Genome-Resolved Metagenomics and Metatranscriptomics Reveal Insights into the Ecology and Metabolism of Anaerobic Microbial Communities in PCB-Contaminated Sediments.

Growth of organohalide-respiring bacteria such as Dehalococcoides mccartyi on halogenated organics (e.g., polychlorinated biphenyls (PCBs)) at contaminated sites or in enrichment culture requires interaction and support from other microbial community members. To evaluate naturally occurring interactions between Dehalococcoides and key supporting microorganisms (e.g., production of H2, acetate, and corrinoids) in PCB-contaminated sediments, metagenomic and metatranscriptomic sequencing was conducted on DNA and RNA extracted from sediment microcosms, showing evidence of both Dehalococcoides growth and PCB dechlorination. Using a genome-resolved approach, 160 metagenome-assembled genomes (MAGs), including three Dehalococcoides MAGs, were recovered. A novel reductive dehalogenase gene, distantly related to the chlorophenol dehalogenase gene cprA (pairwise amino acid identity: 23.75%), was significantly expressed. Using MAG gene expression data, 112 MAGs were assigned functional roles (e.g., corrinoid producers, acetate/H2 producers, etc.). A network coexpression analysis of all 160 MAGs revealed correlations between 39 MAGs and the Dehalococcoides MAGs. The network analysis also showed that MAGs assigned with functional roles that support Dehalococcoides growth (e.g., corrinoid assembly, and production of intermediates required for corrinoid synthesis) displayed significant coexpression correlations with Dehalococcoides MAGs. This work demonstrates the power of genome-resolved metagenomic and metatranscriptomic analyses, which unify taxonomy and function, in investigating the ecology of dehalogenating microbial communities.

Deciphering the role of granular activated carbon (GAC) in anammox: Effects on microbial succession and communication.

Anaerobic ammonium oxidation (anammox) offered an energy-efficient option for nitrogen removal from wastewater. Granular activated carbon (GAC) addition has been reported that improved biomass immobilization, but the role of GAC in anammox reactors has not been sufficiently revealed. In this study, it was observed that GAC addition in an upflow anaerobic sludge blanket (UASB) reactor led to the significantly shortened anammox enrichment time (shortened by 45 days) than the reactor without GAC addition. The nitrogen removal rate was 0.83 kg N/m3/day versus 0.76 kg N/m3/day in GAC and non-GAC reactors, respectively after 255 days' operation. Acyl-homoserine lactone (AHL) quorum sensing signal molecule C8-HSL had comparable concentrations in both anammox reactors, whereas the signal molecule C12-HSL was more pervasive in the reactor containing GAC than the reactor without GAC. Microbial analysis revealed distinct anammox development in both reactors, with Candidatus Brocadia predominant in the reactor that did not contain GAC, and Candidatus Kuenenia predominant in the reactor that contained GAC. Denitrification bacteria likely supported anammox metabolism in both reactors. The analyses of microbial functions suggested that AHL-dependent quorum sensing was enhanced with the addition of GAC, and that GAC possibly augmented the extracellular electron transfer (EET)-dependent anammox reaction.

Metagenomic insights into direct interspecies electron transfer and quorum sensing in blackwater anaerobic digestion reactors supplemented with granular activated carbon.

The addition of granular activated carbon (GAC) enhanced the performance of up-flow anaerobic sludge blanket (UASB) reactor treating blackwater at 35 °C. DNA were extracted from the sludge and biofilms attached to GAC and submitted for shotgun sequencing. In addition, the acyl-homoserine lactones (AHLs) were quantified. Diverse partners for direct interspecies electron transfer (DIET) were enriched in the sludge or biofilm (GAC-biofilm) of GAC amended UASB. Pedosphaera parvula, Syntrophus aciditrophicus and Syntrophorhabus aromaticivorans were dominant syntrophs. The analysis for type IV pilus assembly genes further suggested DIET may be functioned through GAC for GAC-biofilm, while through conductive pili for sludge aggregates. AHLs quantification and the analysis for quorum sensing (QS) related genes indicated higher QS activity at the population level was induced by GAC. Overall, the work illustrated the different DIET patterns, and suggested that QS played an important role in controlling the performance in GAC amended USAB.

Roles of granular activated carbon (GAC) and operational factors on active microbiome development in anaerobic reactors.

Ambient temperature municipal sewage was treated using two laboratory-scale up-flow anaerobic sludge blanket reactors for 225 days. Granular activated carbon (GAC) was added to one reactor to facilitate the development of direct interspecies electron transfer (DIET). The GAC addition increased total chemical oxygen demand removal by 5% - 18%. In addition to assessing the relative abundance of active amplicon sequence variants (ASVs), the mass balance model, the Mantel test, and the generalized linear models were applied to evaluate the dynamics of the active ASVs and the key operational factors controlling the bioreactor microbial community. These results demonstrated that, in addition to the GAC addition, extrinsic engineering operational factors played important roles in controlling (active) microbial communities. This study underlines the importance of taking a wholistic approach to assess microbial population dynamics. Reactor design and performance prediction should consider key engineering parameters when using DIET-based AD reactors in the future.

Diversity and abundance of the functional genes and bacteria associated with RDX degradation at a contaminated site pre- and post-biostimulation.

Bioremediation is becoming an increasingly popular approach for the remediation of sites contaminated with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Multiple lines of evidence are often needed to assess the success of such approaches, with molecular studies frequently providing important information on the abundance of key biodegrading species. Towards this goal, the current study utilized shotgun sequencing to determine the abundance and diversity of functional genes (xenA, xenB, xplA, diaA, pnrB, nfsI) and species previously associated with RDX biodegradation in groundwater before and after biostimulation at an RDX-contaminated Navy Site. For this, DNA was extracted from four and seven groundwater wells pre- and post-biostimulation, respectively. From a set of 65 previously identified RDX degraders, 31 were found within the groundwater samples, with the most abundant species being Variovorax sp. JS1663, Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Further, 9 RDX-degrading species significantly (p<0.05) increased in abundance following biostimulation. Both the sequencing data and qPCR indicated that xenA and xenB exhibited the highest relative abundance among the six genes. Several genes (diaA, nsfI, xenA, and pnrB) exhibited higher relative abundance values in some wells following biostimulation. The study provides a comprehensive approach for assessing biomarkers during RDX bioremediation and provides evidence that biostimulation generated a positive impact on a set of key species and genes. KEY POINTS: • A co-occurrence network indicated diverse RDX degraders. • >30 RDX-degrading species were detected. • Nine RDX-degrading species increased following biostimulation. • Sequencing and high-throughput qPCR indicated that xenA and xenB were most abundant.

Identification of the phylotypes involved in cis-dichloroethene and 1,4-dioxane biodegradation in soil microcosms.

Co-contamination with chlorinated compounds and 1,4-dioxane has been reported at many sites. Recently, there has been an increased interest in bioremediation because of the potential to degrade multiple contaminants concurrently. Towards improving bioremediation efficacy, the current study examined laboratory microcosms (inoculated separately with two soils) to determine the phylotypes and functional genes associated with the biodegradation of two common co-contaminants (cis-dichloroethene [cDCE] and 1,4-dioxane). The impact of amending microcosms with lactate on cDCE and 1,4-dioxane biodegradation was also investigated. The presence of either lactate or cDCE did not impact 1,4-dioxane biodegradation one of the two soils. Lactate appeared to improve the initiation of the biological removal of cDCE in microcosms inoculated with either soil. Stable isotope probing (SIP) was then used to determine which phylotypes were actively involved in carbon uptake from cDCE and 1,4-dioxane in both soil communities. The most enriched phylotypes for 13C assimilation from 1,4-dioxane included Rhodopseudomonas and Rhodanobacter. Propane monooxygenase was predicted (by PICRUSt2) to be dominant in the 1,4-dioxane amended microbial communities and propane monooxygenase gene abundance values correlated with other enriched (but less abundant) phylotypes for 13C-1,4-dioxane assimilation. The dominant enriched phylotypes for 13C assimilation from cDCE included Bacteriovorax, Pseudomonas and Sphingomonas. In the cDCE amended soil microcosms, PICRUSt2 predicted the presence of DNA encoding glutathione S-transferase (a known cDCE upregulated enzyme). Overall, the work demonstrated concurrent removal of cDCE and 1,4-dioxane by indigenous soil microbial communities and the enhancement of cDCE removal by lactate. The data generated on the phylotypes responsible for carbon uptake (as determined by SIP) could be incorporated into diagnostic molecular methods for site characterization. The results suggest concurrent biodegradation of cDCE and 1,4-dioxane should be considered for chlorinated solvent site remediation.

Abundance of Chlorinated Solvent and 1,4-Dioxane Degrading Microorganisms at Five Chlorinated Solvent Contaminated Sites Determined via Shotgun Sequencing.

Shotgun sequencing was used for the quantification of taxonomic and functional biomarkers associated with chlorinated solvent bioremediation in 20 groundwater samples (five sites), following bioaugmentation with SDC-9. The analysis determined the abundance of (1) genera associated with chlorinated solvent degradation, (2) reductive dehalogenase (RDases) genes, (3) genes associated with 1,4-dioxane removal, (4) genes associated with aerobic chlorinated solvent degradation, and (5) D. mccartyi genes associated with hydrogen and corrinoid metabolism. The taxonomic analysis revealed numerous genera previously linked to chlorinated solvent degradation, including Dehalococcoides, Desulfitobacterium, and Dehalogenimonas. The functional gene analysis indicated vcrA and tceA from D. mccartyi were the RDases with the highest relative abundance. Reads aligning with both aerobic and anaerobic biomarkers were observed across all sites. Aerobic solvent degradation genes, etnC or etnE, were detected in at least one sample from each site, as were pmoA and mmoX. The most abundant 1,4-dioxane biomarker detected was Methylosinus trichosporium OB3b mmoX. Reads aligning to thmA or Pseudonocardia were not found. The work illustrates the importance of shotgun sequencing to provide a more complete picture of the functional abilities of microbial communities. The approach is advantageous over current methods because an unlimited number of functional genes can be quantified.

Enhanced removal of soluble Cr(VI) by using zero-valent iron composite supported by surfactant-modified zeolites.

Zero-valent iron (ZVI) was immobilized onto surfactant-modified zeolites (SMZ) using calcium alginate. Scanning electron microscopy showed that ZVI powder was uniformly immobilized on the surface of the SMZ. The added ZVI powder resulted in enhanced dichromate removal efficiency and the heterogeneous surface of the composite. The adsorption of dichromate onto the ZVI-SMZ composites fitted well to a pseudo-second-order model and the Langmuir adsorption isotherm. The maximum dichromate adsorption capacity of the composite was 2.49 mg/g at the temperature of 293 K. Higher removal efficiency was obtained at pH lower than 7. X-ray photoelectron spectrometry revealed that the composites combined the strong reductive quality of ZVI and superior adsorption of SMZ.