Enzymatische Hydrolyse und Ethanolgärung von Lignocellulosen

The kinetics of enzymatic hydrolysis of different lignocellulosic materials (wheat straw, newspaper and microcrystalline cellulose Avicel PH 101) was studied using the cellulase complexes from Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15 and MHC 22. The maximum yields of hydrolysis were obtained with wheat straw partially delignified with 1% NaOH as substrate, and using the enzyme from the mutants T. reesei M 6 and MHC 22. The possibility of simultaneous enzymatic hydrolysis and ethanol fermentation of wheat straw using the enzyme complex from M 6 and yeasts of the genus Candida and Torulopsis was also investigated. A good conversion of liberated glucose and cellobiose to ethanol was obtained, however, xylose was not fermented.     

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Summary Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.     

Cellulase production using different streams of wheat grain- and wheat straw-based ethanol processes

Pretreatment is a necessary step in the biomass-to-ethanol conversion process. The side stream of the pretreatment step is the liquid fraction, also referred to as the hydrolyzate, which arises after the separation of the pretreated solid and is composed of valuable carbohydrates along with compounds that are potentially toxic to microbes (mainly furfural, acetic acid, and formic acid). The aim of our study was to utilize the liquid fraction from steam-exploded wheat straw as a carbon source for cellulase production by Trichoderma reesei RUT C30. Results showed that without detoxification, the fungus failed to utilize any dilution of the hydrolyzate; however, after a two-step detoxification process, it was able to grow on a fourfold dilution of the treated liquid fraction. Supplementation of the fourfold-diluted, treated liquid fraction with washed pretreated wheat straw or ground wheat grain led to enhanced cellulase (filter paper) activity. Produced enzymes were tested in hydrolysis of washed pretreated wheat straw. Supplementation with ground wheat grain provided a more efficient enzyme mixture for the hydrolysis by means of the near-doubled β-glucosidase activity obtained.     

Direct bioconversion of alkali-pretreated straw using simultanesous enzymatic hydrolysis and acetone-butanol fermentation

Summary Conversion of alkali-pretreated wheat straw into butanol and acetone by Clostridium acetobutylicum has been achieved in a one-step hydrolysis and fermentation process involving the use of cellulase from Trichoderma reesei. In the conditions adopted, the results obtained for solvent concentration (17.3 g.l^-1) solvent yield (18.3% with respect to pretreated wheat straw) and overall conversion time (36 h) demonstrate an improved performance over the separate hydrolysis and fermentation operation.     

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L^?1 for rice straw, wheat bran, peptone, and CaCO₃, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL^?1 were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.     

Enzymatic hydrolysis of wheat straw: Kinetic analysis

Pretreated wheat straw was enzymatically hydrolyzed in 250-ml flasks using the culture filtrate of Trichoderma reesei QM-9414. The influence of the initial enzymatic activity in the liquid phase was studied. The unreacted core model was used to analyze the experimental data obtained at 40, 46 and 50°C. The model adequately describes the data for hydrolysis times lower than 10 h.     

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.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

Summary Rice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO₂). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO₂ resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO₂ (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO₂ (twice).     

Influence of substrate particle size and wet oxidation on physical surface structures and enzymatic hydrolysis of wheat straw

In the worldwide quest for producing biofuels from lignocellulosic biomass, the importance of the substrate pretreatment is becoming increasingly apparent. This work examined the effects of reducing the substrate particle sizes of wheat straw by grinding prior to wet oxidation and enzymatic hydrolysis. The yields of glucose and xylose were assessed after treatments with a benchmark cellulase system consisting of Celluclast 1.5 L (Trichoderma reesei) and Novozym 188 β‐glucosidase (Aspergillus niger). Both wet oxidized and not wet oxidized wheat straw particles gave increased glucose release with reduced particle size. After wet oxidation, the glucose release from the smallest particles (53–149 μm) reached 90% of the theoretical maximum after 24 h of enzyme treatment. The corresponding glucose release from the wet oxidized reference samples (2–4 cm) was ～65% of the theoretical maximum. The xylose release only increased (by up to 39%) with particle size decrease for the straw particles that had not been wet oxidized. Wet oxidation pretreatment increased the enzymatic xylose release by 5.4 times and the glucose release by 1.8 times across all particle sizes. Comparison of scanning electron microscopy images of the straw particles revealed edged, nonspherical, porous particles with variable surface structures as a result of the grinding. Wet oxidation pretreatment tore up the surface structures of the particles to retain vascular bundles of xylem and phloem. The enzymatic hydrolysis left behind a significant amount of solid, apparently porous structures within all particles size groups of both the not wet oxidized and wet oxidized particles. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of xylanase under solid-state fermentation by <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H₂SO₄) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.     

Chemical and biological properties of wheat soil in response to paddy straw incorporation and its biodegradation by fungal inoculants

A field experiment was conducted to evaluate the relative contribution of organic fertilizers (paddy straw, microbial inoculants and vermicompost) and inorganic fertilizers (urea and superphosphate) in improving pH, C, N, humus, microbial biomass, dehydrogenase, phosphatase, cellulase, β-glucosidase and xylanase activities of soil under wheat crop. Vermicompost fertilization resulted in highest microbial biomass, available phosphorus, and nitrogen content of wheat soil. It was also found effective in minimizing the alkalinity of soil compared to other treatments as indicated by pH change. However incorporation of paddy straw in conjunction with N₆₀P₆₀ and T. reesei inoculation resulted in maximum dehydrogenase, alkaline phosphatase and highest humus content of soil. Mixed inoculation of A. awamori and T. reesei did not prove effective in improving the soil biochemical properties in comparison to single inoculation of T. reesei. Results showed that in situ incorporation of paddy straw in combination with N₆₀P₆₀ and T. reesei inoculation can be used as an effective measure for valuable disposal of paddy straw and to improve the soil health by reducing mineral fertilization.     

Use of sorbose to enhance cellobiase activity in a Trichoderma reesei cellulase system produced on wheat hydrolysate

Summary Several methods were investigated for increasing cellobiase activity of T. reesei cellulases produced using an acid-hydrolyzed wheat flour. The use of sorbose in cellulase production is preferred to mixed cultures with Aspergillus niger. Using 13.5 g/L wheat hydrolysate and 1.0 g/L sorbose, the culture of T. reesei produced 3.72 IU/mL of filter paper activity (FPA) and 0.53 IU/mL of cellobiase activity. The addition of sorbose resulted in 89% increase in cellobiase activity and 47% increase in FPA.     

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Saccharification of biomass using whole solid‐state fermentation medium to avoid additional separation steps

The enzymatic hydrolysis of steam‐exploded sugarcane bagasse (SESB) was investigated using enzymatic extracts (EE) and whole fermentation media (WM), produced in‐house, from Aspergillus niger 3T5B8 and Trichoderma reesei Rut‐C30 cultivated on wheat bran under solid‐state fermentation (SSF). A detailed and quantitative comparison of the different hydrolysis conditions tested was carried out using the Chrastil approach for modeling enzymatic reactions by fitting the experimental data of total reducing sugar (TRS) released according to hydrolysis time. Conversion of SESB using A. niger enzymatic complex were up to 3.2‐fold higher (in terms of TRS) than T. reesei at similar enzyme loadings, which could be correlated to the higher β‐glucosidase levels (up to 35‐fold higher) of A. niger enzymatic complex. Conversion yields after 72 h exceeded 40% in terms of TRS when the WM was supplemented with a low dosage of a commercial enzyme preparation. When the combination of WM (from either T. reesei or A. niger) and commercial cellulase was used, the dosage of the commercial enzyme could be reduced by half, while still providing a hydrolysis that was up to 36% more efficient. Furthermore, SESB hydrolysis using either EE or WM resulted in similar yields, indicating that the enzyme extraction/filtration steps could be eliminated from the overall process. This procedure is highly advantageous in terms of reduced enzyme and process costs, and also avoids the generation of unnecessary effluent streams. Thus, the enzymatic conversion of SESB using the WM from SSF is cost‐effective and compatible with the biorefinery concept. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1430–1440, 2013     

Inhibition of cellulose enzymatic hydrolysis by laccase‐derived compounds from phenols

The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat‐straw prehydrolysate after steam‐explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase‐derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and β‐glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam‐exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat‐straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase‐derived products. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:700–706, 2015     

Simultaneous saccharification and fermentation of cellulose: Effect of ethanol and cellulases on particular stages

The effects of ethanol and Trichoderma reesei cellulase on the saccharification and fermentation processes as well as the tolerance of the cellulase complex for ethanol have been investigated. The studies were conducted with respect to their usefulness in the process of simulataneous saccharification and fermentation of cellulose to ethanol. The following results were obtained. (1) Fermentative activity of Kluyveromyces fragilis yeasts was gradually depressed with increasing intial ethanol concentrations and temperature of fermentation between 35–46°C. (2) Crude cellulase preparation introduced to the culture broth to a final enzyme activity of 0.5 to 2.0 FPU/ml had not distinct effect on the biomass production, ethanol yield, and glucose uptake by yeasts in 48 h fermentation at 43°C. On the other hand, only a negligible decrease in the cellulase complex activity was observed during fermentation process. (3) Saccharification of wheat straw was inhibited by at least 1% w/v ethanol. (4) The enzymes of the cellulase system showed a high stability to exposure to ethanol for 48 h at 43°C.     

Autohydrolysis extraction process as a pretreatment of lignocelluloses for their enzymatic hydrolysis

Autohydrolysis was studied as a pretreatment to enhance sugar yields from enzymatic hydrolysis of wheat and rape straw, beech, birch and poplar sawdust. Reaction temperatures were 185°C to 212°C and the reaction time 20 min. The pretreated slurries were hydrolyzed with “Novo” cellulase and Fusarium sp. 27 cellulase at 45°C and pH 4.8 for 24 h with addition of Fusarium sp. 27 cellbound cellobiase. From 85% to 90% sugar content of substrates were converted to reducing sugars after 24 h enzymatic hydrolysis, with exception of poplar wood. 10.8 g biomass was obtained after cultivation of Fusarium sp. 27 with water solution hemicellulose fraction from 100 g beech sawdust autohydrolyzed at 200°C during 20 min.     

Comparison of Feedstock Pretreatment Performance and Its Effect on Soluble Sugar Availability

Cellulosic feedstocks for bioenergy differ in composition and processing requirements for efficient conversion to chemicals and fuels. This study discusses and compares the processing requirements for three lignocellulosic feedstocks??soybean hulls, wheat straw, and de-starched wheat bran. They were ground with a hammer mill to investigate how differences in composition and particle size affect the hydrolysis process. Enzyme hydrolysis was conducted using cellulase from Trichoderma reesei at 50°C and pH 5. Ground fractions were also subjected to dilute sulfuric acid treatment at 125°C, 15 psi for 30 min prior to cellulase treatment. Reducing particle size of biomass resulted in segregated components of feedstock. Grinding wheat straw to particle size <132 ??m resulted in measured lignin content from 20% to ??5% and reduced hemicellulose content. Reducing lignin content increased the effectiveness of enzyme hydrolysis of wheat straw. Particles sized <132 ??m exhibited the highest soluble sugar release upon hydrolysis for all three feedstocks studied. Hemicellulose digestion improved with dilute sulfuric acid treatment with residual hemicellulose content <5% in all three feedstocks after acid treatment. This enhanced the cellulase action and resulted in approximately 1.6-fold increase in sugar availability in de-starched wheat bran and ??1.5-fold for wheat straw and soybean hulls. Higher sugar availability in wheat bran after acid-mediated enzyme treatment correlated to higher ethanol yields during yeast fermentation compared with soybean hulls and wheat straw.