Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system

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Abstract

Degradation of lignocellulosic agro-industrial residues by means of complex microbial community is a promising approach providing efficient biomass decomposition for subsequent conversion to value-added products. In this study, an active thermophilic lignocellulose degrading microbial consortium was bred from high-temperature sugarcane bagasse compost by successive subcultivation under aerobic static conditions. The microbial consortium showed efficient degradation activity on potential biorefinery cellulosic substrates, including bagasse, rice straw, corn stover and industrial eucalyptus pulp sludge. The consortium was structurally stable with the co-existence of eight major microbes, comprising anaerobic bacterial genera Clostridium and Thermoanaerobacterium along with an aerobic/facultative anaerobic Rhodocyclaceae bacterium, bacilli, and uncultured bacteria. Majority of the lignocellullolytic activities including endo-glucanase, xylanase and β-glucanase was present in the crude culture supernatant compared to the cell-bound fraction. Proteomic analysis of cellulose bound fraction of the crude extracellular enzyme revealed a multi-species lignocellulolytic enzyme system composed mainly of cellulosomal components and extracellular cellulases of clostridia along with hemicellulases and a β-glucanase from Clostridium, Bacillus, and Thermobacillus related origins. This work presents the first report on analysis of the complex structurally stable lignocellulose degrading microbial consortium together with the characterization of its lignocellulolytic enzyme system applicable for biomass degradation and conversion in biotechnological industry.

Introduction

Lignocellulosic plant biomass is an important renewable carbon resource for the biorefinery industry, and is thus considered a sustainable and environmentally friendly alternative to the current petroleum platform [1]. Lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, consists mainly of three different types of biopolymers i.e. cellulose (35–50%), hemicellulose (25–30%) and lignin (25–30%) [2]. Cellulose and hemicellulose are degraded to sugars, which are versatile starting materials for further conversion by fermentation, biocatalytic, and chemocatalytic processes to value-added products, including biofuels, biopolymers and chemicals. Improvement of microbial and enzymatic processes on lignocellulosic biomass degradation is thus an important area of research in sustainable “green” biotechnology.

In nature, lignocellulosic biomass is degraded with the cooperation of many microorganisms, mainly including diverse fungal and bacterial genera producing a variety of cellulolytic and hemicellulolytic enzymes under aerobic and anaerobic conditions [3]. The biodegradation of cellulosic biomass through the use of microbial co-cultures or complex communities has been proposed as a highly efficient approach for biotechnological application, since it avoids the problems of feedback regulation and metabolite repression posed by isolated single strains [4], [5], [6]. Symbiosis between cellulolytic and non-cellulolytic microorganisms has been reported to promote cellulose degradation by mixed cultures [7], [8], [9]. Haruta and co-workers obtained a structurally stable and complex lignocellulolytic microbial consortium from successive enrichment culture from rice straw compost with a high activity on various cellulosic materials, including rice straw, paper, and cotton [6]. Studies on microbial consortia and their mixed enzyme systems could thus provide an important basis for understanding complex interactions on lignocellulose degradation in nature and can be a platform for biotechnological application involving biomass degradation in composting, anaerobic digestion, enzymatic biomass saccharification, and also the recently introduced concept on direct microbial conversion of cellulosic biomass to products in the absence of added saccharolytic enzymes [10].

The functional and structural stabilities of microbial consortia are considered to be important factors in their biomass degradation capability, and thus their potential for biotechnological application [11], [12]. In this study, a stable thermophilic lignocellulolytic microbial consortium highly active in cellulosic biomass degradation was established from a high-temperature sugarcane bagasse compost. The structure and dynamics of the microbial community have been investigated together with proteomic characterization of its multi-species lignocellulolytic enzyme system. This represents the first report on analysis of a complex lignocellulose degrading microbial community and its lignocellulose degrading enzymes through a molecular culture-independent approach. The microbial consortium provides a valuable platform for further study on multi-microbial species enzyme interaction, and has potential biotechnological application on lignocellulosic biomass degradation and conversion for the promising biorefinery industry.

Section snippets

Materials

Sugarcane bagasse was obtained from the Mitr Phu Wiang Sugar Mill (Khon Kaen, Thailand). Rice straw and corn stover were obtained from local farm. The agricultural by-products were pretreated using the alkali–peracetic acid method according to Zhao et al. [13]. Industrial eucalyptus pulp sludge was obtained from SCG Paper, PLC (Ratchaburi, Thailand) and used without pretreatment. The filter paper used in this study was Whatman no. 1 (Whatman, Kent, UK). The cellulosic substrates were autoclaved

Isolation of the primary enriched microbial consortium

In this step, successive subcultivation under selective enrichment conditions was used in order to establish the structurally stable microbial consortium with high cellulose degrading capability. A number of microbial communities capable of filter paper degradation with different degradation rates were obtained from subcultivation of seed cultures from high-temperature sugarcane bagasse compost under aerobic static conditions. After several subcultivations, the most active microbial community,

Discussion

Lignocellulytic mixed culture is considered a promising tool for efficient degradation or direct conversion of biomass feedstock to value-added bio-based products. In this study, a structurally and functionally stable thermophilic lignocellulose degrading microbial consortium MC3F was obtained, as shown by 16S rDNA DGGE and cellulolytic activity analyses. The microbial consortium showed efficient degradation on potential biorefinery feedstocks, including bagasse, rice straw, corn stover and

Acknowledgements

This project is under the JST-NRCT-BIOTEC collaborative project supported by a research grant from the National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (P-00-20243). Manuscript proofreading by Dr. Philip J. Shaw is appreciated.

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