Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia Sarunyou Wongwilaiwalin & Thanaporn Laothanachareon & Wuttichai Mhuantong & Sithichoke Tangphatsornruang & Lily Eurwilaichitr & Yasuo Igarashi & Verawat Champreda Received: 2 October 2012 /Revised: 3 January 2013 /Accepted: 7 January 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Decomposition of lignocelluloses by cooperative industrial pulp waste with CMCase, xylanase, and β- microbial actions is an essential process of carbon cycling in glucanase activities in the supernatant. Shotgun pyrosequenc- nature and provides a basis for biomass conversion to fuels ing of the BGC-1 metagenome indicated a markedly high and chemicals in biorefineries. In this study, structurally stable relative abundance of genes encoding for glycosyl hydrolases, symbiotic aero-tolerant lignocellulose-degrading microbial particularly for lignocellulytic enzymes in 26 families. The consortia were obtained from biodiversified microflora pres- enzyme system comprised a unique composition of main- ent in industrial sugarcane bagasse pile (BGC-1), cow rumen chain degrading and side-chain processing hydrolases, domi- fluid (CRC-1), and pulp mill activated sludge (ASC-1) by nated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of successive subcultivation on rice straw under facultative an- enzyme profiles to the specific substrate. Gene mapping oxic conditions. Tagged 16S rRNA gene pyrosequencing showed metabolic potential of BGC-1 for conversion of bio- revealed that all isolated consortia originated from highly mass sugars to various fermentation products of industrial diverse environmental microflora shared similar composite importance. The symbiotic consortium is a promising simpli- phylum profiles comprising mainly Firmicutes, reflecting fied model for study of multispecies mechanisms on consol- convergent adaptation of microcosm structures, however, with idated bioprocessing and a platform for discovering efficient substantial differences at refined genus level. BGC-1 compris- synergistic enzyme systems for biotechnological application. ing cellulolytic Clostridium and Acetanaerobacterium in sta- ble coexistence with ligninolytic Ureibacillus showed the Keywords Biorefinery . Glycosyl hydrolase . highest capability on degradation of agricultural residues and Lignocellulose . Metagenome . Microbial consortium S. Wongwilaiwalin : T. Laothanachareon : W. Mhuantong : L. Eurwilaichitr : V. Champreda (*) Introduction Enzyme Technology Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, Lignocellulosic plant biomass is the most abundant organic 113 Thailand Science Park, Klong Luang, Pathum Thani 12120, Thailand carbon reservoir on earth, providing a renewable carbon e-mail: verawat@biotec.or.th source for the chemosphere and the prospective biorefinery industry. Development of carbon neutral biomass conver- S. Tangphatsornruang sion processes has attracted much public interest in recent Genome Institute, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, years as a promising alternative platform for production of Klong Luang, Pathum Thani 12120, Thailand fuels and chemicals, which could reduce the global depen- dence on petroleum (FitzPatrick et al. 2010). Extensive Y. Igarashi research has been conducted to increase our understanding Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, of the complex natural bioconversion of lignocelluloses, Tokyo 113-8657, Japan particularly on degradation of recalcitrant plant biomass, Reproduced from Appl. Microbiol. Biotechnol. DOI 10.1007/s00253-013-4699-y. 163 which is an essential starting step in biorefinery. Insights Culture-independent direct 454 pyrosequencing of envi- into biomass decomposition by means of intricate biological ronmental metagenomes has previously been applied to processes not only provides us a fundamental basis on study diversity and metabolic capabilities of natural biomass essential roles of microbes in maintaining carbon balance decay microbiomes in various environments, e.g., tropical of the eco-system but is also of great interest for future peat swamp forest (Kanokratana et al. 2010), termite gut establishment of viable biorefineries for our sustainable (Warnecke et al. 2007; Nimchua et al. 2011), cow rumen society. (Brulc et al. 2009), and switch grass-adapted compost com- Lignocellulosic materials consist of mainly three different munity (Allgaier et al. 2010). These works illustrate the types of polymers, i.e., cellulose, hemicelluloses, and lignin, highly diversified nature of unexplored plant biomass- which are associated with each other (Fengel and Wegener degrading microbes and their enzymes awaiting discovery 1984). In nature, lignocelluloses are degraded by a complex for biotechnological uses. In this study, comparative analy- multistep process involving cooperative actions of various sis of structurally stable cellulolytic consortia isolated from microorganisms producing a variety of cellulolytic, hemicel- biodiversified microcosms in various environments active in lulolytic, and ligninolytic enzymes that act specifically and plant biomass deconstruction was reported using tagged 16S synergistically to degrade plant biomass. The concept on rRNA gene pyrosequencing, which demonstrated effects of utilization of simplified multispecies consortia, ranging from the enrichment process on shaping of the community struc- dual-species systems to complex microcosms on plant bio- tures. Lignocellulolytic enzyme systems of the consortium mass decomposition, is a promising strategy to achieve high were further explored using shotgun metagenome pyrose- degradation efficiency compared to using a single cellulolytic quencing, which showed the highly enriched nature of gene isolate (Lynd et al. 2002). A number of structurally stable sets encoding a distinct enzyme profile compared with pre- multispecies consortia with high cellulose-degrading activity viously reported plant biomass degrading microbiomes. The have been obtained by successive culture enrichments using work provides the first in-depth analysis of lignocellulolytic agricultural biomass as the sole carbon source under meso- enzyme systems and metabolic potential of a symbiotic and thermophilic conditions. These symbiotic consortia can biomass degrading consortium by metagenomic approaches. efficiently degrade various cellulosic materials, such as agro- This study gives valuable insights into the mechanism in industrial residues, filter paper, cotton, and pulp wastes consolidated lignocellulose degradation and conversion by a (Haruta et al. 2002; Guo et al. 2010; Wongwilaiwalin et al. symbiotic consortium and provides a promising gene min- 2010;Fengetal.2011;Wangetal.2011). A defined cellulose- ing platform for discovery of efficient synergistic enzyme degrading consortium was assembled from microbial isolates systems for biotechnological application. in a mixed culture microcosm (Kato et al. 2004), and study of this simplified microcosm revealed complex metabolic inter- dependences among the community members, which contrib- Materials and methods ute to the consortium stability (Kato et al. 2005, 2008). These symbiotic consortia typically comprise mainly anaerobic cel- Materials lulolytic bacterial taxa in stable coexistence with various non- cellulolytic members, which play important roles in Sources of microflora for preparation of microbial consortia metabolite dependence and environment modification, lead- in this study were collected from (1) sugarcane bagasse ing to enhancement in biomass hydrolysis efficiency of the decomposed soil from an industrial bagasse collection site multispecies systems. Modification of lignocellulose degrad- at the Phu Kheio Bio-Energy factory (Chaiyapoom, ing consortia was first explored by introducing and acclima- Thailand), (2) cow rumen fluid from Holstein cows housed tion of a cellulolytic clostridial strain into the system, which at the Khon Kaen Agriculture and Training Center (Roi Et, showed a way for alteration of the consortium structures and Thailand), and (3) activated sludge from a local soda pulp functions (Narisawa et al. 2007). The conceptual design on mill (Ratchaburi, Thailand). Rice straw and other agricul- development of symbiotic consortia comprising either natural tural by-products used as substrates in enrichment and bio- or engineered members has been recently reported as a prom- mass degradation efficiency analysis were obtained locally. ising approach for the establishment of microbial systems for The agricultural by-products were pretreated with alkali consolidated bioprocessing (Zuroff and Curtis 2012). Thus, (10 % w/v NaOH with 3:1 liquid/solid ratio at 90 °C for these studies indicate potential of the symbiotic microbial 90 min) for partial delignification. The native rice straw consortia, not only as the highly efficient biomass degrading contained 41.90 % cellulose, 34.24 % hemicelluloses, and systems but also for further modification of interspecies met- 22.44 % lignin, while the alkali-treated rice straw comprised abolic flux in designated consortia aiming for direct conver- of 68.69 % cellulose, 19.41 % hemicellulose, and 4.45 % sion of biomass to fuels and chemicals of industrial lignin according to analysis by Technical Association of importance. Pulp and Paper Industry