PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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CHAPTER II MATERIALS AND METHODS 2.1 Materials 2.1.1 Chemicals All chemicals used were of analytical grades, and were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Difco (Detroit, MI, USA), Carlo Erba Reagenti, Fluka Chemika, Merck (Darmstadf, Germany), Pronadisa (Alcobendas, Madrid, Spain), Unilab (Auburn, Australia), and Boehringer Mannheim (Mannheim, Germany). 2.1.2 Microorganisms Microorganisms screened for their abilities of sugar alcohol production were as follows: type cultures (Saccharomycopsis fibuligera TISTR 5033, Rhodotorula rubra TISTR 5067, Hansenula anomala TISTR 5082, H. anomala TISTR 5113, and Kluyveromyces maxianus TISTR 5270) obtained from the Thailand Institute of Scientific and Technological Research (TISTR); Candida utilis, C. famata, C. krusei, C. tropicalis, Geotricum candidum, Saccharomyces bayanus, S. cerevisiae var. burgandi, S. cerevisiae var. champaign, and S. cerevisiae obtained from stock cultures of the School of Microbiology, Suranaree University of Technology, Thailand; yeast isolates Y24, Y60, Y64, and Y69 were obtained from Chumkhunthod (2000); and yeast strains isolated from sample sources (section 2.2). Stock cultures were maintained using Malt-yeast extract (MY) agar (Appendix B2) and stored at 4°C. Two strains of Bradyrhizobium which were Bradyrhizobium japonicum USDA 110 (U.S. Department of Agricultural, Beltsville, Md, USA), and Bradyrhizobium spp. THA 5 (Ministry of Agriculture and Co-operation, Thailand), were used for the evaluation of media prepared from nutrient sources produced by yeasts.
2.1.3 Equipment and other materials Equipment used was as follows: High-Performance Liquid Chromatography (HPLC) (Thermo Separation Products Inc., USA), Thin-Layer Chromatography (TLC), UVvisible spectrophotometer (Ultrospec 2000, Phamacia Biotech Inc., England) Hot air oven, Incubator, Autoclave, Refrigerator (4°C), Deep freezer (-20°C and -70°C) (Heto Ultrafreeze, USA), Larminar flow hood, Rotary shaker (New Brunswick Scientific Inc., Germany), Water bath, Microwave oven, Centrifuge, Analytical balance, Pan balance, pH meter, Compound microscope, Haemocytometer, Micropipette sets, and Vortex mixer. Other materials were petridish, test tubes, plastic bag, ice box, basic microbiological equipment, glycerol combination test kit, and liquid nitrogen (Thai Industrial Gas Co. Ltd., Thailand). 2.2 Methods 2.2.1 Isolation and collection of yeasts Yeast strains were isolated from fresh fruits, fermented fruits, beverages, Khaomak, solid waste from black soy sauce factories (Nguan Chiang Food Industry Co., Ltd., Thailand), fresh fruits (e.g. pine apple, grape, rambutan etc.), and various agricultural sources such as cassava root, yam bean etc. One gram fresh weight of each sample was suspended in 1 ml of sterile water. The appropriate dilution of the sample was plated on Malt-yeast extract agar medium. The plates were incubated at 30°C for 2 days. Yeast colonies having different morphology were collected and further purified. Yeast isolates were then maintained using Maltyeast extract agar. 2.2.2 Screening and selecting of glycerol- and mannitol-producing yeast strains For screening of glycerol- and mannitol-producing yeast strains, one loop of the 3day yeast culture growing on Yeast extract-peptone-dextrose (YEPD) agar containing 2% glucose (Appendix B4) was inoculated into 8 ml of Yeast extract-peptone-dextrose medium containing 5% glucose (Appendix B3). The culture was incubated at 30°C for 4 days, then analysed for glycerol and mannitol, production. Yeast cells were removed by centrifugation at 8,000 rpm for 10 min at 4°C (Figure 7). Cells were disrupted by using 4 cycles of freezing in liquid nitrogen (-196°C), and thaw at 70°C. Glycerol and mannitol concentrations were primarily determined from both ��
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PRODUCTION OF NUTRIENT SOURCES FOR
Page 3 and 4:
PRODUCTION OF NUTRIENT SOURCES FOR
Page 5 and 6: CHARDCHAI BUROM : PRODUCTION OF NUT
Page 7 and 8: CONTENTS Page ABSTRACT (THAI)……
Page 9 and 10: 3.6.1� Determination of the suita
Page 11 and 12: �� 17� Glycerol concent
Page 13 and 14: 40� Time courses of mannitol prod
Page 15 and 16: 59� Effects of various heat shock
Page 17 and 18: LIST OF TABLES Table Page ��
Page 19 and 20: LIST OF ABBREVIATIONS Aw water avai
Page 21 and 22: CHAPTER I INTRODUCTION For mass cul
Page 23 and 24: available (Burton, 1965). The molec
Page 25 and 26: 1.1.1� Carbon metabolism in Rhizo
Page 27 and 28: strains unable to use the carbon so
Page 29 and 30: Figure 2. The fructose bisphosphate
Page 31 and 32: 1.2�Classification of yeasts Mank
Page 33 and 34: Table 3. (continued). Class Order F
Page 35 and 36: Ustilaginales (Oberwinkler, 1987).
Page 37 and 38: f Cystofilobasidium has thick-walle
Page 39 and 40: Table 5. (continued). Sugar alcohol
Page 41 and 42: specific cases, but Brown (1978) pr
Page 43 and 44: KY 6166 was also studied using larg
Page 45 and 46: Figure 5. Important pathways of gly
Page 47 and 48: may be present in different ratios.
Page 49 and 50: amylase systems of the yeast specie
Page 51 and 52: Table 7. (continued). EC Recommende
Page 53 and 54: Table 8. (continued). Species a D.p
Page 55: Table 9. (continued). Cell product
Page 59 and 60: Spotted either the supernatant of c
Page 61 and 62: amylase action, the actual reaction
Page 63 and 64: A) Determination of the suitable an
Page 65 and 66: B) Salt-stress conditions Fifty mil
Page 67 and 68: 2.2.8 Cultivation of Bradyrhizobium
Page 69 and 70: The cultured medium of yeast. Quant
Page 71 and 72: intervals for accumulation of gas i
Page 73 and 74: CHAPTER III RESULTS AND DISCUSSION
Page 75 and 76: Glycerol Xylitol Glucose Mannitol S
Page 77 and 78: Glycerol Xylitol Glucose Mannitol S
Page 79 and 80: Table 10. Primary screening of glyc
Page 81 and 82: Table 10. (continued). No. Isolate
Page 83 and 84: Glycerol in cell lysate (g/l) A Gly
Page 85 and 86: 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
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g/l g/l 7 6 5 4 3 2 1 0 8 6 4 2 0 0
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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
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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
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The results showed that amounts var
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g/l g/l 4.0 3.5 3.0 2.5 2.0 1.5 1.0
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�� fermentation, but the inc
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g/l g/l 2.0 1.6 1.2 0.8 0.4 0.0 3.2
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g/l g/l 1.2 1.0 0.8 0.6 0.4 0.2 0.0
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�� intracellularly a decreas
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g/l g/l 5.0 4.0 3.0 2.0 1.0 0.0 2.5
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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
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g/l 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7
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�� effected by heat shock po
Page 141 and 142:
Table 15. The comparison of media f
Page 143 and 144:
Table 17. Growth of Bradyrhizobium
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�� Rhizobium (Stowers, 1985;
Page 147 and 148:
�� 3.9 Identification of yea
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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.
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�� 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
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�� 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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Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
Page 207 and 208:
Page 209 and 210:
Page 212 and 213:
E. Additional figures Abs 540 nm (a
Page 214 and 215:
Abs 580 nm 1.4 1.2 1 0.8 0.6 0.4 0.
Page 216 and 217:
Figure 7E. Calibration curves for H
Page 218 and 219:
Figure 9E. Example of HPLC chromato
Page 220:
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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