PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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approximate 1.0x10 7 cells/ ml obtained. Various concentrations: 1%, 2%, 3%, 4%, 6%, 8%, and 10% (v/v) of each inoculum size were tested using 250 ml Erlenmeyer flask contained 50 ml of starch medium containing 2% (w/v) of either cassava starch, rice starch, or Khao-klong starch (three replicates) as carbon sources, and shaken on a rotary shaker operating at 200 rpm/min and incubating at 30°C. Changes in glycerol, mannitol, and reducing sugars concentration were measured each day during fermentation for 7 days. The determination of growth and product analysis was mentioned in Section 2.2.5 A. 2.2.6 Sugar alcohol overproduction using physical and chemical stress To obtain the overproduction of sugar alcohols, stress conditions were applied to select yeast isolates. Various conditions which were heat shock treatment, salt-stress, and pH regulation by the addition of CaCO 3 were investigated. A) Heat shock treatment of yeast cells Heat shock treatment was performed at 45°C for 0, 20, 40, and 60 min (Figure 9). Yeast cells were grown in 50 ml of pre-culture medium (starch medium containing the optimal concentration of either cassava starch, or rice starch, or Khao-klong starch) (three replicates), then shaken on a rotary shaker operating at 200 rpm, and incubating at 30°C for 24-48 hours. Ten millilitres of each pre-culture approximate 10 8 cells per ml were drawn aseptically from culture flask and pipette into the test tubes, heat shocked by raising the temperature from 30°C to 45°C in water bath for the desirable periods. After heat shock treatment, the starch medium were shaken on a rotary shaker operating at 200 rpm, and incubating at 30°C for 6 days. Changes in glycerol, mannitol, and reducing sugars were measured at the maximum yield period during fermentation. Three millilitres of culture medium were drawn aseptically from the fermentation flask and centrifuged for 10 min at 4,500 rpm. The supernatant was analyzed for reducing sugars by the DNS method. Cells were disrupted by using 4 cycles of freezing in liquid nitrogen (-196° C) and thaw at 70°C, then assayed for sugar and sugar alcohol by High-Performance Liquid Chromatography (HPLC). Total cell number was determined using heamocytometer. Viable cell number were determined from the number of colonies that appear on plates of Malt-yeast extract agar. ��
B) Salt-stress conditions Fifty millilitres of starch medium containing optimal concentration of either cassava starch, rice starch, or Khao-klong starch containing 0%, 3%, 5%, and 7% (w/v) of NaCl (three replicates). The appropriate concentration of inocula were inoculated into the starch medium, shaken on a rotary shaker operating at 200 rpm, and incubating at 30°C for 6 days. Changes in glycerol, mannitol, and reducing sugars were measured at the maximum yield period during fermentation. The determination of growth and product analysis was mentioned in Section 2.2.6 A. C) Calcium carbonate (CaCO 3 ) addition Fifty millilitres of starch medium containing optimal concentration of either cassava starch, rice starch, or Khao-klong starch containing 0%, 0.25%, 0.5%, and 0.75% (w/v) of CaCO 3 (three replicates). The appropriate concentration of inocula which was 24-48 hour old was inoculated into the starch medium, shaken on a rotary shaker operating at 200 rpm and incubating at 30°C for 6 days. Changes in glycerol, mannitol, and reducing sugars were measured at the maximum yield period during fermentation. The determination of growth and product analysis was mentioned in Section 2.2.6 A. 50 ml of starch medium inoculated with 3 loopfuls of yeast cells from a 24-48 h culture Incubated with shaking at 200 rpm and 30°C for 24 h Precultured yeasts containing approximate 10 8 cells/ml Treated 10 ml of cultured starch medium at 45°C for 0, 20, 40, and 60 min Transferred into 50 ml of starch medium Incubated with shaking 200 rpm and 30°C for 7 days Figure 9. Outline of heat shock treatment for yeast cells. ��
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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
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 and 56: Table 9. (continued). Cell product
Page 57 and 58: 2.1.3 Equipment and other materials
Page 59 and 60: Spotted either the supernatant of c
Page 61 and 62: amylase action, the actual reaction
Page 63: A) Determination of the suitable an
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
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
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g/l g/l 1.6 1.4 1.2 1.0 0.8 0.6 0.4
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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
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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
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Table 15. The comparison of media f
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Table 17. Growth of Bradyrhizobium
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�� Rhizobium (Stowers, 1985;
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�� 3.9 Identification of yea
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KAY1 PIY2 PUY4 Y69 LIY2 COY1 FAY2 C
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�� assimilations were observ
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Table 20. (continued). Yeast strain
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Table 20. (continued). 15. Pichia k
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�� 3.9.5 Ascospore formation
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(a) (b) (c ) (d) (e) �� Figu
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(a) (b) (c) (d) (e) (f) �� F
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(c) (a) �� Figure 72. Cell m
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�� Metshnikowia (Pitt and Mi
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CHAPTER IV CONCLUSION This study ex
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only a low capital investment. Ther
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�� Barnett, J. A., Payne, R.
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�� De Mot, R., and Verachter
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�� Jennings, D. H. (1995). T
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�� Lie, T. A., Muilenbury M.
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�� Onishi, H., and Suzuki, T
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�� Sinclair, N. A., and Stok
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�� Van Rossum, D., Schuurman
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�� Young, J. P. W., and Hauk
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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 207 and 208:
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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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