Microbial residence time is a controlling parameter of the taxonomic composition and functional profile of microbial communities.

A remaining challenge within microbial ecology is to understand the determinants of richness and diversity observed in environmental microbial communities. In a range of systems, including activated sludge bioreactors, the microbial residence time (MRT) has been previously shown to shape the microbial community composition. However, the physiological and ecological mechanisms driving this influence have remained unclear. Here, this relationship is explored by analyzing an activated sludge system fed with municipal wastewater. Using a model designed in this study based on Monod-growth kinetics, longer MRTs were shown to increase the range of growth parameters that enable persistence, resulting in increased richness and diversity in the modeled community. In laboratory experiments, six sequencing batch reactors treating domestic wastewater were operated in parallel at MRTs between 1 and 15 days. The communities were characterized using both 16S ribosomal RNA and non-target messenger RNA sequencing (metatranscriptomic analysis), and model-predicted monotonic increases in richness were confirmed in both profiles. Accordingly, taxonomic Shannon diversity also increased with MRT. In contrast, the diversity in enzyme class annotations resulting from the metatranscriptomic analysis displayed a non-monotonic trend over the MRT gradient. Disproportionately high abundances of transcripts encoding for rarer enzymes occur at longer MRTs and lead to the disconnect between taxonomic and functional diversity profiles

A bioassay for the detection of benzimidazoles reveals their presence in a range of environmental samples.

Cobamides are a family of enzyme cofactors that include vitamin B12 (cobalamin) and are produced solely by prokaryotes. Structural variability in the lower axial ligand has been observed in cobamides produced by diverse organisms. Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions. Many organisms acquire cobamides by salvaging and remodeling cobamides or their precursors from the environment. These processes require free benzimidazoles for incorporation as lower ligands, though the presence of benzimidazoles in the environment has not been previously investigated. Here, we report a new purification method and bioassay to measure the total free benzimidazole content of samples from microbial communities and laboratory media components. The bioassay relies on the "calcofluor-bright" phenotype of a bluB mutant of the model cobalamin-producing bacterium Sinorhizobium meliloti. The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry. Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples. In addition, benzimidazoles were found to be common contaminants of laboratory media components. These results suggest that benzimidazoles present in the environment and in laboratory media have the potential to influence microbial metabolic activities

Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi.

The acetyl-CoA "Wood-Ljungdahl" pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities

Electron bifurcation and fluoride efflux systems implicated in defluorination of perfluorinated unsaturated carboxylic acids by Acetobacterium spp.

Enzymatic cleavage of C─F bonds in per- and polyfluoroalkyl substances (PFAS) is largely unknown but avidly sought to promote systems biology for PFAS bioremediation. Here, we report the reductive defluorination of α, β-unsaturated per- and polyfluorocarboxylic acids by Acetobacterium spp. The microbial defluorination products were structurally confirmed and showed regiospecificity and stereospecificity, consistent with their formation by enzymatic reactions. A comparison of defluorination activities among several Acetobacterium species indicated that a functional fluoride exporter was required for the detoxification of the released fluoride. Results from both in vivo inhibition tests and in silico enzyme modeling suggested the involvement of enzymes of the flavin-based electron-bifurcating caffeate reduction pathway [caffeoyl-CoA reductase (CarABCDE)] in the reductive defluorination. This is a report on specific microorganisms carrying out enzymatic reductive defluorination of PFAS, which could be linked to electron-bifurcating reductases that are environmentally widespread

Identification of Novel Biomarkers from Supportive Microorganisms in TCE-dechlorinating Microbial Communities

Chlorinated ethenes are toxic and carcinogenic compounds that have contaminated a large quantity of groundwater in the U.S. and other developed countries. In order to protect public health, in situ bioremediation via dehalorespiration by Dehalococcoides bacteria is a promising solution. The overall goal of this research is to understand the ecological relationship between Dehalococcoides mccartyi (Dhc) and supportive microorganisms in a community and identify novel biomarkers for monitoring TCE dechlorination activities during bioremediation. To accomplish these goals, traditional molecular and high throughput microarray techniques, as well as newly established analytical approaches were applied. The first objective of this research was to characterize four Dhc-containing microbial communities enriched from contaminated groundwater under different cobalamin stress and methanogenic conditions. Microarray analyses targeting four Dhc genomes revealed a commonly shared core Dhc genome most similar to strain 195. Physiological characterization revealed that inhibited methanogensis optimized the dechlorination performance. Experimental evidence demonstrated the presence of Bacterial species providing corrinoids to Dhc. The dominance of closely related Pelosinus spp., Dendrosporobacter spp. and Sporotalea spp. and the significant effects of cobalamin addition and methanogenic inhibition on the distribution of Clostridium spp. in the communities suggest that species in these genera are potential corrinoid providers to Dhc. In order to further target corrinoid production in enrichments without exogenous cobalamin, a detection method was established that successfully differentiates various corrinoid and lower ligand forms. Corrinoid and lower ligand profiles of different Dhc-containing enrichments indicate that cobalamin was the major corrinoid utilized by Dhc. Further evidence demonstrated that in enrichments without exogenous cobalamin, p-cresolylcobamide was produced, likely by Pelosinus spp., and then modified by Dhc into cobalamin in the presence of dimethylbenzimidazole (DMB) to support dechlorination reactions. Differential gene expression between enrichments with and without exogenous cobalamin was investigated using microarrays targeting four sequenced Dhc genomes. Results suggest that cobT and btuF genes, the Nuo and Hym operons and the tryptophan operon could serve as biomarkers indicating cobalamin stress and corrinoid salvaging. Temporal global gene expression of the enrichment without exogenous cobalamin corroborates the importance of hydrogenases at an early stage of dechlorination and the close relation between TceA reductive dehalogenase and Hup hydrogenase.Based on the knowledge obtained in the previous studies, defined consortia were constructed by growing Dhc strain 195 with potential supportive microorganisms: Desulfovibrio vulgaris Hildenborough, Methanobacterium congolense, and Pelosinus fermentans strain R7. Physiological, transcriptomic and proteomic analyses of strain 195 containing consortia provide strong evidence supporting the previous hypotheses that Desulfovibrio spp. play important roles in hydrogen transfer, Pelosinus spp. provide corrinoid forms to Dhc but not DMB, while methanogens do not contribute to Dhc cell growth or biological corrinoid generation in the presence of the other two species.The significance of this research is that supportive microorganisms and related chemical and molecular biomarkers have been identified. And they may be useful for optimizing bioremediation processes by obtaining accurate feedback information from cells that are indicative of Dhc physiology. Knowledge developed in this research will aid practitioners to better design, monitor and optimize future in situ bioremediation systems

Comment on “Role of Ammonia Oxidation in Organic Micropollutant Transformation during Wastewater Treatment”: Overlooked Evidence to the Contrary

In their critical review, (1) Su, Smets, and coauthors extensively summarized studies on the role played by ammonia-oxidizing bacteria (AOB) in organic micropollutant (OMP) transformation using three levels of evidence: molecular, cellular, and community. They also comprehensively covered the abiotic reactions with the N-species formed from nitrification. We agree with the authors that some studies do support the important role played by AOB in the transformation of specific OMPs. However, we find that the authors’ conclusion, “AOB are the main drivers of OMP biotransformation during wastewater treatment processes” (p 2173), does not stand up to proper scientific scrutiny. In the following, we will present our main arguments and provide the overlooked evidence contradicting the authors’ conclusion. (In the following, all page numbers and references to graphical elements refer to Su et al. 2020