PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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II. MATERIALS AND METHODS 2.1�Materials…………………………………………………………………………………36 2.1.1� Chemicals………………………………………………………….……….36 2.1.2� Microorganisms………………………………………………….…………36 2.1.3� Equipment and other materials………………………………….………….36 2.2�Methods……………………………………………………………………….…………37 2.2.1� Isolation and collection of yeasts………………………………….……….37 2.2.2� Screening and selecting of glycerol- and mannitol-producing yeast strains…………………………………………………………….………...37 2.2.3� Screening and selecting of starch-utilizing yeast strains……………….…..40 2.2.4� Comparison of sugar alcohol production capabilities of selected starchutilizing yeast strains…………………………………..………...…………41 2.2.5� Optimization of some glycerol and mannitol production conditions.…...…42 2.2.6� Sugar alcohol overproduction using physical and chemical stress………………………………………………………………..….…...44 2.2.7� Production of glycerol and mannitol for Bradyrhizobium cultivation……………………………………………………..……………46 2.2.8� Cultivation of Bradyrhizobium using nutrient sources produced by yeasts…………………………………………………..…………………...47 2.2.9� Identification of selected yeast isolates………………...……………….….50 III. RESULTS AND DISCUSSIONS 3.1�Isolation and collection of yeasts……………………………………………………..……53 3.2�Screening and selecting of glycerol- and mannitol-producing yeast strains………………53 3.3�Screening and selecting of starch-utilizing yeast strains………………….……………….65 3.4�Starch hydrolysis by selected yeast strains……………………………….…………….….66 3.5�Comparison of sugar alcohol production capability of selected starch-utilizing strains.….73 3.6�Optimization of some glycerol and mannitol production conditions………………….…..75 �
3.6.1� Determination of the suitable and optimum concentration of carbon source……………………………………………………………………….75 3.6.2� Determination of the suitable and optimum concentration of nitrogen source……………………………………………………………..………...79 3.6.3� Determination of the suitable and optimum concentration of yeast extract………………………….…………………………………….……...83 3.6.4� Determination of the suitable inoculum size ……………………...….……86 3.7�Sugar alcohol overproduction by physical and chemical stress………………………..….99 3.7.1� Sugar alcohol overproduction by the addition of calcium carbonate………99 3.7.2� Salt-stress condition for sugar alcohol overproduction…………………...103 3.7.3� Heat shock treatment………………………………………….……….….109 3.8�Cultivation of rhizobia using nutrient sources produced by yeasts …….………………..119 3.8.1� Cultivation of Bradyrhizobium using medium containing glycerol produced by yeasts.…………………………………………………………….…….119 3.8.2� Cultivation of Bradyrhizobium using medium containing mannitol produced by yeasts…………………………………………………………………...124 3.9�Identification of yeasts……………………………………………………...……….……127 3.9.1� Fermentation and assimilation of carbon compound……….………….….127 3.9.2� Assimilation of nitrogen compound………………………………….……136 3.9.3� Hydrolysis of urea………………………………………………….……...136 3.9.4� Effect of temperature on cell growth………………..…………………….136 3.9.5� Ascospore formation………………………...……………………….……137 IV. CONCLUSIONS……………………………..…………………………………………..………147 REFERENCES………………………………………………………………………………….……150 APPENDICES.……………………………………………………………………………….…..…..166 BIBLIOGRAPHY………………………………………………………………………….………...200 ��
Page 1 and 2: 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: CONTENTS Page ABSTRACT (THAI)……
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 and 56: Table 9. (continued). Cell product
Page 57 and 58: 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
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These results indicated that the co
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3.6.1 Determination of the suitable
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g/l 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4
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Reducing sugars (g/l) 8 6 4 2 0 0 1
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Reducing sugars (g/l) 4.0 3.5 3.0 2
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g/l 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
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g/l g/l g/l 1.5 1.0 0.5 0.0 1.2 1.0
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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
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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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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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