Alei Geng

The presence of diverse carbohydrate-active enzymes (CAZymes) is crucial for the direct bioconversion of lignocellulose. In this study, various anaerobic microbial consortia were employed for the degradation of 10 g/L of minimally pretreated corncob. The involvement of lactic acid bacteria (LAB) and a CAZyme-rich bacterium (Bacteroides cellulosilyticus or Paenibacillus lautus) significantly enhanced the lactic acid production by Ruminiclostridium cellulolyticum from 0.74 to 2.67 g/L (p < 0.01), with a polysaccharide conversion of 67.6%. The supplement of a commercial cellulase cocktail, CTec 2, into the microbial consortia continuously promoted the lactic acid production to up to 3.35 g/L, with a polysaccharide conversion of 80.6%. Enzymatic assays, scanning electron microscopy, and Fourier transform infrared spectroscopy revealed the substantial functions of these CAZyme-rich consortia in partially increasing enzyme activities, altering the surface structure of biomass, and facilitating substrate decomposition. These results suggested that CAZyme-intensified consortia could significantly improve the levels of bioconversion of lignocellulose. Our work might shed new light on the construction of intensified microbial consortia for direct conversion of lignocellulose.

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 (β-O-4), as well as the C-C bond (β-5 and β-β), 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.

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.

The production of lactic acid (LA) from lignocellulosic biomass is an important route for the exploitation of renewable resources; nevertheless, effective LA production from this feedstock is challenged by several limitations, such as pentose and oligosaccharide utilization. In this study, a new strain, Lactococcus sp. X1, which is capable of fermenting glucose, xylose, and several disaccharides to produce L-lactic acid, was isolated from the gut of a wood-feeding termite, Coptotermes formosanus. Compared to conventional lactic acid bacteria, Lactococcus sp. X1 requires less complex nitrogen sources, which might in turn reduce the cost of LA production. In addition, Lactococcus sp. X1 was able to completely ferment 50 g/L of glucose within 3 days, giving a high LA yield of 99.9%, and its LA yield from 50 g/L of pretreated corncob reached up to 0.34 g/g substrates in the presence of a commercial cellulase. Strain X1 was also capable of excreting two kinds of nutritional factors, namely biotin and vitamin C, indicating its crucial role in the nourishment of the termite. In conclusion, Lactococcus sp. X1 is a new lactic acid bacterium, which may hold promise for application in cost-effective LA production as well as in the field of food additives.

Glycoside hydrolase family 43 (GH43) represents a major source of arabinan- and arabinoxylan-active enzymes. Interestingly, some microbes remarkably enrich

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.

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 (A^{2 + 3}XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency (K_cat/K_m) 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 A^{2 + 3}XX (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 β-xylanase as well as a β-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (β/α)₈-barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.

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. 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. Elucidation of the β-aryl ether cleavage pathway in the strain SP-35 indicates that the β-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.

A β-xylosidase gene, xyl43C, from Clostridium clariflavum was heterogeneously expressed in Escherichia coli BL21. Xyl43C showed strong activity toward xylobiose, with specific activity of 76.6 U/mg and Km of 4.97 mM. The optimal pH and temperature of Xyl43C were pH 6.0 and 60 °C, respectively. Xyl43C retained 94.4% activity after incubation at 55 °C for 1 h, and 75.4% at 60 °C for 1 h. It also showed xylose tolerance with IC50 (half maximal inhibitory concentration) of approximately 100 mM. It nearly completely hydrolyzed 2 g/L of xylobiose at enzyme load of 2.51 mg/g xylobiose within 30 min and converted 40 g/L of corncob xylan into xylose at enzyme load of 1.48 mg/g xylan, with a yield of 60.9%. In conclusion, Xyl43C is an efficient xylose-tolerant β-xylosidase, with promising application potential in saccharification of xylan in biofuels industry.