Cellulase Secretion Research Articles

Distinct but Redundant Roles of ER Cargo Receptors p24 and Erv29 in Facilitating Proper Secretion of Cellulases in Trichoderma reesei.

Trichoderma reesei represents an important industrial workhorse for (hemi)cellulase production. However, relatively little is known about the details of its secretory pathway ensuring the extremely high-level enzyme secretion and how they might be leveraged for engineering improved protein production. Here, the functions of T. reesei ER cargo receptors p24 and Erv29 in trafficking cellulase were characterised. Whereas individual deletion of p24 or erv29 resulted in only a marginal effect on extracellular cellulase secretion, distinct intracellular trafficking pathways exist for individual hydrolytic enzyme in T. reesei. Notably, the simultaneous absence of p24 and Erv29 abolished the secreted production of cellulases but not xylanases. The secretion defect was accompanied by an apparent intracellular accumulation of cellulases. Mutations of residues on the cytosolic side of p24 and Erv29 supposed to mediate COPII coat recognition also compromised cellulase secretion although the overall ER exit sites (ERES) formation did not seem to be affected. We further revealed that a VPL motif following the signal peptide of CBH2 necessitates its efficient secretion mediated by Erv29. These results indicate that two specific ER cargo receptors complement each other to mediate the proper intracellular trafficking of cellulases and thus ensuring their extracellular secretion.

AMPK Activates Cellulase Secretion in Penicillium funiculosum by Downregulating P-HOG1 MAPK Levels.

Cellulase production for hydrolyzing plant cell walls is energy-intensive in filamentous fungi during nutrient scarcity. AMP-activated protein kinase (AMPK), encoded by snf1, is known to be the nutrient and energy sensor in eukaryotes. Previous studies on AMPK identified its role in alternate carbon utilization in pathogenic fungi. However, the precise role of AMPK in cellulase production remains elusive. In the present study, we employed gene-deletion analysis, quantitative proteomics and chemical-genetic approaches to investigate the role of AMPK in cellulase synthesis in Penicillium funiculosum. Gene-deletion analysis revealed that AMPK does not promote transcription and translation but is essential for cellulase secretion in a calcium-dependent manner. Proteomic analysis of the snf1-deleted (Δsnf1) strain confirmed trapped cellulase inside the mycelia and identified HOG1 MAPK activation as the most significant Ca2+-induced signaling event during carbon stress in Δsnf1. Western blot analysis analysis revealed that the phosphorylated HOG1 (P-HOG1)/HOG1 MAPK ratio maintained by Ca2+-signaling/Ca2+-activated AMPK, respectively, forms a secretion checkpoint for cellulases, and disturbing this equilibrium blocks cellulase secretion. The proteomic analysis also indicated a massive increase in mTORC1-activated anabolic pathways during carbon stress in Δsnf1. Our study suggests that AMPK maintains homeostasis by acting as a global repressor during carbon stress.

Quorum Quenching Lactonase Alters Virulence of Pectobacterium atrosepticum and Reduces Maceration in Potatoes.

Pectobacterium atrosepticum is a soft rot phytopathogenic bacterium mainly infecting potatoes. The virulence of P. atrosepticum is controlled by quorum sensing (QS), a communication mechanism which enables bacteria to coordinate their behavior in a population density-dependent manner. Inhibiting QS has gained interest as a sustainable alternative to conventional treatments to control pathogens in agriculture. Here, we investigate the potential of a robust lactonase to inhibit P. atrosepticum virulence in vitro, combining phenotypic and proteomic studies. We report that exogenous lactonase treatment reduced the secretion of pectate lyase, cellulase, and polygalacturonase enzymes, leading to decreased virulence on potato tubers. Major changes in the P. atrosepticum proteome revealed that lactonase affects the abundance of proteins with various functions, including virulence. Taken as a whole, these results suggest that lactonase-mediated disruption of QS in P. atrosepticum is a promising strategy to limit P. atrosepticum infections.

Isolation, Characterization, and Optimization of Culture Medium for Local Straw-Degrading Bacteria from Northeastern Black Soils of China

In order to solve the problem of low and poor straw degradation in typical black soil areas of Northeast China, the present study was carried out to screen the potential of in situ strains with cellulose degradation ability from black soils of Northeast China to play a role in the resourceful utilization of straw and the development of sustainable agriculture. The straw degradation potential of the strains was evaluated by combining sodium carboxymethyl cellulose plate screening and cellulase viability assay; the species identification of the strains was carried out by morphology, physiology, biochemistry, and molecular biology; and the basic medium formulation of the strains was optimized by Box–Behnken response surface methodology. Ten cellulose-degrading strains were identified: ZL-5, ZL-69, ZL-88, ZL-95, ZL-111, ZL-137, ZL-139, ZL-140, ZL-187, and ZL-216, of which ZL-139 had the highest cellulase production capacity, with a cellulase secretion of 7.8781 U/mL in the enzyme-producing medium. ZL-139 was identified as Bacillus cereus; the optimized best formulation was glucose—4.284 g/L, yeast extract—1.454 g/L, MgSO4—0.417 g/L, KH2PO4—0.5 g/L, KH2PO4—0.5 g/L, K2HPO4—1.5 g/L, and NaCl—1.0 g/L. In conclusion, strain ZL-139 has good potential for crop straw degradation and can be a candidate strain for a straw-rotting agent in northeast China, with promising prospects for development and utilization.

Self-adaptable HAc/NaAc buffer system enhanced biohydrogen production from dark fermentation of cellulose

ThepHdecrease caused by potential accumulation and dissociation of organic acidsis considereda major challenge hindering stable and constant operation in hydrogen production. In this study, a self-adaptableHAc/NaAc buffer system was investigated based on batch dark fermentation hydrogen production (DFHP) metabolic typesto controlthe pH of fermentation process. Resultsshowedthat increasing substrate concentration resulted in lower H2 production yield, especially when the substrate concentration exceeded 10 g/L. A maximum H2yield of2326.25 mL/L was achieved at the HAc/NaAc-buffered group; productions were 2.84 times and 57.7 % higher than the control and NaOH control groups. Our buffersystem retardedthe decrease of pH, enhanced the selectivemetabolic flux of acetic acid production, promoted the growth of microorganisms, enhanced microbial secretion of cellulase, andregulatedthe ratio of NADH/NAD+. The research provided a preliminary understanding and reference for the buffer regulatory strategy on organic waste for DFHP.

Evaluating the efficacy of probiotic bacterial strain Lactobacillus plantarum for inhibition of fungal strains associated with historical manuscript deterioration: An experimental study

The aim of this study is to develop safe biological methods for controlling fungal deterioration of historical manuscripts. Therefore, fifteen fungal isolates were obtained from paper sheets and leather skins of a deteriorated historical manuscript (dated back to the 13th century). Those isolates were identified using both traditional methods and ITS-sequencing analysis. Aspergillus niger accounted for seven strains, Penicillium citrinum for one strain, Aspergillus flavus for three, Aspergillus fumigatus for one, Aspergillus nidulans for one, and Penicillium chrysogenum for two of the fungal strains that were obtained. The ability of fungal strains for the secretion of cellulase, amylase, gelatinase, and pectinase as hydrolytic enzymes was evaluated. The capability of the probiotic-bacterial strain Lactobacillus plantarum DSM 20174 for inhibition of fungal strains that cause severe deterioration was studied using ethyl acetate-extract. The metabolic profile of the ethyl acetate-extract showed the presence of both high- and low-molecular-weight active compounds as revealed by GC-MS analysis. The safe dose to prevent fungal growth was determined by testing the ethyl acetate extract's biocompatibility against Wi38 and HFB4 as normal cell lines. The extract was found to have a concentration-dependent cytotoxic impact on Wi38 and HFB4, with IC50 values of 416 ± 4.5 and 349.7 ± 5.9 μg mL−1, respectively. It was suggested that 100 μg mL−1 as a safe concentration could be used for paper preservation. Whatman filter paper treated with ethyl acetate extract was used to cultivate the fungal strain Penicillium citrinum AX2. According to data analysis, fungal inhibition measurement, SEM, ATR-FT-IR, XRD, color change measurement, and mechanical property assessment, the recommended concentration of ethyl acetate extract was adequate to protect paper inoculated with the highest enzymatic producer fungi, P. citrinum AX2.

Fungal Enzymes for Saccharification of Gamma-Valerolactone-Pretreated White Birch Wood: Optimization of the Production of Talaromyces amestolkiae Cellulolytic Cocktail.

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

Mechanism of Brevibacillus brevis strain TR-4 against leaf disease of Photinia×fraseri Dress.

Colletotrichum species are among the most common pathogens in agriculture and forestry, and their control is urgently needed. In this study, a total of 68 strains of biocontrol bacteria were isolated and identified from Photinia × fraseri rhizosphere soil. The isolates were identified as Brevibacillus brevis by 16S rRNA. The inhibitory effect of TR-4 on Colletotrichum was confirmed by an in vitro antagonistic experiment. The inhibitory effect of TR-4 was 98% at a concentration of 10 µl/ml bacterial solution, protection of the plant and inhibition of C. siamense was evident. Moreover, the secretion of cellulase and chitosan enzymes in the TR-4 fermentation liquid cultured for three days was 9.07 mol/L and 2.15 µl/mol, respectively. Scanning electron microscopy and transmission electron microscopy confirmed that TR-4 destroyed the cell wall of C. siamense, resulting in leakage of the cell contents, thus weakening the pathogenicity of the bacteria.

Cellulolytic potentials of aspergillus oryzae and streptomyces griseus isolated from waste dump soil in Nile University Of Nigeria, Abuja

The potential of using microorganisms as biological sources of industrially economic enzymes has stimulated interest in the exploitation of extracellular enzymatic activity in several microorganisms. The aim of this research is to assess the cellulose degrading potentials of two microorganisms, Aspergillus oryzae and Streptomyces griseus using cellulose Congo red agar media. Soil sample collected from waste dump was serially diluted and inoculated in starch casein agar and SDA to isolate S. griseus and A. oryzae respectively. To assess their potentials to utilize cellulose, each of the two microorganisms was inoculated on cellulose Congo-red media and incubated at 30 ºC for 7days. A zone of clearing around the colonies after incubation confirms the secretion of extracellular cellulase, and was used as an indication for cellulose utilization. The zone of clearing was measured with a meter rule. In the results obtained, both microorganisms demonstrated cellulose utilization ability with Aspergillus oryzae showing a zone of clearing of 30.50 ± 0.50 mm while Streptomyces griseus showed a wider zone of clearing of 60.00 ± 1.00 mm. The results indicate that both microorganisms can be potent producers of the enzyme cellulase, with Streptomyces griseus having a higher cellulase-producing ability.

The role of the Flb protein family in the life cycle of Aspergillus niger

Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.

Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum

BackgroundLow-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain.ResultsWe began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum.ConclusionsOur study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.

An extracellular glucose sensor for substrate-dependent secretion and display of cellulose-degrading enzymes.

The efficient hydrolysis of lignocellulosic biomass into fermentable sugars is key for viable economic production of biofuels and biorenewable chemicals from second-generation feedstocks. Consolidated bioprocessing (CBP) combines lignocellulose saccharification and chemical production in a single step. To avoid wasting valuable resources during CBP, the selective secretion of enzymes (independent or attached to the surface) based on the carbon source available is advantageous. To enable enzyme expression and secretion based on extracellular glucose levels, we implemented a G-protein-coupled receptor (GPCR)-based extracellular glucose sensor; this allows the secretion and display of cellulases in the presence of the cellulosic fraction of lignocellulose by leveraging cellobiose-dependent signal amplification. We focused on the glucose-responsiveness of the HXT1 promoter and engineered PHXT1 by changing its core to that of the strong promoter PTHD3 , increasing extracellular enzyme activity by 81%. We then demonstrated glucose-mediated expression and cell-surface display of the β-glucosidase BglI on the surface of Saccharomyces cerevisiae. The display system was further optimized by re-directing fatty acid pools from lipid droplet synthesis toward formation of membrane precursors via knock-out of PAH1. This resulted in an up to 4.2-fold improvement with respect to the baseline strain. Finally, we observed cellobiose-dependent signal amplification of the system with an increase in enzymatic activity of up to 3.1-fold when cellobiose was added.

Investigating the cellular functions of β-Glucosidases for synthesis of lignocellulose-degrading enzymes in Trichoderma reesei

β-glucosidases play an important role in the synthesis of cellulase in fungi, but their molecular functions and mechanisms remain unknown. We found that the 10 putative β-glucosidases investigated in Trichoderma reesei facilitate cellulase production, with cel3j being the most crucial. Transcriptional analysis revealed that the most affected biological processes in △cel3j strain were cellulase synthesis, ribosome biogenesis, and RNA polymerases. Moreover, CEL3J was unconventionally transported through the endoplasmic reticulum, bypassing the Golgi apparatus, whereas cel3j overexpression altered cellulase secretion from conventional to unconventional, likely owing to the activated unconventional protein secretion pathway (UPS), as indicated by the upregulation of genes related to UPS. The mTORC1-GRASP55 signaling axis may modulate the unconventional secretion of CEL3J and cellulase. The transcriptional levels of genes associated with DNA replication, the cell cycle, and meiosis were noticeably affected by overexpressing cel3j. These data give new clues for exploring the roles of β-glucosidases and the molecular mechanisms of their unconventional secretion in fungi.

The TgRas1 Gene Affects the Lactose Metabolism of Trichoderma guizhouense NJAU4742

Trichoderma is one of the fungi commonly used in fermentation engineering. The hydrolytic enzymes secreted by Trichoderma have great economic value. Trichoderma guizhouense NJAU4742 is a branch of Trichoderma harzianum, which also has application potential. Lactose can induce fungi to secrete cellulase. Unfortunately, neither the lactose-inducing effect nor the mechanism of lactose metabolism in the study of Trichoderma guizhouense NJAU4742 is clear. Our study showed that carbon sources such as glucose, galactose, and sucrose could not induce cellulase secretion from Trichoderma guizhouense NJAU4742. Lactose induced the filter paper activity of the cellulase secreted by Trichoderma to reach 4.13 ± 0.11 U·mL−1. The ratio of 0.4% lactose–0.6% straw is the best way to induce cellulase and is better than adding only straw or lactose. TgRas family genes respond differently to different carbon sources at the gene level, and these proteins may be involved in different carbon source metabolisms. The results of transcriptional responses under different growth conditions showed that TgRas1 occupies a dominant position among TgRas family genes. The growth of the ΔTgRas1 mutant on the plate was inhibited, and the hyphae were dense, thick, and swollen. Under the condition of lactose, the biomass of ΔTgRas1 was severely inhibited in liquid fermentation, and its biomass decreased by 91.43% compared with WT. The liquid fermentation of ΔTgRas1 under other carbon source conditions was not affected.

Early cellular events and potential regulators of cellulase induction in Penicillium janthinellum NCIM 1366

Cellulase production by fungi is tightly regulated in response to environmental cues, and understanding this mechanism is a key pre-requisite in the efforts to improve cellulase secretion. Based on UniProt descriptions of secreted Carbohydrate Active enZymes (CAZymes), 13 proteins of the cellulase hyper-producer Penicillium janthinellum NCIM 1366 (PJ-1366) were annotated as cellulases- 4 cellobiohydrolases (CBH), 7 endoglucanases (EG) and 2 beta glucosidases (BGL). Cellulase, xylanase, BGL and peroxidase activities were higher for cultures grown on a combination of cellulose and wheat bran, while EG was stimulated by disaccharides. Docking studies indicated that the most abundant BGL- Bgl2- has different binding sites for the substrate cellobiose and the product glucose, which helps to alleviate feedback inhibition, probably accounting for the low level of glucose tolerance exhibited. Out of the 758 transcription factors (TFs) differentially expressed on cellulose induction, 13 TFs were identified whose binding site frequencies on the promoter regions of the cellulases positively correlated with their abundance in the secretome. Further, correlation analysis of the transcriptional response of these regulators and TF-binding sites on their promoters indicated that cellulase expression is possibly preceded by up-regulation of 12 TFs and down-regulation of 16 TFs, which cumulatively regulate transcription, translation, nutrient metabolism and stress response.

Alleviating vacuolar transport improves cellulase production in Trichoderma reesei

Increasing cellulase production in cellulolytic fungus Trichoderma reesei is of interest for biofuels and biorefineries. Previous studies indicated that secreted protein was occasionally accumulated in vacuoles; this phenomenon has also been reported in T. reesei. Therefore, alleviating vacuolar transport seems to be a promising strategy for improving cellulase production in T. reesei. Herein, we found that knockout of vps10, vps13, and vps21, among 11 vacuolar protein sorting factors, improved cellulase production in T. reesei. The filter paper activity in Δvps10, Δvps13, and Δvps21 increased by 1.28-, 2.45-, and 2.11-fold than that of the parent strain. Moreover, the β-glucosidase activity in Δvps13 and Δvps21 increased by 3.22- and 3.56-fold after 6days of fermentation. Furthermore, we also found that the vacuolar trafficking towards vacuoles was partially impaired in three knockout mutants, and disruption of vps13 alleviated the autophagy process. These results indicated that alleviated transport and degradation towards vacuole in Δvps10, Δvps13, and Δvps21 might improve cellulase production. Of note, the expression of cellulase genes in Δvps13 and Δvps21 was dramatically increased in the late induction phase compared to the parent. These results suggested that Vps13 and Vps21 might influence the cellulase production at transcription level. And further transcriptome analysis indicated that increased cellulase gene expression might be attributed to the differential expression of sugar transporters. Our study unravels the effect of alleviating vacuolar transport through knockout vps10, vps13, and vps21 for efficient cellulase secretion, providing new clues for higher cellulase production in T. reesei. KEY POINTS: • Disruption of vps10, vps13 or vps21 improves cellulase production • Vacuolar transport is impaired in three vps KO mutants • Deletion of vps13 or vps21 increases the transcript of cellulase genes in late stage.

Full-Chain FeCl3 Catalyzation Is Sufficient to Boost Cellulase Secretion and Cellulosic Ethanol along with Valorized Supercapacitor and Biosorbent Using Desirable Corn Stalk

Cellulosic ethanol is regarded as a perfect additive for petrol fuels for global carbon neutralization. As bioethanol conversion requires strong biomass pretreatment and overpriced enzymatic hydrolysis, it is increasingly considered in the exploration of biomass processes with fewer chemicals for cost-effective biofuels and value-added bioproducts. In this study, we performed optimal liquid-hot-water pretreatment (190 °C for 10 min) co-supplied with 4% FeCl3 to achieve the near-complete biomass enzymatic saccharification of desirable corn stalk for high bioethanol production, and all the enzyme-undigestible lignocellulose residues were then examined as active biosorbents for high Cd adsorption. Furthermore, by incubating Trichoderma reesei with the desired corn stalk co-supplied with 0.05% FeCl3 for the secretion of lignocellulose-degradation enzymes in vivo, we examined five secreted enzyme activities elevated by 1.3-3.0-fold in vitro, compared to the control without FeCl3 supplementation. After further supplying 1:2 (w/w) FeCl3 into the T. reesei-undigested lignocellulose residue for the thermal-carbonization process, we generated highly porous carbon with specific electroconductivity raised by 3-12-fold for the supercapacitor. Therefore, this work demonstrates that FeCl3 can act as a universal catalyst for the full-chain enhancement of biological, biochemical, and chemical conversions of lignocellulose substrates, providing a green-like strategy for low-cost biofuels and high-value bioproducts.

Overexpression of a Novel Vacuolar Serine Protease-Encoding Gene (spt1) to Enhance Cellulase Production in Trichoderma Reesei

Trichoderma reesei is widely applied as the major industrial fungus for the production of cellulases used for the conversion of lignocellulosic biomass to biofuels and other biobased products. The protein secretion pathway is vital for cellulase secretion, but few reports are related to the role of the vacuole in cellulase production. Here, we identified a novel vacuolar serine protease gene spt1 and investigated the ability of T. reesei to secrete cellulases by disrupting, complementing and overexpressing the spt1 gene. Amino acid sequence analysis of the Spt1 protein showed that it belongs to the subtilisin S8 family and has the conserved catalytic triples (Asp, His, Ser) of the serine protease. The deletion of spt1 did not lead to a decrease in extracellular protease activity, and the observation of mycelia with the Spt1–eGFP fusion expression and the vacuolar membrane dye FM4-64 staining confirmed that Spt1 was an intracellular protease located in the vacuoles of T. reesei. However, the spt1 gene deletion significantly reduced spore production and cellulase secretion, while the spt1 complementation recovered these traits to those of the parental strain. When spt1 was overexpressed by using its native promoter and introducing multiple copies, the cellulase secretion was improved. Furthermore, a strong promoter, Pcdna1, was used to drive the spt1 overexpression, and it was found that the cellulase production was significantly enhanced. Specifically, the filter paper activity of the spt1 overexpression strain SOD-2 reached 1.36 U/mL, which was 1.72 times higher than that of the parental strain. These findings demonstrated that the spt1 gene can be a powerful target for increasing cellulase production in T. reesei, which suggests a possible important role of the vacuole in the cellulase secretion pathway and provides new clues for improving strains for efficient cellulase production.

Valorisation of the invasive alga Rugulopteryx okamurae through the production of monomeric sugars

Rugulopteryx okamurae is an invasive brown alga causing severe environmental and economic problems on the western Mediterranean coasts. Thus, in addition to the difficulties caused to the fishing and tourism sectors, there is a need to manage its accumulation on the beaches. This work aims to valorise this waste by using it as raw material for producing monosaccharides through a two-stage sequential process. These sugars could be used for different fermentative processes to obtain high-value-added bioproducts. In this work, biological pretreatment of the previously conditioned seaweed with the fungus Aspergillus awamori in solid-state fermentation (SSF), followed by enzymatic hydrolysis with a commercial enzyme cocktail, was performed. The effect of the extension of the biological pretreatment (2, 5, 8 and 12 days) on the subsequent release of total reducing sugars (TRS) in the enzymatic hydrolysis stage was studied. To analyse this effect, experimental data of TRS produced along the hydrolysis were fitted to simple first-order kinetics. Also, the secretion of cellulase and alginate lyase by the fungus, along with the biological pretreatment, was determined. The results suggest that 5 days of biological pretreatment of the macroalgae with A. awamori followed by enzymatic saccharification for 24 h with Cellic CTec2® (112 FP units/g of dry biomass) are the best conditions tested, allowing the production of around 240 g of TRS per kg of dried biomass. The main sugars obtained were glucose (95.8 %) and mannitol (1.5 %), followed by galactose (1 %), arabinose (0.9 %) and fucose (0.5 %).Key points• Five-day SSF by A. awamori was the best condition to pretreat R. okamurae.• Five-day SSF was optimal for alginate lyase production (1.63 ±0.011 IU/g biomass).• A maximum yield of 239 mg TRS/g biomass was obtained (with 95.8 % glucose).Graphical

A pilot-scale sustainable biorefinery, integrating mushroom cultivation and in-situ pretreatment-cum-saccharification for ethanol production

Biomass pretreatment incurs 40% of the overall cost of biorefinery operations. The usage of mushroom cultivation as a pretreatment/delignification technique, and bio-ethanol production from spent mushroom substrates, after subsequent pretreatment, saccharification and fermentation processes, have been reported earlier. However, the present pilot-scale, entirely-organic demonstration is one of the very first biorefinery models, which efficiently consolidates: biomass pretreatment; in-situ cellulase production and saccharification; mushroom cultivation, thereby improving the overall operational economy. During pretreatment, the oyster mushroom, Pluerotus florida VS-6, matures into distinct substrate mycelia and fruiting bodies. Consequential variations in the kinetics of growth, biomass degradation/substrate utilization, oxygen uptake and transfer rates, and enzyme production, have been analyzed. Signifying the first-time usage of a biomass mixture, comprising vegetative waste and e-commerce packaging waste, the 30 day-long, bio-economical, non-inhibitor-generating, catabolite repression-limited, solid-state in-situ pretreatment-cum-saccharification, resulted in: 78% lignin degradation; 13.25% soluble-sugar release; 18.25% mushroom yield; 0.88 FPU/g.ds cellulase secretion. The in-situ saccharified biomass, when sequentially subjected to ex-situ enzymatic hydrolysis and fermentation, showed 37.35% saccharification, and a bio-ethanol yield of 0.425 g per g of glucose, respectively. Apart from yielding engine-ready bio-ethanol, the model doubles as an agripreneurial proposition, and encourages mushroom cultivation and consumption.