Whole proteome analyses on Ruminiclostridium cellulolyticum show a modulation of the cellulolysis machinery in response to cellulosic materials with subtle differences in chemical and structural properties

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the xyl-doc cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.This work was supported by a grant [R2DS 2010-08] from Conseil Regional d'Ile-de-France through DIM R2DS programs (http://www.r2ds-ile-de-france.com/). Irstea (www.irstea.fr/) contributed to the funding of a PhD grant for the first author. The funders provided support in the form of salaries for author [NB], funding for consumables and laboratory equipment, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Omics Services provided support in the form of salaries for authors [VS, MD], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors [NB, VS, MD] are articulated in the 'author contributions' section.info:eu-repo/semantics/publishedVersio

Zeolite favours propionate syntrophic degradation during anaerobic digestion of food waste under low ammonia stress

International audienceZeolite addition has been widely suggested for its ability to overcome ammonia stress occurring during anaerobic digestion. However little is known regarding the underlying mechanisms of mitigation and especially how zeolite influences the microbial structuration. The aim of this study was to bring new contributions on the effect of zeolite on the microbial community arrangement under a low ammonia stress. Replicated batch experiments were conducted. The microbial population was characterised with 16S sequencing. Methanogenic pathways were identified with methane isotopic fractionation. In presence of ammonia, zeolite mitigated the decrease of biogas production rate. Zeolite induced the development of Izimaplasmatales order and preserved Peptococcaceae family members, known as propionate degraders. Moreover methane isotopic fractionation showed that hydrogenotrophic methanogenesis was maintained in presence of zeolite under ammonia low stress. Our results put forward the benefit of zeolite to improve the bacteria-archaea syntrophy needed for propionate degradation and methane production under a low ammonia stress

Co-digestion of wastewater sludge: choosing the optimal blend

International audienceAnaerobic co-digestion (AcoD) is a promising strategy to increase the methane production of anaerobic digestion plants treating wastewater sludge (WAS). In this work the degradability of six different mixtures of WAS with fish waste (FW) or garden-grass (GG) was evaluated and compared to the three mono-digestions. Degradation performances and methanogenic pathways, determined with the isotopic signatures of biogas, were compared across time. Fish and grass mono-digestion provided a higher final methane production than WAS mono-digestion. In co-digestion the addition of 25 % of fish was enough to increase the final methane production from WAS while 50 % of grass was necessary. To determine the optimal blend of WAS co-digestion two indicators were specifically designed, representing the maximum potential production (ODI) and the expected production in mono-digestion conditions (MDI). The comparison between these indicators and the experimental results showed that the most productive blend was composed of 75% of co-substrate, fish or grass, with WAS. Indeed, the final methane production was increased by 1.9 times with fish and by 1.7 times with grass associated to an increase of the methane production rate by 1.5 times. Even if the same succession of methanogenic pathways across time was observed for the different mixtures, their relative proportions were different. Sewage sludge degradation was mostly achieved through hydrogenotrophic pathway as confirmed by the archaeal analysis while acetoclastic archaea were identified for fish and grass degradation

Insights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions.

[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]TED [Axe_IRSTEA]TED-SOWASTEInternational audienceDNA-SIP (stable isotope probing) was conducted on anaerobic municipal solid waste samples incubated with (13)C-cellulose, (13)C-glucose and (13)C-acetate under mesophilic conditions. A total of 567 full-length bacterial and 448 1100-bp-length archaeal 16S rRNA gene sequences were analysed. In the clone libraries derived from 'heavy' DNA fractions, the most abundant sequences were affiliated with the phyla Firmicutes, Bacteroidetes, the gamma-subclass of Proteobacteria and methanogenic orders Methanomicrobiales and Methanosarcinales. Sequences related to the genus Acetivibrio (phylum Firmicutes) were recovered only in the 'heavy' DNA fraction derived from the (13)C-cellulose incubation. An oligonucleotide probe (UCL284) targeting specifically Acetivibrio was designed and used for fluorescent in situ hybridization (FISH) experiments. Interestingly, hybridization of the probe was detected in microorganisms aggregated around cellulose fibres, strengthening the conclusion that these microorganisms were major cellulose degraders. Sequences related to genus Clostridium (phylum Firmicutes) and to the family Porphyromonadaceae (phylum Bacteroidetes) were retrieved in large numbers from the 'heavy' DNA library of (13)C-Glucose incubation, suggesting their involvement in saccharide fermentation. Design and hybridization of specific FISH-probes confirmed the abundant representation of Clostridium (CLO401, CLO1248) and Porphyromonadaceae (BAC1040), which were mostly observed in the planktonic phase. Surprisingly, in the (13)C-acetate experiment, the 'heavy' DNA archaeal library was dominated by sequences related to the strictly hydrogenotrophic methanogenic genus Methanoculleus. One single operational taxonomic unit containing 70 sequences, affiliated to the gamma-subclass of Proteobacteria, was retrieved in the corresponding bacterial library. FISH observations with a newly designed specific probe (UGA64) confirmed the dominance of this bacterial group. Our results show that combination of DNA-SIP and FISH applied with a series of functionally connected substrates can shed light on the networks of uncultured microbes catalysing the methanization of the most abundant chemical renewable energy source on Earth

SIMSISH technique does not alter the apparent isotopic composition of bacterial cells.

In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine - iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific (13)C uptake during labelled methanol anaerobic degradation

Six Years Temperature Monitoring Using Fibre-Optic Sensors in a Bioreactor Landfill

International audienceTemperature is a relevant physical parameter to monitor the biodegradation phases of waste mass. Irstea and the landfill operator SAS Les Champs Jouault have been collaborating since 2011 to study the temporal evolution and the spatial distribution of temperature in a municipal solid waste cell. Using distributed temperature sensing technology, optical fibres were installed in waste mass composed of household waste and industrial waste at different depths during the landfilling period. Temperature distributions were studied from 2012 until 2018 and the same evolutions are observed everywhere with more or less important amplitude variations depending on the location of the measurement point. When landfilled, the waste is at ambient temperature and a significant increase is observed the following year due to the exothermic impact of the aerobic biodegradation phase before a slower decrease during the anaerobic biodegradation phase over several years. Thermal parameters of the waste mass and the surrounding soil, as well as the heat generation function, are calculated using numerical simulation to reproduce the temperature evolution and its spatial distribution. The study of the long-term temperature evolution makes it possible to evaluate the favourable period during which the deposit cell will be in optimal conditions to promote the biodegradation waste processes

Rearrangement of incomplete multi-omics datasets combined with ComDim for evaluating replicate cross-platform variability and batch influence

International audienceMulti-omics studies can highlight the interrelationships among data across different layers of biological information. However, methods for the unsupervised analysis of multi-block data do not take the individual variability across batches into account and cannot deal with omics datasets when they present different numbers of replicates. We have explored three different data arrangement strategies to tackle these limitations. Several multiblock methods can be used to decipher the common variations across blocks and to determine the contribution of each block to each common component. In this study the ComDim method was used to compare these rearrangement strategies for three multi-omics datasets. We found that arranging the data using the 'replicate by blocks' strategy, where each block comprises data from only one replicate independently of its data type, provided the most insightful results. ComDim allowed the evaluation of the variability across the replicate blocks, confirming the existence of batch effects in some of the studies. Moreover, since the contributions of these batch effects were separated from the other contributions, the coordinated biological responses common across the different blocks was characterized for each data type

Simultaneous analysis of microbial identity and function using NanoSIMS.

Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of 13C or 15N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated

Generic mitigating and promoting effect of zeolite on anaerobic digestion: Physicochemical and metataxonomic data

This article provides comprehensive data on degradation performance and microbial dynamics derived from a set of 24 lab-scale batch anaerobic digesters involving various types of inhibitors and the addition of zeolite as a support material. In the first series of 12 digesters, three inhibitors were investigated at the following concentrations: 20 g/L of sodium chloride, 400 mg/L of erythromycin, and 5 mg/L of S-metolachlor. Each inhibitor was tested in triplicate, along with a control condition without inhibition. A parallel series was set up identically, except that 15 g/L of zeolite was introduced into each digester to mitigate the inhibition and promote the degradation process. The provided data comprises information regarding the experimental setup, monitoring measurements that assess the degradation performance (production, composition, and apparent isotopic factor of biogas, pH, dissolved inorganic and organic carbon and volatile fatty acids concentrations), microbial samples information, and 16S rRNA gene sequencing data that decipher changes in microbial structure. This datapaper is associated with research article [1] and presents both the sequencing data and the associated physicochemical data in a structured table format. The sequencing data were generated using the Ion Torrent PGM sequencer and have been deposited in the European Nucleotide Archive (ENA) database at EMBL-EBI under accession number PRJEB65129 (https://www.ebi.ac.uk/ena/browser/view/PRJEB65129), with sample accession numbers ranging from ERS16257742 to ERS16257691 [2]. The data serves as a valuable resource for comparisons with data from other studies on lab-scale batch anaerobic digesters, particularly those utilizing zeolite as a support material or involving inhibition caused by similar types of inhibitors (salts, antibiotics, or pesticides)