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In the production chain of palm oil, large amounts of lignocellulosic subproducts are generated, representing an important neutral carbon renewable resource for the production of biomaterials, however, a pre-treatment method is required to overcome the physical and chemical barriers present in the lignin-carbohydrate compound. The objective of this research was to isolate and identify lignocellulolytic fungi capable of biodegrading palm oil subproducts, derived from palm exploitation in the Cesar department, Colombia. The fungi were isolated on potato dextrose agar and identified based on their macroscopic and microscopic characteristics. The laccase activity (ABTS oxidation) and endoglucanase activity (CMCase) were evaluated during 13 days through solid-state fermentation of three subproducts: palm kernel cake, palm kernel shell, and fiber. The biodegraded subproducts were analyzed using scanning electron microscopy (SEM). A total of 12 isolates were quantified and grouped into 2 genera, Aspergillus sp and Trichoderma sp. Additionally, the commercially acquired Pleurotus ostreatus fungus was also used in the study. The highest endoglucanase activity (0,12 IU/mL) under the studied conditions did not show statistical significance for each of the evaluated fungi (p>0.05), in contrast to the laccase activity (203,33 IU/g) (p<0,05). The isolated fungi have the potential to improve the biodegradability of palm oil subproducts, with a focus on an ecological and sustainable strategy. [ABSTRACT FROM AUTHOR]

ABSTRACTLignocellulolytic enzymes play an important role in various industrial applications as well as the sustainable valorisation of lignocellulosic materials. Enzyme production using lignocellulosic fungi has shown great advantages such as high enzyme diversity, high production efficiency, and the availability of solid waste as raw materials. Agricultural waste, an abundant and non-food competitive feedstock, can be used to produce fungal lignocellulolytic enzymes. Pretreatment helps break down the complex structure of the raw material, thereby significantly improving product yield but also requiring more energy consumption. Multiple fermentation technologies, including submerged fermentation, solid-state fermentation, and co-culture, can be used for producing lignocellulolytic enzymes. Process optimisation may promote the yield and productivity of such enzymes without additional investment. Genetic engineering is also useful for enhancing enzyme production to meet industrial requirements. This review summarises the research progress in the fungal production of lignocellulolytic enzymes from various agricultural wastes via advanced fermentation strategies. It aims to provide technical references for the scale-up production of fungal lignocellulolytic enzymes.

Lignocellulolytic enzymes play an important role in various industrial applications as well as the sustainable valorisation of lignocellulosic materials. Enzyme production using lignocellulosic fungi has shown great advantages such as high enzyme diversity, high production efficiency, and the availability of solid waste as raw materials. Agricultural waste, an abundant and non-food competitive feedstock, can be used to produce fungal lignocellulolytic enzymes. Pretreatment helps break down the complex structure of the raw material, thereby significantly improving product yield but also requiring more energy consumption. Multiple fermentation technologies, including submerged fermentation, solid-state fermentation, and co-culture, can be used for producing lignocellulolytic enzymes. Process optimisation may promote the yield and productivity of such enzymes without additional investment. Genetic engineering is also useful for enhancing enzyme production to meet industrial requirements. This review summarises the research progress in the fungal production of lignocellulolytic enzymes from various agricultural wastes via advanced fermentation strategies. It aims to provide technical references for the scale-up production of fungal lignocellulolytic enzymes. [ABSTRACT FROM AUTHOR]

Pennisetum sinese is a giant grass with a fast-growing and high rooting rate, high sugar, protein content, and high biomass yield, which causes it to be an efficient and economic energy crop of high productivity, application in phytoremediation, and fodder production. The composting system of this grass that is adapted to the simplest formulation is easy and economically feasible in small farms for cultivating oyster mushrooms. In this study, giant grass compost was employed as a substrate for cultivating three Pleurotus species: P. florida, P. pulmonarius, and P. ostreatus to assess their enzyme activities, growth, and yields. Lignin peroxidase (LiP) was the most active enzyme in each species, while other enzymes were differently expressed between species and developmental phases. The average mass of fruiting bodies formed on the giant grass compost was 173.4 g, 166.5 g, and 152.2 g. The biological effectivity was 82.6%, 78.6%, and 72.5% for P. pulmonarius, P. ostreatus, and P. florida, respectively. The obtained results indicate the usefulness of giant grass compost for the cultivation of the three studied Pleurotus species.

Abstract Background Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far. Results In this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells. Conclusions Our results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

An increase in exoenzyme production can be enhanced by environmental stresses such as graphene oxide (GO) stress, but the link between the two events is still unclear. In this work, the effect of GO as an environmental stress factor on exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) biosynthesis was investigated in Bacillus subtilis Z2, and a plausible mechanism by which cytosolic Ca21 regulates lignocellulolytic enzyme production in B. subtilis Z2 subjected to GO stress was proposed. The filter paper-hydrolyzing (FPase [representing total cellulase]), carboxymethylcellulase (CMCase [representing endoglucanase]), and b-glucosidase activities and extracellular protein concentration of the wild-type strain under 10 mg/mL GO stress were 1.37-, 1.64-, 1.24-, and 1.16-fold those of the control (without GO stress), respectively. Correspondingly, the transcription levels of lignocellulolytic enzyme genes, cytosolic Ca21 level, and biomass concentration of B. subtilis were all increased. With lignocellulolytic enzyme from B. subtilis used to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 265.53 mg/g, and the removal rates of cellulose, hemicellulose, and lignin were 52.4%, 30.1%, and 7.5%, respectively. Furthermore, transcriptome data revealed that intracellular Ca21 homeostasis played a key role in regulating the levels of gene transcription related to the synthesis of lignocellulolytic enzymes and exoenzymes. Finally, the use of Ca21 inhibitors (LaCl3 and EDTA) and deletion of spcF (a calmodulin-like protein gene) further demonstrated that the overexpression of those genes was regulated via calcium signaling in B. subtilis subjected to GO stress. IMPORTANCE To effectively convert lignocellulose into fermentable sugars, high lignocellulolytic enzyme loading is needed. Graphene oxide (GO) has been shown to promote exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) production in some microorganisms; however, the regulatory mechanism of the biosynthesis of lignocellulolytic enzymes under GO stress remains unclear. In this work, the lignocellulolytic enzyme production of B. subtilis under GO stress was investigated, and the potential mechanism by which B. subtilis enhanced lignocellulolytic enzyme production through the calcium signaling pathway under GO stress was proposed. This work revealed the role of calcium signaling in the production of enzymes under external environmental stress and provided a direction to facilitate lignocellulolytic enzyme production by B. subtilis. [ABSTRACT FROM AUTHOR]

In this study, the potential of alfalfa pulp as an alternative substrate to wheat straw for the cultivation of oyster mushroom (Pleurotus ostreatus) was investigated. The major components associated with different mushroom stages were evaluated, as well as changes in lignocellulolytic enzyme activities in substrates composed of alfalfa pulp, wheat straw or a combination of both. Based on the results, alfalfa pulp was demonstrated to be a better substrate than wheat straw for the production of oyster mushrooms, with a high biological efficiency of 166.3 ± 25.4%. Compared to the cultivation period on commercial straw (31 days), a shorter lifecycle for oyster mushroom was found on alfalfa pulp (24 days), which could help to reduce the risk of contamination during industrial production. Study of the spent substrate as well as the harvested mushrooms revealed that the biological efficiency was related to the higher protein content (17.42%) in the alfalfa pulp compared to wheat straw, as well as greater degradation of cellulose (57.58%) and hemicellulose (56.60%). This was, by and large, due to greater extracellular hydrolytic and oxidative enzyme activity from the mushroom growth in the alfalfa pulp. The quality and safety of the fruiting bodies produced on alfalfa pulp was evaluated, which showed that the protein content was 20.4%, of which 46.3% was essential amino acids, and levels of trace elements and heavy metals were below acceptable limits. Hence, oyster mushroom cultivation using alfalfa pulp provides an alternative method to produce a value-added product, while reducing the biomass wastes in the green protein bio-refinery, and may contribute to sustainable growth in the agricultural industry. [ABSTRACT FROM AUTHOR]

The biorefining of lignocelluloses using microbial degradation methods based on highly efficient microbial strains has received widespread attention. In the present study, a cellulolytic bacterium Serratia marcescens LY1 was isolated and the complete genome sequence of S. marcescens LY1 was assembled into a ring chromosome of 5,171,649 bp and a circle plasmid of 143,701 bp. Functional annotation revealed that a total of 130 genes were annotated in CAZyme families. Secretome sequencing revealed glycoside hydrolase family 3 protein and l -arabinose isomerase, which were annotated. The enzyme activities showed that the endoglucanase, xylanase, laccase, and lignin peroxidase activities peaked on the third day, while exoglucanase, manganese peroxidase, and β-glucosidase peaked on the fourth day. Furthermore, the secretome of S. marcescens LY1 was extracted for bamboo biomass hydrolysis, resulting in the highest yield of released reducing sugars was detected from the third day of secretome culture, with a value of 42.27 ± 0.46 g/L, and the secretomes of the third and fourth days were combined, resulting in the highest reducing sugar quantity of 51.59 ± 1.04 g/L. These findings indicate that the strain S. marcescens LY1 exhibits efficient saccharification of bamboo biomass and has potential use in bamboo biomass bioconversion and energy utilization. [ABSTRACT FROM AUTHOR]

Background: Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far. Results: In this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells. Conclusions: Our results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs. [ABSTRACT FROM AUTHOR]

In this study, the potential of alfalfa pulp as an alternative substrate to wheat straw for the cultivation of oyster mushroom (Pleurotus ostreatus) was investigated. The major components associated with different mushroom stages were evaluated, as well as changes in lignocellulolytic enzyme activities in substrates composed of alfalfa pulp, wheat straw or a combination of both. Based on the results, alfalfa pulp was demonstrated to be a better substrate than wheat straw for the production of oyster mushrooms, with a high biological efficiency of 166.3 ± 25.4%. Compared to the cultivation period on commercial straw (31 days), a shorter lifecycle for oyster mushroom was found on alfalfa pulp (24 days), which could help to reduce the risk of contamination during industrial production. Study of the spent substrate as well as the harvested mushrooms revealed that the biological efficiency was related to the higher protein content (17.42%) in the alfalfa pulp compared to wheat straw, as well as greater degradation of cellulose (57.58%) and hemicellulose (56.60%). This was, by and large, due to greater extracellular hydrolytic and oxidative enzyme activity from the mushroom growth in the alfalfa pulp. The quality and safety of the fruiting bodies produced on alfalfa pulp was evaluated, which showed that the protein content was 20.4%, of which 46.3% was essential amino acids, and levels of trace elements and heavy metals were below acceptable limits. Hence, oyster mushroom cultivation using alfalfa pulp provides an alternative method to produce a value-added product, while reducing the biomass wastes in the green protein bio-refinery, and may contribute to sustainable growth in the agricultural industry.

[Display omitted] • Decay lignocellulosic waste is a source of diversified potential microbes. • Phenotypic Microarray analysis fleeting the study of metabolic pathway of microbes. • Genomic approaches used for lignocellulolytic enzymes gene activity. • Different parameters applied to optimize maximum degradation rate of BP. • Resourceful transformation of BP waste in to valuable metabolites. The most plentiful carbohydrate present on earth is in the form of cellulose which has a tremendous capacity to manufacture second or third-generation biofuels and other valuable metabolites. Cellulose is a significant part of banana pseudostem (BP) squander that causes numerous environmental issues. Hence, disposal of BP is nowadays a major obstacle for Indian farmers because of immense volume, delay in decay raise in a foul smell during debasement which attracts pests and insects to farm. The present study reports the identification of potential isolates, which efficiently debase BP. Out of many isolates, NAULH-4 showed the uppermost decomposition rate of cellulose, hemicellulose, and lignin at 45 °C and pH 8 up to 28 days incubation. Moreover, the taxonomic characterization using phenotypic microarray (PM) technology directs that the isolate NAULH-4 belongs to Bacillus with 0.71 SIM index in metabolic pathway network which is reinforced by 16 s rRNA analysis. Molecular data reveal that NAULH-4 contain laccase , xylanase, and cellulase gene. Overall, Bacillus wakoensis NAULH-4 can be applicable for promising microbe for the conversion of BP waste into value-added metabolites. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mild ammoniation pretreatment efficiently enhanced enzymatic digestibility of BSG. • Enzyme from P. oxalicum more effectively hydrolyzed BSG than that from T. reesei. • Ammonia removal and pH adjustment of hydrolysate eliminated lag phase of yeast growth. • Enzymatic hydrolysate of BSG was effectively converted into SCP with T. cutaneum. Brewer's spent grain (BSG) is a major low-value by-product of beer industry. To realize the high value application of BSG, this work proposed a strategy to produce single cell protein (SCP) with oligosaccharide prebiotics from BSG, via ammoniation pretreatment, enzymatic hydrolysis, and fermentation. The optimum conditions of ammoniation pretreatment obtained by response surface method were 11 % ammonia dosage (w/w), 63 °C for 26 h. Suitable enzyme and yeast were screened to enhance the conversion of cellulose and hemicellulose in BSG into sugars and maximize the SCP yield. It was shown that using lignocellulolytic enzyme SP from Penicillium oxalicum and Trichosporon cutaneum , about 310 g of SCP with 80 g of arabinoxylo-oligosaccharides were obtained from 1000 g of BSG. This process is low cost, high efficiency, and easy to implement, which has good industrial application prospects. [ABSTRACT FROM AUTHOR]

Because climate change is predicted to have a strong impact on high-altitude ecosystems, a better knowledge of litter decomposition in alpine ecosystems is critical to improve our predictions of the effect of climate change on ecosystem processes and services such as nutrient cycling, carbon sequestration, and below-ground biodiversity. To evaluate the effects of vegetation types [alpine shrubland (AS) and alpine meadow (AM)] and litter quality on litter decomposition and related biochemical processes, the decomposition of leaf litter of two dominant shrub species, Sorbus rufopilosa (SR, high quality) and Rhododendron lapponicum (RL, low quality), was studied using the litterbag method in an alpine treeline ecotone on the eastern Tibetan Plateau. After 1 year of decomposition, cellulolytic enzyme activities and gram-negative bacterial biomass were higher in shrubland than in meadow. However, higher fungal biomass, fungal/bacteria ratio and ligninolytic activity were observed in meadow than in shrubland after 2 years of decomposition. During the first year of decomposition, litter decomposition was faster in shrubland than in meadow probably due to the home-field advantage (HFA) effect and the bacteria-dominated decomposition, whereas in later decomposition stages, litter decomposition was faster in meadow than in shrubland, as the HFA effect diminished and fungal-dominated decomposition of recalcitrant components took over. These results indicated that litter quality effects were generally strongest in the first year and diminished in later stages when the effect of vegetation type in incubation sites developed. [ABSTRACT FROM AUTHOR]

• Competent fungi are able to shorten the composting process of EFB. • Naturally occurring lignocellulose-degrading fungi on EFB were documented. • The physical and chemical changes in EFB were correlated with slow rate of natural composting. • Lichtheimia ramosa and Neurospora crassa are the most dominant fungi. • Both fungi producing various lignocellulolytic enzymes for accelerated composting application. Oil palm empty fruit bunch (EFB) is the most significant waste generated from the agricultural industry in Malaysia. Composting is one of the potential approaches to utilize EFB. However, composting of EFB is a time-consuming process, thus impractical for industrial application. The composting process can be shortened by introducing competent fungi into an optimal EFB composting system. This study was conducted to isolate and identify competent fungi that can naturally compost EFB. Samplings were carried out at eight different time points over a 20-weeks experimental period. The physical properties of EFB samples such as pH, residual oil content, and moisture content were measured and the EFB composting process that was indicated by the contents of cellulose, hemicellulose, and lignin were assessed. The fungal growth, distribution, and lignocellulolytic enzyme activities were evaluated. The results indicated that the changes in physical properties of EFB were correlated to the fungal growth. The gradual reduction in moisture content and residual oil, and the increment in pH values in EFB samples throughout the experimental period resulted in reduced fungal growth and diversity. Such phenomenon delayed EFB composting process as revealed by the changes in EFB lignin, hemicellulose, and cellulose contents. The most dominant and resilient fungi (Lichtheimia ramosa and Neurospora crassa) survived up to 16 weeks and were capable of producing various lignocellulolytic enzymes. Further understanding of these factors that would contribute to effective EFB composting could be useful for future industrial applications. [ABSTRACT FROM AUTHOR]

Omics have given rise to research on sparsely studied microbial communities such as the landfill, lignocellulolytic microorganisms and enzymes. The bacterial diversity of Municipal Solid Waste sediments was determined using the illumina MiSeq system after DNA extraction and Polymerase chain reactions. Data analysis was used to determine the community’s richness, diversity, and correlation with environmental factors. Physicochemical studies revealed sites with mesophilic and thermophilic temperature ranges and a mixture of acidic and alkaline pH values. Temperature and moisture content showed the highest correlation with the bacteria community. The bacterial analysis of the community DNA revealed 357,030 effective sequences and 1891 operational taxonomic units (OTUs) assigned. Forty phyla were found, with the dominant phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidota, while Aerococcus, Stenotrophomonas, and Sporosarcina were the dominant species. PICRUSt provided insight on community’s metabolic function, which was narrowed down to search for lignocellulolytic enzymes’ function. Cellulase, xylanase, esterase, and peroxidase were gene functions inferred from the data. This article reports on the first phylogenetic analysis of the Pulau Burung landfill bacterial community. These results will help to improve the understanding of organisms dominant in the landfill and the corresponding enzymes that contribute to lignocellulose breakdown.

Abstract Filamentous fungus Aspergillus niger has high industrial value due to their lignocellulolytic enzyme activities and ATCC 10864 is one of the few type strains of A. niger which has a unique biofilm forming capability. Here we report the first draft genome sequence of A. niger ATCC 10864 strain. The genome of A. niger ATCC 10864 is 36,172,237 bp long and comprise of 310 scaffolds with 49.5% average GC content. A total of 10,804 protein-coding genes were predicted among which 10,761 genes were with putative functions. A. niger ATCC 10864 genome coded for 709 putative carbohydrate active enzyme families distributed in six functional categories and among them glycoside hydrolases (GHs) represent the most number of families (279). Genes that include pepA, brlA, exgA, LaeA, rodA, GCN have also been identified in this study, which may play a role in biofilm formation. This high-quality draft genome sequence will facilitate our understanding of the mechanisms behind fungal biofilm formation and higher lignocellulolytic enzyme production.

Abstract: The feasibility of spent mushroom substrate (SMS) as an alternative fermentation feedstock for cellulase production has been demonstrated in this work. Utilization of SMS as a substrate has been attempted widely due to its high cellulose content and readily available in smaller particle size. On top of that, the availability of delignified SMS by the action of Pleurotus pulmonarius during mushroom cultivation offers another benefit to its use whereby no chemical pretreatment would be required prior to fermentation. The recovery of crude laccase and manganese peroxidase from delignified SMS were found to be 3 and 1.4 U/g, respectively. Further to this, the cellulase production from SMS by Trichoderma asperellum UPM 1 under solid state fermentation was optimized by applying central composite design, resulted in increment of 1.4-fold in CMCase (171.21 U/g) and 1.5-fold in β-glucosidase (6.83 U/g), with the optimum temperature of 27.5 °C, initial moisture content 81% and initial pH of fermentation 4.5. Therefore, this study showed that the direct utilization of SMS is feasible for promising cellulase production by T. asperellum UPM 1.Graphical Abstract: [ABSTRACT FROM AUTHOR]

Genetic engineering of transcription factors is an efficient strategy to improve lignocellulolytic enzyme production in fungi. In this study, the xylanase transcriptional regulators of Trichoderma reesei (Xyr1) and Neurospora crassa (XLR-1), as well as their constitutively active mutants (Xyr1A824V and XLR-1A828V), were heterologously expressed in Penicillium oxalicum. The two heterologous regulators were identified to be able to activate lignocellulolytic enzyme gene expression in P. oxalicum. Particularly, expression of T. reesei Xyr1 resulted in a higher cellulase production level compared with the expression of native xylanase transcriptional regulator XlnR using the same promoter. Xyr1A824V and XLR-1A828V were found to be able to confer P. oxalicum more enhanced lignocellulolytic abilities than wild-type regulators Xyr1 and XLR-1. Furthermore, introduction of regulatory modules containing Xyr1A824V/XLR-1A828V and their target cellulase genes resulted in greater increases in cellulase production than alone expression of transcriptional regulators. Through the cumulative introduction of three regulatory modules containing regulator mutants and their corresponding target cellulase genes from P. oxalicum, T. reesei, and N. crassa, a 2.8-fold increase in cellulase production was achieved in P. oxalicum. [ABSTRACT FROM AUTHOR]

Black gram husk was used as a solid substrate for laccase production by Pleurotus ostreatus, and various fermentation conditions were optimized based on an artificial intelligence method. A total of six parameters, i.e., temperature, inoculum concentration, moisture content, CuSO4, glucose, and peptone concentrations, were optimized. A total of 50 experiments were conducted, and the obtained data were modeled by a hybrid of artificial neural network (ANN) and genetic algorithm (GA) approaches. ANN was employed to model the experimental data, and the predicted values were further optimized by GA. Employment of ANN–GA hybrid methodology resulted in a significant improvement, as approximately two-fold laccase production (4244 U/gds) was achieved.

Filamentous fungus Aspergillus niger has high industrial value due to their lignocellulolytic enzyme activities and ATCC 10864 is one of the few type strains of A. niger which has a unique biofilm forming capability. Here we report the first draft genome sequence of A. niger ATCC 10864 strain. The genome of A. niger ATCC 10864 is 36,172,237 bp long and comprise of 310 scaffolds with 49.5% average GC content. A total of 10,804 protein-coding genes were predicted among which 10,761 genes were with putative functions. A. niger ATCC 10864 genome coded for 709 putative carbohydrate active enzyme families distributed in six functional categories and among them glycoside hydrolases (GHs) represent the most number of families (279). Genes that include pepA, brlA, exgA, LaeA, rodA, GCN have also been identified in this study, which may play a role in biofilm formation. This high-quality draft genome sequence will facilitate our understanding of the mechanisms behind fungal biofilm formation and higher lignocellulolytic enzyme production. [ABSTRACT FROM AUTHOR]

Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5 mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil. [ABSTRACT FROM AUTHOR]

In this study, with combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assay, a novel cellulase and xylanase-producing strain identified as Bacillus sp. was isolated. Using lactose as the only carbon source, a complete and balanced lignocellulolytic enzyme system containing at least endoglucanase (9.6 U/ml), exoglucanase (0.8 U/ml), Fpase (1.4 U/ml), xylanase (3.8 U/ml) and β-glucosidase (1.2 U/ml) was produced. Interestingly, a zymogram of the crude culture supernatant displayed a multifunctional lignocellulolytic enzyme system including at least four bonds with both endoglucanase activity and xylanase activity at 21.2, 23.8, 28.9 and 31.2 kDa, respectively, indicating that these enzymes might be bifunctional. More gratifyingly, according to the binding affinity analysis and scanning electron microscopy, the crude enzyme complex produced by strain BS-5 was capable of hydrolyzing not only pure insoluble polysaccharides, but also agricultural residues such as corn cob. At 5% substrate concentration and 20 FPU/g enzyme loading, the reducing sugar was 350.8 mg/g of alkali-pretreated corn cob after 72 h enzymatic hydrolysis. These results suggested that this strain could be a good candidate for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail for the saccharification of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

The extracellular lignocellulolytic enzymes secreted by Porodaedalea pini were investigated for their ability to degrade the wood of Picea jezoensis over various time periods. In addition, changes in wood chemical component contents were also investigated. Enzyme production and wood degradation by P. pini significantly increased starting at 60 days of incubation. The total lignin, holocellulose, and α-cellulose contents, as well as pH, decreased during the degradation process, while hot water, 1% NaOH, and ethanol-toluene extract contents significantly increased. These results indicate that P. pini simultaneously degraded the lignin and polysaccharides of P. jezoensis wood. Additionally, P. pini continuously produced xylanase, β-glucosidase, and endoglucanase with higher activities than those of exoglucanase and cellobiose dehydrogenase. Manganese(II)-dependent peroxidase showed the highest ligninolytic activity, followed by lignin peroxidase and laccase. These results indicate that P. pini produces a variety of lignocellulolytic enzymes, and that the produced enzymes contribute to the degradation of P. jezoensis wood components. [ABSTRACT FROM AUTHOR]

Objective: Cost-effective production of industrially important enzymes is a key for their successful utilization on large scale. Present study was conducted to appraise Pleurotus sapidus WC 529 potential to produce oxidative (lignin peroxidase, manganese peroxidase, laccase) and hydrolytic (endoglucanase, exoglucanase and β-glucosidase) enzymes, grown on wheat straw, corn cobs, corn stover, rice straw, banana stalk and sugarcane bagasse in solid state fermentation (SSF) process. The crude extract thus produced was used for the delignification of various agro-industrial residues. Methods: Different agricultural wastes were assessed for lignocellulolytic enzymes production. The process parameters were optimized through response surface methodology (RSM) with central composite design (CCD) for maximum enzyme production. Results: P. sapidus secreted lower levels of hydrolytic enzymes but expressed high laccase, MnP and LiP activities in SSF (228.25, 153.45 and 59.20 UmL-1 respectively) of banana stalk. The optimal conditions for yielding highest enzyme activities were: initial pH, 4; temperature, 35°C; moisture level, 60%; inoculum size, 4 mL, and incubation time, 120 h. The enzyme extract removed 56.54, 51.08, 65.81 and 57.4% lignin from rice straw, sugarcane bagasse, corn cobs and wheat straw, respectively, after 48 h at 35°C. Conclusion: High ligninolytic activities were obtained in SSF of banana stalk by P. sapidus. The enzyme production was considerably improved by optimization through RSM. The results suggest that lignocellulolytic enzymes could be produced using cost-effective carbon sources and implicated to digest lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Native lignocellulolytic enzyme systems secreted by filamentous fungi can be further optimized by protein engineering or supplementation of exogenous enzyme components. We developed a protein production and evaluation system in cellulase-producing fungus Penicillium oxalicum. First, by deleting the major amylase gene amy15A, a strain Δ15A producing few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, Xyn10B, and Cel12A) with originally low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. BGL4 and Cel12A overexpression resulted in increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By switching the culture medium, this platform is convenient to produce originally low-expressed lignocellulolytic enzymes in relatively high purities on starch and to evaluate the effect of their supplementation on the performance of a complex cellulase system on cellulose. [ABSTRACT FROM AUTHOR]

A consortium of four hypercellulolytic fungal cultures, namely Aspergillus awamori, Trichoderma viride, Aspergillus nidulans and Phanerochaete chrysosporium was optimized for compost production on the basis of their lignocellulolytic enzyme production potential. The consortium has been effectively used for composting of diverse agricultural wastes such as paddy straw, soybean trash, pearl millet, maize residues and mustard stover. The inoculum of four fungi needs to be produced in bulk for talc-based consortium formulation. Routinely fungus is grown on potato dextrose agar medium, malt extract agar medium or Martin's Rose Bengal agar medium, which escalates the cost of production because of relatively expensive ingredients. Further, harvesting of fungal biomass from solid surface adds additional cost to the production schedule. [ABSTRACT FROM AUTHOR]

Black gram husk was used as a solid substrate for laccase production by Pleurotus ostreatus, and various fermentation conditions were optimized based on an artificial intelligence method. A total of six parameters, i.e., temperature, inoculum concentration, moisture content, CuSO4, glucose, and peptone concentrations, were optimized. A total of 50 experiments were conducted, and the obtained data were modeled by a hybrid of artificial neural network (ANN) and genetic algorithm (GA) approaches. ANN was employed to model the experimental data, and the predicted values were further optimized by GA. Employment of ANN-GA hybrid methodology resulted in a significant improvement, as approximately two-fold laccase production (4244 U/gds) was achieved. [ABSTRACT FROM AUTHOR]

An increase in exoenzyme production can be enhanced by environmental stresses such as graphene oxide (GO) stress, but the link between the two events is still unclear. In this work, the effect of GO as an environmental stress factor on exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) biosynthesis was investigated in Bacillus subtilis Z2, and a plausible mechanism by which cytosolic Ca 2+ regulates lignocellulolytic enzyme production in B. subtilis Z2 subjected to GO stress was proposed. The filter paper-hydrolyzing (FPase [representing total cellulase]), carboxymethylcellulase (CMCase [representing endoglucanase]), and β-glucosidase activities and extracellular protein concentration of the wild-type strain under 10 μg/mL GO stress were 1.37-, 1.64-, 1.24-, and 1.16-fold those of the control (without GO stress), respectively. Correspondingly, the transcription levels of lignocellulolytic enzyme genes, cytosolic Ca 2+ level, and biomass concentration of B. subtilis were all increased. With lignocellulolytic enzyme from B. subtilis used to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 265.53 mg/g, and the removal rates of cellulose, hemicellulose, and lignin were 52.4%, 30.1%, and 7.5%, respectively. Furthermore, transcriptome data revealed that intracellular Ca 2+ homeostasis played a key role in regulating the levels of gene transcription related to the synthesis of lignocellulolytic enzymes and exoenzymes. Finally, the use of Ca 2+ inhibitors (LaCl 3 and EDTA) and deletion of spcF (a calmodulin-like protein gene) further demonstrated that the overexpression of those genes was regulated via calcium signaling in B. subtilis subjected to GO stress. IMPORTANCE To effectively convert lignocellulose into fermentable sugars, high lignocellulolytic enzyme loading is needed. Graphene oxide (GO) has been shown to promote exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) production in some microorganisms; however, the regulatory mechanism of the biosynthesis of lignocellulolytic enzymes under GO stress remains unclear. In this work, the lignocellulolytic enzyme production of B. subtilis under GO stress was investigated, and the potential mechanism by which B. subtilis enhanced lignocellulolytic enzyme production through the calcium signaling pathway under GO stress was proposed. This work revealed the role of calcium signaling in the production of enzymes under external environmental stress and provided a direction to facilitate lignocellulolytic enzyme production by B. subtilis.

Abstract: Harmful environmental issues of fossil-fuels and concerns about petroleum supplies have spurred the search for renewable alternative fuels such as biofuel. Agricultural crop residues represent an abundant renewable resource for future biofuel. To be a viable alternative, a biofuel should provide a net energy gain, have environmental benefits, be economically feasible, and should also be producible in large quantities without reducing food supplies. We used these criteria to evaluate the white rot basidiomycota-derived fungus Ganoderma lucidum that secretes substantial amounts of hydrolytic and oxidative enzymes useful for the degradation of lignocellulosic biomass that were not described hitherto. The current bottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar. To understand the enzymatic hydrolysis of complex biomasses, we cultured G. lucidum with sugarcane bagasse as substrate and qualitatively analyzed the entire secretome. The secreted lignocellulolytic enzymes were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and diverse enzymes were found, of which several were novel lignocellulosic biomass hydrolyzing enzymes. We further explored G. lucidum-derived cellulose, hemicellulose and lignin degrading enzymes as valuable enzymes for the second generation of biofuel obtained from a lignocellulose substrate such as sugarcane bagasse. [Copyright &y& Elsevier]

Abstract: The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC–MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy. [Copyright &y& Elsevier]

Abstract: Degradation of lignocellulosic agro-industrial residues by means of complex microbial community is a promising approach providing efficient biomass decomposition for subsequent conversion to value-added products. In this study, an active thermophilic lignocellulose degrading microbial consortium was bred from high-temperature sugarcane bagasse compost by successive subcultivation under aerobic static conditions. The microbial consortium showed efficient degradation activity on potential biorefinery cellulosic substrates, including bagasse, rice straw, corn stover and industrial eucalyptus pulp sludge. The consortium was structurally stable with the co-existence of eight major microbes, comprising anaerobic bacterial genera Clostridium and Thermoanaerobacterium along with an aerobic/facultative anaerobic Rhodocyclaceae bacterium, bacilli, and uncultured bacteria. Majority of the lignocellullolytic activities including endo-glucanase, xylanase and β-glucanase was present in the crude culture supernatant compared to the cell-bound fraction. Proteomic analysis of cellulose bound fraction of the crude extracellular enzyme revealed a multi-species lignocellulolytic enzyme system composed mainly of cellulosomal components and extracellular cellulases of clostridia along with hemicellulases and a β-glucanase from Clostridium, Bacillus, and Thermobacillus related origins. This work presents the first report on analysis of the complex structurally stable lignocellulose degrading microbial consortium together with the characterization of its lignocellulolytic enzyme system applicable for biomass degradation and conversion in biotechnological industry. [Copyright &y& Elsevier]

Aims: To obtain strong, carbon source-inducible promoters useful for industrial applications of Corynebacterium glutamicum. Methods and Results: DNA microarray and qRT-PCR enabled identification of the promoters of cgR_2367 ( malE1) and cgR_2459 ( git1) as strong, maltose- and gluconate-inducible promoters, respectively, in C. glutamicum. Promoter probe assays revealed that in the presence of the inducing sugars, P malE1 and P git1, respectively, facilitated 3·4- and 4·2-fold increased β-galactosidase activities compared to the same activity induced by glucose. In addition, P malE1 was not functional in Escherichia coli, in which P git1 function was repressible, which enabled the cloning of a hitherto ‘difficult-to-clone’ heterologous gene of a lignocellulolytic enzyme, whose secretion was consequently induced by the carbon sources. Conclusions: P malE1 and P git1 are strong, carbon source-inducible promoters of C. glutamicum whose characteristics in E. coli are integral to the secretion ability of C. glutamicum to secrete lignocellulolytic enzyme. Significance and Impact of the Study: Corynebacterium glutamicum, like its counterpart industrial workhorses E. coli and Bacillus subtilis, does exhibit strong, carbon source-inducible promoters, and the functionality of two of which was demonstrated in this study. While this study may be most relevant in the ongoing efforts to establish technologies of the biorefinery, it should also be of interest to general microbiologists exploring the versatility of industrial micro-organisms. In so doing, the study should impact future advances in industrial microbiology. [ABSTRACT FROM AUTHOR]

Omics have given rise to research on sparsely studied microbial communities such as the landfill, lignocellulolytic microorganisms and enzymes. The bacterial diversity of Municipal Solid Waste sediments was determined using the illumina MiSeq system after DNA extraction and Polymerase chain reactions. Data analysis was used to determine the community's richness, diversity, and correlation with environmental factors. Physicochemical studies revealed sites with mesophilic and thermophilic temperature ranges and a mixture of acidic and alkaline pH values. Temperature and moisture content showed the highest correlation with the bacteria community. The bacterial analysis of the community DNA revealed 357,030 effective sequences and 1891 operational taxonomic units (OTUs) assigned. Forty phyla were found, with the dominant phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidota, while Aerococcus, Stenotrophomonas, and Sporosarcina were the dominant species. PICRUSt provided insight on community's metabolic function, which was narrowed down to search for lignocellulolytic enzymes' function. Cellulase, xylanase, esterase, and peroxidase were gene functions inferred from the data. This article reports on the first phylogenetic analysis of the Pulau Burung landfill bacterial community. These results will help to improve the understanding of organisms dominant in the landfill and the corresponding enzymes that contribute to lignocellulose breakdown. [ABSTRACT FROM AUTHOR]

Rumen fermentation is known to be effective for lignocellulosic-wastes biodegradation to certain extent but it is still unclear if there exists a termination of the microorganisms' action to further degrade the bio-refractory fractions. In order to illuminate the related microbiological characteristics, experiments were conducted in a prolonged duration of rumen fermentation of mechanically ruptured wheat straw, with inoculation of cow rumen microorganisms in vitro. Although the organic wastes could not be biodegraded quickly, continuous conversion of the lignocellulosic contents to volatile fatty acids and biogas proceeded in the duration of more than three months, resulting in 96–97% cellulose and hemicellulose decomposition, and 42% lignin decomposition. X-ray diffraction and Fourier transform infrared spectroscopy further demonstrated the characteristics of lignocellulosic structure decomposition. Under the actions of cow rumen microorganisms, stable pH was maintained in the fermentation liquid, along with a steady NH 4 +-N, volatile fatty acids accumulation, and a large buffering ability. It was identified by enzyme analysis and Illumina MiSeq sequencing that the rich core lignocellulolytic enzymes secreted by the abundant and diverse rumen bacteria and fungi contributed to the persistent degradation of lignocellulosic wastes. Members of the Clostridiales order and Basidiomycota phylum were found to be the dominant lignocellulolytic bacteria and fungi, respectively. It could thus be inferred that the main lignocellulose degradation processes were a series of catalytic reactions under the actions of lignocellulolytic enzymes secreted from bacteria and fungi. The dominant hydrogenotrophic methanogens (Methanomassiliicoccus , Methanobrevibacter , Methanosphaera , and Methanoculleus) in the rumen could also assist CH 4 production if the rumen fermentation was followed with anaerobic digestion. Unlabelled Image • High WS decomposition was achieved by the persistent action of cow rumen microflora. • Degradation characteristics of raw WS subject to rumen fermentation were analysed. • Rich core lignocellulolytic enzymes were secreted by the rumen bacteria and fungi. • Firmicutes and Basidiomycota were the dominant lignocellulolytic bacteria and fungi. • Degradation mechanisms for the in vitro rumen fermentation of WS were elucidated. [ABSTRACT FROM AUTHOR]

Enzymatic conversion of corn fiber to fermentable sugars is beneficial to improving the economic efficiency of corn processing. In this work, the filamentous fungus Penicillium oxalicum was found to secrete enzymes for efficient saccharification of un-pretreated corn fiber. Separate engineering of transcriptional activators ClrB, XlnR, and AraR led to enhanced production of different sets of lignocellulolytic enzymes. Particularly, the enzymes produced by XlnR- and AraR-engineered strains showed a synergistic effect in corn fiber saccharification. Combinatorial engineering of all three activators generated a strain MCAX with 3.1- to 51.0-fold increases in lignocellulolytic enzyme production compared with the parent strain. In addition, the enzymes of strain MCAX released significantly more fermentable sugars from corn fiber than those of the parent strain at the same protein dosage. The results suggest that this strain has potential for on-site production of enzymes for corn fiber saccharification.

A total of 6,219 positive clones were obtained by constructing a BAC library of uncultured ruminal fungi of gayal, and two clones (xynF1 and eglF2) with lignocellulolytic enzyme activity were selected. The sequencing results showed that xynF1 and eglF2 had 903-bp, and 1,995-bp, open reading frames likely to encode β-xylanase (XynF1) and β-glucosidase (EglF2), respectively. The amino acid sequence of XynF1 had 99% coverage and 95% homology to the endo-β-1,4-xylanase encoded by the cellulase gene of Orpinomyces sp. LT-3 (GenBank accession No. AEO51791.1). The amino acid sequence of EglF2 had 99% coverage and 93% homology to the β-glucosidase encoded by the cellulase gene of Piromyces sp. E2 (GenBank accession No. CAC34952.1). Analysis using the SMART software showed that XynF1 contains a glycoside hydrolase family 11 functional module and a carbohydrate-binding module, while EglF2 contains a glycoside hydrolase family 1 functional module. XynF1 showed the highest relative enzymatic activity, up to 95%, at 45°C and pH 4.2, while EglF2 showed the highest relative enzymatic activity, up to 95%, at 55°C and pH 6.2. In this study, we achieved efficient expression of the xynF1 and eglF2 genes in Pichia pastoris, which laid a foundation for the practical application of the lignocellulolytic enzymes.

Background: Agricultural residue is more efficient than purified cellulose at inducing lignocellulolytic enzyme production in Penicillium oxalicum GZ-2, but in Trichoderma reesei RUT-C30, cellulose induces a more efficient response. To understand the reasons, we designed an artificially simulated plant biomass (cellulose plus xylan) to study the roles and relationships of each component in the production of lignocellulolytic enzymes by P. oxalicum GZ-2., Results: The changes in lignocellulolytic enzyme activity, gene expression involving (hemi)cellulolytic enzymes, and the secretome of cultures grown on Avicel (A), xylan (X), or a mixture of both (AX) were studied. The addition of xylan to the cellulose culture did not affect fungal growth but significantly increased the activity of cellulase and hemicellulase. In the AX treatment, the transcripts of cellulase genes (egl1, egl2, egl3, sow, and cbh2) and hemicellulase genes (xyl3 and xyl4) were significantly upregulated (P <0.05). The proportion of biomass-degrading proteins in the secretome was altered; in particular, the percentage of cellulases and hemicellulases was increased. The percentage of cellulases and hemicellulases in the AX secretome increased from 4.5% and 7.6% to 10.3% and 21.8%, respectively, compared to the secretome of the A treatment. Cellobiohydrolase II (encoded by cbh2) and xylanase II (encoded by xyl2) were the main proteins in the secretome, and their corresponding genes (cbh2 and xyl2) were transcripted at the highest levels among the cellulolytic and xylanolytic genes. Several important proteins such as swollenin, cellobiohydrolase, and endo-beta-1,4-xylanase were only induced by AX. Bray-Curtis similarity indices, a dendrogram analysis, and a diversity index all demonstrated that the secretome produced by P. oxalicum GZ-2 depended on the substrate and that strain GZ-2 directionally adjusted the compositions of lignocellulolytic enzymes in its secretome to preferably degrade a complex substrate., Conclusion: The addition of xylan to the cellulose medium not only induces more hemicellulases but also strongly activates cellulase production. The proportion of the biomass-degrading proteins in the secretome was altered significantly, with the proportion of cellulases and hemicellulases especially increased. Xylan and cellulose have positively synergistic effects, and they play a key role in the induction of highly efficient lignocellulolytic enzymes.