Laurent Mazéas

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.

Absence of stable carbon isotope fractionation of saturated and polycyclic aromatic hydrocarbons during aerobic bacterial biodegradation

Compound-specific stable carbon isotopic composition measurement is a promising discriminative tool for source identification of hydrocarbons in modern environments. Nevertheless, to be considered as a source tracer, the 13C/12C ratio either has to be unmodified or at least modified in a predictable fashion by the different processes affecting hydrocarbons in the environment (photo-oxidation, evaporation, biodegradation, etc.). Possible isotopic fractionations introduced by microbial biodegradation have been investigated in this study. Stable carbon isotopic compositions of saturated hydrocarbons and phenanthrene compounds were stable during the course of a crude oil biodegradation experiment with a marine bacterial community. The 13C/12C ratio of some standard 2-methylphenanthrene also remained constant during the course of a biodegradation experiment with a pure bacterial strain. These results indicate the absence of isotopic fractionation of hydrocarbons during aerobic bacterial biodegradation. Therefore, molecular stable carbon isotopic composition of n-alkanes and PAHs can be used for source identification of these compounds in environmental studies.

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.

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.

Occurrence and Removal of Organic Micropollutants in Landfill Leachates Treated by Electrochemical Advanced Oxidation Processes.

In recent years, electrochemical advanced oxidation processes have been shown to be an effective alternative for the removal of refractory organic compounds from water. This study is focused on the effective removal of recalcitrant organic matter (micropollutants, humic substances, etc.) present in municipal solid waste landfill leachates. A mixture of eight landfill leachates has been studied by the electro-Fenton process using a Pt or boron-doped diamond (BDD) anode and a carbon felt cathode or by the anodic oxidation process with a BDD anode. These processes exhibit great oxidation ability due to the in situ production of hydroxyl radicals ((•)OH), a highly powerful oxidizing species. Both electrochemical processes were shown to be efficient in the removal of dissolved total organic carbon (TOC) from landfill leachates. Regarding the electro-Fenton process, the replacement of the classical anode Pt by the anode BDD allows better performance in terms of dissolved TOC removal. The occurrence and removal yield of 19 polycyclic aromatic hydrocarbons, 15 volatile organic compounds, 7 alkylphenols, 7 polychlorobiphenyls, 5 organochlorine pesticides, and 2 polybrominated diphenyl ethers in landfill leachate were also investigated. Both electrochemical processes allow one to reach a quasicomplete removal (about 98%) of these organic micropollutants.

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.

Methanogenic diversity and activity in municipal solid waste landfill leachates

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.

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.

Simultaneous determination of phenol, methylphenols, chlorophenols and bisphenol-A by headspace solid-phase microextraction-gas chromatography-mass spectrometry in water samples and industrial effluents

A headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) automatic method for simultaneous determination of trace amounts of phenol, methylphenols (MPs), chlorophenols (CPs) and bisphenol-A (BPA) in water samples and industrial effluents has been developed. Prior to SPME extraction, a direct derivatisation step using acetic anhydride was performed. Four different SPME fibre coatings (75 µm CAR-PDMS, 65 µm PDMS-DVB, 100 µm PDMS and 85 µm PA) were tested. The parameters affecting the HS-SPME process and derivatisation step studied were: extraction time (5–60 min) and temperature (40–100°C), derivatisation time (5–10 min), sample agitation (0–500 rpm), addition of sodium chloride (0–40%). The GC-MS quantification was performed by internal standard calibration and the limits of detection (LODs) were in the low ng L−1. The proposed analytical procedure provided a good linearity (r 2 > 0.9931) for standard solutions and a repeatability ranging from 4.8 to 15.2% (n = 5). The obtained results show that the developed method was rapid, simple and efficient for phenol, MPs, CPs and BPA analysis, and provides a good alternative to SPE and LLE. Finally, the proposed method has been applied successfully to analyse water samples, municipal solid waste landfill and industrial effluents.