Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties.

Nelly Badalato,Alain Buléon,Ariane Bize,Nina Pourette,Véronique Monnet,Théodore Bouchez,Sylvie Durand,Alain Guillot,Victor Sabarly,Laurent Mazéas,Gérard Mortha,Marc Dubois,Bruno Pontoire,Paul Robert,Arnaud Bridier,Diana Z Sousa

doi:10.1371/journal.pone.0170524

Abstract

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the “xyl-doc” cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.

Highlights

Conversion of cellulose during the degradation of biomass residues and agricultural waste and products has been extensively studied in the context of biofuel production [1,2,3]
With lower acetate-to-lactate concentration ratios (Fig 1D) as well as lower acetate-to-ethanol concentration ratios compared to both other substrates (Fig 1E). This specific metabolic profile likely results from the higher sugar influx in the cells and the faster pH decrease in the milieu over time, from pH 7.1 to pH ~6.3 (S1 Fig, panel B). It has previously been shown for R. cellulolyticum that the carbon flux partition between acetate, lactate and ethanol is greatly influenced by pH and entering carbon flows [20, 34, 35]
Together with the present work, these observations suggest that natural lignocellulosic substrates, in which hemicellulose is associated to lignin within complex entangled structures, are more likely to induce the expression of the “xyl-doc” cluster than more simple or engineered materials where the growth substrates are more readily available

Summary

Introduction

Conversion of cellulose during the degradation of biomass residues and agricultural waste and products has been extensively studied in the context of biofuel production [1,2,3]. Other sources of lignocellulosic materials, such as waste, are currently emerging as attractive options for biorefinery based on anaerobic digestion [4,5,6]. The cellulosic fraction in municipal solid waste (MSW) accounts for up to 50% weight in developed countries. Using this resource for biofuel or synthon production can potentially cuts down emissions of greenhouse gases while improving resource efficiency. Bioprocesses based on the action of complex microbial communities, such as those classically used for organic waste treatment and valorization (e.g. methanization) could be invaluable options [5, 9]

Methods

Results

Conclusion