Sphingosine d18:1 promotes nonalcoholic steatohepatitis by inhibiting macrophage HIF-2α.

Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] is selected from NASH patients. So(d18:1) inhibits macrophage HIF-2α as a direct inhibitor and promotes the inflammatory factors secretion. Male macrophage-specific HIF-2α knockout and overexpression mice verified the protective effect of HIF-2α on NASH progression. Importantly, the HIF-2α stabilizer FG-4592 alleviates liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2α is a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2α in liver macrophages by suppressing the interaction of HIF-2α with ARNT, suggesting that macrophage HIF-2α may be a potential target for the treatment of NASH.

Gynostemma pentaphyllum Extract Alleviates NASH in Mice: Exploration of Inflammation and Gut Microbiota.

NASH (non-alcoholic steatohepatitis) is a severe liver disease characterized by hepatic chronic inflammation that can be associated with the gut microbiota. In this study, we explored the therapeutic effect of Gynostemma pentaphyllum extract (GPE), a Chinese herbal extract, on methionine- and choline-deficient (MCD) diet-induced NASH mice. Based on the peak area, the top ten compounds in GPE were hydroxylinolenic acid, rutin, hydroxylinoleic acid, vanillic acid, methyl vanillate, quercetin, pheophorbide A, protocatechuic acid, aurantiamide acetate, and iso-rhamnetin. We found that four weeks of GPE treatment alleviated hepatic confluent zone inflammation, hepatocyte lipid accumulation, and lipid peroxidation in the mouse model. According to the 16S rRNA gene V3-V4 region sequencing of the colonic contents, the gut microbiota structure of the mice was significantly changed after GPE supplementation. Especially, GPE enriched the abundance of potentially beneficial bacteria such as Akkerrmansia and decreased the abundance of opportunistic pathogens such as Klebsiella. Moreover, RNA sequencing revealed that the GPE group showed an anti-inflammatory liver characterized by the repression of the NF-kappa B signaling pathway compared with the MCD group. Ingenuity Pathway Analysis (IPA) also showed that GPE downregulated the pathogen-induced cytokine storm pathway, which was associated with inflammation. A high dose of GPE (HGPE) significantly downregulated the expression levels of the tumor necrosis factor-α (TNF-α), myeloid differentiation factor 88 (Myd88), cluster of differentiation 14 (CD14), and Toll-like receptor 4 (TLR4) genes, as verified by real-time quantitative PCR (RT-qPCR). Our results suggested that the therapeutic potential of GPE for NASH mice may be related to improvements in the intestinal microenvironment and a reduction in liver inflammation.

Lactobacillus herbarum sp. nov., a species related to Lactobacillus plantarum.

Strain TCF032-E4 was isolated from a traditional Chinese fermented radish. It shares >99% 16S rRNA sequence identity with L. plantarum, L. pentosus and L. paraplantarum. This strain can ferment ribose, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, trehalose and gentiobiose. It cannot ferment sucrose, which can be used by L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis, as well as most of the L. plantarum strains (88.7%). TCF032-E4 cannot grow at temperature above 32 °C. This strain shares 78.2-83.6% pheS (phenylalanyl-tRNA synthetase alpha subunit) and 89.5-94.9% rpoA (RNA polymerase alpha subunit) sequence identity with L. plantarum, L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis. These results indicate that TCF032-E4 represents a distinct species. This hypothesis was further confirmed by whole-genome sequencing and comparison with available genomes of related species. The draft genome size of TCF032-E4 is approximately 2.9 Mb, with a DNA G+C content of 43.5 mol%. The average nucleotide identity (ANI) between TCF032-E4 and related species ranges from 79.0 to 81.1%, the highest ANI value being observed with L. plantarum subsp. plantarum ATCC 14917T. A novel species, Lactobacillus herbarum sp. nov., is proposed with TCF032-E4T ( = CCTCC AB2015090T = DSM 100358T) as the type strain.

Draft Genome Sequence of Lactobacillus sp. Strain TCF032-E4, Isolated from Fermented Radish.

Here, we report the draft genome sequence of Lactobacillus sp. strain TCF032-E4 (= CCTCC AB2015090 = DSM 100358), isolated from a Chinese fermented radish. The total length of the 57 contigs is about 2.9 Mb, with a G+C content of 43.5 mol% and 2,797 predicted coding sequences (CDSs).

Metagenomic-based study of the phylogenetic and functional gene diversity in Galápagos land and marine iguanas.

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

Enrichment of specific bacterial and eukaryotic microbes in the rhizosphere of switchgrass (Panicum virgatum†L.) through root exudates.

Identification of microbes that actively utilize root exudates is essential to understand plant-microbe interactions. To identify active root exudate-utilizing microorganisms associated with switchgrass - a potential bioenergy crop - plants were labelled in situ with (13) CO2 , and 16S and 18S rRNA genes in the (13) C-labelled rhizosphere DNA were pyrosequenced. Multi-pulse labelling for 5 days produced detectable (13) C-DNA, which was well separated from unlabelled DNA. Methylibium from the order Burkholderiales were the most heavily labelled bacteria. Pythium, Auricularia and Galerina were the most heavily labelled eukaryotic microbes. We also identified a Glomus intraradices-like species; Glomus members are arbuscular mycorrhizal fungi that are able to colonize the switchgrass root. All of these heavily labelled microorganisms were also among the most abundant species in the rhizosphere. Species belonging to Methylibium and Pythium were the most heavily labelled and the most abundant bacteria and eukaryotes in the rhizosphere of switchgrass. Our results revealed that nearly all of the dominant rhizosphere bacterial and eukaryotic microbes were able to utilize root exudates. The enrichment of microbial species in the rhizosphere is selective and mostly due to root exudation, which functions as a nutrition source, promoting the growth of these microbes.

Strains in the genus Thauera exhibit remarkably different denitrification regulatory phenotypes.

Denitrifiers differ in how they handle the transition from oxic to anoxic respiration, with consequences for NO and N2O emissions. To enable stringent comparisons we defined parameters to describe denitrification regulatory phenotype (DRP) based on accumulation of NO2(-) , NO and N2O, oxic/anoxic growth and transcription of functional genes. Eight Thauera strains were divided into two distinct DRP types. Four strains were characterized by a rapid, complete onset (RCO) of all denitrification genes and no detectable nitrite accumulation. The others showed progressive onset (PO) of the different denitrification genes. The PO group accumulated nitrite, and no transcription of nirS (encoding nitrite reductase) was detected until all available nitrate (2 mM) was consumed. Addition of a new portion of nitrate to an actively denitrifying culture of a PO strain (T. terpenica) resulted in a transient halt in nitrite reduction, indicating that the electron flow was redirected to nitrate reductase. All eight strains controlled NO at nano-molar concentrations, possibly reflecting the importance of strict control for survival. Transient N2O accumulation differed by two orders of magnitude between strains, indicating that control of N2O is less essential. No correlation was seen between phylogeny (based on 16S rRNA and functional genes) and DRP.

Impact of different bioenergy crops on N-cycling bacterial and archaeal communities in soil.

Biomass production for bioenergy may change soil microbes and influence ecosystem properties. To explore the impact of different bioenergy cropping systems on soil microorganisms, the compositions and quantities of soil microbial communities (16S rRNA gene) and N-cycling functional groups (nifH, bacterial amoA, archaeal amoA and nosZ genes) were assessed under maize, switchgrass and Miscanthus x giganteus at seven sites representing a climate gradient (precipitation and temperature) in Illinois, USA. Overall, the site-to-site variation in community composition surpassed the variation due to plant type, and microbial communities under each crop did not converge on a 'typical' species assemblage. Fewer than 5% of archaeal amoA, bacterial amoA, nifH and nosZ OTUs were significantly different among these crops, but the largest differences observed at each site were found between maize and the two perennial grasses. Quantitative PCR revealed that the abundance of the nifH gene was significantly higher in the perennial grasses than in maize, and we also found significantly higher total N in the perennial grass soils than in maize. Thus, we conclude that cultivation of these perennial grasses, instead of maize, as bioenergy feedstocks can improve soil ecosystem nitrogen sustainability by increasing the population size of N-fixing bacteria.

Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq.

BACKGROUND: Clostridium beijerinckii is a prominent solvent-producing microbe that has great potential for biofuel and chemical industries. Although transcriptional analysis is essential to understand gene functions and regulation and thus elucidate proper strategies for further strain improvement, limited information is available on the genome-wide transcriptional analysis for C. beijerinckii. RESULTS: The genome-wide transcriptional dynamics of C. beijerinckii NCIMB 8052 over a batch fermentation process was investigated using high-throughput RNA-Seq technology. The gene expression profiles indicated that the glycolysis genes were highly expressed throughout the fermentation, with comparatively more active expression during acidogenesis phase. The expression of acid formation genes was down-regulated at the onset of solvent formation, in accordance with the metabolic pathway shift from acidogenesis to solventogenesis. The acetone formation gene (adc), as a part of the sol operon, exhibited highly-coordinated expression with the other sol genes. Out of the > 20 genes encoding alcohol dehydrogenase in C. beijerinckii, Cbei_1722 and Cbei_2181 were highly up-regulated at the onset of solventogenesis, corresponding to their key roles in primary alcohol production. Most sporulation genes in C. beijerinckii 8052 demonstrated similar temporal expression patterns to those observed in B. subtilis and C. acetobutylicum, while sporulation sigma factor genes sigE and sigG exhibited accelerated and stronger expression in C. beijerinckii 8052, which is consistent with the more rapid forespore and endspore development in this strain. Global expression patterns for specific gene functional classes were examined using self-organizing map analysis. The genes associated with specific functional classes demonstrated global expression profiles corresponding to the cell physiological variation and metabolic pathway switch. CONCLUSIONS: The results from this work provided insights for further C. beijerinckii strain improvement employing system biology-based strategies and metabolic engineering approaches.

Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq.

BACKGROUND: Clostridium beijerinckii is an important solvent producing microorganism. The genome of C. beijerinckii NCIMB 8052 has recently been sequenced. Although transcriptome structure is important in order to reveal the functional and regulatory architecture of the genome, the physical structure of transcriptome for this strain, such as the operon linkages and transcript boundaries are not well understood. RESULTS: In this study, we conducted a single-nucleotide resolution analysis of the C. beijerinckii NCIMB 8052 transcriptome using high-throughput RNA-Seq technology. We identified the transcription start sites and operon structure throughout the genome. We confirmed the structure of important gene operons involved in metabolic pathways for acid and solvent production in C. beijerinckii 8052, including pta-ack, ptb-buk, hbd-etfA-etfB-crt (bcs) and ald-ctfA-ctfB-adc (sol) operons; we also defined important operons related to chemotaxis/motility, transcriptional regulation, stress response and fatty acids biosynthesis along with others. We discovered 20 previously non-annotated regions with significant transcriptional activities and 15 genes whose translation start codons were likely mis-annotated. As a consequence, the accuracy of existing genome annotation was significantly enhanced. Furthermore, we identified 78 putative silent genes and 177 putative housekeeping genes based on normalized transcription measurement with the sequence data. We also observed that more than 30% of pseudogenes had significant transcriptional activities during the fermentation process. Strong correlations exist between the expression values derived from RNA-Seq analysis and microarray data or qRT-PCR results. CONCLUSIONS: Transcriptome structural profiling in this research provided important supplemental information on the accuracy of genome annotation, and revealed additional gene functions and regulation in C. beijerinckii.

Changes in N-transforming archaea and bacteria in soil during the establishment of bioenergy crops.

Widespread adaptation of biomass production for bioenergy may influence important biogeochemical functions in the landscape, which are mainly carried out by soil microbes. Here we explore the impact of four potential bioenergy feedstock crops (maize, switchgrass, Miscanthus X giganteus, and mixed tallgrass prairie) on nitrogen cycling microorganisms in the soil by monitoring the changes in the quantity (real-time PCR) and diversity (barcoded pyrosequencing) of key functional genes (nifH, bacterial/archaeal amoA and nosZ) and 16S rRNA genes over two years after bioenergy crop establishment. The quantities of these N-cycling genes were relatively stable in all four crops, except maize (the only fertilized crop), in which the population size of AOB doubled in less than 3 months. The nitrification rate was significantly correlated with the quantity of ammonia-oxidizing archaea (AOA) not bacteria (AOB), indicating that archaea were the major ammonia oxidizers. Deep sequencing revealed high diversity of nifH, archaeal amoA, bacterial amoA, nosZ and 16S rRNA genes, with 229, 309, 330, 331 and 8989 OTUs observed, respectively. Rarefaction analysis revealed the diversity of archaeal amoA in maize markedly decreased in the second year. Ordination analysis of T-RFLP and pyrosequencing results showed that the N-transforming microbial community structures in the soil under these crops gradually differentiated. Thus far, our two-year study has shown that specific N-transforming microbial communities develop in the soil in response to planting different bioenergy crops, and each functional group responded in a different way. Our results also suggest that cultivation of maize with N-fertilization increases the abundance of AOB and denitrifiers, reduces the diversity of AOA, and results in significant changes in the structure of denitrification community.

Fecal microbiota of calves in the clinical setting: effect of penicillin treatment.

The effect of parenteral penicillin treatment on the intestinal microbiota was determined by monitoring the phenotypic antimicrobial resistance among Escherichia coli in 19 calves (15 calves received treatment and four calves were healthy controls) and by examining changes in the fecal microbial community structure using molecular fingerprinting techniques in a subset of eight calves (five treated calves and three control calves). After five days of penicillin treatment an increased resistance to multiple unrelated antimicrobial agents, including non-ß-lactams, was seen in E. coli from treated calves, and this was not seen in the controls. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) revealed that penicillin treatment causes a significant variation in the microbial structure within an individual calf. The study shows that parenteral administration of penicillin has an impact on the composition of the fecal microbiota in calves, and on the antimicrobial resistance pattern of their fecal E. coli.

Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrous [corrected] oxide reductase in Paracoccus denitrificans.

Denitrification in soil is a major source of atmospheric N(2)O. Soil pH appears to exert a strong control on the N(2)O/N(2) product ratio (high ratios at low pH), but the reasons for this are not well understood. To explore the possible mechanisms involved, we conducted an in-depth investigation of the regulation of denitrification in the model organism Paracoccus denitrificans during transition to anoxia both at pH 7 and when challenged with pHs ranging from 6 to 7.5. The kinetics of gas transformations (O(2), NO, N(2)O, and N(2)) were monitored using a robotic incubation system. Combined with quantification of gene transcription, this yields high-resolution data for direct response patterns to single factors. P. denitrificans demonstrated robustly balanced transitions from O(2) to nitric oxide-based respiration, with NO concentrations in the low nanomolar range and marginal N(2)O production at an optimal pH of 7. Transcription of nosZ (encoding N(2)O reductase) preceded that of nirS and norB (encoding nitrite and NO reductase, respectively) by 5 to 7 h, which was confirmed by observed reduction of externally supplied N(2)O. Reduction of N(2)O was severely inhibited by suboptimal pH. The relative transcription rates of nosZ versus nirS and norB were unaffected by pH, and low pH had a moderate effect on the N(2)O reductase activity in cells with a denitrification proteome assembled at pH 7. We thus concluded that the inhibition occurred during protein synthesis/assembly rather than transcription. The study shed new light on the regulation of the environmentally essential N(2)O reductase and the important role of pH in N(2)O emission.

Structural differentiation of bacterial communities in indole-degrading bioreactors under denitrifying and sulfate-reducing conditions.

The acclimated, anaerobic microbial community is an efficient method for indole-containing wastewater treatment. However, our understanding of the diversity of indole-degrading communities is still limited. We investigated two anaerobic, indole-decomposing microbial communities under both denitrifying and sulfate-reducing conditions. Utilizing a near full-length 16S rRNA gene clone library, the most dominant bacteria in the denitrifying bioreactor identified was ß-proteobacteria. Among these, bacteria from genera Alicycliphilus, Acaligenes and Thauera were abundant and thought responsible for indole degradation. However, in the sulfate-reducing bioreactor, Clostridia and Actinobacteria were the dominant bacterial class found and likely the main degrading species. Microbial communities in these bioreactors shared only two operational taxonomic units (OTUs). Differences in the electron acceptors of denitrification or sulfate reduction may be responsible for the higher indole removal capacity in the denitrifying bioreactor (80%) than the capacity in the sulfate-reducing bioreactor (52%). This study is the first detailed analysis of an anaerobic indole-degrading community.

Versatile aromatic compound-degrading capacity and microdiversity of Thauera strains isolated from a coking wastewater treatment bioreactor.

Bacteria of the Thauera genus have been described as important aromatic compound degraders and have attracted increased attention. In this study, three Thauera strains (Q4, Q20-C, and 3-35) were isolated from a coking wastewater treatment plant (WWTP) with a high abundance of Thauera. The 16S rRNA, nitrite reductase, and phenol hydroxylase (LmPH) genes and pollutant-degrading capacity of these strains were characterized and compared. Their 16S rRNA gene sequences were identical, but the genomic structures differed, as demonstrated by distinct enterobacterial repetitive intergenic consensus sequence PCR profiles with a similarity of less than 0.65. The analysis of degradation of coking wastewater by these strains showed that most of the main organic pollutants--phenol, methylphenol, and indole, but not quinoline--were degraded under aerobic conditions. These strains contained different LmPHs genes and showed different phenol degradation rates (Q4 > 3-35 > Q20-C). The presence of a microdiversity of Thauera spp. implies the existence of various finely differentiated niches in the industrial WWTP. The capacity of the Thauera strains to degrade a wide spectrum of aromatic compounds suggests their potential in bioremediation applications targeting aromatic pollutant-containing wastewater.

Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice.

Both genetic variations and diet-disrupted gut microbiota can predispose animals to metabolic syndromes (MS). This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota and modulating MS-relevant phenotypes in mice. Together with its wild-type (Wt) counterpart, the Apoa-I knockout mouse, which has impaired glucose tolerance (IGT) and increased body fat, was fed a high-fat diet (HFD) or normal chow (NC) diet for 25 weeks. DNA fingerprinting and bar-coded pyrosequencing of 16S rRNA genes were used to profile gut microbiota structures and to identify the key population changes relevant to MS development by Partial Least Square Discriminate Analysis. Diet changes explained 57% of the total structural variation in gut microbiota, whereas genetic mutation accounted for no more than 12%. All three groups with IGT had significantly different gut microbiota relative to healthy Wt/NC-fed animals. In all, 65 species-level phylotypes were identified as key members with differential responses to changes in diet, genotype and MS phenotype. Most notably, gut barrier-protecting Bifidobacterium spp. were nearly absent in all animals on HFD, regardless of genotype. Sulphate-reducing, endotoxin-producing bacteria of the family, Desulfovibrionaceae, were enhanced in all animals with IGT, most significantly in the Wt/HFD group, which had the highest calorie intake and the most serious MS phenotypes. Thus, diet has a dominating role in shaping gut microbiota and changes of some key populations may transform the gut microbiota of Wt animals into a pathogen-like entity relevant to development of MS, despite a complete host genome.

Development of group-specific PCR-DGGE fingerprinting for monitoring structural changes of Thauera spp. in an industrial wastewater treatment plant responding to operational perturbations.

A Thauera-specific nested-PCR denaturing gradient gel electrophoresis (DGGE) method was developed, and its usefulness was demonstrated by monitoring the structural shifts of Thauera spp. in an anaerobic-anoxic-oxic fixed-biofilm coking wastewater treatment plant (WWTP) responding to operational perturbations. The specificity of the PCR method was confirmed by the fact that all 16 S rRNA gene sequences, cloned from the amplicons of a biofilm sample, belonged to Thauera genus. 16 S rRNA gene V3 region was then amplified from the first round Thauera-specific PCR product and applied for DGGE analysis. All Thauera clones, with 13 different V3 regions, migrated into 10 positions on DGGE gel, which demonstrated the high resolution of this DGGE method. When the WWTP experienced a gradual deterioration in chemical oxygen demand (COD) removal function due to a mechanical failure of the recirculation pump, biofilm samples were collected from the reactor and analyzed by this method. Principal component analysis (PCA) of the DGGE fingerprinting data showed that the composition of Thauera group exhibited a time related trajectory when the plant's COD removal rate decreased from 84.1+/-2.7% in the first 4 weeks to less than 75% at week 5 and 6, suggesting a concomitant shift of Thauera composition and the system's COD removal function. This group-specific PCR DGGE fingerprinting technology has the potential to be a profiling tool for monitoring structural shifts of Thauera spp. in industrial WWTPs.

Functional robustness and gene pools of a wastewater nitrification reactor: comparison of dispersed and intact biofilms when stressed by low oxygen and low pH.

The functional robustness of biofilms in a wastewater nitrification reactor, and the gene pools therein, were investigated. Nitrosomonas and Nitrosospira spp. were present in similar amounts (cloning-sequencing of ammonia-oxidizing bacteria 16S rRNA gene), and their estimated abundance (1.1 x 10(9) cells g(-1) carrier material, based on amoA gene real-time PCR) was sufficient to explain the observed nitrification rates. The biofilm also had a diverse community of heterotrophic denitrifying bacteria (cloning-sequencing of nirK). Anammox 16S rRNA genes were detected, but not archaeal amoA. Dispersed biofilms (DB) and intact biofilms (IB) were incubated in gas-tight reactors at different pH levels (4.5 and 5.5 vs. 6.5) while monitoring O(2) depletion and concentrations of NO, N(2)O and N(2) in the headspace. Nitrification was severely reduced by suboptimal O(2) concentrations (10-100 microM) and low pH (IB was more acid tolerant than DB), but the N(2)O/NO(3)(-) product ratio of nitrification remained low (<10(-3)). The NO(2)(-) concentrations during nitrification were generally 10 times higher in DB than in IB. Transient NO and N(2)O accumulation at the onset of denitrification was 10-10(3) times higher in DB than in IB (depending on the pH). The contrasting performance of DB and IB suggests that the biofilm structure, with anoxic/micro-oxic zones, helps to stabilize functions during anoxic spells and low pH.

[Specific-PCR and denaturing gradient gel electrophoresis assistant isolation of Thauera spp. from a coking wastewater treatment plant].

OBJECTIVE: We used specific-PCR and denaturing gradient gel electrophoresis (DGGE) to isolate Thauera spp. from a coking wastewater treatment plant. METHODS AND RESULTS: To isolate Thauera from the denitrifying bioreactor of a coking wastewater treatment, biofilm was inoculated to six different media and cultured them under both aerobic and anaerobic conditions. We then compared the composition of Thauera spp. using Thauera-specific PCR-DGGE method. The media 1/10 NB and MMQ which grew higher diverse Thauera spp. and fewer colonies, were used to isolate Thauera sp. under aerobic condition. The colonies were then screened by Thauera-specific PCR. The purity of the colonies that shown Thauera-specific PCR positive signal was then checked by DGGE. The colonies with multiple species were further streaked on different media. DGGE analysis showed that Thauera in colony Q20 was enriched in medium MMP. The colony was finally purified by streaking on MMP medium for several rounds. The composition of the colonies were tracked by Thauera-specific PCR and DGGE at each step. Finally, three strains were purified, which were identified as Thauera sp. according to their 16S rRNA gene sequences. CONCLUSION: Guiding with specific biomarker, the efficiency and sensitivity of bacteria isolation can be largely improved.