Lignocellulose Degradation Research Articles

Analyses of long-term fungal degradation of spruce bark reveals varying potential for catabolism of polysaccharides and extractive compounds

The bark represents the outer protective layer of trees. It contains high concentrations of antimicrobial extractives, in addition to regular wood polymers. It represents a huge underutilized side stream in forestry, but biotechnological valorization is hampered by a lack of knowledge on microbial bark degradation. Many fungi are efficient lignocellulose degraders, and here, spruce bark degradation by five species, Dichomitus squalens, Rhodonia placenta, Penicillium crustosum, Trichoderma sp. B1, and Trichoderma reesei, was mapped, by continuously analyzing chemical changes in the bark over six months. The study reveals how fungi from different phyla degrade bark using diverse strategies, regarding both wood polymers and extractives, where toxic resin acids were degraded by Basidiomycetes but unmodified/tolerated by Ascomycetes. Proteome analyses of the white-rot D. squalens revealed several proteins, with both known and unknown functions, that were specifically upregulated during growth on bark. This knowledge can accelerate improved utilization of an abundant renewable resource.

The Role of Microbial Diversity in Lignocellulosic Biomass Degradation: A Biotechnological Perspective

AbstractLignocellulosic biomass, such as plant residues and agricultural waste, holds immense potential as a renewable resource for the production of biofuels, chemicals, and animal feed. However, the efficient degradation of lignocellulose into fermentable sugars remains a significant challenge. Recent research has highlighted the critical role of microbial diversity in lignocellulosic biomass degradation, offering new insights from a biotechnological perspective. The comprehension and utilization of microbial diversity are crucial for developing efficient biotechnological strategies for lignocellulosic biomass degradation. By uncovering the intricate relationships between microbial communities and their enzymatic machinery, researchers can optimize degradation processes, enhance biofuel production, and contribute to a more sustainable bio‐based economy. Microorganisms, including bacteria, fungi, and archaea, possess diverse enzymatic capabilities, allowing them to secrete a plethora of lignocellulolytic enzymes. Microbial organisms inhabiting extreme environments, such as the rumen, hot and cold springs, deep sea trenches, and acidic and alkaline pH environments, exhibit significant potential in generating enzymes, including hemicellulolytic and lignocellulolytic enzymes, which possess superior biochemical properties essential for industrial bioconversion applications. This review explores the ability of lignocellulosic enzymes from microbial sources to efficiently break down the lignocellulosic biomass and their potential applications in industrial biotechnology.

Genomic mining of Geobacillus stearothermophilus GF16 for xylose production from hemicellulose-rich biomasses using secreted enzymes

The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and β-xylosidase activities (5 ×103 U/L and 1.7 ×105 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach.

How lignocellulose degradation can promote the quality and function of dietary fiber from bamboo shoot residue by Inonotus obliquus fermentation

Lignocellulose constitutes the primary component of dietary fiber. We assessed how fermenting bamboo shoot residue with the medicinal white-rot fungus Inonotus obliquus affected the yield, composition, and functional attributes of dietary fiber by altering bamboo shoot residue lignocellulose's spatial structure and composition. I. obliquus secretes lignocellulolytic enzymes, which effectively enhance the degradation of holocellulose and lignin by 87.8% and 25.5%, respectively. Fermentation led to a more porous structure and reduced crystallinity. The yield of soluble dietary fiber increased from 5.1 g/100 g raw BSR to 7.1 g/100 g 9-day-fermented bamboo shoot residue. The total soluble sugar content of dietary fiber significantly increased from 9.2% to 13.8%, which improved the hydration, oil holding capacity, in vitro cholesterol, sodium cholate, and nitrite adsorption properties of dietary fiber from bamboo shoot residue. These findings confirm that I. obliquus biotransformation is promising for enhancing dietary fiber yield and quality.

Functional Study of Different Lignocellulases from Trichoderma guizhouence NJAU4742 in the Synergistic Degradation of Natural Straw

The enzyme-based degradation of lignocellulose for bioenergy production is an eco-friendly and sustainable approach. This study aimed to elucidate the enzymatic characteristics of endoglucanase (EGL), β-glucosidase (BGL), and xylanase (XYN) from Trichoderma guizhouence NJAU4742, and to explore the potential mechanisms underlying their synergistic degradation of different natural substrates. The results demonstrated that the three enzymes possessed remarkable high-temperature catalytic activity, broad pH adaptability, and responsiveness to different metal ions. The functional group absorption peaks of different substrates were shifted and altered after the synergistic action, particularly for C=O and O-H. Simultaneously, the crystallinity index of wheat straw, soybean straw, rice straw, and corn straw decreased by 7.40%, 2.37%, 20.60%, and 7.67%, respectively, compared to CK (natural straw). Additionally, the dense structure of different substrates was destroyed, and the inner parenchyma began to be exposed after the synergistic action, as observed by SEM. These findings offer valuable theoretical guidance for the development of lignocellulase applications.

Insight into the molecular transformation pathways of humic acid in the co-composting of bagasse and cow manure after adding compound microorganisms

The inoculation of compound microorganisms effectively processes lignocellulosic biomass and promotes the formation of humic acid (HA). However, the basic mechanism of HA formation in compost remains unclear. In this study, we comprehensively characterized the transformation of HA molecules during co-composting. The addition of compound microorganisms enhanced the degradation of proteinoids and lignocellulose in the dissolved organic matter (OM), thereby accelerating the humification of bagasse cow dung co-compost. Elemental and UV–Vis spectral analyses showed that inoculation with the compound microorganisms enhanced the oxidation level and aromatization of the HA structure. Furthermore, two-dimensional Fourier-transform infrared spectroscopy correlation analysis suggested that the addition of compound microorganisms accelerated the decomposition and oxidation of OM. Accordingly, premature aggregation of HA was avoided, and the side chains bonded to HA were more abundant, promoting the evolution of HA to higher-order structures. By influencing the degree of aromaticity, molecular weight, and the C2 fluorescence peak of HA, the inoculation of compound microorganisms promoted HA synthesis during bagasse composting. These results provide an important basis for revealing the mechanism by which compound microorganisms are inoculated to promote HA formation at the molecular level and serve as a reference for the production of high-quality and high-maturity compost.

Optimised Degradation of Lignocelluloses by Edible Filamentous Fungi for the Efficient Biorefinery of Sugar Beet Pulp.

The degradation of the complex structure of lignocellulosic biomass is important for its further biorefinery to value-added bioproducts. The use of effective fungal species for the optimised degradation of biomass can promote the effectiveness of the biorefinery of such raw material. In this study, the optimisation of processing parameters (temperature, time, and s/w ratio) for cellulase activity and reducing sugar (RS) production through the hydrolysis of sugar beet pulp (SBP) by edible filamentous fungi of Aspergillus, Fusarium, Botrytis, Penicillium, Rhizopus, and Verticillium spp. was performed. The production of RS was analysed at various solid/water (s/w) ratios (1:10-1:20), different incubation temperatures (20-35 °C), and processing times (60-168 h). The Aspergillus niger CCF 3264 and Penicillium oxalicum CCF 3438 strains showed the most effective carboxymethyl cellulose (CMC) degrading activity and also sugar recovery (15.9-44.8%) from SBP biomass in the one-factor experiments. Mathematical data evaluation indicated that the highest RS concentration (39.15 g/100 g d.w.) and cellulolytic activity (6.67 U/g d.w.) could be achieved using A. niger CCF 3264 for the degradation of SBP at 26 °C temperature with 136 h of processing time and a 1:15 solid/water ratio. This study demonstrates the potential of fungal degradation to be used for SBP biorefining.

Enhanced humification of full-scale apple wood and cow manure by promoting lignocellulose degradation via biomass pretreatments

Agroforestry waste and cow manure pollute the environment, of which, agroforestry waste is difficult to degrade. Compost is an effective way to dispose agroforestry waste; however, the low degradation efficiency of lignocellulose in agroforestry waste affects the process of composting humification. This study investigated lignocellulose degradation and composting humification in full-size apple wood and cow manure composting processes by applying different pretreatments (acidic, alkaline, and high-temperature) to apple wood. Simultaneously, physicochemical characterization and metagenome sequencing were combined to analyze the function of carbohydrate-active enzymes database (CAZy). Therefore, microbial communities and functions were linked during the composting process and the lignocellulose degradation mechanism was elaborated. The results showed that the addition of apple wood increased the compost humus (HS) yield, and pretreatment of apple wood enhanced the lignocellulose degradation during composting processes. In addition, pretreatment improved the physicochemical properties, such as temperature, pH, electric conductivity (EC), ammonium nitrogen (NH4+), and nitrate nitrogen (NO3−) in the compost, of which, acid treated apple wood compost (AcAWC) achieved the highest temperature of 58.4 °C, effectively promoting nitrification with NO3− ultimately reaching 0.127 g/kg. In all composts, microbial networks constructed a high proportion of positively correlated connections, and microorganisms promoted the composting process through cooperation. The proportions of glycosyltransferase (GT) and glycoside hydrolase (GH) promoted the separation and degradation of lignocellulose during composting to form HS. Notably, the adverse effects of the alkali-treated apple wood compost on bacteria were greater. AcAWC showed significant correlations between bacterial and fungal communities and both lignin and hemicellulose, and had more biomarkers associated with lignocellulose degradation and humification. The lignin degradation rate was 24.57 % and the HS yield increased by 27.49 %. Therefore, AcAWC has been confirmed to enhance lignocellulose degradation and promote compost humification by altering the properties of the apple wood and establishing a richer microbial community.

Effects of Lignin-Diverted Reductant with Polyphenol Oxidases on Cellulose Degradation by Wild and Mutant Types of Lytic Polysaccharide Monooxygenase.

Establishing a multi-enzyme synergistic lignocellulosic biodegradation system using lytic polysaccharide monooxygenase (LPMO) and polyphenol oxidases is vital for efficiently utilizing plant biomass waste, ultimately benefiting the carbon cycle and promoting environmental protection. Single-residue mutations of LPMO can improve the efficiency of lignocellulosic biomass degradation. However, the activity of mutant-type LPMO in relation to lignin-diverted reducing agents has not been sufficiently explored. In this study, laccase and tyrosinase were initially investigated and their optimal conditions and impressive thermal stability were revealed, indicating their potential synergistic abilities with LPMO in lignocellulose biodegradation. When utilizing gallic acid as a reducing agent, the activities of LPMOs were increased by over 10%, which was particularly evident in mutant-type LPMOs after the addition of polyphenol oxidases. In particular, the combination of tyrosinase with either 4-hydroxy-3-methoxyphenylacetone or p-coumaric acid was shown to enhance the efficacy of LPMOs. Furthermore, the highest activity levels of wild-type LPMOs were observed with the addition of laccase and 3-methylcatechol. The similarities between wild and mutant LPMOs regarding their activities in lignin-diverted phenolic compounds and reducing agents are almost identical, suggesting that the single-residue mutation of LPMO does not have a detrimental effect on its performance. Above all, this study indicates that understanding the performance of both wild and mutant types of LPMOs in the presence of polyphenol oxidases and various reducing agents constitutes a key link in the industrialization of the multi-enzyme degradation of lignocellulose.

The impact of microbial inoculants on large-scale composting of straw and manure under natural low-temperature conditions

Understanding large-scale composting under natural conditions is essential for improving waste management and promoting sustainable agriculture. In this study, corn straw (400 tons) and pig manure (200 tons) were composted with microbial inoculants. The thermophilic phase of composting lasted for fourteen weeks, resulting in an alkaline final product. Microbial systems with low-temperature initiation and high-temperature fermentation played a crucial role in enhancing lignocellulose degradation and humic substances (HS) formation. Adding microbes, including Rhodanobacter, Pseudomonas, and Planococcus, showed a positive correlation with degradation rates of cellulose, hemicellulose, and lignin. Bacillus, Planococcus, and Acinetobacter were positively correlated with HS formation. Microorganisms facilitated efficient hydrolysis of lignocelluloses, providing humic precursors to accelerate composting humification through phenolic protein and Maillard pathways. This study provides significant insights into large-scale composting under natural conditions, contributing to the advancement of waste management strategies and the promotion of sustainable agriculture.

Fungal systems for lignocellulose deconstruction: From enzymatic mechanisms to hydrolysis optimization

AbstractLignocellulosic biomass is an abundant renewable feedstock, but its complex structure of lignocellulose poses barriers to its enzymatic hydrolysis and fermentation. Fungi possess diverse lignocellulolytic enzyme systems that synergistically deconstruct lignocellulose into soluble sugars for fermentation. This review elucidates recent advances in understanding the molecular mechanisms underpinning fungal degradation of lignocellulose. We analyze major enzyme classes tailored by fungi to depolymerize cellulose, hemicellulose, and lignin. Highlighted are the concerted actions and intimate partnerships between these biomass‐degrading enzymes. Current challenges impeding large‐scale implementation of enzymatic hydrolysis are discussed, along with emerging biotechnological opportunities. Advanced pretreatments, high‐throughput enzyme engineering platforms, and machine learning or artificial intelligence‐guided lignocellulolytic enzyme cocktail optimization represent promising ways to improve hydrolytic efficiencies. Elucidating the coordinated interplay and regulation of fungal lignocellulolytic machinery can facilitate optimization of fungal biotechnology platforms. Harnessing the efficiency of fungal biomass deconstruction promises to enhance the development of biorefinery processes for sustainable bioenergy.

Effects of thermophilic bacteria inoculation on maturity, gaseous emission and bacterial community succession in hyperthermophilic composting

Hyperthermophilic composting, characterized by temperatures equal to or exceeding 75 °C, offers superior compost maturity and performance. Inoculation with thermophilic bacteria presents a viable approach to achieving hyperthermophilic composting. This study investigates the effects of inoculating thermophilic bacteria, isolated at different temperatures (50 °C, 60 °C, and 70 °C) into compost on maturity, gaseous emissions, and microbial community dynamics during co-composting. Results indicate that the thermophilic bacteria inoculation treatments exhibited peak temperature on Day 3, with the maximum temperature of 75 °C reached two days earlier than the control treatment. Furthermore, these treatments demonstrated increased bacterial richness and diversity, along with elevated relative abundances of Firmicutes and Proteobacteria. They also fostered mutualistic correlations among microbial species, enhancing network connectivity and complexity, thereby facilitating lignocellulose degradation. Specifically, inoculation with thermophilic bacteria at 60 °C increased the relative abundance of Thermobifida and unclassified-f-Thermomonosporaceae (Actinobacteriota), whereas Bacillus, a thermophilic bacterium, was enriched in the 70 °C inoculation treatment. Consequently, the thermophilic bacteria at 60 °C and 70 °C enhanced maturity by 36 %–50 % and reduced NH3 emissions by 1.08 %–27.50 % through the proliferation of thermophilic heterotrophic ammonia-oxidizing bacteria (Corynebacterium). Moreover, all inoculation treatments decreased CH4 emissions by 6 %–27 % through the enrichment of methanotrophic bacteria (Methylococcaceae) and reduced H2S, Me2S, and Me2SS emissions by 1 %–25 %, 47 %–63 %, and 15 %–53 %, respectively. However, the inoculation treatments led to increased N2O emissions through enhanced denitrification, as evidenced by the enrichment of Truepera and Pusillimonas. Overall, thermophilic bacteria inoculation promoted bacteria associated with compost maturity while attenuating the relationship between core bacteria and gaseous emissions during composting.

Efficient activity screening of new glucuronoyl esterases using a pNP-based assay

Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-β-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (β-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-β-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.

Effect of nitrogen retention composite additives Ca(H2PO4)2 and MgSO4 on the degradation of lignocellulose, compost maturation, and fungal communities in compost.

This study investigated the effects of the nitrogen retention composite additives Ca(H2PO4)2 and MgSO4 on lignocellulose degradation, maturation, and fungal communities in composts. The study included control (C, without Ca(H2PO4)2 and MgSO4), 1% Ca(H2PO4)2 + 2% MgSO4 (CaPM1), 1.5% Ca(H2PO4)2 + 3% MgSO4 (CaPM2). The results showed that Ca(H2PO4)2 and MgSO4 enhanced the degradation of total organic carbon (TOC) and promoted the degradation of lignocellulose in compost, with CaPM2 showing the highest TOC and lignocellulose degradation. Changes in the three-dimensional excitation-emission matrix fluorescence spectroscopy (3D-EEM) of dissolved organic matter (DOM) components in compost indicated that the treatment group with the addition of Ca(H2PO4)2 and MgSO4 promoted the production of humic acids (HAs) and increased the degree of compost decomposition, with CaPM2 demonstrating the highest degree of decomposition. The addition of Ca(H2PO4)2 and MgSO4 modified the composition of the fungal community. Ca(H2PO4)2 and MgSO4 increased the relative abundance of Ascomycota, decreased unclassified_Fungi, and Glomeromycota, and activated the fungal genera Thermomyces and Aspergillus, which can degrade lignin and cellulose during the thermophilic stage of composting. Ca(H2PO4)2 and MgSO4 also increased the abundance of Saprotroph, particularly undefined Saprotroph. In conclusion, the addition of Ca(H2PO4)2 and MgSO4 in composting activated fungal communities involved in lignocellulose degradation, promoted the degradation of lignocellulose, and enhanced the maturation degree of compost.

The bioaugmentation effect of microbial inoculants on humic acid formation during co-composting of bagasse and cow manure

The effective degradation of recalcitrant lignocellulose has emerged as a bottleneck for the humification of compost, and strategies are required to improve the efficiency of bagasse composting. Bioaugmentation is a promising method for promoting compost maturation and improving the quality of final compost. In this study, the bioaugmentation effects of microbial inoculants on humic acid (HA) formation during lignocellulosic composting were explored. In the inoculated group, the maximum temperature was increased to 72.5 °C, and the phenol-protein condensation and Maillard humification pathways were enhanced, thus increasing the HA content by 43.85%. After inoculation, the intensity of the microbial community interactions increased, particularly for fungi (1.4-fold). Macrogenomic analysis revealed that inoculation enriched thermophilic bacteria and lignocellulose-degrading fungi and increased the activity of carbohydrate-active enzymes and related metabolic functions, which effectively disrupted the recalcitrant structure of lignocellulose to achieve a high humification degree. Spearman correlation analysis indicated that Stappia of the Proteobacteria phylum, Ilumatobacter of the Actinomycetes phylum, and eleven genera of Ascomycota were the main HA producers. This study provides new ideas for bagasse treatment and recycling and realizing the comprehensive use of resources.

Pure lignin induces overexpression of cytochrome P450 (CYP) encoding genes and brings insights into the lignocellulose depolymerization by Trametes villosa

Trametes villosa is a remarkable white-rot fungus (WRF) with the potential to be applied in lignocellulose conversion to obtain chemical compounds and biofuels. Lignocellulose breakdown by WRF is carried out through the secretion of oxidative and hydrolytic enzymes. Despite the existing knowledge about this process, the complete molecular mechanisms involved in the regulation of this metabolic system have not yet been elucidated. Therefore, in order to understand the genes and metabolic pathways regulated during lignocellulose degradation, the strain T. villosa CCMB561 was cultured in media with different carbon sources (lignin, sugarcane bagasse, and malt extract). Subsequently, biochemical assays and differential gene expression analysis by qPCR and high-throughput RNA sequencing were carried out. Our results revealed the ability of T. villosa CCMB561 to grow on lignin (AL medium) as the unique carbon source. An overexpression of Cytochrome P450 was detected in this medium, which may be associated with the lignin O-demethylation pathway. Clusters of up-regulated CAZymes-encoding genes were identified in lignin and sugarcane bagasse, revealing that T. villosa CCMB561 acts simultaneously in the depolymerization of lignin, cellulose, hemicellulose, and pectin. Furthermore, genes encoding nitroreductases and homogentisate-1,2-dioxygenase that act in the degradation of organic pollutants were up-regulated in the lignin medium. Altogether, these findings provide new insights into the mechanisms of lignocellulose degradation by T. villosa and confirm the ability of this fungal species to be applied in biorefineries and in the bioremediation of organic pollutants.

Expression of cellobiose dehydrogenase gene in Aspergillus niger C112 and its effect on lignocellulose degrading enzymes.

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, β-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency.

Enhancing biomass conservation and enzymatic hydrolysis of sweet sorghum bagasse by combining pretreatment with ensiling and NaOH.

Lignocellulosic pretreatment is an important stage in biomass utilization, which usually requires high input. In this study, a low-cost method using combined ensiling and NaOH was developed for lignocellulosic pretreatment. Sweet sorghum bagasse (SSB) was ensiled for 21 days and then treated with diluted NaOH (0%, 1%, and 2%) for fermentation. The results showed that the application of Lactobacillus plantarum (L) reduced fermentation losses of the silages, mainly low water-soluble carbohydrate (WSC) and ammonia nitrogen loss. Meanwhile, the application of Lactobacillus plantarum and ensiling enzyme (LE) promoted lignocellulosic degradation, as evidenced by low neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin (ADL), and hemicellulosic (HC) contents. The dominant bacterial genera were Lactobacillus, uncultured_bacterium_f_Enterobacteriaceae, and Pantoea after silage, which corresponded to the higher lactic acid and acetic contents and lower pH. The reducing sugar yields of SSB increased after combined pretreatment of silage and NaOH and were further enhanced by the 2% NaOH application, as evidenced by the high reducing sugar yield and microstructure damage, especially in the L-2% NaOH group and the LE-2% NaOH group, in which the reducing sugar yields were 87.99 and 94.45%, respectively, compared with those of the no additive control (CK)-0 NaOH group. Therefore, this study provides an effective method for SSB pretreatment to enhance biomass conservation.

Construction of cellulose-degrading microbial consortium and evaluation of their ability to degrade spent mushroom substrate.

Spent mushroom substrate (SMS) is a solid waste in agricultural production that contains abundant lignocellulosic fibers. The indiscriminate disposal of SMS will lead to significant resource waste and pollution of the surrounding environment.The isolation and screening of microorganisms with high cellulase degradation capacity is the key to improving SMS utilization. The cellulose-degrading microbial consortiums were constructed through antagonism and enzyme activity test. The effect of microbial consortiums on lignocellulose degradation was systematically evaluated by SMS liquid fermentation experiments. In this study, four strains of cellulose-degrading bacteria were screened, and F16, F, and F7 were identified as B. amyloliquefaciens, PX1 identified as B. velezensis. At the same time, two groups of cellulose efficient degrading microbial consortiums (PX1 + F7 and F16 + F) were successfully constructed. When SMS was used as the sole carbon source, their carboxymethyl cellulase (CMCase) activities were 225.16 and 156.63 U/mL, respectively, and the filter paper enzyme (FPase) activities were 1.91 and 1.64 U/mL, respectively. PX1 + F7 had the highest degradation rate of hemicellulose and lignin, reaching 52.96% and 52.13%, respectively, and the degradation rate of F16 + F was as high as 56.30%. Field emission scanning electron microscopy (FESEM) analysis showed that the surface microstructure of SMS changed significantly after microbial consortiums treatment, and the change of absorption peak in Fourier transform infrared spectroscopy (FTIR) and the increase of crystallinity in X-ray diffraction (XRD) confirmed that the microbial consortiums had an actual degradation effect on SMS. The results showed that PX1 + F7 and F16 + F could effectively secrete cellulase and degrade cellulose, which had practical significance for the degradation of SMS. In this study, the constructed PX1 + F7 and F16 + F strains can effectively secrete cellulase and degrade cellulose, which holds practical significance in the degradation of SMS. The results can provide technical support for treating high-cellulose solid waste and for the comprehensive utilization of biomass resources.

Characterization of a Novel Hyperthermophilic GH1 β-Glucosidase from Acidilobus sp. and Its Application in the Hydrolysis of Soybean Isoflavone Glycosides.

A putative β-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of β-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, Km, Vmax, and Kcat/Km of BglAc toward pNPG were 357.62 U mg-1, 3.41 mM, 474.0 μmol min-1·mg-1, and 122.7 s-1mM-1. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new β-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation.