Prospects for multi-omics in the microbial ecology of water engineering

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions – including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.BT/Industriele Microbiologi

Influence of Kinetic and Metabolic Selection on 17alpha-ethinylestradiol Biodegradation in Activated Sludge Wastewater Treatment Systems

Thesis (Master's)--University of Washington, 2013The potent endocrine-disrupting estrogen hormone, 17alpha-ethinylestradiol (EE2), is primarily removed via biodegradation in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes; however, reported EE2 removal efficiencies in AS WWTPs vary widely. A hypothesis of this research was that EE2 biodegradation kinetics vary as a function of AS process and reactor designs, which select for different microbial population compositions. Bench-scale AS reactors treating municipal wastewater and estrogens at ng/L concentrations were operated to simulate kinetic population selection with high initial food-to-biomass ratio feeding conditions (high-F/Mf) or low substrate growth conditions (low-F/Mf), as well as metabolic selection with substrate uptake and growth under aerobic, anaerobic, and anoxic conditions. The latter two metabolic selectors resulted in enhanced biological phosphorus removal and biological nitrogen removal, respectively. A pseudo first-order biodegradation model was used to examine the effects of metabolic and kinetic selective pressures on EE2 biodegradation kinetics. Aerobic low-F/Mf reactors experienced pseudo first-order EE2 biodegradation rate coefficients (kb) that were 1.4 to 2.2 times greater than high-F/Mf aerobic selectors operated in parallel, suggesting that kinetic selection influences EE2 biodegradation activity in AS systems. No significant difference was observed in the EE2 kb of high-F/Mf metabolic bioselectors (aerobic-only, anoxic/aerobic and anaerobic/aerobic). However, metabolic selection reduced the EE2 kb of a low-F/Mf anoxic/aerobic reactor by 40% relative to a low-F/Mf aerobic reactor, demonstrating that the redox state of growth conditions may affect microbial EE2 biodegradation kinetics in AS. The results of this study suggest that operating conditions in which microbial growth occurs aerobically at low substrate concentrations improve EE2 biodegradation kinetics in AS systems, possibly due to the growth of K-strategist heterotrophs capable of more efficient EE2 biodegradation at low ng/L concentrations. Supplementary files to this dissertation include Appendix B, which is a Microsoft Excel file that provides supporting data to the presented research results

Recovering biomethane from fats, oils, and greases: Examining the impacts of microbial ecology on anaerobic codigester stability and bioconversion kinetics

Thesis (Ph.D.)--University of Washington, 2016-12Recovering biomethane with anaerobic digestion is of global interest to reduce carbon footprints and improve process economics for the treatment of organic wastes. Fats, oils, and greases (FOG) are desirable co-substrates for biomethane recovery because they have a substantially higher energy density than wastewater treatment solids or livestock manure. Yet, biomethane recovery from FOG codigestion at wastewater treatment plants or agricultural digesters can be limited due to process inhibition caused by long-chain fatty acids (LCFA) accumulation. Currently, there is a lack of understanding regarding the role of anaerobic digester microbial communities in maintaining efficient conversion of LCFA into biomethane. The ultimate goal of this research was to improve the reliability of biomethane recovery during FOG codigestion by elucidating relationships between microbial community composition and LCFA bioconversion kinetics. The ability to accurately monitor LCFA-degrading populations in anaerobic digesters was obtained by developing and validating quantitative PCR (qPCR) assays for the syntrophic LCFA β-oxidizing genera of Syntrophomonas and Syntrophus. These qPCR assays were then utilized to measure population changes in a codigester treating FOG and municipal wastewater treatment solids at increasing FOG loadings for over 150 days. A relationship was developed that correlated higher effluent LCFA concentrations with higher influent FOG loading rates normalized to digester Syntrophomonas 16S rRNA gene concentrations. Subsequently, the impacts of LCFA feeding strategy on LCFA bioconversion kinetics were investigated using bench-scale codigesters that were either pulse-fed every two days or continuously-fed daily with oleate. The results showed that Bacteria and Archaea community compositions in the codigesters diverged based on LCFA feeding frequency and LCFA loading. Predictive models for LCFA bioconversion kinetics were developed as a function of absolute concentrations of selected Syntrophomonas taxa. DNA-stable isotope probing (SIP) based metagenomics confirmed that different LCFA-degrading syntrophic bacteria were selected with different codigester LCFA feeding frequencies. Taken together, the results of this study demonstrate that higher codigester FOG loadings can be achieved by developing a higher biomass concentration of LCFA-degrading syntrophic consortia, and that the codigester feeding strategy can be adjusted to biologically select for LCFA-degrading populations with higher LCFA bioconversion kinetics at high FOG loadings

SAOB

Raw and final results files for manuscript on stable-isotope informed genome-resolved multi-omics of an anaerobic enrichment community performing acetate oxidation

Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable-isotope-informed genome-resolved metagenomics

<p>Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in ¹³C after growing on [¹³C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [¹³C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (&lt;95%) with any cultured representative species, indicating that it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for acetoclastic methanogenesis, indicating that it was enriched in ¹³C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to identify in situ interspecies metabolic cooperation within syntrophic consortia important to anaerobic waste treatment as well as global carbon cycling. IMPORTANCE Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C₄ fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve "who is doing what" by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.</p

Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable-isotope-informed genome-resolved metagenomics

Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after growing on [13C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [13C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to identify in situ interspecies metabolic cooperation within syntrophic consortia important to anaerobic waste treatment as well as global carbon cycling. IMPORTANCE Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve "who is doing what" by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.</p

DNA-SIP based genome-centric metagenomics identifies key long-chain fatty acid-degrading populations in anaerobic digesters with different feeding frequencies

Fats, oils and greases (FOG) are energy-dense wastes that can be added to anaerobic digesters to substantially increase biomethane recovery via their conversion through long-chain fatty acids (LCFAs). However, a better understanding of the ecophysiology of syntrophic LCFA-degrading microbial communities in anaerobic digesters is needed to develop operating strategies that mitigate inhibitory LCFA accumulation from FOG. In this research, DNA stable isotope probing (SIP) was coupled with metagenomic sequencing for a genome-centric comparison of oleate (C 18:1)-degrading populations in two anaerobic codigesters operated with either a pulse feeding or continuous-feeding strategy. The pulse-fed codigester microcosms converted oleate into methane at over 20% higher rates than the continuous-fed codigester microcosms. Differential coverage binning was demonstrated for the first time to recover population genome bins (GBs) from DNA-SIP metagenomes. About 70% of the 13 C-enriched GBs were taxonomically assigned to the Syntrophomonas genus, thus substantiating the importance of Syntrophomonas species to LCFA degradation in anaerobic digesters. Phylogenetic comparisons of 13 C-enriched GBs showed that phylogenetically distinct Syntrophomonas GBs were unique to each codigester. Overall, these results suggest that syntrophic populations in anaerobic digesters can have different adaptive capacities, and that selection for divergent populations may be achieved by adjusting reactor operating conditions to maximize biomethane recovery

Feasibility of OFMSW co-digestion with sewage sludge for increasing biogas production at wastewater treatment plants

Sweden has the ambition to increase its annual biogas production from the current level of 1.9 to 15 TWh by 2030. The unused capacity of existing anaerobic digesters at wastewater treatment plants is among the options to accomplish this goal. This study investigated the feasibility of utilizing the organic fraction of municipal solid waste (OFMSW) as a co-substrate, with primary and waste-activated sewage sludge (PWASS) for production of biogas, corresponding to 3:1 ratio on volatile solid (VS) basis. The results demonstrated that co-digestion of OFMSW with PWASS at an organic loading rate of 5 gVS l−1 day−1 has the potential to increase the biogas production approximately four times. The daily biogas production increased from 1.0 ± 0.1 to 3.8 ± 0.3 l biogasl−1 day−1, corresponding to a specific methane production of 420 ± 30 Nml methane gVS−1 during the laboratory experiment. Co-digestion of OFMSW with PWASS showed a 50:50 distribution of hydrogenotrophic and aceticlastic methanogens in the digester and enhanced the turnover kinetics of intermediate products (acetate, propionate, and oleate). Practical limitations potentially include the need for sludge dewatering to maintain a sufficient hydraulic retention time (17 days in this study), as well as additional energy consumption for mixing due to an increased sludge apparent viscosity (from 1.8 ± 0.1 to 45 ± 4.8 mPa*s in this study) at elevated OFMSW-loading rates

Microbial rRNA gene expression and co-occurrence profiles associate with biokinetics and elemental composition in full-scale anaerobic digesters

This study examined whether the abundance and expression of microbial 16S rRNA genes were associated with elemental concentrations and substrate conversion biokinetics in 20 full-scale anaerobic digesters, including seven municipal sewage sludge (SS) digesters and 13 industrial codigesters. SS digester contents had higher methane production rates from acetate, propionate and phenyl acetate compared to industrial codigesters. SS digesters and industrial codigesters were distinctly clustered based on their elemental concentrations, with higher concentrations of NH3-N, Cl, K and Na observed in codigesters. Amplicon sequencing of 16S rRNA genes and reverse-transcribed 16S rRNA revealed divergent grouping of microbial communities between mesophilic SS digesters, mesophilic codigesters and thermophilic digesters. Higher intradigester distances between Archaea 16S rRNA and rRNA gene profiles were observed in mesophilic codigesters, which also had the lowest acetate utilization biokinetics. Constrained ordination showed that microbial rRNA and rRNA gene profiles were significantly associated with maximum methane production rates from acetate, propionate, oleate and phenyl acetate, as well as concentrations of NH3-N, Fe, S, Mo and Ni. A co-occurrence network of rRNA gene expression confirmed the three main clusters of anaerobic digester communities based on active populations. Syntrophic and methanogenic taxa were highly represented within the subnetworks, indicating that obligate energy-sharing partnerships play critical roles in stabilizing the digester microbiome. Overall, these results provide new evidence showing that different feed substrates associate with different micronutrient compositions in anaerobic digesters, which in turn may influence microbial abundance, activity and function.Funding Agencies|Swedish Energy Agency; Biogas Research Centre at Linkoping University, Sweden; NSF [DGE-1256082]</p

An Indirect Indentation Method for Evaluating the Linear Viscoelastic Properties of the Brain Tissue

Indentation experiments offer a robust, fast, and repeatable testing method for evaluating the mechanical properties of the solid-state materials in a wide stiffness range. With the advantage of requiring a minimal sample preparation and multiple tests on a small piece of specimen, this method has recently become a popular technique for measuring the elastic properties of the biological materials, especially the brain tissue whose ultrasoft nature makes its mechanical characterization very challenging. Nevertheless, some limitations are associated with the indentation of the brain tissue, such as improper surface detection, negative initial contact force due to tip-tissue moisture interaction, and partial contact between the tip and the sample. In this study, an indirect indentation scheme is proposed to overcome the aforementioned difficulties. In this way, the indentation force is transferred from a sharp tip to the surface of the tissue slices via a rigid coverslip. To demonstrate the accuracy of this method, the linear viscoelastic properties of the white and gray matters of the bovine brain samples are measured by imposing small cyclic loads at different frequencies. The rate, regional, directional, and postmortem time dependence of the viscoelastic moduli are investigated and compared with the previous results from cyclic shear and monotonic experiments on the brain tissue. While findings of this research present a comprehensive set of information for the viscoelastic properties of the brain at a wide frequency range, the central goal of this paper is to introduce a novel experimentation technique with noticeable advantages for biomechanical characterization of the soft tissue