Saccharification Of Pretreated Sugarcane Bagasse Research Articles

Enhanced reducing sugar production by blending hydrolytic enzymes from Aspergillus fumigatus to improve sugarcane bagasse hydrolysis.

Biomass pretreatment for the production of second-generation (2G) ethanol and biochemical products is a challenging process. The present study investigated the synergistic efficiency of purified carboxymethyl cellulase (CMCase), β-glucosidase, and xylanase from Aspergillus fumigatus JCM 10253 in the hydrolysis of alkaline-pretreated sugarcane bagasse (SCB). The saccharification of pretreated SCB was optimised using a combination of CMCase and β-glucosidase (C + β; 1:1) and addition of xylanase (C + β + xyl; 1:1:1). Independent and dependent variables influencing enzymatic hydrolysis were investigated using response surface methodology (RSM). Hydrolysis using purified CMCase and β-glucosidase achieved yields of 18.72mg/mL glucose and 6.98mg/mL xylose. Incorporation of xylanase in saccharification increased the titres of glucose (22.83mg/mL) and xylose (9.54mg/mL). Furthermore, characterisation of SCB biomass by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy respectively confirmed efficient structural disintegration and revealed the degree of crystallinity and spectral characteristics. Therefore, depolymerisation of lignin to produce high-value chemicals is essential for sustainable and competitive biorefinery development.

Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60–80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

Statistical optimization of saccharificaion of carbohydrate content of alkali pretreated sugarcane bagasse by enzyme cocktail produced by Bacillus vallismortis MH 1 and Bacillus aestuarii UE25

Saccharification of widely available agro-industrial waste product, sugarcane bagasse (SB) can be achieved by microbial enzymes. Since single enzyme cannot completely saccharify SB, therefore, enzyme cocktails are designed for effective saccharification. In this study, alkali (NaOH) pretreated SB was saccharified using enzyme preparations from a halotolerant bacterium, Bacillus vallismortis MH 1 and a thermophilic bacterium, B. aestuarii UE25. The factors affecting the process of saccharification were statistically optimized by Central Composite design (CCD). After analysis, CCD predicted the optimum conditions i.e. 8.08 U of B. vallismortis MH 1 and 1.92 U of B. aestuarii UE25 for 1 g of substrate; temperature, 65℃; pH, 4; and reaction time, 48 h. Under optimal conditions, 501 mg g−1 reducing sugars were obtained after the saccharification of pretreated SB. The structural analysis of substrate by scanning electron microscopy, Fourier transform infrared spectroscopy and gravimetric analysis showed damaged structure, increased porosity and a reduction in cellulosic content, indicating significant degradation of the SB by the enzyme cocktail. Hence, it can be concluded that enzyme cocktail used in this study is effective for the saccharification of alkali pretreated SB.

Sugarcane Bagasse Saccharification by Enzymatic Hydrolysis Using Endocellulase and β-glucosidase Immobilized on Different Supports

The saccharification of sugarcane bagasse by enzymatic hydrolysis is one of the most promising processes for obtaining fermentable sugar to be used in the production of second-generation ethanol. The objective of this work was to study the immobilization and stabilization of two commercial enzymes: Endocellulase (E-CELBA) in dextran coated iron oxide magnetic nanoparticles activated with aldehyde groups (DIOMNP) and β-glucosidase (E-BGOSPC) in glyoxyl agarose (GLA) so that their immobilized derivatives could be applied in the saccharification of pretreated sugarcane bagasse. This was the first time that the pretreated sugarcane bagasse was saccharified by cascade reaction using a endocellulase immobilized on dextran coated Fe2O3 with aldehyde groups combined with a β-glucosidase immobilized on glyoxyl agarose. Both enzymes were successfully immobilized (more than 60% after reduction with sodium borohydride) and presented higher thermal stability than free enzymes at 60, 70, and 80 °C. The enzymatic hydrolysis of the sugarcane bagasse was carried out with 15 U of each enzyme per gram of bagasse in a solid-liquid ratio of 1:20 for 48 h at 50 °C. Under these conditions, 39.06 ± 1.18% of the cellulose present in the pretreated bagasse was hydrolyzed, producing 14.11 ± 0.47 g/L of reducing sugars (94.54% glucose). In addition, DIOMNP endo-cellulase derivative maintained 61.40 ± 1.17% of its enzymatic activity after seven reuse cycles, and GLA β-glucosidase derivative maintained up to 58.20 ± 1.55% of its enzymatic activity after nine reuse cycles.

EFFECTS OF THE INCREASE IN SUBSTRATE LOAD AND HYDROLYSIS TIME IN THE SACCHARIFICATION OF SUGARCANE BAGASSE AND ETHANOL PRODUCTION

In this study, the enzymatic saccharification of alkaline pretreated sugarcane bagasse (PSB) and the production of second generation bioethanol were investigated under different conditions. The effects of percentage of dry substrate (3 and 10% w/v) and time of hydrolysis (24 and 72 h) were evaluated in order to obtain maximum contents of total reducing sugar (TRS), glucose and xylose. The monosaccharide concentrations were quantified by High Performance Liquid Chromatography (HPLC) and TRS by the 3.5-dinitrosalicylic acid (DNS) method. The saccharification of PSB with the Cellic Ctec3, promoted an efficient release of sugars. Among the four evaluated conditions, the hydrolysis HD (10% PSB and 72h) was selected as the most efficient with the highest concentrations of released sugars: TRS (73.96 g/L), glucose (31.78 g/L) and xylose (10.85 g/L). The alcoholic fermentation of hydrolysate HD with the yeast Saccharomyces cerevisiae produced the maximum concentration of 16.15 g/L of ethanol, with a volumetric productivity (QP) of 0.69 g/Lh and yield of ethanol as a function of the substrate (YP/S) of 0.48 gethanol/gsugar. S. cerevisiae was capable of fermenting 99.96% of glucose from hydrolysate HD. These results suggest that the increase of the substrate load and time in the enzymatic hydrolysis can be favourable alternatives to improve the sugar yields in hydrolysates and, consequently, the bioethanol production.

Saccharification of pretreated sugarcane bagasse using enzymes solution from Pycnoporus sanguineus MCA 16 and cellulosic ethanol production

Pycnoporus sanguineus MCA 16 was cultivated by solid state fermentation on lignocellulosic residues, at 40 °C. The highest endoglucanase, exoglucanase, β-glucosidase, xylanase, β-xylosidase and laccase activities (200, 29.1, 52.1, 44.2, 1.81 and 22.04 U g−1, respectively) were obtained when using the mixture of wheat bran and soybean meal (1:1 w/w) as substrates, at 96 h of cultivation. Peroxidases highest activities were observed at 48 h (14.61 and 10.02 U g−1 for manganese and lignin peroxidase, respectively). Enzymatic saccharification of sugarcane bagasse submitted to hydrothermal alkaline pretreatment provided glucose at 7.32 g L−1 when using solid load of 13.5% (dry basis), 288.3 total units of endoglucanase/g of cellulose, 130 h and 57 °C. Ligninases present in the enzymatic solution provided a reduction of 82.3% in the concentration of total phenolic compounds during saccharification. The hydrolysate was used for ethanol production by Saccharomyces cerevisiae CAT-1 and a yield of 73.5% was obtained.

Cellulases and xylanases production by endophytic fungi by solid state fermentation using lignocellulosic substrates and enzymatic saccharification of pretreated sugarcane bagasse

Endophytic fungi are widely studied as producers of secondary metabolites of biotechnological interest. In recent years, the interest in these fungi as new sources of enzymes, especially hydrolytic, has increased. In the present study, 14 strains of endophytic fungi not yet explored as enzymes sources were randomly chosen and prospected for cellulases and xylanases production by solid-state fermentation. Initially, fungi were cultivated in a mixture (1:1 w/w) of sugarcane bagasse and wheat bran for 7 days, at 28 °C. In this initial screening, 4 fungi excelled in endoglucanase activity (U/g): Cladosporium cladosporioides PAJ 03 (88.51 ± 1.0), Phomopsis stipata SC 04 (83.44 ± 7.7), Trichoderma viridae PAJ 01 (64.56 ± 4.0) and Botryosphaeria sp. AM 01 (42.79 ± 1.6). On the other hand, the following 4 fungi stood out in relation to β-glucosidase activity (U/g): Saccharicola sp. EJC 04 (51.56 ± 2.7), Paecilomyces sp. SF 021 (33.19 ± 9.2), Ustilaginoidea sp. CV 04 (29.75 ± 0.8) and Ustilaginoidea sp. XYA 04 (21.72 ± 3.05). Among these fungi, P. stipata SC 04 and Botryosphaeria sp. AM 01 were the best producers of xylanase and β-xilosidase (694,33 and 4,87 U/g, respectively). These 8 fungi were then cultured in new mixtures (1:1 w/w) of lignocellulosic substrates. Botryosphaeria sp. AM01and Saccharicola sp. EJC04 stood out regarding endoglucanase and β-glucosidase activities (184.74 ± 6.0 and 92.28 ± 9.57 U/g, respectively) when cultivated on cotton seed meal and wheat bran and were selected to continue the study. The influence of time cultivation, inoculum amount and substrate initial moisture content was evaluated and the best condition for cellulases production was 192 h, six mycelial plugs and 65%, respectively, for both fungi. Cellulases and xylanases produced under these conditions were characterized and optimum pH and temperature values were between 4.5–6 and 60–75 °C, respectively. The enzymes were stable over a wide pH range and under 30–70 °C. β-glucosidase from both isolates retained about 75–80% of their activity in the presence of glucose at 6 mM. The presence of ethanol stimulated β-glucosidase activity from Botryosphaeria sp. AM01 (about 60% higher in the presence of ethanol at 15%). On the other hand, the activity of β-glucosidase produced by Saccharicola sp. EJC 04 was reduced at ethanol concentrations above 15%. A blend of the enzymatic extracts was used to saccharify pretreated sugarcane bagasse and a face-centered central composite design was used to find the best conditions. Under the predicted optimum condition (50 °C, 5% of sugarcane bagasse, 150 U g−1 of endoglucanase and 20 h), glucose and xylose concentrations obtained were 3.56 and 1.66 mg mL−1, respectively. These results show that the 14 endophytic fungi studied have potential to be explored as producers of plant material degrading enzymes. Botryosphaeria sp. AM01 and Saccharicola sp. EJC 04 are promising in relation to the production of cellulases and xylanases and their enzymatic extracts have potential for application in pre-treated sugarcane bagasse saccharification processes.

Sugarcane bagasse saccharification by purified β-glucosidases from Chrysoporthe cubensis

A set of β-glucosidases from the phytopathogenic fungus Chrysoporthe cubensis was partially purified, characterized and evaluated for its potential application in the process of sugarcane bagasse saccharification. C. cubensis was grown in solid state fermentation (SSF) containing wheat bran as carbon source. The group of β-glucosidases, containing at least three β-glucosidases, was purified through ion exchange and gel filtration chromatography. The set of β-glucosidases exhibited maximum activity at pH 4.0 and 60 °C and showed high thermal stability at 50, 60 and 70 °C. The β-glucosidases were specific for hydrolysis of glucose residues in the β position. The km values of 0.132 and 0.7816 mM were obtained using ρNPβGlc and cellobiose as substrates, respectively. The β-glucosidases were competitively inhibited by glucose, with Ki value of 8.43 mM. Saccharification of pretreated sugarcane bagasse by a commercial enzyme cocktail supplemented with the β-glucosidases promoted efficient release of sugars. Saccharification performed by the Chrysoporthe cubensis extract supplemented with β-glucosidases promoted a higher xylose yield. The results suggest that β-glucosidases from Chrysoporthe cubensis have great potential to be used as a supplement of enzymatic preparations.

Addition of metal ions to a (hemi)cellulolytic enzymatic cocktail produced in-house improves its activity, thermostability, and efficiency in the saccharification of pretreated sugarcane bagasse

High activity and stability are essential for (hemi)cellulolytic enzymes used in biomass conversion, while non-productive binding of cellulases to lignin reduces saccharification efficiency and needs to be avoided. One potential strategy is the addition of inexpensive metal ions. This paper describes the influence of divalent metal ions on the activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes produced in-house by Aspergillus niger under solid-state fermentation (SSF). The use of Mn(2+) provided the best (hemi)cellulolytic activity and stability, with an increase in endoglucanase activity of up to 57%. The use of Mn(2+) was then investigated in the saccharification of sugarcane bagasse submitted to acid, steam-explosion, and hydrothermal pretreatments. The addition of Mn(2+) ions at 10mM in the saccharification of acid-pretreated bagasse resulted in a 34% increase in glucose release. These positive effects appeared to be due to a reduction in non-productive enzyme adsorption. The findings suggest that the addition of inexpensive metal ions can help to improve activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes.

Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-l-arabinofuranosidase

Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-l-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%.