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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.