Investigating the viral ecology and contribution to the microbial ecology in full-scale mesophilic anaerobic digesters.

In an attempt to assess the diversity of viruses and their potential to modulate the metabolism of functional microorganisms in anaerobic digesters, we collected digestate from three mesophilic anaerobic digesters in full-scale wastewater treatment plants treating real municipal wastewater. The reads were analyzed using bioinformatics algorithms to elucidate viral diversity, identify their potential role in modulating the metabolism of functional microorganisms, and provide essential genomic information for the potential use of virus-mediated treatment in controlling the anaerobic digester microbiome. We found that Siphoviridae was the dominant family in mesophilic anaerobic digesters, followed by Myoviridae and Podoviridae. Lysogeny was prevalent in mesophilic anaerobic digesters as the majority of metagenome-assembled genomes contained at least one viral genome within them. One virus within the genome of an acetoclastic methanogen (Methanothrix soehngenii) was observed with a gene (fwdE) acquired via lateral transfer from hydrogenotrophic methanogens. The virus-mediated acquisition of fwdE gene enables possibility of mixotrophic methanogenesis in Methanothrix soehngenii. This evidence highlighted that lysogeny provides fitness advantage to methanogens in anaerobic digesters by adding flexibility to changing substrates. Similarly, we found auxiliary metabolic genes, such as cellulase and alpha glucosidase, of bacterial origin responsible for sludge hydrolysis in viruses. Additionally, we discovered novel viral genomes and provided genomic information on viruses infecting acidogenic, acetogenic, and pathogenic bacteria that can potentially be used for virus-mediated treatment to deal with the souring problem in anaerobic digesters and remove pathogens from biosolids before land application. Collectively, our study provides a genome-level understanding of virome in conjunction with the microbiome in anaerobic digesters that can be used to optimize the anaerobic digestion process for efficient biogas generation.

Stringent Response of Cyanobacteria and Other Bacterioplankton during Different Stages of a Harmful Cyanobacterial Bloom.

We conducted a field study to investigate the role of stringent response in cyanobacteria and coexisting bacterioplankton during nutrient-deprived periods at various stages of bloom in a freshwater lake (Utah Lake) for the first time. Using metagenomics and metatranscriptomics analyses, we examined the cyanobacterial ecology and expression of important functional genes related to stringent response, N and P metabolism, and regulation. Our findings mark a significant advancement in understanding the mechanisms by which toxic cyanobacteria survive and proliferate during nitrogen (N) and phosphorus (P) limitations. We successfully identified and analyzed the metagenome-assembled genomes (MAGs) of the dominant bloom-forming cyanobacteria, namely, Dolichospermum circinale, Aphanizomenon flos-aquae UKL13-PB, Planktothrix agardhii, and Microcystis aeruginosa. By mapping RNA-seq data to the coding sequences of the MAGs, we observed that these four prevalent cyanobacteria species activated multiple functions to adapt to the depletion of inorganic nutrients. During and after the blooms, the four dominant cyanobacteria species expressed high levels of transcripts related to toxin production, such as microcystins (mcy), anatoxins (ana), and cylindrospermopsins (cyr). Additionally, genes associated with polyphosphate (poly-P) storage and the stringent response alarmone (p)ppGpp synthesis/hydrolysis, including ppk, relA, and spoT, were highly activated in both cyanobacteria and bacterioplankton. Under N deficiency, the main N pathways shifted from denitrification and dissimilatory nitrate reduction in bacterioplankton toward N2-fixing and assimilatory nitrate reduction in certain cyanobacteria with a corresponding shift in the community composition. P deprivation triggered a stringent response mediated by spoT-dependent (p)ppGpp accumulation and activation of the Pho regulon in both cyanobacteria and bacterioplankton, facilitating inorganic and organic P uptake. The dominant cyanobacterial MAGs exhibited the presence of multiple alkaline phosphatase (APase) transcripts (e.g., phoA in Dolichospermum, phoX in Planktothrix, and Microcystis), suggesting their ability to synthesize and release APase enzymes to convert ambient organic P into bioavailable forms. Conversely, transcripts associated with bacterioplankton-dominated pathways like denitrification were low and did not align with the occurrence of intense cyanoHABs. The strong correlations observed among N, P, stringent response metabolisms and the succession of blooms caused by dominant cyanobacterial species provide evidence that the stringent response, induced by nutrient limitation, may activate unique N and P functions in toxin-producing cyanobacteria, thereby sustaining cyanoHABs.

Effect of different types of volatile fatty acids on the performance and bacterial population in a batch reactor performing biological nutrient removal.

Different ratios of four volatile fatty acids (VFAs) were used as the primary feed to a laboratory scale biological nutrient reactor during four operational stages. The reactor performed efficiently over 500 days of operation with over 90% dissolved phosphorus and over 98% ammonium-nitrogen (NH4+-N) removal. Through in the first experimental phase, acetate and propionate were present in a significant proportion as carbon sources, the relative abundance of Candidatus Accumulibacter, a potential polyphosphate accumulating organism, increased from 10% to 57% and the Defluviicoccus genus, a known glycogen accumulating organism (GAO), decreased from 41% to 5%. Further tests indicated the presence of denitrifying phosphorus accumulating organisms (DPAO) belonging to Clade IIC, that could use nitrite as the electron acceptor during P-uptake. In general, VFAs favored the increase of the genus Defluviicoccus and Candidatus Accumulibacter. High relative abundance of Defluviicoccus did not affect the stability and the performance of the BNR process.

Retention and recycling of granules in continuous flow-through system to accomplish denitrification and perchlorate reduction.

This study employed a completely anoxic reactor and a gravity-settling design for continuously capturing and separating granules from flocculated biomass, and recycling granules back to the main reactor. The average chemical oxygen demand (COD) removal in the reactor was 98%. Average nitrate (NO3--N) and perchlorate (ClO4-) removal efficiencies of 99% and 74 ± 19% were observed, respectively. Preferential utilization of NO3- over ClO4- led to COD limiting conditions, which resulted in ClO4- in the effluent. The average granule diameter in continuous flow-through bubble-column (CFB) anoxic granular sludge (AxGS) bioreactor was 6325 ± 2434 µm, and the average SVI30/SVI1 was >90 % throughout its operation. 16s rDNA amplicon sequencing revealed Proteobacteria (68.53%-88.57%) and Dechloromonas (10.46%-54.77%) to be the most abundant phylum and the genus present in reactor sludge representing the denitrifying and ClO4- reducing microbial community. This work represents a pioneering development of CFB-AxGS bioreactor.

Mainstream nitrogen removal from low temperature and low ammonium strength municipal wastewater using hydrogel-encapsulated comammox and anammox.

Application of partial nitritation (PN)-anammox to mainstream wastewater treatment faces challenges in low water temperature and low ammonium strength. In this study, a continuous flow PN-anammox reactor with hydrogel-encapsulated comammox and anammox was designed and operated for nitrogen removal from mainstream wastewater with low temperature. Long-term operation with synthetic and real wastewater as the feed demonstrated nearly complete ammonium and total inorganic nitrogen (TIN) removal by the reactor at temperatures as low as 10 °C. A significantly decreased nitrogen removal performance and biomass activity was observed in the reactor at 4 °C before a selective heating strategy was employed. A novel heating technology using radiation to heat carbon black co-encapsulated in the hydrogel matrix with biomass was used to selectively heat biomass but not water in the treatment system. This selective heating technology enabled nearly complete ammonium removal and 89.4 ± 4.3 % TIN removal at influent temperature of 4 °C and reactor temperature 5 °C. Activity tests suggested selective heating brought the biomass activity at influent temperatures of 4 °C and reactor temperature 5 °C to a level comparable to that at 10 °C. Comammox and anammox were consistently present in the system and spatially organized in the hydrogel beads as revealed by qPCR and fluorescence in-situ hybridization (FISH). The abundance of comammox largely decreased by 3 orders of magnitude during the operation at 4 °C, and rapidly recovered after the application of selective heating. The anammox-comammox technology tested in this study essentially enabled mainstream shortcut nitrogen removal, and the selective heating ensured good performance of the technology at temperature as low as 5 °C.

Anoxic granular activated sludge process for simultaneous removal of hazardous perchlorate and nitrate.

An airtight, anoxic bubble-column sequencing batch reactor (SBR) was developed for the rapid cultivation of perchlorate (ClO4-) and nitrate (NO3-) reducing granular sludge (GS) in this study. Feast/famine conditions and shear force selection pressures in tandem with a short settling time (2-min) as a hydraulic section pressure resulted in the accelerated formation of anoxic granular activated sludge (AxGS). ClO4- and NO3- were efficiently (>99.9%) reduced over long-term (>500-d) steady-state operation. Specific NO3- reduction, ClO4- reduction, chloride production, and non-purgeable dissolved organic carbon (DOC) oxidation rates of 5.77 ± 0.54 mg NO3--N/g VSS·h, 8.13 ± 0.74 mg ClO4-/g VSS·h, 2.40 ± 0.40 mg Cl-/g VSS·h, and 16.0 ± 0.06 mg DOC/g VSS·h were recorded within the reactor under steady-state conditions, respectively. The AxGS biomass cultivated in this study exhibited faster specific ClO4- reduction, NO3- reduction, and DOC oxidation rates than flocculated biomass cultivated under similar conditions and AxGS biomass operated in an up-flow anaerobic sludge blank (UASB) bioreactor receiving the same influent loading. EPS peptide identification revealed a suite of extracellular catabolic enzymes. Dechloromonas species were present in high abundance throughout the entirety of this study. This is one of the initial studies on anoxic granulation to simultaneously treat hazardous chemicals and adds to the science of the granular activated sludge process.

Volatile fatty acid production from primary and secondary sludges to support efficient nutrient management.

Enhanced hydrolysis of sludges during fermentation is an important factor to achieve solubilization of complex carbon sources and increase the amount of soluble COD that microorganisms could use as food during biological nutrient removal processes. This research shows that a combination of mixing, bioaugmentation, and co-fermentation can be used to increase the hydrolysis of sludges and enhanced the production of volatile fatty acids (VFA). Mixing of primary sludge (PS) at 350 revolutions per minute (RPM) during fermentation increased the hydrolysis of the sludge and increased the soluble chemical oxygen demand (sCOD) by 72% compared to no mixing. Mixing also increased the production of VFA by 60% compared to no mixing conditions. PS hydrolysis was also evaluated using bioaugmentation with the bacteria Bacillus amyloliquefacients, a known producer of the biosurfactant surfactin. Results showed that bioaugmentation enhanced the hydrolysis of the PS by increasing the amount of soluble carbohydrates and soluble proteins present in the form of sCOD. Methanogenesis experiments performed with co-fermentation of decanted primary sludge (PS) and raw waste-activated sludge (WAS) at 75:25 and 50:50 ratios displayed a decreased in production of total biogas by 25.58% and 20.95% and a reduction on methane production by 20.00% and 28.76% respectively, compared to co-fermentation of raw sludges. Compared to fermentation of the sludges separately, co-fermentation of PS and WAS increased the production of VFA and it was determined that 50:50 was the optimum co-fermentation ratio for production of VFA while reducing the reintroduction of nutrients produced during the fermentation process to BNR processes.

Simultaneous anaerobic carbon and nitrogen removal from primary municipal wastewater with hydrogel encapsulated anaerobic digestion sludge and AOA-anammox coated hollow fiber membrane.

In this study, a one-stage continuous-flow membrane-hydrogel reactor integrating both partial nitritation-anammox (PN-anammox) and anaerobic digestion (AD) was designed and operated for simultaneous autotrophic nitrogen (N) and anaerobic carbon (C) removal from mainstream municipal wastewater. In the reactor, a synthetic biofilm consisting of anammox biomass and pure culture ammonia oxidizing archaea (AOA) were coated onto and maintained on a counter-diffusion hollow fiber membrane to autotrophically remove nitrogen. Anaerobic digestion sludge was encapsulated in hydrogel beads and placed in the reactor to anaerobically remove COD. During the pilot operation at three operating temperature (25, 16 and 10 °C), the membrane-hydrogel reactor demonstrated stable anaerobic COD removal (76.2 ± 15.5 %) and membrane fouling was successfully suppressed allowing a relatively stable PN-anammox process. The reactor demonstrated good nitrogen removal efficiency, with an overall removal efficiency of 95.8 ± 5.0 % for NH4+-N and 78.9 ± 13.2 % for total inorganic nitrogen (TIN) during the entire pilot operation. Reducing the temperature to 10 °C caused a temporary reduction in nitrogen removal performance and abundances of AOA and anammox. However, the reactor and microbes demonstrated the ability to adapt to the low temperature spontaneously with recovered nitrogen removal performance and microbial abundances. Methanogens in hydrogel beads and AOA and anammox on the membrane were observed in the reactor by qPCR and 16S sequencing across all operational temperatures.

A Klebsiella pneumoniae NDM-1+ bacteriophage: Adaptive polyvalence and disruption of heterogenous biofilms.

Bacteriophage KL-2146 is a lytic virus isolated to infect Klebsiella pneumoniae BAA2146, a pathogen carrying the broad range antibiotic resistance gene New Delhi metallo-betalactamase-1 (NDM-1). Upon complete characterization, the virus is shown to belong to the Drexlerviridae family and is a member of the Webervirus genus located within the (formerly) T1-like cluster of phages. Its double-stranded (dsDNA) genome is 47,844 bp long and is predicted to have 74 protein-coding sequences (CDS). After challenging a variety of K. pneumoniae strains with phage KL-2146, grown on the NDM-1 positive strain BAA-2146, polyvalence was shown for a single antibiotic-sensitive strain, K. pneumoniae 13,883, with a very low initial infection efficiency in liquid culture. However, after one or more cycles of infection in K. pneumoniae 13,883, nearly 100% infection efficiency was achieved, while infection efficiency toward its original host, K. pneumoniae BAA-2146, was decreased. This change in host specificity is reversible upon re-infection of the NDM-1 positive strain (BAA-2146) using phages grown on the NDM-1 negative strain (13883). In biofilm infectivity experiments, the polyvalent nature of KL-2146 was demonstrated with the killing of both the multidrug-resistant K. pneumoniae BAA-2146 and drug-sensitive 13,883 in a multi-strain biofilm. The ability to infect an alternate, antibiotic-sensitive strain makes KL-2146 a useful model for studying phages infecting the NDM-1+ strain, K. pneumoniae BAA-2146. GRAPHICAL ABSTRACT.

Biodegradation of insensitive munition (IM) formulations: IMX-101 and IMX-104 using aerobic granule technology.

A laboratory-scale aerobic granular sludge (AGS) sequencing batch bioreactor (SBR) was initiated in this study for the biodegradation of hazardous insensitive munition (IM) formulation constituents; 2,4-dinitroanisole (DNAN), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 1-nitroguanidine (NQ), and 3-nitro-1,2,4-triazol-5-one (NTO). Efficient (bio)transformation of the influent DNAN and NTO was achieved throughout reactor operation with removal efficiencies greater than 95%. An average removal efficiency of 38.4 ± 17.5% was recorded for RDX. NQ was only slightly removed (3.96 ± 4.15%) until alkalinity was provided in the influent media, which subsequently increased the NQ removal efficiency up to an average of 65.8 ± 24.4%. Batch experiments demonstrated a competitive advantage for aerobic granular biofilms over flocculated biomass for the (bio)transformation DNAN, RDX, NTO, and NQ, as aerobic granules were capable of reductively (bio)transforming each IM compound under bulk aerobic conditions while flocculated biomass could not, thus demonstrating the contribution of inner oxygen-free zones within aerobic granules. A variety of catalytic enzymes were identified in the extracellular polymeric matrix of the AGS biomass. 16 S rDNA amplicon sequencing found Proteobacteria (27.2-81.2%) to be the most abundant phyla, with many genera associated with nutrient removal as well as genera previously described in relation to the biodegradation of explosives or related compounds.

Integration of EBPR with mainstream anammox process to treat real municipal wastewater: Process performance and microbiology.

The mainstream application of anaerobic ammonium oxidation (anammox) for sustainable N removal remains a challenge. Similarly, with recent additional stringent regulations for P discharges, it is imperative to integrate N with P removal. This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining biofilm anammox with flocculent activated sludge for enhanced biological P removal (EBPR). This technology was assessed in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with a hydraulic retention time of 8.8 h. After a steady state operation was reached, robust reactor performance was obtained with average TIN and P removal efficiencies of 91.3 ± 4.1% and 98.4 ± 2.4%, respectively. The average TIN removal rate recorded over the last 100 d of reactor operation was 118 mg/L·d, which is a reasonable number for mainstream applications. The activity of denitrifying polyphosphate accumulating organisms (DPAOs) accounted for nearly 15.9% of P-uptake during the anoxic phase. DPAOs and canonical denitrifiers removed approximately 5.9 mg TIN/L in the anoxic phase. Batch activity assays, which showed that nearly 44.5% of TIN were removed by the biofilms during the aerobic phase. The functional gene expression data also confirmed anammox activities. The IFAS configuration of the SBR allowed operation at a low solid retention time (SRT) of 5-d without washing out biofilm ammonium-oxidizing and anammox bacteria. The low SRT, combined with low dissolved oxygen and intermittent aeration, provided a selective pressure to washout nitrite-oxidizing bacteria and glycogen-accumulating organisms, as relative abundances of.

Enhanced Bio-P removal: Past, present, and future - A comprehensive review.

Excess amounts of phosphorus (P) and nitrogen (N) from anthropogenic activities such as population growth, municipal and industrial wastewater discharges, agriculture fertilization and storm water runoffs, have affected surface water chemistry, resulting in episodes of eutrophication. Enhanced biological phosphorus removal (EBPR) based treatment processes are an economical and environmentally friendly solution to address the present environmental impacts caused by excess P present in municipal discharges. EBPR practices have been researched and operated for more than five decades worldwide, with promising results in decreasing orthophosphate to acceptable levels. The advent of molecular tools targeting bacterial genomic deoxyribonucleic acid (DNA) has also helped us reveal the identity of potential polyphosphate-accumulating organisms (PAO) and denitrifying PAO (DPAO) responsible for the success of EBPR. Integration of process engineering and environmental microbiology has provided much-needed confidence to the wastewater community for the successful implementation of EBPR practices around the globe. Despite these successes, the process of EBPR continues to evolve in terms of its microbiology and application in light of other biological processes such as anaerobic ammonia oxidation and on-site carbon capture. This review provides an overview of the history of EBPR, discusses different operational parameters critical for the successful operation of EBPR systems, reviews current knowledge of EBPR microbiology, the influence of PAO/DPAO on the disintegration of microbial communities, stoichiometry, EBPR clades, current practices, and upcoming potential innovations.

Response of mixed community anammox biomass against sulfide, nitrite and recalcitrant carbon in terms of inhibition coefficients and functional gene expressions.

Anaerobic ammonium oxidation (anammox) has evolved as a carbon and energy-efficient nitrogen management bioprocess. However, factors such as inhibitory chemicals still challenge the easy operation of this powerful bioprocess. This research systematically evaluated the inhibition kinetics of sulfide, nitrite, and recalcitrant carbon under a genomic framework. The inhibition at the substrate and genetic levels of sulfide, nitrite and recalcitrant carbon on anammox activity was studied using batch tests. Nitrite inhibition of anammox followed substrate inhibition and was best described by the Aiba model with an inhibition coefficient [Formula: see text] of 324.04 mg N/L. Hydrazine synthase (hzsB) gene (anammox biomarker) expression was increased over time when incubated with nitrite up to 400 mg N/L. However, despite having the highest specific nitrite removal (SNR), the expression of hzsB at 100 and 200 mg N/L of nitrite was more muted than in most other samples with lower SNRs. Sulfide severely inhibited anammox activities. The inhibition was fitted with a Monod-based model with a [Formula: see text] of 4.39 mg S/L. At a sulfide concentration of 5 mg/L, the hzsB expression decreased throughout the experiment from its original value at he beginning. Recalcitrant carbon of filtrate from thermal hydrolysis process pretreated anaerobic digester had a minimal effect on maximum specific anammox activity (MSAA), and thus the value of the inhibition coefficient could not be calculated. At the same time, its hzsB expression profile was similar to that in the control. Resiliency and recovery tests indicated that the inhibition of nitrite (up to 400 mg N/L) and recalcitrant carbon (in 100% filtrate) were reversible. About 32% of MSAA was recovered after repeated exposures to sulfide at 2.5 mg/L, while at 5 mg/L, the inhibition was irreversible. Findings from this study will be helpful for the successful design and implementation of anammox in full-scale applications.

Nitrogen fixation contribution to nitrogen cycling during cyanobacterial blooms in Utah Lake.

Nitrogen (N) cycling is an essential process in lake systems and N-fixation is an important component of it. Recent studies have also found that nitrate reduction through heterotrophic denitrification in lake systems did not prevent harmful cyanobacterial blooms, but instead, may have favored the dominance of N2-fixing cyanobacteria. The overall objective of this study was to estimate nitrogen fixation rates and the expressions of associated nitrogenase (nif gene) functional gene at several sites at different occasions in freshwater Utah Lake. For comparison purposes, one time sampling was also conducted in the brackish Farmington Bay of Great Salt Lake (GSL). The microbial ecology of the top 20-cm of surface water was investigated to assess the dominant cyanobacterial communities and N-related metabolisms. Our study revealed that Dolichospermum and Nodularia were potential N2-fixers for Utah Lake and brackish Farmington Bay, respectively. The in situ N2-fixation rates were 0-0.73 nmol N hr-1L-1 for Utah Lake and 0-0.85 nmol N hr-1L-1 for Farmington Bay, and these rates positively correlated with the abundance and expressions of the nif gene. In addition, nitrate reduction was measured in sediment (0.002-0.094 mg N VSS-1 hr-1). Significantly positive correlations were found among amoA, nirS and nirK abundance (R = 0.56-0.87, p < 0.05, Spearman) in both lakes. An exception was the lower nirK gene abundance detected at one site in Farmington Bay where high ammonium retentions were also detected. Based on a mass balance approach, we concluded that the amount of inorganic N loss through denitrification still exceeded the N input by N2-fixation, much like in most lakes, rivers, and marine ecosystems. This indicates that N cycling processes such as denitrification mediated by heterotrophic bacteria contributes to N-export from the lakes resulting in N limitations.

Integrating Virus Monitoring Strategies for Safe Non-potable Water Reuse.

Wastewater reclamation and reuse have the potential to supplement water supplies, offering resiliency in times of drought and helping meet increased water demands associated with population growth. Non-potable water reuse represents the largest potential reuse market. Yet economic constraints for new water reuse infrastructure and safety concerns due to microbial water quality, and especially viral pathogen exposure, limit widespread implementation of water reuse. Cost-effective, real-time methods to measure or indicate viral quality of recycled water would do much to instill greater confidence in the practice. This manuscript discusses advancements in monitoring and modeling of viral health risks in the context of water reuse. First, we describe the current wastewater reclamation processes and treatment technologies with an emphasis on virus removal. Second, we review technologies for the measurement of viruses, both culture- and molecular-based, along with their advantages and disadvantages. We introduce promising viral surrogates and specific pathogenic viruses that can serve as indicators of viral risk for water reuse. We suggest metagenomic analyses for viral screening and flow cytometry for quantification of virus-like particles as new approaches to complement more traditional methods. Third, we describe modeling to assess health risks through quantitative microbial risk assessments (QMRAs), the most common strategy to couple data on virus concentrations with human exposure scenarios. We then explore the potential of artificial neural networks (ANNs) to incorporate suites of data from wastewater treatment processes, water quality parameters, and viral surrogates. We recommend ANNs as a means to utilize existing water quality data, alongside new complementary measures of viral quality, to achieve cost-effective strategies to assess risks associated with infectious human viruses in recycled water. Given the review, we conclude that technologies are ready for identifying and implementing viral surrogates for health risk reduction in the next decade. Incorporating modeling with monitoring data would likely result in more robust assessment of water reuse risk.

Simultaneous reduction of perchlorate and nitrate using fast-settling anoxic sludge.

Fast-settling, anoxic sludge (FAS) was cultivated and utilized in this study to simultaneously reduce elevated levels of perchlorate and nitrate in an anaerobic sequencing batch reactor (AnSBR). Average perchlorate and nitrate removal efficiencies of 96.5 ± 8.44 % and 99.8 ± 0.32 %, respectively, were achieved from an average perchlorate and nitrate loading rate of 159 ± 101 g ClO4-/m3·d and 10.8 ± 7.25 g NO3--N/m3·d, respectively, throughout long-term operation (>500-d). Batch activity tests revealed a preferential utilization of nitrate over perchlorate, where significant perchlorate reduction inhibition occurred when nitrate was present as a competing electron acceptor under carbon-limiting conditions. Specific perchlorate and nitrate reduction rates were shown to increase as the hydraulic retention time (HRT) of the AnSBR was step-wise decreased and subsequently the perchlorate and nitrate loading rates were step-wise increased. Functional, mRNA-based expression of the nitrite reductase (nirS and nirK), nitrous oxide reductase (nosZ), perchlorate reductase subunit A (pcrA), and the chlorite dismutase (cld) genes illustrated the simultaneous activity of heterotrophic denitrification and perchlorate reduction occurring throughout a complete standard reactor operational cycle, and allowed for expression trends to be documented as the HRT of the AnSBR was reduced from 5-d to 1.25-d. Nitrous oxide (N2O) production was detected as a result of incomplete denitrification, where the largest N2O production occurred at the highest nitrate loading rates investigated in this study. Thauera species were heavily enriched at a longer HRT of 5-d, but were out-competed by Dechloromonas species as the HRT of the AnSBR was step-wise reduced to 1.25-d.

Viruses and Their Interactions With Bacteria and Archaea of Hypersaline Great Salt Lake.

Viruses play vital biogeochemical and ecological roles by (a) expressing auxiliary metabolic genes during infection, (b) enhancing the lateral transfer of host genes, and (c) inducing host mortality. Even in harsh and extreme environments, viruses are major players in carbon and nutrient recycling from organic matter. However, there is much that we do not yet understand about viruses and the processes mediated by them in the extreme environments such as hypersaline habitats. The Great Salt Lake (GSL) in Utah, United States is a hypersaline ecosystem where the biogeochemical role of viruses is poorly understood. This study elucidates the diversity of viruses and describes virus-host interactions in GSL sediments along a salinity gradient. The GSL sediment virosphere consisted of Haloviruses (32.07 ± 19.33%) and members of families Siphoviridae (39.12 ± 19.8%), Myoviridae (13.7 ± 6.6%), and Podoviridae (5.43 ± 0.64%). Our results demonstrate that salinity alongside the concentration of organic carbon and inorganic nutrients (nitrogen and phosphorus) governs the viral, bacteria, and archaeal diversity in this habitat. Computational host predictions for the GSL viruses revealed a wide host range with a dominance of viruses that infect Proteobacteria, Actinobacteria, and Firmicutes. Identification of auxiliary metabolic genes for photosynthesis (psbA), carbon fixation (rbcL, cbbL), formaldehyde assimilation (SHMT), and nitric oxide reduction (NorQ) shed light on the roles played by GSL viruses in biogeochemical cycles of global relevance.

Differences in chlorine and peracetic acid disinfection kinetics of Enterococcus faecalis and Escherichia fergusonii and their susceptible strains based on gene expressions and genomics.

This study was conducted to investigate mechanisms of cross-resistance to chlorine and peracetic acid (PAA) disinfectants by antibiotic-resistant bacteria. Our study evaluated chlorine and PAA based disinfection kinetics of erythromycin-resistant Enterococcus faecalis, meropenem-resistant Escherichia fergusonii, and susceptible strains of these species. Using the integrated second-order disinfectant decay model and first-order Chick-Watson's Law, it was found that the meropenem-resistant Escherichia fergusonii strain showed significantly less log inactivation compared to the susceptible E. fergusonii strain in response to both chlorine and PAA disinfection (p-value = 0.059, 3.5 × 10-6). On the other hand, the susceptible Enterococcus faecalis strain showed similar log inactivation compared to the erythromycin-resistant strain in response to either treatment (p-value = 0.075, 0.28). Meropenem-resistant E. fergusonii showed an increase in gene expression of New Delhi metallo-ß-lactamase (blaNDM-1) gene to chlorine, but there was no increase in expression to PAA. Whole genome sequencing (WGS) was then conducted to elucidate the differences in genes among both resistant and susceptible table E. fergusonii strains. The average nucleotide identity (ANI) analysis of the draft genomes (>97% similarity) suggests that meropenem-resistant E. fergusonii (S1) and meropenem-susceptible E. fergusonii (S2) are the same species but different strains. Both strains have the same genes for oxidative stress pathways, oxidative scavenger genes, and nearly 40 different antibiotic efflux pump genes. The chromosomal and plasmid draft genomes of meropenem-resistant and susceptible E. fergusonii strains each have 65 and 52 antibiotic resistance genes, respectively. Of these, the resistant E. fergusonii strain harbored the extended-spectrum beta-lactamases blaCTX-M-15 and blaTEM-1 genes located on the chromosome, and a blaTEM-1 gene on the plasmid. The overall findings of this study are significant, as they reveal that antibiotic-resistant and susceptible strains of E. fergusonii exhibit different responses towards chlorine and PAA disinfection.

Prevalence of SARS-CoV-2 genes in water reclamation facilities: From influent to anaerobic digester.

Several treatment plants were sampled for influent, primary clarifier sludge, return activated sludge (RAS), and anaerobically digested sludge throughout nine weeks during the summer of the COVID-19 pandemic. Primary clarifier sludge had a significantly higher number of SARS-CoV-2 gene copy number per liter (GC/L) than other sludge samples, within a range from 1.0 × 105 to 1.0 × 106 GC/L. Gene copy numbers in raw influent significantly correlated with gene copy numbers in RAS in Silver Creek (p-value = 0.007, R2 = 0.681) and East Canyon (p-value = 0.009, R2 = 0.775) WRFs; both of which lack primary clarifiers or industrial pretreatment processes. This data indicates that SARS-CoV-2 gene copies tend to partition into primary clarifier sludges, at which point a significant portion of them are removed through sedimentation. Furthermore, it was found that East Canyon WRF gene copy numbers in influent were a significant predictor of daily cases (p-value = 0.0322, R2 = 0.561), and gene copy numbers in RAS were a significant predictor of weekly cases (p-value = 0.0597, R2 = 0.449). However, gene copy numbers found in primary sludge samples from other plants significantly predicted the number of COVID-19 cases for the following week (t = 2.279) and the week after that (t = 2.122) respectively. These data indicate that SARS-CoV-2 extracted from WRF biosolids may better suit epidemiological monitoring that exhibits a time lag. It also supports the observation that primary sludge removes a significant portion of SARS-CoV-2 marker genes. In its absence, RAS can also be used to predict the number of COVID-19 cases due to direct flow through from influent. This research represents the first of its kind to thoroughly examine SARS-CoV-2 gene copy numbers in biosolids throughout the wastewater treatment process and the relationship between primary, return activated, and anaerobically digested sludge and reported positive COVID-19 cases.