Response of nitrite accumulation to elevated C/NO- 3-N ratio during partial denitrification process: Insights of extracellular polymeric substance, microbial community and metabolic function.

This study investigated the response of nitrite accumulation to elevated COD/NO3--N ratio (C/N) during partial denitrification (PD). Results indicated nitrite was gradually accumulated and remained stable (C/N = 1.5 âˆ¼ 3.0), while that rapidly declined after reaching the peak (C/N = 4.0 âˆ¼ 5.0). The polysaccharide (PS) and protein (PN) content of tightly-bound extracellular polymeric substances (TB-EPS) reached the maximum at C/N of 2.5 âˆ¼ 3.0, which might be stimulated by high level of nitrite. Illumina MiSeq sequencing showed Thauera and OLB8 were dominated denitrifying genera at C/N of 1.5 âˆ¼ 3.0, while Thauera was further enriched with fading OLB8 at C/N of 4.0 âˆ¼ 5.0. Meanwhile, the highly-enriched Thauera might enhance the activity of nitrite reductase (nirK) promoting further nitrite reduction. Redundancy analysis (RDA) showed positive correlations between nitrite production and PN content of TB-EPS, denitrifying bacteria (Thauera and OLB8) and nitrate reductases (narG/H/I) in low C/N. Finally, their synergistic effects for driving nitrite accumulation were comprehensively elucidated.

Thauera sp. Sel9, a new bacterial strain for polyhydroxyalkanoates production from volatile fatty acids.

Thauera is one of the main genera involved in polyhydroxyalkanoate (PHA) production in microbial mixed cultures (MMCs) from volatile fatty acids (VFAs). However, no Thauera strains involved in PHA accumulation have been obtained in pure culture so far. This study is the first report of the isolation and characterization of a Thauera sp. strain, namely Sel9, obtained from a sequencing batch reactor (S-SBR) set up for the selection of PHA storing biomass. The 16S rRNA gene evidenced a high sequence similarity with T. butanivorans species. Genome sequencing identified all genes involved in PHA synthesis, regulation and degradation. The strain Sel9 was able to grow with an optimum of chemical oxygen demand-to-nitrogen (COD:N) ratio ranging from 4.7 to 18.9. Acetate, propionate, butyrate and valerate were used as sole carbon and energy sources: a lag phase of 72 h was observed in presence of propionate. Final production of PHAs, achieved with a COD:N ratio of 75.5, was 60.12 ± 2.60 %, 49.31 ± 0.7 %, 37.31 ± 0.43 % and 18.06 ± 3.81 % (w/w) by using butyrate, acetate, valerate and propionate as substrates, respectively. Also, the 3-hydroxybutyrate/3-hydroxyvalerate ratio reflected the type of carbon sources used: 12.30 ± 0.82 for butyrate, 3.56 ± 0.02 for acetate, 0.93 ± 0.03 for valerate and 0.76 ± 0.02 for propionate. The results allow a better elucidation of the role of Thauera in MMCs and strongly suggest a possible exploitation of Thauera sp. Sel9 for a cost-effective and environmentally friendly synthesis of PHAs using VFAs as substrate.

Advanced Insights into Catalytic and Structural Features of the Zinc-Dependent Alcohol Dehydrogenase from Thauera aromatica.

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.

Inducing high nitrite accumulation via modulating nitrate reduction power and carbon flux with Thauera spp. selection.

Partial-denitrification (PD, NO3--N â†’ NO2--N) is emerging as a promising approach for application of anaerobic ammonium oxidation (anammox) process. In this study, stable PD with high nitrite (NO2--N) accumulation was achieved by modulating nitrate (NO3--N) reduction activity and carbon metabolism. With the influent NO3--N increasing from 30 to 200 mg/L, specific NO3--N reduction rates (rno3) were significantly improved, corresponding to the nitrate-to-nitrite transforming ratio (NTR) increasing rapidly to 80.0% within just 70 days. The required COD/NO3--N decreased from 4.5 to 2.0 and the carbon flux was more shared in NO3--N reduction to NO2--N. Notably, Thauera spp. as core denitrifying bacteria was highly enriched with the relative abundance of 70.5%∼82.1% despite different inoculations. This study provided a new insight into inducing high NO2--N accumulation and promoting practical application of anammox technology.

Cross-Feeding between Members of Thauera spp. and Rhodococcus spp. Drives Quinoline-Denitrifying Degradation in a Hypoxic Bioreactor.

The complex bacterial community in a quinoline-degrading denitrifying bioreactor is predominated by several taxa, such as Thauera and Rhodococcus However, it remains unclear how the interactions between the different bacteria mediate quinoline metabolism under denitrifying conditions. In this study, we designed a sequence-specific amplification strategy to isolate the most predominant bacteria and obtained four strains of Thauera aminoaromatica, a representative of a key member in the bioreactor. Tests on these isolates demonstrated that all were unable to degrade quinoline but efficiently degraded 2-hydroxyquinoline, the hypothesized primary intermediate of quinoline catabolism, under nitrate-reducing conditions. However, another isolate, Rhodococcus sp. YF3, corresponding to the second most abundant taxon in the same bioreactor, was found to degrade quinoline via 2-hydroxyquinoline. The end products and removal rate of quinoline by isolate YF3 largely varied according to the quantity of available oxygen. Specifically, quinoline could be converted only to 2-hydroxyquinoline without further transformation under insufficient oxygen conditions, e.g., less than 0.5% initial oxygen in the vials. However, resting YF3 cells aerobically precultured in medium with quinoline could anaerobically convert quinoline to 2-hydroxyquinoline. A two-strain consortium constructed with isolates from Thauera (R2) and Rhodococcus (YF3) demonstrated efficient denitrifying degradation of quinoline. Thus, we experimentally verified that the metabolic interaction based on 2-hydroxyquinoline cross-feeding between two predominant bacteria constitutes the main quinoline degradation mechanism. This work uncovers the mechanism of quinoline removal by two cooperative bacterial species existing in denitrifying bioreactors.IMPORTANCE We experimentally verified that the second most abundant taxon, Rhodococcus, played a role in degrading quinoline to 2-hydroxyquinoline, while the most abundant taxon, Thauera, degraded 2-hydroxyquinoline. Metabolites from Thauera further served to provide metabolites for Rhodococcus Hence, an ecological guild composed of two isolates was assembled, revealing the different roles that keystone organisms play in the microbial community. This report, to the best of our knowledge, is the first on cross-feeding between the initial quinoline degrader and a second bacterium. Specifically, the quinoline degrader (Rhodococcus) did not benefit metabolically from quinoline degradation to 2-hydroxyquinoline but instead benefited from the metabolites produced by the second bacterium (Thauera) when Thauera degraded the 2-hydroxyquinoline. These results could be a significant step forward in the elucidation of the microbial mechanism underlying quinoline-denitrifying degradation.

An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria.

The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and ortho-phthalate (hereafter, phthalate). In aerobic bacteria and fungi, oxygenases are involved in the conversion of phthalate to protocatechuate, the substrate for ring-cleaving dioxygenases. In contrast, anaerobic bacteria activate phthalate to the extremely unstable phthaloyl-coenzyme A (CoA), which is decarboxylated by oxygen-sensitive UbiD-like phthaloyl-CoA decarboxylase (PCD) to the central benzoyl-CoA intermediate. Here, we demonstrate that the facultatively anaerobic, denitrifying Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. In vitro assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to benzoyl-CoA. In T. chlorobenzoica 3CB-1, we identified PCD as a highly abundant enzyme in both aerobically and anaerobically grown cells, whereas genes for phthalate dioxygenases are missing in the genome. PCD was highly enriched from aerobically grown T. chlorobenzoica cells and was identified as an identical enzyme produced under denitrifying conditions. These results indicate that the initial steps of aerobic phthalate degradation in denitrifying bacteria are accomplished by the anaerobic enzyme inventory, whereas the benzoyl-CoA oxygenase-dependent pathway is used for further conversion to central intermediates. Such a hybrid pathway requires intracellular oxygen homeostasis at concentrations low enough to prevent PCD inactivation but sufficiently high to supply benzoyl-CoA oxygenase with its cosubstrate.IMPORTANCE Phthalic acid esters (PAEs) are industrially produced on a million-ton scale per year and are predominantly used as plasticizers. They are classified as environmentally relevant xenobiotics with a number of adverse health effects, including endocrine-disrupting activity. Biodegradation by microorganisms is considered the most effective process to eliminate PAEs from the environment. It is usually initiated by the hydrolysis of PAEs to alcohols and o-phthalic acid. Degradation of o-phthalic acid fundamentally differs in aerobic and anaerobic microorganisms; aerobic phthalate degradation heavily depends on dioxygenase-dependent reactions, whereas anaerobic degradation employs the oxygen-sensitive key enzyme phthaloyl-CoA decarboxylase. We demonstrate that aerobic phthalate degradation in facultatively anaerobic bacteria proceeds via a previously unknown hybrid degradation pathway involving oxygen-sensitive and oxygen-dependent key enzymes. Such a strategy is essential for facultatively anaerobic bacteria that frequently switch between oxic and anoxic environments.

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.

Identification and nitrogen removal characteristics of Thauera sp. FDN-01 and application in sequencing batch biofilm reactor.

A strain FDN-01 was isolated from the sequencing batch biofilm reactor (SBBR) which was seeded with wasted activated sludge from a municipal wastewater treatment plant in Shanghai. Bacterium FDN-01 was identified as Thauera sp., and Genbank Sequence_ID was KY393097. By comparing inorganic total nitrogen (TN) removal efficiency by strain FDN-01 under different conditions, the optimal initial pH, carbon source and the ratio of carbon to nitrogen were 7.5, sodium succinate and 4.0, respectively. Inorganic TN removal efficiency was 93% within 3 d while the concentration of nitrate was 100 mg/L, and the type of substrates affected extracellular polymeric substances (EPS) production and the ratio of protein to polysaccharide in the EPS. Further investigation for the application of strain FDN-01 in the SBBRs showed that anoxic ammonia oxidation occurred at room temperature, and the removal efficiencies of inorganic TN were noticeably enhanced by the augmentation of bacterium FDN-01 back into the SBBR. This study provided a promising method of TN removal requiring less carbon source in the wastewater.

Enhancement of the denitrification activity by exoelectrogens in single-chamber air cathode microbial fuel cells.

Single-chamber microbial fuel cells (MFCs) can efficiently treat wastewater containing nitrate, probably because the interaction between exoelectrogens and denitrifying bacteria may enhance the denitrification activity of MFCs. In this study, the denitrification of nitrate with a wide range of concentrations was investigated by using single-chamber air cathode MFCs. The maximum average denitrification rate of the MFCs inoculated and operated under closed-circuit conditions (Group N-CC) was up to 12.2 ±â€¯0.6 kg NO3--N m-3 d-1 at a high nitrate concentration of 2000 mg NO3-N L-1, which was 74.3% higher than that of the MFCs inoculated and operated under open-circuit conditions and which was significantly higher than those of other MFC systems and many traditional bioreactors. The high denitrification activity of the MFCs of Group N-CC was attributed to the significant reduction of nitrite accumulation through the possible bioelectrochemical nitrite reduction by exoelectrogens that were only enriched at the anodes of the MFCs of Group N-CC. In addition, the MFCs of Group N-CC showed good stability (over 3.5 years) and low apparent activation energy (34.0 kJ mol-1) of the denitrification, indicating the good coexistence of exoelectrogens (Geobacter) and denitrifying bacteria (Thauera) with high performance on denitrification during the long-term operation.

Achieving partial denitrification using carbon sources in domestic wastewater with waste-activated sludge as inoculum.

Partial denitrification (PD, nitrate → nitrite) using carbon sources in domestic wastewater with waste-activated sludge as inoculum was firstly achieved in this study. Through controlling influent pH at about 9.0 and anoxic reaction time of 1 h in the start-up, the nitrite (NO2--N) production reached as high as 25.2 mg/L, with influent nitrate (NO3--N) of about 30 mg/L and chemical oxygen demand (COD) to NO3--N ratio of 5.9. Furthermore, PD performance remained stable without pH control during subsequent operations. Efficient NO2--N production was closely related to the consumed amount of readily biodegradable COD (Ss) fraction, with optimal Ss/NO3--N ratio of about 3.5. Thauera (19.1%), norank_f__Xanthomonadaceae (5.2%), and Thiobacillus (5.0%) were enriched during the 208-day operation, which may be responsible for high NO2--N production. These findings provided a novel strategy for promoting mainstream PD/Anammox application, without additional nitrite-accumulating denitrifying sludge and external carbon sources.

Long-term effect of pH on denitrification: High pH benefits achieving partial-denitrification.

Partial-denitrification (nitrate to nitrite) can supply nitrite for anammox which can reduce organic matter consumption in wastewater treatment plants (WWTPs). In order to achieve stable partial-denitrification, the effect of pH on denitrification were investigated for 420 days in three reactors with influent pH of 5.0, 7.0 and 9.0. The results indicate that the nitrite accumulation rate (NAR) increased with pH, with average effluent NARs being 21%, 38% and 57% in the above reactors, respectively. The sludge cultivated at a high pH of 9.0 was resistant to pH shock, with a high NAR being maintained at 83% when it was exposed to a low pH of 5.0. Metagenomic analysis showed that the higher NAR at pH 9.0 was correlated with an enrichment of Thauera, which harbored more nitrate reductase (8098 hits) than nitrite reductase (2950 hits). Based on these findings, a novel process was proposed for achieving partial-denitrification/anammox in mainstream WWTPs.

pH control of an upflow pyrite-oxidizing denitrifying bioreactor via electrohydrogenesis.

Maintenance of stable pH during pyrite-oxidizing denitrification process is important. Here, we demonstrated effective pH control (7.80 ±â€¯0.20-8.40 ±â€¯0.30) in an electrochemical-H2 and pyrite-oxidizing denitrifying bioreactor (HPR) through in situ electrohydrogenesis. HPR achieved a higher nitrate removal activity (maximum:19.66 ±â€¯0.63 mg NO3--N/(L·h)) with excellent resistance to high nitrate loading (up to 400 mg/L NO3--N) compared to that of the control groups. Nitrate removal rate of HPR fitted the Michaelis-Menten kinetic model (R2 = 0.98, p < 0.01) well, and the denitrification followed the zero-order rate law. The results of the biofilm community analyses suggested that Thauera was the dominant bacteria in the cathode biofilm of HPR and may prefer hydrogen as an electron donor for autotrophic denitrification, while the relative abundance of Pseudomonas were similar in the cathode biofilm and pyrite biofilm. This study provides a new alternative for effective pH control in denitrifying bioreactors with pyrite as a packing material.

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.

Stereochemical Insights into the Anaerobic Degradation of 4-Isopropylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1.

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of anaerobic degradation of p-cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4-isopropylbenzoyl-CoA formed by methyl group hydroxylation and subsequent oxidation of p-cymene. Proteogenomics indicated 4-isopropylbenzoyl-CoA degradation involves (i) a novel member of class I benzoyl-CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified ß-oxidation pathway yielding 3-isopropylpimeloyl-CoA analogously to benzoyl-CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate as well as both enantiomers of 3-isopropylpimelate were obtained by stereoselective syntheses via methyl 4-isopropyl-2-oxocyclohexane-1-carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium-catalyzed asymmetric conjugate addition. X-ray crystallography revealed that the thermodynamically most stable stereoisomer of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate is formed during p-cymene degradation. Our findings imply that the reductive dearomatization of 4-isopropylbenzoyl-CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)-4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA.

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.

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.

A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria.

Class I benzoyl-CoA (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of the central BzCoA intermediate and analogues of it to conjugated cyclic 1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduction mechanism. Discovered in 1995, the enzyme from the denitrifying bacterium Thauera aromatica (BCRTar) has so far remained the only isolated and biochemically accessible BCR, mainly because BCRs are extremely labile, and their heterologous production has largely failed so far. Here, we describe a platform for the heterologous expression of the four structural genes encoding a designated 3-methylbenzoyl-CoA reductase from the related denitrifying species Thauera chlorobenzoica (MBRTcl) in Escherichia coli This reductase represents the prototype of a distinct subclass of ATP-dependent BCRs that were proposed to be involved in the degradation of methyl-substituted BzCoA analogues. The recombinant MBRTcl had an αßγδ-subunit architecture, contained three low-potential [4Fe-4S] clusters, and was highly oxygen-labile. It catalyzed the ATP-dependent reductive dearomatization of BzCoA with 2.3-2.8 ATPs hydrolyzed per two electrons transferred and preferentially dearomatized methyl- and chloro-substituted analogues in meta- and para-positions. NMR analyses revealed that 3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl-1,5-dienoyl-CoA. The unprecedented reductive dechlorination of 4-chloro-BzCoA to BzCoA probably via HCl elimination from a reduced intermediate allowed for the previously unreported growth of T. chlorobenzoica on 4-chlorobenzoate. The heterologous expression platform established in this work enables the production, isolation, and characterization of bacterial and archaeal BCR and BCR-like radical enzymes, for many of which the function has remained unknown.

Liquid chromatography/isotope ratio mass spectrometry analysis of halogenated benzoates for characterization of the underlying degradation reaction in Thauera chlorobenzoica CB-1T.

RATIONALE: Halogenated benzoic acids occur in the environment due to their widespread agricultural and pharmaceutical use. Compound-specific stable isotope analysis (CSIA) has developed over the last decades for investigation of in situ transformation and reaction mechanisms of environmental pollutants amenable by gas chromatography (GC). As polar compounds are unsuitable for GC analysis we developed a method to perform liquid chromatography (LC)/CSIA for halogenated benzoates. METHODS: LC/isotope ratio mass spectrometry (IRMS) utilizing a LC-Surveyor pump coupled to a MAT 253 isotope ratio mass spectrometer via a LC-Isolink interface was applied. For chromatographic separation a YMC-Triart C18 column and a potassium hydrogen phosphate buffer (150 mM, pH 7.0, 40°C, 200 µL mL-1 ) were used, followed by wet oxidation deploying 1.5 mol L-1 ortho-phosphoric acid and 200 g L-1 sodium peroxodisulfate at 75 µL mL-1 . RESULTS: Separation of benzoate and halogenated benzoates could be achieved in less than 40 min over a concentration range of 2 orders of magnitude. Under these conditions the dehalogenation reaction of Thauera chlorobenzoica 3CB-1T using 3-chloro-, 3-bromo- and 4-chlorobenzoic acid was investigated resulting in inverse carbon isotope fractionation for meta-substituted benzoic acids and minor normal fractionation for para-substituted benzoic acids. Together with the respective growth rates this led to the assumption that dehalogenation of para-halobenzoic acids follows a different mechanism from that of meta-halobenzoic acids. CONCLUSIONS: A new LC/IRMS method for the quantitative determination of halogenated benzoates was developed and used to investigate the in vivo transformation pathways of these compounds, providing some insights into degradation and removal of these widespread compounds by T. chlorobenzoica 3CB-1T .

Functional redundancy ensures performance robustness in 3-stage PHA-producing mixed cultures under variable feed operation.

Polyhydroxyalkanoates (PHA) are biopolymers that can be produced by mixed microbial cultures using wastes or industrial by-products, which represent an economical and environmental advantage over pure culture processes. The use of alternate feedstocks enables using seasonal by-products, providing that the process is resilient to transient conditions. The mixed microbial communities of a 3-stage PHA producing system fed initially with molasses and then cheese whey were investigated through amplicon sequencing of the 16S rRNA gene. The transition in feedstock resulted in an adaptation of the acidogenic community, where Actinobacteria dominated with sugarcane molasses (up to 93% of the operational taxonomic units) and Firmicutes, with cheese whey (up to 97%). The resulting fermentation products profile also changed, with a higher fraction of HV precursors obtained with molasses than cheese whey (7.1±0.5 and 1.7±0.7 gCOD/L, respectively). As for the PHA storing culture, the genera Azoarcus, Thauera and Paracoccus were enriched with fermented molasses (average 89% of Bacteria). Later, fermented cheese whey fostered a higher diversity, including some less characterised PHA-storers such as the genera Paenibacillus and Lysinibacillus. Although the microbial community structure was significantly affected by the feedstock shift, the acidogenic and PHA storing performance of the 3-stage system was very similar once a pseudo steady state was attained, showing that a reliable level of functional redundancy was attained in both mixed cultures.

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).