Thiobacillus sedimenti sp. nov., a chemolithoautotrophic sulphur-oxidizing bacterium isolated from freshwater sediment.

A sulphur-oxidizing bacterium, designated strain SCUT-2T, was isolated from freshwater sediment collected from the Pearl River in Guangzhou, PR China. This strain was an obligate chemolithoautotroph, utilizing reduced sulphur compounds (elemental sulphur, thiosulphate, tetrathionate and sulphite) as the electron donor. Growth of strain SCUT-2T was observed at 20-40 â„ƒ (optimum at 30 °C), pH 5.0-9.0 (optimum at 6.0), and NaCl concentration range of 0-9 g L-1 (optimum at 1 g L-1). The major cellular fatty acids were C16:0 ω7c and cyclo-C17:0. The DNA G + C content of the complete genome sequence was 66.8 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain SCUT-2T formed a lineage within the genus Thiobacillus, showing gene sequence identity of 98.0% with its closest relative Thiobacillus thioparus THI 115. The genome of strain SCUT-2T contains multiple genes encoding sulphur-oxidizing enzymes that catalyse the oxidation of reduced sulphur compounds, partial genes that are necessary for denitrification, and the genes encoding cbb3-type cytochrome c oxidase, aa3-type cytochrome c oxidase and bd-type quinol oxidase. Facultative anaerobic growth occurs when using nitrate as the electron acceptor and thiosulphate as the electron donor. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic analysis, strain SCUT-2T (= GDMCC 1.4108T = JCM 39443T) is deemed to represent a novel Thiobacillus species, for which we propose the name Thiobacillus sedimenti sp. nov.

Bioaugmentation with Thiobacillus sp. H1 in an autotrophic denitrification desulfurization microbial reactor: Microbial community changes and relationship.

Thiobacillus sp. H1 was isolated and made into solid bacterial agent. The Thiobacillus sp. H1 agent was dosed into two reactor (all the agent dosed one-time, and multi-dosing bacteria evenly) and run for 40 days, a start-up with no microbial agent bioreactor as control. We found that the operational performance of multi-dosing inoculum reactor was stable, and the amount of elemental sulfur produced remained stable at 143.2-152.3 mg/L. The amount of elemental sulfur generated in the reactor without the addition of the inoculum was gradually increased, and the amount of elemental sulfur generated in the reactor with the inoculum added at one-time was decreased. Two kinds of Thiobacillus gen. and unclassified betaproteobacteria that coordinated the overall community function in the autotrophic denitrification desulfurization system with high-throughput sequencing. The trend of FccAB gene in each bioreactor was similar with the trend of elemental sulfur in the effluent. On the 5th day, the copy number of FccAB in bioreactor II was the highest among the three bioreactors, reaching 11.8 log copies L/g. This study explores the possibility of artificially synthesized denitrifying desulfurization flora in the future.

Identification of Thiobacillus bacteria in agricultural soil in Iran using the 16S rRNA gene.

Thiobacillus, as useful soil bacteria, plays an important role in sulfur cycling. The purpose of this study was to identify the species Thiobacillus thioparus, Thiobacillus novellas and Thiobacillus denitrificans in rainfed and irrigated lands soil in Ajabshir, Ilam, Qorveh, Rojintaak, Sonqor, Kermanshah and Research Farm of Razi University in Iran. Sampling was performed as randomized completely with three replications at depth of 0-30 cm. The Thiobacillus species were determined via 16S rRNA characteristics. The results of agarose gel electrophoresis indicated that T. thioparus was the highest amount in the irrigated land in Research Farm and its lowest amount was in the Rojintaak rainfed land. These species not found in four locations and conditions including the Ajabshir irrigated, Qorveh rainfed, Research Farm rainfed and Rojintaak irrigated lands. The results of the T. novellas indicated that this was found in Ilam irrigated, Qorveh rainfed, Research Farm irrigated, Rojintaak irrigated and Rojintaak rainfed lands. The highest and lowest amount of T. novellas was indicated in the Rojintaak and Ilam irrigated lands respectively. The T. denitrificans gene showed that this bacterium was observed only in both samples of Ajabshir. Our study showed that Thiobacillus was not detected in all of the soils. If sulfur fertilizer is given to the soil without this bacterium, it is necessary to use sulfur fertilizer with Thiobacillus bacteria inoculation for better sulfur oxidation.

A comparative study of eubacterial communities by PCR-DGGE fingerprints in anoxic and aerobic biotrickling filters used for biogas desulfurization.

Biological desulfurization has proven to be a process that is technically and economically feasible on using biotrickling filters that can be performed under aerobic and anoxic conditions. However, microbial communities are different mainly due to the use of different final electron acceptors. The analysis of microbial communities in these systems has not been addressed with regard to the anoxic process. The aim of the work reported here was to analyse the eubacterial community in the two types of bioreactor along the packed bed and during the operation time. The analysis was carried out using the 16S PCR-DGGE molecular fingerprint technique. The microbial profile analysis in the aerobic bioreactor revealed that the community was more diverse and stratified compared to those obtained in the two anoxic bioreactors, influenced by environmental factors. The main OTU involved in this process is genus Thiobacillus, although different species were detected depending on each operational condition.

Development of PCR primer systems for amplification of 16S-rDNA to detect of Thiobacillus spp.

Thiobacillus is a genus of Gram-negative, rod-shaped and autotrophic Betaproteobacteria. They catalyze the dissimilatory oxidation of elemental sulfur and reduced inorganic sulfur compounds. Whereas more than 30 species have been known in this genus, most were never reliably or effectively published. The rest were either reclassified into Thiomonas, Paracoccus, Starkeya, Sulfuriferula, Halothiobacillus, Thermithiobacillus or Acidithiobacillus, were lost from culture. Most of Thiobacillus species are obligate autotrophs via elementary sulfur, thiosulfate or polythionates as energy sources. Based on 16S ribosomal RNA sequence analysis, many members of Thiobacillus have been reclassified. A system was developed for the detection of Thiobacillus bacteria by the amplification of specific 16S ribosomal RNA sequence gene (16S rDNA) fragments with PCR. Primer sequences were designed for the amplification of fragments of 16S rDNA.

Spatial variation in bacterial community in natural wetland-river-sea ecosystems.

Wetland-estuarine-marine environments are typical oxic/anoxic transition zones and have complex water flow-paths within the zone of mixing where freshwater interacts with ocean water. Little is known about the impact of this interaction on bacterial community structures or the relationship between bacterial community and geochemical factors in such transitional mixing environments. Hence, we investigated the distribution patterns and diversity in bacterial communities in the Yellow River estuary-coastal wetland-Bohai Sea transition zone by analyzing 39 samples from 13 ordered sites. High-throughput sequencing of the 16S rRNA gene revealed significant shifts in diversity and distribution of bacterial community in sediments from the Yellow River estuary to the Bohai Sea. Yellow River sediment was dominated by hydrogen-, nitrogen-, and iron-cycling bacteria, such as Hydrogenophaga, Nitrospira, Pseudomonas, and Thiobacillus. The coastal wetland had a haloduric community associated with different functions, such as Planctomyces, Marinobacter, Halomonas, Salinivibrio, and Salinibacter. The Bohai Sea sediment had a higher relative abundance of Lutimonas, Desulfococcus, Photobacterium, Propionigenium, and Vibrio. Spatial variation in bacterial community was correlated with pH, salinity and sulfate (SO42-) concentration in such coastal environments. The major bacterial taxa were significantly different across the wetland, estuary, and coastal marine ecosystems, indicating substantial spatial heterogeneity among the three ecosystems. Statistical analysis revealed strong links between variation in bacterial community structure and ecosystem type. Our results demonstrate the importance of geographic and geochemical factors in structuring the bacterial community in natural environments.

Thiosulfate oxidation by Thiomicrospira thermophila: metabolic flexibility in response to ambient geochemistry.

Previous studies of the stoichiometry of thiosulfate oxidation by colorless sulfur bacteria have failed to demonstrate mass balance of sulfur, indicating that unidentified oxidized products must be present. Here the reaction stoichiometry and kinetics under variable pH conditions during the growth of Thiomicrospira thermophila strain EPR85, isolated from diffuse hydrothermal fluids at the East Pacific Rise, is presented. At pH 8.0, thiosulfate was stoichiometrically converted to sulfate. At lower pH, the products of thiosulfate oxidation were extracellular elemental sulfur and sulfate. We were able to replicate previous experiments and identify the missing sulfur as tetrathionate, consistent with previous reports of the activity of thiosulfate dehydrogenase. Tetrathionate was formed under slightly acidic conditions. Genomic DNA from T. thermophila strain EPR85 contains genes homologous to those in the Sox pathway (soxAXYZBCDL), as well as rhodanese and thiosulfate dehydrogenase. No other sulfur oxidizing bacteria containing sox(CD)2 genes have been reported to produce extracellular elemental sulfur. If the apparent modified Sox pathway we observed in T. thermophila is present in marine Thiobacillus and Thiomicrospira species, production of extracellular elemental sulfur may be biogeochemically important in marine sulfur cycling.

Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome-resolved metagenomics.

Gold ore processing uses cyanide (CN(-) ), which often results in large volumes of thiocyanate- (SCN(-) ) contaminated wastewater requiring treatment. Microbial communities can degrade SCN(-) and CN(-) , but little is known about their membership and metabolic potential. Microbial-based remediation strategies will benefit from an ecological understanding of organisms involved in the breakdown of SCN(-) and CN(-) into sulfur, carbon and nitrogen compounds. We performed metagenomic analysis of samples from two laboratory-scale bioreactors used to study SCN(-) and CN(-) degradation. Community analysis revealed the dominance of Thiobacillus spp., whose genomes harbour a previously unreported operon for SCN(-) degradation. Genome-based metabolic predictions suggest that a large portion of each bioreactor community is autotrophic, relying not on molasses in reactor feed but using energy gained from oxidation of sulfur compounds produced during SCN(-) degradation. Heterotrophs, including a bacterium from a previously uncharacterized phylum, compose a smaller portion of the reactor community. Predation by phage and eukaryotes is predicted to affect community dynamics. Genes for ammonium oxidation and denitrification were detected, indicating the potential for nitrogen removal, as required for complete remediation of wastewater. These findings suggest optimization strategies for reactor design, such as improved aerobic/anaerobic partitioning and elimination of organic carbon from reactor feed.

Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph.

Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important drivers of mineral weathering in oxic and anoxic zones of many aquatic environments, including those beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O3 (2-)), which represents an electron donor capable of supporting microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada, over a seasonal melt cycle revealed concentrations of S2O3 (2-) that were typically below the limit of detection, despite the presence of available pyrite and concentrations of the FeS2 oxidation product sulfate (SO4 (2-)) several orders of magnitude higher than those of S2O3 (2-). Here we report on the physiological and genomic characterization of the chemolithoautotrophic facultative anaerobe Thiobacillus sp. strain RG5 isolated from the subglacial environment at RG. The RG5 genome encodes genes involved with pathways for the complete oxidation of S2O3 (2-), CO2 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicated that the energy required to synthesize a cell under oxygen- or nitrate-reducing conditions with S2O3 (2-) as the electron donor was lower at 5.1°C than 14.4°C, indicating that this organism is cold adapted. RG sediment-associated transcripts of soxB, which encodes a component of the S2O3 (2-)-oxidizing complex, were closely affiliated with soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. The consumption of S2O3 (2-) by RG5-like populations may accelerate abiotic FeS2 oxidation, thereby enhancing mineral weathering in the subglacial environment.

Sulfuriferula multivorans gen. nov., sp. nov., isolated from a freshwater lake, reclassification of 'Thiobacillus plumbophilus' as Sulfuriferula plumbophilus sp. nov., and description of Sulfuricellaceae fam. nov. and Sulfuricellales ord. nov.

A sulfur-oxidizing bacterium, strain TTN(T), was isolated from a Thioploca sample obtained from a freshwater lake in Japan. The isolate shared 97.1% 16S rRNA gene sequence similarity with an obligately aerobic chemolithoautotroph, 'Thiobacillus plumbophilus' Gro7(T). Cells were rods, motile, and Gram-stain-negative. The G+C content of the genomic DNA was approximately 66 mol%. Strain TTN(T) grew over a temperature range of 8-32 °C (optimum 22-25 °C), an NaCl concentration range of 0-133.3 mM (optimum 0-3.3 mM) and a pH range of 5.3-8.6 (optimum pH 6.4-7.0). Strain TTN(T) was facultatively anaerobic and could utilize nitrate as an electron acceptor. The isolate oxidized tetrathionate, thiosulfate and elemental sulfur as the sole energy sources for autotrophic growth, and could also grow heterotrophically on a number of organic substrates. Based on its phylogenetic and phenotypic properties, strain TTN(T) represents a novel species of a novel genus, for which the name Sulfuriferula multivorans gen. nov., sp. nov. is proposed. The type strain is TTN(T) ( =NBRC 110683(T) =DSM 29343(T)). Along with this, the reclassification of 'Thiobacillus plumbophilus' as Sulfuriferula plumbophilus sp. nov. (type strain Gro7(T) =NBRC 107929(T) =DSM 6690(T)) is proposed. Based on the data obtained in this study, we describe the designations Sulfuricellaceae fam. nov. and Sulfuricellales ord. nov.

Effect of S/N ratio on sulfide removal by autotrophic denitrification.

In this study a completely stirred tank reactor was used to study the effect of sulfide to nitrate (S/N) ratio on sulfide removal while nitrate was used as electron acceptor. Several S/N ratios were studied for this purpose ranging from 0.3 to 2.4 mol/mol. The complete sulfide removal was achieved when S/N ratio 0.85 mol/mol was used with the autotrophic denitrification efficiency up to 80 %. No nitrite accumulation was observed, and the main product of sulfide oxidation was sulfate. Dissimilatory nitrogen reduction to ammonia occurred and subsequently, elemental sulfur accumulated while S/N ratio was higher than 1.3 mol/mol. The specific autotrophic denitrification rates under S/N ratios 0.8 and 1.2 were 5 and 26 mg g(-1) h(-1) (N-NO3 (-), VSS), respectively. Thiobacillus denitrificans and Thiomicrospira denitrificans were detected in the reactor by fluorescent in situ hybridization, but their overall representation was not more than 5 % of the entire microbial populations.

Potentialities of coupling biological processes (biotrickler/biofilter) for the degradation of a mixture of sulphur compounds.

This study deals with the potential of biological processes combining a biotrickler and a biofilter to treat a mixture of sulphur-reduced compounds including dimethyl sulphide (DMS), dimethyl disulphide (DMDS) and hydrogen sulphide (H2S). As a reference, duplicated biofilters were implemented, and operating conditions were similar for all bioprocesses. The first step of this work was to determine the efficiency removal level achieved for each compound of the mixture and in a second step, to assess the longitudinal distribution of biodegradation activities and evaluate the total bacteria, Hyphomicrobium sp. and Thiobacillus thioparus densities along the bed height. A complete removal of hydrogen sulphide is reached at the start of the experiment within the first stage (biotrickler) of the coupling. This study highlighted that the coupling of a biotrickling filter and a biofilter is an interesting way to improve both removal efficiency levels (15-20% more) and kinetics of recalcitrant sulphur compounds such as DMS and DMDS. The total cell densities remained similar (around 1 × 10(10) 16S recombinant DNA (rDNA) copies g dry packing material) for duplicated biofilters and the biofilter below the biotrickling filter. The relative abundances of Hyphomicrobium sp. and T. thioparus have been estimated to an average of 10 ± 7.0 and 0.23 ± 0.07%, respectively, for all biofilters. Further investigation should allow achieving complete removal of DMS by starting the organic sulphur compound degradation within the first stage and surveying microbial community structure colonizing this complex system.

Isolation, identification and arsenic-resistance of Acidithiobacillus ferrooxidans HX3 producing schwertmannite.

Schwertmannite, a ubiquitous mineral present in iron oxyhydroxides formed in iron- and sulfate-rich acid media, favors incorporation of some toxic anions in its structure. We reported an iron-oxidizing bacterial strain HX3 from a municipal sludge that facilitates the formation of pure schwertmannite in cultures. Ferrous iron oxidation by the isolated strain HX3 was optimum at an initial pH of 2.0-3.3 and temperature of 28-35°C. Pure schwertmannite was found through bacterial oxidation of ferrous iron at an initial pH2.8 and temperature 28°C. Following 16S rDNA gene sequence analysis the bacterial strain HX3 was identified as Acidithiobacillus ferrooxidans. The arsenic-resistance A. ferrooxidans HX3 showed the potential of environmental application in arsenic removal from the As(III)- and iron-rich acid sulfate waters directly by As(III) adsorption or the formation of schwertmannite in the environment.

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201.

Strain THI201, a member of the alphaproteobacteria, is a novel thiocyanate (SCN(-))-degrading bacterium isolated from lake water enriched with potassium thiocyanate (KSCN). This bacterium carries the enzyme thiocyanate hydrolase (SCNase) that hydrolyses thiocyanate to carbonyl sulfide and ammonia. Characterization of both native and recombinant SCNase revealed properties different from known SCNases regarding subunit structure and thermostability: SCNase of strain THI201 was composed of a single protein and thermostable. We cloned and sequenced the corresponding gene and determined a protein of 457 amino acids of molecular mass 50 267 Da. Presence of a twin-arginine (Tat) signal sequence of 32 amino acids was found upstream of SCNase. The deduced amino acid sequence of SCNase showed 83% identity to that of a putative uncharacterized protein of Thiobacillus denitrificans ATCC 25259, but no significant identity to those of three subunits of SCNase from Thiobacillus thioparus strain THI115. The specific activities of native and recombinant enzyme were 0.32 and 4-15 µmol min(-1) (mg protein)(-1), respectively. The maximum activity of SCNase was found in the temperature range 30-70 °C. The thiocyanate-hydrolysing activity in both enzymes was decreased by freeze-thawing, although 25-100% of the activity of recombinant protein could be retrieved by treating the enzyme at 60 °C for 15 min. Furthermore, both native and recombinant enzymes retained the activity after pre-treatment of the protein solution at temperatures up to 70 °C.

Impact of different sulfur sources on the structure and function of sulfur autotrophic denitrification bacteria.

Nitrate pollution in surface water has become a significant environmental concern. Sulfur autotrophic denitrification (SAD) technology is gaining attention for its cost-effectiveness and efficiency in nitrate removal. This study aimed to investigate the structure and function of sulfur autotrophic denitrification microbial communities in systems using sodium thiosulfate (Group A) and elemental sulfur (Group B) as the sole electron donors. Metagenomic amplicon sequencing and physicochemical analysis were performed to examine the microbial communities. The results revealed that on day 13, the nitrate nitrogen removal rate in Group A was significantly higher (89.2%) compared to Group B (74.4%). The dominant genus in both Groups was Thiobacillus, with average abundances of 34.15% and 16.34% in Groups A and B, respectively. ß-diversity analysis based on species level showed significant differences in bacterial community structure between the two Groups (P < 0.001). Group A exhibited a greater potential for nitrate reduction and utilized both thiosulfate and elemental sulfur (P < 0.01) compared to Group B. This study provides a sufficient experimental basis for improving the start-up time and operating cost of SAD system through sulfur source switching and offers new prospects for in-depth mechanistic analysis.

Thiobacillus spp. and Anaeromyxobacter spp. mediate arsenite oxidation-dependent biological nitrogen fixation in two contrasting types of arsenic-contaminated soils.

As(III) oxidation-dependent biological nitrogen fixing (As-dependent BNF) bacteria use a novel biogeochemical process observed in tailings recently. However, our understanding of microorganisms responsible for As-dependent BNF is limited and whether such a process occurs in As-contaminated soils is still unknown. In this study, two contrasting types of soils (surface soils versus river sediments) heavily contaminated by As were selected to study the occurrence of As-dependent BNF. BNF was observed in sediments and soils amended with As(III), whereas no apparent BNF was found in the cultures without As(III). The increased abundances of the nitrogenase gene (nifH) and As(III) oxidation gene (aioA) suggest that an As-dependent BNF process was catalyzed by microorganisms harboring nifH and aioA. In addition, DNA-SIP demonstrated that Thiobacillus spp. and Anaeromyxobacter spp. were putative As-dependent BNF bacteria in As-contaminated soils and sediments, respectively. Metagenomic analysis further suggested that these taxa contained genes responsible for BNF, As(III) oxidation, and CO2 fixation, demonstrating their capability for serving as As-dependent BNF. These results indicated the occurrence of As-dependent BNF in various As-contaminated habitats. The contrasting geochemical conditions in different types of soil suggested that these conditions may enrich different As-dependent BNF bacteria (Thiobacillus spp. for soils and Anaeromyxobacter spp. for sediments).

Phylogenetic assessment of culture collection strains of Thiobacillus thioparus, and definitive 16S rRNA gene sequences for T. thioparus, T. denitrificans, and Halothiobacillus neapolitanus.

The 16S rRNA gene sequences of 12 strains of Thiobacillus thioparus held by different culture collections have been compared. A definitive sequence for the reference type strain (Starkey; ATCC 8158(T)) was obtained. The sequences for four examples of the Starkey type strain were essentially identical, confirming their sustained identity after passage through different laboratories. One strain (NCIMB 8454) was reassigned as a strain of Halothiobacillus neapolitanus, and a second (NCIMB 8349) was a species of Thermithiobacillus. These two strains have been renamed in their catalog by the National Collection of Industrial and Marine Bacteria. The 16S rRNA gene sequence of the type strain of Halothiobacillus neapolitanus (NCIMB 8539(T)) was determined and used to confirm the identity of other culture collection strains of this species. The reference sequences for the type strains of Thiobacillus thioparus and Halothiobacillus neapolitanus have been added to the online List of Prokaryotic Names with Standing in Nomenclature. Comparison of the 16S rRNA gene sequences available for strains of Thiobacillus denitrificans indicated that the sequence for the type strain (NCIMB 9548(T)) should always be used as the reference sequence for new and existing isolates.

The inhibitory effects and underlying mechanism of high ammonia stress on sulfide-driven denitrification process.

Sulfide-driven denitrification (SD) process has been widely studied for treating wastewater containing sulfate and ammonia in recent years. But influence of high ammonia stress on the SD process and microbial community remained unclear. In this work, a series of tests were conducted to investigate effects of different ammonia stress (200-3000 mg-total ammonia nitrogen (TAN)/L) on denitrification efficiency, byproduct accumulation and microbial community of the SD process. According to our results, the SD process was severely inhibited, and 32.67 ± 5.15 mg/L NO2--N was accumulated when ammonia stress reached 3000 mg TAN/L. But the inhibited SD process could recover in about 40 days when ammonia stress was decreased to 200 mg TAN/L. After analyzing the microbial community, Thiobacillus sp. (Thiobacillus sp. 65-29, Thiobacillus sp. SCN 64-317, Thiobacillus sp. 63-78 and Thiobacillus denitrificans) was confirmed as dominant bacteria responsible for the SD process. Further, expression of narG, napA, nirK and nirS were inhibited under high ammonia stress, thus making the SD process stuck in NO3- and NO2- reduction step. This study reveals the inhibitory effects of high ammonia stress on the SD process and its possible underlying mechanism with discussion in gene level.

Microbial community analysis in the autotrophic denitrification process using spent sulfidic caustic by denaturing gradient gel electrophoresis of PCR-amplified genes.

Spent sulfidic caustic (SSC) produced from petrochemical plants contains a high concentration of hydrogen sulfide and alkalinity, and some almost non-biodegradable organic compounds such as benzene, toluene, ethylbenzene and xylenes (BTEX). SSC is mainly incinerated with auxiliary fuel, leading to secondary pollution problems. The reuse of this waste is becoming increasingly important from economic and environmental viewpoints. To denitrify wastewater with low COD/N ratio, additional carbon sources are required. Thus, autotrophic denitrification has attracted increasing attention. In this study, SSC was injected as an electron donor for sulfur-based autotrophic denitrification in the modified Ludzack-Ettinger (MLE) process. The efficiencies of nitrification, COD, and total nitrogen (TN) removal were evaluated with varying SSC dosage. Adequate SSC injection exhibited stable autotrophic denitrification. No BTEX were detected in the monitored BTEX concentrations of the effluent. To analyse the microbial community of the MLE process, PCR-DGGE based on 16 S rDNA with EUB primers, TD primers and nirK gene with nirK primers was performed in order to elucidate the application of the MLE process to SSC.

Metagenomic insights into mixotrophic denitrification facilitated nitrogen removal in a full-scale A2/O wastewater treatment plant.

Wastewater treatment plants (WWTPs) are important for pollutant removal from wastewater, elimination of point discharges of nutrients into the environment and water resource protection. The anaerobic/anoxic/oxic (A2/O) process is widely used in WWTPs for nitrogen removal, but the requirement for additional organics to ensure a suitable nitrogen removal efficiency makes this process costly and energy consuming. In this study, we report mixotrophic denitrification at a low COD (chemical oxygen demand)/TN (total nitrogen) ratio in a full-scale A2/O WWTP with relatively high sulfate in the inlet. Nitrogen and sulfur species analysis in different units of this A2/O WWTP showed that the internal sulfur cycle of sulfate reduction and reoxidation occurred and that the reduced sulfur species might contribute to denitrification. Microbial community analysis revealed that Thiobacillus, an autotrophic sulfur-oxidizing denitrifier, dominated the activated sludge bacterial community. Metagenomics data also supported the potential of sulfur-based denitrification when high levels of denitrification occurred, and sulfur oxidation and sulfate reduction genes coexisted in the activated sludge. Although most of the denitrification genes were affiliated with heterotrophic denitrifiers with high abundance, the narG and napA genes were mainly associated with autotrophic sulfur-oxidizing denitrifiers. The functional genes related to nitrogen removal were actively expressed even in the unit containing relatively highly reduced sulfur species, indicating that the mixotrophic denitrification process in A2/O could overcome not only a shortage of carbon sources but also the inhibition by reduced sulfur of nitrification and denitrification. Our results indicate that a mixotrophic denitrification process could be developed in full-scale WWTPs and reduce the requirement for additional carbon sources, which could endow WWTPs with more flexible and adaptable nitrogen removal.