Lignin Degradation by Klebsiella aerogenes TL3 under Anaerobic Conditions.

Lignin, the largest non-carbohydrate component of lignocellulosic biomass, is also a recalcitrant component of the plant cell wall. While the aerobic degradation mechanism of lignin has been well-documented, the anaerobic degradation mechanism is still largely elusive. In this work, a versatile facultative anaerobic lignin-degrading bacterium, Klebsiella aerogenes TL3, was isolated from a termite gut, and was found to metabolize a variety of carbon sources and produce a single kind or multiple kinds of acids. The percent degradation of alkali lignin reached 14.8% under anaerobic conditions, and could reach 17.4% in the presence of glucose within 72 h. Based on the results of infrared spectroscopy and 2D nuclear magnetic resonance analysis, it can be inferred that the anaerobic degradation of lignin may undergo the cleavage of the C-O bond (ß-O-4), as well as the C-C bond (ß-5 and ß-ß), and involve the oxidation of the side chain, demethylation, and the destruction of the aromatic ring skeleton. Although the anaerobic degradation of lignin by TL3 was slightly weaker than that under aerobic conditions, it could be further enhanced by adding glucose as an electron donor. These results may shed new light on the mechanisms of anaerobic lignin degradation.

Host-Specific Diversity of Culturable Bacteria in the Gut Systems of Fungus-Growing Termites and Their Potential Functions towards Lignocellulose Bioconversion.

Fungus-growing termites are eusocial insects that represent one of the most efficient and unique systems for lignocellulose bioconversion, evolved from a sophisticated symbiosis with lignocellulolytic fungi and gut bacterial communities. Despite a plethora of information generated during the last century, some essential information on gut bacterial profiles and their unique contributions to wood digestion in some fungus-growing termites is still inadequate. Hence, using the culture-dependent approach, the present study aims to assess and compare the diversity of lignocellulose-degrading bacterial symbionts within the gut systems of three fungus-growing termites: Ancistrotermes pakistanicus, Odontotermes longignathus, and Macrotermes sp. A total of 32 bacterial species, belonging to 18 genera and 10 different families, were successfully isolated and identified from three fungus-growing termites using Avicel or xylan as the sole source of carbon. Enterobacteriaceae was the most dominant family represented by 68.1% of the total bacteria, followed by Yersiniaceae (10.6%) and Moraxellaceae (9%). Interestingly, five bacterial genera such as Enterobacter, Citrobacter, Acinetobacter, Trabulsiella, and Kluyvera were common among the tested termites, while the other bacteria demonstrated a termite-specific distribution. Further, the lignocellulolytic potential of selected bacterial strains was tested on agricultural waste to evaluate their capability for lignocellulose bioconversion. The highest substrate degradation was achieved with E. chengduensis MA11 which degraded 45.52% of rice straw. All of the potential strains showed endoglucanase, exoglucanase, and xylanase activities depicting a symbiotic role towards the lignocellulose digestion within the termite gut. The above results indicated that fungus-growing termites harbor a diverse array of bacterial symbionts that differ from species to species, which may play an inevitable role to enhance the degradation efficacy in lignocellulose decomposition. The present study further elaborates our knowledge about the termite-bacteria symbiosis for lignocellulose bioconversion which could be helpful to design a future biorefinery.

Genomic insights into the metabolic potential of a novel lignin-degrading and polyhydroxyalkanoates producing bacterium Pseudomonas sp. Hu109A.

Lignin is the most abundant heterogeneous aromatic polymer present on planet Earth and is recalcitrant to degradation due to its complex structure, therefore, imposing a challenge to biorefinery procedures. Identifying new microbial strains with the potential to valorize lignin into useful compounds is indispensable to achieving green sustainable consumption. In this study, a novel Pseudomonas strain designated as Hu109A was isolated from the termite gut and the genome was sequenced and analyzed further. The genome contains a circular chromosome with the size of 5,131,917 bp having a GC content of 62.6% and 4698 genes. Genome annotation reveals that the strain possesses lignin-oxidizing enzymes such as DyP-type peroxidases, laccase, dioxygenase, and aromatic degradation gene clusters. The genome also contains O-methyltransferases which function in accelerating the lignin degradation by methylating the free hydroxyl phenolic compounds which in high concentration can inhibit the lignin peroxidase. Furthermore, the genome exhibits two gene clusters encoding the enzymes related to polyhydroxyalkanoates (PHA) synthesis. Pseudomonas strains are generally assumed to produce medium chain length PHAs (mcl-PHAs) only, however, strain Hu109A contains both Class II PHA synthase genes involved in mcl-PHAs and Class III PHA synthase gene involved in short-chain length PHAs (scl-PHAs). Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that using 1 g/L lignin as the sole carbon source, the maximum production of PHA observed was 103.68 mg/L, which increased to 186 mg/L with an increase in lignin concentration to 3 g/L. However, PHA production while using glucose as the sole carbon source was significantly lower than the lignin source, and maximum production was 125.6 mg/L with 3 g/L glucose. The strain Hu109A can tolerate a broad range of solvents including methanol, isopropanol, dimethylformamide, and ethanol, revealing its potential for industrial applications.

Arabinan hydrolysis by GH43 enzymes of Hungateiclostridium clariflavum and the potential synergistic mechanisms.

Glycoside hydrolase family 43 (GH43) represents a major source of arabinan- and arabinoxylan-active enzymes. Interestingly, some microbes remarkably enriched GH genes of this family, with the reason unknown. Hungateiclostridium clariflavum DSM 19,732 is an efficient lignocellulose degrader, which harbors up to 7 GH43 genes in its genome. We cloned three of the seven GH43 genes, and found that Abn43A is a unique endoarabinanase, which unprecedently showed approximately two times larger activity on sugar beet arabinan (116.8 U/mg) than that on linear arabinan, and it is efficient in arabinooligosaccharide production. Abn43B is an exoarabinanase which directly releases arabinose from linear arabinan. Abn43C is an α-L-arabinofuranosidase which is capable of splitting the arabinose side-chains from arabinooligosaccharides, arabinoxylooligosaccharides, and arabinoxylan. Most importantly, the three GH43 enzymes synergized in hydrolyzing arabinan. Compared to Abn43B alone, a supplement of Abn43A increased the arabinose production from linear arabinan by 150%, reaching 0.44 g/g arabinan. Moreover, an addition of Abn43C to Abn43A and Abn43B boosted the arabinose production from sugar beet arabinan by 15 times, reaching 0.262 g/g arabinan. Our work suggested the intensified functions of multiple GH43 enzymes toward arabinan degradation in H. clariflavum, and a potential synergetic mechanism among the three GH43 enzymes is suggested. KEY POINTS: • Endoarabinanase GH43A prefers branched substrate to linear one • Exoarabinanase GH43B can directly release arabinose from linear arabinan • The three GH43 enzymes synergized in arabinan hydrolysis.

New Insights into the Co-Occurrences of Glycoside Hydrolase Genes among Prokaryotic Genomes through Network Analysis.

Glycoside hydrolase (GH) represents a crucial category of enzymes for carbohydrate utilization in most organisms. A series of glycoside hydrolase families (GHFs) have been classified, with relevant information deposited in the CAZy database. Statistical analysis indicated that most GHFs (134 out of 154) were prone to exist in bacteria rather than archaea, in terms of both occurrence frequencies and average gene numbers. Co-occurrence analysis suggested the existence of strong or moderate-strong correlations among 63 GHFs. A combination of network analysis by Gephi and functional classification among these GHFs demonstrated the presence of 12 functional categories (from group A to L), with which the corresponding microbial collections were subsequently labeled, respectively. Interestingly, a progressive enrichment of particular GHFs was found among several types of microbes, and type-L as well as type-E microbes were deemed as functional intensified species which formed during the microbial evolution process toward efficient decomposition of lignocellulose as well as pectin, respectively. Overall, integrating network analysis and enzymatic functional classification, we were able to provide a new angle of view for GHs from known prokaryotic genomes, and thus this study is likely to guide the selection of GHs and microbes for efficient biomass utilization.

Highly thermostable GH51 α-arabinofuranosidase from Hungateiclostridium clariflavum DSM 19732.

Arabinofuranosidase plays an essential role in the process of hydrolysis of arabinoxylan (AX). Thermostable, versatile, and efficient arabinofuranosidase is thus of great interest for the biorefinery industry. A GH51 arabinofuranosidase, Abf51, from Hungateiclostridium clariflavum DSM 19732 was heterogeneously expressed in Escherichia coli. Abf51 was found to have an optimal pH and temperature of 6.5 and 60 °C, respectively, with very high thermostability. At the optimal working temperature (60 °C), Abf51 retained over 90% activity after a 2-day incubation and over 60% activity after a 6-day incubation. Abf51 could effectively remove the arabinofuranosyls from three kinds of AX oligosaccharides [23-α-L-arabinofuranosyl-xylotriose (A2XX), 32-α-L-arabinofuranosyl-xylobiose (A3X), and 2333-di-α-L-arabinofuranosyl-xylotriose (A2 + 3XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency (Kcat/Km) was observed using p-nitrophenyl-α-L-arabinofuranoside (pNPAF) as a substrate (205.0 s-1 mM-1), followed by using A3X (22.8 s-1 mM-1), A2XX (6.9 s-1 mM-1), and A2 + 3XX (0.5 s-1 mM-1) as substrates. Moreover, the presence of Abf51 significantly stimulated the saccharification level of AX (18.5 g L-1) up to six times along with a ß-xylanase as well as a ß-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (ß/α)8-barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.

Transcriptome analysis of the digestive system of a wood-feeding termite (Coptotermes formosanus) revealed a unique mechanism for effective biomass degradation.

BACKGROUND: Wood-feeding termite, Coptotermes formosanus Shiraki, represents a highly efficient system for biomass deconstruction and utilization. However, the detailed mechanisms of lignin modification and carbohydrate degradation in this system are still largely elusive. RESULTS: In order to reveal the inherent mechanisms for efficient biomass degradation, four different organs (salivary glands, foregut, midgut, and hindgut) within a complete digestive system of a lower termite, C. formosanus, were dissected and collected. Comparative transcriptomics was carried out to analyze these organs using high-throughput RNA sequencing. A total of 71,117 unigenes were successfully assembled, and the comparative transcriptome analyses revealed significant differential distributions of GH (glycosyl hydrolase) genes and auxiliary redox enzyme genes in different digestive organs. Among the GH genes in the salivary glands, the most abundant were GH9, GH22, and GH1 genes. The corresponding enzymes may have secreted into the foregut and midgut to initiate the hydrolysis of biomass and to achieve a lignin-carbohydrate co-deconstruction system. As the most diverse GH families, GH7 and GH5 were primarily identified from the symbiotic protists in the hindgut. These enzymes could play a synergistic role with the endogenous enzymes from the host termite for biomass degradation. Moreover, twelve out of fourteen genes coding auxiliary redox enzymes from the host termite origin were induced by the feeding of lignin-rich diets. This indicated that these genes may be involved in lignin component deconstruction with its redox network during biomass pretreatment. CONCLUSION: These findings demonstrate that the termite digestive system synergized the hydrolysis and redox reactions in a programmatic process, through different parts of its gut system, to achieve a maximized utilization of carbohydrates. The detailed unique mechanisms identified from the termite digestive system may provide new insights for advanced design of future biorefinery.

Characterization of a laccase from a wood-feeding termite, Coptotermes formosanus.

Coptotermes formosanus Shiraki is a wood-feeding termite which secretes a series of lignolytic and cellulolytic enzymes for woody biomass degradation. However, the lignin modification mechanism in the termite is largely elusive, and the characteristics of most lignolytic enzymes in termites remain unknown. In this study, a laccase gene lac1 from C. formosanus was heterogeneously expressed in insect Sf9 cells. The purified Lac1 showed strong activities toward hydroquinone (305 mU/mg) and 2,6-dimethoxyphenol (2.9 mU/mg) with low Km values, but not veratryl alcohol or 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). Lac1 could function well from pH 4.5 to 7.5, and its activity was significantly inhibited by H2 O2 at above 4.85 mmol/L (P < 0.01). In addition, the lac1 gene was found to be mainly expressed in the salivary glands and foregut of C. formosanus, and seldom in the midgut or hindgut. These findings suggested that Lac1 is a phenol-oxidizing laccase like RflacA and RflacB from termite Reticulitermes flavipes, except that Lac1 was found to be more efficient in phenol oxidation, and it did not require H2 O2 for its function. It is suspected that this kind of termite laccase might only be able to directly oxidize low redox-potential substrates, and the high redox-potential groups in lignin might be oxidized by other enzymes in the termite or by using the Fenton reaction.

Genomics and biochemistry investigation on the metabolic pathway of milled wood and alkali lignin-derived aromatic metabolites of Comamonas serinivorans SP-35.

BACKGROUND: The efficient depolymerization and utilization of lignin are one of the most important goals for the renewable use of lignocelluloses. The degradation and complete mineralization of lignin by bacteria represent a key step for carbon recycling in land ecosystems as well. However, many aspects of this process remain unclear, for example, the complex network of metabolic pathways involved in the degradation of lignin and the catabolic pathway of intermediate aromatic metabolites. To address these subjects, we characterized the deconstruction and mineralization of lignin with milled wood lignin (MWL, the most representative molecule of lignin in its native state) and alkali lignin (AL), and elucidated metabolic pathways of their intermediate metabolites by a bacterium named Comamonas serinivorans SP-35. RESULTS: The degradation rate of MWL reached 30.9%, and its particle size range was decreased from 6 to 30 µm to 2-4 µm-when cultured with C. serinivorans SP35 over 7 days. FTIR analysis showed that the C-C and C-O-C bonds between the phenyl propane structures of lignin were oxidized and cleaved and the side chain structure was modified. More than twenty intermediate aromatic metabolites were identified in the MWL and AL cultures based on GC-MS analysis. Through genome sequencing and annotation, and from GC-MS analysis, 93 genes encoding 33 enzymes and 5 regulatory factors that may be involved in lignin degradation were identified and more than nine metabolic pathways of lignin and its intermediates were predicted. Of particular note is that the metabolic pathway to form the powerful antioxidant 3,4-dihydroxyphenylglycol is described for the first time in bacteria. CONCLUSION: Elucidation of the ß-aryl ether cleavage pathway in the strain SP-35 indicates that the ß-aryl ether catabolic system is not only present in the family of Sphingomonadaceae, but also other species of bacteria kingdom. These newly elucidated catabolic pathways of lignin in strain SP-35 and the enzymes responsible for them provide exciting biotechnological opportunities for lignin valorization in future.

Characterization of cold adapted and ethanol tolerant ß-glucosidase from Bacillus cellulosilyticus and its application for directed hydrolysis of cellobiose to ethanol.

A ß-glucosidase gene from Bacillus cellulosilyticus was expressed in Escherichia coli BL21 and characterized. The recombinant enzyme (BcBgl1A) showed the optimal activity at 40°C, and the half-life of BcBgl1A activity at 40°C was 24h. BcBgl1A was sensitive to heat and exhibited obvious cold-adapted activity, retained 27.2% and 46.5% of the optimal activity at 4 and 20°C, respectively. The best activity was observed at pH 7.0 and stable over the range of pH 6.0-8.0. The activity was significantly enhanced by Fe2+, Fe3+ and Mn2+, also showed a high level of NaCl tolerance. BcBgl1A exhibited high activity using both pNPG and cellobiose as substrates, the Vmax for pNPG and cellobiose was 66.2 and 151.5U/mg of protein, respectively, and its Km values were 2.97 and 10.4mM, respectively. Catalysis efficiency (Kcat/Km) of 45.8s-1 (cellobiose) and 70.3s-1 (pNPG). It displayed high tolerance to glucose and ethanol, the Ki and IC50 value was 200mM and 15% (v/v), respectively. Additionally, the BcBgl1A was found to be highly efficient for conversion of cellobiose (100g/L) to ethanol by simultaneous saccharification and fermentation process, achieved an ethanol content of 47.2g/l corresponding to 87.9% of the theoretical ethanol yield. The results suggest that BcBgl1A has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes.

Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.

Purification and characterization of a hemocyanin (Hemo1) with potential lignin-modification activities from the wood-feeding termite, Coptotermes formosanus Shiraki.

Coptotermes formosanus Shiraki is a well-known wood-feeding termite, which can degrade not only cellulose and hemicellulose polysaccharides, but also some aromatic lignin polymers with its enzyme complex to the woody biomass. In this study, a very abundant protein was discovered and purified, using a three-step column chromatography procedure, from the tissue homogenate of the salivary glands and the gut of C. formosanus. Mass spectrometric analysis and the following peptide searching against the mRNA database toward this termite species indicated that the novel protein was a hemocyanin enzyme, termed as Hemo1, which further exhibited a strong oxidase activity in the substrate bioassays toward ABTS [2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], as well as other aromatic analogues, such as catechol and veratryl alcohols. This oxidative protein was an acid-favored enzyme with a molecular weight at 82 kDa, and highly active at 80 °C. These findings indicated that the novel protein, hemocyanin, discovered from the gut system of C. formosanus, might be an important ligninolytic enzyme involved in the biomass pretreatment processing, which will potentially enhance the digestibility and utilization of biomass polysaccharides in termite digestive systems.

Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing.

In this study, 341, 246, and 386 positive clones with endo-ß-1,4-glucanase, ß-glucosidase, and endo-ß-1,4-xylanase activities, respectively, were identified by screening from a metagenomic fosmid library constructed from a biogas digester. Subsequently, pools of 4, 10, and 16 positive clones were subjected to 454 pyrosequencing in different subruns. In total, 21 unique glycosyl hydrolase (GH) genes were predicted by bioinformatic analysis, which showed similarities to their nearest neighbors from 39 % to 72 %. In addition to bioinformatics prediction, nine GH genes were expressed and purified to identify their activity with four kinds of substrates. The activities of the most expressed proteins were consistent with their annotation based on bioinformatics prediction; however, three GH genes belonging to the GH5 family showed different activities from their annotation. An efficient acidic cellulase En1 had an optimal condition at 55 °C, pH 5.5, with a specific activity toward carboxymethylcellulose at 118 U/mg and K m at 12.8 g/L. This study demonstrated that there are diverse GHs in the biogas digester system with potential industrial application in lignocellulose hydrolysis, and their activities should be investigated with different substrates before their application. Additionally, pool sequencing of positive fosmid clones might be a cost-effective approach to obtain functional genes from metagenomic libraries.

Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei.

A metagenomic fosmid library containing 1 × 10(5) clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and ß-D-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-ß-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi.

Effect of key factors on hydrogen production from cellulose in a co-culture of Clostridium thermocellum and Clostridium thermopalmarium.

A cellulolytic, hydrogen-producing bacterium (Clostridiumthermocellum DSM 1237) and a non-cellulolytic, hydrogen-producing bacterium (Clostridiumthermopalmarium DSM 5974) were co-cultured at 55 degrees C, using cellulose as the sole substrate. At a low load of cellulose (filter paper, 4.5g/L), yeast extract had a significant effect on cellulose degradation and hydrogen production. The extent of cellulose utilization and hydrogen production displayed a linear relationship with the logarithm of the yeast extract concentration, and the optimal weight ratio of yeast extract to cellulose was 1:1. At a high load of filter paper (9g/L), an alkali chemical was required to maintain efficient cellulose degradation. As the KHCO3 concentration increased from 0 to 60mM, the utilized cellulose increased from 1.23g/L (13.5%) to 8.59g/L (94.3%), and maximum hydrogen production (1387ml/L of culture) occurred at 40mM KHCO(3). Increasing the inoculation ratio of C. thermopalmarium to C. thermocellum from 0.05:1 to 0.17:1 had little influence on hydrogen production, probably because of the limited availability of soluble sugar in the medium during the early stages of the co-culture.