Keywords: Bio-fuel, Ethanol, Co-culture, Ethanol tolerant yeast, Simultaneous Saccharification and Fermentation. (SSF),

Transcription

1 Title: Bioethanol Fuel Production from Citrus Fruit Waste by Co- Culture of Ethanol Resistant Saccharomyces cerevisiae And Aspergillus niger. Late Dr. Simendra Singh HOD, Department of Biotechnology, SET, Sharda University Kushal paudel ) (Mentor) Ritu Ninthoujam Nyangoma Natasha Idah-mary Ankit Thapar Department of Biotechnology, School of Engineering and Technology, Sharda University, Knowledge Park III, Greater Noida, UP, India Abstract: Keywords: Bio-fuel, Ethanol, Co-culture, Ethanol tolerant yeast, Simultaneous Saccharification and Fermentation. (SSF), 1 Presently, the most cost effective way of producing commercial ethanol is by the hydration of petroleum-derived ethylene. As petroleum becomes scarce, more expensive, and have many other environmental disadvantages, this will probably change. Then, ethanol derived from an organic substrate, may become an important industrial feedstock. On the other hand, citrus juice extraction companies generates large amount of cellulosic citrus peel wastes. These peels were collected and used as feedstock for ethanol production. While the ethanol production from biomass by Consolidated Bioprocess (CBP) is considered to be the most ideal process, Simultaneous Saccharification and Fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass of citrus fruits remnant to biofuel by co-culture of two different microorganisms such as a cellulase producer Aspergillus niger and an ethanol producer Saccharomyces cerevisiae. Furthermore, the wild strain of Saccharomyces cerevisiae was made high ethanol tolerant by natural selection method and was used for fermentation which increased the yield by 3 fold than that by wild variety.

2 1. INTRODUCTION The demand of alternative fuel has reached the pinnacle as the non-renewable resources are depleting rapidly. In recent years ethanol has been earning increasing attention as a potentially cleaner, renewable, and domestically produced alternative to fossil fuels for transportation. The traditional means or first generation of biofuel production is based upon starch crops like corn and wheat and from sugar crops like sugar cane and sugar beet etc. While first generation method of ethanol production has so many disadvantage as it uses the food of humans and animals, now research is more focused on conversion of lingo-cellulosic feedstock into ethanol. This technology is seen to be developing in the 2 nd generation of biofuel production. On the other hand, citrus juice extraction companies generate high amount of waste which causes significant disposal difficulties. In 2010 the production was estimated to reach 66.4 million metric tons, this is an increase with 14% compared with the levels of [1]. This waste contains lingo-cellulosic citrus fruit remnant like peels and pulps, which can be converted into the ethanol. Still this process is expensive than traditional method, but with simultaneous saccharification of lingo-cellulose by hyper cellulose producer Aspergillusniger and fermentation by ethanol producer Saccharomyces cerevisiae by consolidated bioprocess in single reactor is comparatively cheaper and effective. Furthermore, ethanol is known as microbial inhibitor as it cause mitochondrial DNA damage and degrades bio-membranes [2] of Saccharomyces cerevisiae and inactivate some enzymes such as hexokinase and dehydrogenase [3]. Therefore, in industrial production, ethanol tolerance of Saccharomyces cerevisiae is one of important factor for product optimization. [3] The wild type of Saccharomyces cerevisiae can be made somewhat tolerance to high ethanol concentration by natural selection method and resistant variety can be used to increase production. 2. MATERIALS AND METHODS i. Collection of materials Culture of Saccharomyces cerevisiae and Aspergillus niger in Potato Dextrose Agar media culture media was collected from Division of Plant Pathology, Indian Agricultural Research Institute in New Delhi. The culture was incubated at 25 C. Substrate for ethanol production (citrus fruit peels) was collected from the local juice vendors. iii. Preparation of inoculum 250 ml of each inoculum seed was prepared by culturing A. niger (in 2% peptone and 2% dextrose sugar solution), wild and resistant variety Saccharomyces cerevisiae (in 2% peptone, 2% dextrose and 1% yeast extract) in two different flask. Then it was incubated in rotator shaker till it was ready for use. 2 ii. Development of ethanol tolerant yeast. The wild type Saccharomyces cerevisiae was subcultured every weekend in Potato Dextrose Agar media with increasing ethanol concentration. Starting from 1ml of 1% ethanol everyday for first week, 1ml of 5% ethanol for second week and likewise up to 45% ethanol concentration till 10 weeks was added to the subculture. As at 45% ethanol concentration, the culture was not showing growth, then the culture was forced to grow in 45% ethanol concentration media by adding few nutrition (2% peptone, 2% dextrose and 1% yeast extract) and similar procedure (1ml of 45% of ethanol per day and subculture every weekend) was followed for next 5 weeks. To check if yeast had developed high ethanol tolerance, both wild type and experimented variety of yeast were culture at 45% ethanol.

3 iv. Optimization of ph. The substrate was grinded and made slurry by adding distilled water. The substrate was autoclaved. Two conical flasks with each 50ml of slurry were taken and ph was set to 5 and 6. 5 ml inoculum each of A. niger and wild type S. cerevisiae was added and incubated at 35 C in rotatory shaker for 4 days. The ethanol concentration was determined by spectrometric micro-method for determination of ethanol [4]. v. Preparation of substrate The citrus peels were thoroughly washed and blended with distilled water using clean kitchen blender. Approximately 2 liters of blended slurry was prepared. vii. Estimation of ethanol The produced ethanol concentration was estimated using spectrometric micro-method for determination of ethanol [4]. When Chromium (VI) present in potassium dichromate in the presence of perchloric acid reacts quantitatively with ethanol to form Chromium (III) and acetic acid. The reaction is completed in about 15 minutes at room temperature and the Chromium (VI) consumed during the reaction can be measured by the decrease of absorbance at 267 nm [4]. Then ethanol concentration can be calculated using following formula. 3 vi. Fermentation A 5-litre-capacity fermenter was used. 2 litres of slurry as substrate was added in it and ph was set to 5. All the seals were tightened, and the fermenter was autoclaved. 250 ml inoculum seed of each A. niger and S. cerevisiae was inoculated into it under aseptic condition. ph and temperature probes were connected and calibrated. 35 C temperature and 8000 rmp agitation speed was maintained. The fermentation system was left for 4 days before harvesting. Similarly, the fermentation was carried out with resistant variety of S. Cerevisiae. The fermentation parameters, volumes, and time were maintained same in both the cases. Volumetric Alcohol Content (%) = [4] Where, D = Dilution factor V = volume in ml of the sample used for analysis = equivalent weight of ethanol in the oxidation reaction to acetic acid (M0-M1) = amount (mol L-1) of chromium (VI) reduced to chromium (III) in the oxidation reaction of ethanol to acetic acid. The values Mi are calculated using the measured absorbance Ai applying the following the following expression in which the apparent molar absorption activities (wavelength= 267nm) of Chromium (6) in monomer ε1 =1690 mol L-1 cm-1 and dimer ε2 =4790 mol L-1 cm-1 forms and the equilibrium quotient for the dimerization reaction (k 2, 2 =277 L mol-1) appear:

4 Where: a = ε2 2 ci ) I = 0 or 1 The ethanol concentration was then estimated by taking standard deviation. viii. Distillation The single distillation was carried out with approximately 800 ml of fermented broth. The temperature was set to 80 C and left for overnight. Next morning the condensed ethanol was collected in a bottle. 3. RESULT Comparison of different ph for bioethanol production is shown in Table 1. Fermentation at ph 5 showed the highest concentration of bioethanol, compared with fermentation at ph 6 with ethanol concentration of 0.041% and 0.01% respectively. The concentration of bioethanol increased as ph reached towards 5. The absorbance reading for spectrometric micro method of estimation of ethanol for both the ph 5 and 6 is shown in following table: Table 1: Absorbance at different ph and concentration of ethanol produced. Absorbance (267 nm) ph Before evaporation After evaporation Ethanol concentration(v/v) % % 4

5 Figure 1: Graph showing ethanol concentration at different ph While growing the both types of yeast in media containing 45% (v/v) of ethanol, the developed variety of yeast had shown some growth while the wild variety of yeast did not. This experiment verifies that the supposed variety of yeast has developed some sort of ethanol tolerance characteristics in it and thus, could survive at high ethanol concentration in its environment. Because of this property, yeast biomass in fermentation can retain for long time, therefore more will be the conversion of substrate to ethanol than that by wild variety. In our study, following data shows the percentage ethanol produced using both variety of yeast. Table 2: Absorbance, ethanol concentration and SD (Standard deviation) of the fermentation broth using wild type yeast. Absorbance (267 nm) Concentration (v/v) SD Before Evaporation After Evaporation % % 5

6 Table 3: Absorbance, ethanol concentration and SD (Standard deviation) of the fermentation broth using resistant variety yeast. Absorbance (267 nm) Concentration (v/v) SD Before Evaporation After Evaporation % % Volumetric estimation of ethanol present in citrus feed fermented with co-culture of wild-type Saccharomyces cerevisae and Aspergillusniger was found to be % (v/v). On the other hand, ethanol yield increased to % when wild-type yeast strain was replaced with ethanol-resistant strain. This implied to 3-fold higher ethanol production when yeast was made tolerant to the presence of ethanol. Figure 2: Graph showing the concentration of ethanol produces (v/v) in both the cases. (Wild-type yeast and resistant-type yeast) From the distillation of 800 ml fermented broth, approximately 4 ml of bioethanol was collected from fermentation with wild variety of yeast whereas with resistant variety of yeast approximately 12 ml of bioethanol was collected. 6

7 4. CONCLUSION The findings of this project suggest that peel waste from citrus fruits can be utilized to produce ethanol. Further, the fruit waste (substrate) generated in industries after juice extraction can directly be used without any expensive pretreatment. The co-culture method optimized in this study, does not require additional supply of growth nutrients for growth of microorganisms. The two microorganisms used in this study do not affect each other s growth. Another major objective of this study was to develop high ethanol resistant variety of yeast. Our study came to the result that the ethanol produced by resistant variety of yeast is approximately 3 fold higher than that with using wild variety of yeast. Therefore, ethanol-resistant strains can be developed and used at an industrial scale for production of ethanol using any cellulosic containing waste as substrate like apple remnant, banana peels, tomatoes etc. This concept is fast and easy to handle because of consolidated bioprocess where both the fungi can be manipulated at same time. Research efforts are required to understand the changes taking place at molecular level in the ethanol-resistant strains. In future, this would help us to develop high tolerant and efficient yeast strains and it would be not only highly productive but also very cost effective production of bioethanol. 7

8 ACKNOWLEDGEMENT We are thankful to lab Department of Biotechnology, Sharda University for providing us lab and equipment for conducting of research and also to IARI (Indian Agricultural Research Institute), New Delhi, India for providing us culture of Saccharomyces cerevisiae and Aspergillus niger. We would also like to express our gratitude to Dr. Shahana Majumdar, department of biotechnology, Sharda University for her important help and support. 8

9 REFERENCE [1] Fungal multienzyme production on industrial by-products of citrus-processing industry, Mamma D., Kourtoglou E., Christakopoulos P., Bioresource Technology 99 (2008) [2] Alcohol and Mitochondria: A Dysfunctional Relationship Jan B. Hoek, Alan Cahill, and John G. Pastorino. Gastroenterology Jun; 122(7): doi: /gast [3] Effect of cellular inositol content on ethanol tolerance of Sacchromyces cerevisiae in sake brewing. Furukava K, Kitano H, Mizoguchi H, Hara S. J Bioscience Bioengineering 2004;98; [4] Spectrophotometric micro method for the determination of ethanol in commercial beverages. Andrea D. Magri, Antonio L.Magri, Fabbrizio Balestrieri, Amaliasacchini. D.Marini. Fresenius J. Anal. Chem (1997) 357: [5] One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae. Park EY1, Naruse K, Kato T. Biotechnol Biofuels Aug 31;5(1):64. doi: / [6] Bioethanol from Lignocellulosic Biomass. Xin-Qing Zhao, Li-Han Zi, Feng-Wu Bai, Hai-Long Lin, Xiao- Ming Hao, Guo-Jun Yue and Nancy W. Y. Ho. Adv Biochem Engin/Biotechnol (2012) 128: DOI: /10_2011_129. Springer-Verlag Berlin Heidelberg 2011 Published Online: 3 December [7] Hydrolysis of Lignocellulosic Biomass for Bioethanol Production. Parameswaran Binod*, K.U. Janu, Raveendran Sindhu, Ashok Pandey Centre for Biofuels, Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum , India [8] Bioethanol Production From Cellulosic Materials Umamaheswari, M., Jayakumari, M., Maheswari, K., Subashree, M., Mala, P., Sevanthi, T., and Manikandan, T.* International Journal of Current Research Vol. 1, pp , January, ISSN: X. PG and Research Department of Botany, Arignar Anna Government Arts College, Villupuram , Tamilnadu, INDIA. 9

10 FIGURES: Figure 1: A.niger, wild type yeast and resistant yeast inoculums. Figure 2: Grinding of citrus peels and setting the ph. 10

11 Figure 4: The ongoing fermentation process. 11