J. Microbiol. Biotechnol. (2017), 27(9), 1670–1680 https://doi.org/10.4014/jmb.1705.05008 Research Article Review jmb Metagenomic SMRT Sequencing-Based Exploration of Novel Lignocellulose-Degrading Capability in Wood Detritus from Torreya nucifera in Bija Forest on Jeju Island Han Na Oh1, Tae Kwon Lee2, Jae Wan Park1, Jee Hyun No2, Dockyu Kim3, and Woo Jun Sul1* 1Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 2Department of Environmental Engineering, Yonsei University, Wonju 26493, Republic of Korea 3Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea Received: May 4, 2017 Revised: June 12, 2017 Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through Accepted: June 19, 2017 secondary growth of woody plant, is considered as promising resources for biofuel. In order to First published online use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were June 21, 2017 applied, but knowledge on the decomposition of lignocellulose occurring in a natural *Corresponding author environment is insufficient. We analyzed the 16S rRNA gene and metagenome to understand Phone: +82-31-670-4707; how the lignocellulose is decomposed naturally in decayed Torreya nucifera (L) of Bija forest Fax: +82-31-670-3108; E-mail: [email protected] (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using single molecules real-time sequencing revealed that the assembled contigs determined originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy)- and Protein families (Pfam)-based analysis showed that Proteobacteria was involved in degrading whole lignocellulose, and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrates. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass, and the microbial enzymes in Bija pISSN 1017-7825, eISSN 1738-8872 forest can be useful natural resources in industrial processes. Copyright© 2017 by The Korean Society for Microbiology Keywords: Lignocellulose degradation, Bija forest, metagenome, 16S rRNA, CAZy, Pfam and Biotechnology Introduction transform (in terms of chemical composition or structure) lignocellulosic biomass into value-added products by Lignocellulose, a major compartment of plant dry matter, chemical or biological approaches. High temperature and is the most promising feedstock as a renewable natural acidic conditions are expected to convert lignocellulosic resource for biofuels [1]. Lignocellulose consists mostly of biomasses into such degradation products as 5-hydroxymethyl three components (i.e., cellulose, hemicellulose, and lignin) furfural, xylose, sorbitol, succinic acid, and ethanol [4]. contributing to the hydrolytic stability of the plant cell Complex processes like dilute-acid pretreatment, hydrothermal wall, which is resistant to deconstruction and hydrolysis pretreatment, and liquid hot water pretreatment are applied [2]. The cross-linking of sugar monomers in hemicellulose to reduce lignocellulose recalcitrance and to solubilize each and cellulose and of phenolic units in lignin leads to component in lignocellulose [5]. Lignocellulose transformation recalcitrance of this biomass [3]. Thus, it is important to into value-added products can be a feasible means for understand the enzymatic hydrolysis process in order to reduction of greenhouse gas emissions and for increasing J. Microbiol. Biotechnol. Lignocellulose Degradation Capability in Wood Detritus Metagenome 1671 the security of the energy supply. Recently, biological delignification has gained increasing research attention and importance because it involves more economical, eco- friendly, and less hazardous procedures than do the physicochemical treatments [6]. Thus, an understanding of microbial enzymatic mechanisms has provided insights into improvement of the efficiency of lignocellulosic biomass decomposition. To study microbial metabolic pathways of lignocellulosic biomass degradation and hydrolysis, metagenomic approaches have recently been applied in various environments, including sugarcane bagasse [7], peat swamp forest [8], wet tropical forest soils [9], and wood-feeding insects [10, 11]. These studies on short reads from the Illumina platform have revealed that various bacterial species and functional genes are strongly linked to degradation of lignocellulose at different temperatures, pH, and oxygen availability levels. Nonetheless, short-read sequencing could not directly encompass entire regions of lignocellulosic biomass degradation operons in the microbial genome, and has limitations in terms of Fig. 1. Picture of the sampling site (33° 29’ N, 126° 48’ E) in resolving the complex and mixed metagenome sequences. Bija forest, Jeju Island. These limitations can be overcome by a technology providing Samples were collected from the detritus of rotten branches of Torreya nucifera (L). long-read sequences: PacBio RS II sequencing, based on single-molecule and real-time (SMRT) detection, dramatically improves the assembly of a metagenome from long-read in plant dry matter exposed to natural conditions for sequences, on average 3,000 bp or longer than 20,000 bp, obtaining appropriate genetic information on the degradation compared with the conventional next-generation sequencing of biomass under moderate conditions. technologies [12, 13]. One study showed that PacBio sequencing Torreya nucifera (L) (Bija) on Jeju Island, Korea, is located can provide high-quality information on lignocellulose- in the Bija forest (33° 29’ N, 126° 48’ E) at altitudes of 280- degrading bacteria and fungi via genes and canonical 380 m and the Bija forest is located in a region with a special pathways involved in lignocellulose decomposition [14]. geographical feature called Gotjawal (Fig. 1). T. nucifera (L) Most metagenomic studies have focused on screening for (Bija) is a coniferous tree, which contains 40-50% of microbial enzymes for lignocellulose decomposition in cellulose, 25-30% of hemicellulose, and 25-35% of lignin, extreme microenvironments: low pH, high moisture, or high in general. The annual temperature of Bija forest ranges temperature [9-11]. Industrial bagasse collection serves as from 14oC to 21oC, the annual precipitation is 1,474.9 mm a suitable ecological niche for studying microbial lignocellulose according to the Korea Meteorological Administration deconstruction owing to its physiologically amenable (2001–2010), and the forest has been reported as a valuable conditions [7]. Nevertheless, because the industrial bagasse repository of biological diversity [15]. It is important to collection is conducted after various types of extreme study the microbial decomposition of plant biomass and pretreatments (e.g., high-temperature processing) that change lignocellulose in Bija forest because it is located in Gotjawal, the natural conditions, it may not accurately represent the which is under moderate environmental conditions, and natural microbial metabolic processes or allow us to therefore it is predicted that lignocellulose degradation by discover microbial enzymes with decomposition activities microbes occurs naturally. under moderate conditions that do not require any external Here, we focused on the microbial metabolic capacity for energy supply for biotechnological exploitation. To explore degrading lignocellulose in wood detritus of T. nucifera (L) the lignocellulose-degrading enzymes in microbes, it may (Bija). We examined the following topics: (i) lignocellulose- be one method for studying dead plant matter or its debris, degrading microbial community structure based on 16S which are more readily decomposed by microbes. Thus, it is rRNA gene sequencing (Illumina), (ii) the lignocellulose necessary to study a microbial community or its functionality degradation process by a microbial enzyme detected in a September 2017 ⎪ Vol. 27⎪ No. 9 1672 Oh et al. metagenome via the long-read SMRT sequencing technology, value of 0.5 was conducted according to the script assign_taxonomy.py and (iii) new candidates for lignocellulose-degradative [20]. Among the 16,283 OTUs, all OTUs identified as belonging to enzymes under moderate conditions without pretreatments. class “Chloroplast” (105 OTUs, 1,239 sequences) or “Mitochondria” The characterization of the metagenome in Bija’s wood (801 OTUs, 6,614 sequences), which comes from Eukaryotic cell, detritus will provide basic knowledge on the ecological role were removed using the scripts filter_taxa_from_otu_tably.py and filter_fasta.py. Before we inferred a phylogenetic tree relating the of bacteria and their enzymatic activities for lignocellulosic sequences, align_seqs.py aligned the sequences in a FASTA file to biomass degradation. each other using PyNAST as an aligner [21]. The aligned sequences were filtered to generate a useful phylogenetic tree when aligning Materials and Methods against a template using filter_alignment.py and a phylogenetic tree relating the OTUs from a multiple sequence alignment by Sampling Site and DNA Extraction make_phylogeny.py.