Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori

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1 Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori THESIS Submitted to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur In partial fulfillment of the requirement for the Degree of MASTER OF SCIENCE In AGRICULTURE (MOLECULAR BIOLOGY AND BIOTECHNOLOGY) AASHUTOSH PRASAD SAHU Biotechnology Centre Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh 2016

2 CERTIFICATE I This is to certify that the thesis entitled Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori. submitted in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE (Agriculture) in Molecular Biology and Biotechnology of Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur is a record of the bonafide research work carried out by Mr. Aashutosh Prasad Sahu under my guidance and supervision. The subject of the thesis has been approved by the Student s Advisory Committee and the Director of Instructions. All the assistance and help received during the course of the investigation has been acknowledged by him. Place : Jabalpur Date : 14/06/2016 Dr. L. P. S. Rajput (Chairman of the Advisory Committee) (THESIS APPROVED BY THE STUDENT S ADVISORY COMMITTEE) Committee Name Signature Chairman Dr. L. P. S. Rajput Member Dr. S. Nema Member Dr. S. Kumar

3 CERTIFICATE II This is to certify that the thesis entitled Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori submitted by Mr. Aashutosh Prasad Sahu, to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur in partial fulfillment of the requirements for the degree of Master of Science (Agriculture) in Molecular Biology and Biotechnology has been, after evaluation, approved by the External Examiner and by the Student s Advisory Committee after an oral examination on the same. Place : Jabalpur Date : 14/06/2016 Dr. L. P. S. Rajput (Chairman of the Advisory Committee) (MEMBERS OF THE ADVISORY COMMITTEE) Committee Name Signature Chairman Dr. L. P. S. Rajput... Member Dr. S. Nema... Member Dr. S. Kumar... Director Biotechnology Centre Dr. S. Tiwari Director of Instructions Dr. D. Khare...

4 Declaration and Undertaking by the Candidate I, Aashutosh Prasad Sahu S/o Shri Bhuneshwar Prasad Sahu certify the work embodied in thesis entitled Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori is my own first hand bonafide work carried out by me under the guidance of Dr. L. P. S. Rajput (Professor, Biotechnology Centre), at Biotechnology Centre, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur M.P. during The matter embodied in the thesis has not been submitted for the award of any other degree/diploma. Due credit has been made to all the assistance and help. I, undertake the complete responsibility that any act of misinterpretation, mistakes, and errors of fact are entirely of my own. I, also abide myself with the decision taken by my advisor for the publication of material extracted from the thesis work and subsequent improvement, on mutually beneficial basis, provided the due credit is given, thereof. Place: Jabalpur Date: 14/06/2016 (Aashutosh Prasad Sahu)

5 Copyright Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur Madhya Pradesh 2016 Title of the Thesis : Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori Name of the candidate : Aashutosh Prasad Sahu Subject : Department : Molecular Biology and Biotechnology Biotechnology Centre College : College of Agriculture, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh Year of thesis submission : 2016 Copyright Transfer The undersigned Aashutosh Prasad Sahu assigns to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh, all rights under Copyright Act, that may exists in and for the thesis entitled: Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori submitted for the award of M.Sc. (Ag.) Molecular Biology and Biotechnology. Date: 14/06/2016 Place: Jabalpur Dr. L. P. S. Rajput (Chairman of Advisory Committee) Aashutosh Prasad Sahu Student

6 Acknowledgment I am grateful to the God for the good health and wellbeing that were necessary to complete this thesis. I would like to thank all those people who made this thesis possible and an unforgettable experience for me. There are no proper words to convey my deep gratitude and respect for my research advisor, Dr. L.P.S Rajput Professor, Biotechnology Centre, JNKVV, Jabalpur. He has inspired me to become an independent researcher and realize the power of critical reasoning. He also demonstrated what a brilliant and hard-working scientist can accomplish. Besides my advisor, I would like to thank my thesis committee members Dr. S. Nema, Professor, Biotechnology Centre, JNKVV, Jabalpur, Dr. S. Kumar Professor and Head Department of Food Science and Technology, JNKVV, Jabalpur, for their valuable suggestions, guidance and steady encouragement. I wish to express my sincere gratitude, to Dr. Sharad Tiwari, Director, Biotechnology centre JNKVV, Jabalpur for his ever wiling help and goodwill. I am sincerely thankful to Dr. V.S. Tomar, Hon ble Vice Chancellor, JNKVV, Jabalpur, Dr. D. Khare, Director of Instruction, JNKVV, Jabalpur and Dr. Om Gupta, Dean, College of Agriculture, Jabalpur, for providing necessary facilities for this research work. I am also extremely indebted to Mrs. Keerti Tantwai, Dr. Iti Gontia-Mishra and Dr. Neeraj Tripathi (SRF) for their full collaboration and help during the course of investigation. I would like to express my sincere thanks to my (Ph.D.) seniors Mr. Swapnil Sapre, Mr. Vishwa Vijay Thakur, and Mr. Satish Kachare, Mr. Sandeep Rahangdale, Mr. Laalit Jeena for their co-operative help during my research work. I am also thankful to my seniors Mr.Umasankar Prajapati, Mr.Sagar Indhane, dear friends Krishnakant, Sajjan, Rameshwar, Nutan and Nistha for their friendly support. I have no such words to express my indebtedness, reverence and regard to my parents Mrs. Pramila Devi Sahu, Mr. Bhuneshwar Prasad Sahu and my elder brother Mr. Abhishek Prasad Sahu and my loving nephew Toshan and Swati and all my family members, whose love and cheerful presence, joy and energy, filled in my life with success and prosperity. Finally I want to thank God for giving me this opportunity and all those persons who directly and indirectly supported me in my life. Place: Jabalpur Date: (Aashutosh Prasad Sahu)

7 LIST OF CONTENTS CHAPTER NO Title Page No. 1. Introduction Review of Literature Materials and Methods Results Discussion Summary, Conclusion and Suggestions for further work Summary, Conclusion Suggestions for further work Bibliography Curriculum Vitae

8 LIST OF TABLES TABLE NO. Title Page No. 4.1 Chemical composition of waste potato tubers for suitability in the production of bioethanol. 4.2 Effect of incubation temperature on bioethanol yield at different incubation period in SSF method. 4.3 Effect of ph 5.5 on yield of bioethanol in SiSF method at different incubation temperatures and incubation periods Effect of ph 6.0 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods Effect of ph 6.5 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods. 4.6 Effect of ph 7.0 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods. 4.7 Effect of incubation temperature on residual sugar in solid state fermentation (SSF) at different i ncubation periods. 4.8 Effect of ph 5.5 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. 4.9 Effect of ph 6.0 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods Effect of ph 6.5 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods Effect of ph 7.0 on residual sugar in Simultaneous Saccharificaiton and Fermentation (Si SF) at different incubation temperatures and incubation periods

9 4.12 Quality attributes of bioethanol produced from co-culture of two different strains using SSF and SiSF methods. 38

10 LIST OF FIGURES Fig. No. Title Between Page Flow chart of SSF method for bioethanol production Flow chart of SiSF method for bioethanol production Effect of incubation temperature on bioethanol yield at different incubation periods in SSF method 4.2 Effect of ph 5.5 on yield of bioethanol in SiSF method at different incubation temperatures and incubation periods 4.3 Effect of ph 6.0 on yield of bioethanol in SiSF method at different incubation temperatures and incubation periods 4.4 Effect of ph 6.5 on yield of bioethanol in SiSF method at different incubation temperatures and incubation periods 4.5 Effect of ph 7.0 on yield of bioethanol in SiSF method at different incubation temperatures and incubation periods Effect of incubation temperature on residual sugar in solid state fermentation (SSF) at different incubation temperatures and incubation periods. Effect of ph 5.5 on residual sugar in Simultaneous saccharification and fermentation (SiSF) at different incubation temperatures and incubation periods. Effect of ph 6.0 on residual sugar in Simultaneous saccharification and fermentation (SiSF) at different incubation temperatures and incubation periods. Effect of ph 6.5 on residual sugar in Simultaneous saccharification and fermentation (SiSF) at different incubation temperatures and incubation periods. Effect of ph 7.0 on residual sugar in Simultaneous saccharification and fermentation (SiSF) at different incubation temperatures and incubation periods

11 LIST OF PLATES PLATE NO. Title Between Page 1. Experimental material used for bioethanol production A Waste potatoes B Strain of Saccharomyces cerevisiae (MTCC 170) C Strain of Aspergillus awamori (MTC 8840) D Incubator cum shaker used in investigation Methods used for fermentation of waste potato A Biomass of potato under under solid state fermentation(ssf) B Biomass of potato under under (SiSF) 15-16

12 LIST OF ABBREVIATIONS Abbreviations Stands for % Percent C Degree centigrade Fig. g g/ml hr i.e. l mg min ml Mg/ml nm OD rpm Sec. SiSF SSF Viz. v/v w/v w/w Figure Gram Gram/milliliter Hour that is Liter Milligram minute Milliliter Milligram/ milliliter nanometer Optical density Rotation per minute second Simultaneous saccharification and fermentation Solid state fermentation which is/are Volume/volume Weight/volume Weight/weight

13 INTRODUCTION Ethanol fermented from renewable resources or biomass-based material for fuel is considered as bio-ethanol. Bioethanol as an alternative source of energy has received special attention over the world due to depletion of fossil fuels. Ethanol fermentation is a biological process in which organic material is converted by microorganisms to simpler compounds such as carbon dioxide, lactic acid and ethanol etc. Ethanol production from starch feedstocks such as corn, wheat and potato requires an additional step. Starches must first be catalyzed into simple sugars. This process, called saccharification, requires more energy and may increase the cost of production. Ethanol is one of the bio-energy sources with high efficiency and low environmental impact. Among Asian countries, India is a country with a positive outlook towards renewable energy technologies. In 2010, India registered 85% increase in bio-fuel production from the previous year. Though India holds only 0.3% share of the global production in 2010 (Patni et al. 2011), yet it continues to be the second largest producer of ethanol in Asia with an annual production of 2170 million liters in the year 2012 (USDA GAIN, India Biofuel Annual 2012). The scientific tactics for bioethanol production can be roughly separated into four major generations based on the substrate utilization, technology involvement and microorganism involved in the process. Bioethanol produced from fermentation of food crops (sugary and starchy crops) is called first generation bioethanol. Second -generation biofuels have been developed to overcome the limitation of first generation biofuels. These fuels are produced from non-food crops such as wood, organic waste, food crop waste and specific biomass crops. The major limitation behind production of second generation bioethanol is its high cost process. Third-generation biofuels are generally produced from algal biomass while fourth generation bioethanol is produced from genetically modified carbon negative crops. Fourth generation biofuels are helpful not only for producing sustainable energy but also in capturing and storing CO 2. The varied raw materials used for ethanol production via fermentation are conveniently classified into three main types: sugars, starches and 1

14 cellulose materials. Sugars from sugarcane, sugar beets, molasses and fruits can be converted into ethanol directly. Starches from corn, cassava, potatoes and root crops must first be hydrolyzed to fermentable sugars by the action of enzymes from malt or molds. Cellulose from wood, agricultural residues, waste sulfite liquor from pulp and paper mills must likewise be converted into sugars, generally by the action of mineral acids. Once simple sugars are formed, enzymes from microorganisms can readily ferment them to ethanol. Potatoes are the second most used food in world. The total world potato production is estimated at 364,808,768 tons in 2012 ( FAO, STAT, 2014). India is a second most potato producer country after china which ranks first. China produces 85,920,000 tons and India 45,000,000 tons in 2012 (FAOSTAT, 2014). Potatoes are starchy crop which do not require complex treatment. Although, it is also a high value crop, but 5 to 20% of crops that are waste potato by-products from potato cultivation could be utilized for bioethanol production (Adarsha et al. 2010; Limatainen et al. 2004). Approximately 18% of the potatoes are generated as a waste during processing of potato, particularly in the potato chip industry (Duhan et al 2013). Above facts suggest the impact of waste potato to be used as important source for bioethanol production. Bioethanol production through waste potato fermentation is a cumulative task in which several factors plays important role to obtain maximum yield like substrate and inoculum size, temperature, ph, incubation period and combination of microbes for saccharification and fermentation. Keeping these all facts and increasing demand of renewable sources of energy and efficient utilization of available resources in consideration, present study was conducted with following objectives. Objectives 1. To analyze the major chemical constituents of waste potatoes collected from different locations. 2. To optimize the fermentation variables for maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori. 3. To evaluate the quality of bioethanol produced. 2

15 REVIEW OF LITERATURE Available literature pertaining to the present investigation has been summarised and presented below in order to review the work done by various workers on bioethanol production from waste potato tubers. 2.1 Substrates used for production of bioethanol Various studies carried out on substrates containing starch used in the production of bioethanol have been presented below: Abouzied et al. (1986) reported the direct fermentation of potato starch to ethanol by co-culture of Aspergillus niger and Saccharomyces cerevisiae. Optimum ethanol yields were obtained in the ph range 5 to 6. Ethanol yield was maximal when fermentation was conducted anaerobically. These results indicated that simultaneous fermentation of starch to ethanol can be conducted by using coculture of A. niger and S. cerevisiae. Amutha and Gunasekaran (2001) reported that the co -immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h. Liimatainen et al. (2004) obtained bioethanol production from waste potatoes using different cultivars of potato. It was reported that alcohol yield varied significantly between cultivars. The highest alcohol yield was 9.5g/100g and the lowest yield 6.5 g/100g. The average alcohol yield was 7.6g/100g. Potato skin proved to have a little effect on the alcohol yield. The yield was higher for the potatoes which had a skin. Kroumov et al. (2006) developed a new unstructured model for simultaneous saccharification and fermentation of starch to ethanol by 3

16 recombinant strain. The first level was enzymatic hydrolysis of starch to glucose by bifunctional protein and the second level included utilization and bioconversion of glucose to ethanol by yeasts. The second level unified the enzymatic degradation of starch and glucose metabolization to ethanol by microorganisms. The response surface analysis was used to develop the rates models. A hybrid genetic algorithm and a decomposition approach were used in the nonlinear parameters identification procedure. Wang et al. (2007) studied dry-grind corn process using (raw starch hydrolyzing) RSH enzyme and compared with two combinations (DG1 and DG2) of commercial liquefaction and saccharification enzymes. During SSF, the highest glucose concentration for RSH treatment was 7% (w/v), whereas for DG1 and DG2 treatments, glucose concentrations had maximum of 19% (w/v). Glycerol concentrations were 0.5% (w/v) for RSH treatment and 0.8% (w/v) for DG1 and DG2 treatments. Ghobadian et al. (2008) studied production of bioethanol and sunflower methyl ester oil for investigating fuel blend properties. Bioethanol was produced from potato waste. The suitable blending proportion of bioethanol and diesel fuel was determined to be 12 to 88 and then for maintaining fuel stability at temperatures lower than 15 C, the sunflower methyl ester was added to the mixture.the findings showed that ethanol played an important role on the flash point of the blends, With the addition of 3% bioethanol to diesel and sunflower methyl ester, the flash point was reduced to 16 C. Nikolic et al. (2008) used microwave-assisted liquefaction as a pretreatment for the bioethanol production by the simultaneous saccharification and fermentation (SSF) of corn meal using Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system. The result indicated that the microwave pre-treatment could increase the maximum ethanol concentration produced in the SSF process for 13.4 %. Consequently, a significant increase of the ethanol productivity on substrate (YP, s), as well as the volumetric ethanol productivity (P) in this process could be achieved. Ado et al. (2009) studied bioconversion of cassava starch into ethanol by a simultaneous sacchrification and fermentation process with co-culture of 4

17 Aspergillus niger (GS4) and Saccharomyces cerevisiae (BK6) and found that ethanol yield was 0.35g/100ml at 1% substrate concentration while the ethanol yield increased to a maximum of 3.60g/100ml at 8% substrate concentration. Under optimized culture conditions, the ethanol yield further increased to 4.30g/100ml at a temperature of 35 C, ph of 5.0, 300rpm agitation rate and reduced fermentation period of 4 days. Hoskins et al. (2009) improved bioethanol yield using solid state fermentation products grown on DDGS. Solid-state fermentation products were produced using DDGS from a distillery and DDGS from a fuel ethanol plant as the fungal growth substrate. The spent grains were inoculated with GRAS (Generally Regarded as Safe) organisms Aspergillus oryzae and Rhizopus oligosporus. After growth, the resultant SSF product was dried and used as an enzyme complex supplement that was added to laboratory scale standard fuel ethanol corn mash fermentations. Improved ethanol yields were consistently observed. Ming-Xiong et al. (2009) reported the direct production of ethanol from sweet potato starch by Zymomonas mobilis having glucoamylase genes from Aspergillus awamori through recombinant polymerase chain reaction (PCR). Kinetics of this transformant was also investigated including growth curve, total sugar consumption and ethanol production. Rakin et al. (2009) carried out study to investigate the immobilization of Sacharomyces cerevisiae var. ellipsoideus yeast cells for bioethanol production from corn meal hydrolyzates. The maximum ethanol concentration of 10.05% (w/w) was obtained in the fermentation of corn meal hydrolyzates by 5% (v/v) of inoculum concentration of the yeast immobilized in Ca-alginate using a method of electrostatic droplet generation. Izmirlioglu and Demirci (2010 ) reported the ethanol production from waste potato mash chosen as a carbon source using pretreatment process needed to convert starch of potato to fermentable carbon sources through liquefaction and saccharification process. Then the effect of ph, inoculum size and various nitrogen sources to obtain maximum ethanol from waste potato mash was studied. The maximum ethanol concentration and production rates were 27.7 g/l and 5.47 g/l/h, respectively at controlled ph 5.5, whereas 5

18 22.75 g/l and 2.22 g/l/h were obtained at uncontrolled ph. Optimum inoculum size was determined as 3% for maximum ethanol concentration and production rate. Furthermore, five different nitrogen sources (yeast extract, poultry meal, hull and fines mix, feather meal, and meat and bone meal) were evaluated to determine an economical alternative nitrogen source to yeast extract. In conclusion, this study demonstrated the potential for utilization of potato waste for ethanol production. Manikandan and Viruthagiri (2010) studied the kinetics and optimization of ethanol production from corn flour by simultaneous saccharification and fermentation (SSF) using starch digesting glucoamylase enzyme derived from Aspergillus niger and non starch digesting and sugar fermenting Saccharomyces cerevisiae in a batch fermentation. It was reported that the optimum values of substrate concentration, ph, temperature and enzyme concentration were found to be 160 g/l, 5.5, 30 C and 50 IU respectively. The corn flour solution equivalent to 16% initial starch concentration gave the highest ethanol concentration of g/l after 48 h of fermentation at optimum conditions of ph and temperature. Monod model and Logistic model were used for growth kinetics and Leudeking Piret model was used for product formation kinetics. Rani et al. (2010) studied f lour prepared from potato tubers (Solanum tuberosum) after cooking and drying at 70 C was used for ethanol production. Homogenous slurry of potato flour was prepared in water at solid liquid ratio 1:4. Liquefaction of potato flour slurry with α-amylase (2.05 DUN U/g starch) at 80 C for 30 min followed by saccharification with glucoamylase (20.5 GA U/g starch) at 60 C for 2 h generated 15.2% total reducing sugars in the hydrolysate. Fermentation of hydrolysate with Saccharomyces cerevisiae HAU-1 at 30 C for 48 h resulted in production of 56.8 g/l ethanol. Supplementation of nitrogen sources to potato flour did not contribute significantly to ethanol yield. Simultaneous saccharification and fermentation of hydrolysate was as effective as separate hydrolysis and fermentation. Chohnan et al. (2011) showed that ethanol was efficiently produced from three varieties of sweet sorghum using repeated-batch fermentation without pasteurization or acidification. Saccharomyces cerevisiae cells could 6

19 be recycled in 16 cycles of the fermentation process with good ethanol yields. This technique would make it possible to use a broader range of sweet sorghum varieties for ethanol production. Magdy et al. (2011) Investigated to produce ethanol from industrial solid potato wastes and optimize the fermentation efficiency by commercial bakery yeast. Saccharomyces cerevisiae was selected and applied in the fermentation system under sterilized conditions for ethanol production. This utilization process was proved to be feasible. The substrate concentration of 10 g/l, incubation temperature of 25 C, and ph 3.5 were found optimum for maximum ethanol production from industrial solid potato wastes after 3 days of incubation under sterilized anaerobic fermentation conditions when ammonium sulfate was used as nitrogen source. Farvin et al. (2012) reported that potato peel extract as a natural antioxidant in chilled storage of minced horse mackerel (Trachurus trachurus) which effect on lipid and protein oxidation. The present work was undertaken to examine the utilisation of potato peel, a waste material, as a source of natural antioxidants for retarding lipid and protein oxidation in minced mackerel. Mackerel mince with two different concentrations (2.4 or 4.8 g/kg) of water or ethanol extracts of potato peel and a control with no added extracts were prepared. The samples were stored at 5 C for 96 h and the sampling was done at time points 0, 24, 48 and 96 h. The ethanol extracts, which contained high amounts of phenolic compounds, was found to be very effective in retarding lipid and protein oxidation. Khan et al. (2012) reported that due to gradual decrease in petroleum resources and impacts of these wastes on the environment, there is a need to utilize the wastes of potatoes to get wealth out of wastes and clean the environment. In this study potato waste was used as substrate for bioethanol production. 100 g potato powder was mixed with L 1 distilled water in two separate beakers to form potato slurry. This investigation also revealed that mixture of enzymes significantly enhanced (p<0.05) bioethanol production compared to non treated mixture. Maximum bioethanol productions were due to the presence of sugar in potatoes. 7

20 Izmirlioglu and Demirci ( 2012) optimized hydrolysis of waste potato mash and growth parameters of the ethanol fermentation were optimized to obtain maximum ethanol production. The optimum combination of temperature, dose of enzyme (α amylase) and amount of waste potato mash was 95 C, 1 ml of enzyme (18.8 mg protein/ml) and 4.04 g dry-weight/100 ml distilled water, with a 68.86% loss in dry weight for liquefaction. After optimization of hydrolysis of the waste potato mash, ethanol fermentation was studied. Results showed that ph of 5.5 and 3% inoculum size were optimum ph and inoculum size, respectively for maximum ethanol concentration and production rate. The maximum bio-ethanol production rate was obtained at the optimum conditions of g/l ethanol. Swain et al. (2013) studied the optimization of co-culturing of Trichoderma sp. and Saccharomyces cerevisiae (1:4 ratio) on sweet potato (Ipomoea batatas L.) flour (SPF) for the production of bio -ethanol in solidstate fermentation (SSF). Maximum ethanol (172 g/kg substrate) was produced in a medium containing 80% moisture, ammonium sulphate 0.2%, ph 5.0, inoculuted with 10% inoculum size and fermented at 30ºC for 72h..Concomitant with highest ethanol concentration, maximum ethanol productivity (2.8 g/kg substrate/h), microbial biomass ( CFU/ g substrate), ethanol yield (47 g/100g sugar consumed) and fermentation efficiency (72%) were also obtained under these conditions. Rai et al. (2013) carried o ut studies to assess bioethanol production from waste potatoes and optimize some fermentation variables for Zymomonas mobilis MTCC The waste potatoes contained 76% starch. Under solid state fermentation (SSF) maximum bioethanol of g/100 mll medium was achieved at 35 C when incubated for 120 hr. Under simultaneous saccharification and fermentation (SiSF), highest ethanol yield of g/100 ml medium was attained at 35 C, ph 5.5 and 120 hr. The ethanol produced was volatile, flammable, colourless and miscible in water and organic solvents having pleasant smell. SiSF fermentation gave more ethanol recovery than SSF method Lim et al. (2013) studied the production of high concentration of ethanol from viscous potato tuber mash; Potato tuber mash containing high contents 8

21 of solids (28%) was prepared by grinding the potato tuber without the addition of water. The viscosity of the potato mash was reduced by using Viscozyme (0.1%) at 50 C for 30 min. The potato mash was then liquefied using Liquozyme (0.1%) at 90 C for 30 min. Using response surface methodology, the optimal saccharifying-enzyme dosage and incubation temperature were determined to be 1.45 AGU/g dry matter and 31.3 C, respectively. Under these optimal conditions for SSF, 14.92% (v/v) ethan ol with 91.0% of theoretical yield was produced after 60 h, and all the starch was completely used up. Duhan et al. (2013) studied Kufri Bahar (KB) variety of potato and chosen as a carbon source for bioethanol production. For saccharification process, the optimum dose of amyloglucosidase was 0.35% w/v (300 U/ml) with 16.95% glucose production at ph 5.0 and temperature 60 C after 1 h. The maximum ethanol concentration 7.89% (v/v) was obtained with 10% inoculum size at ph 6.0 after 48 h. Azad et al. (2014) reported the optimum conditions for bioethanol production from potato of Bangladesh. From this study, optimum growth of yeast ( Saccharomyces cerevisiae CCD) was observed at ph 6.0 and temperature 31 C. Optimum concentration of potato in fermentation process was determined using five different potato solutions (5%, 10%, 15%, 20% and 30%). A highest production of bioethanol was found in 20% potato treatment. Therefore, 20% potato solution is recommended for high-scale production of bioethanol from potato starch. Joshi (2014) studied potato paste made from red potatoes grown in hilly regions of Nepal used as carbon source. Prior to fermentation, all carbon sources were saccharified enzymatically using α- amylase at ph 5 and temperature 55 C. Maximum yield of ethanol 5.2% was obtained at a temperature of 30 C and ph 5.0 without exogenous supply of nitrogen. Ammonium sulphate was found as best nitrogen supplement among them. Maximum ethanol percentage of 8.3 was observed at ph 5.0 and temperature 30 C with ammonium sulphate concentration of 2%. Rath et al. (2014) reported fermentation of unhydrolyzed potato waste to ethanol by co-cultures of Aspergillus niger and Saccharomyces cerevisiae at different temperatures (20 C to 50 C) and at different ph (4 to 7). The 9

22 optimum ph and temperature for the fermentation of waste potatoes was found 6 and 30 C. With the optimized ph and temperature, fermentation was then carried out at different yeast concentration 3% to 12%. Using a 12%, 9%, 6%, 3% yeast inoculum, maximum ethanol production was completely achieved in 2, 3, 5, 7 days respectively. The maximum ethanol yield from waste potatoes was %. Aspergillus niger strain B gave a higher production of ethanol. The amount of ethanol content increased with the increase in fermentation time. Meenakshi and Kumaresan (2014) studied the optimum conditions for the production of ethanol. The parameters like, ph, substrate concentration and particle size were optimized using response surface method in the MINITAB 16 software. Both solid and submerged fermentation were studied. Submerged fermentation turned out to be favourable. Yeast fermentation was employed simultaneously with the saccharification process (SSF) for 72 hours. An attempt was made to produce ethanol from Potato peel waste, however it proved corn as an efficient substrate. There was a considerable yield of ethanol of 15.88g/l using ph 5.5, an intermediate particle size of 0.157mm and at a substrate concentration of 10% (W/V). A process development for the entire production was made involving the reactor design and the equipments to be used at an industrial scale. Pranavya et al. (2015) produced ethanol from agricultural wastes using two enzymes namely amylase from Aspergillus niger and cellulase from Trichoderma viridae to hydrolyse the starch and cellulose present in the raw materials. The hydrolysed and filtered extracts were fermented using Saccharomyces cerevisiae and Zymomonas mobilis and found that the Zymomonas mobilis yielded maximum ethanol where as minimum ethanol yield was recorded with Saccharomyces cerevisiae. It was found that the percentage of residual sugar was more in banana peel waste when compared to others. The total sugar percentage was carried out by using untreated substrates and the maximum percentage was found to be high in both sugar cane wastes (1.4%) and banana peel (1.1%) where as in waste paper (0.07%), the total sugar percentage was low when compared to the other two substrates. Ferro at al. (2015) produced bioethanol from Cistus ladanifer (rockrose) pretreated by steam explosion (SE) employing separate enzymatic hydrolysis 10

23 and fermentation (SHF) or si multaneous saccharification and fermentation (SiSF) approaches. Two strains of Saccharomyces cerevisiae, namely NCYC 1119 for SHF experiments and the thermo tolerant strain PYCC 2613 for SiSF assays were used. Comparing with saccharification of rockrose pretreated with diluted acid hydrolysis that produced 313 mg glucose/g dry biomass with an enzyme loading of 60 FPU/g dry biomass. The results obtained after 72 h of simultaneous saccharification and fermentation of rockrose, the maximum ethanol yield was 9.1 g per 100 g of untreated rockrose. The ethanol concentration obtained in SiSF fermentation mode (16.1 g/l) was higher than the ethanol concentration obtained by SHF mode (15.0 g/l). Maximum ethanol concentration achieved for rockrose in SiSF mode was 16.1 g/l with fermentation efficiency of 69.8% and the yield of 22.1 g of ethanol per 100 g of dry rockrose. Thapa et al. (2015) improved bioethanol production from metabolic engineering of Enterobacter aerogenes ATCC The bioethanolproducing strain, E. aerogenes ATCC 29007, was engineered by deleting the D-lactate dehydrogenase (ldha) gene to block the production of lactic acid. Glycerol, a useful byproduct in the biodiesel industry, was employed to convert into bioethanol, using engineered E. aerogenes SUMI014. Under optimal conditions of fermentation (34 C, ph 7.5, 78 h), bioethanol production by the mutant strain was g/l, 1.5 times greater than that produced by its wild type (13.09 g/l). The production of bioethanol increased with time and reached a maximum after 78 h of fermentation, in case of recombinant E. aerogenes SUMI2008 and mutant E. aerogenes SUMI014, and eventually started to decrease. In the case of wild type E. aerogenes ATCC 29007, however, bioethanol production reached maximum after 48 h of fermentation and subsequently decreased. Hou et al. (2015) used a wild-growing glucose-rich (i.e. 56.7% glucose content) brown seaweed species Laminaria digitata used as feedstock for integrated bioethanol and protein production. Two strategies of fermentation i.e. Simultaneous Saccharification and Fermentation (S isf) and Separate Hydrolysis and Fermentation (SHF) were compared. S. cerevisiae was inoculated and the fermentation flasks were incubated in the orbital shaker 11

24 incubator at 150 rpm under 32 C. Only minor pretreatment of milling was used on the biomass to facilitate the subsequent enzymatic hydrolysis and fermentation. The Separate Hydrolysis and Fermentation (SHF) resulted in obviously higher ethanol yield than the Simultaneous Saccharification and Fermentation (SSF). High conversion rate at maximum of 84.1% glucose recovery by enzymatic hydrolysis and overall ethanol yield at maximum of 77.7% theoretical were achieved. Choi et al. (2015) obtained ethanol production from soybean waste (okara) as a valorization biomass. The in-house enzymes were produced from fungi using okara as a carbon source and tested on okara biomass for their hydrolytic activity. The okara biomass was used raw or pretreated in an autoclave (moist heating) for 20 min at 121 C. The chemical compositions content of raw and autoclaved biomass exhibited little difference; however, the enzymatic conversion rate increased significantly from 21.9% for the raw okara to 82.9% for the pretreated okara. The ethanol conversion yield (based on sugar content) from enzymatic hydrolysis after S. cerevisiae fermentation was 96.2%. Sheikh et al. (2016) studied the production of biofuel (bioethanol) from potato peel wastes by Saccharomyces cerevisiae. The possible best fermentation period was between 72h and 96h for both species under approximately all different yeast extract concentrations with the highest bioethanol production of around 2.17% and 2.01% for both commercial and genetically modified S. cerevisiae species, respectively. The suitable yeast extracts were 2 g/l as the best minimum prepared concentration added to the fermentation medium. Consequently, the bioethanol production elevated to 2.83% and 2.64% for both commercial and genetically modified S. cerevisiae species, respectively. 12

25 MATERIALS AND METHODS The present investigation entitled "Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori was conducted in the Fermentation Technology Laboratory, Biotechnology Centre, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur (MP). This chapter deals with various experimental techniques used to understand the production of bioethanol from waste potatoes using Saccharomyces cerevisiae and Aspergillus awamori. 3.1 Materials Waste potato tubers were purchased from different shops of Adhartal vegetable market and krishi upaj mandi Jabalpur (M P). The bioethanol producing microorganisms i.e. co-culture viz. Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 were obtained from Institute of Microbial Technology (IMTECH) Chandigarh, Punjab (Plate 1). 3.2 Methods Selection of Microbes and suitability of starch source (substrate) for bioethanol production Selection of Microbes The most important variable which is responsible for bioethanol production is the type of strain used in the bioconversion of starch into the desirable end products. The strain must have high yielding capacity and should not produce any undesirable substances. For the purpose of production of bioethanol, Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 were selected and taken in the present investigation Suitability of starch sources (Substrates) In order to know the availability of potato, market survey of Jabalpur city in different locations was conducted to assess the stage of wastage to the potatoes. Based on it, potatoes at particular stage, not fit for human 13

26 consumption, were selected in order to get higher yield and better quality of bioethanol. In this experiment, waste potatoes were taken as starch source (substrate) for bioethanol production using two different methods of fermentation namely solid state fermentation (SSF) and simultaneous saccharification and fermentation (SiSF) Preparation of substrate for bioethanol production Before processing, waste potatoes were cleaned gently with potable water thoroughly washed unpeeled potatoes were boiled in distilled water containing 0.5% potassium metabisulphite for 30 minutes. Boiled potatoes were mashed and dried at 70 C for about 7 hours in a hot air oven. After drying, it was ground to fine powder and sieved to remove big particles Cultivation and maintenance of microorganisms (strains) Two different microorganisms namely, Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 were used for cultivation in the present investigation. The culture of Saccharomyces cerevisiae and Aspergillus awamori were grown and maintained on Yeast Extract Peptone Dextrose (YEPD) and Malt Extract Agar (MEA) media respectively. (Plate 1.B and C) Table 3.1. Composition of YEPD medium Components Yeast extract Peptone Dextrose Agar Amount 3 gm 10 gm 20 gm 15 gm All components were added in a conical flask under continuous shaking on magnetic stirrer. Media was sterilised using an autoclave at 121 C for 20 min and then used for research work. 14

27 Table 3.2. Composition of MEA medium Components Malt extract Agar Distilled water Amount 20.0 gm 20.0gm 1 lit. All components were added in a conical flask under continuous shaking on magnetic stirrer and ph of YEPD adjusted at 6.0 and MEA at 6.5. Media was sterilised using an autoclave at 121 C for 20 min and then used for research work. The culture of Saccharomyces cerevisiae and Aspergillus awamori were maintained by sub culturing them every 15 days on YEPD and MEA agar plates, incubating for 24 hrs and 7 days respectively at 30 C and thereafter storing in a refrigerator at 4 C until further use Inoculum and inoculation Inoculum of Saccharomyces cerevisiae and Aspergillus awamori was prepared separately in YEPD and malt extract broth. A loopful of 24 and 7 days old culture of Saccharomyces cerevisiae and Aspergillus awamori was inoculated and incubated at 30 C on a rotary shaker at 200 rpm for 24 hours. These inoculums were used to inoculate sterilized potatoes samples Production techniques used Two different fermentation methods were used for production of bioethanol from waste potatoes by employing solid state fermentation (SSF) and simultaneous saccharification and fermentation (SiSF) using co-culture of Saccharomyces cerevisiae and Aspergillus awamori Solid state fermentation (SSF) method The method described by Rani et al. (2010) was adopted for conducting the experiment on solid state fermentation using waste potatoes. In this method, 20 g potato powder were taken in 250 ml Erlenmeyer flask and initial moisture level was set at 60% by adding the requisite amount of distilled water. The flask was autoclaved at 121 C at 15 psi for 20 min and cooled at 15

28 room temperature or 30 C add then 25% ( v/v) of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 were added. The process of fermentation was carried out on NBS shaker incubator at different temperatures viz. 28 C, 30 C and 32 C for different incubation periods viz.120,144,168 and 192 hrs. After the fermentation period, the fermented mass was centrifuged at rpm for 15 min and then supernatant was collected by filtration which was further used for distillation process (Plate 1. C and Plate 2. A) Simultaneous saccharification and fermentation (SiSF) method The method described by Rath et al. (2014) was adopted for conducting the experiment on simultaneous saccharification and fermentation (SiSF) from waste potatoes using co-culture of Saccharomyces cerevisiae and Aspergillus awamori. In this procedure, 5 gm of potato flour was taken in 250 ml Erlenmeyer flask and 96 ml distilled water was added and then the ph was adjusted with 0.1N HCl and 1N NaOH. These potato solutions were subjected to autoclaving at 121 C at 15psi for 20 min and then cooled at room temperature. Thereafter in each sample, inoculums of Saccharomyces cerevisiae ( cfu/ml) and Aspergillus awamori ( cfu/ml) were added under controlled condition. In order to optimize fermentation variables, the process of fermentation was carried out at different ph viz.5.5, 6.0, 6.5 and 7.0 and different ranges of temperature viz. 28 C, 30 C and 32 C for different incubation periods viz. 120, 144, 168, and 192 hours. After the fermentation period, the fermented mass was centrifuged at rpm for 15 min and supernatant was collected for distillation purpose (Plate 2. B). 3.4 Optimization of different fermentation variables For solid state fermentation (SSF) and simultaneous saccharification and fermentation (SiSF) method, different variables viz. temperature, ph and incubation periods were studied for better recovery of bioethanol Solid state fermentation (SSF) method Solid state fermentation (SSF) method was used for carrying out the different experiments for optimization of process parameters (incubation temperature and incubation period) for attaining the maximum yield of 16

29 bioethanol. By maintaining the optimum condition of moisture content at 60% level, production of bioethanol was carried out at different incubation temperatures viz. 28, 30 and 32 C for different incubation periods viz. 120, 144, 168 and 192 hours in order to attain for maximum recovery of bioethanol using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC Simultaneous saccharification and fermentation (SiSF) method Similar to solid state fermentation method, simultaneous saccharification and fermentation method was also used for carrying out the experiments on optimization of different fermentation variables (ph, incubation temperature and incubation period) in order to get maximum yield of bioethanol. The process of fermentation was carried out at different temperatures viz. 28, 30 and 32 C for different incubation periods viz. 120,14 4, 168 and 192 days with different ranges of ph viz. 5.5, 6.0,6.5 and 7.0 ph for maximum recovery of bioethanol using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC Yield of bioethanol The yield of bioethanol was determined by distillation and dehydration process adopted by O'Leary (2000). Distillation and dehydration was done using rotory evaporator at 78±2 C under vacuum. 3.6 Analytical method Moisture The method prescribed by AOAC (1980) was used for determination of moisture content in potato tubers. Moisture content of potato tubers was determined using oven dry method. A known quantity of chopped potatoes was taken on a preweighed aluminium foil (W 1 ) and recorded the weight of sample and Aluminium foil (W 2 ). It was kept in an oven at 100±1 C temperature for period of about 8 hr until all the moisture present goes off. After the drying period, sample was kept in a desiccator to bring down the temperature to room condition. After this, sample with Aluminium foil was weighed (W 3 ) and the difference in weight of sample was calculated as moisture loss. Using the following formula, moisture content in sample was 17

30 calculated. By determining the moisture content in sample, dry matter content was calculated. Weight of empty Aluminium foil = W 1 Weight of empty foil + chopped potatoes = W 2 Weight of foil + oven dried potatoes = W 3 Weight of moisture lost = (W 2 W 3 ) gm. % Moisture = W 2 - W W 2 - W Estimation of starch content Total starch content of potato tubers was measured as per the procedure of Keer (1950) Glucose content Glucose content in potato tubers was determined by phenol sulphuric acid method (AOAC 1980). A standard curve was prepared using the known concentration of glucose. For this, 100 mg of glucose was dissolved in 100 ml of distilled water and then diluted 10 times so that solution contained 100 µg/ml. Different dilutions were prepared by taking 0.1 ml of glucose stock solution and volume made up to 1.0 ml by adding 0.9 ml of distilled water. Likewise, different volumes of stock glucose solutions (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 ml) were taken separately in test tubes and total volume in each tube was made up to 1.0 ml by adding distilled water. To each tube, 1 ml of 5% phenol and 5 ml of 96% conc. H 2 SO 4 were added and allowed to cool for 20 min. After cooling, O.D. of the sample was measured at 490 nm using spectrophotometer. Using a graph paper, standard curve was prepared by plotting concentration of glucose on one axis and recorded O.D. on the other axis. From this graph, the concentration of glucose in potato samples was determined and starch content was calculated using the following formula. Glucose (g/100ml) 0.93 = Starch (g/100ml). 18

31 Determination of amylose and amylopectin The procedure given in AOAC (1980) was used f or determination of amylose and amylopectin in the potato tubers. To determine amylose content in potatoes, 100 mg of dried mesh potatoes sample was taken in a conical flask and to this 1 ml of distilled alcohol was added shaking gently and then 10 ml of 1N NaOH was added. After adding NaOH, it was left overnight. On next day, 89 ml distilled water was added and shaken well. Then 1 ml of extract was taken in test tube, and 2-3 drops of 0.1% phenolphthalein were added. To this, 3 ml of distilled water was added with 0.1N HCl (added drop wise) to light pink colour and then 2 ml of 0.2% iodine was added and developed colour was read at 600 nm using spectrophotometer. Similarly, standard amylose solution was prepared by taking 100mg of pure amylose and proceeded exactly as above. For preparing iodine blank, 0.2 ml of iodine reagent was made up to 100 ml. The amylose content was calculated using the following formula: Amylose on dry basis O.D. of Sample mg dry amylose present in standard solution = (%) O.D. of Standard 5 dry matter present Using the following formula, amylopectin content in potato tubers was calculated. Amylopectin content = starch content amylose content Estimation of Residual sugar The DNS method of Miller (1959) was used to es timate total sugar in the potato tubers and residual sugar in the fermented broth after the fermentation period was over Reagents Substrate solution- Standard glucose solution of 1000 µg/ml concentration was prepared by dissolving 100 mg of glucose in 100 ml of distilled water. 3, 5 dinitrosalicylic acid (DNS) solution - Reagent was prepared by dissolving 10 gm of 3, 5-DNS, 2 gm phenol and 0.5 g of sodium sulphite in 19

32 500 ml of 2% NaOH solution and then diluting it with distilled water. The reagent was filled and stored in dark colored bottle. Potassium sodium tartarate (Rochelle salt) - 40 gm of potassium sodium tatarate was dissolved in about 50 ml distilled water and the volume was made up to 100 ml Procedure One ml of appropriately diluted sample solution was taken in a test tube in which 3 ml of DNS reagent was added. The tubes were boiled in a boiling water bath for 15 min. Then 1 ml of Rochelle salt was added to the test tubes and then tubes were cooled at room temperature for measuring optical density (O.D.) at 575 nm. A standard curve of glucose was prepared by using mg concentration prepared in distilled water. Based on the O.D. of sample recorded from the standard curve, amount of sugar was calculated Quality evaluation of bioethanol produced from co-culture Quality evaluation of produced bioethanol was determined by three different methods: Density determination using pycnometer The method prescribed by Caylak and Sukan (1998) was used for determination of the density by pycnometer. Density of bioethanol produced from SSF and SiSF method using co-culture of yeast and fungi was measured with the application of pycnometer having a capacity of 25 ml. Before using, pycnometer was cleaned and dried to determine its weight (W). First of all, pycnometer was filled with distilled water and its weight was recorded (W 1 ). Thereafter, water was drained out from pycnometer and it was dried again. After drying, the pycnometer was filled with distilled bioethanol and its weight (W 2 ) was determined at room temperature (25 C). The density (d) of distilled bioethanol was calculated using the following formula and expressed as g/ml. Density of bioethanol (d) (g/ml) 20 = W 2 W W 1 - W Determination of viscosity by Ostwald viscosimeter The procedure prescribed by Bernnan and Tipper (1967) was adopted for viscosity determination. Viscosity of produced bioethanol from co-culture

33 using SSF and SiSF methods was measured by Ostwald viscosimeter. To determine the viscosity, the viscosimeter was rinsed well with acetone. First of all, distilled water was applied through narrow tube of viscosimeter using pipette for avoiding air bubbles and noted down the time (sec.) of water flow through capillary tube up to a certain mark. After rinsing the viscosimeter, distilled bioethanol was applied through narrow tube of viscosimeter and then time of flow taken up to a certain mark through capillary tube was noted down. The viscosity of bioethanol was calculated with the following formula and expressed in terms of centipoise with the help of conversion table. Viscosity (bioethanol) Density (bioethanol ) x Time (bioethanol) x Viscosity = (centipoise) Density (water) x Time (water) Boiling point of produced bioethanol. The procedure given by O'Leary (2000) was used to determine the boiling point of bioethanol produced from co-culture using the methods of SSF and SiSF. The boiling point of bioethanol was determined using rotary flash evaporator by applying temperature in the range of C. 21

34 RESULTS The findings recorded on various experiments during the course of investigation have been presented in this chapter along with tables, graphs and photographs under different heads. 4.1 To analyze the major chemical constituents of waste potatoes collected from different locations. 4.2 To optimize the fermentation variables for maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori. 4.3 To evaluate the quality of bioethanol produced. 4.1 To analyze the major chemical constituents of waste potatoes collected from different locations. In this part of investigation, various experiments were conducted to know the chemical composition of waste potato (substrate) for suitability in the production of bioethanol using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC Data obtained on the amount of various important chemical constituents such as moisture, dry matter, starch, amylose and amylopectin present in waste potato tubers are presented in Table 4.1. The observations recorded revealed that waste potato tubers contained moisture 80.60%, dry matter 19.40%, starch 76.1%, amylose 60.3% and amylopectin 15.8%. Table 4.1: Chemical composition of waste potato tubers for suitability in the production of bioethanol. S. No. Constituents Amount (%) 1. Moisture 80.60± Dry matter 19.40± Starch (on dry weight basis) 76.1± Amylose 60.3± Amylopectin 15.8±1.21 * Values presented are average of triplicates ± Standard deviation 22

35 4.2 To optimize the fermentation variables for maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori. Selection of best suitable substrate containing mainly carbohydrate source such as starch for the production of bioethanol is a primary key factor and an extremely significant step. It is known that substrate provides the required energy and substratum for co-culture to grow and produce the desired end product. For attaining the objective, various experiments were conducted with waste potato tubers as substrate in both the techniques of fermentation i.e. Solid State Fermentation (SSF) and Simultaneous Saccharification and Fermentation (SiSF) at different temperatures, ph and incubation periods for achieving the maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTTC The efficacy of substrate i.e. potato was assessed by the yield of bioethanol as expressed in terms of percentage Effect of incubation temperature on bioethanol yield at different incubation periods in SSF Effect at incubation temperature of 28 o C Observations recorded and presented in Table 4.2 on yield of bioethanol using co-culture i.e. Scaccharomyces cerevisiae and Aspergillus awamori from the process of solid state fermentation (SSF) showed that a t an incubation temperature of 28 o C, co-culture produced maximum yield (5.8%) of bioethanol at an incubation period of 168 hr. The lowest yield of bioethanol (4.4%) was recorded at a incubation period of 120 hr whereas at a incubation period of 144 hr, it was found to be 5.3%. The yield of bioethanol was further recorded which declined to 5.5% at a incubation of period of 192hr. (Fig 4.1) Effect at incubation temperature of 30 o C At an incubation temperature of 30 o C, the bioethanol yield using coculture was found to be maximum 6.7% at a incubation period of 168 hr whereas, the lowest was found to be 4.5% at a incubation period of 120 hr. The bioethanol yield was recorded as 5.5 % at incubation period of 144 hr. It 23

36 was also found that the yield of bioethanol got decreased to 6.3 % at a incubation period of 192 hr Effect at incubation temperature of 32 o C At an incubation temperature of 32 o C, the bioethanol yield employing co-culture was found to be maximum and recorded as 5.6% after a incubation period of 168 hr. The bioethanol yield was found to be the lowest 4.2% at a incubation period of 192 hr whereas, it was recorded as 5.4% at a incubation period of 144 hr. A relatively low yield of bioethanol was observed and recorded as 4.6% at a incubation period of 120 hr. Table 4.2: Effect of incubation temperature on bioethanol yield at Substrate taken - 20 g Water added S. No different incubation period in SSF method ml Incubation period (hr) Yield of bioethanol (%) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ± * Values presented are average of triplicates ± Standard deviation 4.3 Effect of ph 5.5 on yield of bioethanol in Simultaneous Saccharification and Fermentation (SiSF) at different incubation temperatures and incubation periods. Different observations recorded on yield of bioethanol by taking 5 g substrate with 96 ml distilled water at initial ph of 5.5 on different incubation temperatures viz. 28, 30 and 32 C and incubation periods viz. 120,144,168, and 192 hr are presented in Table

37 4.3.1 Effect at incubation temperature of 28 C Observations recorded showed that at a incubation temperature of 28 C, co-culture gave maximum yield of 4.3% after 168 hr of incubation period and minimum yield of 3.0% resulted at initial120 hr of incubation period whereas after 144 and 192 hr of incubation period, the values were recorded as 3.4 and 3.7 % respectively (Fig 4.2) Effect at incubation temperature of 30 C The maximum yield of bioethanol using co-culture was found to be 4.8% at a incubation period of 168 hr and thereafter it got decreased to 4.6% at a incubation period of 192 hr. The bioethanol yield at a incubation period of 120 hr was recorded 3.5% whereas it was 3.8% at incubation period of 144 hr Effect at incubation temperature of 32 C The maximum yield of bioethanol using co-culture at a incubation temperature of 32 C and incubation period of 168 hr was recorded highest 3.8 % whereas it was recorded lowest to 2.8% at a incubation period of 192 hr. The values of bioethanol yield were found to be 3.3 and 3.5% at a incubation period of 120 and 144 hr respectively. Table 4.3: Effect of ph 5.5 on yield of bioethanol in SiSF method at Substrate taken - 5 g Water added S. No different incubation temperatures and incubation periods ml Incubation period (hr) Yield of bioethanol (%) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ± * Values presented are average of triplicates ± Standard deviation 25

38 4.4 Effect of ph 6.0 on yield of bioethanol in Simultaneous Saccharification and Fermentation (SiSF) at different incubation temperatures and incubation periods. Different observations made on yield of bioethanol by taking 5 g substrate with 96 ml distilled water at initial ph 6.0 on different incubation temperatures viz. 28, 30 and 32 C with different incubation periods viz. 120, 144, 168, and 192 hr are presented in Table Effect at incubation temperature of 28 C Observations recorded showed that by maintaining incubation temperature of 28 C, co-culture gave maximum yield of 4.8% at a incubation period of 168 hr whereas lowest yield of 4.4% was recorded at 120 hr of incubation period. The values of bioethanol yield were found to be 4.5% and 4.6% respectively at 144 and 192 hr of incubation period (Fig 4.3) Effect at incubation temperature of 30 C The maximum yield of bioethanol by employing co-culture was recorded as 5.2% at a incubation period of 168 hr whereas minimum yield was found to be 4.5% at incubation period 120 hr. The value of bioethanol yield at incubation period of 144 hr and 192 hr was recorded as 4.9% and 5.1% respectively Effect at incubation temperature of 32 C The maximum yield of bioethanol by using co-culture was recorded as 4.3% at a incubation period of 168 hr whereas minimum value of 3.7% was recorded at incubation period 120 hr. The values of bioethanol yield were found to be 3.9% and 4.0% at incubation period of 144 hr and incubation period of 192 hr respectively. 26

39 Table 4.4: Effect of ph 6.0 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added S. No - 96 ml Incubation period (hr) Yield of bioethanol (%) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ± * Values presented are average of triplicates ± Standard deviation 4.5. Effect of ph 6.5 on yield of bioethanol in Simultaneous Saccharification and Fermentation (SiSF) at a different incubation temperatures and incubation periods. Various observations recorded on yield of bioethanol by taking 5 g substrate with 96 ml of distilled water at initial ph of 6.5 on different incubation temperatures viz. 28, 30 and 32 C at varied incubation periods viz. 120, 144, 168, and 192 hr are presented in Table Effect at incubation temperature of 28 C Data obtained showed that with the advancement in incubation period from 120 to 168 hr, there was a relative increase in the bioethanol yield using co-culture as a microbe for bioconversion. The values of bioethanol yield were recorded as 4.0 and 4.3% at respective incubation periods of 120 and 144 hrs. The highest yield ( 4.5%) of bioethanol was recorded at a incubation period of 168 hr where as it was 4.3% at the incubation period of 192 hr. (Fig 4.4) 27

40 Effect at incubation temperature of 30 C With application of co-culture, the maximum yield of bioethanol was found to be 5.0% at a incubation period of 168 hr. The values of bioethanol yield were recorded as 3.1, 4.4 and 4.6% at incubation period of 120, 144 and 192 hrs Effect at incubation temperature of 32 C The results obtained on the yield of bioethanol using co-culture showed that maximum yield (4.2%) was recorded at incubation period of 168 hr. It was observed that with the advancement in incubation period from 120 hr to 168 hr, there was a relative increase in the yield of bioethanol. The values of bioethanol yield were recorded as 3.6, 3.8 and 4.2% at incubation period of 120, 144 and 168 hr respectively. The bioethanol yield got reduced from 4.2% to 3.3% with the advancement in incubation period from 168 to 192 hr. Table 4.5: Effect of ph 6.5 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added S. No - 96 ml Incubation period (hr) Yield of bioethanol (%) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ± * Values presented are average of triplicates ± Standard deviation 28

41 4.6 Effect of ph 7.0 on yield of bioethanol in Simultaneous Saccharification and Fermentation (SiSF) at a different incubation temperatures and incubation periods. Different observations made on yield of bioethanol by taking 5 g substrate with 96 ml distilled water at initial ph 7.0 on different incubation temperatures viz. 28 C, 30 C and 32 C with different incubation periods viz. 120, 144, 168, and 192 hrs. are presented in Table Effect at incubation temperature of 28 C Results obtained showed that by maintaining a incubation temperature of 28 C, co-culture gave maximum yield (3.9%) at a incubation period of 168 hr whereas lowest yield of 2.8% was recorded at 192 hr of incubation period. The values of bioethanol yield were found to be 3.0% and 3.2% respectively at 120 and 144 hr of incubation period (Fig 4.5) Effect at incubation temperature of 30 C The maximum yield of bioethanol by employing co-culture was recorded as 4.6% at a incubation period of 168 hr whereas minimum yield was found to be 3.0% at incubation period 120 hr. The values of bioethanol yield at incubation periods of 144 and 192 were recorded as 4.3 and 3.5% respectively Effect at incubation temperature of 32 C The maximum yield of bioethanol by using co-culture was recorded as 3.7% at a incubation period of 168 hr whereas minimum value of 2.5% was recorded at incubation period of 192 hr. The bioethanol yield was found to be 2.58 % at incubation period of 120 hr whereas it was recorded as 3.1% at incubation period of 144 hr. 29

42 Table 4.6: Effect of ph 7.0 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added S. No - 96 ml Incubation period (hr) Yield of bioethanol (%) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ± * Values presented are average of triplicates ± Standard deviation 4.7 Effect of incubation temperatures on residual sugar in Solid State Fermentation (SSF) at different incubation period. Various observations made on residual sugar level present in solid state fermentation (SSF) at different i ncubation temperatures viz. 28, 30 and 32 C and incubation periods viz. 120, 144, 168 and 192 hr are presented in Table Effect at incubation temperature of 28 C The finding recorded showed that by using co-culture, the amount of residual sugar got reduced from to mg/ml of fermented broth with the advancement in incubation period from 120 hr to 144 hr. The amount of residual sugar was recorded as mg/ml of fermented broth at incubation period of 168 hr followed by the amount of residual sugar ( mg/ml of fermented broth) which got reduced with the advancement in incubation period up to 192 hr. (Fig 4.6) Effect at incubation temperature of 30 C The observation showed that the values of initial sugar (1.468mg/ml) in solid state fermentation got reduced with the advancement in incubation 30

43 period and recorded as 0.816, 0.387, and mg/ml of fermented broth at the end of incubation periods of 120, 144, 168 and 192 hr. The lowest amount of residual sugar ( 0.143mg/ml of fermented broth) was recorded at the end of incubation period of 192 hr. Table 4.7: Effect of incubation temperature on residual sugar in solid state fermentation (SSF) at different incubation periods. Substrate taken - 20 g Water added Initial sugar S. No - 80 ml mg /ml in sample Incubation period (hr) Residual sugar after Fermentation (mg/ml of fermented broth) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ±0.049 * Values presented are average of triplicates ± Standard deviation Effect at incubation temperature of 32 C Using co-culture in the process of fermentation, it was observed that residual sugar in solid state fermentation got reduced from initial mg/ml to the extent of mg/ml of fermented broth at the end of incubation period of 192 hr. The values of residual sugars were recorded as 0.978, and mg/ml of fermented broth at the end of incubation periods of120, 168 and 192 hr respectively. 31

44 4.8. Effect of ph 5.5 on residual sugar in Simultaneous Saccharification and Fermentation (SiSF) at different incubation temperatures and incubation periods. Various observations made on amount of residual sugar levels at initial ph of 5.5 on different incubation temperatures viz. 28, 30 and 32 C and incubation periods viz. 120, 144, 168 and 192 hr are presented in Table Effect at incubation temperature of 28 C The result obtained showed that with the application of co-culture in the fermentation process, the amount of 0.489mg/ml initial sugars present at 0 day in sample which got reduced to mg/ml with increase of incubation period upto 192 hr. Data obtained showed that the values of , , and mg/ml of residual sugar were found at 120,144,168 and 192 hr respectively. The lowest value of residual sugar ( mg/ml of fermented broth) was observed in incubation period of 192 hr of incubation period. (Fig 4.7) Effect at incubation temperature of 30 C Using co-culture for fermentation, the values of residual sugars were significantly reduced up to 192 hr of incubation periods. Observation showed that the values of , , and mg/ml residual sugar were found in fermented broth at incubation periods of 120, 144, 168 and 192 hr respectively. These observations indicate that during fermentation the amount of initial sugar (0.489) got reduced to mg/ml after 192 hr of incubation periods Effect at incubation temperature of 32 C The amount of residual sugar present in the fermented broth at 32 C showed that residual sugar got decreased with increase in incubation period up to 192 hr. The observations showed that the values of mg/ml at 120 hr and mg/ml were obtained after 144 hr of incubation period. Later on these values were decreased with increase in incubation periods. Data recorded that the values of mg/ml and mg/ml residual sugar 32

45 were found after 168 and 192 hr respectively. The minimum value mg/ml of residual sugar was found at incubation period of 192 hr. Table 4.8: Effect of ph 5.5 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added - 96 ml Initial sugar mg /ml in sample S. No Incubation period (hr) Residual sugar after Fermentation (mg/ml of fermented broth) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ±0.013 * Values presented are average of triplicates ± Standard deviation 4.9. Effect of ph 6.0 on residual sugar in Simultaneous Saccharification and Fermentation (SiSF) at different incubation temperatures and incubation periods. Various observations made on the contents of residual sugar left after fermentation on different incubation temperatures viz. 28, 30 and 32 C and incubation periods viz. 120, 144, 168 and 192 hr are presented in Table Effect at incubation temperature of 28 C Data obtained on the amount of residual sugar present in the fermented broth using co-culture showed that the values of residual sugars got decreased with the advancement in the incubation period up to 192 hr. The amount of residual sugar recorded as , , , and mg/ml of fermented broth at incubation periods of 120, 144, 168 and 192 hr. (Fig 4.8) 33

46 Effect at incubation temperature of 30ºC With the application of co-culture, the level of residual sugar also got reduced from mg/ml to mg/ml with the increase in the incubation period from 120 to 192 hr. The amount of residual sugar found ( mg/ml) at 120 hr was reduced to mg/ml at 192 hr of incubation period. The amount of initial sugar (0.489) mg/ml at zero day got reduced to mg/ml at 192 hr of incubation periods Effect at incubation temperature of 32ºC The values of residual sugars obtained during the process of fermentation using co-culture showed the decreasing trend of values with the increase in incubation period up to 192 hr. The values were recorded as , , and mg/ml of fermented broth at incubation periods of 120, 144, 168 and 192 hr. A relatively higher value ( mg/ml of residual sugars in fermented broth) was obtained after incubation of 120 hr. Table 4.9: Effect of ph 6.0 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added - 96 ml Initial sugar mg /ml in sample S. No Incubation period (hr) Residual sugar after Fermentation (mg/ml of fermented broth) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ± * Values presented are average of triplicates ± Standard deviation 34

47 4.10. Effect of ph 6.5 on residual sugar in Simultaneous Saccharification and Fermentation (Si SF) at different incubation temperatures and incubation period. Various observations made on residual sugars left unused in the fermentation process on a ph of 6.5 at different incubation temperatures viz. 28, 30 and 32ºC and incubation periods viz. 120, 144, 168 and 192 hr are presented in Table Effect at incubation temperature of 28ºC The residual sugars present in fermented broth during the process of fermentation with the application of co-culture revealed that the residual sugar content got reduced with the progressive increase in the incubation period from 120 to 192 hr. The values were recorded as , , and mg/ml in fermented broth at 120, 144,168 and 192 hr respectively. As a result of better fermentation, the value of residual sugar showed a little lower value ( mg/ml of fermented broth) after incubation at 192 hr (Fig 4.9) Effect at incubation temperature of 30ºC The residual sugars present in the fermented broth using co-culture during the process of fermentation showed the similar pattern as seen earlier under different conditions. The values of residual sugars were recorded as, , , and mg/ml of fermented broth at the incubation periods of 120, 144, 168 and 192 hr respectively Effect at incubation temperature of 32ºC The values of residual sugars present in the fermented broth during the process of fermentation using co-culture showed that the minimum value ( mg/ml) of residual sugar was obtained in fermented broth at a incubation period of 192 hr whereas maximum amount ( mg/ml) of residual sugar was present in the fermented broth after incubation period of 120 hr. The values of residual sugar were recorded as and

48 mg/ml of residual sugar obtained in fermented broth at the incubation periods of 144 and 168 hr. Table 4.10: Effect of ph 6.5 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 55 g Water added - 96 ml Initial sugar mg /ml in sample S. No Incubation period (hr) Residual sugar after Fermentation (mg/ml of fermented broth) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ±0.009 * Values presented are average of triplicates ± Standard deviation Effect of ph 7.0 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Different observations made on the contents of residual sugar left after fermentation on different incubation temperatures viz. 28, 30 and 32 C and incubation periods viz. 120, 144, 168 and 192 hr have been presented in Table Effect at incubation temperature of 28 C Data obtained on the amount of residual sugar present in the fermented broth using co-culture showed that the value of residual sugars got decreased with the advancement in the incubation period and recorded as 36

49 0.2493, , and mg/ml of fermented broth at incubation periods of 120, 144, 168 and 192 hr. (Fig 4.10) Effect at incubation temperature of 30 C With the application of co-culture, the level of residual sugar also got reduced with the increase in the incubation period from 120 to192 hr and it got decreased at 120 and 144 hr and recorded respectively as , , and mg/ml of fermented broth Effect at incubation temperature of 32 C The values of residual sugars obtained during the process of fermentation using co-culture showed the decreasing trend of values with the increase in incubation period up to 192 hr. The values were recorded as , , and mg/ml of fermented broth at incubation periods of 120, 144, 168 and 192 hr. A relatively higher value ( mg/ml of residual sugars) in fermented broth was obtained at incubation of 120 hr. Table 4.11: Effect of ph 7.0 on residual sugar Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added - 96 ml Initial sugar mg /ml in sample S. No Incubation period (hr) Residual sugar after Fermentation (mg/ml of fermented broth) Temperature ( C) ± ± ± ± ± ± ± ± ± ± ± ±0.059 * Values presented are average of triplicates ± Standard deviation 37

50 4.12. To evaluate the quality of bioethanol produced The quality of bioethanol produced from co-culture (yeast and fungi) using two different methods, SSF and SiSF of fermentation was assessed by determining the various quality attributes such as density, viscosity and boiling point. Data presented in Table 4.12 revealed that the bioethanol produced was a volatile, flammable, colourless liquid with a pleasant smell. It was found to be completely miscible with water and organic solvents Density: Data presented in Table revealed that the values of density of bioethanol produced from co-culture of S. cerevisiae MTCC 170 and A. awamori MTCC 8840 using SSF and SiSF methods of fermentation were found respectively as ±0.232 and ± g/ml at a temperature of 28 C Viscosity: Data presented in Table 4.12 indicated that the values of viscosity of bioethanol produced from co-culture using SSF and SiSF methods of fermentation were found 0.98±0.011 and 0.99±0.106 centipoise respectively Boiling point: Data presented in Table 4.10 showed that the values of boiling point of bioethanol produced from co-culture using SSF and SiSF method of fermentation were recorded as 78.1±0.305 and 78.2±0.208 C respectively. Table 4.12: Quality attributes. Quality attributes SSF SiSF Density (g/ml) ± ±0.164 Viscosity (centipoise) 0.98± ±0.106 Boiling point ( C) 78.1± ±0.208 * Values presented are average of triplicates ± Standard deviation 38

51 DISCUSSION The discussion on the results of following objectives has been presented under the heads as mentioned below. 5.1 To analyze the major chemical constituents of waste potatoes collected from different locations. 5.2 To optimize the fermentation variables for maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori 5.3 To evaluate the quality of bioethanol produced. 5.1 Chemical composition of waste potato tubers for suitability in the production of bioethanol. The data presented in Table 4.1 on chemical composition of waste potato revealed that various chemical constituents viz. moisture, dry matter, starch, amylose and amylopectin were having the similar composition as reported in the literature, although some variations in the values were observed (Izmirlioglu and Demirci 2010, 2012 ; Rani et al. 2010; Rai et al. 2013; Indhane 2014). In this investigation, the minor difference in the values of various chemical constituents observed in the substrate (potato t ubers) might be due to the genetic variability and purity of the materials taken by various workers in earlier studies. In addition to these, environmental conditions and other factors might have also played some role in influencing the composition of various constituents. On the basis of results obtained on chemical composition of waste potatoes, it was concluded that the contents of carbohydrate source present in substrate were found to be optimum required for bioconversion into bioethanol by undergoing fermentation using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC

52 5.2 Optimization of fermentation variables for maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori. In this investigation, various experiments were conducted on waste potato using both the method of fermentation i.e. Solid State Fermentation (SSF) and Simultaneous Saccharification and Fermentation (SiSF) at different temperatures, ph and incubation periods for obtaining the maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori Effect of incubation temperatures on bioethanol yield at different incubation periods in SSF. In the present study, bioethanol yields varied to a great extent using co-culture of the yeast and fungi. The observations presented in Table 4.2 indicate the maximum bioethanol concentration 6.7% was obtained at a incubation period of 168 hr having maintained optimum incubation temperature of 30 C from co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC The values of bioethanol yield were found minimum and recorded as 4.2% from the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 at incubation temperature of 32 C and incubation period of 192 hr. It was interesting to note that with the advancement in incubation period from 120 to 168 hr, there was a relative increase in bioethanol yield and thereafter it got reduced at incubation period of 192 hr using co-culture of yeast and fungi. Various workers have also reported the similar pattern of bioethanol yield using yeast and fungi (Manikandan and Viruthagiri, 2010; Rani et al. 2010; Magdy et al. 2011; Praveenkumar et al. 2014; Choonut et al. 2014; Belal et al. 2015). Manikandan and Viruthagiri (2010) reported the maximum ethanol yield of g/l at the optimum temperature of 30 C from SSF using corn flour. Rani et al. (2010) also observed 59.9 g/l bioethanol obtained at 30 C temperature after 48 hr incubation periods from potato flour using yeast. Similarly, Choonut et at. (2014) recorded maximum bioethanol found 9.69 g/l at 30 C from pineapple peel using Saccharomyces cerevisiae and Enterobacter aerogenes. Likewaise, Belal et al. (2015) observed 9.1% bioethanol yield produced from 40

53 the hydrolysis of waste potato powder at 30 C under solid state fermentation condition. In the present investigation, it was observed that the bioconversion efficiency of starch into bioethanol was greater due to optimum growth, metabolism and survival of the fermenting organism. Hence it was concluded that fermentation at 30 C temperature with 168 hr incubation period was found optimum for maximum bioethanol production under solid state condition with co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC The findings obtained in the present investigation showed that these are in agreement with the reported observations by earlier workers. Although some variations observed in the values in present investigation might be due to the genetic variability of the strains used and culture conditions maintained Optimization of ph, incubation temperature and incubation period in Simultaneous Saccharification and Fermentation (SiSF) usi ng the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC In the present study, various observations have been made on yield of bioethanol under different ph conditions (5.5, 6.0, 6.5 and 7.0) with different incubation temperatures (28, 30 and 32 C) and incubation periods (120, 144, 168 and 192 hr) for achieving the maximum yield of bioethanol using the coculture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC The findings in the present investigation revealed that bioethanol yield varied to a great extent employing co-culture of yeast and fungi in the process of fermentation. The observations recorded in Table 4.3, 4.4, 4.5 and 4.6 indicated that the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 gave maximum yield of bioethanol (5.2%) at a incubation temperature of 30 C with incubation period of 168 hr and having maintained the ph at 6.0. It is presumed that the rate of fermentation typically increased at 30 with increase in incubation period upto 168 hr. However the bioethanol yield further got decreased at a incubation period of 192 hr. It is also presumed that when the temperature and incubation period increase after the optimum condition, the percentage of bioethanol might also decrease 41

54 as the enzymes begin to denature and unfold and thus become inactive. Several workers have also reported the bioethanol yield almost in the similar range from bioconversion of starch rich substrates using yeast and fungi (Abouzied et al. 1986; Itelima et al.2013; Rath et al. 2014; Azad et al. 2014). Abouzied et al. (1986) observed the effect of initial ph on direct fermentation of potato starch to ethanol by co-culture of A. niger and S. cerevisiae and found maximum yield 1.59 g/100 ml medium of ethanol at incubation period of 168 hr with 5.5 ph using 10% starch under anaerobic condition whereas the lowest yield 0.78 g/100 ml medium was reported at a incubation period of 24 hr under aerobic condition. Similarly, Itelima et al. (2013) reported that optimal bioethanol yield v/v was obtained at 7 days of fermentation using Simultaneous Saccharification and Fermentation of Corn Cobs employing Co- Culture of Aspergillus niger and Saccharomyces cerevisiae. Rath et al. (2014) also evaluated the efficacy of two methods of fermentations and found that SSF method was relatively more effective than SiSF method in the bioconversion of potato starch into bioethanol Effect of incubation temperatures on residual sugar in Solid State Fermentation (SSF) at different incubation periods. In the present investigation, various observations recorded in Table 4.7 showed that initial sugar present in the fermentation medium got reduced relatively with the progressive increase in incubation period up to 192 hr irrespective of the incubation temperature (28, 30 and 32 C) used in the solid state fermentation. It was also observed that there was a relatively more decrease in the level of residual sugar upto fermentation period of 168 hr proceeding to 192 hr using this co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC It was also observed that the level of residual sugar after fermentation was found to be minimum (0.143 mg/ml of medium) at a incubation temperature of 30 C and incubation period of 192 hr. These observations indicated that the enzymatic hydrolysis of sugar must have taken place at a higher rate under the above mentioned fermentation conditions resulting in maximum reduction of sugar and in turn giving rise to maximum production of bioethanol. Various workers have also 42

55 reported the level of residual sugars with respect to initial sugar level, incubation temperature and period of incubation (Hoskins and Lyons, 2009 ; Rai et al. 2013). The similar trend of bioconversion of sugar into ethanol resulting in the reduction of residual sugar level at different time intervals was obtained in present investigation Effect of ph on residual sugar in Simultaneous Saccharification and Fermentation (SiSF) at diffe rent incubation temperatures and incubation periods. In the present investigation, observations recorded in Table 4.8, 4.9, 4.10 and 4.11 on different levels of ph (5.5, 6.0, 6.5 and 7.0 ) at different incubation temperatures (28 C, 30 C and 32 C) and incubation periods (120, and 192 hr) showed that the bioconversion of sugar into bioethanol was relatively low at a ph of 5.5 and 7.0 as compared to ph of 6.0 and 6.5 irrespective of the incubation temperature, incubation period used in the process of fermentation. It was also observed that ph of 6.0 was found to be optimum for better conversion of sugar using co-culture at a incubation temperature of 30 C and incubation period of 168 hr. The findings in the present investigation showed that the levels of residual sugar were found minimum under above mentioned fermentation conditions. The reason for higher efficiency of conversion of sugar might be the higher activity of enzymes involved in the hydrolysis of sugar into bioethanol. Several reports have also been published in the literature on the utilization of sugar in simultaneous saccharification and fermentation (SiSF) process under varied fermentation conditions (Ado et al. 2009; Rai et al. 2013). Ado et al. (2009) reported that using co-culture of A. niger and S. cerevisiae under optimized conditions such as incubation temperature, incubation period and ph, the sugar concentration present in cassava starch reduced from 0.24 g/100 ml on first day of fermentation to 0.01 g/100 ml on the seventh day in simultaneous saccharification and fermentation (SiSF) method. It was further reported that the concentration of sugar got reduced rapidly and consistently during 24 hr of fermentation and thereafter decrease was found to be gradual upto 96 hr of incubation period. Rai et al (2013) also 43

56 reported that Initial sugar mg/ml present at zero day was converted into bioethanol and mg/ml minimum residual was found in fermented broth at ph 5.5 and 35 C temperature after 120 hr of incubation periods. These findings in the present investigations are in agreement with the result of earlier workers as reported above. 5.3 Quality evaluation of bioethanol produced In this investigation, various quality parameters viz. density, viscosity and boiling point were evaluated on bioethanol production by solid state fermentation (SSF) and simultaneous saccharification and fermentation (SiSF) techniques using the co-culture of yeast Saccharomyces cerevisiae MTCC 170 and fungi Aspergillus awamori MTCC Density In this experiment, density of bioethanol, produced by SSF and SiSF methods using the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 was estimated. The observations recorded in Table 4.12 showed that the density of bioethanol produced by SSF method using the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 was ±0.232 g/ml whereas it was recorded as ±0.164 g/ml for bioethanol produced by the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 using SiSF method. Some reports have also been published in the literature on density of bioethanol under varied fermentation conditions (Caylak and Sukan 1996; Meenakshi and Kumaresan 2014; Patil 2014). Caylak and Sukan (1996) reported the final ethanol concentration of g/l equivalent to g/ml density of bioethanol. The findings in present investigation are in agreement with the reported observations by earlier workers. Patil (2014) reported that the values of density of bioethanol produced by SSF and SiSF methods using the co-culture of Saccharomyces cerevisiae MTCC 170 and Zymomonas mobilis MTCC 2427 were g/ml and g/ml respectively. 44

57 5.3.2 Viscosity The observations recorded in Table 4.12 showed that the viscosity value of bioethanol produced by SSF method using the co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 was recorded as 0.98±0.011 centipoise whereas it was recorded as 0.99±0.106 centipoise for the bioethanol produced from the same co-culture using SiSF method. Several workers have also reported the viscosity of bioethanol under varied fermentation conditions (Ghobadian et al. 2008; Rai et al. 2013; Meenakshi and Kumaresan 2014). Ghobadian et al. (2008) studied the production of bioethanol and sunflower methyl ester and investigated fuel blend properties and reported the viscosity of ethanol as 1.10 centipoise. Rai et al. (2013) also observed that viscosity of bioethanol was found 1.02 and 1.07 centipoise at SSF and SiSF method respectively. The values of viscosity of bioethanol in the present investigation also indicated the similar pattern as reported by earlier workers Boiling point The observations recorded in Table 4.12 showed that the values of boiling point of bioethanol produced using the co-culture were found to be 78.1±0.305 and 78.2±0.208 C respectively for SSF and SiSF fermentation methods. Several reports (O Leary 2000; Ghobadian et al. 2008; Zainab and Fakhra 2014) have also been published in the literature on similar range of boiling point of bioethanol as recorded in the present investigation. Zainab and Fakhra (2014) reported that boiling point of the bioethanol produced by Saccharomyces cerevisiae was found at 79± 1 C. 45

58 SUMMARY, CONCLUSION AND SUGGESTIONS FOR FURTHER WORK 6.1 Summary The present investigation entitled " Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori was carried out in the Fermentation Technology Laboratory of Biotechnology Centre, JNKVV, Jabalpur with the objectives to analyse major chemical constituents of waste potatoes collected from different locations, optimization of fermentation variables for maximum yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori and quality evaluation of bioethanol produced from the co-culture. In the first part of this study on chemical composition of waste potatoes for suitability in the production of bioethanol, different chemical constituents viz. moisture, dry matter, starch, amylose and amylopectin present in waste potatoes were analysed. The results obtained showed that the waste potatoes were found to contain a good amount of carbohydrate/starchy source required for bioconversion into bioethanol. In second part of this study on optimization of fermentation variables for maximum recovery of bioethanol using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840, key factors were optimized in solid state fermentation (SSF) and simultaneous saccharification and fermentation (SiSF) method for obtaining better recovery of bioethanol. In order to get maximum recovery of bioethanol, three levels of incubation temperatures (28, 30 and 32 C) and four levels of incubation periods (120, 144, 168 and 192 hr) with 20 g substrate and 80 ml distilled water were employed for solid state fermentation (SSF) whereas in simultaneous saccharification and fermentation (SiSF) four levels of ph (5.5, 6.0,6.5 and 7.0), three levels of temperatures (28, 30 and 32 C) and four levels of incubation periods (120, 144, 168 and 192 hr) were employed with 5 g substrate and 96 ml distilled water for bioethanol production using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 46

59 8840. The results of various experiments revealed that with the SSF technique, the highest yield of bioethanol ( 6.7%) using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 was obtained at incubation temperature of 30 C after 168 hr of incubation period. In case of simultaneous saccharification and fermentation (SiSF), the findings of various experiments revealed that by employing co-culture of yeast and fungi the highest yield of bioethanol (5.2%) was obtained at a ph of 6.0 with incubation temperature of 30 C after 168 hr of incubation period with the residual sugar left to a minimum of ±0.004 mg/ml of fermented broth under a set of above mentioned fermentation variables. In the third part of this study on quality evaluation of bioethanol produced from solid state fermentation (SSF) and simultaneous saccharification and fermentation (SiSF) using co -culture of the yeast (Saccharomyces cerevisiae MTCC 170) and fungi ( Aspergillus awamori MTCC 8840), various quality attributes viz. density, viscosity and boiling point of bioethanol were analysed. The results analysed showed that with the SSF technique, the density of bioethanol produced by co-culture was found to be ±0.232 g/ml, whereas the viscosity value for bioethanol produced by co-culture was found to be 0.98±0.011 centipoise. Likewise, the value of boiling point of bioethanol produced by co-culture was found to be 78.1±0.305 C. In case of SiSF method, the density of bioethanol produced from co-culture was found to be ±0.164 g/ml whereas the viscosity of bioethanol produced by co-culture was found to be 0.99±0.106 centipoise. Similarly, the value of boiling point of bioethanol produced from co-culture was found to be 78.2±0.208 C. 47

60 6.2 Conclusion The salient features of this study are presented below: 1. The major chemical constituents showed that the waste potatoes contained a good amount of starch and could be used as substrate for bioconversion into bioethanol. 2. Using the method of solid state fermentation (SSF), highest yield (6.7%) was obtained at incubation temperature of 30 C after incubation period of 168 hr using co-culture of Saccharomyces cerevisiae MTCC 170 Aspergillus awamori MTCC In simultaneous saccharification and fermentation (SiSF) method, highest yield (5.2%) of bioethanol was obtained using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 at incubation temperature of 30 C after incubation period of 168 hr at ph of Using the method of solid state fermentation (SSF), the lowest residual sugar level (0.143 mg/ml of fermented broth) was obtained at incubation temperature of 30 C after incubation period of 192 hr using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC In simultaneous saccharification and fermentation (SiSF), the lowest residual sugar level ( mg/ml of fermented broth) was obtained using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 at incubation temperature of 30 C with ph maintained at 6.0 after incubation period of 192 hr. 6. Various quality attributes viz. density, viscosity and boiling point of bioethanol produced from both the methods of fermentation (SSF and SiSF) using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 indicated the desirable characteristics required for better quality bioethanol. 48

61 6.3 Suggestions for further work Although the present study has generated many useful information on various aspects of fermentation process for attaining the maximum yield of bioethanol. This investigation has also thrown some light on the feasibility of fermentation techniques for bioconversion of carbohydrate source into bioethanol. Yet, some more work on bioethanol should be conducted keeping in view the following aspects: 1. Different strains of microbes (bacteria, fungi and yeast) other than those used in the present study should be used. 2. Work on strain improvement should be carried out using genetic engineering for bioconversion of starch into bioethanol. 3. The process of scaling up should be carried out for higher recovery at large scale production. 4. Combination of different substrate should be used for better recovery of bioethanol. 49

62 Bioethanol yield (%) Fig 4.1: Effect of incubation temperature on bioethanol yield at different incubation period in SSF method. Substrate taken - 20 g Water added - 80 ml

63 Bioethanol Yield (%) Fig 4.2: Effect of ph 5.5 on yield of bioethanol in SiSF method at different incubation temperatures and incubation periods Substrate taken - 5 g Water added - 96 ml

64 Bioethanol Yield %) Fig 4.3: Effect of ph 6.0 on yield of bioethanol in SiSF at different Substrate taken - 5 g Water added - 96 ml incubation temperatures and incubation periods

65 Bioethanol Yield (%) Fig 4.4: Effect of ph 6.5 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods Substrate taken - 5 g Water added - 96 ml

66 Bioethanol Yield (%) Fig 4.5: Effect of ph 7.0 on yield of bioethanol in SiSF at different incubation temperatures and incubation periods Substrate taken - 5 g Water added - 96 ml

67 Residual sugar after fermentation (mg/ml of fermented broth) Fig 4.6: Effect of incubation temperature on residual sugar in solid state fermentation (SSF) at different incubation periods Substrate taken - 20 g Water added - 80 ml

68 Residual sugar after fermentation (mg/ml of fermented broth) Fig 4.7: ph 5.5 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken 5 g Water added - 96 ml

69 Residual sugar after fermentation (mg/ml of fermented broth) Fig 4.8: Effect of ph 6.0 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 5g Water added - 96 ml

70 Residual sugar after fermentation (mg/ml of fermented broth) Fig 4.9: Effect of ph 6.5 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 5g Water added - 96 ml

71 Residual sugar after fermentation (mg/ml of fermented broth) Fig 4.10: Effect of ph 7.0 on residual sugar in Simultaneous Saccharificaiton and Fermentation (SiSF) at different incubation temperatures and incubation periods. Substrate taken - 5 g Water added - 96 ml

72 Take potato powder (20g) Add 80 ml distilled water Autoclave the sample at 121 C at 15 psi for 20 min Add one day old culture of Saccharomyces cerevisiae MTCC 170 Aspergillus awamori MTCC 25 ml 1:1 in each sample under laminar air flow Incubate samples for 120,144,168 and 192 hr at different incubation temperature viz. 28, 30 and 32 C at 140 rpm Centrifuge fermented mass Filtration Distillation Fig 3.3.1: Flow chart of SSF method for bioethanol production

73 Take potato powder (5 g) Add 96 ml distilled water Adjust the ph 5.5, 6.0,6.5 and 7.0 Autoclave the sample at 121 C at 15 psi for 20 min Add one day old culture of Saccharomyces cerevisiae MTCC 170 Aspergillus awamori MTCC 25 ml 1:1 in each sample under laminar air flow Incubate samples for 120,144,168 and 192 hr at different incubation temperature viz. 28, 30 and 32 C at 140 rpm Centrifuge fermented mass Distillation Fig 3.3.2: Flow chart of SiSF method for bioethanol production

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75 trachurus): Effect on lipid and protein oxidation. Food Chemistry 131: FAOSTAT FAO Statistical data, Ferro MD, Fernandes MC, Paulino AFC, Prozil SO, Gravitis J, Evtuguin DV and Xavier AMRB Bioethanol production from steam explosion pretreated and alkali extracted Cistus ladanifer (rockrose). Biochemical Engineering Journal 104 (2015): Ghobadian B, Rahimi H, Hashjin TT and Khatamifar M Production of bioethanol and sunflower methyl ester and investigation of fuel blend properties. Journal of Agriculture Science and Technology 10: Hoskins B and Lyons M Improving bioethanol yield: The use of solid-state fermentation products grown on DDGS. Journal of the Institute Brewing 115: Hou X, Hansen JH and Bjerre AB Integrated bioethanol and protein production from brown seaweed Laminaria digitata. Bioresource Technology 197 (2015): Indhane SS Bioethanol production from waste potatoes using co-culture of Saccharomyces cerevisiae and Aspergillus niger. M.Sc. Thesis, JNKVV, Jabalpur. 24p. Itelima J, Ogbonna A, Pandukur S, Egbere J, and Salami A Simultaneous Saccharification and Fermentation of Corn Cobs to Bio-Ethanol by Co- Culture of Aspergillus niger and Saccharomyces cerevisiae. International Journal of Environmental Science and Development 4(2): Izmirlioglu G and Demirci A Ethanol production from waste potato mash by using Saccharomyces cerevisiae. American Society of Agricultural and Biological Engineers. Izmirlioglu G and Demirci A Ethanol production from waste potato mash by using Saccharomyces Cerevisiae. Applied Science 2: Joshi J Enhanced production of ethanol from red potatoes grown in hilly regions of Nepal using various nitrogen sources. International Journal of Applied Sciences Biotechnology 2(1): Khan RA Nawaz A, Ahmed M, Khan MR, Dian F, Azam N, Ullah S, Sadullah F, Ahmad A, Shah MS and Khan N Production of bioethanol through enzymatic hydrolysis of potato. African Journal of Biotechnology 11(25): Keer RW Chemistry and industry of starch. Academic press, Inc New York Kroumov AD, Modenes AN and de Araujo Tait MC Development of new unstructured model for simultaneous saccharification and fermentation of starch to ethanol by recombinant strain. Biochemical Engineering Journal 28: Lim Y, Jang Y and Kim K Production of a high concentration of ethanol from potato tuber by high gravity fermentation. Food Science and Biotechnology 22 (2):

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78 ABSTRACT The present study entitled Utilization of waste potatoes for bioethanol production using co-culture of Saccharomyces cerevisiae and Aspergillus awamori was carried out at Biotechnology Centre, JNKVV, Jabalpur with the objectives to analyze the major chemical constituents of waste potatoes collected from different locations, optimize the fermentation variables for better yield of bioethanol using co-culture of Saccharomyces cerevisiae and Aspergillus awamori and quality evaluation of bioethanol produced. In the first of study, major chemical constituents of waste potatoes were analysed. The result analysed showed that the waste potatoes were found to contain good amount of carbohydrate source required for bioconversion into bioethanol. In second part of study, main fermentation variables were optimized in solid state fermentation (SSF) and simultaneous Saccharification and fermentation (SiSF) method s. For obtaining better recovery of bioethanol, three levels of incubation temperatures (28,30 and 32 C) and four levels of incubation periods (120, 144, 168 and192 hr) with 20 g substrate and 80 ml distilled water were employed for solid state fermentation (SSF) w hereas in simultaneous Saccharification and fermentation (SiSF), four levels of ph (5.5, 6.0,6.5 and 7.0), three levels of temperatures (28,30 and 32 C) and four levels of incubation periods ( 120, 144, 168 and 192 hrs) were employed with 5 g substrate and 96 ml distilled water for bioethanol production using co-culture. The results of various experiments revealed that with the SSF technique the highest yield of bioethanol (6.7 %) using co-culture of Saccharomyces cerevisiae MTCC 170 and Aspergillus awamori MTCC 8840 was obtained at incubation temperature of 30 C after 168 hr of incubation. In case of simultaneous Saccharification and fermentation (SiSF), the results of various experiments revealed that by employing co-culture the highest yield of bioethanol (5.2%) was obtained at a ph of 6.0 with incubation temperature of 30 C after 168 hr of incubation period. In third part of this study on quality evaluation of bioethanol produced from above mentioned, various quality attributes such as density, viscosity, and boiling point of the bioethanol produced were assessed.

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