مطالعه کینتیکی هیدرولیز آنزیمی نشاسته توسط قارچ آسپرژیلوس نایجر و تولید اتانول با استفاده از مخمر ساکارومایسس سرویسیه
محورهای موضوعی : زیست فناوری میکروبیمسعود حاتمی منش 1 , موسی نظری 2
1 - دانشجوی دکتری، دانشکده منابع طبیعی و محیط زیست، دانشگاه ملایر، همدان
2 - دانشجوی دکتری، دانشگاه آزاد اسلامی واحد علوم تحقیقات تهران
کلید واژه: هیدرولیز آنزیمی, اتانول, آسپرژیلوس نایجر, ساکارومایسس سرویسیه, مدل کینتیک میکائلیس-منتن,
چکیده مقاله :
سابقه و هدف: استفاده از فرآیندهای بیولوژیکی و بهنیه سازی آنها به منظور تولید سوخت های زیستی نظیر اتانول و به دلیل مسایل اقتصادی و محیط زیستی از اهمیت بسیار بالایی برخوردار است. به همین دلیل در مطالعه حاضر به بررسی مدل کینتیکی فرآیند هیدرولیز آنزیمی نشاسته توسط قارچ آسپرژیلوس نایجر و تولید اتانول با استفاده از مخمر ساکارومایسس سرویسیه پرداخته شد.مواد و روش ها: این مطالعه در مقیاس آزمایشگاهی انجام گرفت. در این مطالعه، تاثیر پارامترهایی مانند pH و دما بر روی فرآیند هیدرولیز آنزیمی نشاسته توسط قارچ آسپرژیلوس نایجر مورد بررسی قرار گرفت. همچنین برای بررسی مدل کینتیکی فرآیند هیدرولیز آنزیمی از مدل کینتیکی میکائلیس-منتن استفاده گردید. به منظور تولید اتانول از نشاسته هیدرولیز شده از مخمر ساکارومایسس سرویسیه در دمای 25 درجه سلیسیوس و 4.5 pH استفاده شد. برای اندازه گیری غلظت اتانول تولیدی از دستگاه کروماتوگرافی گازی استفاد گردید.یافته ها: نتایج نشان داد که 5 pH و دمای 35 درجه سلیسیوس بیشترین تاثیر را بر روی تولید گلوکز توسط قارچ آسپرژیلوس نایجر دارند. همچنین بررسی مدل کینتیکی و ثابت های مدل، به ترتیب Vmax و Km برابر 10.41 و 73.23 را نشان دادند. مقایسه دادهای تجربی با داده های مدل به دست آمده، همبستگی بالای داده های تجربی و داده های مدل کینتیکی را تایید نمود (0.93 = R2). نتایج تولید اتانول توسط مخمر ساکارومایسس سرویسیه از محیط کشت هیدرولیز شده با غلظت گلوکز اولیه به میزان 32 گرم بر لیتر نشان داد که بیشترین میزان اتانول تولیدی و وزن خشک سلولی آن به ترتیب برابر 10.40 و 3.08 گرم بر لیتر می باشد.نتیجه گیری: بر اساس نتایج مطالعه حاضر می توان نتیجه گرفت که مدل کینتیکی میکائلیس-منتن قادر به پیش بینی هیدرولیز آنزیمی نشاسته می باشد. نشاسته هیدرولیز شده می تواند به عنوان یک سوبسترای مناسب برای تولید اتانول به کار گرفته شود.
Background & Objectives: The use of biological processes and optimizing them in order to produce biofuels such as ethanol is very important due to economic and environmental issues. Therefore, this study was conducted to investigate the kinetic model of starch enzymatic hydrolysis by Aspergillus niger and ethanol production using Saccharomyces cerevisiae.Materials & Methods: The study was performed on a laboratory scale. The effect of different parameters such as pH and temperature on starch enzyme hydrolysis by A. niger was investigated. Also, the Michaelis-Menten’s model was used to assess the Kinetic model of the enzymatic hydrolysis process. In order to produce ethanol from the hydrolyzed starch, S. cerevisiae (yeast) was used at the temperature of 25 °C and pH of 4.5.Results: The results showed that the pH of 5 and the temperature of 35 °C have the highest effect on glucose production by A. niger. In addition, assessment of the kinetics and the constants of the model measured Vmax and Km as 10.41 and 73.23, respectively. Comparison between the experimental data and those predicted from the rate model indicated good agreement (R2= 0.93). The results of ethanol production by S. cerevisiae on hydrolyzed medium with glucose initial concentration of 32 g/l showed the maximum ethanol production and cell dry weight as 10.04 g/l and 3.08 g/l, respectively.Conclusion: According to the results of this study Michaelis–Menten kinetics model is able to predict enzymatic hydrolysis of starch. Furthermore, hydrolyzed starch can be used as an appropriate substrate for ethanol production.
MW. Ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by
expression of bacterial bifunctional alcohol dehydrogenases. Microb Biotechnol. 2017; 3. DOI:
10.1111/1751-7915.12486.
2. Lavudi S, Oberoi HS, Mangamoori LN. Ethanol production from sweet sorghum bagasse
through process optimization using response surface methodology. Biotech. 2017; 7: 233.
3. Hatami ou si H ah ami a . imu ta ous saccha ificatio a d m tatio ( ) o
rice cooker wastewater by using Aspergillus niger and Saccharomyces cerevisiae for ethanol
production. J Appl Res Water Wastewater. 2015; 2(1): 103-107.
4. Liu, Z, Inokuma K, Shih-Hsin H, Riaanden H, Willem H Tomohisa H, Akihiko K. Improvement
of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic
Saccharomyces cerevisiae. Biotechnol Bioeng. 2017; 114: 1201-1207.
5. Moshi AP, Hosea KM, Elisante E, Mamo G, Mattiasson B. High temperature simultaneous
saccharification and fermentation of starch from inedible wild cassava (Manihot glaziovii) to
bioethanol using Caloramator boliviensis. Bioresour Technol. 2015; 180: 128-136.
6. Okonkwo CC, Azam MM, Ezeji TC, Qureshi N. Enhancing ethanol production from cellulosic
sugars using Scheffersomyces (Pichia) stipitis. Bioprocess Biosyst Eng. 2016; 39(7): 1023-1032.
7. Verma G, Nigam P, Singh D, Chaudhary K. Bioconversion of starch to ethanol in a single-step
process by coculture of amylolytic yeasts and Saccharomyces cerevisiae 21. Bioresour Technol.
2000; 72(3): 261-266.
8. Talebnia F, Karakashev D, Angelidaki I. Production of bioethanol from wheat straw: an
overview on pretreatment, hydrolysis and fermentation. Bioresour Technol. 2010; 101(13):
4744-4753.
9. Sebayang AH, Masjuki HH, Ong HC, Silitonga AS, Kusumo F, Milano J. Optimization of
bioethanol production from sorghum grains using artificial neural networks integrated with
ant colony. Ind Crops Prod. 2017; 97(2): 146-155
10. Lin Y, Tanaka S. Ethanol fermentation from biomass resources: current state and prospects.
Appl Microbiol Biotechnol. 2006; 69(6): 627-642.
11. Oberoi HS, Vadlani PV, Brijwani K, Bhargav VK, Patil RT. Enhanced ethanol production via
fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC 13803. Process
Biochem. 2010; 45(8): 1299-1306.
12. Sakthi SS, Saranraj P, Rajasekar M. Optimization for cellulase production by Aspergillus niger
using paddy straw as substrate. Int J Adv Sci Tech Res. 2011; 1: 68-85.
13. Liu K, Zhang J, Bao J. Two stage hydrolysis of corn stover at high solids content for mixing
power saving and scale-up applications. Bioresour Technol. 2015; 196 (2): 716-720.
14. Aderemi BO, Abu E, Highina BK. The kinetics of glucose production from rice straw by
Aspergillus niger. Afr J Biotechnol. 2008; 7(11): 1745-1752.
15. Hatamimanesh M, Younesi H, Bahramifar N. The production of glucose used in ethanol
production through an enzymatic hydrolysis of rice mill by Aspergillus niger. J Microb World.
2015; 8(3): 231-240. [In Persian]
16. Ardestani F, Kasebkar R. Non-structured kinetic model of Aspergillus niger growth and
substrate uptake in a batch submerged culture. Brit Biotechnol J. 2014; 4(9): 970-977.
17. Amini M, Younesi H, Bahramifar N. Biosorption of nickel (II) from aqueous solution by
Aspergillus niger: response surface methodology and isotherm study. Chemosphere. 2009; 75:
1483-1491.
18. Marangoni AG. Enzyme kinetics: a modern approach. Published Online. John Wiley & Sons;
2003. doi: 10.1002/0471267295.
19. Kothari R, Kumar V, Pathak VV, Ahmad S, Aoyi O, Tyagi VV. A critical review on factors
influencing fermentative hydrogen production. Front Biosci. 2017; 22: 1195-1220.
20. Chen HC. Non-aseptic, multi-stage, multi-feeding, continuous fermentation of cane molasses
to ethanol. Process Biochem. 1990; 25(3): 87-92.
21. Ghorbani F, Younesi H., Esmaeili Sari A, Najafpour G. Cane molasses fermentation for
continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae.
Renew Energy. 2011; 36(2): 503-509.
22. Miller G L. Use of dinitrosalicylic acid reagent for dtermination of reducing sugar. Anal
Chem. 1959; 31(3): 426-428.
23. Gonçalves FA, Ruiz HA, Silvino dos Santos E, Teixeira JA, de Macedo GR. Bioethanol
production by Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis from
delignified coconut fibre mature and lignin extraction according to biorefinery concept. Renew
Energy. 2016; 94: 353-365.
24. Bailey JE, Ollis DF. Biochemical engineering fundamentals. Chemical engineering education.
1976.
25. Grous W, Converse A, Grethlein H, Lynd L. Kinetics of cellobiose hydrolysis using cellobiase
composites from Ttrichoderma reesei and Aspergillus niger. Biotechnol Bioeng. 1985; 27(4):
463-470.
26. Sohail M, Siddiqi R, Ahmad A, Khan SA. Cellulase production from Aspergillus niger MS82:
effect of temperature and pH. N Biotechnol. 2009; 25(6): 437-541.
27. Pedersen H, Nielsen J. The influence of nitrogen sources on the α-amylase productivity of
Aspergillus oryzae in continuous cultures. Appl Microbiol Biotechnol. 2000; 53(3): 278-281.
28. Karimi k, Beagi A. Effect of temperature, pH and glucose concentration on ethanol production
by the fungus Mucor indicu. Iran Chem Eng J. 2010; 9(50): 38-43. [In Persian]
29. Saha BC, Nichols NN, Qureshi N, Cotta MA. Comparison of separate hydrolysis and
fermentation and simultaneous saccharification and fermentation processes for ethanol
production from wheat straw by recombinant Escherichia coli strain FBR5. Appl Microbiol
Biotechnol. 2011; 92(4): 865-874.
_||_
MW. Ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by
expression of bacterial bifunctional alcohol dehydrogenases. Microb Biotechnol. 2017; 3. DOI:
10.1111/1751-7915.12486.
2. Lavudi S, Oberoi HS, Mangamoori LN. Ethanol production from sweet sorghum bagasse
through process optimization using response surface methodology. Biotech. 2017; 7: 233.
3. Hatami ou si H ah ami a . imu ta ous saccha ificatio a d m tatio ( ) o
rice cooker wastewater by using Aspergillus niger and Saccharomyces cerevisiae for ethanol
production. J Appl Res Water Wastewater. 2015; 2(1): 103-107.
4. Liu, Z, Inokuma K, Shih-Hsin H, Riaanden H, Willem H Tomohisa H, Akihiko K. Improvement
of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic
Saccharomyces cerevisiae. Biotechnol Bioeng. 2017; 114: 1201-1207.
5. Moshi AP, Hosea KM, Elisante E, Mamo G, Mattiasson B. High temperature simultaneous
saccharification and fermentation of starch from inedible wild cassava (Manihot glaziovii) to
bioethanol using Caloramator boliviensis. Bioresour Technol. 2015; 180: 128-136.
6. Okonkwo CC, Azam MM, Ezeji TC, Qureshi N. Enhancing ethanol production from cellulosic
sugars using Scheffersomyces (Pichia) stipitis. Bioprocess Biosyst Eng. 2016; 39(7): 1023-1032.
7. Verma G, Nigam P, Singh D, Chaudhary K. Bioconversion of starch to ethanol in a single-step
process by coculture of amylolytic yeasts and Saccharomyces cerevisiae 21. Bioresour Technol.
2000; 72(3): 261-266.
8. Talebnia F, Karakashev D, Angelidaki I. Production of bioethanol from wheat straw: an
overview on pretreatment, hydrolysis and fermentation. Bioresour Technol. 2010; 101(13):
4744-4753.
9. Sebayang AH, Masjuki HH, Ong HC, Silitonga AS, Kusumo F, Milano J. Optimization of
bioethanol production from sorghum grains using artificial neural networks integrated with
ant colony. Ind Crops Prod. 2017; 97(2): 146-155
10. Lin Y, Tanaka S. Ethanol fermentation from biomass resources: current state and prospects.
Appl Microbiol Biotechnol. 2006; 69(6): 627-642.
11. Oberoi HS, Vadlani PV, Brijwani K, Bhargav VK, Patil RT. Enhanced ethanol production via
fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC 13803. Process
Biochem. 2010; 45(8): 1299-1306.
12. Sakthi SS, Saranraj P, Rajasekar M. Optimization for cellulase production by Aspergillus niger
using paddy straw as substrate. Int J Adv Sci Tech Res. 2011; 1: 68-85.
13. Liu K, Zhang J, Bao J. Two stage hydrolysis of corn stover at high solids content for mixing
power saving and scale-up applications. Bioresour Technol. 2015; 196 (2): 716-720.
14. Aderemi BO, Abu E, Highina BK. The kinetics of glucose production from rice straw by
Aspergillus niger. Afr J Biotechnol. 2008; 7(11): 1745-1752.
15. Hatamimanesh M, Younesi H, Bahramifar N. The production of glucose used in ethanol
production through an enzymatic hydrolysis of rice mill by Aspergillus niger. J Microb World.
2015; 8(3): 231-240. [In Persian]
16. Ardestani F, Kasebkar R. Non-structured kinetic model of Aspergillus niger growth and
substrate uptake in a batch submerged culture. Brit Biotechnol J. 2014; 4(9): 970-977.
17. Amini M, Younesi H, Bahramifar N. Biosorption of nickel (II) from aqueous solution by
Aspergillus niger: response surface methodology and isotherm study. Chemosphere. 2009; 75:
1483-1491.
18. Marangoni AG. Enzyme kinetics: a modern approach. Published Online. John Wiley & Sons;
2003. doi: 10.1002/0471267295.
19. Kothari R, Kumar V, Pathak VV, Ahmad S, Aoyi O, Tyagi VV. A critical review on factors
influencing fermentative hydrogen production. Front Biosci. 2017; 22: 1195-1220.
20. Chen HC. Non-aseptic, multi-stage, multi-feeding, continuous fermentation of cane molasses
to ethanol. Process Biochem. 1990; 25(3): 87-92.
21. Ghorbani F, Younesi H., Esmaeili Sari A, Najafpour G. Cane molasses fermentation for
continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae.
Renew Energy. 2011; 36(2): 503-509.
22. Miller G L. Use of dinitrosalicylic acid reagent for dtermination of reducing sugar. Anal
Chem. 1959; 31(3): 426-428.
23. Gonçalves FA, Ruiz HA, Silvino dos Santos E, Teixeira JA, de Macedo GR. Bioethanol
production by Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis from
delignified coconut fibre mature and lignin extraction according to biorefinery concept. Renew
Energy. 2016; 94: 353-365.
24. Bailey JE, Ollis DF. Biochemical engineering fundamentals. Chemical engineering education.
1976.
25. Grous W, Converse A, Grethlein H, Lynd L. Kinetics of cellobiose hydrolysis using cellobiase
composites from Ttrichoderma reesei and Aspergillus niger. Biotechnol Bioeng. 1985; 27(4):
463-470.
26. Sohail M, Siddiqi R, Ahmad A, Khan SA. Cellulase production from Aspergillus niger MS82:
effect of temperature and pH. N Biotechnol. 2009; 25(6): 437-541.
27. Pedersen H, Nielsen J. The influence of nitrogen sources on the α-amylase productivity of
Aspergillus oryzae in continuous cultures. Appl Microbiol Biotechnol. 2000; 53(3): 278-281.
28. Karimi k, Beagi A. Effect of temperature, pH and glucose concentration on ethanol production
by the fungus Mucor indicu. Iran Chem Eng J. 2010; 9(50): 38-43. [In Persian]
29. Saha BC, Nichols NN, Qureshi N, Cotta MA. Comparison of separate hydrolysis and
fermentation and simultaneous saccharification and fermentation processes for ethanol
production from wheat straw by recombinant Escherichia coli strain FBR5. Appl Microbiol
Biotechnol. 2011; 92(4): 865-874.