Research on the combination of esterases and hydrolases from fungi to convert agro - Industrial by - products into bioethanol

eening of Feruloyl esterase activity The feruloyl esterase synthesis of the 44 fungal species selected was evaluated by the ethyl ester (ethyl 4-hydroxy-3-methoxycinnamate) cleavage on agar plates. Alt.tenuissima SP66 for high feruloyl esterase activity should be selected for subsequent studies to determine culture conditions such as cultivation-time, substrate/carbon source, nitrogen source, temperature and pH. 3.2.2. Screening of acetyl esterase activity After fermentation of 44 fungal species, centrifugation used to remove biomass and other components and then determine acetyl esterase activity (AE) by ability of p-nitrophenyl acetate hydrolysis. X.polymorpha A35 should be selected for further studies due to high acetyl esterase activity. After screening activity of feruloyl esterase and acetyl esterase, two highly active fungal species Alt.tenuissima SP66 and X.polymorpha A35 were selected. Then studies on optimum fermentation conditions for enzyme synthesis, purification and characterization of AE and FAE as well as the use of enzymes for lignocellulose catalytic conversion will be carried out. 6 3.3. Identification of fungi by molecular biology method Figure 3.1. (A) Total DNA electrophoresis on 1% agarose gel and (B) PCR product of SP66 and A35 From the classification results by molecular biology method and morphological analysis method, it can be concluded that the fungal isolate SP66 is the Alternaria tenuissima SP66 in the family Pleosporaceae and the A35 fungal strain is Xylaria Polymorpha A35 in the family Xylariaceae. 3.4. Kinetics of biosynthesis of esterase Optimization of the acetyl esterase and feruloyl esterase biosynthesis of the two strains of X. polymorpha A35 and Alt.tenuissima Sp66 as follows: Acetyl esterase enzyme activity: X.polymorpha A35 cultured on a basic medium supplemented with straw substrate, nitrogen source is pepton, culture time is 10 days, temperature is 25ºC, pH 7, stirring rate 200 rpm, then the highest acetyl esterase activity was 135.4 U/l. Feruloyl esterase enzyme activity: Alt.tenuissima was cultured on a basic medium supplemented with straw substitute, culture time is 12 days, temperature is 25ºC, pH 7, stirring rate 200 rpm, then the highest acetyl esterase activity was 1154.4 U/l. 3.5. Purification of enzyme from culture medium 3.5.1. Purification and properties of esterase from X. polymorpha A35 (XpoAE) Crude enzyme extraction from culture medium after 3 weeks was concentrated by ultrafiltration (10 kDa cut-off filter). Then, the enzyme protein which expresses the XpoAE activity for the p- nitrophenyl acetate substrate is purified by liquid chromatography. The first step of the protein elution was carried out via an anaerobic DEAE Sepharose anion exchange chromatograph and the results were obtained by three active fractions of XpoAE (Sections I, II and III). The eluent volume of the highest enzyme activity fraction (III) was added to the SuperdexTM 75 column. After the gel filtration chromatography step, XpoAE enzyme activity fraction was collected (Figure 3.2). After purification, the amount of purified enzyme protein was 20.6 mg, equivalent to 27 U and 26.8% efficiency with purity of 18.3 times. This purified enzyme fraction is used for further studies of enzymatic protein properties as well as the in vitro transformation of lignocellulose-rich material (Table 3.1). SP66 A35 M SP66 A35 A B 7 Figure 3.2. Purified X.polymorpha A35 acetyl esterase via liquid chromatography steps (A) DEAE Sepharose anion exchange chromatography and (B) SuperdexTM 75 gel filtration chromatography; (─) absorption at λ = 280 nm and (●) XpoAE activity on p-nitrophenyl acetate Table 3.1. Purification of the acetyl esterase activity Purification steps Total protein (mg) Total activity (U) Specific activity (U mg-1) Yield (%) Purification fold Crude extracts 1408,0 1009 0.7 100 1,1 30-10 kDa filter 872,1 944 1.1 93,6 1,5 DEAE Sepharose (Faction III) 86,0 432 5.0 42,8 7 SuperdexTM 75 20,6 270,0 13.1 26,8 18,3 Physical-chemical properties of AltFAE: The SDS-PAGE of faction III via the purification steps above shows a protein band corresponding to XpoAE with MW = 44 kDa after staining with a colloidal blue Staining Kit. IEF electrodes showed two adjacent protein bands with pI values of 3.5 and 3.6 respectively (Figure 3.3). The physical and chemical characteristics of XpoAE correspond to the characteristics of published AE (34-56 kDa). 0 50 100 150 Elutionsvolumen (ml) 0 450 900 1350 1800 2250 2700 A bs or pt io n (2 80 n m ) 0 1 2 3 4 5 A kt iv itä t ( U m l-1 ) 0 0.2 0.4 0.6 N aC l ( M ) I II A Xp oA E a ct iv ity (U ) m l-1 ) Volume of elution (ml) 0 50 100 150 Elutionsvolumen (ml) 0 700 1400 2100 2800 A bs or pt io n (2 80 n m ) 0 0.4 0.8 1.2 1.6 Ak tiv itä t ( U m l-1 ) B A ct iv ity (U m l-1 ) Elution volume (ml) V lume of lution (ml) Xp oA E a ct iv ity (U ) m l-1 ) A bs or ba nc e (2 80 n m ) 8 Figure 3.3. Purified protein (1,3) expresses acetyl esterase activity (XpoAE) on SDS-PAGE (A) and IEF (B) gel cartilage; (2,4) protein maker Optimal temperature and pH: To determine the optimal reaction temperature, the reaction between the purified XpoAE enzyme and the p-nitrophenyl acetate substrate is carried out at a temperature of 35-70°C. Results showed that the XpoAE activity increased from 40% at 35°C to the maximum at 42°C (100%). Then, as the temperature increased, the activity of the enzyme decreased to 51% at 61 ° C (Fig. 3.4-A). The pH value is in the range of 5.0-5.5. Relative activity decreases from 100% at pH 6.5 to 57% at pH 7.0 (Figure 3.4-B) Figure 3.4. Effects of temperature (B) and pH (A) on the activity of XpoAE from X.polymorpha A35 Thermal stability and pH of enzyme XpoAE: Enzymes are relatively stable at 400C after 3 hours of incubation. After that, the activity decreased by more than 50% when incubated for 4 hours and longer. Enzyme activity decreased rapidly at 600C and lost most of the activity after 1 hour of incubation at this temperature. Purified enzymes show active stability at pH 5.0 but lost over 90% of the activity during 1 hour incubation under strong acid conditions (pH 3; Figure 3.5). Purified XpoAE enzyme from X.polymorpha A35 is relatively stable at a temperature of 40-42°C at pH 5. 0 20 40 60 80 100 120 4 4,5 5 5,5 6 6,5 7 R el at iv e ac tiv ity (% ) Value pH A 0 20 40 60 80 100 120 35 40 42 45 50 60 70 R el at iv e a ct iv ity (% ) Temperature(0C)B 9 Figure 3.5. The purity of acetyl esterase from X.polymorpha A35 (XpoAE) at different temperature conditions (A) and pH (B): (A): ♦ 40 oC, ▲60 oC; (B): ▲ pH 3, ● pH 5, ♦ pH 6 3.5.2. Purification and properties of feruloyl esterase from Alt.tenuissima SP66 (AltFAE) Crude enzyme from the culture medium after 2 weeks was cut-off 5 kDa, through which small molecular weight proteins and impurities were also removed. Then, the enzyme protein expresses the feruloyl esterase activity for methyl ferulate substrate by purification by liquid chromatography. The first step in the protein elution process is carried out via the DEAE Sepharose anion exchange chromatograph. After the first step of purification, the purity increased slightly (from 1.4 times) but most of the pigments (possibly polyphenolic compounds) were removed from the target FAE active protein. The total elution volume of the highest enzyme activity fraction was added to the SuperdexTM 75 column. The highest purification efficiency at the gel filtration step, expressed in purity, increased from ~ 12 to ~ 30 times with performance ~ 44%). Meanwhile, using strong anionic chromatography (Q Sepharose; Figure 3.6) in the next step the purity increased to 35.8 times, but the amount of protein and total activity corresponding to the purified efficiency decreased to nearly ½ (last performance was 28.9%; Thus, the final purification step is required for basic research (requiring maximum purity), in the AltFAE enzyme study of Alt.tenuissima SP66 can be used immediately after gel filter chromatography to ensure high recovery efficiency and save time and costs. Figure 3.6. Purification of feruloyl esterase enzyme from Alt.tenuissima SP66 (AltFAE) via Q Sepharose® anion exchange chromatography (●) AltFAE activity for methyl ferulate substrate, (─) absorption protein at λ = 280 nm A ltF A E ac tiv ity (U /m l) R el at iv e ac tiv ity (% ) Time (hours) Time (hours) 10 Table 3.2. Purification of feruloyl esterase from Alt.tenuissima SP66 (AltFAE) Purification steps Total protein (mg) Total activity (U) Specific activity (U mg-1) Yield ( %) Purification fold Crude extracts 1673,2 523,7 0,3 100 1,1 10 kDa filter 259,5 421,7 1,6 80,5 5,2 DEAE Sepharose 79,1 305,4 3,9 58,3 12,3 SEC SuperdexTM 75 24,1 228,3 9,5 43,6 30,3 Q Sepharose® 13,5 151,2 11,2 28,9 35,8 Physical and chemical properties of the AltFAE: The SDS-PAGE electrode after the final purification step through the Q Sepharose® column shows a protein band that expresses FAE activity (methyl ferulate) after staining with Colloidal Blue Staining Kit MW = 30.3 kDa (Figure 3.7). Figure 3.7. SDS-PAGE of the AltFAE under denaturing conditions Lane 1 - AltFAE after Q Sepharose elution; lane 2 - protein markers Optimal pH and temperature: The temperature and pH of the AltFAE are tested from 30-80°C and pH 4.0-9.0. Relative activity decreases from 100% at pH 7.0 to 84% at pH 9.0 (Fig. 3.8-A). The enzyme was purified for 2 hours in culture, the remaining 97% at pH 6.0 and 89% at pH 8.0. Conversely, the reaction at pH 4.0 resulted in a loss of 57% activity after 2 hours. The temperature of 60°C causes the operation to lose more than 50% within 2 hours, when the temperature rises to 70°C, the enzyme activity decreases to only 30%. Thus, the optimum temperature and pH with the corresponding purified enzyme are 42-45°C, pH 6-6.5. 11 Figure 3.8. Effect of temperature (A) and pH (B) on the AltFAE Residual activities were determined at temperature intervals of 20–80°C and pH values of 4.0–9.0. All experiments were performed in triplicates, standard deviation (SD) < 5%. Thermal stability and pH of enzyme AltFAE: Enzymes are relatively stable at 25°C and 40°C after 2 hours of incubation, followed by a reduction of more than 10% in 4 hours at 25°C and 35% by incubation in 2 hours at 40ºC. Enzyme activity decreases rapidly at 60ºC and loses most of its activity after 1 hour of incubation at this temperature. The enzyme was stable at neutral (pH 5) but lost more than 75% of its activity during the 2 hours incubation under strong acid conditions (Figure 3.9). Figure 3.9. Stability of the AltFAE at different temperatures (A) and pH-values (B) in dependence of incubation time (A): 25°C (diamond), 40°C (circle), 60°C (triangle) and (B): pH 10 (diamond), PH 8.0 (circle), pH 7.0 (star), pH 5.0 (triangle) Peptide analysis results of AltFAE: The peptide sequence of the protein expresses FAE activity from Alt. tenuissima SP66 (AltFAE) was successfully determined by ESI-MS mass spectra (Figure 3.10 & Table 3.3) as the basis for initial classification and could use data for primer determination of the coding gene for this protein. R el at iv e ac tiv ity (% ) Time (hours) Time (hours) 12 Figure 3.10. ESI-MS/MS spectrum of peptide fragments of purified enzyme protein from Alt.tenuissima SP66 (AltFAE) Table 3.3. Peptides of purified enzyme protein from Alt.tenuissima SP66 (AltFAE) determined by hydrolysis with trypsin and LC-ESI-MS/MS Peptide* Molecular weight (Da) GAYSLSLR GFFLFVEGGR GSSIFGLAPGK GKVALDDLLTQR LNTLETEEWFFK LNTLETEEWFFK 865,99 1128,3 1033,19 1327,75 1556,75 1556,75 * Symbols for amino acids in accordance with IUPAC (International Union of Pure and Applied Chemistry) 3.5.3. Fermentation, extraction and purification of esterase from fungi + Acetyl esterase from Xylaria polymorpha A35 (XpoAE): The procedure of fermentation for biosynthesis and purification of esterase consists of the following main steps: Cultivation of fungi and enzyme biosynthesis on the medium (5 L/batch): Xylaria polymorpha A35 strain is fermented 5 liters/batch on a basic medium (for 1 liter) with the following composition: MgSO4: 0.5 g; KH2 PO4: 1.5 g; High yeast: 2.0 g; Micro-trace elements (trace): 1 mL. Base medium supplemented with straw substrate, nitrogen source is pepton, temperature 25ºC, pH 7 under culture conditions of 200 rpm for 10 days. Simultaneously with liquid fermentation, the fungus was fermented using 2-3 kg of dry straw, soaked in water overnight and then placed in heat- resistant plastic bags and sterilized at 121ºC for 30 minutes. Use 2-3 petri of peptic algae-malt agar with homogenized mycelium. Next, transfer the whole broth to each plastic bag with a straw substrate, and the entire culture is carried out under sterile conditions. The mycelium was incubated at 23ºC for 10-14 days, then the surface fermentation medium was extracted with distilled water (d.H2O) overnight on a shaker. 13 Extraction of crude enzyme by ultrafiltration: After culture under appropriate conditions on solid and liquid medium, the enzyme will be pre-filtered through crude and filter paper (GF6 and RC 55, WhatmanTM, China). Activated esterase enzyme is centrifuged at 5.000-10.000 rpm and 10 and 30 kDa cut-off ultrafiltration (LongerPump K235 with UFP 30MW membrane, Amersham BioScience, Westborough MA, USA, or Vivaflow200, 10MW Polyethersulfon membrane, Sartorius, Goettingen, Germany, or amicon Ultra Centrifugal Filters, 5MW, Millipore, Bedford, USA) at 11ºC. Purification of enzyme by chromatography: To obtain pure acetyl esterase from X. polymorpha A35, the crude enzyme expressing the esterase activity was purified by ion exchange chromatography and gel filtration chromatography using DEAE Sepharose and SuperdexTM columns. 75 (GE Healthcare, Freiburg, Germany). The first step was performed on the DEAE Sepharose ion exchange column at pH 4.5 with 50 mM Na-acetate Buffer. After filtration, the active fractions can be obtained with 86.0 mg of active protein AE (43.2 U). The next step is to use gel filtration chromatography through SuperdexTM 75 at pH 6.5 with 50 mM Na-acetate buffer and 100 mM NaCl acetate, active fraction acetyl esterase (for p- nitrophenyl acetate substrate). The enzyme activity fractions were mixed, concentrated and dialyzed through 10 kDa filter as above with 10 mM Na-acetate buffer and stored at -20°C. After purification, 20.6 mg of enzyme/batch was obtained, and the total enzyme activity was 270 U. Protein after each step of ultrafiltration and chromatography was measured for activity, purity and molecular weight determination by SDS-PAGE (Novex Xcell SureLock mini cell; Laemmli 1970) and Native-IEF (Novex IEF gel ) (Figure 3.11). 14 Figure 3.11. Process of extracting and purifying acetyl esterase from culture medium X. polymorpha A35 Fungal culture broth X. polymorpha A35 (5 L liquid and 2-3 kg solid, total protein 1408 mg) Centrifuged fermentation - Vacuum filter (Whatman GF6) - Centrifugal 5000 rpm for 10 minute minutes Extract solid Ultrafiltration 30kDa cut-off (1000 mL) Ultrafiltration 10kDa cut-off (872 mg protein) DEAE Sepharose column SuperdexTM 75 column D et er m in at io n of e nz ym e ac tiv ity ; p ro te in q ua nt ifi ca tio n SD S- PA G E an d IE F Acetyl esterase Purification (20,6 mg protein, 270 U enzyme) 15 + Feruloyl esterase from Alternaria tenuissima SP66 (AltFAE) The biosynthesis and purification process of esterase followed the main steps: Fungal culture and enzyme biosynthesis on the medium (5 L/batch): Alt.tenuissima SP66 was cultured on the following medium, supplemented with straw substrate, culture time of 12 days at 25°C, pH 7 in 1000 ml Erlenmeyer flasks or on AmAr fermentor (Mumbai, India) under aeration and continuous shaking 200 rpm. Extraction of crude enzyme by ultrafiltration: After culture under optimum conditions, the enzyme will be preliminarily filtered through coarse and filter paper (GF6 and RC 55, WhatmanTM, China). The extraction of esterase was excluded mycelium and substrates by centrifuging at 5.000-10.000 rpm and 10 and 30 kDa cut-off (UFP 30MW, Amersham BioScience, or Vivaflow200, Polyethersulfon 10MW, Sartorius or 5MW, Millipore) at 11oC. Purification of the enzyme by chromatography: The crude extraction of esterase was purified by ion exchange chromatography and gel filtration chromatography using DEAE Sepharose, Q sepharose and SuperdexTM 75 columns (GE Healthcare, Freiburg, Germany) with Na-acetate elution buffer (10mM, pH 4.5), NaCl (0-1M) and Superdex 75 column, Na-acetate Buffer (50mM, pH 6.5). Soluble proteins were directly determined using probes at λ = 280nm or by colorimetric method according to Bradforf (1976) at λ = 590/450nm. The Fractions containing the esterase activity were mixed, concentrated to remove water and reconstituted with 10 mM Na-acetate Buffer, pH 6.0. Samples are kept at -20ºC. The final purification step was performed with anion exchange column at pH 5.0 with 50 mM Na-acetate Buffer. From 5L fermentation after purification yield 13.5 mg protein and total activity 151.2 U. Protein after each step of ultrafiltration and chromatography was measured for activity, purity evaluation and molecular weight determination by SDS-PAGE (Novex Xcell SureLock mini cell; Laemmli 1970) (Figure 3.12). 16 Figure 3.12. Process for the extraction and purification of feruloyl esterase enzyme from fermentation media of Alt.tenuissima SP66 3.6. Screening of lignocellulose-rich substances in biotransformation Carrying out a post-transformation degradation of 5 substrates: raw straw (RR), agasse (MIA), coffee grounds (CAF), meal wood (MG), seaweed (RT) (XpoAE, AltFAE and Cell/Xyl) according to different experiments in 24h, at 40ºC on TLC plate chromatography. Fungal culture broth Alt.tenuissima SP66 (5 L; total protein 1673 mg) Centrifuged fermentation - Vacuum filter (Whatman GF6) - Centrifugal 5000 rpm for 10 minute Extract solid Ultrafiltration 10 kDa cut-off (500 mL; protein 259,5 mg) DEAE Sepharose column SuperdexTM 75 column Anion-exchange Q Sepharose D et er m in at io n of e nz ym e ac tiv ity ; p ro te in q ua nt ifi ca tio n SD S- PA G E an d IE F Feruloyl esterase Purification (13,5 mg protein; 151,2 U enzyme) 17 Figure 3.13. Smears appear on thin sheets standard substances are fermentable reducing sugars Glucose (Rf (Glu) = 0.34), galactose (Rf (Gla) = 0.28), xylose (Rf (Xyl) = 0.48) and mannose (Rf (Man) = 0.325) Composition, reducing sugar content after bio-transformation: Use thin-layer chromatography (TLC) to determine smear of monosaccharide (reduction sugar) in the solution after transformation by enzyme mixture (XpoAE, AltFAE and Cell/xyl) on 5 substrates: raw straw, bagasse, coffee grounds, meal wood and seaweed (the thin sheets appear the bold smears of monosaccharide and the highest total reduction sugar obtained 154.7 mg/g). Figure 3.14. The total reducing sugar formed after sugarcane bagasse conversion by single enzymes and "enzyme cocktail" 3.7. Optimization of “enzyme cocktail’’ by experimental planning Applying the experimental planning to optimize the conditions of the “enzyme cocktail” that convert bagasse (10%, w/v) to fermentable sugars is kept at 40ºC, pH 5 in 48 hours. Using a flexible nonlinear scheduling algorithm, the recovery equation describing the efficiency of hydrolysis (expressed as the total reduction sugar) depends on the composition of the transformed enzymes Cell/Xyl (x1), AltFAE (x2), XpoAE (x3): y = 206,946 + 29,954x1 + 5,501x2 + 7,323x3 + 2,288x2x3 – 7,011 21x .. From there, the optimal cocktail (U) activity for the metabolism is determined as follows: Cell/Xyl: 100/160 U/gds; AltFAE: 7.56 U/gds and XpoAE: 10.8 U/gds. 0 20 40 60 80 100 120 140 160 180 AltFAE XpoAE Cell/Xyl XpoAE + Cell/Xyl AltFAE & XpoAE AltFAE & Cell/Xyl Cell/Xyl, AltFAE & XpoAE R ed uc ed s ug ar c on te nt ( m g /g) Enzyme cocktail 18 3.8. Combination of chemical treatment to improve efficiency 3.8.1. Combined with alkaline bagasse and "enzyme cocktail" Combination of alkaline and "enzyme cocktail" in bagasse treatment: improve efficiency on treatment of sugarcane bagasse before hydrolysis with cocktail under optimum conditions. Bagasse was treated with NaOH at different concentrations. Table 3.4. Total reducing sugar after treating bagasse by alkaline and "enzyme cocktail" (*) Bagasse (10%; w/v) is crushed and incubated with NAOH solution in 2.5 hours at 85ºC. The conversion process use "enzyme cocktail" (100U Cell/Xyl/gds, 7.56U AltFAE/gds, 10.8U XpoAE/gds), incubated in 48 hours, at 40ºC, pH 5.0. The data presented is the average of two repetitions. 3.8.2. The combination of acid and "enzyme cocktail" hydrolysis of bagasse Table 3.5. Total reducing sugar after treating bagasse by acid and "enzyme cocktail" No. H2SO4 (C%) Total reducing sugar content (mg/g) Treatment by acid Treatment by acid and “enzyme cocktail” (*) 1 0 0 198,2 2 0,05 42,51 253,1 3 0,1 74,23 319,5 4 0,15 81,58 304,8 5 0,2 97,32 294,9 6 0,3 90,15 269,2 7 0,4 74,81 227,2 8 0,5 70,97 193,7 9 0,6 68,07 156,8 (*) Bagasse (10%; w/v) is crushed and incubated with H2SO4 solution in 2.5 hours, at 85ºC. The conversion process use "enzyme cocktail" (100U Cell/Xyl/gds, 7.56U AltFAE/gds, 10.8U XpoAE/gds), incubated in 48 hours, at 40ºC, pH 5.0. The data presented is the average of two repetitions. No. NaOH (M) Total reducing sugar content (mg/g) Treatment by alkaline Treatment by alkaline and “enzyme cocktail” (*) 1 0 0 198,2 2 0,1 34,8 231,5 3 0,15 42,8 265,8 4 0,2 49,6 271,5 5 0,25 64,8 277,5 6 0,3 78,6 287,4 7 0,35 86,2 265,9 8 0,4 91,4 236,3 9 0,5 74,8 187,3 19 3.8.3. The combination of bagasse treatment by heating equipment and "enzyme cocktail" Table 3.6. The total reducing sugar content after treatment with heating equipment and enzyme Total reducing sugar content (mg/g) Heating equipment Heating equipment + “enzyme cocktail” (*) 32,18 221,73 (*) Bagasse (10%; w/v) is finely crushed and soaked in distilled water, treated with a heating device, at 121ºC in 30 minutes, 1 atm. The conversion process of bagasse using “enzyme cocktail” (100U Cell/Xyl/gds, 7.56U AltFAE/gds, 10.8U XpoAE/gds), the reaction was incubated in 48 hours, at 40ºC, pH 5.0. The data presented were average of two repetitions. 3.8.4. The content of monosaccharide in solution after conversion Table 3.7. Carbohydrate content after biological conversion Enzyme Products of reaction (Carbohydrate; mg/g) Galactose Glucose Mannose Xylose Total carbohydrate Cell/Xyl, AltFAE & XpoAE Unknown 195,4 Unknown 60,5 255,9 In the treatment with alkaline, acid, heat combined with hydrolysis of "enzyme cocktail" mixture (Cell/Xyl, AltFAE & XpoAE). Determining the method of treating bagasse with dilute H2SO4 (0.1%) at 85ºC, in 2.5 hours incubation combined with hydrolysis with "enzyme cocktail" for the best effect with total reducing sugar reaching 319.5 mg/g substrate. Results of concentration analysis of monosaccharide in post-conversion solution by high- performance liquid chromatography (HPLC) system determined that concentration of glucose and xylose in pretreatment of bagasse with dilute H2SO4 (0.1%) combined with enzyme cocktail (Cell/Xyl, AltFAE & XpoAE) reached 195.4 mg, respectively and 60.4 mg/g and the contents of other fermentation sugars are 18%. 3.9. Study of bioethanol production 3.9.1. Fermentation media Nutritional sources are one of the factors that directly affect the growth and development of microorganisms in general and Saccharomyces cerevisiae SH1 in particular. Conduct surveys and comparisons to find the optimal medium for fermentation. The experiment was conducted on two medium BM and BM+ medium. The yeast supplement was 3% on the total volume of fermentation, pH 4.8, temperature 30ºC. Table 3.8. Effect of nutritional ingredients (BM and BM+) Time (hour) Results 12 hours