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).     

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation (R(2)=0.99) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaerobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.     

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO₂ chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO₂ was required in comparison to the enzymatically untreated samples.     

Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.     

Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment

Rice straw was irradiated using an electron beam at currents and then hydrolyzed with cellulase and beta-glucosidase to produce glucose. The pretreatment by electron beam irradiation (EBI) was found to significantly increase the enzyme digestibility of rice straw. Specifically, when rice straw that was pretreated by EBI at 80 kGy at 0.12 mA and 1 MeV was hydrolyzed with 60 FPU of cellulase and 30 CBU of beta-glucosidase, the glucose yield after 132 h of hydrolysis was 52.1% of theoretical maximum. This value was significantly higher than the 22.6% that was obtained when untreated rice straw was used. In addition, SEM analysis of pretreated rice straw revealed that EBI caused apparent damage to the surface of the rice straw. Furthermore, EBI pretreatment was found to increase the crystalline portion of the rice straw. Finally, the crystallinity and enzyme digestibility were found to be strongly correlated between rice straw samples that were pretreated by EBI under different conditions.     

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.     

Production of cellulases and xylanase by Trichoderma reesei F-522 on pretreated wheat straw

Autohydrolyzed and ethanol-alkali pulped wheat straw was examined as a candidate feedstock for both cellulase and xylanase production and enzymatic hydrolysis. Submerged cultures of Trichoderma reesei F-522 grown on hydrothermally modified straw provided culture supernatants of the highest enzymatic activities, whereas the maximal efficiency of enzymatic hydrolysis was recorded in straw treated with ethanol-NaOH mixture. Some culture conditions were optimized to improve the growth and cellulase production by T. reesei on autohydrolyzed wheat straw.     

The effect of combined chemical and enzyme treatments on the saccharification and in vitro digestion rate of wheat straw

Wheat straw was pretreated with sodium hydroxide, ozone, and sulfur dioxide, and subsequently treated with four sources of cellulase, T. reesei, T. reesei, T. viride, A. niger, and Oxyporus sp. The effect of the combined chemical + enzyme treatments on the extent of saccharification and on the digestion rate by rumen microorganisms was studied. Cellulases were applied at an equal but low concentration (0.5 FPU/ml) on the pretreated straw. The combined treatments, SO₂ + T. reesei cellulase nd SO₂ + T. viride cellulase, produced the highest and significant levels of reducing sugars (RS), 577 and 597 mg RS/g straw organic matter. The highest enzyme efficiency, 44.7 mg RS/mg enzyme, was found with T. reesei cellulase when applied on SO₂ pretreated straw. The in vitro organic matter digestibility was affected significantly only by the chemical pretreatments, whereas the effect of the cellulases was expressed mainly in increasing the fermentability of the hydrolyzed straw. The in vitro digestion pattern of the saccharified straw was found to be typical of a highly fermentable feed and comparable to a starchy mixture such as used in concentrate ruminant diets.     

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.     

Effect of different gas environments on bench-scale solid state fermentation of oat straw by white-rot fungi

Three white-rot fungi, Phanerochaete chrysosporium, Polyporus tulipiferae, and Polyporus sp. A336 were grown on 100-g amounts of chopped oat straw in gassed 4.5 L (diameter 16 cm, height 23 cm) solid-state reactors for two weeks. The different gas atmospheres were regulated by (1) air diffusion through foam plugs, (2) intermittent or continuous air flow, (3) intermittent oxygen, 50 or 100% continuous oxygen flow, and (4) continuous 10% carbon dioxide in oxygen flow. The fermented straw was analyzed for total weight loss, Klason lignin loss, and enzymatic (cellulase) hydrolysis. P. chrysosporium grown on straw in continuous oxygen at 35 degrees C caused a 41% weight loss and 33.5% hydrolysis was obtained when the pretreated straw was hydrolyzed with cellulase enzyme. P. tulipiferae caused a 27% weight loss and 34.3% cellulase hydrolysis in the straw at 30 degrees C. Polyporus sp. A336 selectively degraded lignin of the straw and under intermittent oxygen resulted in an 18% weight loss and 33.6% cellulase hydrolysis at 35 degrees C. When the straw was supplemented with 10% xylose (straw basis) and was continuously gassed with 50% oxygen, Polyporus sp. A336 produced a 14.5% weight loss and 38.7% cellulase hydrolysis. Oxygen and carbon dioxide exchange rates were measured for some of these bench-scale fermentations.     

Sorption von Cellulase an Weizenstroh und seine Komponenten

By comparing different activity data of the buffered cellulase solution before and after contact with the substrate the interaction between Penicillium janthinellum cellulase and wheat straw, resp. its components (holocellulose and isolated lignin) has been investigated. The loss of activity due to sorption or denaturation has been found to differ widely between the different activity data and between the various substrates. A remarkable loss of enzyme activity was observed after contact with isolated straw lignin. The differences in activity decrease between the cellulose and the lignin moiety were found to be largent with the cellobiase activity.     

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.     

Effect of peracetic acid, sodium hydroxide and phosphoric acid on cellulosic materials as a pretreatment for enzymatic hydrolysis

Crystalline cellulose and cellulosic wastes have been treated with various concentrations of peracetic acid and other reagents at 100°C for various times, washed with water, ethanol and air dried. For each treated cellulose, the degree of enzymatic solubilization was measured with Trichoderma viride cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4]. Cellulosic wastes such as sunflower stalks, wheat straw and sugar-cane bagasse were solubilized effectively by the enzyme. Delignification of wheat straw with 1% sodium hydroxide and treatment of this straw with peracetic acid enhanced the degree of enzymatic solubilization. Infrared spectra of the untreated and treated cellulosic wastes were recorded.     

Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100

Abstract

To prepare a smart biocatalyst, cellulase was immobilized on the reversibly soluble matrix Eudragit L-100 by non-covalent and covalent methods. Covalent immobilization using carbodiimide coupling exhibited superior enzyme loading and reusability compared with non-covalent immobilization, and the covalent loading was increased by almost 20% through the addition of N-hydroxysuccinimide. The temperature optimum of the cellulase was not improved apparently by immobilization but the pH optimum increased from 4.75 to 5.25. Immobilized cellulase was more active than free cellulase above pH 5.0. Immobilized cellulase was more stable than free cellulase during storage at 4°C, room temperature and 50°C. K_m values of immobilized and free cellulase were 85.55 and 73.84 g L^?1, respectively. About 50% productivity was retained after five cycles for hydrolysis of steam-exploded straw.     

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.     

稻草秸秆预处理方法对烟曲霉产纤维素酶的影响

采用机械粉碎、高温、酸碱处理等方法对稻草秸秆进行预处理,以烟曲霉为实验菌株,研究预处理方法对菌株产纤维素酶的影响。结果表明,取机械粉碎后的稻草（30~120目）进行121℃高压蒸汽处理20min（即灭菌处理）,有利于菌株的生长与纤维素酶的产生;与未粉碎的稻草秸秆相比,烟曲霉羧甲基纤维素钠（CMC）酶、微晶纤维素酶、β-葡萄糖苷酶和滤纸（FPA）酶的活力分别提高了63.2%、164.0%、10.2%和14.1%。而采用不同种类、不同浓度的酸碱常温处理稻草秸秆4d或100℃高温处理30min,纤维素酶活力均出现了不同程度的下降。     

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.     

Efficient Recovery of Glucose and Fructose via Enzymatic Saccharification of Rice Straw with Soft Carbohydrates

Soft carbohydrates, defined as readily-recoverable carbohydrates via mere extraction from the biomass or brief enzymatic saccharification, were found in significant amounts in rice straw as forms of free glucose, free fructose, sucrose, starch, and β-1,3-1,4-glucan. In this study, we investigated their amounts in rice straw (defined as culm and leaf sheath), and developed an easy method for glucose and fructose recovery from them with heat-pretreatment and subsequent 4-h enzymatic saccharification with an enzyme cocktail of cellulase and amyloglucosidase. The recovery of glucose and fructose exhibited good correlation with the amounts of soft carbohydrates. The maximum yields of glucose and fructose in the rice straw per dry weight at the heading stage and the mature stage were 43.5% in cv. Habataki and 34.1% in cv. Leafstar. Thus, rice straw with soft carbohydrates can be regarded as a novel feedstock for economically feasible production of readily-fermentable glucose and fructose for bioethanol.     

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.     

Hydrolytic properties of extracellular cellulases fromPleurotus ostreatus

Pleurotus ostreatus mycelium produced extracellular cellulases when grown on ground wheat straw. No cellulase activity was detectable when glucose or aqueous extracts of vegetable material were used as substitutes for straw in culture media. The specific activity of excreted cellulases did not vary significantly by increasing the straw concentration from 1 to 6%. Chromatographic fractionation of extracellular proteins gave rise to five fractions with cellulolytic activity. The hydrolytic properties of these partially purified fractions were analysed by using several substrates (carboxymethylcellulose, cellobiose,p-nitrophenyl--d-cellobioside,p-nitrophenyl--d-lactoside). The results indicate that the cellulase system ofP. ostreatus includes at least a -glucosidase, two endocellulases, an exoglucohydrolase and an exocellobiohydrolase.