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Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-8004-8) contains supplementary material, which is available to authorized users.

12 Páginas, Aloreña de Málaga is a table olive especially characterised by its natural freshness and short shelf-life. In this work, we applied a metataxonomic approach to unravel the microbial diversity of bacterial and fungi populations through the shelf-life of traditionally packed Aloreña de Málaga. A significant increase in lactic acid bacteria and mesophilic aerobic populations was observed during shelf-life, reaching the maximum population levels (4–5 log10 CFU) at the end of the study (260 days). On the contrary, a rapid reduction in yeast and mould populations was reported. The use of a metataxonomic analysis based on the amplification of 16S (bacteria) and internal transcribed spacer (ITS) region (fungi) regions revealed a low diversity for both microbial groups. Lactiplantibacillus (65.05 ± 8.65% in brine vs. 58.70 ± 15.70% in fruit), Pediococcus (28.17 ± 7.36% in brine vs. 27.20 ± 15.95% in fruit), and Celerinatantimonas (4.64 ± 1.08% in brine vs. 11.82 ± 18.17% in fruit) were the main genera found among bacteria, and an increase in Lactiplantibacillus and a reduction in Celerinatantimonas populations during the shelf-life were observed. On the other hand, Citeromyces was the dominant fungi genus (54.11 ± 2.00% in brine vs. 50.91 ± 16.14% in fruit), followed by Candida (8.80 ± 2.57% in brine vs. 12.32 ± 8.61% in fruit) and Penicillium (6.48 ± 1.87% vs. 8.48 ± 4.43% in fruit). No food-borne pathogen genera were detected in any of the samples analysed, indicating the high level of food safety found in this ready-to-eat fermented vegetable. Data obtained in this work will help in the design of new strategies for the control of microbial populations during the shelf-life of Aloreña de Málaga., This research was funded by the Spanish Government through the TOBE project (RTI2018-100883-B-I00).

Biostimulation is widely used to enhance reductive dechlorination of chlorinated ethenes in contaminated aquifers. However, the knowledge on corresponding biogeochemical responses is limited. In this study, glycerol was injected in an aquifer contaminated with cis-dichloroethene (cDCE), and geochemical and microbial shifts were followed for 265 days. Consistent with anoxic conditions and sulfate reduction after biostimulation, MiSeq 16S rRNA gene sequencing revealed temporarily increased relative abundance of Firmicutes, Bacteriodetes and sulfate reducing Deltaproteobacteria. In line with 13 C cDCE enrichment and increased Dehalococcoides mccartyi (Dcm) numbers, dechlorination was observed toward the end of the field experiment, albeit being incomplete with accumulation of vinyl chloride. This was concurrent with (i) decreased concentrations of dissolved organic carbon (DOC), reduced relative abundances of fermenting and sulfate reducing bacteria that have been suggested to promote Dcm growth by providing electron donor (H2 ) and essential corrinoid cofactors, (ii) increased sulfate concentration and increased relative abundance of Epsilonproteobacteria and Deferribacteres as putative oxidizers of reduced sulfur compounds. Strong correlations of DOC, relative abundance of fermenters and sulfate reducers, and dechlorination imply the importance of syntrophic interactions to sustain robust dechlorination. Tracking microbial and environmental parameters that promote/preclude enhanced reductive dechlorination should aid development of sustainable bioremediation strategies.

Background Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes. However, several studies have shown that the methodology rather than the biological variation is responsible for the observed sample composition and distribution. This compromises true meta-analyses, although this fact is often disregarded. Results To facilitate true meta-analysis of microbiome studies, we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities and benchmarked against QIIME as the currently most frequently used pipeline. The microbial composition of 49 independently amplified mock samples was characterized by sequencing two variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform. This allowed evaluating important factors of technical bias in taxonomic classification: 1) run-to-run sequencing variation, 2) PCR–error, and 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 96%. On average 99.94% and 92.02% of the reads could be assigned to at least family or genus level, respectively, while assignment to ‘spurious genera’ represented on average only 0.02% of the reads per sample. Analysis of α- and β-diversity confirmed conclusions guided by biology rather than the aforementioned methodological aspects, which was not the case when samples were analysed using QIIME. Conclusions Different biological outcomes are commonly observed due to 16S rRNA region-specific performance. NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies, diminishing their impact and providing accurate qualitative and quantitative representation of the true sample composition. This will improve comparability between studies and facilitate efforts towards standardization.

The microbiota of the mammalian gut is a complex ecosystem, the composition of which is greatly influenced by host genetics and environmental factors. In this study, we aim to investigate the influence of occupancy (a geographical area of habitation), species, age and sex on intestinal microbiota composition of the three lemur species: Eulemur fulvus, E. rubriventer and E. rufifrons. Faecal samples were collected from a total of 138 wild lemurs across Madagascar, and microbial composition was determined using next-generation sequencing of PCR-amplified 16S rRNA gene fragments. Consistent with reports from other primate species, the predominant phyla were Firmicutes (43 ± 6.4% [s.d.]) and Bacteroidetes (30.3 ± 5.3%). The microbial composition was strongly associated with occupancy in the E. fulvus population, with up to 19.9% of the total variation in microbial composition being explained by this factor. In turn, geographical differences observed in faecal microbiota of sympatric lemur species were less pronounced, as was the impact of the factors sex and age. Our findings showed that among the studied factors occupancy had the strongest influence on intestinal microbiota of congeneric lemur species. This suggests adaptation of microbiota to differences in forest composition, climate variations and correspondingly available diet in different geographical locations of Madagascar.

Background Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. Results The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. Conclusion This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas. Electronic supplementary material The online version of this article (10.1186/s40168-018-0607-0) contains supplementary material, which is available to authorized users.

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01341/full#supplementary-material, Metals play an important role in microbial metabolism by acting as cofactors for many enzymes. Supplementation of biological processes with metals may result in improved performance, but high metal concentrations are often toxic to microorganisms. In this work, methanogenic enrichment cultures growing on H2/CO2 or acetate were supplemented with trace concentrations of nickel (Ni) and cobalt (Co), but no significant increase in methane production was observed in most of the tested conditions. However, high concentrations of these metals were detrimental to methanogenic activity of the cultures. Cumulative methane production (after 6 days of incubation) from H2/CO2 was 40% lower in the presence of 8 mM of Ni or 30 mM of Co, compared to controls without metal supplementation. When acetate was used as substrate, cumulative methane production was also reduced: by 18% with 8 mM of Ni and by 53% with 30 mM of Co (after 6 days of incubation). Metal precipitation with sulphide was further tested as a possible method to alleviate metal toxicity. Anaerobic sludge was incubated with Co (30 mM) and Ni (8 mM) in the presence of sulphate or sulphide. The addition of sulphide helped to mitigate the toxic effect of the metals. Methane production from H2/CO2 was negatively affected in the presence of sulphate, possibly due to competition of hydrogenotrophic methanogens by sulphate-reducing bacteria. However, in the enrichment cultures growing on acetate, biogenically produced sulphide had a positive effect and more methane was produced in these incubations than in similar assays without sulphate addition. The outcome of competition between methanogens and sulphate-reducing bacteria is a determinant factor for the success of using biogenic sulphide as detoxification method., The research was financially supported by the People Program (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013 under REA agreement 289193. Research of AS and DS is supported by a ERC grant (project 323009) of the European Union Seventh Framework Program FP7 and a Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)., info:eu-repo/semantics/publishedVersion

Hyporheic zones mediate vinyl chloride (VC) biodegradation in groundwater discharging into surface waters. At the oxic/anoxic interface (OAI) of hyporheic zones subjected to redox oscillations, VC is degraded via coexisting aerobic ethenotrophic and anaerobic reductive dechlorination pathways. However, the identity of aerobic VC degradation pathways (cometabolic vs metabolic) and their interactions with reductive dechlorination in relation to riverbed sediment geochemistry remain ill-defined. We addressed this using microcosms containing OAI sediments incubated under fluctuating oxic/anoxic atmosphere. Under oxic atmosphere, aerobic metabolic VC oxidation was absent in sediments with high total organic carbon (TOC) and VC was reductively dechlorinated to ethene. Ethene was oxidized by ethenotrophs that can degrade VC cometabolically. Contrastingly, VC was metabolically oxidized by ethenotrophs in low-TOC sediments with low reductive dechlorination potential. Accordingly, enrichment and isolation of metabolic VC-oxidizing ethenotrophs was successful only from the low-TOC sediment. Sequence analysis of etnE genes from the microcosms as well phylogenetic typing of the isolates showed that ethenotrophs in the sediments were facultative anaerobic Proteobacteria capable of coping with OAI-associated redox fluctuations. Our results suggest that local sediment heterogeneity supports/selects divergent VC degradation processes at the OAI and that high reductive dechlorination potential suppresses development of aerobic metabolic VC oxidation potential.

Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g) were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis): soybean meal (SBM), casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic) target of rapamycin (mTOR) activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05) concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05) abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM.

This study investigated the stability and the activity of the microbiota from a single and a pool of donors in the TNO in vitro model of the colon (TIM-2 system). Our findings demonstrate the suitability of the preparation of a pool of fecal sample to be used for fermentation experiments. Chemicals/CAS: isobutyric acid, 5711-69-3, 79-31-2; isovaleric acid, 503-74-2, 539-66-2

The exposure of fish to environmental free-living microbes and its effect on early colonization in the gut have been studied in recent years. However, little is known regarding how the host and environment interact to shape gut communities during early life. Here, we tested whether the early microbial exposure of tilapia larvae affects the gut microbiota at later life stages. The experimental period was divided into three stages: axenic, probiotic and active suspension. Axenic tilapia larvae were reared either under conventional conditions (active suspension systems) or exposed to a single strain probiotic (Bacillus subtilis) added to the water. Microbial characterization by Illumina HiSeq sequencing of 16S rRNA gene amplicons showed the presence of B. subtilis in the gut during the seven days of probiotic application. Although B. subtilis was no longer detected in the guts of fish exposed to the probiotic after day 7, gut microbiota of the exposed tilapia larvae remained significantly different from that of the control treatment. Compared with the control, fish gut microbiota under probiotic treatment was less affected by spatial differences resulting from tank replication, suggesting that the early probiotic contact contributed to the subsequent observation of low inter-individual variation.

Biological markers that measure gut health and diagnose functional gastro-intestinal (GI) disorders, such as irritable bowel syndrome (IBS), are lacking. The objective was to identify and validate a biomarker panel associated with the pathophysiology of IBS that discriminates IBS from healthy controls (HC) and correlates with GI symptom severity. In a case-control design, various plasma and fecal markers were measured in a cohort of 196 clinical IBS patients and 160 HC without GI symptoms. A combination of biomarkers, which best discriminates between IBS and HC was identified and validated in an independent internal validation set and by permutation testing. The correlation between the biomarker panel and GI symptom severity was tested in IBS patients and in a general population cohort of 958 subjects. A set of 8 biomarker panel was identified to discriminate IBS from HC with high sensitivity (88.1%) and specificity (86.5%). The results for the IBS subtypes were comparable. Moreover, a moderate correlation was found between the biomarker panel and GI symptom scores in the IBS (r = 0.59, p r = 0.51, p = 0.003). A novel multi-domain biomarker panel has been identified and validated, which correlated moderately to GI symptom severity in IBS and general population subjects.

Background Next generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy. Results The metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems. Conclusions A reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.