and Thauera. Finally, the integration potential of ISC-driven PdN with mainstream deammonification and sludge densification is evaluated, and priority research needs are identified.

Bacteria detection through the polymerase chain reaction (PCR) often requires extraction and purification of the analyte DNA sequence, with the total time for analysis that is typically 5 to 8 h. In this contribution, we demonstrate that this workflow can be greatly simplified, reducing the total time to detection to 17 min. The on-electrode (oE) PCR technique conducts a surface-based 5-cycle PCR amplification by covalent attachment of a right primer to a pseudo-graphite fiber electrode. This foregoes electrophoretic separation steps. The target is a 225-base sequence of the 16S rDNA gene of Nitrobacter genomic DNA (3.4 million base pairs) and extracellular environmental (e) DNA. The oE-PCR technique overcomes Taq polymerase inhibition associated with untreated matrices of raw sewage and chicken juice. These are challenging media for all other variations of PCR. The detection of surface-based amplicons is through the introduction of the electrochemically active cobalt(III) tris-1,10-phenanthroline intercalator, followed by square-wave voltammetric (SWV) analysis. Detection was positive relative to a blank and no-template control (NTC). The total time required for the oE-PCR process is 17 min with a limit of detection of less than 20 aM. The LOD of this method surpasses the nM required for the detection of Nitrobacter DNA in wastewater matrices. While qPCR offers quantitative results, it is time-consuming. However, oE-PCR may act as a rapid screening tool for qPCR. The ability to detect rapidly Nitrobacter in such a complex matrix, from sample to result, may have important applications in medical, environmental, and forensic sciences.

With passage of the CHIPS (Creating Helpful Incentives to Produce Semiconductors) and Science Act of 2022, growth of semiconductor facilities in the U.S. will increase, which will demand the need for treating industrial wastewater rich in xenobiotic compounds. One wastewater management option is disposal to municipal water resource recovery facilities (WRRFs). However, WRRFs are not conventionally designed for xenobiotics removal specifically associated with semiconductor facilities. Research herein interrogated the effects of reverse osmosis concentrate (ROC) derived from a water recycling system treating semiconductor wastewater on wastewater treatment. ROC impaired nitrification at the bench and eliminated nitrification at the pilot scale; impact at the pilot scale was almost immediate. Ammonia oxidizing bacteria were eliminated once ROC was added; also, no ammonia oxidizing archaea, Crenarchaeota, or comammox were detected. Bench-scale data suggested ROC might impair biological phosphorus removal; more critically, pilot scale effluent phosphorus increased 670%, while the relative fraction of phosphorus bacteria, interrogated at the Clade level, decreased 705%. Metabolomic data revealed key indicators of nitrification – fructose, 3-phosphoglyceric acid, and pyruvate – decreased with ROC addition. Moreover, the ROC-impacted metabolome exhibited the strongest correlations with amino acids. While no direct linkage between the amino acids and nitrification could be inferred, the predominance of these peptide building blocks combined with a significant loss of biomass suggests protein hydrolysis associated with bacterial die-off. Conversely, correlations without ROC were dominated by TCA cycle metabolites, indicating the predominance of oxidative reactions supporting energy production. Mitigation is possible; research focused on the acute effects of ROC addition, and attenuation in the sanitary sewer collection system may mitigate toxicity. Ultimately, municipal WRRFs need to approach receiving semiconductor wastewater with caution and careful study.

Wastewater-based epidemiology (WBE) can provide critical early warnings to aid public health, which can be particularly beneficial in rural communities with limited access to health care. Spikes of SARS-CoV-2 RNA concentration in wastewater have been used to represent infections in a community, but wastewater holds a wealth of information that has not been explored yet. The objectives of this research were to expand the use of WBE to 1) determine the dynamic of SARS-CoV-2 variants in rural communities, and 2) evaluate the relationship between community vaccination status and the outbreak of a variant. We quantified the concentration of SARS-CoV-2 RNA, as well as specific mutations that are consistent with Delta and Omicron in influent raw wastewater samples collected from wastewater treatment facilities (WWTFs) for five populations with <1000 residents and one larger population in Latah County, ID. A binomial generalized linear model using the percent of the population with protection against Omicron from the initial vaccines and the booster shot was able to predict the probability of an uptick in Omicron concentration in wastewater with an accuracy of 0.96. Evaluation of vaccination data indicate that the spike in Omicron infections in December 2021 in the studied towns was linked to low levels of population protection from the initial shots of the COVID-19 vaccine against Omicron infection and limited uptake of booster shots in these communities. Despite difficulties with applying WBE in rural regions, this study shows that beyond evaluating spikes of viral infections, WBE can be used to evaluate the effect of a population's vaccine coverage on SARS-CoV-2 variant dynamics.

Wastewater has emerged as a crucial tool for infectious disease surveillance, offering a valuable means to bridge the equity gap between underserved communities and larger urban municipalities. However, using wastewater surveillance in a predictive manner remains a challenge. In this study, we tested if detecting SARS-CoV-2 in wastewater can forecast outbreaks in rural communities.
Under the CDC National Wastewater Surveillance program, we monitored the SARS-CoV-2 in the wastewater of five rural communities and a small city in Idaho (USA). We then used a particle filter method coupled with a stochastic susceptible-exposed-infectious-recovered (SEIR) model to infer active case numbers using quantities of SARS-CoV-2 in wastewater.
Our findings revealed that while high daily variations in wastewater viral load made real-time interpretation difficult, the SEIR model successfully factored out this noise, enabling accurate forecasts of the Omicron outbreak in five of the six towns shortly after initial increases in SARS-CoV-2 concentrations were detected in wastewater. The model predicted outbreaks with a lead time of 0 to 11 days (average of 6 days +/- 4) before the surge in reported clinical cases.
This study not only underscores the viability of wastewater-based epidemiology (WBE) in rural communities—a demographic often overlooked in WBE research—but also demonstrates the potential of advanced epidemiological modeling to enhance the predictive power of wastewater data. Our work paves the way for more reliable and timely public health guidance, addressing a critical gap in the surveillance of infectious diseases in rural populations.

Forest water reclamation is a decades-old practice of repurposing municipal reclaimed water using land application on forests to filter nutrients and increase wood production. However, long-term application may lead to nutrient saturation, leaching, and potential impairment of ground and surface water quality. We studied long-term effects of reclaimed water application on nutrient leaching potential in a four-decade time series of forest water reclamation facilities in northern Idaho. Our approach compared reclaimed water treated plots with untreated control plots at each of the forest water reclamation facilities. We measured soil nitrifier abundance and net nitrification rates and used tension lysimeters to sample soil matrix water and drain gauges to sample from a combination of matrix and preferential flow paths. We determined nutrient leaching as the product of soil water nutrient concentrations and model-estimated drainage flux. There was more than 450-fold increase in nitrifier abundance and a 1000-fold increase in net nitrification rates in treated plots compared with control plots at long-established facilities, indicating greater nitrate production with increased cumulative inputs. There were no differences in soil water ammonium, phosphate, and dissolved organic nitrogen concentrations between control and effluent treatments in tension lysimeter samples. However, concurrent with increased nitrifier abundance and net nitrification, nitrate concentration below the rooting zone was 2 to 4-fold higher and nitrate leaching was 4 to 10-fold higher in effluent treated plots, particularly at facilities that have been in operation for over two decades. Thus, net nitrification and nitrifier abundance assays are likely indicators of nitrate leaching potential. Inorganic nutrient concentrations in drain gauge samples were 2 to 11-fold higher than lysimeter samples, suggesting nutrient losses occurred predominantly through preferential flow paths. Nitrate was vulnerable to leaching during the wet season under saturated flow conditions. Although nitrogen saturation is a concern that should be mitigated at long-established facilities, these forest water reclamation facilities were able to maintain average soil water nitrate concentrations to less than 2 mg L−1, so that nitrogen and phosphorous are effectively filtered to below safe water standards.
•Forest water reclamation (FWR) is a cost-effective approach to manage wastewater.•Annual osccilation in nutrient leaching depended on seasonal rate of drainage.•Long-term FWR increased nitrifier abundance, net nitrification, and leaching.•Nitrifier abundance and net nitrification are precursors to N saturation.•Phosphate leaching was minimal; occurring mainly through preferential flow.

Wastewater phosphorus removal achieved biologically is associated with the process known as enhanced biological phosphorus removal (EBPR). In contrast with canonical EBPR operations that employ alternating anaerobic-aerobic conditions and achieve asynchronous carbon and phosphorus storage, research herein focused on phosphorus removal achieved under aerobic conditions synchronously with volatile fatty acid (VFA) storage as polyhydroxybutyrate-co-valerate (PHBV). 90.3 ± 3.4 % soluble phosphorus removal was achieved from dairy manure fermenter liquor; influent and effluent concentrations were 38.6 ± 9.5 and 3.7 ± 0.8 mgP/L, respectively. Concurrently, PHBV yield ranged from 0.17 to 0.64 mgCOD/mgCOD, yielding 147-535 mgCOD
/L. No evidence of EBPR mechanisms was observed, nor were canonical phosphorus accumulating organisms present; additionally, the polyphosphate kinase gene was not present in the microbial biomass. Phosphorus removal was primarily associated with biomass growth and secondarily with biomass complexation. Results demonstrate that concurrent PHBV synthesis and phosphorus recovery can be achieved microbially under aerobic dynamic feeding conditions when fed nutrient rich wastewater.

Research focused on interrogating post-anoxic enhanced biological phosphorus removal (EBPR) at bench and pilot scales. Average bench-scale effluent ranged from 0.33 to 1.4 mgP/L, 0.35 to 3.7 mgNH -N/L, and 1.1 to 3.9 mgNO -N/L. Comparatively, the pilot achieved effluent (50th percentile/average) of 0.13/0.2 mgP/L, 9.7/8.2 mgNH -N/L, and 0.38/3.3 mgNO -N/L under dynamic influent and environmental conditions. For EBPR process monitoring, P:C ratio data indicated that 0.2-0.4 molP/molC will result in stable EBPR; relatedly, a target design influent volatile fatty acid (VFA):P ratio would exceed 15 mgCOD/mgP. Post-anoxic EBPR was enriched for Nitrobacter spp. at 1.70%-20.27%, with Parcubacteria also dominating; the former is putatively associated with nitritation and the latter is a putative fermenting heterotrophic organism. Post-anoxic specific denitrification rates (SDNRs) (20°C) ranged from 0.70 to 3.10 mgN/gVSS/h; there was a strong correlation (R  = 0.94) between the SDNR and %Parcubacteria for systems operated at a 20-day solids residence time (SRT). These results suggest that carbon substrate potentially generated by this putative fermenter may enhance post-anoxic EBPR. PRACTITIONER POINTS: Post-anoxic EBPR can achieve effluent of <0.2 mgP/L and <12 mgN/L. The P:C and VFA:P ratios can be predictive for EBPR process monitoring. Post-anoxic EBPR was enriched for Nitrobacter spp. over Nitrospira spp. and also for Parcubacteria, which is a putative fermenting heterotrophic organism. Post-anoxic specific denitrification rates (20°C) ranged from 0.70 to 3.10 mgN/gVSS/h. BLASTn analysis of 16S rDNA PAO primer set was shown to be improved to 93.8% for Ca. Accumulibacter phosphatis and 73.2%-94.0% for all potential PAOs.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with various 3-hydroxyvalerate (3HV) contents biosynthesized by mixed microbial consortia (MMC) fed fermented dairy manure at the large-scale level was assessed over a 3-month period. The thermal, mechanical, and rheological behavior and the chemical structure of the extracted PHBV biopolymers were studied. The recovery of crude PHBV extracted in a large Soxhlet extractor with CHCl3 for 24 h ranged between 20.6% to 31.8% and purified to yield between 8.9% to 26.9% all based on original biomass. C-13-NMR spectroscopy revealed that the extracted PHBVs have a random distribution of 3HV and 3-hydroxybutyrate (3HB) units and with 3HV content between 16% and 24%. The glass transition temperature (T-g) of the extracted PHBVs varied between -0.7 and -7.4 degrees C. Some of the extracted PHBVs showed two melting temperatures (T-m) which the lower T-m(1) ranged between 126.1 degrees C and 159.7 degrees C and the higher T-m(2) varied between 152.1 degrees C and 170.1 degrees C. The weight average molar mass of extracted PHBVs was wide ranging from 6.49 x 10(5) g center dot mol(-1) to 28.0 x 10(5) g center dot mol(-1). The flexural and tensile properties were also determined. The extracted polymers showed a reverse relationship between the 3HV content and Young's modulus, tensile strength, flexural modulus, and flexural strength properties.

Water resource recovery facilities (WRRFs) increasingly must maximize nitrogen and phosphorus removal, but concurrently face challenges to reduce their energy usage and environmental footprint. In particular, biological nutrient removal (BNR), which targets removal of phosphorus and nitrogen, exhibits a large energy demand. However, a BNR process achieving partial oxidation of NH3 to NO2 (nitritation) could reduce energy demands, with secondary environmental emission benefits. Research was conducted on bench-scale systems performing nitritation and nitrification to better understand how mixed microbial consortia, cultured on real wastewater, can sustain nitritation. BNR configurations achieved nitrite accumulation ratios of 64-82%, with excellent overall effluent quality. Applying phylogenetic, transcriptomic, and metabolomic methods, coupled with process monitoring, results indicate that partial nitritation may be induced through a combination of: (1) Employing ammonia-based aeration control, with an ammonia setpoint of 2, 3 mgN/L; (2) Maintaining an aerobic period DO of 1.0-2.0 mg/L; and (3) Operating BNR post-anoxically, integrated within enhanced biological phosphorus removal (EBPR). Significant nitritation was achieved despite the presence Nitrobacter spp., but nitrite oxidoreductase must be functionally impaired or structurally incomplete. Overall, this research demonstrated the value of interrogating a mixed microbial consortia at a macro and molecular level to explore unique metabolic responses such as nitritation.