Manuscript accepted on :April 22, 2011
Published online on: 26-11-2015
Views: (Visited 674 times, 1 visits today)
PDF Downloads:
458N. Uma Maheswari and Kalaikodi M
PG and Research Department of Microbiology, Sengamala Thayaar Educational Trust Women’s College, Mannargudi - 614 001 (India)
Abstract
Ethanol has been known for a long time, being perhaps the oldest product obtained through traditional biotechnology. Ethanol production from cassava starch by co-cultures of selected such as Aspergillus oryzae and Rhizopus oryzae under submerged fermentation. The present study was aimed to evaluate the role of some fermentation parameters like ethanol yield, residual sugar, cell density, pH and temperature on ethanol production from cassava waste by Aspergillus oryzae and Rhizopus oryzae. At 1%, 1% and 10% substrate concentration, 10% gave a better ethanol production. Aspergillus oryzae inoculated medium gave better ethanol production compare to Rhizopus oryzae inoculated medium. The maximum ethanol yield was noted in the combination of Aspergillus oryzae and Rhizopus oryzae 6.2±0.17, residual sugar 2.7±0.17, celldensity 2.9±0.57. The highest ethanol yield was achieved at pH 6(8.7±0.17) and temperature (7.4±0.17) for Aspergillus oryzae.
Keywords
Ethanol; submerged fermentation; Aspergillus oryzae; Rhizopus oryzae; cassava
Download this article as:
| Copy the following to cite this article: Maheswari N. U, Kalaikodi M. Ethanol Production from Cassava by Co-Cultures of Aspergillus oryzae and Rhizopus oryzae. Biomed Pharmacol J 2011;4(1) |
| Copy the following to cite this URL: Maheswari N. U, Kalaikodi M. Ethanol Production from Cassava by Co-Cultures of Aspergillus oryzae and Rhizopus oryzae. Biomed Pharmacol J 2011;4(1). Available from: http://biomedpharmajournal.org/?p=1776 |
Introduction
Total demand for ethanol is expected to reach amount 3X1011 in 20101 . However due to increasing petroleum shortage, fermentation production of ethanol from renewable resources has received considerable attention2 . Now-a-days, fermentation technology produces nearly 80% ethanol as clean fuel. Ethanol production by fermentation process has been studied extensively for several decades. Over the last few years, new approach with great potential for ethanol production from starchy materials and lignocellulosic biomass have been used, that is simultaneous saccharification fermentation (SSF) process3 and Separate Hydrolysis and fermentation (SHF) process4.
Cassava (Manihotesculentacrantz), a species of sub-bush, is perennial plant which grows a circular roots that are greatly different from quantity ant size because of different variety. Cassava root contains high starch content while it holds less protein, lipids and ash. The fresh root of some good variety of cassava includes 25%-32% starch content on which cassava is shown as a good raw material for starch. Cassava pulp, a fibrous residual material, is a by product of starch manufacturing; It generates as solid waste and result in creation of around 10-25% of the original cassava root weight5. It is used as a carbohydrate source in balanced feed, replacing wheat flour and synthetic binders and forming depending on the manufacture. Ethanol is generally produced by the fermentation of sugar, cellulose, or converted starch and has a long history. The first type of plant will produce a strong alcohol from cassava, but this will have an odor as the distilling process is very crude. It is potential source of ethanol production based on the high yields and low cost6 reported the ethanol production by two steps from cassava pulp.
Submerged fermentation define does not have the problem of contamination, pH adjustment etc., which is a great problem in solid state fermentation7 . Supplementation with medium components during growth of the organism is easy8. Suggested that the advantages of Submerged fermentation. They are high water content and more dilute nature makes temperature control easier. Product purification may be easier. Fermentation is life without air9.
Aspergillusoryzae is a group of molds, which is found everywhere world wide, especially in the autumn and winter in the northern hemisphere. Cassava was successfully converted to ethanol by separate enzymatic hydrolysis with the aid of fermentation by Aspergillusoryzae.
The ability of Rhizopusoryzae strains of ethanol production 80% of initial moisture content consisted of 16g cassava. The amylolyticfungus, Rhizopusoryzae produces extracellular amylase and liquefy starch to oligosaccharides and glucose. Respectively ethanol under oxygen limiting condition10 . The present study was aimed at ethanol production by the co-cultures of Aspergillusoryzae and Rhizopusoryzae under Submerged Fermentation and optimized fermentation parameters also.
Material and Methods
Source of Organisms
The soil samples were collected from a paddy field at Mannargudi, Thiruvarur (DT) Tamil Nadu, India and stored in a sterile polythene bags. Then the soil sample was immediately brought to the laboratory for the isolation of fungi11 .Aspergillusoryzae and Rhizopusoryzae strains were obtained from serial dilution technique. The organisms were maintained on potato dextrose agar slants at 4°C.
Sample Preparation and Pretreatment
Cassava samples were collected from milling centers in Mannargudi, Thiruvarur (DT), Tamil Nadu. The samples were converted into fine powder by milling and sieving. Pretreatment of the sample was then carried out by refluxing the powder with 0.2M NaoH for 2 hours and then neutralized with HCL. The pretreated sample was dried in an oven at 65° C.
Culture condition for ethanol production
A synthetic medium containing yeast extract base glucose broth and a complex medium containing the pretreated cassava were used for ethanol production under Submerged Fermentation. The synthetic medium was prepared by dissolving 6.7 g of yeast extract and 10 g of glucose in a liter of distilled water. The complex medium was prepared with the cassava at a concentration of 10 g / liter. Supplemented with 3g FeNH4 (SO4)2, 5g (NH4)2 HPO4, 6g urea and 10g peptone. The pH of the medium was adjusted to 5.0, sterilized in an autoclave and filtered. Another set of media containing 100g/L of glucose and cassava were similarly prepared. The media were dispensed into 500 ml Erlenmeyer flasks each containing 250ml of the medium. The flask containing synthetic medium were inoculated with Aspergillusoryzae and the another synthetic medium were inoculated with Rhizopusoryzae while these containing complex medium were inoculated with both Rhizopusoryzae and Aspergillusoryzae. The flasks were incubated at 25°C for 5 days12.
Effect of pH on ethanol production
10g of cassava was prepared and dispensed into different flasks. The pH of the broth was set as 4, 5 and 6 in a flask and sterilized. A loopful of Aspergillusoryzae was inoculated into 3 flask and Rhizopusoryzae was inoculated into 3 flask. All the 6 flasks and incubated at 28° C for 5 days. After incubation to determine the percentage of ethanol production.
Effect of temperature on ethanol production
10g of cassava was prepared and dispensed into different flasks. The temperature of the broth was set as 25° C, 30° C and 35° C in a flask and sterilized. A loopful of Aspergillusoryzae was inoculated into 3 flasks and Rhizopusoryzae was inoculated into 3 flasks and incubated at 28° C for 5 days. After incubation to determine the percentage of ethanol production.
Analytical methods
At 24 hour intervals, samples were taken aseptically from the fermentation media determine growth, residual sugar and ethanol concentration.
The growth was determined by measuring the cell density (optical density) at 650nm, The residual sugar was determined using dinitrosalicylic acid (DNS) method described by13 and ethanol was determined after standard distillation using the method described by14.
Statistical analysis
The results obtained in the present investigation were subjected to statistical analysis like mean (X) and standard deviation 15
Results and Discussion
In this present study producing organisms, Aspergillusoryzae and Rhizopusoryzae were isolated from paddy field soil sample. Ethanol production was estimated by Aspergillusoryzae and Rhizopusoryzae using cassava waste. High ethanol production was observed in 10% cassava containing medium than the 10% gucose containing medium.
The results of the ethanol yield from the synthetic medium containing glucose and complex medium containing cassava are shown in (Table1). At 1%, 1% and 10% glucose concentration, the synthetic medium gave a maximum ethanol yield of 4.4±0.05, 3.9±0.12 and 5.6±0.28 respectively. While at the same concentrations of cassava, the complex medium yielded 5.0±0.05, 4.7±0.17 and 6.2±0.17 ethanol. It was observed that at all concentrations of substrates. The ethanol yield increased steadily reaching the peak after 72 hrs of fermentation and then declined. The Cassava at 1% and 10% concentration gave a better ethanol yield compared to glucose. This shows that the cassava as one of the cheaper substrates for ethanol production16.
Table 1: Ethanol Yield from Synthetic and Complex Media
| Fermentation SM (Glucose) CM (Glucose) |
| Time 1% A.oryzae 1% R.oryzae 10% A.oryzae& 1% A.oryzae 1% R.oryzae 10% A.oryzae& |
| R.oryzae R.oryzae |
| 24 2.3± 005 1.9± 0.05 3.5± 0.11 3.5± 0.11 3.3± 0.11 5.3± 0.22 |
| 48 3.7± 0.05 3.3±0.17 4.8±0.05 4.4± 0.11 4.3 ±0.09 5.9± 0.11 |
| 72 4.4±0.05 3.9± 0.12 5.6±0.28 5.0± 0.05 4.7± 0.17 6.2 ± 0.17 |
| 96 3.8±0.09 3.5± 0.05 4.5± 0.05 4.7± 0.11 4.2± 0.17 5.7± 0.17 |
| 120 3.2±0.12 2.9±0.11 4.0± 0.05 4.1± 0.17 3.7± 0.05 5.2±0.19 |
| Values are expressed as Mean ± Standard deviation |
| SM ® Synthetic medium |
| CM ® Complex medium |
The results in Table 2 showed the pattern of residual sugar during fermentation period. At 1%, 1% and 10% glucose concentration, the synthetic medium gave a maximum residual sugar was 1.4±0.17, 0.9±0.17 and 1.7±0.17 respectively. While at the same concentrations of cassava, the complex medium yielded 2.4±0.17, 2.1 ±0.17 and 2.7±0.17. The residual sugar in the fermentation media was observed to decrease with increase in fermentation time. This could be attributed to the utilization of the sugar as carbon source for the growth and subsequent ethanol production. Cassava at 1% and 10% concentration gave a better residual sugar compared to glucose. The both Aspergillusoryzae and Rhizopusoryzae inoculated medium gave a better residual sugar compare than individually inoculated medium.
Table 2: Residual sugar obtained during fermentation of the synthetic and complex media
| Fermentation SM (Glucose) CM (cassava) |
| Time 1% A.oryzae 1% R.oryzae 10% A.oryzae& 1% A.oryzae 1% R.oryzae 10% A.oryzae& |
| R.oryzae R.oryzae |
| 24 0.2±0.05 0.1±0.05 0.4±0.05 1.1±0.05 1.0±0.05 1.5±0.05 |
| 48 0.5± 0.11 0.4±0.01 0.7±0.11 1.6±0.11 1.4±0.11 2.1±0.11 |
| 72 1.4±0.17 0.9±0.17 1.7±0.17 2.4± 0.17 2.1±0.17 2.7±0.17 |
| 96 0.9±0.05 0.7±0.05 1.4±0.05 2.0±0.09 1.9±0.09 2.4±0.05 |
| 120 0.6±0.09 0.4± 0.1 0.8±0.09 1.8±0.05 1.6±0.05 2.2±0.09 |
| Values are expressed as Mean ± Standard deviation |
| SM ® Synthetic medium |
| CM ® Complex medium |
The growth (cell density) of the organisms was also observed to increase steadily during the fermentation period (Table 3). At 1%, 1% and 10% glucose concentration, the synthetic medium gave a maximum cell density was 0.43±0.03, 0.4±0.03 and 2.0±0.28. While at the same concentrations of cassava, the complex medium yielded 0.57±0.03, 0.57±0.03 and 2.9±0.57. This may be due to the fact that the organisms are utilizing the nutrients present in the media for growth and ethanol production. However, the media with 10 % substrate concentrations supported more growth of the organisms. Also the cassava seem to support higher growth of the organisms compared to glucose. The both Aspergillusoryzae and Rhizopusoryzae inoculated medium gave a better Growth compare than individually inoculated medium. Similarly reported by17 in corn cobs. This may be due to the presence of other compounds in the cassava such as non starch carbohydrates, proteins, amino acids and other compounds which support more growth
Simultaneous Saccharification Fermentation (SSF) process using co-cultures between starch decomposing microbe and ethanol producing microbe is an alternative route for ethanol production from starch. Because this procedure leads to save for energy and low cost in fuel ethanol production from renewable feedstock. Ethanol production from an efficient microbes will be an important role in the new area.
Table 3: Growth (cell density) obtained during fermentation of the synthetic and complex media
| Fermentation SM (Glucose) CM (cassava) |
| Time 1% A.oryzae 1% R.oryzae 10% A.oryzae& 1% A.oryzae 1% R.oryzae 10% A.oryzae& |
| R.oryzae R.oryzae |
| 24 0.27± 0.01 0.24± 0.01 1±0.07 0.39±0.01 0.35± 0.01 1.6± 0.77 |
| 48 0.29± 0.11 0.27± 0.02 1.1±0.40 0.42±0.02 0.41± 0.02 1.9± 0.11 |
| 72 0.34± 0.13 0.31±0.12 1.4±0.17 0.49±0.02 0.46± 0.02 2.1± 0.33 |
| 96 0.38± 0.03 0.36± 0.03 1.6±0.23 0.56± 0.03 0.53± 0.03 2.3 ± 0.23 |
| 120 0.43±0.03 0.42± 0.03 2.0± 0.28 0.59± 0.02 0.57± 0.03 2.9 ± 0.57 |
| Values are expressed as Mean ± Standard deviation |
| SM® Synthetic medium |
| CM® Complex medium |
Effect of pH concentration of Ethanol yield
The ethanol yield significantly influenced by different pH such as 4,5 and 6. The maximum ethanol yield was 8.7±0.17, 5.4±0.77 at pH 6 such as Aspergillusoryzae and Rhizopusoryzae respectively. The Aspergillusoryzae gave a better ethanol yield compared to Rhizopusoryzae.(Table 4). The similar results were reported by18 .
Table 4: Optimization of parameters
| S. Organisms pH Temperature |
| No 4 5 6 25°C 30°C 35°C |
| 1. Aspergillusoryzae 7.2±0.05 8.0±0.09 81±0.17 6.6±0.05 6.9±0.09 7.4±0.17 |
| 2. Rhizopusoryzae 4.1±0.04. 8±0.09 5.4±0.17 5.9±0.05 6.4±0.09 7.1±0.17 |
Effect of temperature concentration of Ethanol yield
The ethanol yield significantly influenced by different temperature such as 25°C, 30°C and 35°C. The maximum ethanol yield was 7.4±0.17, 7.1±0.17 at 35°C. The Aspergillusoryzae gave a better ethanol yield compared to Rhizopusoryzae.(Table4). The optimal temperature of ethanol production was determined to be around 32° C. The related results were observed by19 reported the maximum ethanol production was found to be 30° C.
Conclusion
The results of this study showed that cassava are good substrate for ethanol production compared to glucose. The substrates at 10% concentrations supported higher yield of ethanol and increases with fermentation time and peaked at 72 hours. Therefore findings of this work suggest that ethanol can be produced from agricultural wastes such as cassava. Cassava is the cheapest raw materials for ethanol production. Ethanol production from an efficient microbes will be an another important role in the area.
References
- Inoue T., H. Lefuji., T. Fujii., H. Soga and K. Satoh. Cloning and characterization of a gene complementing the mutation of an ethanol sensitive mutant of sake yeast. Biosci.Biotechnol.Biochem.64: 229-236 (2000).
- Ames, B.N. DNA damage from micronutrient deficiencies is likely to be a major cause of cancer. Mut. Res. 475: 7-20 (2001).
- Wingren A., M.Galbc and G.Zacchi. Techno-electronic evaluation of producing ethanol from soft wood a comparison of SSF and SHF and identification of bottlenecks, Biotechnol.Prog.19: 1109-1117 (2003).
- Montesinos T and J.M. Nararro. Production of alcohol from raw wheat flour by amyloglucosidase and S.cerevisiae enzyme.Microbial. Technol. 27: 362-370 (2000).
- Kosugi., N.Kondo.,M.Ueda., Y.Murata., P.Vaithanomsat., W.Thanapase., T.Arai., and Y.Mori. Production of ethanol from cassava pulp via fermentation with a surface engineered yeast strain displaying glucoamylase. Renew Energ. 271 -278 ( 2008).
- Suesat C., B. Pitiyont., T. Srinorakutara and V. Kitpreechavanich. Utilization of waste from cassava starch plant for ethanol production In the 42ndKasetsart University Annual Conference, Bangkok, Thailand. 450-458 (2004).
- Kaliz H.M., Microbial protease Adv, Biochem. Engg.3(1): 51-62 (1988).
- Mitchell B.M., David, A., and Krizer, N. Biochemical engineering aspects of solid state bioprocessing. Adv. Biochem. Eng. 68: 61-138 (2000).
- Louis Pasteur, Influence de I oxygene Sur le development de la levure at al fermentation alcoolique. Journal of the History of Biology. 4 (1971): 35– 62 (1857).
- Ronald, H.W.M., R.B. Bakker., G. Eggink and R.A. Weusthuis. Lactic acid production from xylose by the fungus Rhizopusoryzae.Appl. Microbiol. Biotechnol.72(5), 861-868 (2006).
- Dring, D M. Techniques for microscopic preparation. In methods in Microbiology, 4, booth (Eds) Academic press, London. 95–111 (1976).
- Ado. S.A., G.U. Kachala., M.P. Tijjani and M.S. Aliyu.ethanol production from corn cobs. Journal of Pure and appliedMicrobiology. 4(1): 91-94 (2010).
- Miller, G.C. Use of the dinitrosalicylic acid reagent for the determination of reducing sugar. Analytical Chemistry.1959: 31: 426-428.
- AOAC ,Official Methods of Analysis ,Harwits ,W (Ed) 12thEdn. Association of Official Analytical Chemist ,Washington DC (1974).
- Zar.J.H. In: Biostatistical Analysis, EngleviodCliffs,N.J:Prenticehall,Inc. New York 56(2):358-360 (1984).
- TeerapatrSrinorakutara, LerdlukKaewvimol and VichienKipreechvanich., Cassava wastes pretreatments for fuel ethanol production. Journal of Agricultual Research.7: 245-249(1993) (2002).
- AchiHata, O.K. and Njoku – Obi, A.N.U. Production of a raw saccharifying amylase by Bacillus alvei grown on dtfferent agricultural substrates.WorldJournalofMicrobiologyandBiotechnology.8: 206-207 (1992).
- Godia, F., C. Casas and C. Sola. Asurvey of continuous ethanol fermentation systems using immobilized cells. ProcessBiochem.22: 43-48 (1987).
- Rocha M., V.P.Souza and R. Machiel, Appl. Biochem. Biotechnol., 136-140: 185-194 (2007).
