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.     

Thermostable cellulase production of Aspergillus fumigatus Z5 under solid-state fermentation and its application in degradation of agricultural wastes

A lignocellulosic decomposing fungus Z5 was isolated and identified as Aspergillus fumigatus, its capacity to produce cellulase was assessed under solid-state fermentation (SSF) using lignocellulosic materials as substrates. Cultivation conditions of A. fumigatus Z5 for cellulase production were optimized, results showed that for carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase), the best condition was 50 °C, 80% initial moisture, initial pH 4.0 and 7% initial inoculum, the average activity of CMCase activity, FPase activity reached 526.3 and 144.6 U g⁻¹ dry weight (dw) respectively, much higher than most of previous reports of this genus. Optimal temperature and pH for the CMCase activity of the crude enzyme were found to be 50 °C and 5.0, respectively. Zymogram analysis showed that eight kinds of CMCase were secreted by A. fumigatus Z5 when cellulose-containing materials were supplied in the culture. The crude enzyme secreted by the strain was further applied to hydrolyze pretreated corn stover and the enzymatic hydrolysate was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of bio-ethanol was 0.112 g g⁻¹ dry substrate (gDS), suggesting that it is a promising fungus in the bio-ethanol production process.     

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.     

Immobilization of cellulase onto electrospun polyacrylonitrile (PAN) nanofibrous membranes and its application to the reducing sugar production from microalgae

This study demonstrates a method to prepare an immobilized cellulase by using an electrospun polyacrylonitrile (PAN) nanofibrous membrane as the support. To obtain an immobilized cellulase with high hydrolytic activity, the immobilization conditions including activation time, enzyme concentration, immobilization time, and temperature were optimized. Under those conditions, the immobilized cellulase possessed a protein loading of 30 mg/g-support and a specific activity of 3.2 U/mg-protein. After immobilization, the enzymatic stability of cellulase against pH and thermal stresses was improved. Fourier transform infrared spectroscopy (FTIR) measurements also revealed that the cellulase was covalently bonded to the supports. The immobilized cellulase was then used to hydrolyze cell wall of microalgae for the production of reducing sugars. Analyses using response surface methodology (RSM) show that the hydrolysis yield was affected by the reaction temperature, pH, and substrate/cellulase mass ratio, and a hydrolysis yield of 60.86% could be obtained at 47.85 °C, pH 5.82, and a substrate/cellulase mass ratio of 40 g-substrate/g-cellulase. This result suggests that the proposed scheme for the cellulase immobilization has great potential for the application to the reducing sugar production.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali–peracetic acid pretreatment

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)–peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 °C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 °C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali–PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali–PAA pretreated bagasse after enzymatic hydrolysis for 120 h.     

The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498

Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H(2)SO(4) (3.0%,v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%,w/v) and sodium chlorite (3.0%,w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of beta-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 degrees C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.     

Purification and characterization of cellulase from a marine Bacillus sp. H1666: A potential agent for single step saccharification of seaweed biomass

In this study, 115 marine bacterial isolates were screened for cellulase enzymatic activity and enzyme with a molecular mass of 40 kDa was purified from culture supernatant of the marine bacterium Bacillus sp. H1666 using ion exchange and size exclusion chromatography method. Growth of bacterial strain H1666 with efficient cellulase enzyme production was observed on untreated wheat straw and rice bran. The biochemical properties of the extracted cellulase were studied and enzyme was found active over a range of pH 3–9. The optimum cellulase activity was observed at pH 7 and temperature 50 °C. The enzyme was also shown to be slightly thermo-stable with 40% residual activity at 60 °C for 4 h. The potential applicability of enzyme was tested on dried green seaweed (Ulva lactuca) and 450 mg/g increase in glucose yield was obtained after saccharification. MALDI TOF–TOF analysis of cellulase peptide fingerprint showed similarity to the sequence of the glycoside hydrolase family protein.     

The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods

Selective white-rot fungi have shown potential for lignocellulose pretreatment. In the study, a new fungal isolate, Echinodontium taxodii 2538, was used in biological pretreatment to enhance the enzymatic hydrolysis of two native woods: Chinese willow (hardwood) and China-fir (softwood). E. taxodii preferentially degraded the lignin during the pretreatment, and the pretreated woods showed significant increases in enzymatic hydrolysis ratios (4.7-fold for hardwood and 6.3-fold for softwood). To better understand effects of biological pretreatment on enzymatic hydrolysis, enzyme–substrate interactions were investigated. It was observed that E. taxodii enhanced initial adsorption of cellulase but which did not always translate to high initial hydrolysis rate. However, the rate of change in hydrolysis rate declined dramatically with decreasing irreversible adsorption of cellulase. Thus, the enhancement of enzymatic hydrolysis was attributed to the decline of irreversible adsorption which may result from partial lignin degradation and alteration in lignin structure after biological pretreatment.     

Production of a cellulolytic enzyme system in mixed-culture solid-state fermentation of soybean hulls supplemented with wheat bran

Solid-state fermentation of soybean hulls supplemented with wheat bran using a co-culture of Trichoderma reesei and Aspergillus oryzae was performed. Three parameters — initial moisture content, incubation temperature, and initial pH — were optimized in culture flasks using response surface methodology. Parameter optimization was carried out with respect to filter paper activity and β-glucosidase activity in the culture. Temperature of 30 °C, pH of 5, and moisture content of 70% were found to be optimum. Optimized parameters were used for laboratory scale-up in static tray fermenters. The maximum filter paper activity of 10.7 FPU/g-ds and β-glucosidase of 10.7 IU/g-ds were obtained after 96-h incubation period in static tray fermenters in agreement with optimized activities at shake flask level. The results of static tray fermentation also highlighted the importance of mixed-culture fermentation. Both enzyme activities and volumetric productivities of enzyme produced were significantly higher in mixed-culture fermentation as compared to mono-culture static tray fermentation. Expression profile of cellulase system was characterized using SDS-PAGE and it indicated the presence of all the five major activities corresponding to β-glucosidase, CBH I, CBH II, EG I and xylanase. Enzyme broth was centrifuged and concentrated in an ultrafiltration cell. The concentrate was used for enzymatic saccharification of pretreated wheat straw and the potential of an indigenously developed enzyme concoction was reported in terms of saccharification efficiency. Pretreatment using both acid and alkali was carried out, and differences in sugar yield due to differences in composition as a result of pretreatment were reported. Results showed that alkali treatment generated higher sugars as compared to acid pretreatment. This was due to lignin removal and concentration of the cellulosic fraction. Present work showed that solid-state fermentation in a static tray bioreactor is a valuable technique for producing a system of enzymes with balanced activities that can efficiently saccharify lignocellulosic biomass like wheat straw.     

Using low temperature to balance enzymatic saccharification and furan formation during SPORL pretreatment of Douglas-fir

Comparing analytical results for Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL) of Douglas-fir (Pseudotsuga menziesii) at two different temperatures shows that the apparent activation energy of sugar degradation is higher than that of hemicellulose hydrolysis, approximately 161 kJ/mole versus 100 kJ/mole. Thus, one can balance the production of degradation products against hemicellulose hydrolysis and therefore the enzymatic saccharification efficiency of the resultant substrate by changing pretreatment temperature and duration. Specifically, pretreatment at 165 °C for 75 min significantly reduced furan formation compared with the pretreatment at 180 °C for 30 min while maintaining the same pretreatment severity and therefore the same substrate enzymatic digestibility (SED). Obtaining high SED with Douglas-fir is also limited by lignin content. Fortunately, the bisulfite in SPORL provides delignification activity. By combining kinetic models for hemicelluloses hydrolysis, sugar degradation, and delignification, the performance of pretreatment can be optimized with respect to temperature, duration, acid, and bisulfite loading. The kinetic approach taken in this study is effective to design viable low temperature pretreatment processes for effective bioconversion of lignocelluloses.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Enzymatic digestion of liquid hot water pretreated hybrid poplar

Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200^oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot‐washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot‐washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar‐to‐ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production

The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29 %) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61 %). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3–18.6 %) and lignin (35.2–22.1 %) content and increase cellulose content (40.5–59.3 %) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae.     

Supercritical CO2 pretreatment of lignocellulose enhances enzymatic cellulose hydrolysis

The supercritical carbon dioxide (SC-CO2) pretreatment of lignocellulose for enzymatic hydrolysis of cellulose was investigated. Aspen (hardwood) and southern yellow pine (softwood) with moisture contents in the range of 0-73% (w/w) were pretreated with SC-CO2 at 3100 and 4000 psi and at 112-165 degrees C for 10-60 min. Each pretreated lignocellulose was hydrolyzed with commercial cellulase to assess its enzymatic digestibility. Untreated aspen and southern yellow pine (SYP) gave final reducing sugar yields of 14.5 +/- 2.3 and 12.8 +/- 2.7% of theoretical maximum, respectively. When no moisture was present in lignocellulose to be pretreated, the final reducing sugar yield from hydrolysis of SC-CO2-pretreated lignocellulose was similar to that of untreated aspen. When the moisture content of lignocellulose was increased, particularly in aspen, significantly increased final sugar yields were obtained from enzymatic hydrolysis of SC-CO2-pretreated lignocellulose. When the moisture content of lignocellulose was 73% (w/w) before pretreatment, the sugar yields from the enzymatic hydrolysis of aspen and southern yellow pine pretreated with SC-CO2 at 3100 psi and 165 degrees C for 30 min were 84.7 +/- 2.6 and 27.3 +/- 3.8% of theoretical maximum, respectively. The SC-CO2 pretreatments of both aspen and SYP with moisture contents of 40, 57, and 73% (w/w) showed significantly higher final sugar yields compared to the thermal pretreatments without SC-CO2.     

Production of diosgenin from yellow ginger (<Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> C. H. Wright) saponins by commercial cellulase

A commercial cellulase was first assessed to be effective in hydrolyzing glycosyl at the C-3 and C-26 positions in steroidal saponins from yellow ginger (Dioscorea zingiberensis C. H. Wright) to diosgenin, a very important chemical in the pharmaceutical industry. The effect of different parameters on enzyme hydrolysis was further investigated by systematically varying them. The highest yield was achieved when the hydrolysis ran at 55°C and pH 5.0 with an enzyme to substrate ratio of 15 × 10³ U/g. The biotransformed products identified using TLC and HPLC confirmed that the cellulase was capable of releasing diosgenin from steroidal saponins. Moreover, the biotransformation process was explored by LC-MS and LC-MS/MS analysis. Enzymatic hydrolysis together with 40 % of the original sulphuric acid used increased the diosgenin yield by 15.4 ± 2.7% than traditional method. Therefore, the commercial cellulase may serve as a promising tool for industrial diosgenin production and for further use in saponin modification.     

Cellulases as an aid to enhance saccharification of cassava root

Summary The conventional saccharification of cassava root by enzymatic hydrolysis is improved by using a little amount of cellulase and cellobiase in addition to conventional enzyme, glucoamylase. With new saccharification[glucoamylase 0.45SGU/g cassava, cellulase 4.5NCU/g cassava, cellobiase 0.09U/g cassava, pH 4.3, temperature 60°C, total volume 465ml : 100g of cassava/400ml of water], the reaction time was reduced by about 5 hours, the concentration of reducing sugar was increased by 40%, glucose production was enhanced by 10% , and the viscosity was reduced by 30%.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

超临界CO2流体对纤维素酶催化反应的影响

超临界二氧化碳流体预处理对纤维素超分子结构及纤维素酶催化反应有重要影响。一定含水量的微晶纤维素用SC-CO2在10MPa,50℃处理30min,其结构发生了有利于进一步被酶解的变化。上述超临界条件单独作用于纤维素酶时,并未造成酶催化活力的降低;但与纤维素共同进行SC—CO2处理时,纤维素酶则失去催化活性,但这种处理却能提高纤维素进一步被酶解的效率。一定范围内处理时的酶用量与酶解效率的增加正相关。纤维素的含水量对SC-CO2处理后的酶解效率有显影响。