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Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

ABSTRACT: The engineering of complex communities can be a successful path to understand the ecology of microbial systems and improve biotechnological processes. Here, we developed a strategy to assemble a minimal and effective lignocellulolytic microbial consortium (MELMC) using a sequential combination of dilution-to-stimulation and dilution-to-extinction approaches. The consortium was retrieved from Andean forest soil and selected through incubation in liquid medium with a mixture of three types of agricultural plant residues. After the dilution-to-stimulation phase, approximately 50 bacterial sequence types, mostly belonging to the Sphingobacteriaceae, Enterobacteriaceae, Pseudomonadaceae, and Paenibacillaceae, were significantly enriched. The dilution-to-extinction method demonstrated that only eight of the bacterial sequence types were necessary to maintain microbial growth and plant biomass consumption. After subsequent stabilization, only two bacterial species (Pseudomonas sp. and Paenibacillus sp.) became highly abundant (>99%) within the MELMC, indicating that these are the key players in degradation. Differences in the composition of bacterial communities between biological replicates indicated that selection, sampling, and/or priority effects could shape the consortium structure. The MELMC can degrade up to ?13% of corn stover, consuming mostly its (hemi)cellulosic fraction. Tests with chromogenic substrates showed that the MELMC secretes an array of endoenzymes able to degrade xylan, arabinoxylan, carboxymethyl cellulose, and wheat straw. Additionally, the metagenomic profile inferred from the phylogenetic composition along with an analysis of carbohydrate-active enzymes of 20 bacterial genomes support the potential of the MELMC to deconstruct plant polysaccharides. This capacity was mainly attributed to the presence of Paenibacillus sp.IMPORTANCE The significance of our study mainly lies in the development of a combined top-down enrichment strategy (i.e., dilution to stimulation coupled to dilution to extinction) to build a minimal and versatile lignocellulolytic microbial consortium. We demonstrated that mainly two selectively enriched bacterial species (Pseudomonas sp. and Paenibacillus sp.) are required to drive the effective degradation of plant polymers. Our findings can guide the design of a synthetic bacterial consortium that could improve saccharification (i.e., the release of sugars from agricultural plant residues) processes in biorefineries. In addition, they can help to expand our ecological understanding of plant biomass degradation in enriched bacterial systems.

SUBMITTER: Diaz-Garcia L

PROVIDER: S-EPMC7783344 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

Díaz-García Laura L Huang Sixing S Spröer Cathrin C Sierra-Ramírez Rocío R Bunk Boyke B Overmann Jörg J Jiménez Diego Javier DJ

Applied and environmental microbiology 20210104 2

The engineering of complex communities can be a successful path to understand the ecology of microbial systems and improve biotechnological processes. Here, we developed a strategy to assemble a minimal and effective lignocellulolytic microbial consortium (MELMC) using a sequential combination of dilution-to-stimulation and dilution-to-extinction approaches. The consortium was retrieved from Andean forest soil and selected through incubation in liquid medium with a mixture of three types of agri ...[more]

PMID: 33127812

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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:Traditionally, starting inoculants have been applied to improve ensiling of forage used for livestock feed. Here, we aimed to build up a bioinoculant composed of lactic acid-producing and lignocellulolytic bacteria (LB) derived from the Megathyrsus maximus (guinea grass) phyllosphere. For this, the dilution-to-stimulation approach was used, including a sequential modification of the starting culture medium [Man, Rogosa, and Sharpe (MRS) broth] by addition of plant biomass (PB) and elimination of labile carbon sources. Along 10 growth-dilution steps (T1-T10), slight differences were observed in terms of bacterial diversity and composition. After the sixth subculture, the consortium started to degrade PB, decreasing its growth rate. The co-existence of Enterobacteriales (fast growers and highly abundance), Actinomycetales, Bacillales, and Lactobacillales species was observed at the end of the selection process. However, a significant structural change was noticed when the mixed consortium was cultivated in higher volume (500ml) for 8days, mainly increasing the proportion of Paenibacillaceae populations. Interestingly, Actinomycetales, Bacillales, and Lactobacillales respond positively to a pH decrease (4-5), suggesting a relevant role within a further silage process. Moreover, gene-centric metagenomic analysis showed an increase of (hemi)cellulose-degrading enzymes (HDEs) during the enrichment strategy. Reconstruction of metagenome-assembled genomes (MAGs) revealed that Paenibacillus, Cellulosimicrobium, and Sphingomonas appear as key (hemi)cellulolytic members (harboring endo-glucanases/xylanases, arabinofuranosidases, and esterases), whereas Enterococcus and Cellulosimicrobium have the potential to degrade oligosaccharides, metabolize xylose and might produce lactic acid through the phosphoketolase (PK) pathway. Based on this evidence, we conclude that our innovative top-down strategy enriched a unique bacterial consortium that could be useful in biotechnological applications, including the development/design of a synthetic bioinoculant to improve silage processes.

Project description:Assembling a functional consortium and identifying novel degraders from contaminated environments are still challenging due to the large diversity of microorganisms and the difficulty in isolating pure cultures. Here, we constructed a relatively simple functional consortium by enriching 1,4-dioxane-degrading-consortia using a culture-dependent dilution-to-extinction (DTE) method and reported a new key dioxane-degrader Shinella sp. Our results showed that serial dilution and reculture led to a divergence in the degradation ability of each consortium. Next-generation sequencing data revealed that the divergence in degradation performance was due to the reassembly of microbiota in the DTE process, which occurred most notably in 10-8 and 10-9 dilutions. The shift in community structure at 10-9 prevented the recovery of 1,4-dioxane degradation capacity, and the newly dominant taxa, Xanthobacter and Acinetobacter, struggled to replace the original dominant genus Shinella for 1,4-dioxane biodegradation. Combining differential analysis of community structure and metabolic function, we confirmed that Shinella species have a stronger 1,4-dioxane degradation ability than Xanthobacter species in the enriched consortium. In addition, we verified our findings using our isolated dioxane-degrading bacteria, Shinella yambaruensis, resulting in the rapid recovery of degradation performance of a 10-9 dilution consortium with Xanthobacter and Acinetobacter as the dominant microbiota. Taken together, this study provides a strategy for self-assembling functional consortiums and identifying the key degraders to explore the underlying biological mechanisms of enriched contaminant-degrading consortia.IMPORTANCEAssembling a functional microbial consortium and identifying key degraders involved in the degradation of 1,4-dioxane are crucial for the design of synergistic consortia used in enhancing the bioremediation of 1,4-dioxane-contaminated sites. However, due to the vast diversity of microbes, assembling a functional consortium and identifying novel degraders through a simple method remain a challenge. In this study, we reassembled 1,4-dioxane-degrading microbial consortia using a simple and easy-to-operate method by combining dilution-to-extinction and reculture techniques. We combined differential analysis of community structure and metabolic function and confirmed that Shinella species have a stronger 1,4-dioxane degradation ability than Xanthobacter species in the enriched consortium. In addition, a new dioxane-degrading bacterium was isolated, Shinella yambaruensis, which verified our findings. These results demonstrate that DTE and reculture techniques can be used beyond diversity reduction to assemble functional microbial communities, particularly to identify key degraders in contaminant-degrading consortia.

Project description: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.

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.

Dataset Information

Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

Publications

Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

Similar Datasets

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