Enzymatic saccharification of cornstalk by onsite cellulases produced by Trichoderma viride for enhanced biohydrogen production

Lignocellulosic biomass, if properly saccharified, could be an ideal feedstock for biohydrogen production. However, the high cellulases cost is the key obstacle to its development. In this work, cost‐effective enzyme produced by Trichoderma viride was used to saccharify cornstalk. To obtain high sugar yield, a central composite design of response surface method was used to optimize enzymatic saccharification process. Experimental results showed that the enzymatic saccharification rate reached the highest of 81.2% when pH, temperature, cellulases and substrate concentration were 5, 49.7 °C, 35.7 IU g^?1, and 38.5 g L^?1, respectively. The cornstalk hydrolysate was subsequently introduced to fermentation by Thermoanaerobacterium thermosaccharolyticum W16, the yield of hydrogen reached the highest level of 90.6 ml H₂ g^?1 pretreated cornstalk. The present results indicate the potential of using T. thermosaccharolyticum W16 for high yield conversion of cornstalk hydrolysate, which was saccharified by onsite enzyme produced by T. viride.     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Induction and production of cellulases by l-sorbose in Trichoderma reesei

Summary Cellulase production in Trichoderma reesei mutants was induced by l-sorbose, known to be an inhibitor of -1,3-glucan synthesis. In the experiments the washed mycelia were used as resting cells. For CMCase induction over 24 h using T. reesei PC-3-7, the most effective pH, temperature and l-sorbose concentration were 2.8, 28° C and 0.3 mg/ml, respectively. Comparison with other cellulase inducers showed that the inductive level of CMCase by l-sorbose was similar to that by sophorose, known to be the most potent inducer of cellulases. Since the induction of CMCase was inhibited completely by 10 g of cycloheximide per ml, the induction process was considered to involve de novo synthesis. Although l-sorbose had the effective inducibility of CMCase, the assimilation rate of l-sorbose was very low in T. reesei PC-3-7.Production of Ethanol from Biomasses. Part III.Production of Ethanol from Biomasses. Part III.     

拟康氏木霉(Trichoderma pseudokoningii)UV III纤维素酶合成的诱导与阻遏

拟康氏木霉 (T .pseudokoningii)TH经紫外诱变获得一抗高浓度葡萄糖阻遏突变株UVIII,纤维素酶产量显著提高。研究表明 ,UVIII对诱导物的敏感性增加了 10 0倍 ,并且对葡萄糖的吸收能力明显下降 ,导致部分解除了葡萄糖阻遏作用 ,这可能都是该突变株产酶提高的原因     

Biochemical and Proteomic Characterization of a Novel Extracellular β-Glucosidase from Trichoderma citrinoviride

β-Glucosidases are of pivotal importance in bioconversion of carbonic biomass into fermentable and other useful metabolites, food industry, biotransformation, glyco-trimming of metabolome, etc. Trichoderma citrinoviride when grown on delignified Lantana camara produced a β-glucosidase and secreted it out in the medium. The extracellularly secreted β-glucosidase of T. citrinoviride was homogeneity purified and then characterized for its kinetic properties and proteomic characteristics. The 90 kDa enzyme was monomeric in nature, optimally active at pH 5.5 and the catalytic reaction rate was highest at 55°C. Uniquely, the enzyme was insensitive to inhibition by glucose (up to 5 mM). It also possessed catalytic ability of transglycosylation, as it could catalyze conversion of geraniol into its glucoside. MALDI-TOF assisted proteomic analysis revealed its high degree of sequence similarity with family 3 glycoside hydrolases.     

Optimal control strategy for the enhanced production of cellulase enzyme using the new mutant Trichoderma reesei E-12

Cellulase enzyme production was enhanced using the mutant strain Trichoderma reesei, E-12, which was shown to be partially resistant to catabolite repression. An optimal profile for pH, which was found to be the critical environmental parameter, was determined using a rigorous mathematical optimization procedure. Semi-empirical models were used to minimize complications in the computation. A 30% increase in enzyme activity and productivity was obtained using the optimal pH strategy as compared to the pH cycling strategy.List of Symbols a ₁ , a ₂ , a ₃ d^–1, d^–2, d^–3 coefficients of the polynomial in the generalized logistic growth model - a ₄, a ₅, a ₆ d^–1, d^–2, d^–3 coefficients of the polynomial in the generalized logistic product model - b ₁ d^–1 enzyme synthesis rate constant - b ₂ d ^–1 enzyme decay rate constant - b ₃ power coefficient in the polynomial model for enzyme synthesis - H Hamiltonian function - J Objective function of the maximization procedure - K ₁ kg/m³ limiting cell mass concentration in biomass logistic model - K _s kg/m³ saturation constant - K ^s kg/m³ saturation death rate constant - q power coefficient in polynomial model - s kg/m³ substrate concentration - t d fermentation time - T d total fermentation time (=7 d) - x ₁₀ kg/m³ initial biomass concentration - x ₁ kg/m³ biomass concentration at time t - x ₂ F.P.A enzyme activity at time t - x ₃ d state variable replacing time term on the right hand side of biomass equation - x _f kg/m³ final biomass concentration - z ₁, z ₂, z ₃ adjoint variable corresponding to state variable x ₁, x ₂, x ₃ - d^–1 specific death rate - d^–1 specific growth rate     

Strain improvement for enhanced production of cellulase in Trichoderma viride

The filamentous fungi Trichoderma species produce extracellular cellulase. The current study was carried out to obtain an industrial strain with hyperproduction of cellulase. The wild-type strain, Trichoderma viride TL-124, was subjected to successive mutagenic treatments with UV irradiation, low-energy ion beam implantation, atmospheric pressure non-equilibrium discharge plasma (APNEDP), and N-methyl-N'-nitro-N-nitrosoguanidine to generate about 3000 mutants. Among these mutants, T. viride N879 strain exhibited the greatest relevant activity: 2.38-fold filter paper activity and 2.61-fold carboxymethyl cellulase, 2.18-fold beta-glucosidase, and 2.27-fold cellobiohydrolase activities, compared with the respective wild-type activities, under solid-state fermentation using the inexpensive raw material wheat straw as a substrate. This work represents the first application of APNEDP in eukaryotic microorganisms.     

Production of cellulases in batch culture using a mutant strain of Trichoderma reesei growing on soluble carbon source

Trichoderma reesei Rut-C30 is a highly derepressed mutant which synthesised active cellulases in culture media containing glucose and lactose as the only carbon sources. The maximum biomass, filter paper and specific filter paper activities for cell growth on 20 g glucose l^–1 were 20 g dry cell wt l^–1, 1.9 FPU ml^–1 and 4.8 FPU mg^–1 protein respectively, while on 40 g glucose l^–1 were 25 g dry cell wt l^–1, 4.5 FPU ml^–1 and 6.2 FPU mg^–1 protein, respectively. This strain had a higher specific filter paper activity (6.2 FPU mg^–1 protein) than was produced by other T. reesei mutants (3.6 FPU mg^–1 protein).     

The use of cellulases from a beta-glucosidase-hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw

Conidia of the cellulolytic strain Trichoderma reesei F522 were mutagenized with UV irradiation and N-methyl|-N'-nitro-N-nitrosoguanidine (NTG). A visual agar plate detection system was developed, using esculin and ferric ions, to identify mutants of T. reesei with increased beta-glucosidase activity. Selected mutants were tested for production of extracellular cellulases in shake flasks on autohydrolyzed wheat straw as carbon source. The most active mutant V-7 showed about 6-times higher activity of beta-glucosidase than the parent strain F-522, whereas the filter paper degrading and endo-1,4-beta-D-glucanase activities increased by 45% and by almost 31%, respectively. Cellulase preparations obtained from the parent and mutant strains were then used along with Kluyveromyces fragilis cells for ethanol production from ethanol-alkali pulped straw in the simultaneous saccharification and fermentation (SSF) process. From 10% (w/v) of straw pulp (dry matter), 2.5% (w/v) ethanol was obtained at 43 degrees C after 48 h using cellulase derived from the parent strain of T. reesei. When the beta-glucosidase-hyperproducing mutant V-7 was employed, the ethanol yield in the SSF process increased to 3.4% (w/v), the reaction time was shortened to 24 h and no cellobiose was detected in straw hydrolyzates.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

Characterization of a Saccharomyces cerevisiae mutant with enhanced production of beta-D-fructofuranosidase

The present study focused on the improvement of Saccharomyces cerevisiae through random mutagenesis for enhanced production of beta-D-fructofuranosidase (FFase) using sucrose salt media. Sixty strains of S. cerevisiae were isolated from different fruits and soil samples and screened for FFase production. Enzyme productivity of different yeast isolates ranged from 0.03 to 1.10 U/ml. The isolate with the highest activity was subjected to ultraviolet (UV) radiation and mutagenesis using N-methyl N-nitro N-nitroso guanidine (MNNG). One mutant produced FFase at a level of 17.8+/-0.9 U/ml. The MNNG-treated isolate was exposed to ethyl methane sulphonate (EMS), and a mutant with an enzyme activity of 25.56+/-1.4 U/ml was obtained. Further exposure to UV radiation and chemicals yielded a mutant exhibiting an activity of 34.12+/-1.8 U/ml. After optimization of incubation time (48 h), sucrose concentration (5.0 g/L), initial pH (6.0) and inoculum size (2.0% v/v), enzyme production reached 45.65+/-4.6 U/ml with a noticeable greater than 40-fold increase compared to the wild-type culture. On the basis of kinetic variables, notably Q(p) (0.723+/-0.2U/g/h), Y(p/s) (2.036+/-0.05 U/g) and q(p) (0.091+/-0.02 U/g yeast cells/h), the mutant S. cerevisiae UME-2 was found to be a hyperproducer of FFase (LSD 0.054, p0.05).     

Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor

With the aim to produce cellulases and to study the effect of mechanical agitation, a 35 L draft-tube airlift bioreactor equipped with a mechanical impeller was developed and validated to grow Trichoderma reesei RUT-C30 in a cellulose culture medium with lactose and lactobionic acid as fed batch. Cultures carried out without mechanical agitation resulted in higher volumetric enzyme productivity (200 U L⁻¹ h⁻¹), filter paper activity (17 U mL⁻¹), carboxymethyl cellulase activity (11.8 U mL⁻¹) and soluble proteins (3.2 mg mL⁻¹) when compared to those with agitation. Stereo and polarized light microscopy analyses reveal that mechanical agitation resulted in shorter mycelial hyphae and larger numbers of tips.     

Trichoderma reesei cellulases in the bleaching of kraft pulp

In this work the effects of individual purified cellulases of Trichoderma reesei were studied in the enzyme-aided bleaching of kraft pulps. The cellobiohydrolases I and II, when used alone, had no positive effect on the bleachability of kraft pulps. The endoglucanase I (EG I), however, acted on pulp similarly to xylanases and with an enzyme dosage of 0.1 mg/g a clear increase in pulp brightness could be observed. Due to the unspecificity of this enzyme, the viscosity of the pulp was simultaneously decreased. Of the cellulases, EG II was clearly most detrimental in reducing the pulp viscosity. Hence, the action of purified cellulases of T. reesei on pulp as a substrate differs profoundly, and all cellulases are not detrimental to the pulp properties. Correspondence to: J. Buchert     

Synergism of Trichoderma reesei cellulases while degrading different celluloses

Summary The synergistic action of purified cellulases from Trichoderma reesei in hydrolysis of cellulose decreased with increasing substrate concentration, depended strongly on the the type of cellulose used, and was maximal on crystalline cellulose. Contrarily, the activity of the individual cellulases was highest on amorphous cellulose. The binary combinations CBH I/EG III and CBH I/CBH II exhibited the greatest degree of synergism on crystalline cellulose.     

Production of Trichoderma reesei cellulases on glucose-containing media.

The filamentous fungus Trichoderma reesei was shown to secrete active cellobiohydrolase I and the endoglucanase I catalytic core domain into the culture medium when the fungus was grown on glucose-containing medium. The expression of the proteins was driven by the promoters of the elongation factor 1 alpha, tef1, and the unidentified gene for cDNA1. The cDNA1 promoter gave the best yields. The highest amounts of cellobiohydrolase I and the endoglucanase I core, being 50 to 100 mg/liter, accounted for more than half of the total protein secreted by the fungus. The levels obtained with the tef1 promoter were 20 to 50 times lower.     

Production of cellulases from alkali-treated bagasse in Trichoderma reesei

Summary Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X-31, in which strong cellulase activity is inducible by l-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1 jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.Production of Ethanol from Biomasses Part IV.Production of Ethanol from Biomasses Part IV.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.