Solid-state fermentation in multi-well plates to assess pretreatment efficiency of rot fungi on lignocellulose biomass Simeng Zhou, Sana Raouche, Sacha Grisel, David Navarro, Jean-Claude Sigoillot, Isabelle Gimbert To cite this version: Simeng Zhou, Sana Raouche, Sacha Grisel, David Navarro, Jean-Claude Sigoillot, et al.. Solid-state fermentation in multi-well plates to assess pretreatment efficiency of rot fungi on lignocellulose biomass. Microbial Biotechnology, Wiley, 2015, 8 (6), pp.940-949. 10.1111/1751-7915.12307. hal-01269512 HAL Id: hal-01269512 https://hal.archives-ouvertes.fr/hal-01269512 Submitted on 27 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. bs_bs_banner Solid-state fermentation in multi-well plates to assess pretreatment efficiency of rot fungi on lignocellulose biomass Simeng Zhou,1,2 Sana Raouche,1,2 Sacha Grisel,1,2 Introduction David Navarro,1,2,3 Jean-Claude Sigoillot1,2 and Fungi play a dominant role in lignocellulose conversion. Isabelle Herpoël-Gimbert1,2 * Wood-decay fungi, including white-rot and brown-rot fungi 1INRA, UMR 1163 Biodiversity and Biotechnology of (WRFs and BRFs), have been extensively studied for Fungi, F-13009 Marseille, France. their abilities to efficiently modify, degrade and 2Aix-Marseille Université, Polytech Marseille, UMR 1163 depolymerize major plant cell wall components. Among Biodiversity and Biotechnology of Fungi, F-13009 them, some WRFs are able to mineralize the more recal- Marseille, France. citrant lignin with little consumption of cellulose (Martinez 3International Centre for Microbial Resources et al., 2005). This unique ability lends great potential inter- collection-Filamentous Fungi, CIRM-CF, F-13009 est for them in the pretreatment of biomass in many Marseille, France. biotechnological applications, such as wood pulping and bleaching, and biofuel production. Summary Solid-state fermentation (SSF) is defined by Barrios-Gonzalez (2012) as when a microbial culture The potential of fungal pretreatment to improve develops on the surface and inside of a solid matrix in the fermentable sugar yields from wheat straw or absence of free water. SSF has been shown to be of Miscanthus was investigated. We assessed 63 fungal interest in at least two types of applications: the produc- strains including 53 white-rot and 10 brown-rot fungi tion of lignocellulolytic enzymes and the degradation of belonging to the Basidiomycota phylum in an original lignocellulose itself. SSF mimics the natural habitat of the 12 day small-scale solid-state fermentation (SSF) fungus to facilitate enzyme secretion. Industrial enzymes experiment using 24-well plates. This method offers for biofuel applications have been successfully produced the convenience of one-pot processing of samples at the commercial level by SSF (Singhania et al., 2010), from SSF to enzymatic hydrolysis. The comparison of and differences in enzyme production by fungus between the lignocellulolytic activity profiles of white-rot fungi SSF and submerged fermentation (SmF) have been and brown-rot fungi showed different behaviours. The reviewed by Barrios-Gonzalez (2012). Enzymes are gen- hierarchical clustering according to glucose and erally produced in much higher concentrations in SSF, reducing sugars released from each biomass after some of them display higher optimal temperature or pH 72 h enzymatic hydrolysis splits the set of fungal stability and some are secreted only in SSF. Regarding strains into three groups: efficient, no-effect and the degradation of lignocellulose, the enzymes secreted detrimental-effect species. The efficient group con- along mycelia are close to their substrates, and the rapid tained 17 species belonging to seven white-rot genera diffusion of oxygen in SSF creates favourable conditions and one brown-rot genus. The yield of sugar released to achieve high performance in the oxidative degradation increased significantly (max. 62%) compared with of lignocellulose. These findings have led to fungi to be non-inoculated controls for both substrates. considered as a suitable alternative for lignocellulose pre- treatment in the second-generation ethanol production processes due to their low cost, environmental friendli- ness and adaptability to both continuous and batch pro- Received 18 December, 2014; revised 1 June, 2015; accepted 10 cesses. Moreover, cellulose and hemicellulose become June, 2015. *For correspondence. E-mail isabelle.gimbert@univ- more accessible for hydrolysis by the breakdown of the amu.fr; Tel. (+33) 4 91 82 86 00; Fax (+33)491828601. Microbial Biotechnology (2015) 8(6), 940–949 lignin–carbohydrate complexes, and so avoid substantial doi:10.1111/1751-7915.12307 holocellulose (cellulose and hemicellulose) loss, thereby Funding Information This study was supported by the French increasing conversion rates and productivity of ferment- National Research Agency Program Bio-ME as part of the ‘Stockage actif de la biomasse pour faciliter sa transformation industrielle’ able sugar. Recent studies have demonstrated the (STOCKACTIF) project ANR-12-BIME-0009. efficiency of some fungal strains [e.g. Ceriporiopsis © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Multi-well plate SSF to assess fungal pretreatment 941 subvermispora (Cianchetta et al., 2014), Pycnoporus BRFs belonging to the Basidiomycota phylum (Martinez cinnabarinus (Gupta et al., 2011), Trametes hirsuta et al., 2005). (Saritha et al., 2012), Irpex lacteus (Salvachua et al., The new 24-well plate-based screening method 2013), Gloeophyllum trabeum and Postia placenta was used to evaluate the potential of several strains of (Schilling et al., 2012)] in such pretreatment steps for basidiomycetes simultaneously (Fig. 1). SSF of improving the production of bioethanol or biogas from lignocellulosic substrates and subsequent enzymatic various substrates. Two distinguishable patterns of hydrolysis of the pretreated biomasses were performed. biomass breakdown, namely simultaneous lignin and This allows one-pot multistep treatments including fungal polysaccharide degradation or selective delignification growth, mild alkaline wash and enzymatic hydrolysis. This have been described among WRFs (Martinez et al., procedure was refined from to an experimental design 2005). The simultaneous attack of cellulose and lignin previously described in studies on the evaluation of bio- used as a preferred strategy by some strains could logical pretreatment (Tian et al., 2012; Lopez-Abelairas result in the concurrent production of cellulases, et al., 2013). A culture time of 12 days combined with a hemicellulases and ligninolytic activities; whereas fungal high inoculum loading allowed extensive and fast fungal strains acting as selective delignifiers secrete very low colonization. The mild alkaline wash performed before levels of holocellulolytic enzymes. However, selective enzymatic hydrolysis is reported to improve the enzymatic delignification ranges among fungal species according to digestibility of the biopretreated substrate (Yu et al., 2010; pretreatment time and the type of lignocellulosic biomass Salvachua et al., 2011). The main effect of sodium (Wan and Li, 2012). hydroxide pretreatment on lignocellulosic biomass was A biodiversity screening step is a prerequisite in choos- shown to be delignification through the cleavage of the ing the best fungus for efficient pretreatment. Natural ester bonds cross-linking lignin and xylan, thus increasing fungal diversity offers a huge array of new fungal strains the porosity of the biomass (McIntosh and Vancov, 2011; with high biotechnological potential (Blackwell, 2011; Maurya et al., 2015). In addition, fungal hyphae grow on Berrin et al., 2012). Fungal biodiversity has been investi- the surface of the lignocellulosic materials forming a gated previously for biomass pretreatment, but these dense hyphal coat that hinders cellulases’ access to cel- studies were often restricted to a limited number of strains lulose. As suggested for washing or heating steps before (Li et al., 2008; Salvachua et al., 2011). Traditional screen- enzymatic hydrolysis, the very mild alkaline conditions ing is performed with multiple fungal SSF in flasks and it is applied were expected to remove this biological barrier or difficult to apply these culture conditions to large-scale inhibitors of saccharification (Shi et al., 2009). The addi- strain screening. Scaled-down screening methods hold tional effects of these actions were expected to render the great promise for accelerating progress in biomass con- biomass more susceptible and accessible to saccharifica- version as they lead to the selection of a narrower number tion, and increase the production of fermentable sugar. of most effective strains to be later tested in a scaled-up Compared with traditional cultures