A comparison of the performance of bacterial biofilters and fungal-bacterial coupled biofilters in BTEp-X removal.

Background: Conventional biofilters, which rely on bacterial activity, face challenges in eliminating hydrophobic compounds, such as aromatic compounds. This is due to the low solubility of these compounds in water, which makes them difficult to absorb by bacterial biofilms. Furthermore, biofilter operational stability is often hampered by acidification and drying out of the filter bed. Methods: Two bioreactors, a bacterial biofilter (B-BF) and a fungal-bacterial coupled biofilter (F&B-BF) were inoculated with activated sludge from the secondary sedimentation tank of the Sinopec Yangzi Petrochemical Company wastewater treatment plant located in Nanjing, China. For approximately 6 months of operation, a F&B-BF was more effective than a B-BF in eliminating a gas-phase mixture containing benzene, toluene, ethylbenzene, and para-xylene (BTEp-X). Results: After operating for four months, the F&B-BF showed higher removal efficiencies for toluene (T), ethylbenzene (E), benzene (B), and para-X (p-Xylene), at 96.9%, 92.6%, 83.9%, and 83.8%, respectively, compared to those of the B-BF (90.1%, 78.7%, 64.8%, and 59.3%). The degradation activity order for B-BF and F&B-BF was T > E > B > p-X. Similarly, the rates of mineralization for BTEp-X in the F&B-BF were 74.9%, 66.5%, 55.3%, and 45.1%, respectively, which were higher than those in the B-BF (56.5%, 50.8%, 43.8%, and 30.5%). Additionally, the F&B-BF (2 days) exhibited faster recovery rates than the B-BF (5 days). Conclusions: It was found that a starvation protocol was beneficial for the stable operation of both the B-BF and F&B-BF. Community structure analysis showed that the bacterial genus Pseudomonas and the fungal genus Phialophora were both important in the degradation of BTEp-X. The fungal-bacterial consortia can enhance the biofiltration removal of BTEp-X vapors.

In-situ treatment of gaseous benzene in fixed-bed biofilter with polyurethane foam: Functional population response and benzene transformation pathway.

Volatile organic compounds emitted from landfills posed adverse effect on health. In this study, gaseous benzene was biologically treated using an in-situ biofilter without air pump. Its performance was investigated and the removal efficiency of benzene reached over 90 %. The decrease in the average benzene concentration was consistent with first-order reaction kinetics. Mycolicibacterium dominated the bacterial consortium (41-57 %) throughout the degradation. Annotation of genes by metagenomic analysis helped to deduce the degradation pathways (benzene degradation, catechol ortho-cleavage and meta-cleavage) and to reveal the contribution of different species to the degradation process. In total, 21 kinds of key genes and 13 enzymes were involved in the three modules of benzene transformation. Mycolicibacter icosiumassiliensis and Sphingobium sp. SCG-1 carried multiple functional genes critically involved in benzene biodegradation. These findings provide technical and theoretical support for the in-situ bioremediation of benzene-contaminated soil and waste gas reduction in landfills.

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.

New perspectives on the anaerobic degradation of BTEX: Mechanisms, pathways, and intermediates.

Aromatic hydrocarbons like benzene, toluene, xylene, and ethylbenzene (BTEX) can escape into the environment from oil and gas operations and manufacturing industries posing significant health risks to humans and wildlife. Unlike conventional clean-up methods used, biological approaches such as bioremediation can provide a more energy and labour-efficient and environmentally friendly option for sensitive areas such as nature reserves and cities, protecting biodiversity and public health. BTEX contamination is often concentrated in the subsurface of these locations where oxygen is rapidly depleted, and biodegradation relies on anaerobic processes. Thus, it is critical to understand the anaerobic biodegradation characteristics as it has not been explored to a major extent. This review presents novel insights into the degradation mechanisms under anaerobic conditions and presents a detailed description and interconnection between them. BTEX degradation can follow four activation mechanisms: hydroxylation, carboxylation, methylation, and fumarate addition. Hydroxylation is one of the mechanisms that explains the transformation of benzene into phenol, toluene into benzyl alcohol or p-cresol, and ethylbenzene into 1-phenylethanol. Carboxylation to benzoate is thought to be the primary mechanism of degradation for benzene. Despite being poorly understood, benzene methylation has been also reported. Moreover, fumarate addition is the most widely reported mechanism, present in toluene, ethylbenzene, and xylene degradation. Further research efforts are required to better elucidate new and current alternative catabolic pathways. Likewise, a comprehensive analysis of the enzymes involved as well as the development of advance tools such as omic tools can reveal bottlenecks degradation steps and create more effective on-site strategies to address BTEX pollution.

Synchronous removal of gaseous toluene and benzene and degradation process shifts in microbial fuel cell-biotrickling filter system.

Illustrating the biodegradation processes of multi-component volatile organic compounds (VOCs) will expedite the implication of biotechnology in purifying industrial exhaust. Here, performance shifts of microbial fuel cell and biotrickling filter combined system (MFC-BTF) are investigated for removing single and dual components of toluene and benzene. Synchronous removal of toluene (95 %) and benzene (97 %) are achieved by MFC-BTF accompanied with the output current of 0.41 mA. Elevated content of extracellular polymeric substance facilitates the mass transfer of benzene with the presence of toluene. Strains of Bacteroidota, Proteobacteria and Chloroflexi contribute to the removal of dual components VOCs. Empty bed reaction time and the VOCs concentration are the important factors influencing their dissolution in the system. The biodegradation of toluene and benzene proceeds with 2-hydroxymuconic semialdehyde and o-hydroxybenzoic acid as the main intermediates. These results provide a comprehensive understanding of multi-component VOCs removal by MFC-BTF and guide the system design, optimization, and scale-up.

Mechanisms of benzene and benzo[a]pyrene biodegradation in the individually and mixed contaminated soils.

There is a lack of knowledge on the biodegradation mechanisms of benzene and benzo [a]pyrene (BaP), representative compounds of polycyclic aromatic hydrocarbons (PAHs), and benzene, toluene, ethylbenzene, and xylene (BTEX), under individually and mixed contaminated soils. Therefore, a set of microcosm experiments were conducted to explore the influence of benzene and BaP on biodegradation under individual and mixed contaminated condition, and their subsequent influence on native microbial consortium. The results revealed that the total mass loss of benzene was 56.0% under benzene and BaP mixed contamination, which was less than that of individual benzene contamination (78.3%). On the other hand, the mass loss of BaP was slightly boosted to 17.6% under the condition of benzene mixed contamination with BaP from that of individual BaP contamination (14.4%). The significant differences between the microbial and biocide treatments for both benzene and BaP removal demonstrated that microbial degradation played a crucial role in the mass loss for both contaminants. In addition, the microbial analyses revealed that the contamination of benzene played a major role in the fluctuations of microbial compositions under co-contaminated conditions. Rhodococcus, Nocardioides, Gailla, and norank_c_Gitt-GS-136 performed a major role in benzene biodegradation under individual and mixed contaminated conditions while Rhodococcus, Noviherbaspirillum, and Phenylobacterium were highly involved in BaP biodegradation. Moreover, binary benzene and BaP contamination highly reduced the Rhodococcus abundance, indicating the toxic influence of co-contamination on the functional key genus. Enzymatic activities revealed that catalase, lipase, and dehydrogenase activities proliferated while polyphenol oxidase was reduced with contamination compared to the control treatment. These results provided the fundamental information to facilitate the development of more efficient bioremediation strategies, which can be tailored to specific remediation of different contamination scenarios.

Prefeeding of Clarias gariepinus with Spirulina platensis counteracts petroleum hydrocarbons-induced hepato- and nephrotoxicity.

Petroleum aromatic hydrocarbons are considered one of the most dangerous aquatic pollutants due to their widespread across water bodies, persistence, and extension to the food chain. To our knowledge, there hasn't been any research investigating the hepatorenoprotective effects of Spirulina platensis (SP) against toxicity induced by these environmental toxicants in fish. Thus, we decided to explore its potential safeguarding against benzene and toluene exposure in adult Clarias gariepinus. To achieve this objective, fish were divided into five groups (60 per group; 20 per replicate). The first group served as a control. The second and third groups were intoxicated with benzene and toluene at doses of 0.762 and 26.614 ng/L, respectively for 15 days. The fourth and fifth groups (SP + benzene and SP + toluene, respectively) were challenged with benzene and toluene as previously mentioned following dietary inclusion of SP at a dose of 5 g/kg diet for 30 days. The marked increase in liver metabolizing enzymes, glucose, total protein, albumin, globulin, albumin/globulin ratio, and creatinine confirmed the hepato- and nephrotoxic impacts of benzene and toluene. These outcomes were coupled with cytopathological affections and excessive collagen deposition. The incorporation of SP in ration formulation, on the contrary, restored the previously mentioned toxicological profile due to its antioxidant and cytoprotective attributes. Regardless of SP intervention, the renal tissues still displayed histo-architectural lesions, because of insufficient dose and timeframe. Additional research will be required to identify the ideal SP remediation regimen.

Production of Biobased Ethylbenzene by Cascade Biocatalysis with an Engineered Photodecarboxylase.

Production of commodity chemicals, such as benzene, toluene, ethylbenzene, and xylenes (BTEX), from renewable resources is key for a sustainable society. Biocatalysis enables one-pot multistep transformation of bioresources under mild conditions, yet it is often limited to biochemicals. Herein, we developed a non-natural three-enzyme cascade for one-pot conversion of biobased l-phenylalanine into ethylbenzene. The key rate-limiting photodecarboxylase was subjected to structure-guided semirational engineering, and a triple mutant CvFAP(Y466T/P460A/G462I) was obtained with a 6.3-fold higher productivity. With this improved photodecarboxylase, an optimized two-cell sequential process was developed to convert l-phenylalanine into ethylbenzene with 82 % conversion. The cascade reaction was integrated with fermentation to achieve the one-pot bioproduction of ethylbenzene from biobased glycerol, demonstrating the potential of cascade biocatalysis plus enzyme engineering for the production of biobased commodity chemicals.

Bioaerosols released from multistage biofilter for gaseous benzene removal: Escape behavior and pathogenicity.

Biological deodorization systems are widely used to control odors and volatile organic compounds. However, the secondary contamination of bioaerosol emissions is a noteworthy issue in the operation of biofilters for off-gas purification. In this study, a multistage biofilter for benzene treatment was utilized to investigate the bioaerosol emissions under different flow rates and spray intervals. At the outlet of the biofilter, 99-7173 CFU/m3 of bioaerosols were detected, among which pathogens accounted for 8.93-98.73 %. Proteobacteria and Firmicutes dominated bioaerosols at the phylum level. The Mantel test based on the Bray-Curtis distance revealed strong influences of flow rate introduced to the biofilter and biomass colonized on the packing materials (PMs) on bioaerosol emissions. The non-metric multidimensional scaling results suggested a correlation between the bioaerosol community and bacteria on the PMs. Bacillus and Stenotrophomonas were the two main genera stripped from the biofilm on PMs to form the bioaerosols. SourceTracker analysis confirmed that microorganisms from the PMs near outlet contributed an average of 22.3 % to bioaerosols. Pathogenic bacteria carried by bioaerosols included Bacillus, Serratia, Stenotrophomonas, Achromobacter, Enterococcus, and Pseudomonas. Bioaerosols were predicted to cause human diseases, with antimicrobial drug resistance and bacterial infectious disease being the two main pathogenic pathways. Stenotrophomonas sp. LMG 19833, Pseudomonas sp., and Stenotrophomonas sp. were the keystone species in the bioaerosol co-occurrence network. Overall, results of present study promote the insight of bioaerosols, particularly pathogen emissions, and provide a basis for controlling bioaerosol contamination from biofilters.

Tim-3 facilitates immune escape in benzene-induced acute myeloid leukemia mouse model by promoting macrophage M2 polarization.

Benzene poisoning can cause acute myeloid leukemia (AML) through a variety of passways. Tim-3 has gained prominence as a potential candidate in mediating immunosuppression in tumor microenvironments. The macrophage polarization is also related to immune escape. Herein, we reported that Tim-3 and macrophage M2 polarization play a vital role in benzene-induced AML. First, the benzene-induced AML C3H/He mouse model was constructed by subcutaneously injecting 250 mg/kg of benzene. After six months, macrophage phenotype, cytokines, and Tim-3 expression levels were investigated. Flow cytometry assay revealed that the T-cell inhibitory receptor Tim-3 was significantly upregulated in both bone marrow and spleen of the benzene-induced AML mouse model. Elisa's results displayed a decreased serum level of IL-12 while increased TGF-ß1. Mechanistically, changes in cytokine secretion promote the growth of M2-type macrophages in the bone marrow and spleen, as determined by immunofluorescence assay. The increased levels of PI3K, AKT, and mTOR in the benzene-exposure group further proved the crucial role of Tim-3 in regulating the functional status of macrophages in the AML microenvironment. These results demonstrate that Tim-3 and macrophage polarization may play a vital role during the immune escape of the benzene-induced AML. This study provides a new potential intervention site for immune checkpoint-based AML therapeutic strategy.

Fennel affects porcine ovarian cell functions: The interrelationships with the environmental contaminant benzene.

The objective of this study was to examine the direct effects of the medicinal plant fennel on basic functions of ovarian cells, including proliferation, apoptosis, and release of progesterone and insulin-like growth factor I (IGFI), as well as to prevent the influence of the environmental contaminant benzene on these cells. Porcine ovarian granulosa cells were cultured with or without fennel extract alone or in combination with benzene. The expression of the proliferation marker PCNA and the apoptosis marker bax was analyzed by quantitative immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). Fennel was able to promote proliferation and IGF-I release, but to suppress apoptosis and progesterone release. Benzene promoted the accumulation of both the proliferation and apoptosis markers, as well as IGF-I release, but it inhibited progesterone secretion. The presence of fennel did not prevent the effects of benzene on any of the measured parameters, while benzene prevented the effects of fennel on cell proliferation, apoptosis, and IGF-I but not progesterone output. These observations demonstrate the direct influence of fennel and benzene on basic ovarian cell functions. Furthermore, they show the inability of fennel to prevent the effects of benzene on these cells. On the other hand, the environmental contaminant benzene can block the response of ovarian cells to the medicinal plant fennel.

CYP72D19 from Tripterygium wilfordii catalyzes C-2 hydroxylation of abietane-type diterpenoids.

KEY MESSAGE: CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.

Novel BTEX-degrading strains from subsurface soil: Isolation, identification and growth evaluation.

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and o, m, and p-xylenes (BTEX) are high-risk pollutants because of their mutagenic and carcinogenic nature. These pollutants are found with elevated levels in groundwater and soil in Canada at several contaminated sites. The intrinsic microbes present in the subsurface have the potential to degrade pollutants by their metabolic pathways and convert them to non-toxic products. However, the low subsurface temperature (5-10 °C) limits their growth and degradation ability. This study examined the feasibility of subsurface heat augmentation using geothermal heating for BTEX bioremediation. Novel potent BTEX-degrading bacterial strains were isolated from soil at 3.0, 42.6, and 73.2 m depths collected from a geothermal borehole during installation and screened using an enrichment technique. The selected strains were identified with Sanger sequencing and phylogenetic tree analysis, revealing that all the strains except Bacillus subtilis are novel with respective to BTEX degradation. The isolates, Microbacterium esteraromaticum and Bacillus infantis showed the highest degradation with 67.98 and 65.2% for benzene, 72.8 and 71.02% for toluene, 77.52 and 76.44% for ethylbenzene, and 74.58 and 74.04% for xylenes respectively. Further, temperature influence at 15 ± 1 °C, 28 ± 1 °C and 40 ± 1 °C was observed, which showed increased growth by two-fold and on average 35-49% more biodegradation at higher temperatures. Results showed that temperature is a positive stimulant for bioremediation, hence geothermal heating could also be a stimulant for in-situ bioremediation.

Efficiency, mechanism, influencing factors, and integrated technology of biodegradation for aromatic compounds by microalgae: A review.

Aromatic compounds have received widespread attention because of their threat to ecosystem and human health. However, traditional physical and chemical methods are criticized due to secondary pollution and high cost. As a result of ecological security and the ability of carbon sequestration, biodegradation approach based on microalgae has emerged as a promising alternative treatment for aromatic pollutants. In light of the current researches, the degradation efficiency of BTEX (benzene, toluene, ethylbenzene, and xylene), polycyclic aromatic hydrocarbons (PAHs), and phenolic compounds by microalgae was reviewed in this study. We summarized the degradation pathways and metabolites of p-xylene, benzo [a]pyrene, fluorene, phenol, bisphenol A, and nonylphenol by microalgae. The influence factors on the degradation of aromatic compounds by microalgae were also discussed. The integrated technologies based on microalgae for degradation of aromatic compounds were reviewed. Finally, this study discussed the limitations and future research needs of the degradation of these compounds by microalgae.

Influence of growth substrate and contaminant mixtures on the degradation of BTEX and MTBE by Rhodococcus rhodochrous ATCC strain 21198.

The degradation of the prevalent environmental contaminants benzene, toluene, ethylbenzene, and xylenes (BTEX) along with a common co-contaminant methyl tert-butyl ether (MTBE) by Rhodococcus rhodochrous ATCC Strain 21198 was investigated. The ability of 21198 to degrade these contaminants individually and in mixtures was evaluated with resting cells grown on isobutane, 1-butanol, and 2-butanol. Growth of 21198 in the presence of BTEX and MTBE was also studied to determine the growth substrate that best supports simultaneous microbial growth and contaminants degradation. Cells grown on isobutane, 1-butanol, and 2-butanol were all capable of degrading the contaminants, with isobutane grown cells exhibiting the most rapid degradation rates and 1-butanol grown cells exhibiting the slowest. However, in conditions where BTEX and MTBE were present during microbial growth, 1-butanol was determined to be an effective substrate for supporting concurrent growth and contaminant degradation. Contaminant degradation was found to be a combination of metabolic and cometabolic processes. Evidence for growth of 21198 on benzene and toluene is presented along with a possible transformation pathway. MTBE was cometabolically transformed to tertiary butyl alcohol, which was also observed to be transformed by 21198. This work demonstrates the possible utility of primary and secondary alcohols to support biodegradation of monoaromatic hydrocarbons and MTBE. Furthermore, the utility of 21198 for bioremediation applications has been expanded to include BTEX and MTBE.

LncRNA OBFC2A modulated benzene metabolites-induced autophagy and apoptosis by interacting with LAMP2.

Exposure to benzene results in peripheral blood cell reduction, aplastic anemia, and leukemia. We previously observed that the lncRNA OBFC2A was upregulated significantly in benzene-exposed workers and correlated with reduced blood cell counts. However, the role of lncRNA OBFC2A in benzene hematotoxicity remains unclear. In this study, we discovered that lncRNA OBFC2A was regulated by oxidative stress and played roles in cell autophagy and apoptosis caused by the benzene metabolite 1,4-Benzoquinone (1,4-BQ) in vitro. Mechanistically, protein chip, RNA pull-down, and FISH colocalization uncovered that lncRNA OBFC2A directly bound to LAMP2, a regulator of chaperone-mediated autophagy (CMA), and upregulated its expression in 1,4-BQ-treated cells. LncRNA OBFC2A knockdown alleviated LAMP2 overexpression caused by 1,4-BQ, which confirmed their regulatory relationship. In conclusion, we demonstrate that lncRNA OBFC2A mediates 1,4-BQ-induced apoptosis and autophagy by interacting with LAMP2. LncRNA OBFC2A could serve as a biomarker for hematotoxicity caused by benzene.

Low-dose blood BTEX are associated with pulmonary function through changes in inflammatory markers among US adults: NHANES 2007-2012.

The effects of blood benzene, toluene, ethylbenzene, and xylenes (BTEX) on lung function among general adults remain unknown. We enrolled 5519 adults with measured blood BTEX concentrations and lung function from the US National Health and Nutrition Examination Survey 2007-2012. Weighted linear models were fitted to assess the associations of BTEX with lung function and inflammation parameters (white blood cell five-part differential count and C-reactive protein). The mediating effect of inflammation between BTEX and lung function was also examined. Blood BTEX concentrations decreased yearly from 1999 and were extremely low from 2007 to 2012. Benzene and toluene exerted the greatest influence on lung function in terms of forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), calculated FEV1:FVC ratio, peak expiratory flow rate (PEFR), and forced mid expiratory flow (FEF25-75%). Both ethylbenzene and all xylene isomers had no effects on FVC but reduced FEV1, FEV1:FVC ratio, PEFR, and FEF25-75%. Weighted quantile analyses demonstrated that BTEX mixture was associated with decreases in FVC, FEV1, FEV1:FVC ratio, PEFR, and FEF25-75%, with benzene weighted most heavily for all lung function parameters. BTEX also increased the levels of inflammation indicated by white blood cell five-part differential count and C-reactive protein, and increased levels of inflammation also reduced lung function. From multiple mediation analysis, inflammation mediated the effects of benzene on FEV1 and PEFR, the effects of toluene on FEV1, and the effects of ethylbenzene on FEV1 and PEFR. Low-dose exposure to BTEX was associated with reduced pulmonary function both in large and small airways. Inflammation could be involved in this pathogenesis.

Genome sequencing of Cladophialophora exuberans, a novel candidate for bioremediation of hydrocarbon and heavy metal polluted habitats.

Cladophialophora exuberans is a filamentous fungus related to black yeasts in the order Chaetothyriales. These melanized fungi are known for their 'dual ecology', often occurring in toxic environments and also being frequently involved in human infection. Particularly Cladophialophora exuberans, C. immunda, C. psammophila, and Exophiala mesophila have been described with a pronounced ability to degrade aromatic compounds and xenobiotic volatiles, such as benzene, toluene, ethyl-benzene, and xylene, and are candidates for bioremediation applications. The objective of the present study is the sequencing, assembly, and description of the whole genome of C. exuberans focusing on genes and pathways related to carbon and toxin management, assessing the tolerance and bioremediation of lead and copper, and verifying the presence of genes for metal homeostasis. Genomic evaluations were carried out through a comparison with sibling species including clinical and environmental strains. Tolerance of metals was evaluated via a microdilution method establishing minimum inhibitory (MIC) and fungicidal concentrations (MFC), and agar diffusion assays. Heavy metal bioremediation was evaluated via graphite furnace atomic absorption spectroscopy (GFAAS). The final assembly of C. exuberans comprised 661 contigs, with genome size of 38.10 Mb, coverage of 89.9X and a GC content of 50.8%. In addition, inhibition of growth was shown at concentrations of 1250 ppm for copper and at 625 ppm for lead, using the MIC method. In the agar tests, the strain grew at 2500 ppm of copper and lead. In GFAAS tests, uptake capacities were observed of 89.2% and 95.7% for copper and lead, respectively, after 21 experimental days. This study enabled the annotation of genes involved in heavy metal homeostasis and also contributed to a better understanding of the mechanisms used in tolerance of and adaptation to extreme conditions.

Comparative Genomic Analysis and BTEX Degradation Pathways of a Thermotolerant Cupriavidus cauae PHS1.

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.