Microbial diversity and metabolic pathways linked to benzene degradation in petrochemical-polluted groundwater.

The rapid advance in shotgun metagenome sequencing has enabled us to identify uncultivated functional microorganisms in polluted environments. While aerobic petrochemical-degrading pathways have been extensively studied, the anaerobic mechanisms remain less explored. Here, we conducted a study at a petrochemical-polluted groundwater site in Henan Province, Central China. A total of twelve groundwater monitoring wells were installed to collect groundwater samples. Benzene appeared to be the predominant pollutant, detected in 10 out of 12 samples, with concentrations ranging from 1.4 µg/L to 5,280 µg/L. Due to the low aquifer permeability, pollutant migration occurred slowly, resulting in relatively low benzene concentrations downstream within the heavily polluted area. Deep metagenome sequencing revealed Proteobacteria as the dominant phylum, accounting for over 63 % of total abundances. Microbial α-diversity was low in heavily polluted samples, with community compositions substantially differing from those in lightly polluted samples. dmpK encoding the phenol/toluene 2-monooxygenase was detected across all samples, while the dioxygenase bedC1 was not detected, suggesting that aerobic benzene degradation might occur through monooxygenation. Sequence assembly and binning yielded 350 high-quality metagenome-assembled genomes (MAGs), with 30 MAGs harboring functional genes associated with aerobic or anaerobic benzene degradation. About 80 % of MAGs harboring functional genes associated with anaerobic benzene degradation remained taxonomically unclassified at the genus level, suggesting that our current database coverage of anaerobic benzene-degrading microorganisms is very limited. Furthermore, two genes integral to anaerobic benzene metabolism, i.e, benzoyl-CoA reductase (bamB) and glutaryl-CoA dehydrogenase (acd), were not annotated by metagenome functional analyses but were identified within the MAGs, signifying the importance of integrating both contig-based and MAG-based approaches. Together, our efforts of functional annotation and metagenome binning generate a robust blueprint of microbial functional potentials in petrochemical-polluted groundwater, which is crucial for designing proficient bioremediation strategies.

Photo-Activated Direct Catalytic Oxidation of Gaseous Benzene with a Cu-Connected Serial Heterojunction Array of Co3 O4 /Cux O/Foam Cu Assembled via Layer upon Layer Oxidation.

The foam copper (FCu) has been first used as a promising supporting material to prepare a photo-activated catalyst of Co3 O4 /Cux O/FCu, in which the fine Co3 O4 particles are inlayed on the Cux O nanowires to form a Z-type heterojunction array connected by substrate Cu. The prepared samples have been used as a photo-activated catalyst to directly decompose gaseous benzene and the optimized Co3 O4 /Cux O/FCu demonstrates a 99.5% removal efficiency and 100% mineralizing rate within 15 min in benzene concentration range from 350 to 4000 ppm under simulate solar light irradiation. To track the reactive mechanism, a series of MOx /Cux O/FCu (M = Mn, Fe, Co, Ni, Cu, Zn) is prepared and a novel photo-activated direct catalytic oxidation route is proposed based on the comparative investigation of material properties. Moreover, the approach grew in situ via layer upon layer oxidation on FCu dedicates to the extra lasting reusability and the easy accessibility in the diverse situations. This work provides a novel strategy for the preparation of Cu connected series multidimensional heterojunction array and a promising application for the quick abatement of the high-leveled concentration gaseous benzene and its derivatives from the industrial discharged flow or the accident scene's leakage.

Kinetic Aspects of Benzene Degradation over TiO2-N and Composite Fe/Bi2WO6/TiO2-N Photocatalysts under Irradiation with Visible Light.

In this study, composite materials based on nanocrystalline anatase TiO2 doped with nitrogen and bismuth tungstate are synthesized using a hydrothermal method. All samples are tested in the oxidation of volatile organic compounds under visible light to find the correlations between their physicochemical characteristics and photocatalytic activity. The kinetic aspects are studied both in batch and continuous-flow reactors, using ethanol and benzene as test compounds. The Bi2WO6/TiO2-N heterostructure enhanced with Fe species efficiently utilizes visible light in the blue region and exhibits much higher activity in the degradation of ethanol vapor than pristine TiO2-N. However, an increased activity of Fe/Bi2WO6/TiO2-N can have an adverse effect in the degradation of benzene vapor. A temporary deactivation of the photocatalyst can occur at a high concentration of benzene due to the fast accumulation of non-volatile intermediates on its surface. The formed intermediates suppress the adsorption of the initial benzene and substantially increase the time required for its complete removal from the gas phase. An increase in temperature up to 140 °C makes it possible to increase the rate of the overall oxidation process, and the use of the Fe/Bi2WO6/TiO2-N composite improves the selectivity of oxidation compared to pristine TiO2-N.

Structure and functional capacity of a benzene-mineralizing, nitrate-reducing microbial community.

AIMS: How benzene is metabolized by microbes under anoxic conditions is not fully understood. Here, we studied the degradation pathways in a benzene-mineralizing, nitrate-reducing enrichment culture. METHODS AND RESULTS: Benzene mineralization was dependent on the presence of nitrate and correlated to the enrichment of a Peptococcaceae phylotype only distantly related to known anaerobic benzene degraders of this family. Its relative abundance decreased after benzene mineralization had terminated, while other abundant taxa-Ignavibacteriaceae, Rhodanobacteraceae and Brocadiaceae-slightly increased. Generally, the microbial community remained diverse despite the amendment of benzene as single organic carbon source, suggesting complex trophic interactions between different functional groups. A subunit of the putative anaerobic benzene carboxylase previously detected in Peptococcaceae was identified by metaproteomic analysis suggesting that benzene was activated by carboxylation. Detection of proteins involved in anaerobic ammonium oxidation (anammox) indicates that benzene mineralization was accompanied by anammox, facilitated by nitrite accumulation and the presence of ammonium in the growth medium. CONCLUSIONS: The results suggest that benzene was activated by carboxylation and further assimilated by a novel Peptococcaceae phylotype. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the hypothesis that Peptococcaceae are important anaerobic benzene degraders.

Degradation of benzene present in wastewater using hydrodynamic cavitation in combination with air.

The degradation of benzene present in wastewater using hydrodynamic cavitation (HC) alone as well as in combination with air has been studied using nozzles as cavitating device of HC reactor. Initially, the energy efficiency of the HC reactor operated at different inlet pressures was determined using the calorimetric studies. Maximum energy efficiency of 53.4% was obtained at an inlet pressure of 3.9 bar. The treatment processes were compared under adiabatic as well as isothermal conditions and it was observed that under the adiabatic condition, the extent of degradation is higher as compared to isothermal condition. Studies related to the understanding the effect of inlet pressure (range of 1.8-3.9 bar) revealed that the maximum degradation as 98.9% was obtained at 2.4 bar pressure using the individual operation of HC under adiabatic conditions and in 70 min of treatment. The combination of HC with air was investigated at different air flow rates with best results for maximum degradation of benzene achieved at air flow rate of 60 mL/sec. A novel approach of using cavitation for a limited fraction of total treatment time was also demonstrated to be beneficial in terms of the extent of degradation as well as energy requirements and cost of operation. Based on the cavitational intensity, the resonant radius of aggregates of cavitation bubbles was also determined for distilled water as well as for aqueous solution of benzene. Overall, significant benefits of using HC combined with air have been demonstrated for degradation of benzene along with fundamental understanding into cavitation effects.

The Co-occurrence of DNRA and Anammox during the anaerobic degradation of benzene under denitrification.

It was revealed that Anammox process promotes the anaerobic degradation of benzene under denitrification. This study investigates the effect of dissimilatory nitrate reduction to ammonium (DNRA) and exogenous ammonium on anaerobic ammonium oxidation bacteria (AnAOB) during the anaerobic degradation of benzene under denitrification. The results indicate that anammox occurs synergistically with organisms using the DNRA pathway, such as Draconibacterium and Ignavibacterium. Phylogenetic analysis showed 64% (16/25) and 36% (5/25) hzsB gene sequences, a specific biomarker of AnAOB, belonged to Candidatus 'Brocadia fuldiga' and Candidatus 'Kuenenia', respectively. Exogenous ammonium addition enhanced the anammox process and accelerated benzene degradation at a 1.89-fold higher average rate compared to that in the absence of exogenous ammonium and AnAOB belonged to Ca. 'Kuenenia' (84%) and Ca. 'Brocadia fuldiga' (16%). These results indicate that Ca. 'Brocadia fuldiga' could also play a role in DNRA. However, the diversity of abcA and bamA, the key anaerobic benzene metabolism biomarkers, remained unchanged. These findings suggest that anammox occurrence may be coupled with DNRA or exogenous ammonium and that anammox promotes anaerobic benzene degradation under denitrifying conditions. The results of this study contribute to understanding the co-occurrence of DNRA and Anammox and help explore their involvement in degradation of benzene, which will be crucial for directing remediation strategies of benzene-contaminated anoxic environment.

Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing.

Constructed wetlands (CWs) are effective ecological remediation technologies for various contaminated water bodies. Here, we queried for benzene-degrading microbes in a horizontal subsurface flow CW with reducing conditions in the pore water and fed with benzene-contaminated groundwater. For identification of relevant microbes, we employed in situ microcosms (BACTRAPs, which are made from granulated activated carbon) coupled with 13C-stable isotope probing and Illumina sequencing of 16S rRNA amplicons. A significant incorporation of 13C was detected in RNA isolated from BACTRAPs loaded with 13C-benzene and exposed in the CW for 28 days. A shorter incubation time did not result in detectable 13C incorporation. After 28 days, members from four genera, namely Dechloromonas, Hydrogenophaga, and Zoogloea from the Betaproteobacteria and Arcobacter from the Epsilonproteobacteria were significantly labeled with 13C and were abundant in the bacterial community on the BACTRAPs. Sequences affiliated to Geobacter were also numerous on the BACTRAPs but apparently those microbes did not metabolize benzene as no 13C label incorporation was detected. Instead, they may have metabolized plant-derived organic compounds while using the BACTRAPs as electron sink. In representative wetland samples, sequences affiliated with Dechloromonas, Zoogloea, and Hydrogenophaga were present at relative proportions of up to a few percent. Sequences affiliated with Arcobacter were present at < 0.01% in wetland samples. In conclusion, we identified microbes of likely significance for benzene degradation in a CW used for remediation of contaminated water.

Anaerobic Benzene Mineralization by Nitrate-Reducing and Sulfate-Reducing Microbial Consortia Enriched From the Same Site: Comparison of Community Composition and Degradation Characteristics.

Benzene mineralization under nitrate-reducing conditions was successfully established in an on-site reactor continuously fed with nitrate and sulfidic, benzene-containing groundwater extracted from a contaminated aquifer. Filling material from the reactor columns was used to set up anoxic enrichment cultures in mineral medium with benzene as electron donor and sole organic carbon source and nitrate as electron acceptor. Benzene degradation characteristics and community composition under nitrate-reducing conditions were monitored and compared to those of a well-investigated benzene-mineralizing consortium enriched from the same column system under sulfate-reducing conditions. The nitrate-reducing cultures degraded benzene at a rate of 10.1 ± 1.7 µM d-1, accompanied by simultaneous reduction of nitrate to nitrite. The previously studied sulfate-reducing culture degraded benzene at similar rates. Carbon and hydrogen stable isotope enrichment factors determined for nitrate-dependent benzene degradation differed significantly from those of the sulfate-reducing culture (ΛH/C nitrate = 12 ± 3 compared to ΛH/C sulfate = 28 ± 3), indicating different benzene activation mechanisms under the two conditions. The nitrate-reducing community was mainly composed of Betaproteobacteria, Ignavibacteria, and Anaerolineae. Azoarcus and a phylotype related to clone Dok59 (Rhodocyclaceae) were the dominant genera, indicating an involvement in nitrate-dependent benzene degradation. The primary benzene degrader of the sulfate-reducing consortium, a phylotype belonging to the Peptococcaceae, was absent in the nitrate-reducing consortium.

Benzene degradation in a denitrifying biofilm reactor: activity and microbial community composition.

Benzene is an aromatic compound and harmful for the environment. Biodegradation of benzene can reduce the toxicological risk after accidental or controlled release of this chemical in the environment. In this study, we further characterized an anaerobic continuous biofilm culture grown for more than 14 years on benzene with nitrate as electron acceptor. We determined steady state degradation rates, microbial community composition dynamics in the biofilm, and the initial anaerobic benzene degradation reactions. Benzene was degraded at a rate of 0.15 µmol/mg protein/day and a first-order rate constant of 3.04/day which was fourfold higher than rates reported previously. Bacteria belonging to the Peptococcaceae were found to play an important role in this anaerobic benzene-degrading biofilm culture, but also members of the Anaerolineaceae were predicted to be involved in benzene degradation or benzene metabolite degradation based on Illumina MiSeq analysis of 16S ribosomal RNA genes. Biomass retention in the reactor using a filtration finger resulted in reduction of benzene degradation capacity. Detection of the benzene carboxylase encoding gene, abcA, and benzoic acid in the culture vessel indicated that benzene degradation proceeds through an initial carboxylation step.

Pulsed (13)C2-Acetate Protein-SIP Unveils Epsilonproteobacteria as Dominant Acetate Utilizers in a Sulfate-Reducing Microbial Community Mineralizing Benzene.

In a benzene-degrading and sulfate-reducing syntrophic consortium, a clostridium affiliated to the genus Pelotomaculum was previously described to ferment benzene while various sulfate-reducing Deltaproteobacteria and a member of the Epsilonproteobacteria were supposed to utilize acetate and hydrogen as key metabolites derived from benzene fermentation. However, the acetate utilization network within this community was not yet unveiled. In this study, we performed a pulsed (13)C2-acetate protein stable isotope probing (protein-SIP) approach continuously spiking low amounts of acetate (10 µM per day) in addition to the ongoing mineralization of unlabeled benzene. Metaproteomics revealed high abundances of Clostridiales followed by Syntrophobacterales, Desulfobacterales, Desulfuromonadales, Desulfovibrionales, Archaeoglobales, and Campylobacterales. Pulsed acetate protein-SIP results indicated that members of the Campylobacterales, the Syntrophobacterales, the Archaeoglobales, the Clostridiales, and the Desulfobacterales were linked to acetate utilization in descending abundance. The Campylobacterales revealed the fastest and highest (13)C incorporation. Previous experiments suggested that the activity of the Campylobacterales was not essential for anaerobic benzene degradation in the investigated community. However, these organisms were consistently detected in various hydrocarbon-degrading and sulfate-reducing consortia enriched from the same aquifer. Here, we demonstrate that this member of the Campylobacterales is the dominant acetate utilizer in the benzene-degrading microbial consortium.

Benzene and sulfide removal from groundwater treated in a microbial fuel cell.

Sulfidic benzene-contaminated groundwater was used to fuel a two-chambered microbial fuel cell (MFC) over a period of 770 days. We aimed to understand benzene and sulfide removal processes in the anoxic anode chamber and describe the microbial community enriched over the operational time. Operated in batch feeding-like circular mode, supply of fresh groundwater resulted in a rapid increase in current production, accompanied by decreasing benzene and sulfide concentrations. The total electron recoveries for benzene and sulfide were between 18% and 49%, implying that benzene and sulfide were not completely oxidized at the anode. Pyrosequencing of 16S rRNA genes from the anode-associated bacterial community revealed the dominance of δ-Proteobacteria (31%), followed by ß-Proteobacteria, Bacteroidetes, ϵ-Proteobacteria, Chloroflexi, and Firmicutes, most of which are known for anaerobic metabolism. Two-dimensional compound-specific isotope analysis demonstrated that benzene degradation was initiated by monohydroxylation, probably triggered by small amounts of oxygen which had leaked through the cation exchange membrane into the anode chamber. Experiments with [(13)C(6) ]-benzene revealed incorporation of (13)C into fatty acids of mainly Gram-negative bacteria, which are therefore candidates for benzene degradation. Our study demonstrated simultaneous benzene and sulfide removal by groundwater microorganisms which use an anode as artificial electron acceptor, thereby releasing an electrical current.