Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives.

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

Comparative Gut Microbiomes of Four Species Representing the Higher and the Lower Termites.

Aiming at learning the association between the gut microbiota and termites with different diet habits and phylogenetic positions, the gut bacteria of three populations for each of the two higher termites (wood-feeding Mironasutitermes shangchengensis and fungus-feeding Odontotermes formosanus) and two wood-feeding lower termites (Tsaitermes ampliceps and Reticulitermes flaviceps) were analyzed by high-throughput 454 pyrosequencing of 16S V1-V3 amplicons. As results, 132 bacterial genera and some unidentified operational taxonomic units within 29 phyla in the gut bacteria were detected, with Spirochaetes (11-55%), Firmicutes (7-18%), Bacteroidetes (7-31%), and Proteobacteria (8-14%) as the main phyla, and Treponema, TG5, Dysgonomonas, Tannerella, za29, Lactococcus, Pseudomonas, and SJA-88 as the common genera in all the four termites. The diversity of gut bacterial communities in the higher termite guts was significantly greater than that in the lower termites; while the gut microbiota in M. shangchengensis (wood-feeding higher termite) was more similar to those of the wood-feeding lower termites rather than that of O. formosanus (fungus-feeding higher termite), and phylum Spirochaetes and nitrogen-fixing bacteria were super-dominant in the wood-feeding termites, despite of their phylogenetic relations. This study reported for the first time the gut bacterial communities for the termites of M. shangchengensis and T. ampliceps and the comparative analyses showed that the gut microbial communities varied according to the phylogeny and the diet habits of termites.

Reassessment of the role of CaCO3 in n-butanol production from pretreated lignocellulosic biomass by Clostridium acetobutylicum.

In this study, the role of CaCO3 in n-butanol production was further investigated using corn straw hydrolysate (CSH) media by Clostridium acetobutylicum CICC 8016. CaCO3 addition stimulated sugars utilization and butanol production. Further study showed that calcium salts addition to CSH media led to the increase in Ca2+ concentration both intracellularly and extracellularly. Interestingly, without calcium salts addition, intracellular Ca2+ concentration in the synthetic P2 medium was much higher than that in the CSH medium despite the lower extracellular Ca2+ concentrations in the P2 medium. These results indicated that without additional calcium salts, Ca2+ uptake by C. acetobutylicum CICC 8016 in the CSH medium may be inhibited by non-sugar biomass degradation compounds, such as furans, phenolics and organic acids. Comparative proteomics analysis results showed that most enzymes involved in glycolysis, redox balance and amino acids metabolism were up-regulated with CaCO3 addition. This study provides further insights into the role of CaCO3 in n-butanol production using real biomass hydrolysate.

Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity.

A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

Variation in the Gut Microbiota of Termites (Tsaitermes ampliceps) Against Different Diets.

Termites are well recognized for their thriving on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling microbiota; however, the effects of diet changes on termite gut microbiota are poorly understood, especially for the lower termites. In this study, we employed high-throughput 454 pyrosequencing of 16S V1-V3 amplicons to compare gut microbiotas of Tsaitermes ampliceps fed with lignin-rich and lignin-poor cellulose diets after a 2-week-feeding period. As a result, the majority of bacterial taxa were shared across the treatments with different diets, but their relative abundances were modified. In particular, the relative abundance was reduced for Spirochaetes and it was increased for Proteobacteria and Bacteroides by feeding the lignin-poor diet. The evenness of gut microbiota exhibited a significant difference in response to the diet type (filter paper diets < corn stover diets < wood diets), while their richness was constant, which may be related to the lower recalcitrance of this biomass to degradation. These results have important implications for sampling and analysis strategies to probe the lignocellulose degradation features of termite gut microbiota and suggest that the dietary lignocellulose composition could cause shifting rapidly in the termite gut microbiota.

The preparation and ethanol fermentation of high-concentration sugars from steam-explosion corn stover.

In the field of biofuel ethanol, high-concentration- reducing sugars made from cellulosic materials lay the foundation for high-concentration ethanol fermentation. In this study, corn stover was pre-treated in a process combining chemical methods and steam explosion; the cellulosic hydrolyzed sugars obtained by fed-batch saccharification were then used as the carbon source for high-concentration ethanol fermentation. Saccharomyces cerevisiae 1308, Angel yeast, and Issatchenkia orientalis were shake-cultured with Pachysolen tannophilus P-01 for fermentation. Results implied that the ethanol yields from the three types of mixed strains were 4.85 g/100 mL, 4.57 g/100 mL, and 5.02 g/100 mL (separately) at yield rates of 91.6, 89.3, and 92.2%, respectively. Therefore, it was inferred that shock-fermentation using mixed strains achieved a higher ethanol yield at a greater rate in a shorter fermentation period. This study provided a theoretical basis and technical guidance for the fermentation of industrial high-concentrated cellulosic ethanol.

Effects of thermo-chemical pretreatment plus microbial fermentation and enzymatic hydrolysis on saccharification and lignocellulose degradation of corn straw.

In order to increase corn straw degradation, the straw was kept in the combined solution of 15% (w/w) lime supernatant and 2% (w/w) sodium hydroxide with liquid-to-solid ratio of 13:1 (mL/g) at 83.92°C for 6h; and then added with 3% (v/v) H2O2 for reaction at 50°C for 2h; finally cellulase (32.3 FPU/g dry matter) and xylanase (550 U/g dry matter) was added to keep at 50°C for 48 h. The maximal reducing sugars yield (348.77 mg/g) was increased by 126.42% (P<0.05), and the degradation rates of cellulose, hemicellulose and lignin in pretreated corn straw with enzymatic hydrolysis were increased by 40.08%, 45.71% and 52.01%, compared with the native corn straw with enzymatic hydrolysis (P<0.05). The following study indicated that the combined microbial fermentation and enzymatic hydrolysis could further increase straw degradation and reducing sugar yield (442.85 mg/g, P<0.05).

[Effect of byproducts in lignocellulose hydrolysates on ethanol fermentation by Issatchenkia orientalis].

Byproducts in lignocellulose hydrolysates, namely sodium formate (1 to 5 g/L), sodium acetic (2.5 to 8.0 g/L), furfural (0.2-2 g/L), 5-hydroxymethylfurfural (5-HMF, 1 to 1.0 g/L) or vanillin (0.5 to 2 g/L) were used to evaluate their effects on ethanol fermentation by Issatchenkia orientalis HN-1 using single factor test and the response surface central composite experiment. Results showed that most of the byproducts had no obvious inhibition on the production of ethanol, except for the addition of 2 g/L vanillin or 1 g/L of 5-HMF, which reduced the ethanol production by 20.38% and 11.2%, respectively. However, high concentration of some byproducts in lignocellulose hydrolysates, such as sodium formate (1 to 5 g/L), sodium acetic (2.5 to 8.0 g/L), furfural (0.2 to 2 g/L) and vanillin (0.5 to 2 g/L) inhibited the growth of I. orientalis HN-1 significantly. Compared with the control, the dry cell weight of I. orientalis HN-1 decreased by 25.04% to 37.02%, 28.83% to 43.82%, 20.06% to 37.60% and 26.39% to 52.64%, respectively, when the above components were added into the fermentation broth and the fermentation lasted for 36 h. No significant interaction effect of the various inhibitors (sodium formate, sodium acetic, furfural and vanillin) except for vanillin single factor on the ethanol production was observed based on the central composite experiments. The concentrations of byproducts in most lignocellulose hydrolysates were below the initial inhibition concentration on ethanol production by Issatchenkia orientalis HN-1, which indicated that Issatchenkia orientalis HN-1 can be used for ethanol production from lignocellulose hydrolysates.

Biological pretreatment of corn stover with ligninolytic enzyme for high efficient enzymatic hydrolysis.

Aiming at increasing the efficiency of transferring corn stover into sugars, a biological pretreatment was developed and investigated in this study. The protocol was characterized by the pretreatment with crude ligninolytic enzymes from Phanerochete chrysosporium and Coridus versicolor to break the lignin structure in corn stover, followed by a washing procedure to eliminate the inhibition of ligninolytic enzyme on cellulase. By a 2 d-pretreatment, sugar yield from corn stover hydrolysis could be increased by 50.2% (up to 323 mg/g) compared with that of the control. X-ray diffractometry and FT-IR analysis revealed that biological pretreatment could partially remove the lignin of corn stover, and consequently enhance the enzymatic hydrolysis efficiency of cellulose and hemeicellulose. In addition, the amount of microbial inhibitors, such as acetic acid and furfural, were much lower in biological pretreatment than that in acid pretreatment. This study provided a promising pretreatment method for biotransformation of corn stovers.

Effect of steam explosion and microbial fermentation on cellulose and lignin degradation of corn stover.

In order to increase nutrient values of corn stover, effects of steam explosion (2.5 MPa, 200 s) and Aspergillus oryzae (A. oryzae) fermentation on cellulose and lignin degradation were studied. The results showed the contents of cellulose, hemicellulose and lignin in the exploded corn stover were 8.47%, 50.45% and 36.65% lower than that in the untreated one, respectively (P<0.05). The contents of cellulose and hemicellulose in the exploded and fermented corn stover (EFCS) were decreased by 24.36% and 69.90%, compared with the untreated one (P<0.05); decreased by 17.35% and 38.59%, compared with the exploded one (P<0.05). The scanning electron microscope observations demonstrated that the combined steam explosion and fermentation destructed corn stover. The activities of enzymes in EFCS were increased. The metabolic experiment showed that about 8% EFCS could be used to replace corn meal in broiler diets, which made EFCS become animal feedstuff possible.

[Optimization of corn stover hydrolysis by fed-batch process].

High-concentration sugars production from stover is an important perspective technology for the cellulosic ethanol industrialization. Fed-batch process is an effective way to achieve this goal in the fermentation industry. In this study, based on fed-batch process, high-concentration sugars were produced from pretreated corn stover by enzymatic hydrolysis. After being pretreated by the dilute sulphuric acid, the impacts of the ratio of solid raw material to liquid culture, the content of supplementary materials and the refilling time on the saccharification rate were investigated. Results showed that the initial ratio of solid raw material to liquid culture was 20% (W/V) and the initial concentrations of enzymes for xylanase, cellulose and pectinase were 220 U, 6 FPU, and 50 U per gram of substrates, respectively. After 24 hours and 48 hours, 8% pretreated corn stovers were added respectively together with the additions of xylanase (20 U) and cellulose (2 FPU) per gram of substrates. After 72 hours, the final concentration of reducing sugar was increased to 138.5 g/L from 48.5 g/L of the non fed-batch process. The rate of enzyme hydrolysis of the raw material was 62.5% of the thoretical value in the fed-batch process. This study demonstrated that the fed-batch process could significantly improve the concentration of reducing sugar.

Immobilization of Aspergillus niger xylanase on chitosan using dialdehyde starch as a coupling agent.

Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS) beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed to be 5 degrees C higher than that of free enzyme (50 degrees C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability as compared to the CS-GA-immobilized and free xylanase. The apparent K (m) and V (max) values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 mumol/min/mg protein, respectively. The CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose, as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase immobilization application.