Dataset Information

Metasecretome analysis of a lignocellulolytic microbial consortium grown on wheat straw, xylan and xylose.

ABSTRACT: BACKGROUND:Synergistic action of different enzymes is required to complete the degradation of plant biomass in order to release sugars which are useful for biorefining. However, the use of single strains is often not efficient, as crucial parts of the required enzymatic machinery can be absent. The use of microbial consortia bred on plant biomass is a way to overcome this hurdle. In these, secreted proteins constitute sources of relevant enzyme cocktails. Extensive analyses of the proteins secreted by effective microbial consortia will contribute to a better understanding of the mechanism of lignocellulose degradation. RESULTS:Here, we report an analysis of the proteins secreted by a microbial consortium (metasecretome) that was grown on either wheat straw (RWS), xylose or xylan as the carbon sources. Liquid chromatography-tandem mass spectrometry was used to analyze the proteins in the supernatants. Totals of 768 (RWS), 477 (xylose) and 103 (xylan) proteins were identified and taxonomically and functionally classified. In RWS, the proteins were mostly affiliated with Sphingobacterium-like consortium members (~50 %). Specific abundant protein clusters were predicted to be involved in polysaccharide transport and/or sensing (TonB-dependent receptors). In addition, proteins predicted to degrade plant biomass, i.e. endo-1,4-beta-xylanases, alpha-l-arabinofuranosidases and alpha-l-fucosidases, were prominent. In the xylose-driven consortium, most secreted proteins were affiliated with those from Enterobacteriales (mostly Klebsiella species), whereas in the xylan-driven one, they were related to Flavobacterium-like ones. Notably, the metasecretomes of the consortia growing on xylose and xylan contained proteins involved in diverse metabolic functions (e.g. membrane proteins, isomerases, dehydrogenases and oxidoreductases). CONCLUSIONS:An analysis of the metasecretomes of microbial consortia originating from the same source consortium and subsequently bred on three different carbon sources indicated that the major active microorganisms in the three final consortia differed. Importantly, diverse glycosyl hydrolases, predicted to be involved in (hemi)cellulose degradation (e.g. of CAZy families GH3, GH10, GH43, GH51, GH67 and GH95), were identified in the RWS metasecretome. Based on these results, we catalogued the RWS consortium as a true microbial enzyme factory that constitute an excellent source for the production of an efficient enzyme cocktail for the pretreatment of plant biomass.

SUBMITTER: Jimenez DJ

PROVIDER: S-EPMC4666044 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Metasecretome analysis of a lignocellulolytic microbial consortium grown on wheat straw, xylan and xylose.

Jiménez Diego Javier DJ Maruthamuthu Mukil M van Elsas Jan Dirk JD

Biotechnology for biofuels 20151201

<h4>Background</h4>Synergistic action of different enzymes is required to complete the degradation of plant biomass in order to release sugars which are useful for biorefining. However, the use of single strains is often not efficient, as crucial parts of the required enzymatic machinery can be absent. The use of microbial consortia bred on plant biomass is a way to overcome this hurdle. In these, secreted proteins constitute sources of relevant enzyme cocktails. Extensive analyses of the protei ...[more]

PMID: 26628913

Similar Datasets

Project description:BackgroundNew lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched on rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes.ResultsDifferential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora.ConclusionsCoupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.

Project description:BackgroundLignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2.ResultsWith all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71 g/gCDW/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth.ConclusionsLaboratory evolution of strain BP10001 resulted in effectively enhanced qXylose at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes.

Project description: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.

Project description:BackgroundIn a previous study, shaking speed was found to be an important factor affecting the population dynamics and lignocellulose-degrading activities of a synthetic lignocellulolytic microbial consortium composed of the bacteria Sphingobacterium paramultivorum w15, Citrobacter freundii so4, and the fungus Coniochaeta sp. 2T2.1. Here, the gene expression profiles of each strain in this consortium were examined after growth at two shaking speeds (180 and 60 rpm) at three time points (1, 5 and 13 days).ResultsThe results indicated that, at 60 rpm, C. freundii so4 switched, to a large extent, from aerobic to flexible (aerobic/microaerophilic/anaerobic) metabolism, resulting in continued slow growth till late stage. In addition, Coniochaeta sp. 2T2.1 tended to occur to a larger extent in the hyphal form, with genes encoding adhesion proteins being highly expressed. Much like at 180 rpm, at 60 rpm, S. paramultivorum w15 and Coniochaeta sp. 2T2.1 were key players in hemicellulose degradation processes, as evidenced from the respective CAZy-specific transcripts. Coniochaeta sp. 2T2.1 exhibited expression of genes encoding arabinoxylan-degrading enzymes (i.e., of CAZy groups GH10, GH11, CE1, CE5 and GH43), whereas, at 180 rpm, some of these genes were suppressed at early stages of growth. Moreover, C. freundii so4 stably expressed genes that were predicted to encode proteins with (1) β-xylosidase/β-glucosidase and (2) peptidoglycan/chitinase activities, (3) stress response- and detoxification-related proteins. Finally, S. paramultivorum w15 showed involvement in vitamin B2 generation in the early stages across the two shaking speeds, while this role was taken over by C. freundii so4 at late stage at 60 rpm.ConclusionsWe provide evidence that S. paramultivorum w15 is involved in the degradation of mainly hemicellulose and in vitamin B2 production, and C. freundii so4 in the degradation of oligosaccharides or sugar dimers, next to detoxification processes. Coniochaeta sp. 2T2.1 was held to be strongly involved in cellulose and xylan (at early stages), next to lignin modification processes (at later stages). The synergism and alternative functional roles presented in this study enhance the eco-enzymological understanding of the degradation of lignocellulose in this tripartite microbial consortium.

Project description:BackgroundMixed microbial cultures, in which bacteria and fungi interact, have been proposed as an efficient way to deconstruct plant waste. The characterization of specific microbial consortia could be the starting point for novel biotechnological applications related to the efficient conversion of lignocellulose to cello-oligosaccharides, plastics and/or biofuels. Here, the diversity, composition and predicted functional profiles of novel bacterial-fungal consortia are reported, on the basis of replicated aerobic wheat straw enrichment cultures.ResultsIn order to set up biodegradative microcosms, microbial communities were retrieved from a forest soil and introduced into a mineral salt medium containing 1% of (un)treated wheat straw. Following each incubation step, sequential transfers were carried out using 1 to 1,000 dilutions. The microbial source next to three sequential batch cultures (transfers 1, 3 and 10) were analyzed by bacterial 16S rRNA gene and fungal ITS1 pyrosequencing. Faith's phylogenetic diversity values became progressively smaller from the inoculum to the sequential batch cultures. Moreover, increases in the relative abundances of Enterobacteriales, Pseudomonadales, Flavobacteriales and Sphingobacteriales were noted along the enrichment process. Operational taxonomic units affiliated with Acinetobacter johnsonii, Pseudomonas putida and Sphingobacterium faecium were abundant and the underlying strains were successfully isolated. Interestingly, Klebsiella variicola (OTU1062) was found to dominate in both consortia, whereas K. variicola-affiliated strains retrieved from untreated wheat straw consortia showed endoglucanase/xylanase activities. Among the fungal players with high biotechnological relevance, we recovered members of the genera Penicillium, Acremonium, Coniochaeta and Trichosporon. Remarkably, the presence of peroxidases, alpha-L-fucosidases, beta-xylosidases, beta-mannases and beta-glucosidases, involved in lignocellulose degradation, was indicated by predictive bacterial metagenome reconstruction. Reassuringly, tests for specific (hemi)cellulolytic enzymatic activities, performed on the consortial secretomes, confirmed the presence of such gene functions.ConclusionIn an in-depth characterization of two wheat straw degrading microbial consortia, we revealed the enrichment and selection of specific bacterial and fungal taxa that were presumably involved in (hemi) cellulose degradation. Interestingly, the microbial community composition was strongly influenced by the wheat straw pretreatment. Finally, the functional bacterial-metagenome prediction and the evaluation of enzymatic activities (at the consortial secretomes) revealed the presence and enrichment of proteins involved in the deconstruction of plant biomass.

Project description:Morels (Morchella sp.) are economically important edible macro-fungi, which can grow on various synthetic or semi-synthetic media. However, the complex nutritional metabolism and requirements of these fungi remain ill-defined. This study, based on the plant biomass commonly used in the artificial cultivation of morels, assessed and compared the growth characteristics and extracellular enzymes of Morchella importuna cultivated on glucose, rice straw, sawdust, wheat grain, and a mixture of equal proportions of the three latter plant substrates (MIX). M. importuna could grow on all five tested media but displayed significant variations in mycelial growth rate, biomass, and sclerotium yield on the different media. The most suitable medium for M. importuna was wheat and wheat-containing medium, followed by glucose, while rice straw and sawdust were the least suitable. A total of 268 secretory proteins were identified by liquid chromatography coupled with tandem mass spectrometry detection. Functional classification and label-free comparative analysis of these proteins revealed that carbohydrate-active enzyme (CAZYme) proteins were the predominant component of the secretome of M. importuna, followed by protease, peptidase, and other proteins. The abundances of CAZYme proteins differed among the tested media, ranging from 64% on glucose to 88% on rice straw. The CAZYme classes of glycoside hydrolases and carbohydrate-binding module were enriched in the five secretomes. Furthermore, the enzyme activities of CMCase, lignase, amylase, xylase, pNPCase, and pNPGase were detected during the continuous culture of M. importuna in MIX medium, and the relative expression of the corresponding genes were detected by quantitative real-time PCR. The combined data of growth potential, secretome, extracellular enzyme activity, and gene expression on different substrates inferred that M. importuna was weak in lignocellulose degradation but a good starch decomposer. Specifically, in terms of the degradation of cellulose, the ability to degrade cellulose into oligosaccharides was weaker compared with further degradation into monosaccharides, and this might be the speed-limiting step of cellulose utilization in M. importuna. In addition, M. importuna had a strong ability to decompose various hemicellulose glycosidic bonds, especially α- and β-galactosidase. Only a very few lignin-degradation-related proteins were detected, and these were in low abundance, consistent with the presence of weak lignin degradation ability. Furthermore, the presence of lipase and chitinase implied that M. importuna was capable of decomposition of its own mycelia in vitro. The study provides key data that facilitates a further understanding of the complex nutritional metabolism of M. importuna.

Dataset Information

Metasecretome analysis of a lignocellulolytic microbial consortium grown on wheat straw, xylan and xylose.

Publications

Metasecretome analysis of a lignocellulolytic microbial consortium grown on wheat straw, xylan and xylose.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets