Dynamic shifts within volatile fatty acid-degrading microbial communities indicate process imbalance in anaerobic digesters.

Buildup of volatile fatty acids (VFAs) in anaerobic digesters (ADs) often results in acidification and process failure. Understanding the dynamics of microbial communities involved in VFA degradation under stable and overload conditions may help optimize anaerobic digestion processes. In this study, five triplicate mesophilic completely mixed AD sets were operated at different organic loading rates (OLRs; 1-6 g chemical oxygen demand [COD] LR-1day-1), and changes in the composition and abundance of VFA-degrading microbial communities were monitored using amplicon sequencing and taxon-specific quantitative PCRs, respectively. AD sets operated at OLRs of 1-4 g COD LR-1day-1 were functionally stable throughout the operational period (120 days) whereas process instability (characterized by VFA buildup, pH decline, and decreased methane production rate) occurred in digesters operated at ≥ 5 g COD LR-1day-1. Though microbial taxa involved in propionate (Syntrophobacter and Pelotomaculum) and butyrate (Syntrophomonas) degradation were detected across all ADs, their abundance decreased with increasing OLR. The overload conditions also inhibited the proliferation of the acetoclastic methanogen, Methanosaeta, and caused a microbial community shift to acetate oxidizers (Tepidanaerobacter acetatoxydans) and hydrogenotrophic methanogens (Methanoculleus). This study's results highlight the importance of operating ADs with conditions that promote the maintenance of microbial communities involved in VFA degradation.

A microfluidic platform for the simultaneous quantification of methanogen populations in anaerobic digestion processes.

Methanogens are a diverse group of archaea that play a critical role in the global carbon cycle. The lack of appropriate molecular tools to simultaneously quantify numerous methanogenic taxa, however, has largely limited our ability to study these communities in a wide variety of habitats, such as anaerobic digesters (ADs). In this study, 34 probe-based quantitative PCR (qPCR) assays were designed to target all known methanogenic genera within the archaeal phylum Euryarchaeota. These qPCR assays were adapted to a high-throughput microfluidic platform, which allowed for the simultaneous detection and absolute quantification of numerous taxa in a single run. The resulting microfluidic qPCR (MFQPCR) platform was successfully used to decipher structure-function relationships among methanogenic communities in four laboratory-scale digesters exposed to a transient organic overload. Twelve of the 34 genera targeted in the MFQPCR were detected in the ADs, similar to results obtained using high-throughput sequencing. The MFQPCR platform and conventional qPCR assays also generated similar quantitative results. The MFQPCR tool developed here will help optimize AD technologies for efficient waste treatment and enhanced biogas production and can facilitate studies that will increase our understanding of methanogenic communities in other environments.

Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach.

Anaerobic digestion is an important biotechnology treatment process for conversion of waste to energy. In this study, a comparative core microbiome approach, i.e., determining taxa that are shared in functioning digesters but not shared in non-functioning digesters, was used to determine microbial taxa that could play key roles for effective anaerobic digestion. Anaerobic digester functions were impaired by adding the broad-spectrum antimicrobial triclosan (TCS) or triclocarban (TCC) at different concentrations, and the core microbiomes in both functioning and non-functioning anaerobic digesters were compared. Digesters treated with high (2500 mg/kg) or medium (450 mg/kg) TCS and high (850 mg/kg) TCC concentrations lost their function, i.e., methane production decreased, effluent volatile fatty acid concentrations increased, and pH decreased. Changes in microbial community diversity and compositions were assessed using 16S rRNA gene amplicon sequencing. Microbial richness decreased significantly in non-functioning digesters (p < 0.001). Microbial community compositions in non-functioning digesters significantly differed from those in functioning digesters (p = 0.001, ANOSIM). Microbes identified as potentially key taxa included previously known fatty acid-degrading syntrophs and amino acid-degrading syntrophs. A diverse group of syntrophs detected in this study had low relative abundance in functioning digesters, suggesting the importance of rare microbes in anaerobic digester operation. The comparative microbiome approach used in this study can be applied to other microbial systems where a community-driven biological phenomena can be observed directly.

Triclocarban Influences Antibiotic Resistance and Alters Anaerobic Digester Microbial Community Structure.

Triclocarban (TCC) is one of the most abundant organic micropollutants detected in biosolids. Lab-scale anaerobic digesters were amended with TCC at concentrations ranging from the background concentration of seed biosolids (30 mg/kg) to toxic concentrations of 850 mg/kg to determine the effect on methane production, relative abundance of antibiotic resistance genes, and microbial community structure. Additionally, the TCC addition rate was varied to determine the impacts of acclimation time. At environmentally relevant TCC concentrations (max detect = 440 mg/kg), digesters maintained function. Digesters receiving 450 mg/kg of TCC maintained function under gradual TCC addition, but volatile fatty acid concentrations increased, pH decreased, and methane production ceased when immediately fed this concentration. The concentrations of the mexB gene (encoding for a multidrug efflux pump) were higher with all concentrations of TCC compared to a control, but higher TCC concentrations did not correlate with increased mexB abundance. The relative abundance of the gene tet(L) was greater in the digesters that no longer produced methane, and no effect on the relative abundance of the class 1 integron integrase encoding gene (intI1) was observed. Illumina sequencing revealed substantial community shifts in digesters that functionally failed from increased levels of TCC. More subtle, yet significant, community shifts were observed in digesters amended with TCC levels that did not inhibit function. This research demonstrates that TCC can select for a multidrug resistance encoding gene in mixed community anaerobic environments, and this selection occurs at concentrations (30 mg/kg) that can be found in full-scale anaerobic digesters (U.S. median concentration = 22 mg/kg, mean = 39 mg/kg).

Pyrolysis of Dried Wastewater Biosolids Can Be Energy Positive.

Pyrolysis is a thermal process that converts biosolids into biochar (a soil amendment), py-oil and py-gas, which can be energy sources. The objectives of this research were to determine the product yield of dried biosolids during pyrolysis and the energy requirements of pyrolysis. Bench-scale experiments revealed that temperature increases up to 500 °C substantially decreased the fraction of biochar and increased the fraction of py-oil. Py-gas yield increased above 500 °C. The energy required for pyrolysis was approximately 5-fold less than the energy required to dry biosolids (depending on biosolids moisture content), indicating that, if a utility already uses energy to dry biosolids, then pyrolysis does not require a substantial amount of energy. However, if a utility produces wet biosolids, then implementing pyrolysis may be costly because of the energy required to dry the biosolids. The energy content of py-gas and py-oil was always greater than the energy required for pyrolysis.

Quantitative detection of syntrophic fatty acid-degrading bacterial communities in methanogenic environments.

In methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H(2), acetate and CO(2) by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.

An inexpensive, reproducible method to quantify activated sludge foaming potential: Validation through lab-scale studies and year-long full-scale sampling campaign.

Activated sludge is a conventional treatment process for biochemical oxygen demand (BOD) and total suspended solids (TSS) removal at water resource recovery facilities (WRRFs). Foaming events are a common operational issue in activated sludge and can lead to decreased treatment efficiency, maintenance issues, and potential environmental health risks. Stable foaming events are caused by biological and chemical drivers (i.e., microbes and surfactants) during the aeration process. However, foaming events are difficult to predict and quantify. We present an inexpensive and easy-to-use method that can be applied at WRRFs to quantify foaming potential. Subsequently, the method was applied over a year-long full-scale study while data on microbial community composition and functional parameters associated with foaming potential were collected from activated sludge samples at South Shore Water Reclamation Facility (WRF) (Oak Creek, WI). Results from the development of the foaming potential method using linear alkylbenzene sulfonate (LAS) showed that the method was reproducible (relative standard deviation <20%) and able to capture changes in foam-inducing constituents. Using full-scale activated sludge samples, higher relative abundance values for the following genera were associated with foaming events: Zoogloea, Flavobacterium, Variovorax, and Bdellovibrio. This is the first report that Variovorx and Bdellovibrio relative abundance was correlated with foaming events in activated sludge. Furthermore, the foaming potential positively correlated (ρ = 0.24) with soluble total nitrogen. Characterizing foaming events through frequent sampling and monitoring of specific genera and functional parameters may allow for predictions and preemptive mitigation efforts to avoid negative consequences in the future. PRACTITIONER POINTS: A reproducible method to measure foaming potential in activated sludge is available. Genera Zoogloea, Flavobacterium, Variovorax, and Bdellovibrio correlated with foaming events. A year-long sampling campaign of activated sludge measuring foaming potential and microbial community composition was conducted at South Shore Water Reclamation Facility in Oak Creek, WI. More research at other facilities with this method is needed to understand links between microbes and foaming.

Current and future approaches to wet weather flow management: A review.

Sewers can become hydraulically overburdened during high-intensity precipitation resulting in untreated water entering receiving streams. Combined (CSOs) and sanitary sewer overflows (SSOs) cause adverse public health and environmental impacts as well as management challenges for many wastewater utilities. This novel review presents information regarding wet weather flow regulation, impacts, and current management methods, and offers ideas for future approaches in the United States. Currently, storage followed by conventional municipal water reclamation facility treatment after precipitation events is often employed. Stand-alone alternative technologies include high-rate solids removal, rapid disinfection, filtration, and green infrastructure. However, most current stand-alone approaches do not address soluble BOD5 or emerging contaminants in stormwater and wastewater. As the needs for wet weather flow management change, future approaches should include a goal of zero overflows and achieve effluent quality as good as or better than conventional treatment. To help achieve zero overflows and complete treatment, the "peaker facility" concept is proposed. The peaker facility often remains idle but treats excess flow when needed. Considering the challenges of remaining idle for long periods, starting up quickly, and handling high flows, chemical oxidation may be an applicable peaker facility component. However, more research and development are needed to determine best practices. PRACTITIONER POINTS: Combined (CSO) and sanitary sewer overflows (SSOs) pose both environmental and public health risks as untreated water is discharged into lakes and rivers during high-intensity rain events. Current stand-alone approaches for managing or treating CSOs focus on particulate BOD/COD and solids removal, and do not typically address soluble BOD or emerging contaminants in stormwater and wastewater (including pathogens). New wet weather policies and regulations encourage more holistic approaches by wastewater utilities, and future approaches should include a zero-overflow goal for all CSOs and SSOs. To help achieve zero overflows, the concept of the "peaker facility" is proposed. Chemical oxidation may be an applicable component of peaker facilities for its short detention time and ability to remove, oxidize, or inactive water impairment-causing contaminants.

Ion exchange nutrient recovery from anaerobic membrane bioreactor permeate.

Nutrient recovery from municipal wastewater was evaluated using anion exchange media loaded with hydrated ferric oxide (HFO) and copper (Cu2+ ) (Dow-HFO-Cu resin) to selectively capture phosphate, followed by clinoptilolite for ammonium removal and recovery. Nutrients were concentrated in the regenerants and recovered as precipitated struvite. Media exchange capacity after multiple ion exchange cycles was determined using permeate from an anaerobic membrane bioreactor (AnMBR) treating synthetic or actual municipal wastewater from a full-scale water reclamation facility. Regeneration through five ion exchange cycles using relatively low concentration regenerant solution (2% NaCl and 0.5% NaOH) resulted in the highest phosphate exchange capacity and phosphate recovery. This regenerant also provided the most consistent ammonium recovery. Column tests treating AnMBR permeate were performed over five ion exchange cycles; Dow-HFO-Cu resin exchange capacities ranged from 1.6 to 2.8 mg PO4 -P/g dry media. A maximum of 94% of the removed phosphate was recovered during regeneration. The rate and extent of regeneration was insensitive to regenerant salt concentrations in the range investigated. Precipitation using a mixture of the spent regeneration brines from the Dow-HFO-Cu resin and clinoptilolite columns produced low molar ratios of Mg:NH4 :PO4 , suggesting that the recovered product was not pure struvite. PRACTITIONER POINTS: Ion exchange-precipitation for the removal and recovery of PO 4 3 - and NH4 + from AnMBR permeate is a promising technology. 2% NaCl + 0.5% NaOH regeneration solution provided the most consistent exchange performance for both phosphate and ammonium recovery. Regenerated Dow-HFO-Cu resin exchange capacity was consistently less than the virgin resin, likely due to copper leaching during regeneration. Molar ratios in the precipitates suggested that the precipitated material was not pure struvite.

Municipal anaerobic digesters for codigestion, energy recovery, and greenhouse gas reductions.

Codigestion of five wastes and municipal wastewater sludge was evaluated using full-scale testing. Synergistic, antagonistic, and neutral outcomes were observed depending on codigestate identity and concentration, highlighting the value of careful blending. Yeast waste resulted in notable synergism, increasing biogas production by over 50%, whereas aircraft deicing waste resulted in antagonism at high loadings and neutral outcomes at lower loadings. Restaurant waste codigestion resulted in neutral outcomes. The synergisim with yeast codigestates may have resulted from trace nutrients (i.e., iron, nickel, and cobalt) in the wastes that increased microbiological activity. Antagonist outcomes with deicing waste may have been the result of organic overload or inhibitory deicer constituents. Codigestion of wastes at the feed rates investigated was estimated to produce 0.50 ML/d of methane having an energy equivalent of 17 500 MJ/d. This was estimated to reduce net carbon dioxide emissions by 560 tonnes/y.

Thermophilic aerobic granular biomass for enhanced settleability.

Aerobic biological wastewater treatment at thermophilic (ca. 55 degrees C) temperatures notoriously produces biomass that flocculates poorly or not at all. Contrary to this, thermophilic aerobic biomass that settled well in sequencing batch reactors was cultured with sludge volume index (SVI) values as low as 60mL/g. A mixture of granular and flocculant biomass resulted when closed reactors were sparged with recirculated reactor headspace gas containing some air, whereas a conventionally aerated control reactor sparged with air alone contained dispersed growth that did not flocculate. Maximum granule diameter was from 1.2 to 1.9mm, and granule resistance to disintegration was comparable to aerobic mesophilic granules. Two bacteria were isolated and identified as Anoxybacillus flavothermus and Pseudoxanthomonas taiwanensis as determined by partial 16S rDNA sequencing. Anoxybacilli species are alkaliphilic or alkalitolerant, with the type species having an obligate requirement for carbonate, even when grown on glucose. We postulate that high alkalinity and CO(2) may select for a population of aerobic thermophilies that flocculates and granulates.

Microbial source tracking using host specific FAME profiles of fecal coliforms.

The objective of this study was to investigate the host-specific differences in fatty acid methyl ester (FAME) profiles of fecal coliforms (FC). A known-source library was constructed with 314 FC isolates cultured from 6 possible sources of fecal pollution; 99 isolates from sewage; 29 from bovine; 29 from poultry; 50 from swine; 46 from waterfowl; and 61 from deer. It was found that the hydroxy FAMEs 12:0 2 OH, 12:03 OH, and 14:02 OH were exclusively associated with isolates of human origin. On the other hand, 3 saturated FAMEs, 10:0, 15:0, and 18:0 were found only in isolates from non-human sources, 15:0 being associated with livestock samples only. In addition to the presence of these signature FAMEs, the mean relative masses of 16:1 omega7c and 16:1 ISO/14:03 OH were significantly different between the isolates of human and non-human origins. A linear discriminant function differentiated FC isolates of human origin from those of livestock and wildlife origin at 99% accuracy. These results strongly suggest that the FAME profiles of FC show statistically significant host specificity and may have the potential to be used as a phenotypic microbial source tracking tool.

Chronic exposure to triclosan sustains microbial community shifts and alters antibiotic resistance gene levels in anaerobic digesters.

Triclosan, an antimicrobial chemical found in consumer personal care products, has been shown to stimulate antibiotic resistance in pathogenic bacteria. Although many studies focus on antibiotic resistance pertinent to medical scenarios, resistance developed in natural and engineered environments is less studied and has become an emerging concern for human health. In this study, the impacts of chronic triclosan (TCS) exposure on antibiotic resistance genes (ARGs) and microbial community structure were assessed in lab-scale anaerobic digesters. TCS concentrations from below detection to 2500 mg kg(-1) dry solids were amended into anaerobic digesters over 110 days and acclimated for >3 solid retention time values. Four steady state TCS concentrations were chosen (30-2500 mg kg(-1)). Relative abundance of mexB, a gene coding for a component of a multidrug efflux pump, was significantly higher in all TCS-amended digesters (30 mg kg(-1) or higher) relative to the control. TCS selected for bacteria carrying tet(L) and against those carrying erm(F) at concentrations which inhibited digester function; the pH decrease associated with digester failure was suspected to cause this selection. Little to no impact of TCS was observed on intI1 relative abundance. Microbial communities were also surveyed by high-throughput 16S rRNA gene sequencing. Compared to the control digesters, significant shifts in community structure towards clades containing commensal and pathogenic bacteria were observed in digesters containing TCS. Based on these results, TCS should be included in studies and risk assessments that attempt to elucidate relationships between chemical stressors (e.g. antibiotics), antibiotic resistance genes, and public health.

Bioaugmentation for improved recovery of anaerobic digesters after toxicant exposure.

Bioaugmentation was investigated as a method to decrease the recovery period of anaerobic digesters exposed to a transient toxic event. Two sets of laboratory-scale digesters (SRT = 10 days, OLR = 2 g COD/L-day), started with inoculum from a digester stabilizing synthetic municipal wastewater solids (MW) and synthetic industrial wastewater (WW), respectively, were transiently exposed to the model toxicant, oxygen. Bioaugmented digesters received 1.2 g VSS/L-day of an H2-utilizing culture for which the archaeal community was analyzed. Soon after oxygen exposure, the bioaugmented digesters produced 25-60% more methane than non-bioaugmented controls (p < 0.05). One set of digesters produced lingering high propionate concentrations, and bioaugmentation resulted in significantly shorter recovery periods. The second set of digesters did not display lingering propionate, and bioaugmented digesters recovered at the same time as non-bioaugmented controls. The difference in the effect of bioaugmentation on recovery may be due to differences between microbial communities of the digester inocula originally employed. In conclusion, bioaugmentation with an H(2)-utilizing culture is a potential tool to decrease the recovery period, decrease propionate concentration, and increase biogas production of some anaerobic digesters after a toxic event. Digesters already containing rapidly adaptable microbial communities may not benefit from bioaugmentation, whereas other digesters with poorly adaptable microbial communities may benefit greatly.

Nonylphenol ethoxylates and other additives in aircraft deicers, antiicers, and waters receiving airport runoff.

Samples of nine different formulations of aircraft deicer and antiicer fluids (ADAF) were screened for the presence of selected surfactants. Nonylphenol ethoxylates (NPnEO) were identified in three ADAF formulations, octylphenol ethoxylates were identified in two formulations, and six formulations contained alcohol ethoxylates. A preliminary field study was conducted at General Mitchell International Airport, Milwaukee, WI, to quantify NPnEO (n = 1-15) and one of its byproducts, nonylphenol (NP), in airport runoff. Samples were collected from two airport outfalls, from the receiving stream, and from an upstream reference site during intensive ADAF application events. NPnEO was measured at concentrations up to 1190microg/L in airport outfall samples, up to 77 ug/L in samples from the receiving stream and less than 5.0 microg/L from the upstream reference. Concentrations of glycol and other ADAF-related constituents, including NPnEO, were reduced by approximately 1 order of magnitude between the outfall sites and the receiving stream site; however, concentrations of NP in the receiving stream remained similar to those from the outfalls (< 0.04 microg/L at the upstream reference, 0.98 and 7.67 microg/L at outfalls, and 3.89 microg/L in the receiving stream). The field data suggest that NP is generated through degradation of NPnEO from airport runoff.