New Researches in Biotechnology - Facultatea de Biotehnologii ...

Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011The maintenance medium was YPG agar. The yeasts were maintained on a slant ofYPGA medium (yeast extract 1%, peptone 1%, glucose 2%, and agar 2% (w/v)) and storedat 4°C.INOCULUMThe culture medium was portioned in 1000 ml Erlenmeyer flasks, which werecotton-plugged and sterilized at 121°C for 15 min. When the flasks attained roomtemperature, they were inoculated (2% v/v) with the yeasts. The culture medium forinoculum was malt-agar (malt extract 3%, peptone 0.5%, agar 2%).The yeast were cultivated in 1000 ml Erlenmeyer flasks containing 500 ml ofmedium at 30 0 C on a Unimax 1010 orbital rotatory shaker with Incubator 1000 (Heidolph)at 150 rpm for 24 h. Microaerobic conditions were provided by the use of flasks withcottonwool plugs. At the end of the fermentation process, the final pH and the yeastbiomasses were determined.The inoculum (10% of the working volume and roughly 5.2 x 10 7 cells/ml) wastransferred from the flasks to stainless steel bioreactor (total volume of 20 l) fitted with pHmonitoring and controlling equipment and dosage equipment (raw material, nutrient saltsolutions) and which contained approx. 15 l liquid volume (work was performed below75% of bioreactor capacity to avoid foam problems).FERMENTATION MEDIAThe preparation of molasses used as fermentation medium comprises the followinggroups of technological operations: dilution of molasses with potable water at aconcentration of 40% d.m.; acidification of molasses using concentrated sulphuric acid (pHfinal 4.5 – 4.8); sterilisation of molasses; clarification of diluted and sterilized molassesthrough decantation and filtration.The molasses tested were characterized through the following parameters:- Sugar beet molasses - soluble dry matter 80.63%; moisture content 22.58%; total sugar57.71% expressed in glucose; initial reducing sugar 9.25%; sucrose 50.48%; ash 5.7%; pH5.7 (in solution 10%); nitrogen content 1.23%;- Sugar cane molasses - soluble dry matter 82.2%; moisture content 23.7%; total sugar59.24% expressed in glucose; initial reducing sugar 11.3%; sucrose 55%; ash 7.8%; pH 6(in solution 10%); nitrogen content 1.19%.The minerals necessary for yeast were introduced into the culture medium in theform of solutions.The yeast multiplication was tested, with and without addition of vitamins (biotin,calcium panthotenat and inositol) to the medium based on molasses. The vitamins wereadded one by one and as mixture of two or three within the culture medium.The soluble dry matter content for the molasses starting base medium beforeinoculation in the bioreactor was 20 0 Brix.The media based on molasses contained the following ingredients (g/l):Medium I: (NH4) 2 S0 4 , 0.6; KH 2 PO 4 , 0.3; in 1000ml distilled water;Medium II: MgSO 4 x7H 2 O, 0.3; CuSO 4 x7H 2 O, 0.002; ZnSO 4 x7H 2 O, 0.02; CaCl 2 x2H 2 O,0.08; (NH4) 2 S0 4 , 0.6; KH 2 PO 4 , 0.3; in 1000ml distilled water;Medium III: MgSO 4 x7H 2 O, 0.3; CuSO 4 x7H 2 O, 0.002; ZnSO 4 x7H 2 O, 0.02; CaCl 2 x2H 2 O,0.08; (NH4) 2 S0 4 , 0.6; KH 2 PO 4 , 0.3; inositol, 0.23; calcium panthotenat, 0.05; in 1000mldistilled water;126