[Microbial Community Structure of Activated Sludge for Total Nitrogen Upgrading Project].

The activated sludge of a biochemical unit (WLK_OD) and an advanced denitrification unit (WLK_AD) were collected from a municipal wastewater treatment plant (WWTP), in which the TN concentration of effluent was less than 1.5 mg·L-1, and their microbial community structure and function profiles were analyzed using 16S rRNA gene high-throughput sequencing. The microorganisms in WLK_AD had lower evenness compared with that in WLK_OD, which was attributed to environmental selection. Furthermore, PCoA revealed that different incoming wastewaters had an impact on microbial community structure. At the phylum level, Proteobacteria (70.11%) was enriched in WLK_AD. At the genus level, Thauera, Flavobacterium, Hydrogenophaga, and Zoogloea served as distinct-dominant denitrifying bacteria in WLK_AD; however, Trichococcus (3.50%) and Terrimonas (1.10%) were enriched in WLK_OD. Through the comparison between groups (P<0.05), the biomarkers detected in each WWTP were different. Furthermore, the results of the co-occurrence network showed that the bacteria from module I had a higher proportion in WLK_AD; the bacteria from module II had a higher proportion in WLK_OD, and they were common microorganisms in WWTPs, implying that wastewater environments drpve the differences in the microbial community structure. Among the types of environmental parameters, the removal efficiency of COD and TN had the greatest impact on the microbial community by the RDA. The removal efficiency of COD was positively correlated with the dominant bacteria from WLK_OD, such as Saccharibacteria, Thermomarinilinea, Terrimonas, and Comamonas; the removal efficiency of TN was positively correlated with the denitrifying bacteria from WLK_AD, such as Dokdonella, Thauera, Flavobacterium, and Zoogloea. WLK_AD was enriched with Novosphingobium, Dokdonella, Thauera, and Sphingomonas, which synergistically removed TN, leading to the TN of the effluent being less than 1.5 mg·L-1. Moreover, based on the results of function prediction, WLK_AD had a higher proportion of genes that could code the denitrification enzymes.

Thauera sedimentorum sp. nov., Isolated from Coastal Sediment.

A Gram-negative, strictly aerobic, rod-shaped, non-spore-forming bacterium, designated CAU 1555T, was isolated from a sediment sample collected on Jeju Island, Republic of Korea. Growth of the isolate was observed at 20-37 °C (optimum at 30 °C) and pH 5.5-10.0 (optimum at 8.0). Phylogenetic analysis based on the result of 16S rRNA gene sequences revealed that strain CAU 1555T belonged to the genus Thauera and was closely related to Thauera hydrothermalis GD-2T (98.4% sequence similarity), Thauera lacus D20T (96.6%), and Thauera linaloolentis 47LolT (95.5%). Strain CAU 1555T possessed phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid, and one aminophospholipid as the major polar lipids; Q-8 as the predominant respiratory quinone; and C16:0, summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c), and summed feature 8 (comprising C18:1 ω7c/ C18:1 ω6c) as the major fatty acids. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between the new isolate and T. hydrothermalis GD-2T were 84.5%, 86.4%, and 28.0%, respectively. Whole-genome sequencing of strain CAU 1555T revealed 3,955,289 bp with a DNA G + C content of 68.0 mol%. Based on the results of its polyphasic properties and genomic analysis, the isolate represents a novel species within the genus Thauera, for which the name Thauera sedimentorum sp. nov. is proposed, with CAU 1555T (= KCTC 72546T = MCCC 1K04065T) as the type strain.

Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens strain PIV-1.

Quaternary carbon-containing compounds exist in natural and fossil oil-derived products and are used in chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment. Here, we investigated the unknown degradation of the quaternary carbon-containing model compound pivalate (2,2-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens strain PIV-1 (formerly Thauera pivalivorans). We provide multiple evidence for a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed similar to the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA and the cleavage of 3-methyl-3-hydroxyglutaryl-CoA to acetyl-CoA and acetoacetate. The completed genome of Thauera humireducens strain PIV-1 together with proteomic data was used to identify pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. A pivalate-induced gene encoding a putative carboxylic acid CoA ligase was heterologously expressed, and its highly enriched product exhibited pivalate CoA ligase activity. The results provide the first experimental insights into the biodegradation pathway of a quaternary carbon-containing model compound that serves as a blueprint for the degradation of related quaternary carbon-containing compounds.

Genome analysis of Thauera chlorobenzoica strain 3CB-1T, a halobenzoate-degrading bacterium isolated from aquatic sediment.

The genus Thauera is characterized by several species and strains with the ability to degrade a variety of aromatic compounds under denitrifying conditions. Thauera chlorobenzoica strain 3CB-1T, isolated from river sediment, has the unique ability to degrade a variety of halobenzoates, such as 3-chlorobenzoate, 3-bromobenzoate, 3-iodobenzoate, and 2-fluorobenzoate, coupled to nitrate reduction. The genome of T. chlorobenzoica strain 3CB-1T has been sequenced, allowing us to gain insights into the molecular basis for the anaerobic degradation of (halo)aromatic compounds. The 3.77-Mb genome contains 3584 genes; 3514 are protein-coding genes of which 198 are likely associated with degradation of aromatic compounds. It has a G + C content of 67.25%. The genome contains two sets of CoA reductase gene clusters, both belonging to class I benzoate-CoA reductases (BCRs). The genes in one of the two clusters differ from the typical BCRs, with low sequence identities, suggesting they might have different substrate specificities. The genome also contains four benzoate-CoA ligase genes. One likely encodes a 3-hydroxybenzoate-CoA ligase, and two others group together with benzoate-CoA ligases from Thauera aromatica. The fourth has a 77% identity to the mbdA gene from Azoarcus sp. CIB, is absent in the T. aromatica genome, and potentially encodes a halobenzoate-CoA ligase. 3-Chlorobenzoate is reductively dechlorinated in T. chlorobenzoica by a benzoyl-CoA reductase.

Cloning, expression and biochemical characterization of a novel amidase from Thauera sinica K11.

A novel amidase (TAM) was identified and cloned from the genome of Thauera sinica K11. The recombinant protein was purified to homogeneity by one-step affinity chromatography for up to 26.4-fold with a yield of 38.1%. Gel filtration chromatography and SDS-PAGE revealed that the enzyme was a tetramer with a subunit of approximately 37.5 kDa. The amidase exhibited the maximum acyl transfer activity at 45 °C and pH 7.0, and it was highly stable over a wide pH range of 6.0-11.0. Inhibition of enzyme activity was observed in the presence of metal ions, thiol reagents and organic solvents. TAM showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides. For linear aliphatic monoamides, the acyl transfer activity of TAM was decreased with the extension of the carbon chain length, and thus the highest activity of 228.2 U/mg was obtained when formamide was used as substrate. This distinct selectivity of amidase to linear aliphatic monoamides expanded the findings of signature amidases to substrate specificity.

Differential expression of clade I and II N2O reductase genes in denitrifying Thauera linaloolentis 47LolT under different nitrogen conditions.

Nitrous oxide (N2O) is a potent greenhouse gas and its reduction to dinitrogen gas by the N2O reductase (encoded by the nosZ gene) is the only known biological N2O sink. Within the nosZ phylogeny there are two major clades (I and II), which seem to have different ecological niches. However, physiological differences of nosZI and nosZII expression that may impact emissions of N2O are not well understood. Here, we evaluated the differential expression of nosZI and nosZII, both present in Thauera linaloolentis strain 47LolT, in response to N2O concentration and the presence of the competing electron acceptor nitrate (NO3-). Different N2O levels had a negligible effect on the expression of both nosZ clades. Interestingly, nosZII expression was strongly upregulated in the absence of NO3-, while nosZI expression remained constant across the conditions tested. Thus, NO3- possibly inhibited nosZII expression, which suggests that N2O mitigation mediated by nosZII can be restricted due to the presence of NO3- in the environment. This is the first study demonstrating differential expression of nosZI and nosZII genes under the same physiological conditions and their implications for N2O emission under varying environmental conditions in terms of NO3- availability.

Anaerobic degradation of 2-chloro-4-nitroaniline by Geobacter sp. KT7 and Thauera aromatica KT9.

2-chloro-4-nitroaniline is a nitroaromatic compound widely used in industrial and agricultural sectors, causing serious environmental problems. This compound and some of its analogs were utilized by two Fe3+-reducing microbial strains Geobacter sp. KT7 and Thauera aromatica KT9 isolated from contaminated sediment as sole carbon and nitrogen sources under anaerobic conditions. The anaerobic degradation of 2-chloro-4-nitroaniline by the mixed species was increased approximately by 45% compared to that of individual strains. The two isolates' crossfeeding, nutrient sharing and cooperation in the mixed culture accounted for the increase in degradation rates. The determination of degradation pathways showed that Geobacter sp. KT7 transformed the nitro group in 2-chloro-4-nitroaniline to the amino group following by the dechlorination process, while T. aromatica KT9 dechlorinated the compound before removing the nitro group and further transformed it to aniline. This study provided an intricate network of 2-chloro-4-nitroaniline degradation in the bacterial mixture and revealed two parallel routes for the substrate catabolism.

Establishment of BmoR-based biosensor to screen isobutanol overproducer.

BACKGROUND: Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4-C6 linear alcohols and C3-C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library. RESULTS: Firstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment. CONCLUSIONS: This work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.

DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway.

The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (P orf20 ) was only active in the presence of 3,5-DHB, the other two (P dbhL and P orf18 ) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the P dbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCEThauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

Thauera hydrothermalis sp. nov., a thermophilic bacterium isolated from hot spring.

A Gram-stain-negative, non-spore-forming, rod-shaped, motile bacterial strain, designated GD-2T, was isolated from a sediment sample collected from a hot spring in the Tibet Autonomous Region, China. Strain GD-2T grew at a temperature range of 37-55 °C (optimum, 45-50 °C), a pH range of 5.5-11.0 (pH 7.0-7.5) and a NaCl concentration range of 0-4.0 % (0 %). The phylogenetic analysis based on 16S rRNA gene sequencing showed that strain GD-2T represented a member of the genus Thauera within the family Zoogloeaceae. Strain GD-2T was closely related to Thauera linaloolentis 47LolT with the highest 16S rRNA gene sequence similarity of 95.5 %. The whole genomic average nucleotide identity value for GD-2T and 47LolT was 75.3 %. The predominant cellular fatty acids of the strain were C16 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C10 : 0 3-OH and C12 : 0. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids and two unidentified aminolipids. The major isoprenoid quinone was ubiquinone 8. Genome sequencing revealed that the genome size of GD-2T was 3 059 321 bp with a G+C content of 63.57 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain GD-2T is considered to represent a novel species of the genus Thauera, for which the name Thauera hydrothermalis sp. nov. is proposed. The type strain is GD-2T (=NBRC 112472T=CGMCC 1.15527T).

Anaerobic degradation of 2,4-dichlorophenoxyacetic acid by Thauera sp. DKT.

Thauera sp. strain DKT isolated from sediment utilized 2,4-dichlorophenoxyacetic acid (2,4D) and its relative compounds as sole carbon and energy sources under anaerobic conditions and used nitrate as an electron acceptor. The determination of 2,4D utilization at different concentrations showed that the utilization curve fitted well with the Edward model with the maximum degradation rate as 0.017 ± 0.002 mM/day. The supplementation of cosubstrates (glucose, acetate, sucrose, humate and succinate) increased the degradation rates of all tested chemical substrates in both liquid and sediment slurry media. Thauera sp. strain DKT transformed 2,4D to 2,4-dichlorophenol (2,4DCP) through reductive side-chain removal then dechlorinated 2,4DCP to 2-chlorophenol (2CP), 4-chlorophenol (4CP) and phenol before complete degradation. The relative degradation rates by the isolate in liquid media were: phenol > 2,4DCP > 2CP > 4CP > 2,4D ≈ 3CP. DKT augmentation in sediment slurry enhanced the degradation rates of 2,4D and chlorophenols. The anaerobic degradation rates in the slurry were significantly slower compared to the rates in liquid media.

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D3 Synthesis.

Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Aerobic, steroid-degrading bacteria employ oxygenases for isoprenoid side chain and tetracyclic steran ring cleavage. In contrast, a Mo-containing steroid C-25 dehydrogenase (S25DH) of the dimethyl sulfoxide (DMSO) reductase family catalyzes the oxygen-independent hydroxylation of tertiary C-25 in the anaerobic, cholesterol-degrading bacterium Sterolibacterium denitrificans Its genome contains eight paralogous genes encoding active site α-subunits of putative S25DH-like proteins. The difficult enrichment of labile, oxygen-sensitive S25DH from the wild-type bacteria and the inability of its active heterologous production have largely hampered the study of S25DH-like gene products. Here we established a heterologous expression platform for the three structural genes of S25DH subunits together with an essential chaperone in the denitrifying betaproteobacterium Thauera aromatica K172. Using this system, S25DH1 and three isoenzymes (S25DH2, S25DH3, and S25DH4) were overproduced in a soluble, active form allowing a straightforward purification of nontagged αßγ complexes. All S25DHs contained molybdenum, four [4Fe-4S] clusters, one [3Fe-4S] cluster, and heme B and catalyzed the specific, water-dependent C-25 hydroxylations of various 4-en-3-one forms of phytosterols and zoosterols. Crude extracts from T. aromatica expressing genes encoding S25DH1 catalyzed the hydroxylation of vitamin D3 (VD3) to the clinically relevant 25-OH-VD3 with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH1 was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD3 synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.IMPORTANCE Steroids are ubiquitous bioactive compounds, some of which are considered an emerging class of micropollutants. Their degradation by microorganisms is the major process of steroid elimination from the environment. While oxygenase-dependent steroid degradation in aerobes has been studied for more than 40 years, initial insights into the anoxic steroid degradation have only recently been obtained. Molybdenum-dependent steroid C25 dehydrogenases (S25DHs) have been proposed to catalyze oxygen-independent side chain hydroxylations of globally abundant zoo-, phyto-, and mycosterols; however, so far, their lability has allowed only the initial characterization of a single S25DH. Here we report on a heterologous gene expression platform that allowed for easy isolation and characterization of four highly active S25DH isoenzymes. The results obtained demonstrate the key role of S25DHs during anoxic degradation of various steroids. Moreover, the platform is valuable for the efficient enzymatic hydroxylation of vitamin D3 to its clinically relevant C-25-OH form.

Thauera sinica sp. nov., a phenol derivative-degrading bacterium isolated from activated sludge.

A bacterial strain, K11T, capable of degrading phenol derivatives was isolated from activated sludge of a sewage treatment plant in China. This strain, which can degrade more than ten phenol derivatives, was identified as a Gram-stain negative, rod-shaped, asporogenous, facultative anaerobic bacterium with a polar flagellum. The strain was found to grow in tryptic soy broth in the presence of 0-2.5% (w/v) NaCl (optimum 0-1%), at 4-43 °C (optimum 30-35 °C) and pH 4.5-10.5 (optimum 7.5-8). Comparative analysis of nearly full-length 16S rRNA gene sequences showed that this strain belongs to the genus Thauera. The 16S rRNA gene sequence was found to show high similarity (97.5%) to that of Thauera chlorobenzoica 3CB-1T, with lesser similarity to other recognised Thauera strains. The G+C content of the DNA of the strain was determined to be 67.8 mol%. The DNA-DNA hybridization value between K11T and Thauera aromatica DSM6984T was 10.4 ± 4.5%. The genomic OrthoANI values of K11T with the other nine type strains of genus Thauera were less than 81.1%. Chemotaxonomic analysis of strain K11T revealed that Q-8 is the predominant quinone; the polar lipids contain phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and five uncharacterised lipids; the major cellular fatty acid was identified as summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH; 45.9%), followed by C16:0 (20.5%) and C18:1 ω7c (15.8%). Based on the phenotypic and phylogenetic evidence, DNA-DNA hybridisation, OrthoANI, chemotaxonomic analysis and results of the physiological and biochemical tests, a new species named Thauera sinica sp. nov. is proposed with strain K11T (= CGMCC 1.15731T = KACC 19216T) designated as the type strain.

Thauera propionica sp. nov., isolated from downstream sediment sample of the river Ganges, Kanpur, India.

A Gram-stain-negative, non-endospore-producing, short-rod strain, KNDSS-Mac4T, was isolated from a downstream sediment sample of the river Ganges, Kanpur, India and studied by using the polyphasic taxonomic approach. 16S rRNA gene sequence analysis uncovered that the strain had similarity to species of the genus Thauera and formed a distinct phylogenetic cluster with Thauera humireducens KACC16524T. However, KNDSS-Mac4T showed closest phylogenetic affiliation to Thauera aminoaromatica DSM 14742T with 16S rRNA gene sequence similarity of 98.7 % followed by Thauera phenylacetica DSM 14743T (98.6 %), Thauera chlorobenzoica (98.2 %), T. humireducens KACC16524T (98.2 %), Thauera selenatis ATCC 55363T (98.2 %) and Thauera mechernichensis DSM 12266T (98.0 %). The digital DNA-DNA hybridization and average nucleotide identity values between strain KNDSS-Mac4T and the two most closely related taxa, T. aminoaromatica DSM 14742T and T. phenylacetica DSM 14743T, were 26.0, 26.7 and 84.0, 84.3 % respectively. Major lipids present were phosphatidylglycerol, three unknown aminophospholipids, phosphatidylmethylethanolamine, two unidentified lipids and Q-8 as the only ubiquonone. The major cellular fatty acids present were C16 : 1 ω6c/C16 : 1ω7c and C16 : 0. The DNA G+C content of strain KNDSS-Mac4T was 65.9 %. Based on data from phenotypic tests and the genotypic differences of strain KNDSS-Mac4T from its closest phylogenetic relatives, it is evident that this isolate should be regarded as a new species. It is proposed that strain KNDSS-Mac4T should be classified in the genus Thauera as a novel species, Thauerapropionica sp. nov. The type strain is KNDSS-Mac4T (=KCTC 52820T=VTCC-B-910017T).

Thauera phenolivorans sp. nov., a phenol degrading bacterium isolated from activated sludge.

A Gram-stain negative, short rod-shaped and non-motile bacterial strain ZV1CT capable of degrading phenol was isolated from a wastewater treatment system of Huafu mustard tuber salinity preservation factory in Chongqing, China. Aerobic growth was observed at 20-42 °C (optimum, 30 °C) and at pH 5-10 (optimum, pH 8). Cells tolerated NaCl concentrations of 0-2% (w/v) (optimum, 0%). The major respiratory quinone is ubiquinone Q-8 and the major cellular fatty acids are C16:1 ω7c /C16:1 ω6c and C16:0. The 16S rRNA gene sequence of stain ZV1CT is phylogenetically related to the 16S rRNA genes of the type strains of Thauera species (similarity: 96.6-97.7%). The genome of strain ZV1CT was sequenced and the size of the genome is 3.68 Mb. The genomic DNA G+C content is 68.2 mol %. Strain ZV1CT exhibited whole-genome average nucleotide identity values of 82.3, 81.5 and 80.9% with respect to Thauera phenylacetica B4PT, Thauera aminoaromatica S2T and Thauera selenatis AXT, respectively. Accordingly, the genome-to-genome distances between strain ZV1CT and the type strains ranged from 21.5 to 31.3%. Based on the results of this study, it is proposed that strain ZV1CT represents a novel species of the genus Thauera, for which the name Thauera phenolivorans is proposed. The type strain is ZV1CT (=CGMCC 1.15497 = NCBR 112379).

Elemental sulfur recovery and spatial distribution of functional bacteria and expressed genes under different carbon/nitrate/sulfide loadings in up-flow anaerobic sludge blanket reactors.

To characterize the impact of influent loading on elemental sulfur (S0) recovery during the denitrifying and sulfide oxidation process, three identical, lab-scale UASB reactors (30cm in length) were established in parallel under different influent acetate/nitrate/sulfide loadings, and the reactor performance and functional community structure were investigated. The highest S0 recovery was achieved at 77.9% when the acetate/nitrate/sulfide loading was set to 1.9/1.6/0.7kgd-1m-3. Under this condition, the genera Thauera, Sulfurimonas, and Azoarcus were predominant at 0-30, 0-10 and 20-30cm, respectively; meanwhile, the sqr gene was highly expressed at 0-30cm. However, as the influent loading was halved and doubled, S0 recovery was decreased to 27.9% and 45.1%, respectively. As the loading was halved, the bacterial distribution became heterogeneous, and certain autotrophic sulfide oxidation genera, such as Thiobacillus, dominated, especially at 20-30cm. As the loading doubled, the bacterial distribution was relatively homogeneous with Thauera and Azoarcus being predominant, and the nirK and sox genes were highly expressed. The study verified the importance of influent loading to regulate S0 recovery, which could be achieved as Thauera and Sulfurimonas dominated. An influent loading that was too low or too high gave rise to insufficient oxidation or over-oxidation of the sulfide and low S0 recovery performance.

The anaerobic linalool metabolism in Thauera linaloolentis 47 Lol.

BACKGROUND: The betaproteobacterium Thauera linaloolentis 47Lol(T) was isolated on the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. Growth experiments indicated the formation of geraniol and geranial. Thus, a 3,1-hydroxyl-Δ(1)-Δ(2)-mutase (linalool isomerase) activity may initiate the degradation, followed by enzymes of the acyclic terpene utilization (Atu) and leucine/isovalerate utilization (Liu) pathways that were extensively studied in Pseudomonas spp. growing on citronellol or geraniol. RESULTS: A transposon mutagenesis yielded 39 transconjugants that could not grow anaerobically on linalool and nitrate in liquid medium. The deficiencies were apparently based on gene functions required to overcome the toxicity of linalool, but not due to inactivation of genes in the degradation pathway. Growing cultures formed geraniol and geranial transiently, but also geranic acid. Analysis of expressed proteins detected several enzymes of the Atu and Liu pathways. The draft genome of T. linaloolentis 47Lol(T) had atu and liu genes with homology to those of Pseudomonas spp.. CONCLUSION: The in comparison to monoterpenes larger toxicity of monoterpene alcohols is defeated by several modifications of the cellular structure and metabolism in Thauera linaloolentis 47Lol(T). The acyclic terpene utilization pathway is used in T. linaloolentis 47Lol(T) during growth on (R,S)-linalool and nitrate under anoxic conditions. This is the first experimental verification of an active Atu pathway outside of the genus Pseudomonas.

Complete genome of Thauera humireducens SgZ-1, a potential bacterium for environmental remediation and wastewater treatment.

Thauera humireducens SgZ-1(T) (KACC 16524(T)=CCTCC M2011497(T)), isolated from the anode biofilm of a microbial fuel cell, is able to grow under anaerobic conditions via the oxidation of various organic compounds coupled to the reduction of humus, Fe(III) species and nitrate. Addtionally, the strain has the ability to produce exopolysaccharide (EPS). Here, we report the complete genome sequence of T. humiruducens SgZ-1(T), which is relevant to metabolism of electron donors and acceptors for environmental remediation and wastewater treatment.

Metagenomes reveal microbial structures, functional potentials, and biofouling-related genes in a membrane bioreactor.

Metagenomic sequencing was used to investigate the microbial structures, functional potentials, and biofouling-related genes in a membrane bioreactor (MBR). The results showed that the microbial community in the MBR was highly diverse. Notably, function analysis of the dominant genera indicated that common genes from different phylotypes were identified for important functional potentials with the observation of variation of abundances of genes in a certain taxon (e.g., Dechloromonas). Despite maintaining similar metabolic functional potentials with a parallel full-scale conventional activated sludge (CAS) system due to treating the identical wastewater, the MBR had more abundant nitrification-related bacteria and coding genes of ammonia monooxygenase, which could well explain its excellent ammonia removal in the low-temperature period. Furthermore, according to quantification of the genes involved in exopolysaccharide and extracellular polymeric substance (EPS) protein metabolism, the MBR did not show a much different potential in producing EPS compared to the CAS system, and bacteria from the membrane biofilm had lower abundances of genes associated with EPS biosynthesis and transport compared to the activated sludge in the MBR.

Efficient regulation of elemental sulfur recovery through optimizing working height of upflow anaerobic sludge blanket reactor during denitrifying sulfide removal process.

In this study, two lab-scale UASB reactors were established to testify S(0) recovery efficiency, and one of which (M-UASB) was improved from the previous T-UASB by shortening reactor height once S(2-) over oxidation was observed. After the height was shortened from 60 to 30cm, S(0) recovery rate was improved from 7.4% to 78.8%, and while, complete removal of acetate, nitrate and S(2-) was simultaneously maintained. Meanwhile, bacterial community distribution was homogenous throughout the reactor, with denitrifying sulfide oxidization bacteria predominant, such as Thauera and Azoarcus spp., indicating the optimized condition for S(0) recovery. The effective control of working height/volume in reactors plays important roles for the efficient regulation of S(0) recovery during DSR process.