Project description:1,4-Dioxane is one of the most common and persistent artificial pollutants in petrochemical industrial wastewaters and chlorinated solvent groundwater plumes. Despite its possible biological treatment in natural environments, the identity and dynamics of the microorganisms involved are largely unknown. Here, we identified active and diverse 1,4-dioxane-degrading microorganisms from activated sludge by high-sensitivity stable isotope probing of rRNA. By rigorously analyzing 16S rRNA molecules in RNA density fractions of 13C-labeled and unlabeled 1,4-dioxane treatments, we discovered 10 significantly 13C-incorporating microbial species from the complex microbial community. 16S rRNA expression assays revealed that 9 of the 10 species, including the well-known degrader Pseudonocardia dioxanivorans, an ammonia-oxidizing bacterium and phylogenetically novel bacteria, increased their metabolic activities shortly after exposure to 1,4-dioxane. Moreover, high-resolution monitoring showed that, during a single year of operation of the full-scale activated sludge system, the nine identified species exhibited yearly averaged relative abundances of 0.001-1.523%, and yet showed different responses to changes in the 1,4-dioxane removal efficiency. Hence, the co-existence and individually distinct dynamics of various 1,4-dioxane-degrading microorganisms, including hitherto unidentified species, played pivotal roles in the maintenance of the biological system removing the recalcitrant pollutant.
Project description:Tetrahydrofuran (THF) is known to induce the biodegradation of 1,4-dioxane (dioxane), an emerging contaminant, but the mechanisms by which THF affects dioxane biodegradation in microbial communities are not well understood. To fill this knowledge gap, changes in the microbial community structure in microcosm experiments with synthetic medium and landfill leachate were examined over time using 16S rRNA gene amplicon sequencing and functional gene quantitative PCR assays. The overarching hypothesis being tested was that THF promoted dioxane biodegradation by increasing the abundance of dioxane-degrading bacteria in the consortium. The data revealed that in experiments with synthetic medium, the addition of THF significantly increased the abundance of Pseudonocardia, a genus with several representatives that can grow on both dioxane and THF, and of Rhodococ cus ruber, a species that can use THF as the primary growth substrate while cometabolizing dioxane. However, in similar experiments with landfill leachate, only R. ruber was significantly enriched. When the THF concentration was higher than the dioxane concentration, THF competitively inhibited dioxane degradation since dioxane degradation was negligible, while the dioxane-degrading bacteria and the corresponding THF/dioxane monooxygenase gene copies increased by a few orders of magnitude.IMPORTANCE Widespread in groundwater and carcinogenic to humans, 1,4-dioxane (dioxane) is attracting significant attention in recent years. Advanced oxidation processes can effectively remove dioxane but require high energy consumption and operation costs. Biological removal of dioxane is of particular interest due to the ability of some bacteria to mineralize dioxane at a low energy cost. Although dioxane is generally considered recalcitrant to biodegradation, more than 20 types of bacteria can degrade dioxane as the sole electron donor substrate or the secondary electron donor substrate. In the latter case, tetrahydrofuran (THF) is commonly studied as the primary electron donor substrate. Previous work has shown that THF promotes dioxane degradation at a low THF concentration but inhibits dioxane degradation at a high THF concentration. Our work expanded on the previous work by mechanically examining the effects of THF on dioxane degradation in a microbial community context.
Project description:The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.
Project description:The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.
Project description:New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.
Project description:We sequenced the metagenome of a microbial community enriched under strictly anaerobic conditions from wastewater treatment plant-derived digester sludge. The metagenomic analysis of the enrichment revealed that Acetobacterium and methanogenic archaea belonged to the dominant prokaryotes, and genes encoding components of the Wood-Ljungdahl pathway were identified.
Project description:BACKGROUND:As one of the most abundant agricultural wastes, sugarcane bagasse is largely under-exploited, but it possesses a great potential for the biofuel, fermentation, and cellulosic biorefinery industries. It also provides a unique ecological niche, as the microbes in this lignocellulose-rich environment thrive in relatively high temperatures (50°C) with varying microenvironments of aerobic surface to anoxic interior. The microbial community in bagasse thus presents a good resource for the discovery and characterization of new biomass-degrading enzymes; however, it remains largely unexplored. RESULTS:We have constructed a fosmid library of sugarcane bagasse and obtained the largest bagasse metagenome to date. A taxonomic classification of the bagasse metagenome reviews the predominance of Proteobacteria, which are also found in high abundance in other aerobic environments. Based on the functional characterization of biomass-degrading enzymes, we have demonstrated that the bagasse microbial community benefits from a large repertoire of lignocellulolytic enzymes, which allows them to digest different components of lignocelluoses into single molecule sugars. Comparative genomic analyses with other lignocellulolytic and non-lignocellulolytic metagenomes show that microbial communities are taxonomically separable by their aerobic "open" or anoxic "closed" environments. Importantly, a functional analysis of lignocellulose-active genes (based on the CAZy classifications) reveals core enzymes highly conserved within the lignocellulolytic group, regardless of their taxonomic compositions. Cellulases, in particular, are markedly more pronounced compared to the non-lignocellulolytic group. In addition to the core enzymes, the bagasse fosmid library also contains some uniquely enriched glycoside hydrolases, as well as a large repertoire of the newly defined auxiliary activity proteins. CONCLUSIONS:Our study demonstrates a conservation and diversification of carbohydrate-active genes among diverse microbial species in different biomass-degrading niches, and signifies the importance of taking a global approach to functionally investigate a microbial community as a whole, as compared to focusing on individual organisms.
Project description:Biodegradation is found to be a promising, cost-effective and eco-friendly option for the treatment of industrial wastewater contaminated by 1,4-dioxane (1,4-D), a highly stable synthetic chemical and probable human carcinogen. This study aimed to isolate, identify, and characterize metabolic 1,4-D-degrading bacteria from a stable 1,4-D-degrading microbial consortium. Three bacterial strains (designated as strains TS28, TS32, and TS43) capable of degrading 1,4-D as a sole carbon and energy source were isolated and identified as Gram-positive <i>Pseudonocardia</i> sp. (TS28) and Gram-negative <i>Dokdonella</i> sp. (TS32) and <i>Afipia</i> sp. (TS43). This study, for the first time, confirmed that the genus <i>Dokdonella</i> is involved in the biodegradation of 1,4-D. The results reveal that all of the isolated strains possess inducible 1,4-D-degrading enzymes and also confirm the presence of a gene encoding tetrahydrofuran/dioxane monooxygenase (<i>thmA/dxmA</i>) belonging to group 5 soluble di-iron monooxygenases (SDIMOs) in both genomic and plasmid DNA of each of the strains, which is possibly responsible for the initial oxidation of 1,4-D. Moreover, the isolated strains showed a broad substrate range and are capable of degrading 1,4-D in the presence of additional substrates, including easy-to-degrade compounds, 1,4-D biodegradation intermediates, structural analogs, and co-contaminants of 1,4-D. This indicates the potential of the isolated strains, especially strain TS32, in removing 1,4-D from contaminated industrial wastewater containing additional organic load. Additionally, the results will help to improve our understanding of how multiple 1,4-D-degraders stably co-exist and interact in the consortium, relying on a single carbon source (1,4-D) in order to develop an efficient biological 1,4-D treatment system.
Project description:By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d8 and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.