Guillermina Hernandez-Raquet

FROGS: Find, Rapidly, OTUs with Galaxy Solution

Motivation Metagenomics leads to major advances in microbial ecology and biologists need user friendly tools to analyze their data on their own. Results This Galaxy‐supported pipeline, called FROGS, is designed to analyze large sets of amplicon sequences and produce abundance tables of Operational Taxonomic Units (OTUs) and their taxonomic affiliation. The clustering uses Swarm. The chimera removal uses VSEARCH, combined with original cross‐sample validation. The taxonomic affiliation returns an innovative multi‐affiliation output to highlight databases conflicts and uncertainties. Statistical results and numerous graphical illustrations are produced along the way to monitor the pipeline. FROGS was tested for the detection and quantification of OTUs on real and in silico datasets and proved to be rapid, robust and highly sensitive. It compares favorably with the widespread mothur, UPARSE and QIIME. Availability and implementation Source code and instructions for installation: https://github.com/geraldinepascal/FROGS.git. A companion website: http://frogs.toulouse.inra.fr. Supplementary information Supplementary data are available at Bioinformatics online.

Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture.

Polyhydroxybutyrate (PHB) production directly by waste activated sludge (WAS) was investigated in aerobic fed-batch conditions using acetic acid as substrate. PHB production was induced by phosphorus limitation. WAS of different origin were tested with various degrees of phosphorus limitation and PHB contents of up to 70% (gCOD PHB/gCOD particulate) were obtained. This strategy showed the importance of maintaining cell growth for PHB production in order to increase PHB concentration and that the degree of phosphorus limitation has a direct impact on the quantity of PHB produced. Pyrosequencing of 16S rRNA transcripts showed changes in the active bacteria of the WAS microbial community as well as the acclimation of populations depending on sludge origin. The monitoring of the process appeared as the key factor for optimal PHB production by WAS. Different strategies are discussed and compared in terms of carbon yield and PHB content with the feast and famine selection process.

Efficient anaerobic transformation of raw wheat straw by a robust cow rumen-derived microbial consortium.

A rumen-derived microbial consortium was enriched on raw wheat straw as sole carbon source in a sequential batch-reactor (SBR) process under strict mesophilic anaerobic conditions. After five cycles of enrichment the procedure enabled to select a stable and efficient lignocellulolytic microbial consortium, mainly constituted by members of Firmicutes and Bacteroidetes phyla. The enriched community, designed rumen-wheat straw-derived consortium (RWS) efficiently hydrolyzed lignocellulosic biomass, degrading 55.5% w/w of raw wheat straw over 15days at 35°C and accumulating carboxylates as main products. Cellulolytic and hemicellulolytic activities, mainly detected on the cell bound fraction, were produced in the earlier steps of degradation, their production being correlated with the maximal lignocellulose degradation rates. Overall, these results demonstrate the potential of RWS to convert unpretreated lignocellulosic substrates into useful chemicals.

Understanding of polyhydroxybutyrate production under carbon and phosphorus-limited growth conditions in non-axenic continuous culture.

In a waste into resource strategy, a selection of polyhydroxybutyrate (PHB)-accumulating organisms from activated sludge was achieved in an open continuous culture under acetic acid and phosphorus limitation. Once the microbial population was selected at a dilution rate (D), an increase in phosphorus limitation degree was applied in order to study the intracellular phosphorus plasticity of selected bacteria and the resulting capacity to produce PHB. Whatever D, all selected populations were able to produce PHB. At a D, the phosphorus availability determined the phosphorus-cell content which in turn fixed the amount of cell. All the remaining carbon was thus directed toward PHB. By decreasing D, microorganisms adapted more easily to higher phosphorus limitation leading to higher PHB content. A one-stage continuous reactor operated at D=0.023h(-)(1) gave reliable high PHB productivity with PHB content up to 80%. A two-stage reactor could ensure better productivity while allowing tuning product quality.

CAZyChip: dynamic assessment of exploration of glycoside hydrolases in microbial ecosystems.

BackgroundMicroorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans.ResultsThis DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares, and microarrays were directly synthesized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known GHs RNA extracted from recombinant E. coli strains, which were previously identified by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota.ConclusionsThe CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems.

Enrichment and adaptation yield high anammox conversion rates under low temperatures

This study compared two anammox sequencing batch reactors (SBR) for one year. SBRconstantT was kept at 30 °C while temperature in SBRloweringT was decreased step-wise from 30 °C to 20 °C and 15 °C followed by over 140 days at 12.5 °C and 10 °C. High retention of anammox bacteria (AnAOB) and minimization of competition with AnAOB were key. 5-L anoxic reactors with the same inoculum were fed synthetic influent containing 25.9 mg NH4+-N/L and 34.1 mg NO2--N/L (no COD). Specific ammonium removal rates continuously increased in SBRconstantT, reaching 785 mg NH4+-N/gVSS/d, and were maintained in SBRloweringT, reaching 82.2 and 91.8 mg NH4+-N/gVSS/d at 12.5 and 10 °C respectively. AnAOB enrichment (increasing hzsA and 16S rDNA gene concentrations) and adaptation (shift from Ca. Brocadia to Ca. Kuenenia in SBRloweringT) contributed to these high rates. Rapidly settling granules developed, with average diameters of 1.2 (SBRconstantT) and 1.6 mm (SBRloweringT). Results reinforce the potential of anammox for mainstream applications.

Analysis of large 16S rRNA Illumina data sets: Impact of singleton read filtering on microbial community description

Next‐generation sequencing technologies give access to large sets of data, which are extremely useful in the study of microbial diversity based on 16S rRNA gene. However, the production of such large data sets is not only marred by technical biases and sequencing noise but also increases computation time and disc space use. To improve the accuracy of OTU predictions and overcome both computations, storage and noise issues, recent studies and tools suggested removing all single reads and low abundant OTUs, considering them as noise. Although the effect of applying an OTU abundance threshold on α‐ and β‐diversity has been well documented, the consequences of removing single reads have been poorly studied. Here, we test the effect of singleton read filtering (SRF) on microbial community composition using in silico simulated data sets as well as sequencing data from synthetic and real communities displaying different levels of diversity and abundance profiles. Scalability to large data sets is also assessed using a complete MiSeq run. We show that SRF drastically reduces the chimera content and computational time, enabling the analysis of a complete MiSeq run in just a few minutes. Moreover, SRF accurately determines the actual community diversity: the differences in α‐ and β‐community diversity obtained with SRF and standard procedures are much smaller than the intrinsic variability of technical and biological replicates.

Ecofriendly lignocellulose pretreatment to enhance the carboxylate production of a rumen-derived microbial consortium

Innovative dry chemo- and chemo-mechanical pretreatments form an interesting approach for modifying the native physico-chemical composition of lignocellulose facilitating its microbial conversion to carboxylates. Here, the impact of four dry-pretreatment conditions on the microbial transformation of wheat straw was assessed: milling to 2mm and 100µm, and NaOH chemical impregnation at high substrate concentrations combined with milling at 2mm and 100µm. Pretreatment effect was assessed in the light of substrate structure and composition, its impact on the acidogenic potential and the major enzyme activities of a rumen-derived microbial consortium RWS. Chemo-mechanical pretreatment strongly modified the substrate macroporosity. The highest carboxylate production rate was reached after dry chemo-mechanical treatment with NaOH at 100µm. A positive impact of the dry chemo-mechanical treatment on xylanase activity was observed also. These results underline that increasing substrate macroporosity by dry chemo-mechanical pretreatment had a positive impact on the microbial acidogenic potential.

Anaerobic lignocellulolytic microbial consortium derived from termite gut: enrichment, lignocellulose degradation and community dynamics

BackgroundLignocellulose is the most abundant renewable carbon resource that can be used for biofuels and commodity chemicals production. The ability of complex microbial communities present in natural environments that are specialized in biomass deconstruction can be exploited to develop lignocellulose bioconversion processes. Termites are among the most abundant insects on earth and play an important role in lignocellulose decomposition. Although their digestive microbiome is recognized as a potential reservoir of microorganisms producing lignocellulolytic enzymes, the potential to enrich and maintain the lignocellulolytic activity of microbial consortia derived from termite gut useful for lignocellulose biorefinery has not been assessed. Here, we assessed the possibility of enriching a microbial consortium from termite gut and maintaining its lignocellulose degradation ability in controlled anaerobic bioreactors.ResultsWe enriched a termite gut-derived consortium able to transform lignocellulose into carboxylates under anaerobic conditions. To assess the impact of substrate natural microbiome on the enrichment and the maintenance of termite gut microbiome, the enrichment process was performed using both sterilized and non-sterilized straw. The enrichment process was carried out in bioreactors operating under industrially relevant aseptic conditions. Two termite gut-derived microbial consortia were obtained from Nasutitermes ephratae by sequential batch culture on raw wheat straw as the sole carbon source. Analysis of substrate loss, carboxylate production and microbial diversity showed that regardless of the substrate sterility, the diversity of communities selected by the enrichment process strongly changed compared to that observed in the termite gut. Nevertheless, the community obtained on sterile straw displayed higher lignocellulose degradation capacity; it showed a high xylanase activity and an initial preference for hemicellulose.ConclusionsThis study demonstrates that it is possible to enrich and maintain a microbial consortium derived from termite gut microbiome in controlled anaerobic bioreactors, producing useful carboxylates from raw biomass. Our results suggest that the microbial community is shaped both by the substrate and the conditions that prevail during enrichment. However, when aseptic conditions are applied, it is also affected by the biotic pressure exerted by microorganisms naturally present in the substrate and in the surrounding environment. Besides the efficient lignocellulolytic consortium enriched in this study, our results revealed high levels of xylanase activity that can now be further explored for enzyme identification and overexpression for biorefinery purposes.

Short and long term effect of decreasing temperature on anammox activity and enrichment in mainstream granular sludge process

This study investigates the impact of lower temperature on short term and long term (down to 10 °C) on a completely anoxic anammox granular sludge process. This is the first time granular sludge Anammox is operated in pure anoxic condition in SBR and at low temperature. Conversion performance, kinetic parameters, sludge characteristics and microbial community were analyzed.