Théodore Bouchez

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.

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity.

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.

Anaerobic biodegradation of cellulosic material: batch experiments and modelling based on isotopic data and focusing on aceticlastic and non-aceticlastic methanogenesis.

Utilizing stable carbon isotope data to account for aceticlastic and non-aceticlastic pathways of methane generation, a model was created to describe laboratory batch anaerobic decomposition of cellulosic materials (office paper and cardboard). The total organic and inorganic carbon concentrations, methane production volume, and methane and CO(2) partial pressure values were used for the model calibration and validation. According to the fluorescent in situ hybridization observations, three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H(2)/H(2)CO(3) as well as acetate-oxidizing syntrophs, were considered. It was shown that temporary inhibition of aceticlastic methanogens by non-ionized volatile fatty acids or acidic pH was responsible for two-step methane production from office paper at 35 degrees C where during the first and second steps methane was generated mostly from H(2)/H(2)CO(3) and acetate, respectively. Water saturated and unsaturated cases were tested. According to the model, at the intermediate moisture (150%), much lower methane production occurred because of full-time inhibition of aceticlastic methanogens. At the lowest moisture, methane production was very low because most likely hydrolysis was seriously inhibited. Simulations showed that during cardboard and office paper biodegradation at 55 degrees C, non-aceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens were the dominant methanogenic pathways.

Microvirgula aerodenitrificans gen. nov., sp. nov., a new Gram-negative bacterium exhibiting co-respiration of oxygen and nitrogen oxides up to oxygen-saturated conditions

A denitrifier micro-organism was isolated from an upflow denitrifying filter inoculated with an activated sludge. The cells were Gram-negative, catalase- and oxidase-positive curved rods and very motile. They were aerobic as well as anoxic heterotrophs that had an atypical respiratory type of metabolism in which oxygen and nitrogen oxides were used simultaneously as terminal electron acceptors. The G&C content was 65 mol%. Our isolate was phenotypically similar to Comamonas testosteroni, according to classical systematic classification systems. However, a phylogenetic analysis based on the 165 rRNA sequence showed that the aerobic denitrifier could not be assigned to any currently recognized genus. For these reasons a new genus and species, Microvirgula aerodenitrificans gen. nov., sp. nov., is proposed, for which SGLY2T is the type strain.

COMBINED PHOSPHATE AND NITROGEN REMOVAL IN A SEQUENCING BATCH REACTOR USING THE AEROBIC DENITRIFIER, MICROVIRGULA AERODENITRIFICANS

A phosphate removal sludge was bioaugmented with the aerobic denitrifier, Microvirgula aerodenitrificans in order to reduce the nitrate produced during the aerobic nitrifying-phosphate uptake phase. Fluorescent in situ hybridization (FISH) was used to follow the fate of the added strain. In order to maintain the pure strain in the complex ecosystem, diverse physiological and kinetic based strategies of bioaugmentation were tested under the sequencing batch reactor (SBR) type culture. The nature of the M. aerodenitrificans inoculum (adapted to nitrate-aerobic conditions or to anoxic one) had no influence on the SBR performances and did not enhance aerobic denitrifying performances. The optimum quantity of the added strain (10% of the total biomass) seemed to have much more positive influence on the long term maintenance of the pure strain than on the SBR performances. A small but daily supply of M. aerodenitrificans gave exactly the same result than a massive and 1-day supply, i.e. no enhancement of performances and no amelioration of the length of maintenance. A continuous supply of carbon during the first hour of the aerobic phase combined to a 10% supply of M. aerodenitrificans gave the best compromise in terms of phosphate removal, nitrification and aerobic denitrification performances. It was accompanied too by a decreased number of the ammonia and nitrite-oxidizing bacteria and a modification of the nitrite-oxidizing floc structure. FISH on M. aerodenitrificans revealed that (i) before bioaugmentation, the strain was already present in the phosphate removal sludge and (ii) the added bacteria almost disappeared from the reactor after 16 HRT. In a last experiment, M. aerodenitrificans embedded in alginate beads allowed enhancement of both aerobic denitrifying performances and length of strain maintenance.

Members of the uncultured bacterial candidate division WWE1 are implicated in anaerobic digestion of cellulose

Clones of the WWE1 (Waste Water of Evry 1) candidate division were retrieved during the exploration of the bacterial diversity of an anaerobic mesophilic (35 ± 0.5°C) digester. In order to investigate the metabolic function of WWE1 members, a 16S rRNA gene ‐based stable isotope probing (SIP) method was used. Eighty‐seven percent of 16S r rRNA gene sequences affiliated to WWE1 candidate division were retrieved in a clone library obtained after polymerase chain reaction (PCR) amplification of enriched DNA fraction from anaerobic municipal solid waste samples incubated with 13C‐cellulose, at the end of the incubation (day 63) using a Pla46F‐1390R primer pair. The design of a specific WWE1 probe associated with the fluorescence in situ hybridization (FISH) technique corroborated the abundant representation of WWE1 members in our 13C‐cellulose incubations. Secondary ion mass spectrometry–in situ hybridization (SIMSISH) using an iodine‐labeled oligonucleotide probe combined with high‐resolution nanometer‐scale SIMS (NanoSIMS) observation confirmed the isotopic enrichment of members of WWE1 candidate division. The 13C apparent isotopic composition of hybridized WWE1 cells reached the value of about 40% early during the cellulose degradation process, suggesting that these bacteria play a role either in an extracellular cellulose hydrolysis process and/or in the uptake fermentation products.

Combined monitoring of changes in δ13CH4 and archaeal community structure during mesophilic methanization of municipal solid waste

Reconstituted municipal solid waste (MSW) with varying contents of putrescible and cellulosic waste was incubated anaerobically under mesophilic conditions. Standard physicochemical parameters were monitored, together with stable isotopic signatures of produced CH(4) and CO(2). delta(13)C values for CH(4) indicated a change of methanogenic metabolism with time. CH(4) was predominantly produced from H(2)/CO(2) at the beginning of the incubations. This period was associated with important shifts in archaeal communities monitored by automated ribosomal intergenic spacer analysis (ARISA) and FISH of oligonucleotidic probes targeting specifically 16S rRNA gene of various methanogenic groups. The onset of the active methane generation phase was characterized by an increase of CH(4)delta(13)C, indicating a progressive shift toward an aceticlastic metabolism. When the methane production levelled off, a decrease in the isotopic signature was observed toward values characteristics of hydrogenotrophic metabolism. ARISA profiles were, however, found to be stable from the beginning of the active methane generation phase until the end of the experiment. FISH observation indicated that members of the family Methanosarcinaceae were predominant in the archaeal community during this period, suggesting that these methanogens might exhibit a high metabolic versatility during methanization of waste.

Anaerobic digestion of biowaste under extreme ammonia concentration: Identification of key microbial phylotypes

Ammonia inhibition represents a major operational issue for anaerobic digestion (AD). In order to get more insights into AD microbiota resistance, anaerobic batch reactors performances were investigated under a wide range of Total Ammonia Nitrogen (TAN) concentrations up to 50.0g/L at 35°C. The half maximal inhibitory concentration (IC50) value was determined to be 19.0g/L. Microbial community dynamics revealed that above a TAN concentration of 10.0g/L, remarkable modifications within archaeal and bacterial communities occurred. 16S rRNA gene sequencing analysis showed a gradual methanogenic shift between two OTUs from genus Methanosarcina when TAN concentration increased up to 25.0g/L. Proportion of potential syntrophic microorganisms such as Methanoculleus and Treponema progressively raised with increasing TAN up to 10.0 and 25.0g/L respectively, while Syntrophomonas and Ruminococcus groups declined. In 25.0g/L assays, Caldicoprobacter were dominant. This study highlights the emergence of AD key phylotypes at extreme ammonia concentrations.

Methanosarcina as the dominant aceticlastic methanogens during mesophilic anaerobic digestion of putrescible waste

Taking into account isotope 13C value a mathematical model was developed to describe the dynamics of methanogenic population during mesophilic anaerobic digestion of putrescible solid waste and waste imitating Chinese municipal solid waste. Three groups of methanogens were considered in the model including unified hydrogenotrophic methanogens and two aceticlastic methanogens Methanosaeta sp. and Methanosarcina sp. It was assumed that Methanosaeta sp. and Methanosarcina sp. are inhibited by high volatile fatty acids concentration. The total organic and inorganic carbon concentrations, methane production, methane and carbon dioxide partial pressures as well as the isotope 13C incorporation in PSW and CMSW were used for the model calibration and validation. The model showed that in spite of the high initial biomass concentration of Methanosaeta sp. Methanosarcina sp. became the dominant aceticlastic methanogens in the system. This prediction was confirmed by FISH. It is concluded that Methanosarcina sp. forming multicellular aggregates may resist to inhibition by volatile fatty acids (VFAs) because a slow diffusion rate of the acids limits the VFA concentrations inside the Methanosarcina sp. aggregates.

Effect of inoculum to substrate ratio (I/S) on municipal solid waste anaerobic degradation kinetics and potential

The goal of this study is to evaluate the impact of the inoculum to substrate ratio (I/S) on the anaerobic degradation potential of municipal solid waste (MSW). Reconstituted MSW samples were thus incubated under batch anaerobic conditions and inoculated with an increasing amount of inoculum originating from a mesophilic sludge digester. I/S tested values were 0 (no inoculum added), 0.015, 0.03, 0.06, 0.12, 0.25, 1, 2 and 4 (gVM(inoculum)/gVM(waste)). The results indicate that the apparent maximal rate of dissolved organic carbon accumulation is reached at I/S=0.12. Under this level, the hydrolysis process is limited by the concentration of biomass and can thus be described as first order kinetics phenomena with respect to biomass for I/S ratios below 0.12. The maximum methane production rate and the minimal latency are reached at a ratio of 2. In addition to that, both methane signature and ARISA show a shift in the methanogenic populations from hydrogenotrophic to acetoclastic.