Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

Aims: To complement our proteome study, whole-transcriptome analyses were utilized here to identify proteins related to degrading cis-1,2-dichloroethylene (cis-DCE). Methods and Results: Metabolically engineered Escherichia coli strains were utilized expressing an evolved toluene ortho-monooxygenase along with either (i) glutathione S-transferase and altered c-glutamylcysteine synthetase or (ii) a rationally engineered epoxide hydrolase. cis-DCE degradation induced 30 known stress genes and 32 uncharacterized genes. Because of the reactive cis-DCE epoxides formed, we hypothesized that some of these uncharacterized genes may be related to a variety of stresses. Using isogenic mutants, IbpB, YchH, YdeI, YeaR, YgiW, YoaG and YodD were related to hydrogen peroxide, cadmium and acid stress. Additional whole-transcriptome studies with hydrogen peroxide stress using the most hydrogen peroxide-sensitive mutants, ygiW and ychH, identified that FliS, GalS, HcaR, MglA, SufE, SufS, Tap, TnaB, YhcN and YjaA are also involved in the stress response of E. coli to hydrogen peroxide, cadmium and acid, as well as are involved in biofilm formation. Conclusion: Seventeen proteins are involved in the stress network for this organism, and YhcN and YchH were shown to be important for the degradation of cis-DCE. Significance and Impact of the Study: Six previously uncharacterized proteins (YchH, YdeI, YgiW, YhcN, YjaA and YodD) were shown to be stress proteins.

No abstract

In this study, cow manure was hydrothermally carbonized (HTC) at 180°C–260°C for 5 and 30 min. Mass and elemental (carbon and nitrogen) balances were performed for each HTC reaction condition. Fuel, fertilizer, and nutrient recovery potential of HTC products are evaluated and discussed. In terms of mass loss, dehydration, and energy densification, cow manure is shown to be relatively less reactive than lignocellulosic biomass in HTC. Ash‐free HHV for hydrochar increased up to a maximum of 22.1 MJ/kg from 19.1 MJ/kg. NPK values of the hydrochar are approximately 20:20:3. Significant amounts of phosphorous and other minerals were absorbed by the solid char from the liquid phase during reaction between 5 and 30 min reaction time. HTC process liquid pH was slightly acidic to neutral, and contains organic acids, sugars, and some amino acids. About half of the nitrogen and most of the potassium are dissolved in the liquid product. © 2016 American Institute of Chemical Engineers Environ Prog, 35: 1002–1011, 2016

The microbial communities of two field-scale pilot sulfate-reducing bioreactors treating acid mine drainage (AMD), Luttrell and Peerless Jenny King (PJK), were compared using biomolecular tools and multivariate statistical analyses. The two bioreactors were well suited for this study because their geographic locations and substrate compositions were similar while the characteristics of influent AMD, configuration and degree of exposure to oxygen were distinct. The two bioreactor communities were found to be functionally similar, including cellulose degraders, fermenters and sulfate-reducing bacteria (SRB). Significant differences were found between the two bioreactors in phylogenetic comparisons of cloned 16S rRNA genes and adenosine 5'-phosphosulfate reductase (apsA) genes. The apsA gene clones from the Luttrell bioreactor were dominated by uncultured SRB most closely related to Desulfovibrio spp., while those of the PJK bioreactor were dominated by Thiobacillus spp. The fraction of the SRB genus Desulfovibrio was also higher at Luttrell than at PJK as determined by quantitative real-time polymerase chain reaction analysis. Oxygen exposure at PJK is hypothesized to be the primary cause of these differences. This study is the first rigorous phylogenetic investigation of field-scale bioreactors treating AMD and the first reported application of multivariate statistical analysis of remediation system microbial communities applying UniFrac software.