Application of a statistical technique to the production of ethanol from sugar beet molasses by Saccharomyces cerevisiae

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Abstract

In this work, a statistical model which can be used to describe the rate of ethanol production from sugar beet molasses by Saccharomyces cerevisiae was developed. Total sugar concentration, pH, and ammonium dihydrogen phosphate (NH4H2PO4) concentration were chosen as a 23-factorial experimental design and the effects of these factors on ethanol production were examined in repeated shake flask cultures according to the Box–Wilson experimental design method. With the use of the developed model a maximum ethanol production of 1.06 g dm−3 h−1 was obtained when sugar concentration, pH, ammonium dihydrogen phosphate concentration, were 1.6 g dm−3, 4.51, 0.72 g dm−3, respectively.

Introduction

From the view point of industrial ethanol production, the sucrose-based substrates such as sugar cane and sugar beet juices present many advantages including their relative abundance and renewable nature. Molasses, the noncrystallizable residue remaining after sucrose purification, has additional advantages: it is a relatively inexpensive raw material, readily available, and already used for industrial ethanol production. The Saccharomyces cerevisiae is widely used as a biocatalyst in bioconversion processes and is suitable for production of ethanol from molasses under certain conditions (Park and Baratti, 1991). Some countries without known petroleum reserves have started to develop their fermentation industries for producing ethanol. However, significant improvements in alcohol production technology are necessary in order to reduce production costs and make ethanol a competitive resource material.

Experimental design technique Box et al., 1958, Thompson, 1957 is used for the empirical study of relationships between a measured objective on one hand and a number of operating conditions on the other hand. This method is used to find how a particular objective is affected by a given set of operating conditions over some specified region of interest and also the values of operating conditions which will yield a maximum for the specific objective as a result of optimization. The major advantage of applying experimental design is the reduced number of experiments that have to be carried out with the choice of best experimental points to get maximum information.

Several studies Edwards, 1970, Converti et al., 1985, Jones et al., 1981, Jones R.P., 1981, Holzberg et al., 1963 exist in the literature concerning the effects of the sugar concentration, addition of different supplements to the molasses, pH, and some other factors on productivity, but in an independent variable form. In these studies experiments were carried out conventionally, i.e., dependency of production rates on one of the variables was investigated for fixed values of the remaining variables.

It is useful to have a correlation which will yield information concerning the productivity variations of the processes when the productive process conditions are changed. Therefore, the main objective of this paper was to devise a model system in order to study and predict alcohol fermentation by S. cerevisiae as affected by factors such as pH, initial substrate concentration, and the concentration of supplemented ammonium dihydrogen phosphate via the Box–Wilson experimental design technique which has proved very useful in some fields (Güngör, 1993), especially chemical and micro-organism reaction conditions Thompson, 1957, Watson et al., 1984, Horitsu et al., 1992. The conditions which yielded the maximum value of the ethanol productivity for the selected region of interest and also the response surfaces for the predicting model are presented.

Section snippets

Micro-organism

S. cerevisiae ATCC 24860 was obtained from the American Type Culture Collection. It was maintained by transferring to fresh agar-malt slants each month and storing at 4°C. Agar-malt extract contained (g dm−3): malt extract 3.0; yeast extract 3.0; peptone 5.0; glucose 10; agar 20 and had a pH value of 4.5.

Medium

Sugar beet molasses obtained from Ankara Sugar company in Turkey was used for the production of cells under aerobic conditions as well as for ethanol production studies. For the preparation of

Results and discussion

Substrate concentration is an important factor in fermentation, so it was selected as one of the variables in experimental design. High substrate concentrations inhibit growth and product formation and may distort the metabolism of micro-organisms. It is reported that, in general, substrate inhibition becomes significant somewhere between in the range 5–25% (w/v) sugar with complete inhibition of growth at 40% (w/v) glucose (Holeberg and Margalith, 1981, Jones et al., 1981, Thatipamala et al.,

Conclusion

The Box–Wilson experimental design method was seen to be applicable in modeling the productivity of ethanolic fermentation. By the use of the model developed and the response surfaces, variation of the ethanol production rate depending on the changes in system variables, pH, sugar concentration and ammonium dihydrogen phosphate concentrations, can easily be investigated and maximized.

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