PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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�� might conclude PUY4 cultured medium showed the same results, but it gave the amount of bacterial cell at 10 8 cells/ml when compared with G5 medium. It might cause of the amounts of ethanol in cultured medium. Lie et al., (1991) Reported that no inhibitory effect of 5 g/l ethanol in a glycerol-yeast extract medium were observed. However, growth of Bradyrhizobium CB 756 on pure ethanol (0.1, 0.5, 1 and 2.5 g/l) was very poor. The amounts of ethanol in this experiment was not measured or controlled. But the smells of ethanol were observed in the 5% sucrose prefermented by yeast. It was suggested that the amount of ethanol in the medium may effected to the growth of Bradyrhizobium japonicum USDA 110, but not effectd for Bradyrhizobium spp. THA5 growth. 3.8.2 Cultivation of Bradyrhizobium using medium containing mannitol produced by yeasts A total 6000 ml of starch medium (2000 ml of Khao-klong starch medium + 2000 ml of rice starch medium + 2000 ml of cassava starch medium) were utilized by yeast isolate KAY1. The cultivation product used for the rhizobial carbon sources was mannitol which produced in the yeast cells. After centrifugation, the supernatant was discarded. The yeast cells grown from the three starch cultured medium were pooled into one flask. Cell were lysed by using 4 cycles of freeze-thawing. The initial high mannitol concentration in cell lysate was adjusted by the addition of distilled water. The cell lysate medium was adjusted to provide 0.5% (v/v) as same as YMB standard rhizobial culture medium. The quantitation of mannitol were tested by using High-Performance Liquid Chromatography (HPLC). And adjusted again as depicted in Table 18. From the Table 18, The cell lysate medium was adjusted. Other substances: 0.05% K2HPO4 and 0.01% NaCl were added, and pH adjusted 6.8 at room temperature. After heat sterilization, the medium was inoculated with B. japonicum USDA 110 and Bradyrhizobium spp. THA5. The results in Table 19, it clearly showed that the cell lysate medium containing mannitol could supported the growth of B. japonicum USDA 110 and Bradyrhizobium spp. THA5. The best results was obtained with 10 8 cells/ml being almost the same as that found with YMB medium. Mannitol was the traditional carbon and energy source used for small volume cultivation of all
�� Rhizobium (Stowers, 1985; Elkan, 1987). The 5% inoculum size of 6 days cultured was used in this experiment. The recommended inoculum level was between 1 and 10%; however, a higher level of inoculum will decrease the time in a given fermentor and was an advantage with the slow-growing rhizobia because it decreased the opportunity for contamination (Elkan, 1987; Vincent, 1982) To check the contamination in the cultured medium, beside to observed the appearance of colony during Table 18. The comparison of media containing mannitol for Bradyrhizobium cultivation. 2%starch medium YMB medium KAY1-medium Mannitol - 5.0 g 6.0 g Glycerol - - 0.8 g Yeast extract 3.0 g 0.5 g - b MgSO4.7H2O 0.5 g 0.2 g - b K2HPO4 - 0.5 g 0.5 g a KH2PO4 1.0 g - - b NaCl - 0.1 g 0.1 g a Glucose - - - Arabinose - - - Tryptone - - - Sucrose - - - PVP-40 - - - Fe- EDTA - - - Starch 20.0 g - - Distilled water 1000 ml 1000 ml 500 ml pH 6.0 6.8 6.8 Note that a same as standard medium b no add
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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
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were isolated from fresh fruits, Ma
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�third day of incubation (Figure
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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 and 138: g/l 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7
Page 139 and 140: �� effected by heat shock po
Page 141 and 142: Table 15. The comparison of media f
Page 143: Table 17. Growth of Bradyrhizobium
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
Page 189 and 190: � 300 µg/ml, 200 µg/ml, and 100
Page 191 and 192: Soluble Starch 20.0 g Distilled wat
Page 193 and 194: 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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