PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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�� explained that the correlation between growth rate and stress sensitivity such that cells growing quickly in a glucose-rich medium are more sensitive to heat (Walker, 1998). Glycerol acted as the compatible solutes in high osmotic pressure condition such as high salt concentration and high sugar concentration. A little amounts of reducing sugars in this experiment was not enough to enhanced the high glycerol production by yeast. Interestingly, the starch was vigorously utilized, it might correlated to the amylase enzyme that released to catch with substrate. Thus, from this study is necessary for further experiment. Tabel 14. Production of α-amylase, β-amylase, and glucoamylase activity by the yeast isolate KAY1. Tatal Alpha amylase Beta amylase Glucoamylase Day Condition Protein Total act. Specific act. Total act. Specific act. Total act. Specific act. (mg) (Units) (Units/mg) (Units) (Units/mg) (Units) (Units/mg) 1 Heat shock 0.4516 44.70 98.98 64.51 142.84 123.84 274.22 Control 0.4004 46.001.02 114.881.16 91.201.41 227.771.59 131.581.06 328.621.19 2 Heat shock 0.4496 88.621.28 197.101.15 130.801.29 290.921.16 204.011.18 453.751.06 Control 0.4029 68.80 170.76 100.67 249.86 171.94 426.75 Soluble starch medium (1% soluble starch, 0.3% yeast extract, 0.05% MgSo47H2O, and 0.1% KH2PO4) inoculated with 2% (v/v) of precultured yeast cells (heat shock treatment) (approximately 5.3 × 10 7 ) and yeast cells (wihtout heat shock treatment) (1.4 × 10 8 ). U = 1 unit of α, β amylase defined as 1 µmol of maltose that liberated at 30 o C pH 6.9 and 4.8 respectively. U = 1 unit of glucoamylase defined as 1 µmol of glucose that liberated at 30 o C pH 4.5. Yeast isolate KAY1 performed the ability to produced α-amylase, β-amylase and glucoamylase (Table 14). At the first day, the total activity of amylase from without heat shocked cells (control) increased 1.02-to 1.41 fold as compared with that the heat shocked cells and the specific activity of amylase from without heat shocked was higher than the heat shocked cells. In contrast, at the second day the total activity of amylase from heat shocked cells increased 1.18-to 1.29 fold as compared with the control and the specific activity of amylase was also higher than the control. From the results showed that the large amounts of amylase enzyme was produced at the second day. These results compared with the time courses of heat shock treatment (Figure 60, 61 and 62) could explained that the residual starch rapidly decreased at the first day and the second day was
�� effected by heat shock power which stimulated the yeast cell constructed the high amounts of proteins inside the cell. The amylase enzyme was largely produced to catch the substrate and converted starch into the high amounts of reducing sugars. After that the yeast cells were absorbed reducing sugars into the cell. Consequently, the sugar concentration in the medium stimulated the construction of mannitol in the yeast cell. Because high initial starch concentrations were employed for an economical process, amylase production from yeast should be high to hydrolyzation of the substrate. The results suggested that the heat shock treatment might be an effective and simple method for improving the starch hydrolyzation. 3.8 Cultivation of rhizobia using nutrient sources produced by yeast The slow-growing Bradyrhizobium japonicum USDA 110, and Bradyrhizobium spp. THA5, which isolated in Thailand were used in this experiment. The two selected glycerol-producing yeast isolates: PIY2, and PUY4 were able to grow on sucrose as a sole carbon source, converted the sucrose to glycerol. The selected yeast isolate KAY1 was used to change the starch to glycerol, and mannitol. The standard medium for rhizobia cultivation such as G5 and Yeast-mannitol (YM) broth were depicted in Table 15. 3.8.1 Cultivation of Bradyrhizobium using medium containing glycerol produced by yeasts In view of the earlier reports of high glycerol yields by osmophillic yeasts (Spencer and Sallans, 1956; Spencer and Spencer, 1978). A number of Zygosaccharomyces rouxii and Z. bailii strains were first tested for glycerol production. However, it was found that most of these osmophillic strains were unable to metabolized sucrose (Lie et al., 1991). Fortunately, the yeast isolate PIY2, and PUY4 were screened for their ability to produced glycerol by glucose and also produced glycerol by using sucrose as sole source of carbon. Especially, sucrose was cheap and locally available substrate similar to a starch. Bradyrhizobium, The symbiont of many tropical legumes, including soybean, cannot use sucrose (Graham, 1964; Elkan and Kwik, 1968) because it lacks invertase, required for hydrolysing the disaccharide sucrose (Martinez-de Drets and Arias, 1972). For fast-growing Rhizobium and slow-growing Bradyrhizobium, glycerol could supported a very high bacterial density (Ballatti, 1982; Ballatti et al., 1987). In this experiment, in the first stage, sucrose was utilized by the
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PRODUCTION OF NUTRIENT SOURCES FOR
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PRODUCTION OF NUTRIENT SOURCES FOR
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CHARDCHAI BUROM : PRODUCTION OF NUT
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CONTENTS Page ABSTRACT (THAI)……
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3.6.1� Determination of the suita
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�� 17� Glycerol concent
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40� Time courses of mannitol prod
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59� Effects of various heat shock
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LIST OF TABLES Table Page ��
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LIST OF ABBREVIATIONS Aw water avai
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CHAPTER I INTRODUCTION For mass cul
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available (Burton, 1965). The molec
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1.1.1� Carbon metabolism in Rhizo
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strains unable to use the carbon so
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Figure 2. The fructose bisphosphate
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1.2�Classification of yeasts Mank
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Table 3. (continued). Class Order F
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Ustilaginales (Oberwinkler, 1987).
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f Cystofilobasidium has thick-walle
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Table 5. (continued). Sugar alcohol
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specific cases, but Brown (1978) pr
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KY 6166 was also studied using larg
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Figure 5. Important pathways of gly
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may be present in different ratios.
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amylase systems of the yeast specie
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Table 7. (continued). EC Recommende
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Table 8. (continued). Species a D.p
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Table 9. (continued). Cell product
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2.1.3 Equipment and other materials
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Spotted either the supernatant of c
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amylase action, the actual reaction
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A) Determination of the suitable an
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B) Salt-stress conditions Fifty mil
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2.2.8 Cultivation of Bradyrhizobium
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The cultured medium of yeast. Quant
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intervals for accumulation of gas i
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CHAPTER III RESULTS AND DISCUSSION
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Glycerol Xylitol Glucose Mannitol S
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Glycerol Xylitol Glucose Mannitol S
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Table 10. Primary screening of glyc
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Table 10. (continued). No. Isolate
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Glycerol in cell lysate (g/l) A Gly
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average were 4-10 g of glycerol per
Page 87 and 88: were isolated from fresh fruits, Ma
Page 89 and 90: �third day of incubation (Figure
Page 91 and 92: Reducing sugars (g/l) Reducing suga
Page 93 and 94: These results indicated that the co
Page 95 and 96: 3.6.1 Determination of the suitable
Page 97 and 98: g/l 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4
Page 99 and 100: Reducing sugars (g/l) 8 6 4 2 0 0 1
Page 101 and 102: Reducing sugars (g/l) 4.0 3.5 3.0 2
Page 103 and 104: g/l 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Page 105 and 106: g/l g/l g/l 1.5 1.0 0.5 0.0 1.2 1.0
Page 107 and 108: Starch fermentation was conducted i
Page 109 and 110: g/l g/l 7 6 5 4 3 2 1 0 8 6 4 2 0 0
Page 111 and 112: g/l g/l 8 6 4 2 0 8 6 4 2 0 1 2 3 4
Page 113 and 114: g/l g/l 8 6 4 2 0 8 6 4 2 0 0 1 1 2
Page 115 and 116: g/l g/l 1.6 1.4 1.2 1.0 0.8 0.6 0.4
Page 117 and 118: g/l g/l 1.6 1.4 1.2 1.0 0.8 0.6 0.4
Page 119 and 120: The results showed that amounts var
Page 121 and 122: g/l g/l 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Page 123 and 124: �� fermentation, but the inc
Page 125 and 126: g/l g/l 2.0 1.6 1.2 0.8 0.4 0.0 3.2
Page 127 and 128: g/l g/l 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Page 129 and 130: �� intracellularly a decreas
Page 131 and 132: g/l g/l 5.0 4.0 3.0 2.0 1.0 0.0 2.5
Page 133 and 134: g/l g/l 2 1.8 1.6 1.4 1.2 1 0.8 0.6
Page 135 and 136: g/L 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 0
Page 137: g/l 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7
Page 141 and 142: Table 15. The comparison of media f
Page 143 and 144: Table 17. Growth of Bradyrhizobium
Page 145 and 146: �� Rhizobium (Stowers, 1985;
Page 147 and 148: �� 3.9 Identification of yea
Page 149 and 150: KAY1 PIY2 PUY4 Y69 LIY2 COY1 FAY2 C
Page 151 and 152: �� assimilations were observ
Page 153 and 154: Table 20. (continued). Yeast strain
Page 155 and 156: Table 20. (continued). 15. Pichia k
Page 157 and 158: �� 3.9.5 Ascospore formation
Page 159 and 160: (a) (b) (c ) (d) (e) �� Figu
Page 161 and 162: (a) (b) (c) (d) (e) (f) �� F
Page 163 and 164: (c) (a) �� Figure 72. Cell m
Page 165 and 166: �� Metshnikowia (Pitt and Mi
Page 167 and 168: CHAPTER IV CONCLUSION This study ex
Page 169 and 170: only a low capital investment. Ther
Page 171 and 172: �� Barnett, J. A., Payne, R.
Page 173 and 174: �� De Mot, R., and Verachter
Page 175 and 176: �� Jennings, D. H. (1995). T
Page 177 and 178: �� Lie, T. A., Muilenbury M.
Page 179 and 180: �� Onishi, H., and Suzuki, T
Page 181 and 182: �� Sinclair, N. A., and Stok
Page 183 and 184: �� Van Rossum, D., Schuurman
Page 185 and 186: �� Young, J. P. W., and Hauk
Page 187 and 188: Acetone Methanol Solvent for mobile
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� 300 µg/ml, 200 µg/ml, and 100
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Soluble Starch 20.0 g Distilled wat
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13.�Nitrate broth KNO 3 1.0 g Glu
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If the sample had been diluted befo
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Table 1D. (continued). Source of ye
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E. Additional figures Abs 540 nm (a
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Abs 580 nm 1.4 1.2 1 0.8 0.6 0.4 0.
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Figure 7E. Calibration curves for H
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Figure 9E. Example of HPLC chromato
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Cell/mL 1.0E+09 8.0E+08 6.0E+08 4.0
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PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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