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The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, new information was found leading to the withdrawal of the qualification 'absence of aminoglycoside production ability' for Bacillus velezensis. The qualification for Bacillus paralicheniformis was changed to 'absence of bacitracin production ability'. For the other TUs, no new information was found that would change the status of previously recommended QPS TUs. Of 52 microorganisms notified to EFSA between April and September 2022 (inclusive), 48 were not evaluated because: 7 were filamentous fungi, 3 were Enterococcus faecium, 2 were Escherichia coli, 1 was Streptomyces spp., and 35 were taxonomic units (TUs) that already have a QPS status. The other four TUs notified within this period, and one notified previously as a different species, which was recently reclassified, were evaluated for the first time for a possible QPS status: Xanthobacter spp. could not be assessed because it was not identified to the species level; Geobacillus thermodenitrificans is recommended for QPS status with the qualification 'absence of toxigenic activity'. Streptoccus oralis is not recommended for QPS status. Ogataea polymorpha is proposed for QPS status with the qualification 'for production purposes only'. Lactiplantibacillus argentoratensis (new species) is included in the QPS list.

Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon (e.g., sugars) and nitrogen (e.g., ammonia) inputs are required. Gas-fixing microorganisms that can grow on H 2 from renewable water splitting and gaseous CO 2 and N 2 offer a renewable path to overcoming these limitations but confront challenges owing to the scarcity of genetic engineering in such organisms. Here, we demonstrate that the hydrogen-oxidizing carbon- and nitrogen-fixing microorganism Xanthobacter autotrophicus grown on a CO 2 /N 2 /H 2 gas mixture can overproduce the vitamin riboflavin (vitamin B 2 ). We identify plasmids and promoters for use in this bacterium and employ a constitutive promoter to overexpress riboflavin pathway enzymes. Riboflavin production is quantified at 15 times that of the wild-type organism. We demonstrate that riboflavin overproduction is maintained when the bacterium is grown under hybrid inorganic-biological conditions, in which H 2 from water splitting, along with CO 2 and N 2 , is fed to the bacterium, establishing the viability of the approach to sustainably produce food and nutrients.

1,4-Dioxane is a highly toxic and carcinogenic pollutant found worldwide in groundwater and soil environments. Several microorganisms have been isolated by their ability to grow on 1,4-dioxane; however, low 1,4-dioxane tolerance and slow degradation kinetics remain obstacles for their use in 1,4-dioxane bioremediation. We report here the isolation and characterization of a new strain, Xanthobacter sp. YN2, capable of highly efficient 1,4-dioxane degradation. High degradation efficiency and high tolerance to 1,4-dioxane make this new strain an ideal candidate for the biodegradation of 1,4-dioxane in various treatment facilities. The maximum degradation rate of 1,4-dioxane was found to be 1.10 mg-1,4-dioxane/h mg-protein. Furthermore, Xanthobacter sp. YN2 was shown to grow in the presence of higher than 3000 mg/L 1,4-dioxane with little to no degradation inhibition. In addition, Xanthobacter sp. YN2 could grow on and degrade 1,4-dioxane at pH ranges 5 to 8 and temperatures between 20 and 40 °C. Xanthobacter sp. YN2 was also found to be able to grow on a variety of other substrates including several analogs of 1,4-dioxane. Genome sequence analyses revealed the presence of two soluble di-iron monooxygenase (SDIMO) gene clusters, and regulation studies determined that all of the genes in these two clusters were upregulated in the presence of 1,4-dioxane. This study provides insights into the bacterial stress response and the highly efficient biodegradation of 1,4-dioxane as well as the identification of a novel Group-2 SDIMO.

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO

Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl–CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.

A novel, aerobic nitrogen-fixing methylotrophic bacterium, strain 29kT, was enriched and isolated from sludge generated during wastewater treatment at a paper mill in Baikal, Russian Federation. Cells were Gram-stain-variable. The cell wall was of the negative Gram-type. Cells were curved oval rod-shaped, 0.5–0.7×1.7–3.4 µm and formed yellow-coloured colonies. Cells tended to be pleomorphic if grown on media containing succinate or coccoid if grown in the presence of methyl alcohol as the sole carbon source. Cells were non-motile, non-spore-forming and contained retractile (polyphosphate) and lipid (poly-β-hydroxybutyrate) bodies. The major respiratory quinone was ubiquinone Q-10 and the predominant cellular fatty acids were C18:1 ω7, C19:0 cyclo and C16:0. The genomic DNA G+C content was 67.95 mol%. Strain 29kT was able to grow at 4–37 °C (optimum, 30 °C), at pH 6.0–8.5 (optimum, pH 6.5–7.0) and at salinities of 0–0.5% (w/v) NaCl (optimum, 0% NaCl). Catalase and oxidase were positive. Strain 29kT could grow chemolithoautotrophically in mineral media under an atmosphere of H2, O2 and CO2 as well as chemoorganoheterotrophically on methanol, ethanol, n-propanol, n-butanol and various organic acids. The carbohydrate utilization spectrum is limited by glucose and raffinose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the newly isolated strain was a member of the genus Xanthobacter with Xanthobacter autotrophicus 7cT (99.9% similarity) and Xanthobacter viscosus 7dT (99.4 % similarity) as closest relatives among species with validly published names. The average nucleotide identity and digital DNA–DNA hybridization values of 92.7 and 44.9%, respectively, of the 29kT to the genome of the most closely related species, X. autotrophicus 7cT, were below the species cutoffs. Based on genotypic, phenotypic and chemotaxonomic characteristics, it is proposed that the isolate represents a novel species, Xanthobacter oligotrophicus sp. nov. The type strain is 29kT (=KCTC 72777T=VKM B-3453T).

A Gram-stain-negative, rod-shaped, non-motile, aerobic, catalase-negative and oxidase-positive bacterium, designated strain Sn-9-2T, was isolated from a cave soil sample collected from Tiandong cave, Guizhou Province, south-west PR China. Growth occurred at 15–40 °C (optimum, 30 °C), at pH 5.0–9.0 (optimum, pH 7.0–8.0) and with 0–1 % NaCl (w/v). The predominant respiration quinone was ubiquinone-10 (Q-10). The major cellular fatty acids were summed feature 8 (C18 : 1ω7c or C18 : 1ω6c; 83.9 %) and C16 : 0 (5.8 %). The major polar lipids were phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphatidylglycerol, three unidentified phospholipids, two unidentified glycolipids, two unidentified polar lipids and one unidentified aminolipid. The DNA G+C content of strain Sn-9-2T was 67.5 mol%. Based on the results of 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain Sn-9-2T (MF958452) were identified as Aquabacter spiritensis (FR733686) DSM 9035T (97.5 %), Xanthobacter autorophicus (jgi.1053054) DSM 432T (97.2 %) and Xanthobacter tagetidis ATCC 700314T RCTF01000015 (96.9 %). The average nucleotide identity values were 78.0, 77.4 and 77.6 % and the digital DNA–DNA hybridization values were 21.8, 22.0 and 18.8 % between strain Sn-9-2T and A. spiritensis DSM 9035T, X. autotrophicus DSM 432T and X. tagetidis DSM 11105T, respectively. The DNA–DNA hybridization data indicated that strain Sn-9-2T represented a novel genomic species. On the basis of the results of phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA–DNA hybridization data, strain Sn-9-2T should represent a novel species of the genus Aquabacter , for which the name Aquabacter cavernae sp. nov. is proposed. The type strain is Sn-9-2T (=KCTC 62308T=CCTCC AB 2018270T).

The biodegradation of biorecalcitrant opioid drug tramadol (TRAM) was studied in a model biodegradation experiment performed with an enriched activated sludge culture pre-adapted to high concentration of TRAM (20 mg/L). TRAM and its transformation products (TPs) were determined by applying ultrahigh-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS), the sludge culture was characterized using a 16S rRNA gene amplicon sequencing, whereas ecotoxicological evaluation was performed based on determination of toxicity to freshwater algae. Tramadol removal was much faster (t1/2 = 1.3 days) and more efficient in glucose-containing mineral medium (cometabolic conditions) than in a medium without glucose. The elimination of the parent compound resulted in the formation of five TPs, two of which (TP 249 and TP 235) were identified as N-desmethyltramadol (N-DM TRAM) and N,N-didesmethyltramadol (N,N-diDM TRAM). The remaining 3 TPs (TP 277a-c) were isomeric compounds with an elemental composition of protonated molecules C16H24NO3 and a putative structure which involved oxidative modification of the dimethylamino group. Pronounced changes in the taxonomic composition of the activated sludge were observed during the enrichment, especially regarding an enhanced percentage of 8 genera (Bacillus, Mycobacterium, Enterobacter, Methylobacillus, Pedobacter, Xanthobacter, Leadbetterella and Kaistia), which might be related to the observed transformations. The removal of TRAM resulted in proportional reduction of algal toxicity, implying a positive result of the accomplished transformation processes.

An engineered metabolic pathway consisting of reactions that convert fatty acids to aldehydes and eventually alkanes would provide a means to produce biofuels from renewable energy sources. The enzyme aldehyde-deformylating oxygenase (ADO) catalyzes the conversion of aldehydes and oxygen to alkanes and formic acid and uses oxygen and a cellular reductant such as ferredoxin (Fd) as co-substrates. In this report, we aimed to increase ADO-mediated alkane production by converting an unused by-product, formate, to a reductant that can be used by ADO. We achieved this by including the gene (fdh), encoding formate dehydrogenase from Xanthobacter sp. 91 (XaFDH), into a metabolic pathway expressed in Escherichia coli. Using this approach, we could increase bacterial alkane production, resulting in a conversion yield of ∼50%, the highest yield reported to date. Measuring intracellular nicotinamide concentrations, we found that E. coli cells harboring XaFDH have a significantly higher concentration of NADH and a higher NADH/NAD(+) ratio than E. coli cells lacking XaFDH. In vitro analysis disclosed that ferredoxin (flavodoxin):NADP(+) oxidoreductase could use NADH to reduce Fd and thus facilitate ADO-mediated alkane production. As formic acid can decrease the cellular pH, the addition of formate dehydrogenase could also maintain the cellular pH in the neutral range, which is more suitable for alkane production. We conclude that this simple, dual-pronged approach of increasing NAD(P)H and removing extra formic acid is efficient for increasing the production of renewable alkanes via synthetic biology-based approaches.

The application of native enzymes may not be economical owing to the stability factor. A smaller protein molecule may be less susceptible to external stresses. Haloalkane dehalogenases (HLDs) that act on toxic haloalkanes may be incorporated as bioreceptors to detect haloalkane contaminants. Therefore, this study aims to develop mini proteins of HLD as an alternative bioreceptor which was able to withstand extreme conditions. Initially, the mini proteins were designed through computer modeling. Based on the results, five designed mini proteins were deemed to be viable stable mini proteins. They were then validated through experimental study. The smallest mini protein (model 5) was chosen for subsequent analysis as it was expressed in soluble form. No dehalogenase activity was detected, thus the specific binding interaction of between 1,3-dibromopropane with mini protein was investigated using isothermal titration calorimetry. Higher binding affinity between 1,3-dibromopropane and mini protein was obtained than the native. Thermal stability study with circular dichroism had proven that the mini protein possessed two times higher Tm value at 83.73 °C than the native at 43.97 °C. In conclusion, a stable mini protein was successfully designed and may be used as bioreceptors in the haloalkane sensor that is suitable for industrial application.

1,4-Dioxane is an emerging wastewater contaminant with probable human carcinogenicity. Our current understanding of microbial interactions during 1,4-dioxane biodegradation process in mixed cultures is limited. Here, we applied metagenomic, metatranscriptomic and co-occurrence network analyses to unraveling the microbial cooperation between degrader and non-degraders in an efficient 1,4-dioxane-degrading microbial consortium CH1. A 1,4-dioxane-degrading bacterium, Ancylobacter polymorphus ZM13, was isolated from CH1 and had a potential of being one of the important degraders due to its high relative abundance, highly expressed monooxygenase genes tmoABCDEF and high betweenness centrality of networks. The strain ZM13 cooperated obviously with 6 bacterial genera in the network, among which Xanthobacter and Mesorhizobium could be involved in the intermediates metabolism with responsible genes encoding alcohol dehydrogenase (adh), aldehyde dehydrogenase (aldh), glycolate oxidase (glcDEF), glyoxylate carboligase (gcl), malate synthase (glcB) and 2-isopropylmalate synthase (leuA) differentially high-expressed. Also, 1,4-dioxane facilitated the shift of biodiversity and function of CH1, and those cooperators cooperated with ZM13 in the way of providing amino acids or fatty acids, as well as relieving environmental stresses to promote biodegradation. These results provide new insights into our understandings of the microbial interactions during 1,4-dioxane degradation, and have important implications for predicting microbial cooperation and constructing efficient and stable synthetic 1,4-dioxane-degrading consortia for practical remediation.

Polyphosphates (polyPs) are ubiquitous polymers in living organisms from bacteria to mammals. They serve a wide variety of biological functions, ranging from energy storage to stress response. In the last two decades, polyP has been primarily viewed as linear polymers with varying chain lengths. However, recent biochemical data show that small metaphosphates, cyclic oligomers of [PO(3)]((−)), can bind to the enzymes ribonuclease A and NAD kinase, raising the question of whether metaphosphates can occur naturally as products of biological activity. Before the 1980’s, metaphosphates had been reported in polyPs extracted from various organisms, but these results are considered artifactual due to the extraction and purification protocols. Here, we employ non-destructive (31)P solid-state NMR spectroscopy to investigate the chemical structure of polyphosphates in whole cells as well as insoluble fractions of the bacterium Xanthobacter autotrophicus. Isotropic and anisotropic (31)P chemical shifts of hydrated whole cells indicate the coexistence of linear and cyclic phosphates. Under our cell growth condition and the concentrated condition of the solid-state NMR samples, we found substantial amounts of cyclic phosphates in X. autotrophicus, suggesting that in fresh cells metaphosphate concentrations can be significant. The cellular metaphosphates are identified by comparison with the (31)P chemical shift anisotropy of synthetic metaphosphates of known structures. In X. autotrophicus, the metaphosphates have a chemical shift anisotropy that is consistent with an average size of 3–8 phosphate units. These metaphosphates are enriched in insoluble and electron-dense granules. Exogenous hexametaphosphate added to X. autotrophicus cell extracts are metabolized to trimetaphosphates, supporting the presence and biological role of metaphosphates in cells. The definitive evidence for the presence of metaphosphates, reported here in whole bacterial cells for the first time, opens the path for future investigations of the biological function of metaphosphates in many organisms.

Forty-two bacterial strains were isolated from root samples of Sorghum bicolor. The strains spanned 17 genera, including Dechloromonas, Duganella, Dyella, Flavobacterium, Herbaspirillum, Lutibacter, Mucilaginibacter, Novosphingobium, Paraburkholderia, Pedobacter, Pleomorphomonas, Rhizobacter, Rhizobium, Rhizomicrobium, Rugamonas, Variovorax, and Xanthobacter. Their whole-genome sequences revealed diverse metabolic processes, including biological nitrogen fixation, in sorghum root microbiota.

The main aim of this study was to evaluate the performance of an air membrane bioreactor (aMBR) for the treatment of gas-phase methanol. A laboratory-scale hollow fiber aMBR was operated for 150 days, at inlet methanol concentrations varying between 2 and 30 g m−3 and at empty bed residence times (EBRT) of 30, 10 and 5 s. Under steady-state conditions, a maximum methanol removal efficiency (RE) of 98% was obtained at an EBRT of 30 s and a decrease in RE of methanol was observed at lower EBRTs. On increasing the inlet loading rate, some portion of gas-phase MeOH was stripped into the liquid phase due to its solubility in water. Under transient conditions, the MeOH removal efficiency dropped from an average value of 95%–90% after 5 h of 10-fold shock load and dropped from an average value of 95%–88% under 5-fold increase in shock load. During transient-state tests, the aMBR performed well under different upset loading conditions and a drop in RE of ∼ 5–10% was observed. However, the aMBR performance was restored within 1–2 days when pre-shock conditions were restored. The results from microbial structure analysis revealed a big shift of the dominant methanol degrader, from Candida boidinii strain TBRC 217 to Xanthobacter sp. and Fusicolla sp., respectively.

Soil samples were collected from Kuwait, Lebanon, Egypt, and Germany, and artificially polluted with 3% (w/w) crude oil. Cross-bioaugmentation was done among them, and the oil-consumption and the constituent communities of hydrocarbonoclastic bacteria were monitored periodically through 6 months. The results showed that cross-bioaugmentation did not bring about reproducible effects on oil-removal in the four soils. After 6 months, oil-removal values reached between 82 and 95% in most of the samples including the unbioaugmented controls. The numbers of hydrocarbonoclastic bacteria showed significant increases followed by significant decreases during the course of bioremediation also in the unbioaugmented controls. In most cases, the inoculated bacterial taxa failed to colonize the soils, and oil-removal was achieved mainly by the native (autochthonous) soil bacterial communities. those belonged to the genera Mycolicibacterium, Mycobacterium, Xanthobacter, Pseudoxanthomonas, Pseudomonas, Zavarzinia, and others. The microbial communities in the four soils also comprised nitrogen fixing bacteria belonging to the genera Gordonia, Rhizobium, Kocuria, and Azospirillum. Such diazotrophs are known to enrich the soils with fixed nitrogen and thus, contribute to enhancing the microbiological hydrocarbon-consumption. It was concluded that cross-bioaugmentation leads to unpredictable and inconsistent effects on oil removal. Therefore, it could not beregarded as the technology of choice for oil-bioremediation.

The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ~10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus. Furthermore, engineering an enzyme in the CoA detour, the Lactobacillus brevis CoA-acylating aldehyde dehydrogenase, allowed stringent product selectivity. Targeted production of 3-methyl-1-butanol (3-MB) in E. coli mediated by the CoA detour showed a 3-MB:side-product (isobutanol) ratio of >20, an increase over the ratios previously achieved using the conventional Ehrlich pathway.

Recent studies highlighted that hydrophobic microorganisms played key roles in VOC removal performance of two-phase partitioning bioreactors (TPPBs). The objective of this study was to evaluate the performance of a hydrophobic mixed culture for 1,2-dichloroethane (1,2-DCA) biodegradation in both batch and TPPB tests. The hydrophobic mixed culture with cell surface hydrophobicity of 79% was acclimated from an activated sludge able to efficiently degrade 1,2-DCA as sole carbon and energy source. It was primarily composed of Xanthobacter genus (62%). Nearly complete degradation with coefficients of 0.80 mgCO2 mg1,2-DCA−1 and 0.66 mgCl− mg1,2-DCA−1 was achieved for a wide range of 1,2-DCA concentrations (114.1–1141.5 mg L−1) in the presence of silicone oil. The maximum specific growth rate was 0.247 h−1 which was approximately two times the values of previous pure strains. The two-phase partitioning airlift bioreactors inoculated with the hydrophobic mixed culture was robust against step changes of gas flow rates and spike changes of inlet 1,2-DCA concentrations. All data proved the potential of the hydrophobic mixed culture for toxic-VOC removal in the TPPBs.

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe-His. Here, we propose that this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.

The stability of a mixed bacterial population in a fixed bed reactor for atrazine biodegradation under carbon limiting conditions was examined. A one litre reactor was filled with volcanic scoria and operated as a sequencing batch reactor (SBR) for 66 batches over a period of 230 days. The reactor was inoculated with a mixed atrazine degrading bacterial population and given an atrazine growth medium for start-up, followed by batches of 20 mg/L atrazine in 5 mM phosphate buffer with no additional carbon. Complete (100%) atrazine biodegradation was observed in all batches. Until the 10th batch after start-up, traces of cyanuric acid were observed and 66–70% of the theoretical nitrogen available from atrazine was recovered in the form of ammonium. From the 10th batch onwards, 100% of the theoretical nitrogen available from atrazine was recovered in the form of nitrate with no trace of cyanuric acid observed. After reactor start-up the reactor contained 98% bacteria with the relative abundance of the dominant bacteria comprised of Xanthobacter (25.6%), Variovorax (23%), Ralstonia (12.2%), and Comamonas (8.1%). At the end of reactor operation, 70.8% bacteria and 29.2% archaea were found with Xanthobacter, Ralstonia and Variovorax (1.4%) in much smaller amounts, replaced mainly by Candidatus Nitrososphaera (29.2%), Phyllobacterium (5.5%) and Nitrospira (5.4%).

The use of microbial consortia to remove contaminants in industrial systems and in natural environments could be an alternative to the use of unique strains of microorganisms, since microbial consortia have greater robustness to environmental fluctuations. However, it is necessary to evaluate the relationship between the genetic structure and functionality of the consortia. In this work, the functional and structural stability over time of two bacterial consortia (C5 and C6) with the potential to remove CO2 and H2S from biogas was evaluated. Both consortia decreased the dissolved CO2 by over 30% at the end of the incubation period, but C5 presented shorter removal kinetics (3.9 days) than C6 (6.4 days). Additionally, a chemical oxidation of H2S could have occurred in the microcosms. Moreover, both consortia presented a stable genetic structure, measured by terminal restriction fragment length polymorphism profiles of the 16S rRNA gene, characterized by high homogeneity and prevalence of the genus Rhodopseudomonas throughout the incubation period, and an increasing abundance of Xanthobacter during the exponential phase of the growth curve in C5, which would account for the functionality of the consortia.

We demonstrate the synthesis of NH3 from N2 and H2O at ambient conditions in a single reactor by coupling hydrogen generation from catalytic water splitting to a H2-oxidizing bacterium Xanthobacter autotrophicus, which performs N2 and CO2 reduction to solid biomass. Living cells of X. autotrophicus may be directly applied as a biofertilizer to improve growth of radishes, a model crop plant, by up to ∼1,440% in terms of storage root mass. The NH3 generated from nitrogenase (N2ase) in X. autotrophicus can be diverted from biomass formation to an extracellular ammonia production with the addition of a glutamate synthetase inhibitor. The N2 reduction reaction proceeds at a low driving force with a turnover number of 9 × 109 cell–1 and turnover frequency of 1.9 × 104 s–1⋅cell–1 without the use of sacrificial chemical reagents or carbon feedstocks other than CO2. This approach can be powered by renewable electricity, enabling the sustainable and selective production of ammonia and biofertilizers in a distributed manner.

Two new bacterial biopolymers (exopolysaccharides), ancylan and xylophilan, have been isolated and characterized. The optimal parameters for ancylan and xylophilan production under laboratory conditions were selected. Their physicochemical properties and effects on microorganisms (bacteria, fungi, and ciliates) were studied. The results suggest the potential application of these new exopolysaccharides in medicine and veterinary science.

During the summer period (15–25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 μg/mL) and are probably plant symbionts.

Saline anode microbial fuel cell (SA-MFC) equipped with carbon felt as anode and cathode, separated by nafion membrane was used to evaluate the aquaculture wastewater treatment efficiency and energy production under saline condition (4%). Operation of SA-MFC was executed at different organic load (OL) from 0.5 gCOD/L to 1.5 gCOD/L under saline condition. TCOD (Total Chemical Oxygen Demand) removal was 75 ± 1.4%, 81 ± 0.9% and 84 ± 1.1% at 0.5, 0.75 and 1 gCOD/L OL respectively. SCOD (Soluble Chemical Oxygen Demand) removal in SA-MFC was 68 ± 1.2%, 75 ± 0.8% and 81 ± 0.5% at 0.5, 0.75 and 1 g COD/L OL. Among the OL used 1.25 gCOD/L recorded high TCOD (92 ± 1.1%), SCOD (91 ± 0.5%) and TSS (Total Suspended Solids) removal (78 ± 0.7%) with a maximum power production (369 mW/m2). Increase in OL to 1.5 gCOD/L revealed decline in TCOD (79 ± 1.3%), SCOD (74 ± 0.8%), TSS (71 ± 1.5%) removal and power production (310 mW/m2). The predominant bacterial strains responsible for aquaculture wastewater treatment and energy production was identified as Ochrobactrum (53%), Marinobacter (22%) and Rhodococcus (15%). The other strains such as Bacillus, Stenotrophomonas, Xanthobacter, Sphingomonas, Pseudomonas and Sedimentibacter also present in the SA-MFC reactor operated under saline condition.

1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria ( Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene ( dhlA ) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria , which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (IS Xa1 and IS Pme1 ) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes ( dhlB1 and dhlB2 ) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1 . Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes.

We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

Long-term atrazine degradation was studied with Pseudomonas sp. strain ADP (P. ADP) encapsulated in electrospun microtubes with no addition of external carbon source. Experiments were conducted in consecutive 3-day sequential batches under semisterile and nongrowth conditions over a period of 2.5 years. During the entire period, 95.2% atrazine was degraded on average, with 68.2% nitrogen recovery as ammonium. Only a slight buildup of intermediate (approximately 15–20% cyanuric acid) was observed. Confocal microscopy taken after 6 months showed bacteria colonizing outside the microtubes whose origin came from external contamination. Analysis of the microtube microbial community after 1.5 years revealed a mixed population containing P. ADP along with Chitinophagaceae, Variovorax, and Microbacterium. After 2.5 years, P. ADP was not found in the microtube bacterial population comprising Variovorax, Cupriavidus, Comamonas, Xanthobacter, Microbacterium, and Tsukamurella. Presence of pADP-1 plasmid in t...

This study investigates the use of electrically conductive granules as packing material in biotrickling filter (BTF) systems as to provide insights on the specific microbial abundance and functions during the treatment of xylene-containing waste gas. In addition, the effect of applied potential on attached biofilm on conductive granules during xylene degradation was briefly investigated. During stable operation period, the conductive granules packed BTF achieved reactor performance of no less than 80% with a maximum EC of 137.7 g/m3 h. Under applied potential of 1V, the BTF system showed deterioration of xylene removal by ranging from 21 to 76%, which also affected the distribution and relative abundance of the major microorganisms such as Xanthobacter, Acidovorax, Rhodococcus, Hydrogenophaga, Arthrobacter, Brevundimonas, Pseudoxanthomonas, Devosia, Shinella, Sphingobium, Dokdonella, Pseudomonas and Bosea. The acclimation of applied potential led to the enrichment of autotrophic bacteria and strains, which are correlated to improved nitrogen cycling. In general, applying electrical potential is feasible to shape the microbiological structure of biofilms to selectively adjust their biochemical functions.

A hydrogen-based membrane biofilm reactor (MBfR) equipped with separate hydrogen (H2) and carbon dioxide (CO2) adjustable gas-transfer modules was used to explore denitrification in a water containing a high concentration of sulfate (SO42−) (500 mg S/L). CO2 was used for pH control and as the sole carbon source for long-term operation of the MBfR, during which >90% denitrification was achieved. Short-term experiments demonstrated that increasing the SO42− loading had negligible effect on nitrate (NO3−) reduction, but SO42− reduction was strongly suppressed by high NO3− loading regardless of H2 availability. Sulfide oxidation accompanied with SO42− reduction occurred in the biofilm, and the intermediate element sulfur was further oxidized to SO42−. Results of high-throughput sequencing suggest that sulfide oxidation was carried out by the known sulfide-oxidizing bacteria: Starkeya and Xanthobacter. And sulfide oxidation coupled to denitrification was a primary reason for minimal net SO42− reduction when the NO3− loading was high enough. This study demonstrates the promise of effective denitrification without net SO42− reduction in MBfR systems and documents the important sulfur-conversion processes.

Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition—based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose.

The study of aerosol dispersion characteristics in wastewater treatment plants (WWTPs) has attracted extensive attention. Oxidation ditch (OD) is a commonly implemented process during biological wastewater treatment. This study assessed the component characteristics, source apportionment, and exposure risks of aerosols generated from a WWTP using the OD process (AWO). The results indicated that the aeration part of oxidation ditch (ODA) exhibited the highest concentrations and proportions of the respiratory fractions (RF) of bacteria, Enterobacteriaceae, Staphylococcus aureus, and Pseudomonas aeruginosa. Some pathogenic or opportunistic-pathogenic bacteria and carcinogenic metal(loid)s were detected in the AWO. The source apportionment results indicated that the outdoor wastewater treatment processes and ambient air contributed to the constitution of the AWO. The indoor aerosols were mainly constituted by composition of the wastewater treatment process such as the sludge dewatering room (SDR). The pathogenic or opportunistic-pathogenic bacteria with eight genera (Colinsella, Dermatophilus, Enterobactor, Erycherichia-Shigella, Ledionella, Selenomonas, Xanthobacter, and Veillonella) were largely attributed to wastewater or sludge. The risk assessment suggested that inhalation was the main exposure pathway for aerosols (including bacteria and metal(loid)s). Additionally, As indicated the highest non-carcinogenic risks. Furthermore, As, Cd, and Co were associated with high carcinogenic risks. The ODA and sludge dewatering room (SDR) indicated the highest carcinogenic and non-carcinogenic risks of metal(loid)s, respectively. Thus, the AWO should be sufficiently researched and monitored to mitigate their harmful effects on human health, particularly with regard to the health of the site workers.

During the summer period (15–25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 μg/mL) and are probably plant symbionts.

NADPH:2-ketopropyl–coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO(2) fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO(2). The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pK(a) between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C–S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid–base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C–S bond cleavage and selectivity for CO(2) fixation.

Toluene has high toxicity and mutagenicity, thus, the removal of toluene from air is necessary. In this study, two biotrickling filters (BTFs) were constructed and packed with conductive packing material to treat toluene waste gas. BTF-O exhibited good toluene removal performance even under high toluene inlet concentration, and over 80% of removal efficiency was observed. The elimination capacity reached 120.1 g/m3 h corresponding to an inlet concentration of 2.259 g/m3 under 61.5 s of empty bed retention time. During toluene biodegradation, the output voltage was observed in BTF-O and BTF-E, moreover BTF-E also showed slight power storage capacity. The applied voltage inhibited toluene removal and affected the bacterial community. The predominant bacterial genera in BTF-O were Acidovorax, Rhodococcus, Hydrogenophaga, Brevundimonas, Arthrobacter, Pseudoxanthomonas, Devosia, Gemmobacter, Rhizobium, Dokdonella and Pseudomonas. Genera Xanthobacter and Pelomonas accounted for the main bacterial community in BTF-E.

Two previously uncharacterized potential broad-spectrum mercury (Hg) resistance operons ( mer ) are present on the chromosome of the soil Alphaproteobacteria Xanthobacter autotrophicus Py2. These operons, mer1 and mer2 , contain two features which are commonly found in mer operons in the genomes of soil and marine Alphaproteobacteria , but are not present in previously characterized mer operons: a gene for the mercuric reductase (MerA) that encodes an alkylmercury lyase domain typical of those found on the MerB protein, and the presence of an additional gene, which we are calling merK , with homology to glutathione reductase. Here, we demonstrate that Py2 is resistant to 0.2 μM inorganic mercury [Hg(II)] and 0.05 μM methylmercury (MeHg). Py2 is capable of converting MeHg and Hg(II) to elemental mercury [Hg(0)], and reduction of Hg(II) is induced by incubation in sub toxic concentrations of Hg(II). Transcription of the merA genes increased with Hg(II) treatment, and in both operons merK resides on the same polycistronic mRNA as merA . We propose the use of Py2 as a model system for studying the contribution of mer to Hg mobility in soil and marine ecosystems.

Atrazine-mineralizing bacteria were enriched from water and sediment samples from a constructed flow-through wetland that received water from a river with an agricultural watershed. Bio–Sep beads laden with atrazine were submerged in the wetland sediment and water column in an attempt to sorb atrazine-metabolizing microbes on beads. Biometer experiments with in-situ incubated beads did not detect 14CO2 formation from 14C-atrazine. HPLC analysis was used to rank atrazine biodegradation by enrichment cultures from wetland samples. Analysis of the 16S rRNA gene sequences of 60 clones from aerobic enrichment cultures revealed the predominance of the genera Acidovorax (33%), Ralstonia (22%), Azorhizobium (12%), and Xanthobacter (12%) with sequence match ≥0.90 in wetland samples. Similar analysis of 12 clones from anaerobic enrichment cultures revealed the predominance of the genera Hydrogenophaga (42%) and Rhizobium (25%). Pure culture isolates (n = 18) were primarily Arthrobacter spp. (89%) at sequence match ≥0.97. Pure cultures were screened qualitatively for the presence of 8 catabolic genes associated with known pathways of atrazine biodegradation. None of the cultures had all Atz pathway genes and none contained atzD and trzD genes. AtzC was found in all isolates. The data indicate that the diversity of atrazine degraders was comparable to findings in agricultural soil studies, with the notable cluster of Arthrobacter spp. which may be favored by the culture conditions used for isolation.

Xanthobacter autotrophicus GJ10 has been widely studied because of its ability to degrade halogenated compounds, especially 1,2-dichloroethane (1,2-DCA), which is achieved through chromosomal as well as plasmid pAUX1 encoded 1,2-DCA degrading genes. This work described the gene expression and enzyme activity profiles as well as the intermediates formed during the 1,2-DCA degradation by this organism. A correlation between gene expression, enzyme activity and metabolic intermediates, after the induction of GJ10 grown culture with 1,2-DCA, was established at different time intervals. Haloalkane dehalogenase (dhlA) and haloacid dehalogenase (dhlB) were constitutively expressed while the expression of alcohol dehydrogenase (max) and aldehyde dehydrogenase (ald) was found to be inducible. The DhlA and DhlB activities were relatively higher compared to that of the inducible enzymes, Max and Ald. To the best of our knowledge, this is the first study to correlate gene expression profiles with enzyme activity and metabolite formation during 1,2-DCA degradation process in GJ10. Findings from this study may assist in fully understanding the mechanism of 1,2-DCA degradation by GJ10. It could also assist in the design and implementation of appropriate bioaugmentation strategies for complete removal of 1,2-DCA from contaminated environment.

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze β-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes β-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing β-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate β-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.

It is important to construct microbiological treatment systems for organic solvent-contaminated water. We developed a continuous culture supplemented with a biostimulation agent named BD-C, which is formulated from canola oil, and Xanthobacter autotrophicus strain GJ10 for an aerobic dichloromethane (DCM)-dechlorinating microorganism. The continuous culture was a chemostat constructed using a 1 L screw-capped bottle containing artificial wastewater medium with 2.0 mM DCM and 1.0% (v/v) BD-C. The expression of genes for DCM metabolism in the dechlorinating aerobe was monitored and analyzed by reverse transcription-quantitative PCR. Strain GJ10 was able to dechlorinate approximately 74% of the DCM in medium supplemented with BD-C during 12 days of incubation. The DCM dechlorination rate was calculated to be 0.11 mM/day. The

Haloalkane dehalogenases catalyze the hydrolysis of halogen–carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å(3)) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å(3)). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure–dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.

Tetracyclines (TCs), including tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC), are amongst the most common antibiotics used in animal husbandry. Residual amounts of these antibiotics in the environment are a concern because they contribute to selection of resistant bacteria. In this study, we investigated the biodegradation of three TCs in swine wastewater. In batch experiments, OTC and CTC were completely degraded at d 18 and 20, respectively, but TC was remained at 7.1% after 20 d incubation. The degradation rates of TCs in the wastewater were in the order of OTC > CTC > TC. Degradation of the TCs was enhanced by the addition of enzyme extract from spent mushroom compost (SMC) of Pleurotus eryngii. The degradation rates were higher with the addition of extract-containing microcapsules than suspended enzyme extract in swine wastewater. In the bioreactor experiment, the addition of extract-containing microcapsules enhanced the removal rates of the three TCs, and adding TCs twice maintained enzyme activity in the swine wastewater. Of the microorganism strains isolated from the wastewater samples, strain HL2 (identified as Xanthobacter flavus) showed the best degrading ability.

A pilot-scale membrane bioreactor (MBR) was tested for bioremediation of 1,2-dichloroethane (DCA) in groundwater. Pyrosequencing of 16S rDNA was used to study changes in the microbiology of the MBR over 137 days, including a 67 day initial adaptation phase of increasing DCA concentration. The bacterial community in the MBR was distinct from those in soil and groundwater at the same site, and was dominated by alpha- and beta- proteobacteria, including Rhodobacter, Methylibium, Rhodopseudomonas, Methyloversatilis, Caldilinea, Thiobacillus, Azoarcus, Hyphomicrobium, and Leptothrix. Biodegradation of DCA in the MBR began after 26 days, and was sustained for the remainder of the experiment. A quantitative PCR (qPCR) assay for the dehalogenase gene dhlA was developed to monitor DCA-degrading bacteria in the MBR, and a positive correlation was seen between dhlA gene abundance and the cumulative amount of DCA that had entered the MBR. Genera previously associated with aerobic DCA biodegradation (Xanthobacter, Ancylobacter, Azoarcus) were present in the MBR, and the abundance of Azoarcus correlated well with dhlA gene abundance. This study shows that MBRs can be an effective method for removal of DCA from groundwater, and that the dhlA qPCR is a rapid and sensitive method for detection of DCA-degrading bacteria.

(R)- and (S)-hydroxypropyl-coenzyme M dehydrogenases (R- and S-HPCDH) are stereospecific enzymes that are central to the metabolism of propylene and epoxide in Xanthobacter autotrophicus. The bacterium produces R- and S-HPCDH simultaneously to facilitate transformation of R- and S-enantiomers of epoxypropane to a common achiral product 2-ketopropyl-CoM (2-KPC). Both R- and S-HPCDH are highly specific for their respective substrates as each enzyme displays less than 0.5% activity with the opposite substrate isomer. In order to elucidate the structural basis for stereospecificity displayed by R- and S-HPCDH we have determined substrate bound crystal structures of S-HPCDH to 1.6 A resolution. Comparisons to the previously reported product-bound structure of R-HPCDH reveal that although the placement of catalytic residues within the active site of each enzyme is nearly identical, structural differences in the surrounding area provide each enzyme with a distinct substrate binding pocket. These structures demonstrate how chiral discrimination by R- and S-HPCDH results from alternative binding of the distal end of substrates within each substrate binding pocket.

Alkane-degrading bacteria are crucial in the bioremediation of petroleum contamination from soil and groundwater. The alkane monooxygenase (alkB) gene encoding the Alk enzyme involved in aerobic degradation is a potentially functional gene biomarker for the detection of alkane-degrading bacteria. This study describes a denaturing gradient gel electrophoresis (DGGE) assay to facilitate the rapid and sensitive detection of alkB genes in wastewater from oilfield. The results showed that the presence of a considerable genetic diversity of alkB genes in the wastewater as evidenced by a total of 13 unique DNA bands detected. These alkB genes belong to nine genera, including Acinetobacter, Alcanivorax, Acidisphaera, Burkholderia, Geobacillus, Marinobacter, Mycobacterium, Pseudomonas, Rhodococcus and Xanthobacter. The abundance of alkane-degrading bacteria and total bacteria was calculated to range from 1.46 × 103 to 9.89 × 104 cell ml−1 and from 1.18 × 104 to 6.29 × 105 cells ml−1, respectively. The distribution of alkane-degrading bacteria was positively correlated with the environmental temperature and the specific types of n-alkanes in the wastewaters. Our results suggest that alkB-based DGGE methods could describe the diverse alkane-degrading bacteria in oil-degrading community, which may improve the understanding of the treatment for oil-polluted wastewater.

© 2016 American Chemical Society We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

The characteristic His-Glu catalytic dyad of the disulfide oxidoreductase (DSOR) family of enzymes is replaced in 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase (2-KPCC) by the residues Phe-His. 2-KPCC is the only known carboxylating member of the DSOR family and has replaced this dyad potentially to eliminate proton-donating groups at a key position in the active site. Substitution of the Phe-His by the canonical residues results in production of higher relative concentrations of acetone versus the natural product acetoacetate. The results indicate that these differences in 2-KPCC are key to discriminating between carbon dioxide and protons as attacking electrophiles. This article is protected by copyright. All rights reserved.

In this study, antimicrobial properties of both lyophilized and fresh water extracts of the okra pods were assessed against Rhodococcus erythropolis and R. opacus, Mycobacterium sp. and M. aurum, Staphylococcus aureus, Escherichia coli, and Xanthobacter Py2. The extracts were effective against all bacterial strains tested, except R. erythropolis and the fresh extract displayed better antimicrobial properties than the lyophilized extract. A fresh extract concentration of 97.7 mg/mL was sufficient to kill all S. aureus cells, which is a worldwide source of nosocomial infection. The extract was also effective in inhibiting the growth of both Mycobacterium strains, X. Py2 and S. aureus, but was ineffective against R. erythropolis and E. coli. The lipid fraction of the okra gum was found to be responsible for the antibacterial properties and the protein and polysaccharide fractions displayed no antimicrobial activity. The two major constituents of the lipid fraction, palmitic and stearic acids, were apparently responsible for the antimicrobial properties of the okra extract.

Microbial degradation of the chiral 2-phenylbutyric acid (2-PBA), a metabolite of surfactant linear alkylbenzene sulfonates (LAS), was investigated using both racemic and enantiomer-pure compounds together with quantitative stereoselective analyses. A pure culture of bacteria, identified as Xanthobacter flavus strain PA1 isolated from the mangrove sediment of Hong Kong Mai Po Nature Reserve, was able to utilize the racemic 2-PBA as well as the single enantiomers as the sole source of carbon and energy. In the presence of the racemic compounds, X. flavus PA1 degraded both (R) and (S) forms of enantiomers to completion in a sequential manner in which the (S) enantiomer disappeared much faster than the (R) enantiomer. When the single pure enantiomer was supplied as the sole substrate, a unidirectional chiral inversion involving (S) enantiomer to (R) enantiomer was evident. No major difference was observed in the degradation intermediates with either of the individual enantiomers when used as the growth substrate. Two major degradation intermediates were detected and identified as 3-hydroxy-2-phenylbutanoic acid and 4-methyl-3-phenyloxetan-2-one, using a combination of liquid chromatography-mass spectrometry (LC-MS), and (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. The biochemical degradation pathway follows an initial oxidation of the alkyl side chain before aromatic ring cleavage. This study reveals new evidence for enantiomeric inversion catalyzed by pure culture of environmental bacteria and emphasizes the significant differences between the two enantiomers in their environmental fates.

The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been determined in a state in which CO2 is observed providing insights into the mechanism of carboxylation. In the substrate encapsulated state of the enzyme, CO2 is bound at the base of a narrow hydrophobic substrate access channel. The base of the channel is demarcated by a transition from a hydrophobic to hydrophilic environment where CO2 is located in position for attack on the carbanion of the ketopropyl group of the substrate to ultimately produce acetoacetate. This binding mode effectively discriminates against H2O and prevents protonation of the ketopropyl leaving group. Structured summary 2-KPCC binds to 2-KPCC by x-ray crystallography ( View interaction )