IBG 202

INDUSTRIAL MICROBIOLOGY

LAB 6 Production of Ethanol from Renewable Sources (Potato Starch)

Prepared by : MUHAMMED AZHAR BIN AZIZAN

LOW PIK KUAN TOH SEE MIN

Group

Prepared for : Pn.WAN NADIAH WAN ABDULLAH

Objective To experience designing an experiment which produces the highest yield of ethanol using a certain substrate and bakers yeast. To determine the amount of ethanol that is produced from different types of renewable sources.

Introduction
Ethanol, also known as ethyl ethanol, pure alcohol, grain alcohol or drinking alcohol, is a volatile, flammable, colourless liquid with the structural formula CH3CH2OH (also abbreviated as C2H5OH or C2H6O. Ethanol fermentation is applied in production of alcoholic beverages like beer, wine and bread. For ethanol fermentation, only the simplest form of sugar like glucose is able to be fermented into ethanol by yeasts. Ethanol fermented from renewable sources for fuel or fuel additives are known as bioethanol. Additionally, the ethanol from biomass-based waste materials is considered as bio-ethanol. Currently, there is a growing interest for ecologically sustainable bio-fuels. Bioethanol can be produced through ethanol fermentation. The raw materials used can be from molasses, sugar beet, sugar cane or potatoes. Bio-ethanol production from potatoes is based on the utilization of waste potatoes. Waste potatoes are produced from 5-20 % of crops as by-products in potato cultivation. At present, waste potatoes are used as feedstock only in one plant in Finland.

Materials and methods Materials Potatoes Alpha-amylase enzyme from Aspergillus sp. Glucoamylase enzyme from Aspergillus sp Bakers yeast (Saccharomyces cerevisiae) Reagent Hydrochloric acid Sterile water Distilled water Apparatus Eppendorf tube Filter Stirring hot plate Glass rod pH meter Knife Analytical balance Beakers Conical flask Pipette Thermometer Refractometer

Methods 1. 4 pieces of potatoes (the skin been peeled off) were used in this experiment. 2. The potatoes were cut into many small cubes and then cooked in a boiling hot water bath for one hour. 3. During the 1-hour cooking, the cube-shaped potatoes were continuously stirred in order to mash it. 4. 3 ml of alpha-amylase was added into the mixture before cooking. 5. After one hour, the potato mash was cooled to 80-90 C while another 3ml of alpha- amylase was added into the mash. 6. The mash was stirred to mix well and left for an hour. 7. After that, the mash was allowed to cool to 60C for 30 minutes, then to 30C. 8. Potato starch was squeezed from the mash, using a filter, into a 250ml beaker. 9. With the help of the pH meter, the overall pH of the broth was adjusted to around 4 4.50. 10. The broth is then transferred into a 1L conical flask for fermentation. 11. 2 g of bakers yeast was previously activated in 100 ml of water with some sugar before the experiment and was then added into the mash. 12. The mouth of the conical flask was sealed with a rubber stopper which was connected to a stoppered test tube using a rubber tube. 13. The test tube was filled with water to seal the escape route of air and to confirm the release of carbon dioxide throughout the fermentation process. 14. During the total of 6 days of fermentation, the mash was mixed regularly at a constant slow speed. 15. A raw sample taken out at Day 6 and 7 were collected to be analysed using the gas chromatography method. 16. After Day 7, the raw samples were poured out and placed in eppendorf tubes and later centrifuged at 3000rpm for 15minutes. 17. The supernatant is collected as sample to be used. 18. Internal standard is prepared from 0.6% (v/v) propan-1-ol. 19. Both the samples were then analysed using Gas Chromatography (GC). 20. The GC sample was prepared by; exactly 0.8 mL of the internal standard solution was pipetted into a GC vial and exactly 0.8mL of the sample solution was added then was capped and mixed well. 21. The standard solution was prepared by; 0.8ml of ethanol was pipetted into a GC vial and exactly 0.8mL of the internal standard was added then was capped and mixed well. Ethanol (% v/v) = [
( )

a = slope of the graph of ratio area for different ethanol concentration b = y-intercept DF = dilution factor Yield = (percentage of ethanol produced / percentage glucose utilized) x 100%

Results and discussion

Figure 1: Calibration Curve of at Ethanol Concentrations (0.02 to 6 %, v/v) GC-FID (using Rt-Q-Bond Column) Method Table 1: Results from Gas Chromatography Method
Peak area of ethanol Peak area of propan-1-ol Data 1 111415 102047 Data 2 149087 48132 Average 130251 75089.5

Ethanol (% v/v) = [

=[

= 1.3497 % (v/v) Yield = (percentage of ethanol produced / percentage glucose utilized) x 100% = (1.3497 / 6.1) x 100% = 22.13% Bakers yeast (Saccharomyces cerevisiae) was chosen because it contributes to efficient alcohol production and also tolerates very high alcohol levels. Dry form of bakers yeast was used because its easier to handle and its activation period can be controlled. Sugarcane juice was the substrate for the yeast to grow. This was because substrates with high level of glucose were preferable over other complex sugars.

Yeast cells grow well in low pH; this allows for inhibition of growth of undesired bacteria. For the fermentation process to occur, the pH of the solution was adjusted to 4.30. This provided an acidic medium to affect membrane permeability and inhibit the further growth of the yeast cells. At higher pH (alkali medium), the metabolic pathway of the yeasts would switch to produce glycerol instead of ethanol in microaerophilic conditions. Temperature of the fermentation process was controlled at 32OC which is around the optimum temperature for fermentation to happen. Higher temperatures would kill the yeast cells without the presence of alcohol while killing temperature decreases as more alcohol is produced. The percentage of alcohol produced was 1.3497 % (v/v) with its percentage of yield 22.13%. The experiment can be improved by increasing the activation time of the yeasts or by increasing the amount of yeasts used. Conclusion The results for ethanol production using potato starch as substrate was not very encouraging but steps can be taken to further improve the results by taking into account other variables affecting the fermentation process. Reference 1. G. Grassi: Modern Bioenergy in the European Union, Renewable Energy, Vol 16, p. 985- 990, 1999. 2. Commission Regulation (EC) No 2870/2000: Laying Down Community Reference Methods for the Analysis of Spirits Drinks, Official Journal of the European Communities, L 333/20, 29.12.2000. 3. Kimmo Vahtola and Liisa Myllykoski: Bioetanolin valmistus jteperunasta Esiselvitys teknisest toteutuksesta ja taloudellinen arviointi, Report 239, Oulun yliopisto, Prosessitekniikan osasto, 1999.