Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling.

BACKGROUND: The global prevalence of vitamin D (VitD) deficiency associated with numerous acute and chronic diseases has led to strategies to improve the VitD status through dietary intake of VitD-fortified foods and VitD supplementation. In this context, the circulating form of VitD3 (cholecalciferol) in the human body, 25-hydroxy-VitD3 (calcifediol, 25OHVitD3), has a much higher efficacy in improving the VitD status, which has motivated researchers to develop methods for its effective and sustainable synthesis. Conventional monooxygenase-/peroxygenase-based biocatalytic platforms for the conversion of VitD3 to value-added 25OHVitD3 are generally limited by a low selectivity and yield, costly reliance on cyclodextrins and electron donor systems, or by the use of toxic co-substrates. RESULTS: In this study, we used a whole-cell approach for biocatalytic 25OHVitD3 synthesis, in which a molybdenum-dependent steroid C25 dehydrogenase was produced in the denitrifying bacterium Thauera aromatica under semi-aerobic conditions, where the activity of the enzyme remained stable. This enzyme uses water as a highly selective VitD3 hydroxylating agent and is independent of an electron donor system. High density suspensions of resting cells producing steroid C25 dehydrogenase catalysed the conversion of VitD3 to 25OHVitD3 using either O2 via the endogenous respiratory chain or externally added ferricyanide as low cost electron acceptor. The maximum 25OHVitD3 titer achieved was 1.85 g L-1 within 50 h with a yield of 99%, which is 2.2 times higher than the highest reported value obtained with previous biocatalytic systems. In addition, we developed a simple method for the recycling of the costly VitD3 solubiliser cyclodextrin, which could be reused for 10 reaction cycles without a significant loss of quality or quantity. CONCLUSIONS: The established steroid C25 dehydrogenase-based whole-cell system for the value-adding conversion of VitD3 to 25OHVitD3 offers a number of advantages in comparison to conventional oxygenase-/peroxygenase-based systems including its high selectivity, independence from an electron donor system, and the higher product titer and yield. Together with the established cyclodextrin recycling procedure, the established system provides an attractive platform for large-scale 25OHVitD3 synthesis.

Toxicological study on ibuprofen and selenium in freshwater mussel Lamellidens marginalis and exploring the microbial cytochrome through modelling and quantum mechanics approaches for its toxicity degradation in contaminated environment.

Toxicological stress in aquatic organisms is caused by the discharge of hundreds of toxic pollutants and contaminants among which the current study concentrates on the toxic effect of non-steroidal anti-inflammatory drug ibuprofen (IBF) and the trace element selenium (Se). In this study, IBF and Se toxicity on freshwater mussel Lamellidens marginalis was studied for 14 days, and in silico predictions for their degradation were made using Molecular modelling and Quantum Mechanical approaches. The degrading propensity of cytochrome c oxidase proteins from Trametes verticillatus and Thauera selenatis (Turkey tail fungi and Gram-negative bacteria) is examined into atom level. The results of molecular modelling study indicate that ionic interactions occur in the T. selenatis-HEME bound complex by Se interacting directly with HEME, and in the T. versicolor-HEME bound complex by IBF bound to a nearby region of HEME. Experimental and theoretical findings suggest that, the toxicological effects of Se and IBF pollution can be reduced by bioremediation with special emphasis on T. versicolor, and T. selenatis, which can effectively interact with Se and IBF present in the environment and degrade them. Besides, this is the first time in freshwater mussel L. marginalis that ibuprofen and selenium toxicity have been studied utilizing both experimental and computational methodologies for their bioremediation study.

Ordinary heterotrophic organisms with aerobic storage capacity provide stable aerobic granular sludge for C and N removal.

The study investigated the mechanisms and microbial communities underlying the long-term stability and removal performances shown by aerobic granular sludge (AGS) reactor involving polyhydroxyalkanoates (PHA) aerobic-storing bacteria. The characteristics of the sludge, removal performances and bacterial community structure were determined. The prevailing metabolic phenotype was similar in the parent conventional activated sludge (CAS) reactor and its upgraded AGS version, showing high COD and NH4 uptake, versus low P and N reduction. Polyphosphate and glycogen accumulating organisms, PAO and GAO, were not enriched in the reactors despite initial targeting of anaerobic-aerobic cycle. Instead, PHA-aerobic storing bacteria (Thauera and Paracoccus) were dominant, but revealing a stable AGS system for BOD and N removal. The PAO/GAO failed selection and Thauera overgrowth were analyzed for beneficial use in developing alternative AGS technology for BOD and N removal applications.

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.

Thauera lacus sp. nov., isolated from a saline lake in Inner Mongolia.

A Gram-stain-negative, facultatively anaerobic, motile and rod-shaped bacterium, designated D20T, was isolated from the saline Lake Dai in Inner Mongolia, PR China. Growth of strain D20T occurred at 25-45 °C (optimum, 40 °C), pH 4.0-12.0 (optimum, 8.0) and with 0-3 % NaCl (w/v); (optimum, 0-1 %). The results of 16S rRNA gene sequence analysis revealed that strain D20T was most closely related to three Thauera species, Thaueraselenatis AXT, Thaueraaminoaromatica S2T and Thaueraaromatica K172T, with a similarity value of 96.2 %. The major respiratory quinone of strain D20T was ubiquinone-8 (Q-8), and the dominant fatty acids (>10 %) were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 39.8 %), C16 : 0 (30.9 %) and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c; 13.5 %). The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, one aminophospholipid and five unidentified lipids. The DNA G+C content was 67.2 mol% (data from the genome sequence). The estimated genome size was 3.7 Mb. The phenotypic, genotypic and chemotaxonomic differences between strain D20T and its phylogenetic relatives indicated that strain D20T should be regarded as a novel species in the genus Thauera, for which the name Thaueralacus sp. nov. is proposed. The type strain is D20T (=MCCC 1H00305T=KCTC 62586T).

Effective removal of nitrate by denitrification re-enforced with a two-stage anoxic/oxic (A/O) process from a digested piggery wastewater with a low C/N ratio.

The combined process of a long-term biogas digester and double anoxic/oxic tanks is very commonly used in piggery wastewater treatment in South China, but the effluent does not meet the discharge standard of total nitrogen (TN) and chemical oxygen demand (CODCr) due to a low C/N ratio and insufficient organic carbon in digested piggery wastewater. Thus, a typical two-stage anoxic/oxic (A1/O1/A2/O2) process, which is widely used to treat digested piggery wastewater in the engineering application, was selected for study on a laboratory scale. Finally, the average removal efficiency of ammonia nitrogen in the two-stage AO process was 98.7%; at the same time, the content of nitrate increased to 180-190 mg/L. To further eliminate nitrogen, an anaerobic tank (S1), which was equipped the sludge that was acclimated in our laboratory by a high nitrogen loading slurry, was employed to treat the effluent from the two-stage AO process and contributed more than 70% removal efficiency. Further analysis showed that ammonia-oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in the O1 and O2 tanks together contributed to the conversion of ammonia nitrogen to nitrate, but the process of heterotrophic denitrification was inhibited in the A1 and A2 tanks because of insufficient carbon sources. In addition, most of the nitrate concentration was reduced under conditions with insufficient carbon sources, while Thauera-dominated the bacterial population in the sludge sample of the S1 tank.

The difference of morphological characteristics and population structure in PAO and DPAOgranular sludges.

We examined how long-term operation of anaerobic-oxic and anaerobic-anoxic sequencing batch reactors (SBRs) affects the enhanced biological phosphorus removal (EBPR) performance and sludge characteristics. The microbial characteristics of phosphorus accumulating organism (PAO) and denitrifying PAO (DPAO) sludge were also analyzed through a quantitative analysis of microbial community structure. Compared with the initial stage of operation characterized by unstable EBPR, both PAO and DPAO SBR produced a stable EBPR performance after about 100-day operation. From day 200 days (DPAO SBR) and 250 days (PAO SBR) onward, sludge granulation was observed, and the average granule size of DPAO SBR was approximately 5 times larger than that of PAO SBR. The DPAO granular sludge contained mainly rod-type microbes, whereas the PAO granular sludge contained coccus-type microbes. Fluorescence in situ hybridization analysis revealed that a high ratio of Accumulibacter clade I was found only in DPAO SBR, revealing the important role of this organism in the denitrifying EBPR system. A pyrosequencing analysis showed that Accumulibacter phosphatis was present in PAO sludge at a high proportion of 6%, whereas it rarely observed in DPAO sludge. Dechloromonas was observed in both PAO sludge (3.3%) and DPAO sludge (3.2%), confirming that this organism can use both O2 and NO3- as electron acceptors. Further, Thauera spp. was identified to have a new possibility as denitrifier capable of phosphorous uptake under anoxic condition.

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).

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).

Identification and characterization of a novel esterase from Thauera sp.

A novel esterase gene TLip was identified from the strain Thauera sp. and expressed at high levels in Escherichia coli. The TLip protein shared the highest identity (48%) to esterase TesA from Pseudomonas aeruginosa when compared to enzymes with reported properties. Phylogenetic analysis showed that TLip belongs to the GDSL family of bacterial lipolytic enzymes. TLip was an alkaline esterase with a broad optimal temperature range 37-50 °C and an optimal pH of 8.0. Substrate specificity assays showed that TLip preferred medium chain p-nitrophenyl esters (C6 -C12 ). Besides, the activity of TLip was strongly inhibited by Cu2+ but greatly enhanced by Triton X-100 and Tween 80. Thermostability assay revealed that TLip was stable without loss of activity at 37 °C and still retained 69% activity at 50 °C after 2 H of incubation. Together, these provided a good candidate for further exploration of TLip as a promising biocatalyst in industry.

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.

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.

Catechol 2,3-dioxygenase from a new phenolic compound degrader Thauera sp. K11: purification and biochemical characterization.

Catechol 2,3-dioxygenase (C23O) from a new phenolic compound degrader Thauera sp. K11 was purified and characterized. The native form of the enzyme was determined as a homotetramer with a molecular weight of 140 kDa, and its isoelectric point was close to 6.4. One iron per enzyme subunit was detected using atom absorption spectroscopy, and the effective size of C23O in its dilute solution (0.2 g L-1 , pH 8.0) was 14.5 nm. The optimal pH and temperature were 8.4 and 45 °C, respectively. The addition of Mg2+ , Cu2+ , Fe2+ , and Mn2+ could improve the enzyme activity, while Ag+ was found to be a strong inhibitor. C23O was stable in alkali conditions (pH 7.6-11.0) and thermostable below 50 °C. The final purified C23O had a sheet content of 53%, consistent with the theoretical value. This showed that the purified catechol 2,3-dioxygenase folded with a reasonable secondary structure.

Effect of air-exposed biocathode on the performance of a Thauera-dominated membraneless single-chamber microbial fuel cell (SCMFC).

To investigate the effect of air-exposed biocathode (AEB) on the performance of single-chamber microbial fuel cell (SCMFC), wastewater quality, bioelectrochemical characteristics and the electrode biofilms were researched. It was demonstrated that exposing the biocathode to air was beneficial to nitrogen removal and current generation. In Test 1 of 95% AEB, removal rates of ammonia, total nitrogen (TN) and chemical oxygen demand (COD) reached 99.34%±0.11%, 99.34%±0.10% and 90.79%±0.12%, respectively. The nitrogen removal loading rates were 36.38gN/m3/day. Meanwhile, current density and power density obtained at 0.7A/m3 and 104mW/m3 respectively. Further experiments on open-circuit (Test 2) and carbon source (Test 3) indicated that this high performance could be attributed to simultaneous biological nitrification/denitrification and aerobic denitrification, as well as bioelectrochemical denitrification. Results of community analysis demonstrated that both microbial community structures on the surface of the cathode and in the liquid of the chamber were different. The percentage of Thauera, identified as denitrifying bacteria, maintained at a high level of over 50% in water, but decreased gradually in the AEB. Moreover, the genus Nitrosomonas, Alishewanella, Arcobacter and Rheinheimera were significantly enriched in the AEB, which might contribute to both enhancement of nitrogen removal and electricity generation.

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).

Linalool isomerase, a membrane-anchored enzyme in the anaerobic monoterpene degradation in Thauera linaloolentis 47Lol.

BACKGROUND: Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ(1)-Δ(2)-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool. RESULTS: The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42 ± 0.28 nkat mg * protein(-1) and a kM value of 455 ± 124 µM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C. CONCLUSION: The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

Integration of the sulfate reduction and anammox processes for enhancing sustainable nitrogen removal in granular sludge reactors.

The Anammox and Sulfate Reduction Ammonium Oxidation processes were compared in two granular sequencing batch reactors operated for 160 days under anammox conditions. It was hypothesized that increasing the concentration of SO42- may positively influence the rate of N removal under anaerobic conditions and it was tested whether SO42- reduction and anammox occur independently or are related to each other. The cooperation of N-S cycles by increasing the concentration of influent SO42- to 952 mg S/L in the second reactor, a higher ammonium utilization rate and sulfate utilization rate was achieved compared to the first reactor, i.e., 2.1-fold and 15-fold, respectively. Nitrosomonas played the dominant role in the N metabolism, while Thauera - in the S metabolism. This study highlights the benefits of linking the N-S cycles as an effective approach for the treatment of NH4+ and SO42- - rich wastewater, including lower substrate removal cost and reduced energy consumption.

Valorization of food waste derived anaerobic digestate into polyhydroxyalkanoate (PHA) using Thauera mechernichensis TL1.

Polyhydroxyalkanoate (PHA) is a biopolymer that can be used as a bioplastic, offering a green alternative to petroleum-based plastics. In this study, we investigated PHA production using Thauera mechernichensis TL1. The optimal molar C/N ratio was determined to be 20 from among the ratios of 4, 20, 40, 80, and 200 and in the absence of nitrogen. Food waste anaerobic digestate, mainly comprised of acetate and propionate, was used as the carbon source for PHA production by T. mechernichensis TL1, resulting in a maximum PHA content of 23.98 ± 0.52 wt% (0.52 ± 0.02 g/L PHA) with a PHA productivity of 0.043 g/L-h PHA. In addition, when using acetate and propionate, T. mechernichensis TL1 produced PHA with a maximum PHA content of 57.43 ± 2.84 wt% (2.04 ± 0.11 g/L PHA) and 50.94 ± 1.61 wt% (2.62 ± 0.16 g/L PHA), with a PHA productivity of 0.092 g/L-h PHA and 0.070 g/L-h PHA, respectively. Proton nuclear magnetic resonance spectroscopy (1H NMR) confirmed polyhydroxybutyrate (PHB) production using acetate as a carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production using propionate or food waste anaerobic digestate as the carbon source. The whole-genome analysis of T. mechernichensis TL1 confirmed the existence of a PHA biosynthesis pathway, with the presence of phaA, phaB, phaC (Class I and Class II), and phaJ genes. This study was the first to demonstrate Thauera sp.'s ability to produce PHA from food waste anaerobic digestate, rendering it as a promising candidate for PHA-producing bacteria for the valorization of food waste.