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

ABSTRACT: 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.

SUBMITTER: Lu F

PROVIDER: S-EPMC3869005 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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

Lü Fan F Bize Ariane A Guillot Alain A Monnet Véronique V Madigou Céline C Chapleur Olivier O Mazéas Laurent L He Pinjing P Bouchez Théodore T

The ISME journal 20130815 1

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 infer ...[more]

PMID: 23949661

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Project description:UNLABELLED: BACKGROUND:Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. RESULTS:Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. CONCLUSIONS:T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.

Project description:Closing water loops in chemical industries result in hot and highly saline residual streams, often characterized by high strength and the presence of refractory or toxic compounds. These streams are attractive for anaerobic technologies, provided the chemical compounds are biodegradable. However, under such harsh conditions, effective biomass immobilization is difficult, limiting the use of the commonly applied sludge bed reactors. In this study, we assessed the long-term phenol conversion capacity of a lab-scale anaerobic membrane bioreactor (AnMBR) operated at 55°C, and high salinity (18 gNa+.L-1). Over 388 days, bioreactor performance and microbial community dynamics were monitored using specific methanogenic activity (SMA) assays, phenol conversion rate assays, volatile fatty acids permeate characterization and Illumina MiSeq analysis of 16S rRNA gene sequences. Phenol accumulation to concentrations exceeding 600 mgPh.L-1 in the reactor significantly reduced methanogenesis at different phases of operation, while applying a phenol volumetric loading rate of 0.12 gPh.L-1.d-1. Stable AnMBR reactor performance could be attained by applying a sludge phenol loading rate of about 20 mgPh.gVSS-1.d-1. In situ maximum phenol conversion rates of 21.3 mgPh.gVSS-1 .d-1 were achieved, whereas conversion rates of 32.8 mgPh.gVSS-1 .d-1 were assessed in ex situ batch tests at the end of the operation. The absence of caproate as intermediate inferred that the phenol conversion pathway likely occurred via carboxylation to benzoate. Strikingly, the hydrogenotrophic SMA of 0.34 gCOD-CH4 .gVSS-1 .d-1 of the AnMBR biomass significantly exceeded the acetotrophic SMA, which only reached 0.15 gCOD-CH4 .gVSS-1 .d-1. Our results indicated that during the course of the experiment, acetate conversion gradually changed from acetoclastic methanogenesis to acetate oxidation coupled to hydrogenotrophic methanogenesis. Correspondingly, hydrogenotrophic methanogens of the class Methanomicrobia, together with Synergistia, Thermotogae, and Clostridia classes, dominated the microbial community and were enriched during the three phases of operation, while the aceticlastic Methanosaeta species remarkably decreased. Our findings clearly showed that highly saline phenolic wastewaters could be satisfactorily treated in a thermophilic AnMBR and that the specific phenol conversion capacity was limiting the treatment process. The possibility of efficient chemical wastewater treatment under the challenging studied conditions would represent a major breakthrough for the widespread application of AnMBR technology.

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

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

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