The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor.

The microbes responsible for anaerobic benzene biodegradation remain poorly characterized. In this study, we identified and quantified microbial populations in a series of 16 distinct methanogenic, benzene-degrading enrichment cultures using a combination of traditional 16S rRNA clone libraries (four cultures), pyrotag 16S rRNA amplicon sequencing (11 cultures), metagenome sequencing (1 culture) and quantitative polymerase chain reaction (qPCR; 12 cultures). An operational taxonomic unit (OTU) from the Deltaproteobacteria designated ORM2 that is only 84% to 86% similar to Syntrophus or Desulfobacterium spp. was consistently identified in all enrichment cultures, and typically comprised more than half of the bacterial sequences. In addition to ORM2, a sequence belonging to Parcubacteria (candidate division OD1) identified from the metagenome data was the only other OTU common to all the cultures surveyed. Culture transfers (1% and 0.1%) were made in the presence and absence of benzene, and the abundance of ORM2, OD1 and other OTUs was tracked over 415 days using qPCR. ORM2 sequence abundance increased only when benzene was present, while the abundance of OD1 and other OTUs increased even in the absence of benzene. Deltaproteobacterium ORM2 is unequivocally the benzene-metabolizing population. This study also hints at laboratory cultivation conditions for a member of the widely distributed yet uncultivated Parcubacteria (OD1).

The initial metabolic reactions for anaerobic benzene biodegradation remain uncharacterized. Isotopic data for carbon and hydrogen fractionation from nitrate-reducing, sulfate-reducing, and methanogenic benzene-degrading enrichment cultures and phylogenic information were used to investigate the initial reaction step in anaerobic benzene biodegradation. Dual parameter plots of carbon and hydrogen isotopic data (deltadelta2H/ deltadelta13C) from each culture were linear, suggesting a consistent reaction mechanism as degradation proceeded. Methanogenic and sulfate-reducing cultures showed consistently higher slopes (m = 29 +/- 2) compared to nitrate-reducing cultures (m = 13 +/- 2) providing evidence for different initial reaction mechanisms. Phylogenetic analyses confirmed that culture conditions were strictly anaerobic, precluding any involvement of molecular oxygen in the observed differences. Using published kinetic data, we explored the possibility of attributing such slopes to reaction mechanisms. The higher slopes found under methanogenic and sulfate-reducing conditions suggest against an alkylation mechanism for these cultures. Observed differences between the methanogenic and nitrate-reducing cultures may not represent distinct reactions of different bonds, but rather subtle differences in relative reaction kinetics. Additional mechanistic conclusions could not be made because kinetic isotope effect data for carboxylation and other putative mechanisms are not available.

Chlorobenzene is a widespread groundwater contaminant found at many industrial sites. Reductive dechlorination of chlorobenzene requires input of electron donor and results in problematic accumulation of benzene, which is more toxic than chlorobenzene. We hypothesized that coupling a culture capable of reductive dechlorination of chlorobenzene to benzene with a second benzene-degrading methanogenic culture would completely detoxify chlorobenzene. To this end, active chlorobenzene-dechlorinating microcosms that were producing benzene were inoculated with a previously described enriched methanogenic benzene-degrading consortium. The combination resulted in the transformation of chlorobenzene via benzene to the nontoxic degradation products, CO2 and CH4. Sustainable degradation of chlorobenzene and benzene was observed in the microcosms and was further confirmed by shifts in the carbon isotopic ratios of chlorobenzene and benzene during degradation. Moreover, we could show that benzene derived electrons fueled chlorobenzene dechlorination removing the need to provide exogenous electron donor. The results have promising implications for sustainable bioremediation of sites contaminated with chlorinated benzenes and benzene.

Mine tailings present major environmental issues in the mining industry. However due to the depletion of high-grade sulfide ores for metal recovery, tailings could also be a potential resource for certain valuable metals. The present study investigates the potential to recover nickel from pyrrhotite tailings. Leaching tests were performed in acidic ferric sulfate media with 0.14 wt % solids to keep the ferric concentration essentially constant. The temperature was varied between 30 and 55 °C, and the ferric concentration was in a range 0.02–0.3 M. The results showed that both temperature and ferric sulfate concentration had significant effects on the nickel extraction kinetics. The shrinking core model (SCM) was applied to the nickel extraction data. The rate controlling step was found to be product layer diffusion. The Arrhenius plot yielded an activation energy of Ea = 62.12 kJ/mol based on apparent reaction rates obtained by the SCM. The reaction order with respect to ferric ion was found to be 1 at the high concentration range. SEM images of partially leached tailings confirmed the presence of elemental sulfur around the pyrrhotite particles, which was responsible for the observed non-linear leaching kinetics (diffusion control).