PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

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2.2.3 Screening and selecting of starch-utilizing yeast strains Yeast strains (Section 2.2.1) were screened for their amylase production capabilities. The yeast isolates were cultured by patching cells on starch agar (Appendix B5) plate surface (two replicates), and allowed to grow for 3 days at 30°C. Amylolytic zones corresponding to enzymatic degradation of starch were detected by staining the plates with iodine solution (Hesseltine et al., 1963, Appendix A5). Colonies exhibiting large clear zones comparing to each other were selected, and maintained using soluble starch agar medium (Appendix B6). The starch hydrolysis and sugar alcohol production of by selected yeast strains was also determined in liquid medium. Yeast isolates were cultivated in 50 ml of starch broth (Appendix B7) containing 2% (w/v) of either cassava starch (Korat Cassava Industrial, Nakhon Ratchasima, Thailand), or rice starch (Rongsenmechoheng Co., Ltd., Nakhon Ratchasima, Thailand), or Khao-klong starch (Rongngan Pochanahanchainsiri Co., Ltd. Bangkok, Thailand) as their carbon sources with shaking at 200 rpm and incubating at 30°C for 72 hours (two replicates). Samples were collected. 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 (Miller, 1959) (Appendix A6). 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 alcohols by High-Performance Liquid Chromatography (HPLC). Growth as cell numbers was determined using haemocytometer (Appendix C1). Starch hydrolysis enzymes were determined. � The measurement of α-amylase activity was performed in test tubes by adding 0.5 ml enzyme solution (diluted if necessary) to a 0.5 ml substrate solution containing 0.0067 M NaCl and 1% soluble starch in 100 ml of 0.02 M phosphate buffer (pH 6.9) (two replicates). After 10 mins incubation at 30°C, the reaction was terminated by adding 0.5 ml 3,5-dinitrosalicylic acid solution and quickly placing the reaction tubes into a boiling water bath for 5 min. The reaction tubes were then cooled in running tap water and diluted with 4 ml doubled-distilled water. The extent of product formation by enzyme reaction was monitored at 540 nm absorbance (A 540). Glucose was used as a standard. One unit of α-amylase activity was defined as the amount of enzyme required to liberate one µM of glucose/min or in terms of milligram of maltose liberated in 1 minute at 30°C by 1 ml of the enzyme solution (Berfeld, 1951), even though, in the case of ��
amylase action, the actual reaction products are dextrin rather than maltose. Specific activity was expressed as amylase activity per milligram of protein. Protein is determined according to Lowry et al., (1951) (Appendix A8). For β-amylase 0.016 M acetate buffer, pH 4.8 was investigated. For glucoamylase activity, standard was assayed in the same manner as α-amylase except that the reaction was carried out in 0.05 M sodium acetate buffer, pH 4.5. One glucoamylase unit was defined as the amount of enzyme that released one µM of glucose/min (Tan et al., 1984). � �2.2.4 Comparison of sugar alcohol production capabilities of selected starch-utilizing yeast strains Selected yeast isolates were tested for their abilities to convert starch into glycerol and mannitol. A) Preparation of inocula Two loopfuls of selected yeast isolates grown for 2 days at 30°C on soluble starch agar medium were inoculated into 100 ml of starch broth. The inoculated starch broth was incubated on a rotary shaker with operating at 200 rpm and incubating at 30°C for 24-48 hours until the culture grow approximate 10 7 cells/ml. B) Sugar alcohol production from starch One millilitre of inocula (Section 2.2.4 A) was inoculated into 50 ml of starch broth. Three types of starch: cassava starch, rice starch, and Khao-klong starch were tested as microbial carbon sources. The inoculated starch media were incubated on a rotary shaker operating at 200 rpm and incubating at 30°C for 3 days. C) Analysis of yeast products The progress of yeast product formation was determined by their reducing sugars, and glycerol and mannitol production of some starch. Three millilitres of each culture medium (Section 2.2.4 B) 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. Cell number was determined with a haemocytometer. And the cell viability was examined by using 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)……
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 and 56: Table 9. (continued). Cell product
Page 57 and 58: 2.1.3 Equipment and other materials
Page 59: Spotted either the supernatant of c
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
Page 109 and 110: 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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Page 205 and 206:
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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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