INTRODUCTION:

Microorganisms require specific minerals for growth and metabolic activities. This requirement varies with the type of organisms as well as the nature of the basal medium used. Yeast has a complex nutrient requirement¹.To overcome the limitation of low productivity of ethanol, some conditions are to the established to favour the multiplication and successive functioning of the organisms².Each enzyme catalysed a different reaction and also has specific nutrient requirement for optimum performance. So the studies of the optimized combinations of trace elements are thus of great practical importance. It has been reported that Zn²⁺ is an important element for yeast cells³.

At a concentration of 0.2 ppm Zn²⁺promotes and provides optimal growth of yeast. Considerable studies have been made in the requirements of trace elements for growth of yeast and production of ethanol ⁴.0.1 g/L MnSO₄ and 0.024 g/L FeSO₄ were detrimental to yeast growth⁵.Yoshinori Ohsumi et al.⁶ have described the specific effect Cu²⁺ eliciting selective changes in the permeability of intact S. cerevisiae cells.

They found that when 100µM CuCl₂ was added to cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min. at 25°C.Christopher White and Geoffrey M. Gadd⁷ have studied on the uptake and cellular distributions of Zn²⁺ in S. cerevisiae.They have examined and characterized external concentration which are probably of the greatest physiological importance and have also examined the influence of toxicity an uptake at higher Zn²⁺ concentration.Considering all these reports, in the present work an extensive study had been made to find out the trace element requirements for the selection of suitable medium for Bioethanol production from alkali hydrolysed water hyacinth by Saccharomyces cerevisiae AB₈₁₀.

MATERIALS AND METHODS:

Microorganism used:

Saccharomyces cerevisiae AB₈₁₀, a newly isolated ethanol resistant strain in our laboratory has been used in these studies⁸.

Medium and cultured condition:

The alcohol and temperature resistant strain of Saccharomyces cerevisiae AB₈₁₀ was maintained in YPD agar medium containing yeast extract 1%, peptone 2%, dextrose 2%, agar 4%, pH was adjusted to 4.5.Organism was maintained at 28⁰Cfor 48 hours. Inoculums were harvested by washing the slant with sterile distilled water; the cell density was adjusted to 2.6×10⁷ cell/ml of the suspension. The cell suspension (3.0 ml) was used the inoculation of fermentation medium. Surface culture fermentation was carried out using 500 ml conical flasks each containing 200 ml of medium. The flasks were then incubated at 28 ⁰C for 48 hours. The fermentation medium used for alcohol production contained glucose 15 %( obtained from hydrolysed water hyacinth), KH₂PO4 0.1%, NaNO₃0.3%, MgSO₄.7H₂O 0.05%, yeast exteract1% and pH4.5.

Addition of trace elements to the basal medium:

Initially the basal medium did not contain the trace elements to be investigated. The impurities present in the inorganic salts were further purified by the method of Majumder and Bose et al.⁹ The solutions of all trace elements prepared in triple glass distilled water, sterilised in autoclave at 15 lbs pressure for 15 min. and added separately to the medium to attain the required concentration.

Determination of ethanol concentration:

After alcohol fermentation the ethanol produce was determined by the Gas chromatography (GC) (Pye Unicam Series ion) with flame ionization detector (FID) on a column of Propak-O using N₂ as carrier gas. The Column temperature and detector temperature was 190ºC and 230ºC respectively .In each case 5µL sample was injected. The quantitative calculation of ethanol concentration was determined by measuring the peak areas sample in calibration relative to the internal standard n-Propanal used as internal standard¹⁰.

Statistical analysis:

All data were expressed as mean ±SEM, where n=6.The data were analysed by one way ANOVA followed by Dunett’s post-hoc multiple comparison test using “Prism 4.0” software (graph pad Ind., USA). A ‘p’ value less than 0.005 was considered significant and less than 0.001 as a highly significant.

RESULTS AND DISCISSION:

The effect of different trace elements on ethanol production by Saccharomyces cerevisiae AB₈₁₀ was depicted in the Table 1-5.

Table 1: Effect of zinc (added as ZnSO₄. 7H₂O) and Copper (added asCuSO₄.5H₂O) on ethanol production by S.cerevisiaeAB₈₁₀

Concentration (µg/ml)	ZnSO₄. 7H₂O	CuSO₄. 5H₂O
Concentration (µg/ml)	Ethanol production (%)	Ethanol production (%)
0	12.0	12.0
1	12.0	10.0
5	13.0	9.2
10	11.7	8.0
15	10.5	7.1
20	9.0	6.2