Investigating the Production of Alcohol by Rice Wine Yeast (Saccharomycopsis fibuligera)

Project Proposal

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Hwa Chong Institution (High School)

Mentor: YYY

Introduction

Rationale

Biofuels like ethanol play a major role in reducing reliance on fossil fuels. However, ethanol derived from food crops has driven up the prices of food crops. The development of biofuels should not compete with crops used for food.

Thus, this project will focus on producing ethanol from starchy wastes, which will reduce reliance on food crops. Hence, instead of using crops for fuel, starch wastes is actually being recycled. However, fermentation from starchy wastes is not economically feasible as pure amylase has to be added to break down the starchy wastes into simple sugars before fermentation can occur. Therefore, the yeast, Saccharomycopsis fibuligera, is used instead of the yeast commonly used in fermentation, Saccharomyces cerevisiae¸ as Saccharomycopsis fibuligera, produces extracellular amylase, thus saving costs.

Objectives

1.  Investigate the production of ethanol from starchy wastes (e.g. rice, noodle and barley wastewaters)

2.  Compare the amount of ethanol produced by Saccharomyces cerevisiae and Saccharomycopsis fibuligera

Hypotheses

1.  Saccharomycopsis fibuligera is able to produce ethanol from starchy wastes.

2.  Saccharomyces cerevisiae is able to produce ethanol from amylase-treated starchy wastes.

3.  Saccharomycopsis fibuligera is able to produce a higher amount of ethanol than Saccharomyces cerevisiae.

Literature Review

Recently, there has been growing interests and investments in the research on Saccharomycopsis fibuligera. According to Chi, Liu, Wang, Ju, Zhang (2009), S. fibuligera has the ability to assimilate glucose, sucrose, cellobiose, trehalose and soluble starch. S. fibuligera has been considered in the realm of ascomycetous yeast as it is a good producer of extracellular amylolytic enzymes (Reiser Gasperik, 1995). These enzymes, which are associated with the degradation of starch, are excellent and widely used raw materials in many fermentation processes (Reiser Gasperik, 1995). On top of that, S. fibuligera has very high amylolytic activity as compared to the many other different types of amylolytic yeasts (Saelim, Dissara and H-Kittikun 2007).

The saccharifying properties of S. fibuligera are of interest because of their various applications: accumulate trehalose from starch, secrete amylases, acid protease and β-glucosidase used in industrial applications, and the production of ethanol from starch (Chi et al., 2009). Initially, it was assumed that only one amylase was produced and that glucose is the predominant hydrolytic end product of the enzyme. However, Lemmel, Heimsch, Korus (1980) reported that S. fibuligera is actually able to produce an amylolytic complex that consists of an α-amylase, a glucoamylase, and a maltase. Studies by Waghorn, TalõÂa, Elba, Liliana (2003) have shown that most starch is being hydrolysed by α-amylase. On top of that, several studies (Lemmel et al., 1980; Chi et al., 2009) have also shown that glucoamylase is an industrially important enzymes that catalyse the release of glucose from the non-reducing ends of starch molecules.

An important application of S. fibuligera is the production of ethanol. Chi et al. (2009) reported that ethanol is a popular liquid biofuel and also an excellent raw material used for synthetic chemicals. Starch, existing in abundance and low in cost, is a very good substrate for ethanol production (Chi et al., 2009).

Based on several studies (Chi et al., 2009; Machida et al., 1988), Saccharomyces cerevisiae was initially used in the production of ethanol from starch. Unfortunately, the disadvantage of using S. cerevisiae is that it would require a high-energy input. This is because S. cerevisiae cannot produce amylase. Artificial amylases are hence required to be added manually in the fermentation process, which results in high overall costs (Chi et al., 2009).

Outline of Methods

Materials

·  Potato dextrose broth / ·  Yeast extract
·  α-amylase / ·  Peptone
·  Phosphate-buffered saline / ·  Potassium Dichromate (K2Cr2O7)
·  Starchy wastes (Rice, barley and noodle wastewater) / ·  Ethanol
Apparatus
·  Centrifuge
·  Spectrophotometer

Variables

Independent Variables / ·  Starchy wastes (noodle, rice and barley waster)
·  Yeast (S.cerevisiae and S.fibuligera)
Dependent Variables / ·  Amount of ethanol produced
Controlled Variables / ·  Amount of yeast cells transferred to setups
·  Amount of starchy waste in each of the setups
·  Growth medium (potato dextrose broth)

Preparation of Medium with Starchy Wastewaters

Yeast extract peptone broth (YEP) containing 1% yeast extract and 0.5% peptone will first be prepared. 20ml of each of the wastewaters are then prepared and 1ml of 1% amylase is then added to 10ml of each of the wastewaters and left in room temperature for 15 minutes. YEP and the amylase-treated and untreated wastewaters will be autoclaved at 10psi for 10 minutes to sterilize. 10ml of each of the wastewaters are added to 20ml of YEP and a control, consisting of 20ml YEP and 10ml sterile water is set up.

Growth of Yeasts

Saccharomycopsis fibuligera and Saccharomyces cerevisiae are inoculated into 10ml of potato dextrose broth. The cultures are then incubated at 30°C overnight. 1ml of the cultures will then be transferred into each of the setups as below.

·  20ml YEP+10ml amylase-treated noodle wastewater

·  20ml YEP+10ml amylase-treated rice wastewater

·  20ml YEP+10ml amylase-treated barley wastewater

·  20ml YEP+10ml untreated noodle wastewater

·  20ml YEP+10ml untreated rice wastewater

·  20ml YEP+10ml untreated barley wastewater

·  20ml YEP+10ml sterile water

The rationale is that the starchy wastes act as a carbon source for the yeasts to

grow.

The setups are then incubated at 30°C for 7 days with shaking.

The data will then be analysed as below.

The following data will be collected:

1.  The density of yeast cells in the samples

2.  The concentration of ethanol in the samples

The density of yeast cells are compared between each wastewater (barley, rice and noodle wastewaters) and between the two yeasts (Saccharomyces cerevisiae and Saccharomycopsis fibuligera). Absorbance readings for each sample are taken at 660nm. The density of yeast cells are found out by counting the cells using a hemocytometer.

Determination of Ethanol Concentration

The yeast cultures are centrifuged at 7000rpm for 10 minutes and the supernatant collected. Acidified potassium dichromate, K2Cr2O7, is added to the supernatants in a 3:1 ratio and the solutions are then heated over a boiling water bath for 15 minutes. Absorbance readings for each of the solutions are taken at 590nm with a spectrophotometer.

The concentration of ethanol in the samples are compared between each of the wastewaters (barley, rice and noodle wastewaters) and between the two yeasts (Saccharomyces cerevisiae and Saccharomycopsis fibuligera). The ethanol content are then found by the following method:

Different concentrations of ethanol are then prepared and acidified potassium dichromate will be added into the ethanol in a 3:1 ratio. The solutions are heated over a boiling water bath for 15 minutes and the absorbance readings for each concentration of ethanol will be taken at 590nm with a spectrophotometer.

A calibration curve is then plotted with the absorbance readings from each concentrations of ethanol and the amount of ethanol in each of the products are then found.

Safety Precautions

During experiments, latex gloves and lab-coats will be worn. When working with the yeasts, laminar flow hoods will be used. All microbial cultures and apparatus used to contain the yeasts will be sterilized with an autoclave before disposal.

References

Chi, Z., Liu, G., Wang, F., Ju, L., Zhang, T. (2009). Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnology Advances, 1, 1-9. Retrieved February 20, 2010 from http://www.elsevier.com/locate/biotechadv

Waghorn, J.J, TalõÂa del Pozo, Elba A. Acevedo, Liliana A. Cardemil (2003). The role of two isoenzymes of a-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination. Journal of Experimental Botany, 54, 1-11. Retrieved May 1, 2010 from http://jxb.oxfordjournals.org/cgi/reprint/54/384/901

Lemmel, S.A., Heimsch, R.C. & Korus, R.A. (1980). Kinetics of growth and Amylase production of Saccharomycopsis fibuligera on potato processing Wastewater. Applied and Environmental Microbiology, 39, 1-7. Retrieved February 20, 2010 from http://aem.asm.org/cgi/reprint/39/2/387

Machida, M., Ohtsuki, I., Fukui, S. & Yamashita, I. (1988). Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular r-glucosidases as expressed in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 54, 1-9. Retrieved on February 20, 2010 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC204441/pdf/aem00117-0273.pdf/?tool=pmcentrez

Reiser, V. Gasperik, J. (1995). Purification and characterization of the cell-wall-associated and extracellular x-glucosidases from Saccharomycopsis fibuligera. Biochemistry Journal, 753, 1-8. Retrieved On February 20, 2010 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1136789/pdf/biochemj00061-0061.pdf/?tool=pmcentrez

Saelim, K., Dissara, Y. & H-Kittikun, A. (2007). Saccharification of cassava starch by Saccharomycopsis fibuligera YCY1 isolated from Loog-Pang (rice cake starter). Songklanakarin Journal of Science and Technology, 1, 1-7. Retrieved February 20, 2010 from http://rdo.psu.ac.th/sjst/ejournal/journal/30-Suppl-1/0125-3395-30-S1-65-71.pdf

Timeline

January 2010 / ·  Research on Project Topics
February 2010 / ·  Literature Review
·  Write up Proposal
March 2010 / ·  Confirmation of Methods
·  Begin Experiments
July 2010 / ·  Complete Experiments
·  Analyse Results
·  Write Up Proposal

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