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Recently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacterium Iocasia fonsfrigidae strain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-D-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (KM = 0.27 mM; kcat = 0.36 s-1; kcat/KM = 1.34 mM-1 s-1) compared to the enzymatic hydrolysis of barley b-D-glucan (KM: 0.04 mM, kcat: 0.51 s-1, kcat/KM = 0.08 mM-1 s-1). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid-pretreated cellulosic feedstock. KEY POINTS: • IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance • IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass • IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis.

BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Lignin can affect the enzymatic hydrolysis efficiency of lignocellulose. In this study, the lignin isolated from sugarcane bagasse (SCB) pretreated with p-toluenesulfonic acid (PL) was firstly aminated, and then the effects of PL and aminated PL (APL) on the bagasse enzymatic hydrolysis efficiency (EHE) were investigated. The results showed that the addition of PL and APL promoted the EHE, and EHE with APL (73.82 %) was higher than PL (51.39 %). To explore the reason, the data were further analyzed including cellulase adsorption capacity, enzyme activity, cellulase-lignin interaction, and molecular docking. It was found that APL adsorbed more cellulase (27.83 mg protein/g lignin) than PL (4.96 mg protein/g lignin), resulting from the greater interaction force and lower binding free energy between APL and cellulase. The addition of APL more remarkably enhanced the cellobiohydrolase and endoglucanase activities than PL due to more effectively inducing cellulase conformation optimization. [Display omitted] • Enzymatic hydrolysis of cellulosic substrate was enhanced from 45.83 % to 73.82 % by aminated lignin. • Aminated lignin adsorbed more cellulase molecules. • Activities of cellobiohydrolase (CBH) and endoglucosidase (EG) were promoted by aminated lignin. • Aminated lignin owned lower binding energy with cellulase. [ABSTRACT FROM AUTHOR]

Introduction: Several species of cellulolytic bacteria display cellulosomes, massive multi-cellulase containing complexes that degrade lignocellulosic plant biomass (LCB). A greater understanding of cellulosome structure and enzyme content could facilitate the development of new microbial-based methods to produce renewable chemicals and materials. Methods: To identify novel cellulosome-displaying microbes we searched 305,693 sequenced bacterial genomes for genes encoding cellulosome proteins; dockerin-fused glycohydrolases (DocGHs) and cohesin domain containing scaffoldins. Results and discussion: This analysis identified 33 bacterial species with the genomic capacity to produce cellulosomes, including 10 species not previously reported to produce these complexes, such as Acetivibrio mesophilus. Cellulosome-producing bacteria primarily originate from the Acetivibrio, Ruminococcus, Ruminiclostridium , and Clostridium genera. A rigorous analysis of their enzyme, scaffoldin, dockerin, and cohesin content reveals phylogenetically conserved features. Based on the presence of a high number of genes encoding both scaffoldins and dockerin-fused GHs, the cellulosomes in Acetivibrio and Ruminococcus bacteria possess complex architectures that are populated with a large number of distinct LCB degrading GH enzymes. Their complex cellulosomes are distinguishable by their mechanism of attachment to the cell wall, the structures of their primary scaffoldins, and by how they are transcriptionally regulated. In contrast, bacteria in the Ruminiclostridium and Clostridium genera produce 'simple' cellulosomes that are constructed from only a few types of scaffoldins that based on their distinct complement of GH enzymes are predicted to exhibit high and low cellulolytic activity, respectively. Collectively, the results of this study reveal conserved and divergent architectural features in bacterial cellulosomes that could be useful in guiding ongoing efforts to harness their cellulolytic activities for bio-based chemical and materials production. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this experiment was to explore the effect of adding cellulase and laccase on fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains. Try to a new approach for the proper preservation and utilization of the agro-industrial by-products (corn stover and wet brewer's grains). Methods: The experiment was divided into four groups: CK (control), C (cellulase, 120 U/g fresh matter [FM]), L (laccase, 50 U/g FM), CL (cellulase 120 U/g FW and laccase 50 U/g FM), and the chemical composition, fermentation quality, microbial population and microbial community in mixed silage of corn stover and wet brewer's grains after 30 day's fermentation were determined. Results: Compared to control, the addition of cellulase significantly increased crude protein (CP), water-soluble carbohydrate (WSC), lactic acid bacteria (LAB) counts, while significantly decreased the neutral detergent fiber (NDF), acid detergent fiber (ADF) content (P < 0.05). Adding laccase significantly decreased the acid detergent lignin (ADL) content (P < 0.05). Combined application of cellulase and laccase significantly increased the CP, WSC content and LAB counts, while significantly decreased pH value, NDF, ADF and ADL content (P < 0.05), thereby improving fermentation quality. In addition, the application of cellulase and laccase increased the abundance of Firmicutes and LAB genera, and decreased microbial diversity level of the sample. Conclusion: The combined application of cellulase and laccase further improved fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains. [ABSTRACT FROM AUTHOR]

The fate of biobased and biodegradable cellulose-derived plastics in landfills represents an important topic from economic and environmental points of view. Anaerobic digestion is a cost-effective waste-to-energy technology. The behaviour of six polymer types—that is, cellulose (C), cellulose acetate (CA), viscose (V), nanocellulose (NC), acetate textile (AT), and heteropolysaccharide pectin (P)—was studied under anaerobic batch mesophilic conditions in a landfill leachate for 147 days. The cumulative biogas production was as follows: C>V=CA>>AT>>NC=P. Metagenomic analysis revealed notable variations in the proportion of bacterial and archaeal domains with the highest archaeal abundance in the presence of CA (80.2%) and C (78.5%). At the end of digestion, cellulolytic, hydrolytic, and dehydrogenase activities were measured in the intact samples, as well as the liquid and solid fractions, under aerobic and anaerobic conditions. Cellulolytic activity in P was detected only in the pellet, while in NC, activity was mostly in the supernatant under both aerobic and anaerobic conditions. Scanning electron microscopy and confocal scanning laser microscopy showed a defragmentation and degradation of polymeric substrates as well as microbial colonisation. Based on the results, landfill leachate is appropriate for the anaerobic biodegradation of cellulose-derived polymers; however, the process is polymer specific. [ABSTRACT FROM AUTHOR]

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in Δ creA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in Δ creA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus. [ABSTRACT FROM AUTHOR]

The enzyme-assisted approaches for plant phenolics extraction are more eco-friendly methods compared to acid or alkaline hydrolysis. Carbohydrase enzymes can release free phenolics from plant materials by cleaving the glycosidic bonds between phenolic compounds and cell wall polymers. In this study, the efficiency of carbohydrase-assisted treatment approaches was evaluated to extract bioactive phenolics from hawthorn (Crataegus orientalis) fruit residues. Enzymatic treatment of the fruits was operated by using a crude cellulolytic enzyme cocktail from Rhizomucor miehei NRRL 5282 and a pectinase preparate from Aspergillus niger. Both cellulase and combined cellulase–pectinase treatments improved the total phenolic content (TPC) and antioxidant activity of extracts. The TPC increased to 1899 ± 27 mg gallic acid equivalents/100 g dry matter during the combined enzyme treatment, showing a strong correlation with the average antioxidant capacity determined by ferric-reducing antioxidant power (1.7-fold increment) and 2,2-diphenyl-1-picrylhydrazyl (1.15-fold increment) reagents. The major phenolics in enzyme-treated extracts were vanillic and ferulic acids, the concentrations of which increased 115.6-fold and 93.9-fold, respectively, during carbohydrase treatment. The planktonic growth of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Chromobacterium violaceum was slightly inhibited by the extracts with minimum inhibitory concentration values between 15.0 and 77.9 mg/mL, while the yeasts tested were quite resistant to the samples. B. subtilis and yeast biofilms were sensitive to the enzyme-treated extracts, which also showed quorum-sensing inhibitory effects against C. violaceum. The obtained bioactive hawthorn extracts hold potential as a natural source of antioxidants and antimicrobials. [ABSTRACT FROM AUTHOR]

High-value metabolites, such as enzymes and biofuels, can be produced from various agro-industrial waste containing high percentages of cellulose and hemicellulose. Aspergillus niger ITV02 demonstrates high potential in cellulases production, the key enzyme for converting lignocellulosic materials into fermentable sugars to produce second-generation bioethanol (bioethanol 2G). This study evaluated five lignocellulosic residues of agricultural importance: sugarcane bagasse (SCB), sorghum bagasse (SB), corn stubble (CS), barley straw (BS) and rice husk (RH) as substrates for cellulase production. The temperature, pH and stirring conditions were optimized using a Box–Behnken design to identify the most suitable conditions for cellulase production while minimizing nitrogen concentrations. The results indicate that the best way of propagation A. niger ITV02 is through the use of spores as an inoculant, in conjunction with the use of materials with a high cellulose/lignin ratio, such as CS and SB for the generation of cellulases. These conditions promote the expression of cellulases towards β-glucosidase production, unlike materials with lower cellulose/lignin ratios like BS and RH, which exhibited lower cellulase activity. The optimal conditions for cellulase production by A. niger ITV02 were determined to be 33 °C, pH 5.3, and 200 rpm, resulting in a 1.7-fold increase in Exoglucanase (FPase activity) (from 0.127 to 0.215 U/mL). These findings demonstrate the potential to enhance FPase activity by utilizing substrates with high cellulose/lignin content and implementing optimal operational conditions without the need to raise the nitrogen content of the basal medium, thus mitigating the economic impact of cellulase production. [ABSTRACT FROM AUTHOR]

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Background: The microbial landscape within termite guts varies across termite families. The gut microbiota of lower termites (LT) is dominated by cellulolytic flagellates that sequester wood particles in their digestive vacuoles, whereas in the flagellate-free higher termites (HT), cellulolytic activity has been attributed to fiber-associated bacteria. However, little is known about the role of individual lineages in fiber digestion, particularly in LT. Results: We investigated the lignocellulolytic potential of 2223 metagenome-assembled genomes (MAGs) recovered from the gut metagenomes of 51 termite species. In the flagellate-dependent LT, cellulolytic enzymes are restricted to MAGs of Bacteroidota (Dysgonomonadaceae, Tannerellaceae, Bacteroidaceae, Azobacteroidaceae) and Spirochaetota (Breznakiellaceae) and reflect a specialization on cellodextrins, whereas their hemicellulolytic arsenal features activities on xylans and diverse heteropolymers. By contrast, the MAGs derived from flagellate-free HT possess a comprehensive arsenal of exo- and endoglucanases that resembles that of termite gut flagellates, underlining that Fibrobacterota and Spirochaetota occupy the cellulolytic niche that became vacant after the loss of the flagellates. Furthermore, we detected directly or indirectly oxygen-dependent enzymes that oxidize cellulose or modify lignin in MAGs of Pseudomonadota (Burkholderiales, Pseudomonadales) and Actinomycetota (Actinomycetales, Mycobacteriales), representing lineages located at the hindgut wall. Conclusions: The results of this study refine our concept of symbiotic digestion of lignocellulose in termite guts, emphasizing the differential roles of specific bacterial lineages in both flagellate-dependent and flagellate-independent breakdown of cellulose and hemicelluloses, as well as a so far unappreciated role of oxygen in the depolymerization of plant fiber and lignin in the microoxic periphery during gut passage in HT. Ak1UNwnHwhcNN-Fj12kHob Video Abstract [ABSTRACT FROM AUTHOR]

The production of lignocellulytic enzymes by microwave-radiated Pleurotus sajor-caju was assayed. Wheat straw was employed as substrate to P. sajor-caju for production of laccase, manganese peroxidase (MnPase), filter-paperase (FPase), carboxmethyl cellulase (CMCase), and cellulase (as evaluated using microcrystalline cellulose). P. sajor-caju exposed to 10 s of microwave radiation (MR) showed maximum growth with colony radius of 7.17 ± 0.45 cm, while with increasing the exposure time up to 50 s the growth decreased up to 2.67 ± 0.22 cm. Moreover, it failed to grow at 80 s of exposure time. Cellulase, MnPase, FPase, CMCase, and laccase activities were induced to 37 ± .0.54, 49 ± 2.36, 189 ± 2.12, 0.37 ± 0.06, and 1.58 ± 0.03 U/mL compared to that at control 31 ± 0.25, 46 ± 1.25, 177 ± 1.65, 0.28 ± 0.03, and 1.37 ± 0.12 U/mL, respectively as a result of P. sajor-caju exposure to 10 s of MR. As the exposure time increased, these enzymes activity decreased. Different levels of moisture with surfactant (polysorbate 80) were applied to optimize the enzymes activities at 10 s of exposure time. The optimum activities 3.15 ± 0.23, 0.62 ± 0.06, 269 ± 5.36, 65 ± 1.63, and 48 ± 0.98 U/mL were recorded for cellulase, MnPase, FPase, CMCase, and laccase, respectively at 70% of moisture and 0.15 mL/L of polysorbate 80. [ABSTRACT FROM AUTHOR]

The effects of laboratory beating on unbleached regular grade pine kraft pulp (UPKP), pre-treated with commercial cellulase/hemicellulase enzymatic preparation (C/HEP) were assessed using degrees of Schopper-Riegler (°SR) and fibre-quality numbers (FQN). The tests showed a significant increase in the °SR (used in papermaking laboratories and industry to assess the degree of beating) of the UPKP after its enzymatic pre-treatment in the amount of 0.25 to 5.0 mL/kg of pulp and beating. However, only a several percent reduction of beating time was observed when the effect of this pretreatment of pulp on the speed of its beating was determined using another method. The treatment of the pulp selected for research with C/HEP before beating negatively influenced its strength properties after this process. The results correlated well with the most important basic fiber properties from the standpoint of papermaking. [ABSTRACT FROM AUTHOR]

In order to alleviate environmental pollution and the waste of resources caused by improper disposal of fungal residue, this study used fungal residue waste, which was treated with NaClO alkaline solution and nitric acid ethanol method for rough fiber preparation. The enzymatic hydrolysis of cellulase conditions were optimized using response surface optimization method, and the optimal preparation parameters were: enzyme addition of 5000 U/g, enzymatic hydrolysis temperature of 52 °C, enzymatic hydrolysis time of 2.65 h, and solid-liquid ratio of 1:20. The Fr-MCC purity reached over 97%. The Fr-MCC obtained had an irregular granular or lamellar aggregation morphology, typical I-type cellulose crystal structure and molecular features, good thermal stability, and was similar in properties to commercial MCC. It was judged to be suitable for further processing and manufacturing of biological base materials. This approach was shown to improve the utilization efficiency of cultivation residues, reducing environmental pollution caused by the accumulation of cultivation residues, and providing new methods and ideas for the preparation of MCC and other bio-based materials. [ABSTRACT FROM AUTHOR]

In the last few decades, the increasing levels of environmental pollution have prompted a shift towards alternative energy sources and biobased solutions, such as lignocellulosic biomass. Lignocellulosic biomass (LB) is primarily derived from plants and is composed mainly of polysaccharides, namely cellulose, hemicellulose, and the aromatic polymer lignin. Hazelnut shells (HS), with a high lignin content of 43%, hemicellulose of 30%, and cellulose of 26%, hold promise as a valuable source of LB. In order to process those LB, lignin and hemicellulose are separated using various treatment methods. However, instead of being used solely for combustion, lignin-containing materials can be valorized for a range of purposes, from biomedical applications to the energy sector. In this study, the enzymatic hydrolysis of HS was conducted over different time periods (24, 48, 72, and 96 hours), at different temperature values with varying enzyme concentrations (0.05, 0.1, and 0.25 mL of cellulase/xylanase enzyme cocktail). To enhance the enzymatic hydrolysis, an alkaline pretreatment method using sodium hydroxide (NaOH) was employed. The results demonstrate that the maximum sugar concentration was achieved at 50°C, after 72 hours, and with a cellulase/xylanase cocktail concentration of 0.1 mL. [ABSTRACT FROM AUTHOR]

Trichoderma species have been reported as masters in producing cellulolytic enzymes for the biodegradation of lignocellulolytic biomass and biocontrol agents against plant pathogens and pests. In our previous study, a novel Trichoderma strain LZ117, which shows potent capability in cellulase production, was isolated. Herein, we conducted multilocus phylogenetic analyses based on DNA barcodes and performed time-scaled phylogenomic analyses using the whole genome sequences of the strain, annotated by integrating transcriptome data. Our results suggest that this strain represents a new species closely related to T. atrobrunneum (Harzianum clade). Genes encoding carbohydrate-active enzymes (CAZymes), transporters, and secondary metabolites were annotated and predicted secretome in Trichoderma sp. LZ117 was also presented. Furthermore, genetic manipulation of this strain was successfully achieved using PEG-mediated protoplast transformation. A putative transporter gene encoding maltose permease (Mal1) was overexpressed, which proved that this transporter does not affect cellulase production. Moreover, overexpressing the native Cre1 homolog in LZ117 demonstrated a more pronounced impact of glucose-caused carbon catabolite repression (CCR), suggesting the importance of Cre1-mediated CCR in cellulase production of Trichoderma sp. LZ117. The results of this study will benefit further exploration of the strain LZ117 and related species for their applications in bioproduction. [ABSTRACT FROM AUTHOR]

Simple Summary: The black soldier fly (Hermetia illucens L.) is a saprophagous insect belonging to the Stratiomyidae family that can degrade organic waste and convert it into high-quality insect protein and lipid. Corn straw is a kind of prevalent agricultural waste. In this study, black soldier fly larvae were reared with corn straw to obtain larval biomass and consume corn straw. The results showed that most of the black soldier fly larvae could survive and gain weight when fed with corn straw. Gut microorganisms could respond to the substrates, and four cellulase-producing strains isolated from the gut were beneficial to digesting corn straw. Corn straw is considered a renewable biomass energy source, and its unreasonable disposal leads to resource waste and environmental pollution. Black soldier fly (Hermetia illucens L.) larvae (BSFL) facilitate the bioconversion of various types of organic wastes. In this study, we found that 88% of BSFL survived, and 37.4% of corn straw was digested after 14 days of feeding with corn straw. Contrary to expectations, the pretreatment of corn straw with alkaline hydrogen peroxide did not promote its digestion but rather reduced the growth and survival rates of BSFL. Acinetobacter, Dysgonomonas, and unclassified Enterobacteriaceae were the abundant genera in the BSFL gut fed with corn straw. Compared with the standard diet, the relative abundances of carbohydrate metabolism genes, such as the gene abundances of β-glucosidase and α-glucosidase, were higher with corn straw as the substrate. These results suggested that the gut microbial community could regulate suitable and functional microorganisms in response to the substrates. Furthermore, four cellulase-producing strains, namely Klebsiella pneumoniae, Proteus mirabilis, Klebsiella oxytoca, and Providencia rettgeri, were isolated from the guts of corn straw BSFL. These four strains helped increase the conversion rates of corn straw, the weights of BSFL, and survival rates. In summary, we reared BSFL with corn straw and discovered the functions of gut microorganisms in adapting to the substrates. We also isolated four cellulase-producing strains from the BSFL guts and declared the benefits of BSFL digesting corn straw. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the cellulase production of four fungi: Aspergillus terreus NIH2624, Aspergillus clavatus NRRL1, Aspergillus versicolor CBS583.65 and Aspergillus phoenicis ATCC3157, under submerged cultivation conditions. When these fungi were cultured in shake flasks using Mandels and Weber's minimal medium with 1% sugarcane bagasse as a carbon source and 1.8 g/L of rice bran extract as a nitrogen source, A. terreus showed maximum cellulase production (filter paper activity (FPase) 3.35 U/mL; carboxymethyl cellulase activity (CMCase) 1.69 U/mL). Consequently, A. terreus was selected for the optimization study for cellulase production. Among the different tested carbon sources, A. terreus showed higher CMCase activity when it was cultivated on delignified sugarcane bagasse (1.64 U/mL) and higher FPase activity on sugarcane straw (7.95 U/mL). Regarding the nitrogen sources, the maximum FPase activity was observed when using rice bran (FPase, 8.90 U/mL) and soybean meal (FPase, 9.63 U/mL). The optimized fermentation medium (minimal medium with delignified sugarcane bagasse and rice bran as carbon and nitrogen sources, respectively) resulted in an enzymatic cocktail mainly composed of xylanases, with a maximum activity of 1701.85 U/mL for beechwood xylan, 77.12 U/mL for endoglucanase and 21.02 U/mL for cellobiohydrolase. Additionally, the enzymatic cocktail showed efficient activities for β-glucosidase, β-xylanase, arabinofuranosidase and lytic polysaccharide monoxygenases (LPMOs). This cellulase enzyme solution has the potential to efficiently hydrolyze lignocellulosic biomass, producing second-generation sugars in biorefineries. [ABSTRACT FROM AUTHOR]

As a large agricultural country, China produces a large number of agricultural and sideline products while harvesting agricultural products every year. Crop straw is one of them. Broom sorghum is a traditional crop in China, which produces a large amount of straw resources every year. These straw resources are placed in the field and cannot be used efficiently. The purpose of this study was to solve the problem of straw utilization of Broom sorghum, one of the main food crops in arid and semi-arid areas of northern China. Broom sorghum is not only a nutritious food crop, its straw is also rich in crude fiber and mineral elements, which has high utilization value. However, due to the high content of lignocellulose in straw, the texture is hard, which limits its digestion and utilization efficiency as feed. In this study, the broom sorghum straw was used as the research object, and the straw raw materials were treated with Lactobacillus plantarum, cellulase and xylanase, respectively. After silage fermentation for 30 d and 60 d, the bags were opened to determine the nutritional quality, fermentation quality, microbial community structure and other indicators. The best fermentation time and additives for broom sorghum straw silage were comprehensively screened to improve the nutritional value of straw and animal production performance. The results showed that the nutritional quality of silage straw increased with the extension of fermentation time. Compared with silage straw after 30 days of fermentation, the nutritional quality and fermentation quality of straw were significantly improved after 60 days of fermentation. Lactobacillus plantarum, cellulase and xylanase could improve the silage performance of broom sorghum straw by improving the microbial community structure in straw, and the effect of cellulase was the best. When cellulase was used in straw at the standard of 20 U/g FM, the content of water-soluble carbohydrates could be significantly increased to 31.35 g/kg FM, and the concentration of lactic acid was also significantly increased to 23.79 g/kg FM. Therefore, in actual production, it is recommended to use cellulase at a dose of 20 U/g FM in broom sorghum silage and open the bag after 60 days of silage fermentation. The results of this study provided a scientific basis for the efficient utilization of broom sorghum straw as feed. [ABSTRACT FROM AUTHOR]

The escalating prevalence of drug-resistant pathogens not only jeopardizes the effectiveness of existing treatments but also increases the complexity and severity of infectious diseases. Escherichia coli is one the most common pathogens across all healthcare-associated infections. Enzymatic treatment of bacterial biofilms, targeting extracellular polymeric substances (EPS), can be used for EPS degradation and consequent increase in susceptibility of pathogenic bacteria to antibiotics. Here, we characterized three recombinant cellulases from Thermothelomyces thermophilus: a cellobiohydrolase I (TthCel7A), an endoglucanase (TthCel7B), and a cellobiohydrolase II (TthCel6A) as tools for hydrolysis of E. coli and Gluconacetobacter hansenii biofilms. Using a design mixture approach, we optimized the composition of cellulases, enhancing their synergistic activity to degrade the biofilms and significantly reducing the enzymatic dosage. In line with the crystalline and ordered structure of bacterial cellulose, the mixture of exo-glucanases (0.5 TthCel7A:0.5 TthCel6A) is effective in the hydrolysis of G. hansenii biofilm. Meanwhile, a mixture of exo- and endo-glucanases is required for the eradication of E. coli 042 and clinical E. coli biofilms with significantly different proportions of the enzymes (0.56 TthCel7B:0.44 TthCel6A and 0.6 TthCel7A:0.4 TthCel7B, respectively). X-ray diffraction pattern and crystallinity index of E. coli cellulose are comparable to those of carboxymethyl cellulose (CMC) substrate. Our results illustrate the complexity of E. coli biofilms and show that successful hydrolysis is achieved by a specific combination of cellulases, with consistent recurrence of TthCel7B endoglucanase. [ABSTRACT FROM AUTHOR]

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes. Therefore, we designed hybrid cellulase complexes from 49 biotinylated catalytic domain (CD) and 30 biotinylated cellulose-binding domain (CBD) libraries on streptavidin-conjugated nanoparticles to enhance cellulose hydrolysis by mimicking the cellulosome structure. The hybrid cellulase complex, incorporating both native CD and CBD, significantly improved reducing sugar production from cellulose compared to free native modular enzymes. The optimal CBD for each hybrid cellulase complex differed from that of the native enzyme. The most effective hybrid cellulase complex was observed with the combination of CD6-4 from Thermobifida fusca YX and CBD46 from the Bacillus halodurans C-125. The hybrid cellulase complex/CD6-4-CBD46 and -CD6-4-CBD2-5 combinations showed increased reducing sugar production. Similar results were also observed in microcrystalline cellulose degradation. Furthermore, clustering on nanoparticles enhanced enzyme thermostability. Our results demonstrate that hybrid cellulase complex structures improve enzyme function through synergistic effects and extend the lifespan of the enzyme. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis contributes to obtaining fermentable sugars using pretreated lignocellulose materials for bioethanol generation. Unfortunately, the pretreatment of lignocellulose causes low substrate enzymatic hydrolysis, which is due to the structure changes of lignin to produce main phenolic by-products and non-productive cellulase adsorption. It is reported that modified lignin enhances the speed of enzymatic hydrolysis through single means to decrease the negative effects of fermentation inhibitors or non-productive cellulase adsorption. However, a suitable modified lignin should be selected to simultaneously reduce the fermentation inhibitors concentration and non-productive cellulase adsorption for saving resources and maximizing the enzymatic hydrolysis productivity. Meanwhile, the adsorption micro-mechanisms of modified lignin with fermentation inhibitors and cellulase remain elusive. In this review, different pretreatment effects toward lignin structure, and their impacts on subsequent enzymatic hydrolysis are analyzed. The main modification methods for lignin are presented. Density functional theory is used to screen suitable modification methods for the simultaneous reduction of fermentation inhibitors and non-productive cellulase adsorption. Lignin-fermentation inhibitors and lignin-cellulase interaction mechanisms are discussed using different advanced analysis techniques. This article addresses the gap in previous reviews concerning the application of modified lignin in the enhancement of bioethanol production. For the first time, based on existing studies, this work posits the hypothesis of applying theoretical simulations to screen efficient modified lignin-based adsorbents, in order to achieve a dual optimization of the detoxification and saccharification processes. We aim to improve the integrated lignocellulose transformation procedure for the effective generation of cleaner bioethanol. [Display omitted] • Effects of pretreatment in lignin structure and enzymatic hydrolysis are analyzed. • Lignin modification is the promising enhancement process to bioethanol production. • DFT calculation contributes to screening the suitable lignin modification methods. • Advanced methods to study inhibitors-lignin-cellulase interactions are reviewed. [ABSTRACT FROM AUTHOR]

Soil biota and functions are impacted by various anthropogenic stressors, including climate change, chemical pollution or microplastics. These stressors do not occur in isolation, and soil properties and functions appear to be directionally driven by the number of global change factors acting simultaneously. Building on this insight, we here hypothesize that co-acting factors with more diverse effect mechanisms, or higher dissimilarity, have greater impacts on soil properties and functions. We created a factor pool of 12 factors and calculated dissimilarity indices of randomly-chosen co-acting factors based on the measured responses of soil properties and functions to the single factors. Results show that not only was the number of factors important, but factor dissimilarity was also key for predicting factor joint effects. By analyzing deviations of soil properties and functions from three null model predictions, we demonstrate that higher factor dissimilarity and a larger number of factors could drive larger deviations from null models and trigger more frequent occurrence of synergistic factor net interactions on soil functions (decomposition rate, cellulase, and β-glucosidase activity), which provides mechanistic insights for understanding high-dimensional effects of factors. Our work highlights the importance of considering factor similarity in future research on interacting factors. Soil properties and functions are both affected by many global change factors. Here, the authors use a microcosm experiment to demonstrate that the number of these factors as well as their dissimilarity were important for predicting the impacts of global change factors. [ABSTRACT FROM AUTHOR]

This paper introduces an enzymatic approach to estimate internal mass-transfer resistances during food digestion studies. Cellulase has been used to degrade starch cell walls (where cellulose is a significant component) and reduce the internal mass-transfer resistance, so that the starch granules are released and hydrolysed by amylase, increasing the starch hydrolysis rates, as a technique for measuring the internal mass-transfer resistance of cell walls. The estimated internal mass-transfer resistances for granular starch hydrolysis in a beaker and stirrer system for simulating the food digestion range from 2.2 × 107 m−1 s at a stirrer speed of 100 rpm to 6.6 × 107 m−1 s at 200 rpm. The reaction rate constants for cellulase-treated starch are about three to eight times as great as those for starch powder. The beaker and stirrer system provides an in vitro model to quantitatively understand external mass-transfer resistance and compare mass-transfer and reaction rate kinetics in starch hydrolysis during food digestion. Particle size analysis indicates that starch cell wall degradation reduces starch granule adhesion (compared with soaked starch samples), though the primary particle sizes are similar, and increases the interfacial surface area, reducing internal mass-transfer resistance and overall mass-transfer resistance. Dimensional analysis (such as the Damköhler numbers, Da, 0.3–0.5) from this in vitro system shows that mass-transfer rates are greater than reaction rates. At the same time, SEM (scanning electron microscopy) images of starch particles indicate significant morphology changes due to the cell wall degradation. [ABSTRACT FROM AUTHOR]

The objective of this study was to develop an efficient method for enhancing the production of bioethanol in a single‐reactor system (SRS) by implementing acid–base pretreatment and enzymatic hydrolysis techniques, thereby eliminating differentiation and removal processes. The aim was to establish a process for bioethanol synthesis using hydrolyzed palm oil mill effluent (POME) and a locally sourced cellulase enzyme. The pretreatment and enzymatic hydrolysis methods were successfully optimized, resulting in a maximum yield of reducing sugars of 26.6 g/L (53.14%). To achieve the highest bioethanol yield, fermentation was carried out using both the one‐factor‐at‐a‐time (OFAT) and face‐centered central composite design (FCCCD) approaches. The OFAT approach was employed to obtain the maximum bioethanol production, which yielded 6.75% v/v of bioethanol from the hydrolyzed POME using the same bioreactor. In the case of the FCCCD process, the optimal conditions led to a bioethanol production of 7.64% v/v during the fermentation stage. Kinetic analyses of the bioethanol produced revealed a specific growth rate (μ) of 0.198 h−1 and a specific product formation rate (qp) of 0.239 h−1 after 3 days of fermentation. These findings highlight a promising strategy for efficient management of POME through the production of biofuels, which could contribute to the economic growth of the country. [ABSTRACT FROM AUTHOR]

Bamboo is a perennial rapid-growing plant that is given preference for renewable biosources for biofuels and bio-based chemical conversion. Bamboos are rich in cellulose and have highly recalcitrant biomass due to high lignin. Bamboo is abundantly available in Northeastern India and can be utilized as a feedstock biofuels. Here, we evaluated the pretreatment of bamboo residues Dendrocalamus strictus with different concentrations of alkali, hydrogen peroxide, and alkaline hydrogen peroxide and its influence on biomass digestibility for enhancement of sugar recovery with Celic C cellulase enzyme blend. Enzymatic hydrolysis data indicated untreated raw biomass showed a digestibility of 40% after 48 h of incubation. The biomass pretreated with alkali showed a maximum digestibility of 61% obtained from 10% loaded with 0.5% w/v NaOH. Pretreatment of the bamboo with H2O2 shows a maximum digestibility of 75% from biomass loaded with 1% w/v of H2O2. Combinational pretreatment of alkaline hydrogen peroxide showed a maximum efficiency of biomass digestibility of 83% attained from biomass loaded with 1% w/v NaOH-H2O2. Crystallinity index (CrI) analysis showed that CrI increased from 64% to 70.75% in pretreated biomass. FTIR and SEM analysis show changes in functional groups, morphology, and surface of biomass in pretreated biomass. Compositional analysis shows that 68% of lignin removal is obtained from alkaline hydrogen peroxide pretreatment. Cellulose content increased from 52% to 65%, and hemicellulose decreased from 18.6% to 8.6%. Results indicated that the potential possibility of bamboo waste biomass as feedstock for biorefinery products and alkaline hydrogen peroxide pretreatment methods is an efficient strategy for sugar recovery for bioethanol production. [ABSTRACT FROM AUTHOR]

The utilisation of enzymes in the industry has brought numerous benefits and advantages to production processes. Enzymes serve as biocatalysts, efficiently catalyzing reactions and hydrolysis in biochemical processes. However, there are challenges in applying enzymes in the industry, particularly concerning enzyme stability. The obstacle encountered in the production processes involving industrial enzyme applications is the low stability of enzymes when used at high temperatures. Heat-sensitive enzymes undergo damage or denaturation. Thermophilic microorganisms are chosen because they hold the potential to produce thermophilic enzymes. The thermophilic enzymes exhibit better heat stability compared to other enzymes, making them an effective alternative for future industrial production processes. This study aims to isolate thermotolerant bacteria from Nglimut Hot Spring sediment, screen for cellulase- and amylase-producing isolates, and molecularly identify the best isolate using 16S rRNA barcode. The results show that 22 bacterial isolates were found in the sediment of a hot spring; TS-14 was the best isolate in producing amylase, with the highest average amylolytic index of 2.38, whereas TS-15 had the highest cellulolytic index of 2.11. Based on 16S rRNA identification, TS-14 showed an homological identity of 79% with Bacillus amyloliquefaciens, while TS-15 had a 100% homological identity with Bacillus licheniformis. These results were important as the first step of screening bacterial potential to produce thermophilic enzymes that could be applied in the downstream processing in future industrial and biotechnology companies. [ABSTRACT FROM AUTHOR]

This study aims to evaluate the in silico genomic characteristics of five species of the genus Planotetraspora: P. kaengkrachanensis, P. mira, P. phitsanulokensis, P. silvatica, and P. thailandica, with a view to their application in therapeutic research. The 16S rRNA comparison indicated that these species were phylogenetically distinct. Pairwise comparisons of digital DNA-DNA hybridization (dDDH) and OrthoANI values between these studied type strains indicated that dDDH values were below 62.5%, while OrthoANI values were lower than 95.3%, suggesting that the five species represent distinct genomospecies. These results were consistent with the phylogenomic study based on core genes and the pangenome analysis of these five species within the genus Planotetraspora. However, the genome annotation showed some differences between these species, such as variations in the number of subsystem category distributions across whole genomes (ranging between 1979 and 2024). Additionally, the number of CAZYme (Carbohydrate-Active enZYme) genes ranged between 298 and 325, highlighting the potential of these bacteria for therapeutic research applications. The in silico physico-chemical characteristics of cellulases from Planotetraspora species were analyzed. Their 3D structure was modeled, refined, and validated. A molecular docking analysis of this cellulase protein structural model was conducted with cellobiose, cellotetraose, laminaribiose, carboxymethyl cellulose, glucose, and xylose ligand. Our study revealed significant interaction between the Planotetraspora cellulase and cellotetraose substrate, evidenced by stable binding energies. This suggests that this bacterial enzyme holds great potential for utilizing cellotetraose as a substrate in various applications. This study enriches our understanding of the potential applications of Planotetraspora species in therapeutic research. [ABSTRACT FROM AUTHOR]

Leaves play a crucial role as ornamental organs in Spathiphyllum, exhibiting distinct differences across various Spathiphyllum varieties. Leaf development is intricately linked to processes of cell proliferation and expansion, with cell morphology often regulated by plant cell walls, primarily composed of cellulose. Alterations in cellulose content can impact cell morphology, subsequently influencing the overall shape of plant organs. Although cellulases have been shown to affect cellulose levels in plant cells, genetic evidence linking them to the regulation of leaf shape remains limited. This study took the leaves of Spathiphyllum 'Mojo' and its somatic variants as the research objects. We screened four cellulase gene family members from the transcriptome and then measured the leaf cellulose content, cellulase activity, and expression levels of cellulase-related genes. Correlation analysis pinpointed the gene SpGH9A3 as closely associated with leaf shape variations in the mutant. Green fluorescent fusion protein assays revealed that the SpGH9A3 protein was localized to the cell membrane. Notably, the expression of the SpGH9A3 gene in mutant leaves peaked during the early spread stage, resulting in smaller overall leaf size and reduced cellulose content upon overexpression in Arabidopsis. [ABSTRACT FROM AUTHOR]

A thermo-alkali stable cellulase from Geobacillus sp. TP-1 was isolated from Tapovan hot spring soil sample. The BLASTn sequence analysis of 16S rRNA sequence revealed that the isolate belonged to the Geobacillus genus and shared the highest degree of sequence similarity (99.43%) with the different strains of Geobacillus subterraneus. The neighbour joining method of multiple sequence alignment revealed that the 16S rRNA sequence of Geobacillus sp. TP-1 shows maximum similarity with Geobacillus stearothermophilus strain S_YE6-1017-022. One-Factor-At-a-Time analysis was used to optimize the carbon source, nitrogen source, pH, temperature, inoculum size and growth profile with respect to cellulase production. When compared to un-optimized basal media, optimised medium increased cellulase production by around 3.6 times. The Plackett Burman factorial design was employed to identify the critical medium components influencing cellulase activity and temperature was determined to have a significant effect on overall cellulase production. The current strain was capable of utilising lignocellulosic waste as an alternative carbon source. The use of sugarcane molasses and wheat bran as carbon sources resulted in a significant increase (~ 7.2 fold) in cellulase production in the current study, indicating the bacterium's potential for valorising lignocellulosic biomass into value-added products, which encourages its use in lignocellulosic-based bio refineries. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass, the most abundant natural resource on earth, can be used for cellulosic ethanol production but requires a pretreatment to improve enzyme access to the polymeric sugars while obtaining value from the other components. γ‐Valerolactone (GVL) is a promising candidate for biomass pretreatment since it is renewable and bio‐based. In the present work, the effect of a pretreatment based on GVL on the enzymatic saccharification of white birch was evaluated at a laboratory scale and the importance of the washing procedure for the subsequent saccharification was demonstrated. Both the saccharification yield and the production of cellulosic ethanol were higher using a noncommercial enzyme crude from Talaromyces amestolkiae than with the commercial cocktail Cellic CTec2 from Novozymes. Furthermore, the production of extracellular cellulases by T. amestolkiae has been optimized in 2 L bioreactors, with improvements ranging from 40% to 75%. Finally, it was corroborated by isoelectric focus that optimization of cellulase secretion by T. amestolkiae did not affect the pattern production of the main β‐glucosidases and endoglucanases secreted by this fungus. [ABSTRACT FROM AUTHOR]

Sawdust, a major waste product of the forestry industry, is accumulating along the Lagos Lagoon in Lagos, Nigeria, without it being effectively managed. Besides its use in manufacturing sound-absorbing boards to reinforce concrete beams and for energy purposes, its potential as a renewable energy source and feedstock for bio-product development has not yet been realized. Cellulose, a glucose biopolymer and structural component of cellulose can be hydrolyzed by a hydrolytic enzyme known as cellulase. During the process, the enzyme breaks the B-1,4-glucosidic bond, which keeps the glucose units together, and by acting on this bond, numerous glucose units are released. As part of sawdust, the cellulose molecule is not freely available for the degradation action of the cellulase enzyme as it is strongly associated with lignin, which acts as bio-glue, keeping cellulose and hemicellulose together. Delignification is an effective technique that was used to make the sawdust from ten different trees along the Lagos Lagoon in Nigeria more susceptible to saccharification by cellulase isolated from the fungus Aspergillus niger. Delignified and non-delignified sawdust masses between 2 mg and 10 mg were incubated with the A. niger cellulase solution (2 mg.mL-1), whereafter, the amount of sugar produced by the cellulase action was determined. The percentage saccharification of each sawdust material was also linked with the amount of sugar produced during cellulase action. From these investigations was concluded that delignification increased sugar production when almost all the masses of different sawdust materials were degraded. It was also observed that the ratio of sawdust mass to enzyme concentration is an important variable that influences the effectiveness of the saccharification process. The percentage saccharification of the various sawdust materials was also determined, and it indicated that the highest percentage of saccharification was not obtained when the highest amount of sawdust was degraded, producing the highest amount of sugar. The saccharification of sawdust could contribute to the development of renewable energy sources and feedstock for bioproduct development. The process is, however, not that straightforward as variables such as the type of cellulase enzyme, pretreatment of the cellulose substrate, and optimizing of cellulase to cellulose ratio are a few that need to be optimized for the process to be effective in terms of glucose production. [ABSTRACT FROM AUTHOR]

The Victoria Falls rainforest is a protected site whose forest floors harbor a host of cellulolytic microorganisms involved in biomass degradation. This study collected decaying logs and soil from the rainforest for bioprospecting cellulases from their metagenomes. Metagenomic DNA was isolated from the compound sample. Degenerate cellulase primers were used to amplify cellulase genes in the metagenome. The resulting amplicons cloned into Z-competent Escherichia coli DH5α were analyzed by functional screening for the production of cellulase extracellularly. Functional screening of the clones resulted in one clone (Clone-i) testing positive for extracellular cellulase production. Submerged fermentation of Clone-i was carried out for cellulase production. The cellulases were characterized to determine their activity's optimum pH and temperature. The diversity of the cellulases produced by Clone-i was determined. Clone-i's optimum enzyme activity was observed after 72 hours of incubation at 50°C and pH 5. Clone-i produced 80% more exoglucanases as compared to endoglucanases. The cellulolytic Clone-i' isolate shows Victoria Falls rainforest's potential as an enzyme bioprospecting site, reflecting that metagenomics is a valuable tool in microbial ecology. [ABSTRACT FROM AUTHOR]

β-glucosidase is a key component of cellulase for its function in hydrolyzing cellobiose to glucose in the final step of cellulose degradation. The high-level expression of β-glucosidase is essential for cellulose conversion. Aspergillus niger ATCC 20611 has the potential for efficient protein expression because of its ability to secret enzymes for the industrial production of fructooligosaccharides, but it lacks robust promoters for high-level protein expression. Here, the development of A. niger 20611 as a powerful protein expression system exploited the conserved constitutive promoter Pgpd1 of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene from Trichoerma reesei to drive the expression of the enhanced green fluorescent protein in A. niger ATCC 20611. The mycelium of the transformant AGE9 exhibited intense fluorescence. Then, the promotor Pgpd1 was used to drive the expression of β-glucosidase and the enzyme activity of transformants AGB1 and AGB33 were 1.02 and 0.51 U/mL, respectively. These results demonstrate that the promotor Pgpd1 from T. reesei was applicable for A. niger ATCC 20611. Furthermore, the T. reesei-specific robust promoter Pcdna1 was used to drive the expression of β-glucosidase. The β-glucosidase exhibited a high-level expression with a yield of 15.2 U/mL, which was over 13.9 times higher than that driven by the promoter Pgpd1. The β-glucosidase was thermally stable and accounted for 85% of the total extracellular proteins. Subsequently, the fermentation broth including β-glucosidase was directly added to the cellulase mixture of T. reesei for saccharification of the acid-treated corncob residues and the delignified corncob residues, which increased the saccharification efficiency by 26.21% and 29.51%, respectively. Thus, β-glucosidase exhibited a high level of expression in A. niger ATCC 20611 and enhanced cellulose degradation by addition in vitro. In addition, the robust promoter Pcdna1 of T. reesei could drive the high-level expression of protein in A. niger ATCC 20611. These results demonstrate that the promoters in filamentous fungi could be employed across species in A. niger ATCC 20611 and further facilitated the efficient expression of β-glucosidase to optimize cellulases for efficient cellulose transformation. [ABSTRACT FROM AUTHOR]

Enzymatic degradation of cellulosic biomass represents the most sustainable and environmentally friendly method for producing liquid biofuel, widely utilized in various commercial processes. While cellulases are predominantly produced by bacteria and fungi, the enzymatic potential of cellulase-producing yeasts remains significantly less explored. In this study, the yeast strain Trichosporon insectorum, isolated from the gut of the coprophagous beetle Gymnopleurus sturmii, was utilized for cellulase production in submerged fermentation. A central composite design was employed to optimize cellulase production, with substrate concentration, temperature, and pH as dependent variables. The highest CMCase activity of 0.71 IU/mL was obtained at 1% substrate concentration, pH 5, and an incubation temperature of 40 °C for 72 h of fermentation using cellulose as a carbon source. For FPase production, the high value was 0.23 IU/mL at 0.5% CMC, pH 6, and an incubation temperature of 40 °C for 72 h. After purification, the enzymes produced by T. insectorum represent 39% of the total proteins. The results of this study offer an alternative strategy for utilizing various carbon sources, both soluble (CMC, carboxymethylcellulose) and insoluble (cellulose), to efficiently produce cellulase for the degradation of lignocellulosic materials. This approach holds promising benefits for sustainable waste management. [ABSTRACT FROM AUTHOR]

The genus Trichoderma is widely recognized for its ability to secrete lignocellulosic enzymes, establishing it as a crucial technological resource for the bioconversion of agro-industrial waste biomass via fermentation. This review explores the potential of utilizing lignocellulosic waste from diverse industries as a sustainable nutrient source for producing Trichoderma spp. and various desirable metabolites via fermentation. Significant emphasis is placed on the enzymatic activities of Trichoderma species in two critical stages of second-generation biofuel production. Firstly, in the pre-treatment stage to break down complex polysaccharides of lignocellulosic biomass, thereby enhancing production efficiency and yield, and, secondly, during the hydrolysis process to produce fermentable sugars essential for biofuel production. Additionally, this review discusses other applications of Trichoderma fermentation, such as enhancing animal feedstock nutrition and employing its spores as biocontrol agents. Ongoing research efforts are directed at optimizing fermentation protocols, identifying suitable waste substrates, and genetic manipulation of strains to enhance the economic viability of Trichoderma's biotechnological applications. This manuscript contributes to the field of circular biotechnology by offering a detailed review of recent progress on the integration of agro-industrial waste materials in Trichoderma-based bioconversion technologies, highlighting both current achievements and future research directions necessary to enhance the economic and environmental sustainability of waste biomass utilization. [ABSTRACT FROM AUTHOR]

Xylanase is commonly thought to effectively cooperate with cellulase to promote the bioconversion of lignocellulose. In this study, a novel xylanase, SipoEnXyn10A (Xyn10A), previously identified from Streptomyces ipomoeae, was employed to investigate its synergetic effects on sugarcane bagasse (SCB) transformation. It was shown that the relative increase in reducing sugars reached up to 65%, with enhanced yields of glucose and xylose by 78% and 50%, respectively, in the case of the replacement of cellulase with an equivalent amount of Xyn10A at an enzyme loading of 12.5%. The highest degrees of synergy (DS) for glucose and xylose could reach 2.57 and 1.84. Moreover, the hydrolysis rate increased evidently, and the reaction time to reach the same yield of glucose and xylose was shortened by 72 h and 96 h, respectively. This study on synergistic mechanisms demonstrated that the addition of Xyn10A could cause the destruction of substrates' morphology and the dissolution of lignin components but could not change the accessibility and crystallinity of substrate cellulose. The joint effect of cellulase and xylanase during the hydrolysis process was thought to result in a synergistic mechanism. [ABSTRACT FROM AUTHOR]

Mandarin peel (MP) has gained attention as a feedstock for flavonoid recovery via the extraction process based on the biorefinery concept, but residues remain after the extraction. Toward an integrated biorefinery concept, this study aimed to valorize extracted MP (eMP) by using it in bioethanol production. For efficient fermentable sugar production, the effect of enzymatic hydrolysis conditions on sugar conversion from eMP was investigated, and the results showed that combining cellulase and cellobiase resulted in a higher enzymatic glucose conversion (78.2%) than the use of the individual enzymes (37.5% and 45.6%). Pectinase played an essential role in enhancing enzymatic arabinose conversion, and the optimal conditions were determined to be pH 4 and 90 units of the three enzymes. Under optimal conditions, the sugar yield was 199 g glucose and 47 g arabinose/kg eMP, and the hydrolysate was used in bioethanol fermentation. The results showed that the bioethanol production was 3.78 g/L (73.9% yield), similar to the control medium (3.79 g/L; 74.2% yield), although the cell growth of the yeast was slightly delayed in the eMP hydrolysate medium. This study highlights the potential of eMP as a low-cost feedstock for sugar and bioethanol production. [ABSTRACT FROM AUTHOR]

A new flour blend (F) composed of selected milling and leaving passages with a high content of non-starch polysaccharides underwent thermal (T), hydrothermal (H) or hybrid processing and was used along with cellulase (C) and cellulase-xylanase complex (CX) to produce bread. This modified flour can be considered a clean label product. In this study, blends of common and treated flours were tested for dough properties and rheology. The modified flours were added at 10 and 20% to the base wheat flour. A pan bread was then prepared to test their suitability for bread baking. Dough and bread properties were subsequently assessed. Accordingly, dough with added thermally, hydrothermally, and hybrid modified flours revealed differences in rheology. Addition of hybrid enzymatic-hydrothermal treated flour increased dough tenacity by 23% and baking strength by 26%, but decreased dough extensibility by 19%, whereas hybrid enzymatic-thermal modification increased water absorption by 6% and bread yield from 146.77% to 150.02% when modified flour was added at 20%. Breads with added modified flours demonstrated a 16% increase in bread volume, 8% lower baking loss, and 14% greater density, with no negative effect on color and texture. Thus, hybrid thermal-enzymatic treatment of the developed flours can be recommended as a suitable method for enhancing the utilization of waste flour fractions and increasing their value by enabling them to be considered as clean label bread improvers. [ABSTRACT FROM AUTHOR]

This study investigates the effectiveness of hydrothermal and alkaline pretreatment methods in enhancing the concentration of fermentable sugars derived from rice husk waste. After the pretreatments, enzymatic hydrolysis and fermentation processes were executed to evaluate the ethanol production from each pretreatment. Rice husk powder measuring ≤250 µm was used. For the alkaline pretreatment, sodium hydroxide (NaOH) was used at concentrations ranging from 0.5, 1 to 1.5% w/v. The efficacy of the hydrothermal pretreatment method was evaluated after 15, 30 and 45 min at 120 °C. The enzymatic hydrolysis process was performed over 144 h at 50 °C, pH 4.8 with an enzyme loading of 30 FPU (filter paper units). Fermentation was carried out at 37 °C using a strain of Saccharomyces cerevisiae Hansen 1883 (NCYC 366). Results indicated that the optimal conditions for alkaline pretreatment were observed at a 1.5% NaOH, while the best hydrothermal procedure was achieved at 120 °C and 45 min. The impact of these pretreatments was assessed based on the efficiency of enzymatic hydrolysis. The alkaline pretreatment resulted in 81.70% conversion of cellulose to glucose and 96.30% conversion of hemicellulose to xylose. In contrast, the hydrothermal pretreatment achieved 93% cellulose-to-glucose conversion and 83.35% hemicellulose-to-xylose conversion. The ethanol production registered ranged from 13 to 13.23 g·L−1, corresponding to a conversion factor of 0.43 for ethanol from fermentable sugars. [ABSTRACT FROM AUTHOR]

Abstract Land plants, especially those with significant woody biomass, represent the largest source of biomass on Earth, making the biodegradation of lignocellulosic materials critical to understanding the global carbon cycle. Cellulose, a major component of lignocellulose, is notoriously resistant to degradation due to its highly crystalline structure. While the degradation of cellulose by terrestrial microbes has been extensively studied, the mechanisms of cellulose degradation in deep-sea environments remain largely unexplored. The deep-sea ecosystem depends on organic matter, such as cellulose, that is synthesized in terrestrial environments and surface waters and descends to the deep sea. Recent studies suggest that a significant amount of cellulose is likely to reach the deep sea. Here, we present an in-depth study of cellulases from a novel deep-sea γ-proteobacterial strain TOYAMA8, isolated from Toyama Bay, Japan, using Surface Pitting Observation Technology (SPOT), a highly sensitive assay for enzymatic cellulose hydrolysis. The cellulases of strain TOYAMA8 show similarities to those of a previously reported deep-sea cellulolytic microbe, Marinagarivorans cellulosilyticus strain GE09. Genomic and transcriptomic analyses of these strains reveal novel cellulase genes and mechanisms that differ from terrestrial counterparts, shedding light on the unique adaptations of deep-sea microbes to recalcitrant biomass. In particular, these strains produce high-molecular-weight cellulases with unique domain architectures, likely optimized for membrane anchoring, which prevents enzyme diffusion and ensures efficient localized activity. Our findings provide critical insights into the microbial cellulose degradation in the deep sea, highlighting its role in the fate of organic carbon and the potential for biotechnological applications in biorefineries.

Abstract Background The microbial landscape within termite guts varies across termite families. The gut microbiota of lower termites (LT) is dominated by cellulolytic flagellates that sequester wood particles in their digestive vacuoles, whereas in the flagellate-free higher termites (HT), cellulolytic activity has been attributed to fiber-associated bacteria. However, little is known about the role of individual lineages in fiber digestion, particularly in LT. Results We investigated the lignocellulolytic potential of 2223 metagenome-assembled genomes (MAGs) recovered from the gut metagenomes of 51 termite species. In the flagellate-dependent LT, cellulolytic enzymes are restricted to MAGs of Bacteroidota (Dysgonomonadaceae, Tannerellaceae, Bacteroidaceae, Azobacteroidaceae) and Spirochaetota (Breznakiellaceae) and reflect a specialization on cellodextrins, whereas their hemicellulolytic arsenal features activities on xylans and diverse heteropolymers. By contrast, the MAGs derived from flagellate-free HT possess a comprehensive arsenal of exo- and endoglucanases that resembles that of termite gut flagellates, underlining that Fibrobacterota and Spirochaetota occupy the cellulolytic niche that became vacant after the loss of the flagellates. Furthermore, we detected directly or indirectly oxygen-dependent enzymes that oxidize cellulose or modify lignin in MAGs of Pseudomonadota (Burkholderiales, Pseudomonadales) and Actinomycetota (Actinomycetales, Mycobacteriales), representing lineages located at the hindgut wall. Conclusions The results of this study refine our concept of symbiotic digestion of lignocellulose in termite guts, emphasizing the differential roles of specific bacterial lineages in both flagellate-dependent and flagellate-independent breakdown of cellulose and hemicelluloses, as well as a so far unappreciated role of oxygen in the depolymerization of plant fiber and lignin in the microoxic periphery during gut passage in HT. Video Abstract

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (β/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s-1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD. KEY POINTS: • Anaerobic fungi host a wealth of industrially useful enzymes but are understudied. • P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes. • CelDs kinetics do not change with domain fusion, exhibiting high modularity.

Abstract Background In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. Results The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett–Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of −5.71. Conclusions Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Abstract Background Elucidating the mechanism of fiber transformation underlying microbial metabolism is critical for improving fiber-rich silage digestibility and preserving silage energy for ruminant nutrient absorption. However, few studies have combined quantitative microbial function and transformation products in silage to explain this mechanism. Here, we constructed a workflow to detect the substrates and products of fiber transformation in mixed silage of Sesbania cannabina and sweet sorghum (SS) and combined the absolute quantification 16S rRNA sequencing to reveal this mechanism. Results The synergistic effect of Lactobacillus cocktail and cellulase (LC) simplified the microbial diversity and minimized the microbial quantity, making Lentilactobacillus buchneri the dominant species in SS silage. As a result, the LC-treated silage had greater lactic acid content, lower pH value, and less NH3-N content. The indigestible fibers were significantly decreased due to the synergistic effect of the Lactobacillus cocktail and cellulase. Changes in microbial structure during ensiling also resulted in metabolic alterations. The increased levels of microbial enzymes, including β-glucosidase and sucrose phosphorylase, involved in starch and sucrose metabolism led to the enrichment of monosaccharides (including glucose, xylose, mannose, galactose, ribose, rhamnose, and arabinose) in the LC-treated silage. We found that L. buchneri was positively associated with β-glucosidase and sucrose phosphorylase, reflecting the crucial contribution of L. buchneri to fiber decomposition in SS silage. Conclusion Using an absolute quantitative microbiome, we found that LC treatment decreased the microbial biomass in SS silage, which in turn promoted the energy preservation in the SS silage. The cooperative interaction of the Lactobacillus cocktail and cellulase improved the fiber decomposition and in vitro dry matter digestibility rate by changing the microbiome structure and function in the SS silage, providing guidance and support for future fiber-rich silage production in the saline-alkaline region. Graphical Abstract

Because of the association with other complex polysaccharides, extracting and utilizing cellulose from lignocellulosic materials requires the combined action of a broad range of carbohydrate‐active enzymes, including multiple glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The interplay between these enzymes and the way in which Nature orchestrates their co‐existence and combined action are topics of great scientific and industrial interest. To gain more insight into these issues, we have studied the lignocellulose‐degrading abilities of an enzyme from Caldibacillus cellulovorans (CcLPMO10‐Man5), comprising an LPMO domain, a GH5 mannanase domain and two family 3 carbohydrate‐binding modules (CBM3). Using a natural softwood substrate, we show that this enzyme promotes cellulase activity, i.e., saccharification of cellulose, both by removing mannan covering the cellulose and by oxidatively breaking up the cellulose structure. Synergy with CcLPMO10‐Man5 was most pronounced for two tested cellobiohydrolases, whereas effects were smaller for a tested endoglucanase, which is in line with the notion that cellobiohydrolases and LPMOs attack the same crystalline regions of the cellulose, whereas endoglucanases attack semi‐crystalline and amorphous regions. Importantly, the LPMO domain of CcLPMO10‐Man5 is incapable of accessing the softwood cellulose in absence of the mannanase domain. Considering that LPMOs not bound to a substrate are sensitive to autocatalytic inactivation, this intramolecular synergy provides a perfect rationale for the evolution of modular enzymes such as CcLPMO10‐Man5. The intramolecular coupling of the LPMO with a mannanase and two CBMs ensures that the LPMO is directed to areas where mannans are removed and cellulose thus becomes available. [ABSTRACT FROM AUTHOR]

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Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry. [ABSTRACT FROM AUTHOR]

Lime pretreatment on maize stover was conducted with various pretreatment conditions selected by central composite design. Xylan and lignin contents in pretreated maize stover were relatively constant. Concentration of glucose monomer and oligomers decreased while concentration of xylooligomers increased in the pretreatment hydrolysate with increasing pretreatment intensity. The overall carbohydrate recovery was at least 85% for the conditions studied. Xylan removal during pretreatment was found to have a linear correlation with lignin removal. Pretreatment had a higher effect in enhancing carbohydrate solubilization for enzymatic hydrolysis than that for C. thermocellum fermentation. For all the pretreated solids, Clostridium thermocellum fermentation was found to result in much higher carbohydrate solubilization than enzymatic hydrolysis with a cellulase loading of 8 mg/g solids and a xylanase loading of 2 mg/g solids. Carbohydrate solubilization was found to have a linear correlation with lignin removal during lime pretreatment for both enzymatic hydrolysis and C. thermocellum fermentation. Considering the current challenges, this research provides a new idea for the industrial application of lignocellulosic biorefinery. [ABSTRACT FROM AUTHOR]

Agrocybe cylindracea dietary fiber (ADF) contains 95% water-insoluble dietary fiber, resulting in poor application performance. To address this issue, ADF was modified by four methods (cellulase, sodium hydroxide, high-temperature, and Lactobacillus fermentation) in this paper. By comparing the physicochemical properties, microstructures, monosaccharide compositions, and functional characteristics (antioxidant and α-glucosidase inhibitory activities in vitro) of all modified ADF samples, the optimal modification method was selected. Results showed that sodium hydroxide treatment was deemed the most effective modification method for ADF, as alkali-treated ADF (ADF-A) revealed a higher oil-holding capacity (2.02 g/g), swelling capacity (8.38 mL/g), cholesterol adsorption (6.79 mg/g), and α-glucosidase inhibitory activity (more than 70% at 0.4–0.6 mg/mL) than the other modified samples. The looser microstructure in ADF-A might be attributed to molecular rearrangement and spatial structure disruption, which resulted in smaller molecular sizes and decreased viscosity, hence improving ADF's physicochemical and functional qualities. All these findings indicate the greater application potential of modified ADF products in food and weight-loss industries, providing a comprehensive reference for the industrial application of ADF. [ABSTRACT FROM AUTHOR]