臺灣博碩士論文加值系統

在利用米根黴 (Rhizopus oryzae ATCC 9363) 生產L型乳酸的過程中，菌絲乾重和幾丁聚醣受尿素添加量的影響。用5公升攪拌式醱酵槽中進行醱酵，當尿素濃度從每公升2.5公克增加到10.0公克，並以氫氧化鈣控制pH值在6.0，菌絲乾重從每公升4.35公克增加到7.25公克，同時幾丁聚醣從0.290公克增加到0.841公克；在尿素濃度每公升5.0公克時，乳酸有最高產量每公升101.3公克。以氫氧化鈉和碳酸氫鈉混合液控制pH值在6.0，添加尿素濃度每公升5.0公克為氮源，在此條件下，以每公升120公克的葡萄糖為受質，菌絲乾重每公升8.15公克，萃取出幾丁聚醣最高為1.362公克。

During L-lactic acid fermentation by culture of Rhizopus oryzae ATCC 9363, the production of biomass and chitosan was affected by the urea concentration. When fermentation was carried out in a 5-L stirred fermenter by adding calcium hydroxide to control the pH at 6.0, biomass increased from 4.35 g/L to 7.25 g/L as urea from 2.5 to 10.0 g/L was used as N-source; meanwhile, the produced chitosan increased from 0.290 g to 0.841 g. The highest yield of L-lactic acid was 101.3 g/L as 5.0 g/L of urea was used. Upon 5.0 g/L of urea as N-source and 120 g/L of glucose as substrate, 8.15 g of biomass and the highest yield of chitosan at 1.362 g were obtained when pH was controlled at 6.0 by adding a mixture of sodium hydroxide and sodium bicarbonate.

ACKNOWLEDGEMENT………………………………..i
ABSTRACT........................................................................ii
CHINESE ABSTRACT....................................................iii
TABLE OF CONTENTS………..………………………iv
LIST OF TABLES.............................................................ix
LIST OF FIGURES............................................................x
CHAPTER 1 INTRODUCTION......................................1
1.1 Lactic acid………………………….…………………………1
1.1.1 Characterization of lactic acid………………………….…...………1
1.1.2 Commercial production of lactic acid…..………………………..….4
1.1.2.1 Chemical synthesis……………………………………..…..4
1.1.2.2 Fermentation…………………………...…….……..………6
1.1.3 Applications of lactic acid……………………………….………….9
1.1.3.1 Applications of processed food………...….……..……….10
1.1.3.2 The industrial applications…………….……….………….11
1.1.4 Polylactic acid (PLA)…………….…………………….….……....12
1.2 Chitosan……………….………….………….…….………...14
1.2.1 Fungal chitosan………………………………………….….……..14
1.2.2 Structure of chitin and chitosan……………..…………….……….15
1.2.3 Characteristics of chitosan…………………....….…………..…….17
1.2.4 Deacetylation of chitosan……………………………….…………18
1.2.5 Molecular weight……………………………………….………….20
1.3 Rhizopus oryzae…………………….……………….……….21
1.3.1 Rhizopus species………………….…………………….………….21
1.3.2 Rhizopus oryzae…….………………………………….…………..22
1.3.3 The mycelial morphology…………………………….……………25
1.4 Purpose of this study………………………………………..30
CHAPTER 2 MATERIALS AND METHODS.............31
2.1 Microorganisms………………………….…………….……31
2.2 Instruments………………………………………………….31
2.3 Chemicals……………………………….…………………...33
2.4 Cultivation………………………………………….………..34
2.4.1 Culture medium……………….…………………………….……..34
2.4.2 Culture of Rhizopus oryzae ATCC 9363……………….……….....34
2.4.3 Batch fermentation……………………………………..…………..35
2.4.4 Control of fermentation……………………………..……………..37
2.5 Determination of biomass......................................................40
2.6 Analysis of sample compounds by HPLC………….……...40
2.7 Assay of urea………...………………………………...…….41
2.8 Isolation chitosan from the fungal cell wall………….……42
2.9 Analysis of chitosan molecular weight by HPLC……...….43
CHAPTER 3 RESULTS AND DISCUSSION...............44
3.1 Influence of different urea concentration on submerged culture of R. oryzae………………………………………...44
3.1.1 Controlling pH at 6.0 by adding calcium hydroxide during fermentation………………………………………..…........……..45
3.1.1.1 Lactic acid production by R. oryzae using 2.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter…………….….….46
3.1.1.2 Lactic acid production by R. oryzae using 5.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………..….48
3.1.1.3 Lactic acid production by R. oryzae using 7.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter……………...……50
3.1.1.4 Lactic acid production by R. oryzae using 10.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter……..…………….52
3.1.2 Controlling pH at 6.0 by a mixture of sodium hydroxide and sodium hydrogen carbonate…………………………………………..……54
3.1.2.1 Lactic acid production by R. oryzae using 2.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………...…55
3.1.2.2 Lactic acid production by R. oryzae using 5.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………..….57
3.1.2.3 Lactic acid production by R. oryzae using 7.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter…………….….….59
3.1.2.4 Lactic acid production by R. oryzae using 10.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter…………….….61
3.2 Effect of urea concentration on the production of lactic
acid and chitosan……………………..…………………….63
3.3 Effect of urea concentration on molecular weight of
chitosan…………………………………...…………..…….65
CHAPTER 4 CONCLUSION.........................................67
REFERENCES.................................................................69
APPENDIXES…………………………………………..76
Appendixes 1: Standard Curve of Chitosan Molecular Weight………….……………………………76
Appendixes 2: Polymer Average Molecular Weight…………..77
Appendixes 2.1 Number-average molecular weight (Mn)………...77
Appendixes 2.2 Weight-average molecular weight (Mw)………...77
Appendixes 3: The HPLC Diagram of Glucose and Lactic acid…………………………………………….78
Appendixes 4: Pictures of R. oryzae……………………………79














LIST OF TABLES
Table 1.1 Physical properties of lactic acid.....................................................3
Table 3.1 Effect of urea concentration on the production of lactic acid and chitosan by adding Ca(OH)2 to control pH during the fermentation……………………………………………………….…......64
Table 3.2 Effect of urea concentration on the production of lactic acid and chitosan by adding a mixture of NaOH and NaHCO3 to control pH during the fermentation.........................................................................................64
Table 3.3 Effect of urea concentration on molecular weight of chitosan by adding Ca(OH)2 to control pH during the fermentation……..………..…66
Table 3.4 Effect of urea concentration on molecular weight of chitosan by adding a mixture of NaOH and NaHCO3 mixture solution to control pH during the fermentation…………………………………………..……....66



LIST OF FIGURES
Figure 1.1 Lactic acid exists in two optically active isomeric forms…….….3
Figure 1.2 Structures of cellulose, chitin and chitosan……………...……...16
Figure 1.3 Model of glucose metabolism in the filamentous fungus Rhizopus oryzae……………………………………………………………...……..24
Figure 2.1 Scheme of procedures in the fermentative experiments…...........36
Figure 2.2 A display of the original ADVENTECH GENIE strategy of fermentation (main board)………………………………...………….….38
Figure 2.3 A display of original ADVENTENCH GENIE strategy for fermentation (connecting system)…………………………………..……39
Figure 3.1 Time course of lactic acid fermentation by R. oryzae using 2.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………………..47
Figure 3.2 Time course of lactic acid fermentation by R. oryzae using 5.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter…..……..……………..49
Fugure 3.3 Time course of lactic acid fermentation by R. oryzae using 7.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter……….....……..….51
Figure 3.4 Time course of lactic acid fermentation by R. oryzae using 10.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter...…….................…53
Figure 3.5 Time course of lactic acid fermentation by R. oryzae using 2.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………………..56
Figure 3.6 Time course of lactic acid fermentation by R. oryzae using 5.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………………..58
Figure 3.7 Time course of lactic acid fermentation by R. oryzae using 7.5 g/L of urea as N-source at pH 6.0 in a 5-L fermenter……………………..…60
Figure 3.8 Time course of lactic acid fermentation by R. oryzae using 10.0 g/L of urea as N-source at pH 6.0 in a 5-L fermenter………………..…..62

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