Influence of solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive tree biomass

Olive tree pruning biomass, pretreated by either liquid hot water or steam explosion under selected conditions, was used as a substrate for enzymatic hydrolysis. The pretreated material was further submitted to alkaline delignification, the objective being to improve hydrolysis yields as well as increasing cellulose content in the pretreated feedstock. The enzymatic hydrolysis of pretreated residues was performed using a commercial cellulase mixture supplemented with β-glucosidase, using a solid loading range from 2 to 30% (w/v). The influence of substrate concentration on the enzymatic hydrolysis yield and on glucose concentration was studied. Comparative results with and without a delignification step are presented. Enzymatic hydrolysis at high substrate concentration (≥20%) is possible, yielding a concentrated glucose solution (>50 g/L). Nevertheless, a cellulose fraction of the pretreated residue remains unaltered.     

Cellulase production from Aspergillus niger MS82: effect of temperature and pH

Fungal cellulases are well-studied enzymes and are used in various industrial processes. Much of the knowledge of enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase system. Species of Trichoderma can produce substantial amounts of endoglucanase and exoglucanase but very low levels of β-glucosidase. This deficiency necessitates screening of fungi for cellulytic potential. A number of indigenously isolated fungi were screened for cellulytic potential. In the present study, the kinetics of cellulase production from an indigenous strain of Aspergillus niger MS82 is reported. Product formation parameters of endoglucanase and β-glucosidase (Q_p + Y_p/s) indicate that A. niger MS82 is capable of producing moderate to high levels of both endoglucanase and β-glucosidase when grown on different carbon containing natural substrates, for example, grass, corncob, bagasse along side purified celluloses. Furthermore, it was observed that the production of endoglucanase reaches its maximum during exponential phase of growth, while β-glucosidase during the Stationary phase. Enzyme production by solid-state fermentation was also investigated and found to be promising. Highest production of cellulase was noted at pH 4.0 at 35 °C under submerged conditions. Growth and enzyme production was affected by variations in temperature and pH.     

High-loading oil palm empty fruit bunch saccharification using cellulases from Trichoderma koningii MF6

Strain Trichoderma koningii D-64 was improved for enhanced cellulase production. A potential mutant MF6 was obtained and its enzymes contained filter paper cellulase (FPase), carboxymethylcellulase (CMCase), β-glucosidase and xylanase with respective activities of 2.0, 1.3, 2.0 and 3.0 folds of those for the parental strain. MF6 cellulases showed enhanced hydrolysis performance for the treated lignocellulosic biomass. Hydrolysis of treated oil palm empty fruit bunch (OPEFB), horticulture wastes (HW) and wood chips (WC) resulted in cellulose to glucose conversion of 96.3 ± 2.2%, 98.2 ± 3.0% and 81.9 ± 1.4%, respectively. The corresponding conversions of xylan to xylose were 96.9 ± 1.5%, 95.0 ± 2.2% and 76.1 ± 3.1%. Consistently, high sugar yield of 770–844 mg/g biomass was obtained for high-loading (10–16%, w/v) of OPEFB hydrolysis and sugar titer of 135.1 g/L was obtained for 16% (w/v) OPEFB loading at 96 h. In addition, MF6 enzymes alone performed equally well for high-loading OPEFB hydrolysis compared to the enzyme mixture of β-glucosidase from Aspergillus niger and cellulase from T. reesei Rut C30.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

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.     

Enzyme adsorption and recycling during hydrolysis of wheat straw lignocellulose

The objective of this research was to investigate cellulase adsorption and recycling during enzymatic hydrolysis of two differently pretreated wheat straws (WS). Dilute acid treated WS showed lower hydrolysis yield of polysaccharides fraction and adsorbed more cellulase with hydrolyzed residue than dilute alkali treated sample. Four methods capable of recovering and recycling the enzyme bound to the residual substrate and the enzyme free in solution were used for three consecutive rounds of hydrolysis to compare their recycling efficiencies. Compared to the absorption recycling method, ultrafiltration recycling method possessed the capacity to retain β-glucosidase, thereby avoiding the supplementation of fresh β-glucosidase in subsequent rounds of hydrolysis. It was found that whatever recycling method was used, better recycling results were obtained for dilute alkali treated substrate than for dilute acid treated substrate. These results suggested that the great difference in the lignin content between acid treated WS and alkali treated WS would significantly affect enzymatic hydrolysis, cellulase adsorption and cellulase recycling efficiencies.     

Enhanced cellulase production through random mutagenesis of Talaromyces pinophilus OPC4-1 and fermentation optimization

Reducing cellulase cost remains a major challenge for lignocellulose to fuel and chemical industries. In this study, mutants of a novel wild-type cellulolytic fungal strain Talaromyces pinophilus OPC4-1 were developed by consecutive UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) and ethylmethane sulfonate (EMS) treatment. A potential mutant EMM was obtained and displayed enhanced cellulase production. Using Solka Floc cellulose as the substrate, through fed-batch fermentation, mutant strain T. pinophilus EMM generated crude enzymes with an FPase activity of 27.0 IU/mL and yield of 900 IU/g substrate. When corncob powder was used, strain EMM produced crude enzymes with an FPase activity of 7.3 IU/mL and yield of 243.3 IU/g substrate. In addition, EMM crude enzymes contained 29.2 and 16.3 IU/mL β-glucosidase on Solka Floc cellulose and corncob power, respectively. The crude enzymes consequently displayed strong biomass hydrolysis performance. For corncob hydrolysis, without supplement of any commercial enzymes, glucose yields of 591.7 and 548.6 mg/g biomass were obtained using enzymes produced from Solka Floc cellulose and corncob powder, respectively. It was 553.9 mg/g biomass using the commercial enzyme mixture of Celluclast 1.5 L and Novozyme 188. Strain T. pinophilus EMM was therefore a potential fungus for on-site enzyme production in biorefinery processes.     

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and β-glucosidase played an important role in mannan hydrolysis. Addition of 10 mg/ml mannan reduced the glucose yield of Avicel (at 20 g/l) from 40.1 to 24.3%. No inhibition of β-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on β-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.     

Enzyme adsorption on SO2 catalyzed steam-pretreated wheat and spruce material

Lignocellulose is widely recognized as a sustainable substrate for biofuels production, and the enzymatic hydrolysis is regarded as a critical step for the development of an effective process for the conversion of cellulose into ethanol. One key factor affecting the overall conversion rate is the adsorption capacity of the cellulase enzymes to the surface of the insoluble substrate. Pretreatment has a strong impact on hydrolysis, which could be related to both chemical changes and morphological changes of the material. In the current work, the accessibility of four differently pretreated wheat straw substrates, two differently pretreated spruce materials, and Avicel cellulose was investigated. Adsorption isotherms (at 4 °C and 30 °C) for a cellulase preparation were obtained, and the rates of hydrolysis were determined for the different materials. Furthermore, the surface area and pore size distribution of the various materials were measured and compared to adsorption and hydrolysis properties, and the structures of the pretreated materials were examined using scanning electron microscopy (SEM).The results demonstrated a positive correlation between enzyme adsorption and the substrate specific surface area within each feedstock. Overall, the amount of enzyme adsorbed was higher for pretreated spruce than for the pretreated wheat straw, but this was not accompanied by a higher initial rate of hydrolysis for spruce. Also, the difference in the measured endoglucanase adsorption and overall FPU adsorption suggests that a larger fraction of the enzyme adsorbed on spruce was unproductive binding. The SEM analysis of the material illustrated the structural effects of pretreatment harshness on the materials, and suggested that increased porosity explains the higher rate of hydrolysis of more severely pretreated biomass.     

Sulfite pretreatment (SPORL) for robust enzymatic saccharification of spruce and red pine

This study established a novel process using sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust and efficient bioconversion of softwoods. The process consists of sulfite treatment of wood chips under acidic conditions followed by mechanical size reduction using disk refining. The results indicated that after the SPORL pretreatment of spruce chips with 8–10% bisulfite and 1.8–3.7% sulfuric acid on oven dry (od) wood at 180 °C for 30 min, more than 90% cellulose conversion of substrate was achieved with enzyme loading of about 14.6 FPU cellulase plus 22.5 CBU β-glucosidase per gram of od substrate after 48 h hydrolysis. Glucose yield from enzymatic hydrolysis of the substrate per 100 g of untreated od spruce wood (glucan content 43%) was about 37 g (excluding the dissolved glucose during pretreatment). Hemicellulose removal was found to be as critical as lignin sulfonation for cellulose conversion in the SPORL process. Pretreatment altered the wood chips, which reduced electric energy consumption for size reduction to about 19 Wh/kg od untreated wood, or about 19 g glucose/Wh electricity. Furthermore, the SPORL produced low amounts of fermentation inhibitors, hydroxymethyl furfural (HMF) and furfural, of about 5 and 1 mg/g of untreated od wood, respectively. In addition, similar results were achieved when the SPORL was applied to red pine. By building on the mature sulfite pulping and disk refining technologies already practiced in the pulp and paper industry, the SPORL has very few technological barriers and risks for commercialization.     

Biochemical characterization of a new β-glucosidase (Cel3E) from Penicillium piceum and its application in boosting lignocelluloses bioconversion and forming disaccharide inducers: New insights into the role of β-glucosidase

Fungal genome sequencing has revealed the presence of multiple putative β-glucosidases; however, information regarding these new β-glucosidases is limited. A new β-glucosidase from Penicillium piceum, designated as PpCel3E, was first isolated and characterized. Using p-nitrophenyl-β-d-glucoside as substrate, PpCel3E showed the lowest Km among the β-glucosidases among all fungi studied. Moreover, PpCel3E exhibited a high transglycosylation activity of 1100 mg gentiobiose/mg and 142 mg sophorose/mg using glucose as the donor. PpCel3E is a novel bifunctional glycoside hydrolase with both β-glucosidase and β-xylosidase activity. Our results show that PpCel3E plays an important role in forming soluble cellulose inducer compounds, as well as in amplifying weak cellulase inducer signal and hemicellulase synthesis via its high transglycosylation activity. Supplementing PpCel3E at low concentrations (40 μg/g substrate) increased the saccharification efficiency of different cellulases by 20% to 27% by removing multiple inhibitors.     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Immobilization of cellulase on polyamidoamine dendrimer-grafted silica

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups, and is suitable to carry biomolecules due to its affinity and biocompatibility. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation. A novel media was developed through immobilizing cellulase onto the prepared PAMAM-grafted silica by adsorption and crosslinking methods and applied in hydrolysis of carboxymethyl cellulose. The results demonstrate that the enzyme binding capacity and enzymolysis efficiency increased with generations of PAMAM. The properties of the immobilized cellulase-PAMAM-grafted silica were investigated, which possessed high enzymatic activity and exhibited better stability with respect to pH, temperature compared with free enzyme. The optimal immobilization conditions for adsorption and crosslinking method were respectively obtained at 5 and 4 mg ml⁻¹ cellulase for 2 h of immobilization. A high enzymolysis efficiency was achieved by employing pH 4.8 and 5.8 substrate solution at 60 °C for adsorbed and crosslinked cellulase, respectively. After repeated three run cycles, the retained activities were found to be 75% and 82%. The results indicate that the PAMAM has a good performance as a carrier, and can be potentially adapted to support other biomacromolecules.     

Cellulase production and oil palm empty fruit bunch saccharification by a new isolate of Trichoderma koningii D-64

A new strain Trichoderma koningii D-64 was isolated from Singapore soil samples. It produced cellulase, xylanase, and laccase on a variety of carbon sources. Enzyme activities of 3.8 ± 0.3, 40.3 ± 5.1, 6.6 ± 0.3 and 98.8 ± 10.3 U/mL were respectively obtained for FPase, CMCase, β-glucosidase and xylanase in a mixture of 1% cellulose and 2% wheat bran. About 70–95% saccharification efficiency of oil palm empty fruit bunch was obtained using T. koningii D-64 enzymes alone without the supplement of any other commercial enzymes. Strain T. koningii D-64 is therefore a potential cellulase producer for the efficient lignocellulosic biomass conversion to fermentable sugars.     

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.     

Three-stage enzymatic hydrolysis of steam-exploded corn stover at high substrate concentration

The feasibility of three-stage hydrolysis of steam-exploded corn stover at high-substrate concentration was investigated. When substrate concentration was 30% and enzyme loading was 15-30 FPU/g cellulose, three-stage (9+9+12 h) hydrolysis could reach a hydrolysis yield of 59.9-81.4% in 30 h. Compared with one-stage hydrolysis for 72 h, an increase of 34-37% in hydrolysis yield could be achieved. When steam-exploded corn stover was used as the substrate for enzyme synthesis and hydrolysis was conducted at a substrate concentration of 25% with an enzyme loading of 20 FPU/g cellulose, a hydrolysis yield of 85.1% was obtained, 19% higher than that the commercial cellulase could reach under the same conditions. The removal of end products was suggested to improve the adsorption of cellulase on the substrate and enhance the productivity of enzymatic hydrolysis.     

Purification and functional characterization of the first stilbene glucoside-specific β-glucosidase isolated from Lactobacillus kimchi

This study aimed to develop viable enzymes for bioconversion of resveratrol-glucoside into resveratrol. Out of 13 bacterial strains tested, Lactobacillus kimchi JB301 could completely convert polydatin into resveratrol. The purified enzyme had an optimum temperature of 30–40 °C and optimum pH of pH 5.0 against polydatin. This enzyme showed high substrate specificities towards different substrates in the following order: isorhaponticin >> polydatin >> mulberroside A > oxyresveratrol-3-O-glucoside. Additionally, it rarely hydrolyzed astringin and desoxyrhaponticin. Based on these catalytic specificities, we suggest this enzyme be named stilbene glucoside-specific β-glucosidase. Furthermore, polydatin extracts from Polygonum cuspidatum were successfully converted to resveratrol with a high yield (of over 99%). Stilbene glucoside-specific β-glucosidase is the first enzyme isolated from lactic acid bacteria capable of bio-converting various stilbene glucosides into stilbene.     

Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts

Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 °C. The regenerated cellulose substrate exhibited a significant improvement about 4.4–6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (CrI) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose I to cellulose II, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the CrI although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl₂ and H₂SO₄ could improve the rate of enzymatic saccharification.     

A response surface methodological study on prediction of glucosylation yields of thiamin using immobilized β-glucosidase

Response surface methodology was used to predict the glucosylation yields of thiamin using immobilized β-glucosidase. A Central Composite Rotatable Design (CCRD) of 32 experiments with immobilized β-glucosidase, thiamin, incubation period, buffer concentration and pH, as five independent variables was employed at five levels. A second-order polynomial equation was developed, the regression coefficient values of which exhibited a R² value of 0.74. Contour plots explained the glucosylation behaviour of the enzyme through a reversal in glucosylation at a cross-over point corresponding to 60% (w/w d-glucose) immobilized β-glucosidase and 0.12 mM buffer concentration at pH 6. The highest conversion yield of 58% obtained experimentally compared well with the predicted yield of 52% under optimum conditions of 40% immobilized β-glucosidase, 0.55 mmol thiamin, 96 h incubation period and 0.16 mM buffer concentration at pH 7. Validation experiments carried out at certain random predictive conditions also showed good correspondence between experimental and predictive yields.