New Researches in Biotechnology - Facultatea de Biotehnologii ...

More documents

Recommendations

Info

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
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011Medium IV: 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; biotin,0.06x10 -3 ; in 1000ml distilled water.Formulations were tested, combining in different rations the sugar beet and sugarcane molasses. The initial pH values of all media were adjusted to 4.5 with H 2 SO 4 conc.FERMENTATION CONDITIONSBefore inoculation, the culture medium in the bioreactor was steam sterilized insitu and after cooling was inoculated with 10% inoculum (24-hours-old).The molasses was fed-batch added following a program, maintaining the sugarconcentration around 8%, in order to avoid sugar fermentation and ethanol production. Thefeeding diagram with molasses is presented in fig. 1.Fig. 1 Molasses addition programs for baking yeast productionFermentation was performed at a temperature of 30 0 C. Agitation speed andaeration rate in the bioreactor were 1000 rpm and 0.6 litres / litres / min, respectively. ThepH was automatically controlled at pH 4.5-4.8 with 2N NaOH. Silicone antifoam (CarlRoth) was used to prevent foam formation. The total fermentation time was 51 hours, andafter completion of the molasses addition, the culture was maintained with aeration for 4hto allow yeast maturation.Parameters such air flow, oxygen concentration in the culture, sugars and ammoniain the medium are controlled in commercial yeast production to improve yield [Kristiansen,1994; Lee et al., 1999; Maqueda et al., 2011], but these parameters were not monitoredduring this work.At the end of multiplication process (55 hours) the biomass was separated bycentrifugation using a laboratory centrifuge ROTINA 38 Hettich with 250 ml tubes. Theyeast biomass was centrifuged for 20 minutes at 4000 rpm. In order to assess the yield inbiomass, the amount of biomass formed after 55 hours of fermentation was determined.Cell growth was measured by determining the optical density at 600 nm using aspectrophotometer. To determine culture dry weight, culture samples (10 ml) were filtered127
Page 1 and 2:
UNIVERSITY OF AGRONOMICAL SCIENCES
Page 3 and 4:
Proceeding of the 4 rd Internationa
Page 5 and 6:
Page 7 and 8:
Page 9 and 10:
Page 11 and 12:
Page 13 and 14:
Page 15 and 16:
Page 17 and 18:
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Page 75 and 76: Proceeding of the 4 rd Internationa
Page 125: Proceeding of the 4 rd Internationa
Page 163 and 164: Day 7Day 10Proceeding of the 4 rd I
Page 177 and 178:
Page 179 and 180:
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
Page 191 and 192:
Page 193 and 194:
Page 195 and 196:
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
Page 207 and 208:
Page 209 and 210:
Page 211 and 212:
Page 213 and 214:
Page 215 and 216:
Page 217 and 218:
Page 219 and 220:
Page 221 and 222:
Page 223 and 224:
Page 225 and 226:
Page 227 and 228:
Page 229 and 230:
Page 231 and 232:
Page 233 and 234:
Page 235 and 236:
show all

New Researches in Biotechnology - Facultatea de Biotehnologii ...

Create successful ePaper yourself

Delete template?

Save as template?