Metagenomic SMRT Sequencing-Based Exploration of Novel Lignocellulose-Degrading Capability in Wood Detritus from Torreya nucifera in Bija Forest on Jeju Island

( Han Na Oh ),( Tae Kwon Lee ),( Jae Wan Park ),( Jee Hyun No ),( Dockyu Kim ),( Woo Jun Sul ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.9

Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through secondary growth of woody plant, is considered as promising resources for biofuel. In order to use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were applied, but knowledge on the decomposition of lignocellulose occurring in a natural environment is insufficient. We analyzed the 16S rRNA gene and metagenome to understand how the lignocellulose is decomposed naturally in decayed Torreya nucifera (L) of Bija forest (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 forest can be useful natural resources in industrial processes.

Antarctic tundra soil metagenome as useful natural resources of cold-active lignocelluolytic enzymes

Han Na Oh,Doyoung Park,Hoon Je Seong,Dockyu Kim,Woo Jun Sul 한국미생물학회 2019 The journal of microbiology Vol.57 No.10

Lignocellulose composed of complex carbohydrates and aromatic heteropolymers is one of the principal materials for the production of renewable biofuels. Lignocellulose-degrading genes from cold-adapted bacteria have a potential to increase the productivity of biological treatment of lignocellulose biomass by providing a broad range of treatment temperatures. Antarctic soil metagenomes allow to access novel genes encoding for the cold-active lignocellulose-degrading enzymes, for biotechnological and industrial applications. Here, we investigated the metagenome targeting cold-adapted microbes in Antarctic organic matter-rich soil (KS 2-1) to mine lignolytic and celluloytic enzymes by performing single molecule, real-time metagenomic (SMRT) sequencing. In the assembled Antarctic metagenomic contigs with relative long reads, we found that 162 (1.42%) of total 11,436 genes were annotated as carbohydrate-active enzymes (CAZy). Actinobacteria, the dominant phylum in this soil’s metagenome, possessed most of candidates of lignocellulose catabolic genes like glycoside hydrolase families (GH13, GH26, and GH5) and auxiliary activity families (AA7 and AA3). The predicted lignocellulose degradation pathways in Antarctic soil metagenome showed synergistic role of various CAZyme harboring bacterial genera including Streptomyces, Streptosporangium, and Amycolatopsis. From phylogenetic relationships with cellular and environmental enzymes, several genes having potential for participating in overall lignocellulose degradation were also found. The results indicated the presence of lignocellulose-degrading bacteria in Antarctic tundra soil and the potential benefits of the lignocelluolytic enzymes as candidates for cold-active enzymes which will be used for the future biofuel-production industry.

Characterization of a Thermophilic Lignocellulose-Degrading Microbial Consortium with High Extracellular Xylanase Activity

( Dongdong Zhang ),( Yi Wang ),( Chunfang Zhang ),( Dan Zheng ),( Peng Guo ),( Zongjun Cui ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.2

A microbial consortium, TMC7, was enriched for the degradation of natural lignocellulosic materials under high temperature. TMC7 degraded 79.7% of rice straw during 15 days of incubation at 65°C. Extracellular xylanase was effectively secreted and hemicellulose was mainly degraded in the early stage (first 3 days), whereas primary decomposition of cellulose was observed as of day 3. The optimal temperature and initial pH for extracellular xylanase activity and lignocellulose degradation were 65°C and between 7.0 and 9.0, respectively. Extracellular xylanase activity was maintained above 80% and 85% over a wide range of temperature (50-75°C) and pH values (6.0-11.0), respectively. Clostridium likely had the largest contribution to lignocellulose conversion in TMC7 initially, and Geobacillus, Aeribacillus, and Thermoanaerobacterium might have also been involved in the later phase. These results demonstrate the potential practical application of TMC7 for lignocellulosic biomass utilization in the biotechnological industry under hot and alkaline conditions.

Hydrolytic performances of different organic compounds in different lignocellulosic biomass during anaerobic digestion

Haifeng Yang,Rui Deng,Junwei Jin,Yuling Wu,Xin Jiang,Jinhua Shi 대한환경공학회 2022 Environmental Engineering Research Vol.27 No.4

Hydrolytic performances of different organic compounds in lignocellulosic biomass (LB) during anaerobic digestion (AD) are worth investigating due to the complex and refractory structure of lignocellulose. This study aimed to clarify the hydrolytic performances of different lignocellulosic components (hemicellulose, cellulose and lignin) and other typical organics (saccharides, protein and lipid) in AD process. Furthermore, an in-depth study of different lignocellulosic components mono-/co-digestive performances, as well as their effects on digestive systems were also designed to explain the mechanism. Kinetic models were specially applied to evaluate the hydrolytic process and make comparison among different lignocellulosic components. Results showed that hemicellulose obtained high degradation ratio (77.2-85.0%) during anaerobic digestion, while cellulose was difficult to hydrolyze without sufficient acidity. And organics (saccharides, protein and lipid) were much easier to be hydrolyzed than lignocellulose. Results also depicted that lignocellulose addition could efficiently enhance the volatile solid (VS) removals of digestive systems, while lignin existing in systems reduced the VS removal. The limited hydrolysis of lignocellulose hindered the degradation of total VS in digester. It is quite important to obtain high bioenergy conversion, pretreatments, which can destroy the lignin wrapping in LBs digestion. This study could provide a reference for the AD of LBs.

목질섭식곤충의 장내 세균 다양성 분석 및 섬유소 분해균 탐색

최민영,안재형,송재경,김성현,배진우,원항연,Choi, Min-Young,Ahn, Jae-Hyung,Song, Jaekyeong,Kim, Seong-Hyun,Bae, Jin-Woo,Weon, Hang-Yeon 한국미생물학회 2015 미생물학회지 Vol.51 No.3

In this study, gut bacterial communities in xylophagous insects were analyzed using the pyrosequencing of 16S rRNA genes for their potential biotechnological applications in lignocelluloses degradation. The result showed that operational taxonomic units (OTUs), species richness and diversity index were higher in the hindgut than in the midgut of all insect samples analyzed. The dominant phyla or classes were Firmicutes (54.0%), Bacteroidetes (14.5%), ${\gamma}-Proteobacteria$ (12.3%) in all xylophagous insects except for Rhinotermitidae. The principal coordinates analysis (PCoA) showed that the bacterial community structure mostly clustered according to phylogeny of hosts rather than their habitats. In our study, the two carboxymethyl cellulose (CMC)-degrading isolates which showed the highest enzyme activity were most closely related to Bacillus toyonensis $BCT-7112^T$ and Lactococcus lactis subsp. hordniae $NCDO\;2181^T$, respectively. Cellulolytic enzyme activity analysis showed that ${\beta}-1,4-glucosidase$, ${\beta}-1,4-endoglucanase$ and ${\beta}-1,4-xylanase$ were higher in the hindgut of Cerambycidae. The results demonstrate that xylophagous insect guts harbor diverse gut bacteria, including valuable cellulolytic bacteria, which could be used for various biotechnological applications. 목질 섭식 곤충에 관한 장내 세균 군집의 연구를 이용한 lignocellulose의 분해는 생명 공학적 응용에 있어 큰 잠재력을 갖는다. 본 연구에서 목질 섭식 곤충의 장내 세균 군집은 16S rRNA 유전자를 기반으로 한 파이로시퀀싱 방법을 이용하여 분석되었다. 분석된 모든 곤충에서 중장보다 후장에서 OTU수, 종 풍부도, 다양성 지수가 높았다. 세균 문 또는 강 수준의 다양성을 분석한 결과, 흰개미를 제외한 곤충의 장내 군집에는 Firmicutes, Bacteroidetes, ${\gamma}-Proteobacteria$가 우점하였다. PCoA (principal coordinates analysis)를 이용하여 세균의 군집 구조를 분석한 결과, 서식지보다는 곤충의 과별로 클러스터링 되는 경향이었다. CMC 분해 활성이 가장 높은 두 균주는 Bacillus toyonensis $BCT-7112^T$와 Lactococcus lactis subsp. hordniae $NCDO\;2181^T$과 유연관계가 높았다. 장 적출물의 섬유소 분해활성 실험 결과, 하늘소 후장에서 ${\beta}-1,4-glucosidase$, ${\beta}-1,4-endoglucanase$, ${\beta}-1,4-xylanase$의 효소활성이 가장 높았다. 본 연구에서는 목질 섭식 곤충의 장내에 다양하고 풍부한 세균이 서식하며, 섬유소를 분해하는 세균이 존재한다는 사실을 확인하였고, 이로부터 다양하고 유용한 섬유소 분해균을 분리할 수 있을 것으로 판단되었다.

Complete genome sequence of Bacillus sp. 275, producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes

Gong, G.,Kim, S.,Lee, S.M.,Woo, H.M.,Park, T.H.,Um, Y. Elsevier Science Publishers 2017 Journal of biotechnology Vol.254 No.-

<P>Technologies for degradation of three major components of lignocellulose (e.g. cellulose, hemicellulose and lignin) are needed to efficiently utilize lignocellulose. Here, we report Bacillus sp. 275 isolated from a mudflat exhibiting various lignocellulolytic activities including cellulase, xylanase, laccase and peroxidase in the cell culture supernatant. The complete genome of Bacillus sp. 275 strain contains 3832 protein cording sequences and an average G + C content of 46.32% on one chromosome (4045,581bp) and one plasmid (6389bp). The genes encoding enzymes related to the degradation of cellulose, xylan and lignin were detected in the Bacillus sp. 275 genome. In addition, the genes encoding glucosidases that hydrolyze starch, mannan, galactoside and arabinan were also found in the genome, implying that Bacillus sp. 275 has potentially a wide range of uses in the degradation of polysaccharide in lignocellulosic biomasses.</P>

Cellobiose dehydrogenase from the agaricomycete Coprinellus aureogranulatus and its application for the synergistic conversion of rice straw

Nghi Do Huu,Kellner Harald,Büttner Enrico,Huong Le Mai,Duy Le Xuan,Giap Vu Dinh,Quynh Dang Thu,Hang Tran Thi Nhu,Verberckmoes An,Diels Ludo,Liers Christiane,Hofrichter Martin 한국응용생명화학회 2021 Applied Biological Chemistry (Appl Biol Chem) Vol.64 No.5

From the biotechnological viewpoint, the enzymatic disintegration of plant lignocellulosic biomass is a promising goal since it would deliver fermentable sugars for the chemical sector. Cellobiose dehydrogenase (CDH) is a vital component of the extracellular lignocellulose-degrading enzyme system of fungi and has a great potential to improve catalyst efficiency for biomass processing. In the present study, a CDH from a newly isolated strain of the agaricomycete Coprinellus aureogranulatus (CauCDH) was successfully purified with a specific activity of 28.9 U mg− 1. This pure enzyme (MW = 109 kDa, pI = 5.4) displayed the high oxidative activity towards β-1–4-linked oligosaccharides. Not least, CauCDH was used for the enzymatic degradation of rice straw without chemical pretreatment. As main metabolites, glucose (up to 165.18 ± 3.19 mg g− 1), xylose (64.21 ± 1.22 mg g− 1), and gluconic acid (5.17 ± 0.13 mg g− 1) could be identified during the synergistic conversion of this raw material with the fungal hydrolases (e.g., esterase, cellulase, and xylanase) and further optimization by using an RSM statistical approach.

Towards a miniaturized culture screening for cellulolytic fungi and their agricultural lignocellulosic degradation

Jantima Arnthong,Chatuphon Siamphan,Charuwan Chuaseeharonnachai,Nattawut Boonyuen,Surisa Suwannarangsee 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.11

The substantial use of fungal enzymes to degrade lignocellulosic plant biomass has widely been attributed to the extensive requirement of powerful enzyme-producing fungal strains. In this study, a two-step screening procedure for finding cellulolytic fungi, involving a miniaturized culture method with shake-flask fermentation, was proposed and demonstrated. We isolated 297 fungal strains from several cellulose-containing samples found in two different locations in Thailand. By using this screening strategy, we then selected 9 fungal strains based on their potential for cellulase production. Through sequence-based identification of these fungal isolates, 4 species in 4 genera were identified: Aspergillus terreus (3 strains: AG466, AG438 and AG499), Penicillium oxalicum (4 strains: AG452, AG496, AG498 and AG559), Talaromyces siamensis (1 strain: AG548) and Trichoderma afroharzianum (1 strain: AG500). After examining their lignocellulose degradation capacity, our data showed that P. oxalicum AG452 exhibited the highest glucose yield after saccharification of pretreated sugarcane trash, cassava pulp and coffee silverskin. In addition, Ta. siamensis AG548 produced the highest glucose yield after hydrolysis of pretreated sugarcane bagasse. Our study demonstrated that the proposed two-step screening strategy can be further applied for discovering potential cellulolytic fungi isolated from various environmental samples. Meanwhile, the fungal strains isolated in this study will prove useful in the bioconversion of agricultural lignocellulosic residues into valuable biotechnological products.

Functional Characteristics and Diversity of a Novel Lignocelluloses Degrading Composite Microbial System with High Xylanase Activity

( Peng Guo ),( Wan Bin Zhu ),( Hui Wang ),( Yu Cai Lu ),( Xiao Fen Wang ),( Dan Zheng ),( Zong Jun Cui ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.2

Effect of pretreatment solutions and conditions on decomposition and anaerobic digestion of lignocellulosic biomass in rice straw

Kim, Moonkyung,Kim, Byung-Chul,Nam, Kyoungphile,Choi, Yongju Elsevier 2018 Biochemical engineering journal Vol.140 No.-

<P><B>Abstract</B></P> <P>This study assessed the effects of hot water, acid, and alkali pretreatments on the lignocellulosic composition of rice straw and methane production potential of the pretreated solids-reagent mixture. Autoclaving (121 °C, 1.45 atm, 60 min) after addition of 2% H<SUB>2</SUB>SO<SUB>4</SUB> showed the highest lignocellulose decomposition efficiency of 65.4%. However, the methane production potential was even smaller than that of the untreated sample, indicating the inhibitory effect of the acid in the biogas production process. On the other hand, hot water- and alkali-pretreated samples showed a factor of 2.1 greater methane production potential than untreated sample despite the relatively lower lignocellulose decomposition efficiency of 17.0–50.4%. By simply keeping the rice straw immersed in water at 100 °C for 30–60 min. or autoclaving prior to anaerobic digestion, the methane production potential of 805.8–824.2 mL of CH<SUB>4</SUB>/g total carbon and the overall lignocellulose degradability of 73.6–84.4% was achieved. The results of this study show the potential of hot water pretreatment as an efficient, chemical-free method to improve the feasibility of methane production from rice straw.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Decomposition of rice straw is observed with all pretreatment methods. </LI> <LI> Sulfuric acid pretreatment inhibits subsequent anaerobic biodegradation. </LI> <LI> Hot water and alkali pretreatment are better for enhancing biodegradability. </LI> <LI> Hot water pretreatment substantially enhances methane production potential. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>