Production Of Cellulolytic Enzymes Research Articles

The transcription factor STE12 influences growth on several carbon sources and production of dehydroacetic acid (DHAA) in Trichoderma reesei

The filamentous ascomycete Trichoderma reesei, known for its prolific cellulolytic enzyme production, recently also gained attention for its secondary metabolite synthesis. Both processes are intricately influenced by environmental factors like carbon source availability and light exposure. Here, we explore the role of the transcription factor STE12 in regulating metabolic pathways in T. reesei in terms of gene regulation, carbon source utilization and biosynthesis of secondary metabolites. We show that STE12 is involved in regulating cellulase gene expression and growth on carbon sources associated with iron homeostasis. STE12 impacts gene regulation in a light dependent manner on cellulose with modulation of several CAZyme encoding genes as well as genes involved in secondary metabolism. STE12 selectively influences the biosynthesis of the sorbicillinoid trichodimerol, while not affecting the biosynthesis of bisorbibutenolide, which was recently shown to be regulated by the MAPkinase pathway upstream of STE12 in the signaling cascade. We further report on the biosynthesis of dehydroacetic acid (DHAA) in T. reesei, a compound known for its antimicrobial properties, which is subject to regulation by STE12. We conclude, that STE12 exerts functions beyond development and hence contributes to balance the energy distribution between substrate consumption, reproduction and defense.

Investigation of the presence and enzymatic activity of fungi in stored corn (Zea mays L.) and soybean [Glycine max (L.) Merrill] grains

Soy [Glycine max (L.) Merrill] and corn (Zea mays L.) are important crops in Brazilian agribusiness. When stored improperly, these grains are susceptible to insects and microorganisms. Among the microorganisms capable of causing grain deterioration, fungi stand out. Despite the harm associated with fungi, they are biotechnologically appreciable due to their potential ability to produce hydrolytic enzymes, such as cellulases and proteases. Fungal hydrolytic enzymes have been applied in various processes in the food, textile, and detergent production industries, among others. The objective of this study was to verify the presence of fungi in samples of corn and soybean grains under storage conditions and characterize them regarding the production of cellulolytic and proteolytic enzymes. Among the results, there was a greater quantity and variety of filamentous fungi (35 morphotypes) in association with the grains evaluated, than of yeasts (three morphotypes). Representatives of the genera Aspergillus spp., Curvularia sp., and Penicillium spp. were detected, corroborating previous reports in the literature. Filamentous fungi that tested positive for cellulase production had an enzyme ratio index (ERI) varying between 1.06 and 2.57 (mean ± standard deviation = 1.37 ± 0.48), while for proteolytic activity, only the fungi isolated from soybean tested positive, with ERI (mean ± standard deviation) = 1.21 ± 0.01. Although the taxonomy of the isolates in this study has not been completely elucidated, it is possible to state that these fungi are promising for producing hydrolytic enzymes, with potential for use in the development of products and/or biotechnological processes.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity

BackgroundLignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process.ResultsThe current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities.ConclusionThe results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Cellulolytic Aerobic Bacteria Isolated from Agricultural and Forest Soils: An Overview.

This review provides insights into cellulolytic bacteria present in global forest and agricultural soils over a period of 11 years. It delves into the study of soil-dwelling cellulolytic bacteria and the enzymes they produce, cellulases, which are crucial in both soil formation and the carbon cycle. Forests and agricultural activities are significant contributors to the production of lignocellulosic biomass. Forest ecosystems, which are key carbon sinks, contain 20-30% cellulose in their leaf litter. Concurrently, the agricultural sector generates approximately 998 million tons of lignocellulosic waste annually. Predominant genera include Bacillus, Pseudomonas, Stenotrophomonas, and Streptomyces in forests and Bacillus, Streptomyces, Pseudomonas, and Arthrobacter in agricultural soils. Selection of cellulolytic bacteria is based on their hydrolysis ability, using artificial cellulose media and dyes like Congo red or iodine for detection. Some studies also measure cellulolytic activity in vitro. Notably, bacterial cellulose hydrolysis capability may not align with their cellulolytic enzyme production. Enzymes such as GH1, GH3, GH5, GH6, GH8, GH9, GH10, GH12, GH26, GH44, GH45, GH48, GH51, GH74, GH124, and GH148 are crucial, particularly GH48 for crystalline cellulose degradation. Conversely, bacteria with GH5 and GH9 often fail to degrade crystalline cellulose. Accurate identification of cellulolytic bacteria necessitates comprehensive genomic analysis, supplemented by additional proteomic and transcriptomic techniques. Cellulases, known for degrading cellulose, are also significant in healthcare, food, textiles, bio-washing, bleaching, paper production, ink removal, and biotechnology, emphasizing the importance of discovering novel cellulolytic strains in soil.

Extraction of Cellulases Produced through Solid-State Fermentation by Trichoderma reesei CCT-2768 Using Green Coconut Fibers Pretreated by Steam Explosion Combined with Alkali

The industrial processing of coconut to produce valuable foods, such as water and milk, generates large volumes of waste, especially the fruit shell. Despite this, material can be used in bioprocess applications, e.g., the production of enzymes, its recalcitrance hinders the cultivation of microorganisms, and low productivity is usually achieved. In this study, the production of cellulolytic enzymes through solid-state fermentation (SSF) and their extraction was investigated using the green coconut fiber pretreated by steam explosion, followed by alkali. The fungus Trichoderma reesei CCT-2768 was cultivated, using an experimental design, to study the effect of the water activity and the amount of biomass in the reactor. The combination of the pretreatment strategies yielded more porous biomass, with less hemicellulose (5.38%, compared to 10.15% of the raw biomass) and more cellulose (47.77% and 33.96% in the pretreated and raw biomasses, respectively). The water activity significantly affected the production of cellulases, with maximum activity yielded at the highest investigated value (0.995). Lastly, the extraction of the enzymes from the cultivation medium was studied, and a 9 g/L NaCl solution recovered the highest CMCase and FPase activities (5.19 and 1.19 U/g, respectively). This study provides an important contribution to the valorization of the coconut residue through (i) the application of the steam explosion technology to optimize the production of cellulases using the SSF technology and (ii) their extraction using different solvents.

Determination of cellulolytic potentials of <i>Aspergillus</i> species isolated from central waste dump site of Nile University of Nigeria

A large number of microorganisms are capable of degrading cellulose but only a few of these microorganisms produce significant quantities of enzymes capable of completely hydrolyzing cellulose. Fungi are the main cellulase-producing microorganisms. This study was aimed to determine the cellulolytic potentials of Aspergillus species isolated from the central waste dump site of Nile University of Nigeria. In this study, fungal species were isolated from soil samples obtained from waste dump site using pour plate technique. The isolates were characterized using cultural and morphological features as well as microscopic examination. Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, which were isolated were further screened on carboxymethylcellulose agar for their ability to degrade cellulose. Screening of fungal isolates was performed by plate method. Cellulolytic fungi were evaluated after 5 days for the production of cellulolytic enzymes by staining with 1% Congo red. The diameter of clear zone on fungal plates, gave an approximate indication of cellulase activities. Aspergillus niger had a zone of clearing of 25.50 mm while Aspergillus flavus had 18.50 mm. Aspergillus terreus did not show any cellulolytic activity. Aspergillus niger had the highest occurrence rate of 50%. Aspergillus flavus and Aspergillus terreus both had 25% occurrence rate.

Different Putative Methyltransferases Have Different Effects on the Expression Patterns of Cellulolytic Genes.

Putative methyltranferase LaeA and LaeA-like proteins, conserved in many filamentous fungi, regulate fungal growth, development, virulence, the biosynthesis of secondary metabolites, and the production of cellulolytic enzymes. Penicillium oxaliucm is a typical fungus that produces cellulolytic enzymes. In this study, we reported the biological function of eight putative methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) containing a methyltransf_23 or methyltransf_25 domain, with a focus on their roles in the production of cellulolytic enzymes. In P. oxalicum, various methyltransferase genes displayed different transcriptional levels. The genes Pomtr23C and Pomtr25A exhibited high transcriptional levels, while Pomtr23D/E/F/G/H and Pomtr25B were transcribed constantly at low levels. The gene deletion mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) were constructed. Various mutants have different patterns in cellulolytic enzyme production. Compared to the WT, the largest increase in filter paper activity (FPA, indicating total cellulase activity) was observed in the Δmtr23G mutant, the only mutant with a cellulolytic halo surrounding the colony. Three mutants (Δmtr23C/D and Δmtr25A) also showed increased cellulolytic enzyme production. The Δmtr23E and Δmtr25B mutants displayed decreased FPA activity, while the Δmtr23F and Δmtr23H mutants displayed similar patterns of cellulolytic enzyme production compared with the WT. The assay of transcriptional levels of cellobiohydrolase gene Pocbh1 and β-1,4-endoglucanase Poeg1 supported that higher cellulolytic gene transcription resulted in higher production of cellulolytic enzymes, and vice versa. The transcriptional levels of two transcription factors, activator XlnR and repressor CreA, were measured. The high transcription level of the PoxlnR gene in the Δmtr23D mutant should be one reason for the increased transcription of its cellulolytic enzyme gene. Both XlnR and CreA transcriptional levels increased in the Δmtr23G mutant, but the former showed a more significant increase than the latter, indicating that the activation effect predominated. The PoMtr25A is localized in the nucleus. The catalytic subunit SNF2 of the SWI/SNF chromatin-remodeling complex was found as one of the interacting proteins of PoMtr25A via tandem affinity purification coupled with mass spectrometry. PoMtr25A may affect not only the transcription of repressor CreA but also by recruiting SWI/SNF complexes that affect chromatin structure, thereby regulating the transcription of target genes.

Date seed waste derived nanocatalyst and its application in production of hydrolytic enzyme, fermentative sugars and biohydrogen

Biofuel production from cellulosic biomass is a promising approach; however, the cost-intensive utilization of cellulolytic enzymes is a major roadblock to economic production. This study reports the preparation of a nanocatalyst using date seed and evaluates the impact of nanocatalysts on cellulolytic enzyme production using solid-state fermentation of date pulp waste through bacterial co-cultivation. Under optimized conditions, 30 IU/gds filter paper activity is produced in the presence of 2 mg of nanocatalyst. Cellulase showed thermal stability at 50 °C and pH 7 up to 10 h in the presence of nanocatalyst, and it produced 32.31 g/L glucose through the hydrolysis of acidic-pretreated date seeds in 24 h. Subsequently, 1788 mL/L of cumulative H2 in 24 h through cocultured dark fermentation could be produced. The approach presented in this study can be effective for multiple value additions, including nanocatalyst preparation, cellulase enzyme, and biohydrogen production.

Al2(SO4)3 alters the antioxidant mitochondrial metabolism of Botritys cinerea and optimizes the production of cellulose and oxidative degrading enzymes

The enzymes produced by pathogenic fungi, especially Botritys cinerea, deserve specific attention due to the diversity of their applications, mainly in biofuel production, food processing, and the pharmaceutical industry. Thus, this work used Al2(SO4)3 as a stressor in order to evaluate if the stress levels caused by the concentrations of 100, 250, 500, and 1000 ppm were sufficient to increase the production of hydrolytic cellulolytic enzymes (FPase, CMCase, Avicelase, β-glucosidase, xylanase) and oxidative (laccase and manganese peroxidase). The study also evaluated the stress levels in previously treated mycelia of B. cinerea and whether they corresponded to the different states of mitochondrial respiration. Our study indicates that Al2(SO4)3 increased the production of cellulolytic and oxidative enzymes in all concentrations in a dose-dependent manner and that Al2(SO4)3 alters the mitochondrial respiratory rate, with lower ATP productions, indicating that less-coupled mitochondria were obtained and that this may be due to the increase of oxidative stress. Thus, it is plausible to suggest the use of Al2(SO4)3 in the production of cellulolytic enzymes, which could be used in the hydrolysis stage of second-generation ethanol production processes, as it reduces the time required for enzymatic expression applications in industrial processes.

Cellulase and Xylanase Production by a Newly Isolated Penicillium crustosum Strain under Solid-State Fermentation, Using Water Hyacinth Biomass as Support, Substrate, and Inducer

Cellulase and xylanase have been widely studied for bioconversion processes and applied in various industries. The high cost of these enzymes remains to be the major bottleneck for large-scale commercial application of lignocellulosic biorefinery. The use of agroindustrial residues and weeds as fermentation substrates is an important strategy to increase cellulolytic enzymes production and reduce costs. Penicillium crustosum was newly isolated and selected to study its enzyme production during solid-state fermentation (SSF). Natural and pretreated water hyacinth (WH) biomass was used as support, substrate and inducer of cellulases and xylanases. Thermochemical pretreatments of WH biomass at 121 °C and sulfuric acid at three concentrations (0.2, 0.6 and 1 M) were assayed. The pretreatments of WH biomass released mono- and oligo-saccharides that favored fungal growth and enzymes production on SSF. WH is a cost-effective substrate-support and inducer, which to be used as a solid medium, was impregnated with a saline solution, containing only (NH4)2SO4, KH2PO4 y MgCl2. Maximum cellulases (carboxymethylcellulase (CMCase)) and xylanases productions of P. crustosum cultured on SSF were reached using the WH pretreated biomass with H2SO4 0.6 M and 121 °C. The simultaneous CMCase and xylanases production reached (647.51 and 4257.35 U/g dry WH, respectively) are among the highest values ever reported.

Sucrose-nonfermenting 1 kinase activates histone acetylase GCN5 to promote cellulase production in Trichoderma.

Trichoderma serves as the primary producer of cellulases and hemicellulases in industrial settings as it readily secretes a variety of cellulolytic enzymes. The protein kinase SNF1 (sucrose-nonfermenting 1) can enable cells to adapt to changes in carbon metabolism by phosphorylating key rate-limiting enzymes involved in the maintenance of energy homeostasis and carbon metabolism within cells. Histone acetylation is an important epigenetic regulatory mechanism that influences physiological and biochemical processes. GCN5 is a representative histone acetylase involved in promoter chromatin remodeling and associated transcriptional activation. Here, the TvSNF1 and TvGCN5 genes were identified in Trichoderma viride Tv-1511, which exhibits promising activity with respect to its ability to produce cellulolytic enzymes for biological transformation. The SNF1-mediated activation of the histone acetyltransferase GCN5 was herein found to promote cellulase production in T. viride Tv-1511 via facilitating changes in histone acetylation. These results demonstrated that cellulolytic enzyme activity and the expression of genes encoding cellulases and transcriptional activators were clearly enhanced in T. viride Tv-1511 mutants in which TvSNF1 and TvGCN5 were overexpressed, with concomitant changes in histone H3 acetylation levels associated with these genes. GCN5 was also found to be directly recruited to promoter regions to alter histone acetylation, while SNF1 functioned upstream as a transcriptional activator that promotes GCN5 upregulation at the mRNA and protein levels in the context of cellulase induction in T. viride Tv-1511. These findings underscore the important role that this SNF1-GCN5 cascade plays in regulating cellulase production in T. viride Tv-1511 by promoting altered histone acetylation, offering a theoretical basis for the optimization of T. viride in the context of industrial cellulolytic enzyme production. KEY POINTS: • SNF1 kinase and GCN5 acetylase promoted cellulase production in Trichoderma by increasing the expression of genes encoding cellulases and transcriptional activators • SNF1 and GCN5 promoted cellulase production by driving H3ac modifications, and GCN5 directly band to the promoter regions to catalyze distinct H3ac modifications • SNF1 acts upstream of GCN5 as a transcriptional activator in the cellulase production of Trichoderma.

Ethanol from cellulose and cellobiose of woody-substrates in a single stage of 3-combined-bioprocesses employing a non-GM yeast cell-factory

The present study aims to prepare a Saccharomyces cerevisiae cell factory, to combine three bioprocesses in a single stage, including cellulolytic enzyme production, cellulose hydrolysis to glucose, and fermentation with low cost of nutrients to ethanol, without any genetic modification. Cell factories are prepared, in which S. cerevisiae cells are covered with starch gel (SG), either containing Trichoderma reesei (SG-T. reesei), or cellulase produced from T. reesei (SG-cellulase). The work comprises the cellulose bioconversion using S. cerevisiae/SG-cellulase, and S. cerevisiae/SG-T. reesei to carry out alcoholic fermentation in one batch with low nutrient cost. In addition, the effect of different concentrations of yeast in the cell factory on fermentation rate was examined. SEM, FTIR spectra analysis and cellulose fermentation were used to prove the successful preparation of cell factory and its activity to ferment cellulose in one combined bioprocess. The cellulose sourced from delignified sawdust was characterized with XRD spectra and porosimetry analysis. Cell factory models of S. cerevisiae/SG-T. reesei and S. cerevisiae/SG-cellulase were able to ferment cellulose from wood, obtaining 32% and 62% ethanol production yield, respectively. S. cerevisiae/SG-T. reesei and S. cerevisiae/SG-cellulase resulted to 4.07 and 7.30 mL ethanol/L, respectively. The final glucose concentration was very low (0–0.07 g glucose/L). It is also concluded that the increase of S. cerevisiae concentration in CF helped an improvement in the bioconversion process.

Biologically derived copper oxide-based nanocatalyst using Moringa oleifera leaves and its applications in hydrolytic enzymes and biohydrogen production

Due to the limited availability of fossil fuels, pollution causing serious environmental issues, and their continuously rising price, the development of low-cost efficient enzymes and their implementation in biomass-based bioenergy industries are highly demanded. In the present work, phytogenic fabrication of copper oxide based nanocatalyst has been performed using moringa leaves and has been characterized using different techniques. Herein, the impact of different dosages of as-prepared nanocatalyst on fungal co-cultured cellulolytic enzyme production under co-substrate fermentation using wheat straw and sugarcane bagasse in 4:2 ratios in solid state fermentation (SSF) has been investigated. An optimal concentration of 25 ppm of nanocatalyst influenced the production of 32 IU/gds of enzyme, which showed thermal stability at 70 °C for 15 h. Additionally, enzymatic bioconversion of rice husk at 70 °C librated 41 g/L of total reducing sugars, which led to the production of 2390 mL/L of cumulative H2 in 120 h.

Production of cellulolytic enzymes from microorganisms isolated from coconut fiber

The worldwide production of microbial enzymes is generally limited by the cost of the substrate used for the cultivation of microorganisms. Thinking of a low-cost and abundant lignocellulosic substrate, coconut fiber is an alternative because it is rich in cellulose, hemicellulose, and lignin, presenting itself as a promising raw material for the production of an enzyme cocktail, which can be used in the production of second-generation biofuels. The objective of this work was to produce cellulolytic enzymes from microorganisms isolated from coconut fiber. In the production of the enzyme, the kinetic profile of the enzymatic complex was evaluated, analyzing the effects of moisture content, temperature, and pH during solid-state fermentation of coconut fiber, using a microorganism isolated from the bagasse itself. Statistica software was used to evaluate the enzyme complex production parameters. Statistical results indicated that the best humidity, temperature, and pH conditions were 80%, 30°C and 3, respectively, with activities of 119.93 U/g CMCase (endoglucanase) and 89.32 U/g FPase (total cellulases ) over a period of 48 h.

Intracellular Nitric Oxide and cAMP Are Involved in Cellulolytic Enzyme Production in Neurospora crassa.

Although molecular regulation of cellulolytic enzyme production in filamentous fungi has been actively explored, the underlying signaling processes in fungal cells are still not clearly understood. In this study, the molecular signaling mechanism regulating cellulase production in Neurospora crassa was investigated. We found that the transcription and extracellular cellulolytic activity of four cellulolytic enzymes (cbh1, gh6-2, gh5-1, and gh3-4) increased in Avicel (microcrystalline cellulose) medium. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) detected by fluorescent dyes were observed in larger areas of fungal hyphae grown in Avicel medium compared to those grown in glucose medium. The transcription of the four cellulolytic enzyme genes in fungal hyphae grown in Avicel medium was significantly decreased and increased after NO was intracellularly removed and extracellularly added, respectively. Furthermore, we found that the cyclic AMP (cAMP) level in fungal cells was significantly decreased after intracellular NO removal, and the addition of cAMP could enhance cellulolytic enzyme activity. Taken together, our data suggest that the increase in intracellular NO in response to cellulose in media may have promoted the transcription of cellulolytic enzymes and participated in the elevation of intracellular cAMP, eventually leading to improved extracellular cellulolytic enzyme activity.

Statistics-Based Optimization of Cellulase and Xylanase Production by the Endophytic Fungus Talaromyces Funiculosus using Agricultural Waste Materials

Lignocellulosic biomass can be utilized as a low-cost, renewable, and sustainable feedstock for obtaining non-fossil energy sources with low CO2 emission. One of the most promising technologies for producing 2G biofuels is the saccharification of agricultural waste materials with the help of cellulolytic enzymes, followed by yeast fermentation of sugars into cellulosic ethanol. Cellulases are multi-component enzymes involved in the degradation of cellulose, which can synergistically degrade cellulose and includes three major categories: endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21). The core enzyme used for the degradation of the xylan skeleton of hemicellulose is endo-β-1,4-xylanase (EC 3.2.1.8). The high cost of enzymes synthesized by fungi is a bottleneck for the production of cellulosic ethanol. Optimization of the nutrient medium composition is an important factor in increasing the production of enzymes and the efficiency of lignocellulosic biomass hydrolysis. The aim of the current study was to optimize the production of cellulolytic and xylanolytic enzymes through cultivation of filamentous fungus Talaromyces funiculosus on low-cost nutrient media with non-pretreated agricultural waste materials. Methods. Filamentous fungus Talaromyces funiculosus was grown on potato-dextrose agar for 10—14 days at 26±2 °С. To obtain the culture filtrate, the fungus was cultivated under submerged conditions in an Erlenmeyer flask for 4 days. The nutrient medium composition was varied according to the factor experiment design. A two-step optimization of the nutrient medium composition was used. A screening experiment with the Plackett-Burman fractional factorial design and response surface methodology with the Box-Behnken design were used to optimize cellulase production. The enzymatic activity was determined by measuring the reduced sugar production after the enzymes hydrolysis with specific substrates: exoglucanase with filter paper, endoglucanase with carboxymethylcellulose, and xylanase with beech wood xylan, using the colorimetric DNS method with glucose or xylose as a standard. The activity of β-glucosidase was determined by the hydrolysis reaction of p-nitrophenyl-β-D-glucopyranoside, which results in the formation of p-nitrophenol, quantified at 410 nm. Results. As a result of experiments with using agricultural waste, including wheat straw, corn stalk, and corn cob as carbon sources of the culture medium, it was shown that T. funiculosus is able to grow and produce cellulase and xylanase on all non-pretreated substrates studied. The two-step sequential optimization of the nutrient medium composition for T. funiculosus cultivation according to the Plackett-Berman and Box-Behnken designs made it possible to increase the activity of cellulolytic and xylanolytic enzymes by 2.4—2.6 times. The optimized cultivation medium does not contain such expensive components as Avicel, peptone, and yeast extract and has the following composition, g/L: corn stalks — 50.0; urea — 0.86; NaNO3 — 1.0; KH2PO4 — 6.0; KCl — 0.25; MgSO4 — 0.25; FeSO4 — 0.01. Conclusions. The studied strain of T. funiculosus produces a lignocellulosic enzyme complex with a high level of β-glucosidase activity when cultivated on an optimized nutrient medium with untreated agricultural waste and is promising for the conversion of lignocellulosic biomass into fermentable sugars.

Integration of agricultural residues as biomass source to saccharification bioprocess and for the production of cellulases from filamentous fungi.

The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70kDa in C. cladosporioides and ~ 21, 63 and 100kDa in P. funiculosum. The best results for saccharification (10.11g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36h of the hydrolytic process at pH 5 and 50°C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.

The ERAD Pathway Participates in Fungal Growth and Cellulase Secretion in Trichoderma reesei.

Trichoderma reesei is a powerful fungal cell factory for the production of cellulolytic enzymes due to its outstanding protein secretion capacity. Endoplasmic reticulum-associated degradation (ERAD) plays an integral role in protein secretion that responds to secretion pressure and removes misfolded proteins. However, the role of ERAD in fungal growth and endogenous protein secretion, particularly cellulase secretion, remains poorly understood in T. reesei. Here, we investigated the ability of T. reesei to grow under different stresses and to secrete cellulases by disrupting three major genes (hrd1, hrd3 and der1) involved in the critical parts of the ERAD pathway. Under the ER stress induced by high concentrations of DTT, knockout of hrd1, hrd3 and der1 resulted in severely impaired growth, and the mutants Δhrd1 and Δhrd3 exhibited high sensitivity to the cell wall-disturbing agents, CFW and CR. In addition, the absence of either hrd3 or der1 led to the decreased heat tolerance of this fungus. These mutants showed significant differences in the secretion of cellulases compared to the parental strain QM9414. During fermentation, the secretion of endoglucanase in the mutants was essentially consistent with that of the parental strain, while cellobiohydrolase and β-glucosidase were declined. It was further discovered that the transcription levels of the endoglucanase-encoding genes (eg1 and eg2) and the cellobiohydrolase-encoding gene (cbh1) were not remarkedly changed. However, the β-glucosidase-encoding gene (bgl1) was significantly downregulated in the ERAD-deficient mutants, which was presumably due to the activation of a proposed feedback mechanism, repression under secretion stress (RESS). Taken together, our results indicate that a defective ERAD pathway negatively affects fungal growth and cellulase secretion, which provides a novel insight into the cellulase secretion mechanism in T. reesei.

Solid-State Fermentation of Steam-Exploded Opuntia ficus-indica Cladodes Using Trichoderma reesei CCT-2768 for the Production of Cellulolytic Enzymes.

The sustainable development of the drylands, i.e., regions with limited availability of water, depends on the exploitation of the few biomass types that can thrive in such conditions, such as the Opuntia ficus-indica, a plant of the Cactaceae family. In the present study, the cladodes of O. ficus-indica were used as a substrate by the fungus Trichoderma reesei CCT-2768 for the production of cellulolytic enzymes through solid-state fermentation. Firstly, the extraction of the mucilage, soluble components of industrial interest, was evaluated. Temperature, water-to-biomass ratio, and time of mixture were varied using an experimental design and impacted, especially, the pectin removal. Then, the lignocellulosic residue was used for the production of enzymes; the effect of the water activity, biomass pretreatment, mineral supplementation, temperature, and inoculum size on the enzymatic production were investigated using two sets of experimental designs. The steam explosion pretreatment exposed the fiber to the microbial action and boosted the enzyme production, provided that the medium was supplemented with salts. This combination has improved the production of xylanase, CMCase, FPase, and polygalacturonase by 27, 62, 98, and 185%, respectively. The temperature of 35°C was determined as the optimal for the production of FPase, xylanase, and polygalacturonase, while no effect was observed on the production of CMCase and β-glucosidase. The optimization of the enzymatic production performed in this study can potentially provide a new application for the Opuntia biomass and improve the sustainable development of the drylands.

Study of Biodeterioration Potential of Microorganisms Isolated in the Paintings Storeroom of Mouze Makhsus Museum, Golestan Palace, Tehran

ABSTRACT Paintings are essential components of cultural heritage all over the world. They consist of organic molecules such as oils, waxes, gums, sugars, polysaccharides, and proteins that can support the growth of various microorganisms. In this study, in order to identify the microbial community possibly involved in deterioration of paintings in the storeroom of Mouze Makhsus, culturable bacteria and fungi colonizing the paintings were isolated. The identification of isolated bacteria and fungi was performed through morphological analyses and either PCR amplification and DNA sequencing of 16srRNA gene, for bacteria, and ITS genomic region, for fungi. Based on the obtained 16S rRNA gene sequence of bacteria and ITS region sequence of fungi, a phylogenetic tree was constructed using the Neighbor-Joining method. The study also evaluated, in vitro, the cellulolytic activity of the bacterial and fungal isolates. The results highlighted the diverse array of microbial groups colonizing the sampled paintings. Culture-based analyses revealed 27 isolates of bacteria and fungi. Detected fungi fell in seven genera including Aspergillus, Penicillium, Cladosporium, Alternaria, Curvularia, Chaetomium, and Trametes. The majority of isolated bacteria belong to Bacillus genus, whereas to a lesser extent Staphylococcus, Micrococcus, Paenibacillus, Arthrobacter, Heyndrickxia, Priestia, and Rathayibacter were isolated. Qualitative and quantitative assessment of cellulolytic enzyme production showed that the fungal isolates present important cellulolytic activity, and they are potentially dangerous to the paintings.