Fungal Cellulase Production Research Articles

Process development for fungal cellulase production and enzymatic saccharification of Parthenium hysterophorus for bioethanol production

Parthenium hysterophorus (PH), often known as parthenium weed, is one of the seventh most devastating weeds in the world.The primary aim of the study was to produce and optimize fungal FPase enzymes under solid-state fermentation (SSF) conditions for application in enzymatic hydrolysis to produce bioethanol. The impact of various physiological factors was verified through a systematic approach, initially using the one-factor-at-a-time (OFAT) method. Further optimization was carried out using statistical tools by response surface methodology (RSM), selecting three variables, i.e., lactose concentration, peptone concentration, and pH level. The outcomes of this research demonstrated that the RSM-based model led to the optimization of enzyme activity, resulting in 4.08U/ml of FPase, with a lactose concentration of 1.62 %, a peptone concentration of 1.5 %, and a pH level of 6 being the ideal conditions. When employing the FPase enzyme for biomass hydrolysis, the maximum yield of reducing sugar, at 0.37 ± 0.01 g/g, was achieved within a 96-hour period, using enzyme and substrate levels of 30 FPU/100 ml and 3 % (w/v), respectively. Fermentation of enzymatic hydrolysate was carried out by Saccharomyces cerevisiae at 30 °C, pH 6.0, and 120 rpm, which resulted in 0.45g/g ethanol yield after 48 h. Overall, our findings reveal thatPH biomass can be introduced into sugar-based biorefineries to produce biofuels and other valuable chemicals.

Bionanofabrication of Cupric oxide catalyst from Water hyacinth based carbohydrate and its impact on cellulose deconstructing enzymes production under solid state fermentation

One of the most important properties of cellulolytic enzyme is its ability to convert cellulosic polymer into monomeric fermentable sugars which are carbohydrate by nature can efficiently convert into biofuels. However, higher production costs of these enzymes with moderate activity-based stability are the main obstacles to making cellulase-based applications sustainably viable, and this has necessitated rigorous research for the economical availability of this process. Using water hyacinth (WH) waste leaves as the substrate for cellulase production under solid state fermentation (SSF) while treating the fermentation production medium with CuO (cupric oxide oxide) bionanocatalyst have been examined as ways to make fungal cellulase production economically feasible. Herein, a sustainable green synthesis of CuO bionanocatalyst has been performed by using waste leaves of WH. Through XRD, FT-IR, SEM, and TEM analysis, the prepared CuO bionanocatalyst's physicochemical properties have been evaluated. Furthermore, the effect of CuO bionanocatalyst on the temperature stability of raw cellulases was observed, and its half-life stability was found to be up to 9 h at 65 °C. The results presented in the current investigation may have broad scope for mass trials for various industrial applications, such as cellulosic biomass conversion.

Facile pretreatment strategies to biotransform Kans grass into nanocatalyst, cellulolytic enzymes, and fermentable sugars towards sustainable biorefinery applications

The present investigation is targeted towards the facile fabrication of a carbon-based nanocatalyst (CNCs) using Kans grass biomass (KGB) and its sustainable application in microbial cellulase enhancement for the alleviation of enzymatic hydrolysis for sugar production. Different pretreatments, including physical, KGB extract-mediated treatment, followed by KOH pretreatment, have been applied to produce CNCs using KGB. The presence of CNCs influences the pretreatment of KGB substrate, fungal cellulase production, stability, and sugar recovery in the enzymatic hydrolysis of KGB. Using 1.0% CNCs pretreated KGB-based solid-state fermentation, 33 U/gds FPA and 126 U/gds BGL were obtained at 72 h, followed by 107 U/gds EG at 48 h in the presence of 0.5% CNCs. Further, 42 °C has been identified as the optimum temperature for cellulase production, while the enzyme showed thermal stability at 50 °C up to 20 h and produced 38.4 g/L sugar in 24 h through enzymatic hydrolysis of KGB.

The putative methyltransferase LaeA regulates mycelium growth and cellulase production in Myceliophthora thermophila

BackgroundFilamentous fungi with the ability to use complex carbon sources has been developed as platforms for biochemicals production. Myceliophthora thermophila has been developed as the cell factory to produce lignocellulolytic enzymes and plant biomass-based biofuels and biochemicals in biorefinery. However, low fungal growth rate and cellulose utilization efficiency are significant barriers to the satisfactory yield and productivity of target products, which needs our further exploration and improvement.ResultsIn this study, we comprehensively explored the roles of the putative methyltransferase LaeA in regulating mycelium growth, sugar consumption, and cellulases expression. Deletion of laeA in thermophile fungus Myceliophthora thermophila enhanced mycelium growth and glucose consumption significantly. Further exploration of LaeA regulatory network indicated that multiple growth regulatory factors (GRF) Cre-1, Grf-1, Grf-2, and Grf-3, which act as negative repressors of carbon metabolism, were regulated by LaeA in this fungus. We also determined that phosphoenolpyruvate carboxykinase (PCK) is the core node of the metabolic network related to fungal vegetative growth, of which enhancement partially contributed to the elevated sugar consumption and fungal growth of mutant ΔlaeA. Noteworthily, LaeA participated in regulating the expression of cellulase genes and their transcription regulator. ΔlaeA exhibited 30.6% and 5.5% increases in the peak values of extracellular protein and endo-glucanase activity, respectively, as compared to the WT strain. Furthermore, the global histone methylation assays indicated that LaeA is associated with modulating H3K9 methylation levels. The normal function of LaeA on regulating fungal physiology is dependent on methyltransferase activity.ConclusionsThe research presented in this study clarified the function and elucidated the regulatory network of LaeA in the regulation of fungal growth and cellulase production, which will significantly deepen our understanding about the regulation mechanism of LaeA in filamentous fungi and provides the new strategy for improvement the fermentation properties of industrial fungal strain by metabolic engineering.

Intracellular Sugar Transporters Facilitate Cellulase Synthesis in Trichoderma reesei Using Lactose.

Sugar transporters play an important role in the cellulase production of lignocellulose-degrading fungi. Nevertheless, the role and function of these transporters are still unclear. Here we first report intracellular sugar transporters assisting cellulase production in Trichoderma reesei (T. reesei) using lactose. The mRNA levels of sugar transporter genes mfs, gst, and lac1 were substantially upregulated in T. reesei cultivated on lactose, with the most abundant mRNA levels at 24 h as compared to glucose. Moreover, the individual deletion of these sugar transporters significantly inhibited cellulase production, solid cell growth, and sporulation of T. reesei, suggesting they play a supporting role in cellulase production when grown in lactose. Surprisingly, MFS, GST, and LAC1 were mainly localized in the cytoplasm, with MFS and LAC1 in the endoplasmic reticulum (ER), representing the first discovery of intracellular sugar transporters involved in cellulase biosynthesis in lactose culture. The absence of the gene lac1 noticeably inhibited most of the crucial genes related to cellulase production, including cellulase-encoding genes, transcription factors, and sugar transporters, at 24 h, which was fully relieved at 48 h or 72 h, indicating that lac1 affects cellulase production more at the early step. This research advances the understanding of the function of intracellular sugar transporters in fungi, particularly for fungal cellulase production.

Enhancement of fungal cellulase production using pretreated orange peel waste and its application in improved bioconversion of rice husk under the influence of nickel cobaltite nanoparticles

Enhancement of fungal cellulase production using pretreated orange peel waste and its application in improved bioconversion of rice husk under the influence of nickel cobaltite nanoparticles

Plasma Promotes Fungal Cellulase Production by Regulating the Levels of Intracellular NO and Ca2.

For the industrial-scale production of useful enzymes by microorganisms, technological development is required for overcoming a technical bottleneck represented by poor efficiency in the induction of enzyme gene expression and secretion. In this study, we evaluated the potential of a non-thermal atmospheric pressure plasma jet to improve the production efficiency of cellulolytic enzymes in Neurospora crassa, a filamentous fungus. The total activity of cellulolytic enzymes and protein concentration were significantly increased (1.1~1.2 times) in media containing Avicel 24–72 h after 2 and 5 min of plasma treatment. The mRNA levels of four cellulolytic enzymes in fungal hyphae grown in media with Avicel were significantly increased (1.3~17 times) 2–4 h after a 5 min of plasma treatment. The levels of intracellular NO and Ca2+ were increased in plasma-treated fungal hyphae grown in Avicel media after 48 h, and the removal of intracellular NO decreased the activity of cellulolytic enzymes in media and the level of vesicles in fungal hyphae. Our data suggest that plasma treatment can promote the transcription and secretion of cellulolytic enzymes into the culture media in the presence of Avicel (induction condition) by enhancing the intracellular level of NO and Ca2+.

Enhancement of Indigenous Fungal Cellulase Production by Gamma Rays

The increasing demand for cellulases causes the need for a high cellulase-producing microbe. Mutagenesis is an efficient way to produce a high-titer cellulase-producing strain. Mutagenesis using gamma rays irradiation has the advantage that it can cause a double strand break of DNA. Repair of double-strand break tends to has an error-prone repair that leads to the alteration of DNA sequence. The aim of this study was to screen high cellulase-producing indigenous fungal mutants produced by mutagenesis. Trichoderma sp. PK1J2 was subjected to gamma irradiation at 300 Gy. The mutants produced were screened using a plate medium containing cellulose as a sole carbon source. After staining with congo red, colonies with wider clear zones were grown in a liquid medium for four days, and the cellulase activities were analyzed. Mutant M8 produces endoglucanase, FPase, and β-glucosidase at 0.46 U/ml, 0.18 U/ml, and 1.10 U/ml, respectively, which were 90%, 50%, and 30% higher than those of the parental strain.

Evaluation of enhanced production of cellulose deconstructing enzyme using natural and alkali pretreated sugar cane bagasse under the influence of graphene oxide

High production cost of cellulase enzyme is one of the main constraints in the practical implementation of biofuels at global scale. Therefore, the present investigation is focused to produce low-cost cellulase via sustainable strategies. This work evaluates to achieve enhanced fungal cellulase production using natural and pretreated sugar cane bagasse (SCB) via Rhizopus oryzae NS5 under the solid state fermentation (SSF) while implementing graphene oxide (GO) as a catalyst. A low alkali treatment showed better performance for cellulase production wherein 14 IU/gds FP activity is observed in 96 h using 0.5% alkali treated SCB, significantly higher as compared to 10 IU/gds FP in case of untreated SCB. Further, the effect of GO has been investigated on cellulase production, incubation temperature and pH of the production medium. Under the influence of 1.5% concentration of GO, alkali pretreated SCB produced maximum 25 IU/gds cellulase in 72 h at pH 5.0 and 40 °C.

Nickel ferrite nanoparticles induced improved fungal cellulase production using residual algal biomass and subsequent hydrogen production following dark fermentation

The present study reports nickel ferrite nanoparticles (NiFe2O4 NPs) induced enhanced production of crude cellulase enzyme using residual algal biomass of cyanobacteria Lyngbya limnetica as substrate. It is noticed that the residual algal substrate and NiFe2O4 NPs mediated crude cellulase exhibits nearly 2.5 fold enhanced filter paper activity after 72 h along with better efficiency in terms of pH and thermal stability as compared to the control system. Further, NiFe2O4 NPs mediated crude cellulase enzyme was employed for the enzymatic hydrolysis of rice straw to produce sugar hydrolyzate. Subsequently, using bacterial strains Bacillus subtilisPF_1 the cumulative hydrogen ~ 1820 mL/L has been produced under the dark fermentation.

Technological advances for improving fungal cellulase production from fruit wastes for bioenergy application: A review

Fruit wastes can be imperative to elevate economical biomass to biofuels production process at pilot scale. Because of the renewable features, huge availability, having low lignin content organic nature and low cost; these wastes can be of much interest for cellulase enzyme production. This review provides recent advances on the fungal cellulase production using fruit wastes as a potential substrate. Also, the availability of fruit wastes, generation and processing data and their potential applications for cellulase enzyme production have been discussed. Several aspects, including cellulase and its function, solid-state fermentation, process parameters, microbial source, and the application of enzyme in biofuels industries have also been discussed. Further, emphasis has been made on various bottlenecks and feasible approaches such as use of nanomaterials, co-culture, molecular techniques, genetic engineering, and cost economy analysis to develop a low-cost based comprehensive technology for viable production of cellulase and its application in biofuels production technology.

Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei.

ABSTRACTTrichoderma reesei has 11 putative β-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many β-glucosidases and the distinct role each β-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular β-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei. Four types of cellular distribution patterns were observed for β-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular β-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi.

Impact of pretreatment severity on fungal cellulase production on sugarcane bagasse substrate

On-site production of cellulases using lignocellulosic materials can improve the economic viability of biorefineries. This, however, requires the pretreatment of substrates using thermochemical conditions that can vary in severity. To understand the effect of pretreatment severity on cellulase production by Aspergillus ustus S3 on sugarcane bagasse, we applied NaOH pretreatment corresponding to 3 severity factors (SF1.32, SF1.79, and SF3.64) to generate SCB that was used as inducing substrate. The highest cellulase activity (0.681 U/mL) was obtained with the intermediate severity (SF1.79) while significantly lower activities of 0.495 and 0.539 U/mL were recorded with low (SF1.32) and high (SF3.64) severities, respectively. Chemical and structural characterization revealed that low and intermediate severities improved cellulose accessibility and cellulase titres while high severity impaired them, thus limiting substrate suitability for enzyme production. These results show that though high SFs might be desirable in other applications, moderate severities may be more appropriate for cellulase production.

Detergent-compatible fungal cellulases

Detergent enzymes are currently added to all powder and liquid detergents that are manufactured. Cellulases, lipases, amylases, and proteases are used in the detergency to replace toxic phosphates and silicates and to reduce high energy consumption. This makes the use of enzymes in detergent formulation cost effective. Fungi are producers of important extracellular enzymes for industrial use. The fungal and bacterial cellulases maintain the shape and color of the washed garments. There is a high demand for cellulases at the market by detergent industries. With this high demand, genetic engineering has been a solution due to its high production of detergent-compatible cellulases. Fungi are the famous source for detergent-compatible cellulases production, but still, there is a lack of the cost-effective process of alkaline fungal cellulase production. Review papers on detergent-compatible bacterial cellulase and amylase and detergent-compatible fungal and bacterial proteases and lipases are available, but there is no review on detergent fungal cellulases. This review aims to highlight the production, properties, stability, and compatibility of fungal cellulases. It will help other academic and industrial researchers to study, produce, and commercialize the fungal cellulases with good aspects.

Establishment of a highly efficient and low cost mixed cellulase system for bioconversion of corn stover by Trichoderma reesei and Aspergillus niger

The high cost of cellulase production is one of the main obstacles for the conversion of lignocellulose into high valued products. As a widely existing biomass resource, corn stover can be used to reduce the cost of cellulase production. In this study, Parameters of producing cellulase by Trichoderma reesei were optimized using Response Surface methodology, the optimum conditions were as follows: 3.84% solid acid pretreated corn stover, 1.19% yeast extract, initial pH of 5.01, 0.12% glycerol monostearate, 0.25% Na2HPO4, with 3% inoculum content for 12 days at 30 °C. Filter paper activity (FPA) of Trichoderma reesei was increased by 180% to 13.44 IU/mL. Enzymatic hydrolysis experiments were performed on different pretreated corn stovers with Trichoderma reesei cellulase produced under optimal conditions, and the highest cellulose conversion efficiency reached 62.39%. In addition, a blend of Trichoderma reesei cellulase at a concentration of 30 FPU/g cellulose and Aspergillus niger beta-glucosidase at a concentration of 50 FPU/g cellulose could effectively hydrolyse alkali pretreated corn stover, resulting in an improvement in the enzymatic hydrolysis efficiency by 17.26% compared to Trichoderma reesei cellulase alone. In parallel, the mixed enzymes achieved an equivalent enzymatic effect with that of commercial cellulase (Novozymes, Cellic Ctec2) at the same concentration. These data suggested that solid acid pretreated corn stover was a promising carbon source for fungal cellulase production for effective saccharification of corn stover biomass. Glycerol monostearate was first found to have a strong induction effect on enzyme production. The development of a high efficiency and low-cost mixed enzyme system had a potential application in future industrialization scale.

Tandem integration of aerobic fungal cellulase production, lignocellulose substrate saccharification and anaerobic ethanol fermentation by a modified gas lift bioreactor

Cellulase production, lignocellulose saccharification and bioethanol fermentation were integrated to efficiently produce bioethanol. A modified gas lift bioreactor was developed for bioethanol production by the integrated process. Cellulase production was achieved using Aspergillus niger mycelia immobilized within the reactor in wire meshes, and Saccharomyces cerevisiae cells were immobilized in resin beads. During four repeated batches fermentation, cellulase activities were more than 6.28 U/mL and bioethanol production was over 45.9 g/L for 48 h. The factual bioethanol conversion efficiency was 86.8%. By the modification of the modified gas lift bioreactor, immobilization of Aspergillus niger mycelia and Saccharomyces cerevisiae cells, aerobic cellulase production, substrate saccharification and anaerobic bioethanol fermentation were successfully integrated in tandem. The integrated processes is of great significance in bioethanol production.

Green seaweeds (Ulva fasciata sp.) as nitrogen source for fungal cellulase production.

In developing countries, local enzyme production can help decrease the dependency of imported enzymes for bioconversion of e.g. cellulosic feedstocks, but the use of conventional nitrogen sources contributes significantly to such enzyme production cost. Use of local resources is therefore important to consider. Green seaweeds are marine macroalgae that are rich in nitrogen, but not exploited for their nitrogen content. Cellulase production was accomplished by using cocoa pod husk (CPH) and green seaweed (GS) (Ulva fasciata sp.) as growth substrates for Polyporus ciliatus CBS 366.74 in submerged cultivation. The nitrogen concentration of GS was comparable to that of CPH with 0.6%w/v peptone at 4%w/v substrate concentration. A decline of cellulase activity in peptone supplemented GS growth media indicated nitrogen sufficiency of GS to serve as a potential nitrogen source for the fungal growth and cellulase production. Comparison of enzyme production on CPH growth media supplemented with either GS or peptone based on equivalent carbon to nitrogen ratios was done for two Polyporus strains namely; P. ciliatus CBS 366.74 and P. brumalis CBS 470.77. Peptone could be substituted by up to 0.6% w/v with GS at inclusion levels of 50-100% of substrate concentration to attain satisfactory cellulase productivity. However, the cellulase productivity response varied among the two Polyporus species. This study demonstrated that green seaweeds may be used as alternative nitrogen sources for fungal cellulase production.

Optimisation of fungal cellulase production from textile waste using experimental design

Abstract Abundant textile waste has raised increasing concerns worldwide in developing novel circular textiles approach. This study investigated the optimum cellulase production from textile waste by Aspergillus niger CKB. Textile wastes consisting of cotton and polyester in various ratios were used as low-cost feedstock. Three types of cultivation media were compared in solid state fermentation and Mandels medium with yeast extract selected due to its superior cellulase production. Conditions including moisture, pH, inoculum size and organic nitrogen were evaluated and optimised via response surface methodology. Supplementary carbon sources and cellulase inducers were also employed to enhance fungal growth and cellulase production. The results indicated that the optimised fermentation method significantly improved cellulase producing efficiency and enzyme activity by 88.7% and 25.8%, respectively. The maximum cellulase activity reached 1.56 FPU g−1 in 6 days. The outcomes led to the efficient recovery of glucose and polyester, which could contribute to a closed-loop recycling strategy for the textile industry, and enable the transition to an advanced circular textiles economy.

Culturable fungal diversity and cellulase production by mixed culture Aspergillus fungi from Sanya mangrove.

Fungi are the most suitable cellulase producers attributing to its ability to produce a complete cellulase system. 33 Genus, 175 Species fungi were isolated from Sanya mangrove, Hainan, China. Using congo red cellulose (CMC) medium, five fungi of cellulose-degrading were selected for further study. Molecular biology and morphological identification showed that all of these five fungi belong to Aspergillus fungi. The cellulase produced by these fungi were monitored during liquid state fermentation. The optimum conditions study for enzyme production illustrated that the highest activities appeared at pH 3.0, 35°C after fermentation for 3 days. Beyond that, the enzyme activity of mixed fungi is 11-26% higher than pure. The study demonstrated that mixed culture improved the hydrolysis of fungi cellulase.

In vitro Optimisation of Fungal Cellulase Production from Fruit Waste for Handmade Paper Industries

In vitro Optimisation of Fungal Cellulase Production from Fruit Waste for Handmade Paper Industries