Thauera sp. for efficient nitrate removal in continuous denitrifying moving bed biofilm reactor.

Thauera is the most widely found dominant denitrifying genus in wastewater. In earlier study, MBBR augmented with a specially developed denitrifying five-membered bacterial consortium (DC5) where Thauera was found to be the most abundant and persistent genus. Therefore, to check the functional potential of Thauera in the removal of nitrate-containing wastewater in the present study Thauera sp.V14 one of the member of the consortium DC5 was used as the model organism. Thauera sp.V14 exhibited strong hydrophobicity, auto-aggregation ability, biofilm formation and denitrification ability, which indicated its robust adaptability short colonization and nitrate removal efficiency. Continuous reactor studies with Thauera sp.V14 in 10 L dMBBR showed 91% of denitrification efficiency with an initial nitrate concentration of 620 mg L-1 within 3 h of HRT. Thus, it revealed that Thauera can be employed as an effective microorganism for nitrate removal from wastewater based on its performance in the present studies.

Bioaugmentation of Thauera mechernichensis TL1 for enhanced polyhydroxyalkanoate production in mixed microbial consortia for wastewater treatment.

Polyhydroxyalkanoate (PHA) is a fully biodegradable bioplastic. To foster a circular economy, the integration of PHA production into wastewater treatment facilities can be accomplished using mixed microbial consortia. The effectiveness of this approach relies greatly on the enrichment of PHA-accumulating microorganisms. Hence, our study focused on bioaugmenting Thauera mechernichensis TL1 into mixed microbial consortia with the aim of enriching PHA-accumulating microorganisms and enhancing PHA production. Three sequencing batch reactors-SBRctrl, SBR2.5%, and SBR25%-were operated under feast/famine conditions. SBR2.5% and SBR25% were bioaugmented with T. mechernichensis TL1 at 2.5%w/w of mixed liquor volatile suspended solids (MLVSS) and 25%w/w MLVSS, respectively, while SBRctrl was not bioaugmented. SBR2.5% and SBR25% achieved maximum PHA accumulation capacities of 56.3 %gPHA/g mixed liquor suspended solids (MLSS) and 50.2 %gPHA/gMLSS, respectively, which were higher than the 25.4 %gPHA/gMLSS achieved by SBRctrl. The results of quantitative polymerase chain reaction targeting the 16S rRNA gene specific to T. mechernichensis showed higher abundances of T. mechernichensis in SBR2.5% and SBR25% compared with SBRctrl in the 3rd, 17th, and 31st cycles. Fluorescence in situ hybridization, together with fluorescent staining of PHA with Nile blue A, confirmed PHA accumulation in Thauera spp. The study demonstrated that bioaugmentation of T. mechernichensis TL1 at 2.5%w/w MLVSS is an effective strategy to enhance PHA accumulation and facilitate the enrichment of PHA-accumulating microorganisms in mixed microbial consortia. The findings could contribute to the advancement of PHA production from wastewater, enabling the transformation of wastewater treatment plants into water and resource recovery facilities.

Synergistic and additive interactions of Shewanella sp., Pseudomonas sp. and Thauera sp. with chlorantraniliprole and emamectin benzoate for controlling Spodoptera litura (Fabricius).

The imprudent use of insecticides causes the development of resistance in insect pest populations, contamination of the environment, biological imbalance and human intoxication. The use of microbial pathogens combined with insecticides has been proposed as an alternative strategy for insect pest management. This IPM approach may offer effective ways to control pests, in addition to lowering the risk of chemical residues in the environment. Spodoptera litura (Fabricius) is a major pest of many crops like cotton, maize, tobacco, cauliflower, cabbage, and fodder crops globally. Here, we evaluated the combined effects of new chemistry insecticides (chlorantraniliprole and emamectin benzoate) and entomopathogenic bacterial strains, Shewanella sp. (SS4), Thauera sp. (M9) and Pseudomonas sp. (EN4) against S. litura larvae inducing additive and synergistic interactions under laboratory conditions. Both insecticides produced higher larval mortality when applied in combination with bacterial isolates having maximum mortality of 98 and 96% with LC50 of chlorantraniliprole and emamectin benzoate in combination with LC50 of Pseudomonas sp. (EN4) respectively. The lower concentration (LC20) of both insecticides also induced synergism when combined with the above bacterial isolates providing a valuable approach for the management of insect pests. The genotoxic effect of both the insecticides was also evaluated by conducting comet assays. The insecticide treatments induced significant DNA damage in larval hemocytes that further increased in combination treatments. Our results indicated that combined treatments could be a successful approach for managing S. litura while reducing the inappropriate overuse of insecticides.

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.

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids.

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.

The nitrogen removal performance and microbial community on mixotrophic denitrification process.

Sulfur autotrophic denitrifiers and heterotrophic denitrifiers widely exist in aquatic ecosystem, however, the response of sulfide to the microbial community structure in mixotrophic denitrification ecosystem is unknown yet. In this study, the denitrification performance and microbial community were explored by changing the molar ratio of influent C/N/S. From the level of genus, the joint action of Thauera, Pacacoccus, Fusibacter Pseudoxanthomonas, Thiobacillus, Sulfurovum and Sulfurimonas brought about the efficient denitrification performance in the mixotrophic system. Thauera increased from from 0.97% to more than 13%, and the relative abundances of Thiobacillus and Sulfurimonas were about 4.14% and 3.89% separately after adding S2-. The results of this study showed that the denitrification performance could be indeed intensified in the mixotrophic system, among which provided a theoretical basis for establishing an efficient biological nitrogen removal system.

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

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

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.

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

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

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

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

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.

Omics and mechanistic insights into di-(2-ethylhexyl) phthalate degradation in the O2-fluctuating estuarine sediments.

Di-(2-ethylhexyl) phthalate (DEHP) represents the most used phthalate plasticizer with an annual production above the millions of tons worldwide. Due to its inadequate disposal, outstanding chemical stability, and extremely low solubility (3 mg/L), endocrine-disrupting DEHP often accumulates in urban estuarine sediments at concentrations above the predicted no-effect concentration (20-100 mg/kg). Our previous study suggested that microbial DEHP degradation in estuarine sediments proceeds synergistically where DEHP side-chain hydrolysis to form phthalic acid represents a bottleneck. Here, we resolved this bottleneck and deconstructed the microbial synergy in O2-fluctuating estuarine sediments. Metagenomic analysis and RNA sequencing suggested that orthologous genes encoding extracellular DEHP hydrolase NCU65476 in Acidovorax sp. strain 210-6 are often flanked by the co-expressed composite transposon and are widespread in aquatic environments worldwide. Therefore, we developed a turbidity-based microplate assay to characterize NCU65476. The optimized assay conditions (with 1 mM Ca2+ and pH 6.0) increased the DEHP hydrolysis rate by a factor of 10. Next, we isolated phthalic acid-degrading Hydrogenophaga spp. and Thauera chlorobenzoica from Guandu estuarine sediment to study the effect of O2(aq) on their metabolic synergy with strain 210-6. The results of co-culture experiments suggested that after DEHP side-chain hydrolysis by strain 210-6, phthalic acid can be degraded by Hydrogenophaga sp. when O2(aq) is above 1 mg/L or degraded by Thauera chlorobenzoica anaerobically. Altogether, our data demonstrates that DEHP could be degraded synergistically in estuarine sediments via divergent pathways responding to O2 availability. The optimized conditions for NCU65476 could facilitate the practice of DEHP bioremediation in estuarine sediments.

Inactive pseudoenzyme subunits in heterotetrameric BbsCD, a novel short-chain alcohol dehydrogenase involved in anaerobic toluene degradation.

Anaerobic toluene degradation proceeds by fumarate addition to produce (R)-benzylsuccinate as first intermediate, which is further degraded via ß-oxidation by five enzymes encoded in the conserved bbs operon. This study characterizes two enzymes of this pathway, (E)-benzylidenesuccinyl-CoA hydratase (BbsH), and (S,R)-2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD) from Thauera aromatica. BbsH, a member of the enoyl-CoA hydratase family, converts (E)-benzylidenesuccinyl-CoA to 2-(α-hydroxybenzyl)succinyl-CoA and was subsequently used in a coupled enzyme assay with BbsCD, which belongs to the short-chain dehydrogenases/reductase (SDR) family. The BbsCD crystal structure shows a C2-symmetric heterotetramer consisting of BbsC2 and BbsD2 dimers. BbsD subunits are catalytically active and capable of binding NAD+ and substrate, whereas BbsC subunits represent built-in pseudoenzyme moieties lacking all motifs of the SDR family required for substrate binding or catalysis. Molecular modeling studies predict that the active site of BbsD is specific for conversion of the (S,R)-diastereomer of 2-(α-hydroxybenzyl)succinyl-CoA to (S)-2-benzoylsuccinyl-CoA by hydride transfer to the re-face of nicotinamide adenine dinucleotide (NAD)+ . Furthermore, BbsC subunits are not engaged in substrate binding and merely serve as scaffold for the BbsD dimer. BbsCD represents a novel clade of related enzymes within the SDR family, which adopt a heterotetrameric architecture and catalyze the ß-oxidation of aromatic succinate adducts.

Diversified metabolism makes novel Thauera strain highly competitive in low carbon wastewater treatment.

Thauera, as one of the core members of wastewater biological treatment systems, plays an important role in the process of nitrogen and phosphorus removal from low-carbon source sewage. However, there is a lack of systematic understanding of Thauera's metabolic pathway and genomics. Here we report on the newly isolated Thauera sp. RT1901, which is capable of denitrification using variety carbon sources including aromatic compounds. By comparing the denitrification processes under the conditions of insufficient, adequate and surplus carbon sources, it was found that strain RT1901 could simultaneously use soluble microbial products (SMP) and extracellular polymeric substances (EPS) as electron donors for denitrification. Strain RT1901 was also found to be a denitrifying phosphate accumulating bacterium, able to use nitrate, nitrite, or oxygen as electron acceptors during poly-ß-hydroxybutyrate (PHB) catabolism. The annotated genome was used to reconstruct the complete nitrogen and phosphorus metabolism pathways of RT1901. In the process of denitrifying phosphorus accumulation, glycolysis was the only pathway for glycogen metabolism, and the glyoxylic acid cycle replaced the tricarboxylic acid cycle (TCA) to supplement the reduced energy. In addition, the abundance of conventional phosphorus accumulating bacteria decreased significantly and the removal rates of total nitrogen (TN) and chemical oxygen demand (COD) increased after the addition of RT1901 in the low carbon/nitrogen (C/N) ratio of anaerobic aerobic anoxic-sequencing batch reactor (AOA-SBR). This research indicated that the diverse metabolic capabilities of Thauera made it more competitive than other bacteria in the wastewater treatment system.

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

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

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

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

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

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

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Functional Species and Metabolic Characteristics of an Enriched Denitratation Community.

Nitrite supply for mainstream anammox via denitratation has attracted increasing attention. The functional species responsible for denitratation and their metabolic characteristics were unravelled in this study. A highly stable denitratation community was enriched from activated sludge by combined control of C/N and pH. Nitrite accumulation and nitrate removal efficiencies were both higher than 80% during long-term operation (>100 d). The genotypic complete denitrifier Thauera aminoaromatica MZ1T was identified to be mainly responsible for acetate consumption, polyhydroxybutyrate (PHB) accumulation, and nitrate reduction. The presence of nitrate restricted the transcription and electron allocation of downstream denitrifying enzymes due to low expression of their electron transport modules (cytochrome bc1 and cytochrome c). Metabolic reconstruction of this strain indicated that the reducing power generated via the tricarboxylic acid (TCA) cycle was mainly provided for PHB synthesis and nitrate reduction in the exogenous feast phase. After the depletion of acetate, PHB was degraded and then entered the TCA cycle, providing reducing power for nitrate reduction. This allocation strategy of reducing power with priority given to carbon storage instead of nitrite reduction might favor their survival in oligotrophic and weak alkaline habitats. These results updated our understanding of the causes underlying nitrite accumulation and its physiological benefits.

[Rapid Start-up and Stability of Partial Denitrification Based on Different Waste Sludge Sources].

To explore the feasibility of the rapid start-up of partial denitrification and the stable accumulation of NO2--N in different waste sludge sources, three identical SBR reactors (S1, S2, and S3) were inoculated respectively with sludge discharged from a laboratory municipal wastewater denitrifying phosphorus removal system, surplus sludge from a municipal wastewater treatment plant, and river sediment sludge. The characteristics of the partial denitrification start-up and NO2--N accumulation were compared, and the partial denitrification activity of the system or NO3--N→NO2--N transformation performance were investigated by analyzing the characteristics of the functional bacteria genera of the reactor from the perspective of microbiology. The results showed that all three SBR partial denitrification reactors could be launched successfully in a short time with sodium acetate as the sole carbon source, under a high alkalinity, and by using a suitable COD/NO3--N ratio. The average NO3--N→NO2--N transformation ratio of the system was ranked as:S1 > S2 > S3 (75.92% > 73.36% > 69.90%). It was found that S1 and S2 had different degrees of partial denitrification performance deterioration under a continuous low temperature, but that S3 could maintain a good NO2--N accumulation performance. High throughput sequencing showed that Proteobacteria and Bacteroidetes were dominant in the partial denitrification system, and that the abundance of Thauera was significantly different in the three PD reactors:S3 > S1 > S2 (25.09% > 4.71% > 3.60%), thus indicating that S3 had stable and efficient NO2--N accumulation performance and that a high abundance of Thauera might play a significant role in maintaining low temperature partial denitrification activity.