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研究生：	何奈卡 Heru, Naomi Oktarina
論文名稱：	以大型藻為料源進行乳酸之醱酵生產 Fermentative Lactic Acid Production from Macroalgae Feedstock using Lactobacillus sp. and Weissella sp.
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	115
中文關鍵詞：	乳酸桿菌、乳酸醱酵、可再⽣料源、聚⼄烯醇、⽯莼
外文關鍵詞：	L.plantarum 23, lactic acid fermentation, renewable feedstock, polyvinyl alcohol, macroalgae
相關次數：	點閱：96 下載：1
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乳酸是⼀種天然有機酸，是⼀種有價值的⼯業化學品，最常⽤於食品和製藥⾏業。乳酸還⽤於⽣產⽣物可降解塑料聚乳酸，這是⼀種有前途的⽣物相容性和⽣態友好型替代品，可替代化⽯燃料衍⽣的塑料。可再⽣原料是作為乳酸⽣產底物的潛在替代品，因為乳酸的當前⼯業⽣產成本受到原材料/醱酵底物⾼成本的極⼤影響。使⽤可再⽣原料的優點包括廉價的獲取和⾼碳⽔化合物含量。⼤型藻類或海藻是可醱酵碳⽔化合物的豐富⽽廉價的來源，可通過醱酵將其轉化為乳酸。在這項研究中，各種乳酸菌如乳酸桿菌屬(Lactobacillus sp.)和魏斯⽒菌屬(Weissella sp.)被⽤於乳酸的醱酵⽣產。為了獲得⾼的乳酸⽣產率和產量，將乳酸菌固定在聚⼄烯醇（PVA）中。本研究探討了三種⼤型藻類，即綠藻（Ulva sp.）、紅藻（Gracillaria sp.）和褐藻（Sargassum cristaefolium），以評估它們作為乳酸醱酵原料的適⽤性。我們之前的研究（Atika.2018）報告了最佳發酵條件：pH 5.5，溫度30度5，PVA顆粒負載量為12.5％，細胞負載量濃度為5.25 g cell/L，還原糖濃度為40 g/L。相應的⼤型藻類使⽤熱酸⽔解⽅法進⾏了預處理。來⾃預處理的⽔解產物⽤作乳酸醱酵的碳源。與懸浮細胞醱酵相比，使⽤固定有PVA的細胞進⾏醱酵可獲得更⾼的⽣產率。使⽤綠⾊⼤型藻類（Ulva sp.）進⾏的分批乳酸醱酵，鼠李糖乳桿菌(L. rhamnosus)的最⼤乳酸產量為5.22±0.34 g/L/h，最⼤乳酸濃度為31.30±0.58 g/L。另⼀⽅⾯，使⽤L. rhamnosus 24的紅⾊⼤型藻類（Gracillaria sp.）的最⼤乳酸濃度為32.11±0.21 g/L，最⼤⽣產率為6.34±0.19 g/L/h。使⽤L. plantarum 23的⼤型藻（Sargassum cristaefolium），產⽣的最⼤乳酸濃度和⽣產⼒分別為30.91±0.45 g/L和7.091±.64 g/L/h。

Lactic acid (LA), a natural organic acid and a valuable industrial chemical, is most commonly used in food and pharmaceutical industry. LA is also used for the production of biodegradable plastics (PLA), which is a promising biocompatible and eco-friendly alternative for fossil fuel derived plastics. Renewable feedstock is a potential alternative as a substrate for lactic acid production, because current industrial production costs of LA is highly affected by the high cost of raw materials/fermentation substrates. The advantages of using renewable feedstock include inexpensive acquirement and high carbohydrate content. Macroalgae or seaweed are abundant and inexpensive source of fermentable carbohydrates, which could be converted to lactic acid via fermentation In this study, various lactic acid bacteria (LAB) such as Lactobacillus sp. and Weissella sp. were used for fermentative LA production. To obtain high LA productivity and yield, LAB were immobilized in polyvinyl alcohol (PVA). Three varieties of macroalgae namely, green algae (Ulva sp.), red algae (Gracillaria sp.), and brown algae (Sargassum cristaefolium) were investigated to evaluate their suitability as lactic acid fermentation feedstock. The optimal fermentation conditions were reported in our previous study (Atika.2018): pH 5.5, temperature 30°C, PVA particle loading 12.5%, cell loading concentration 5.25gcell/L, and reducing sugar concentration 40 g/L. The corresponding macroalgae were pretreated using thermal-acid hydrolysis method. The hydrolysate from the pretreatment were used as the carbon source for LA fermentation.
Fermentation using PVA-immobilized cell obtained better productivity compared to suspended cell fermentation. Batch LA fermentation using green macroalgae (Ulva sp.), attained a maximum lactic acid productivity of 5.22±0.34 g/L/h with L. rhamnosus and the maximum lactic acid concentration was 31.30±0.58 g/L. On the other hand, maximum lactic acid concentration using red macroalgae (Gracillaria sp.) using L. rhamnosus 24 was 32.11 ±0.21 g/L and maximum productivity was 6.34±0.19 g/L/h. The highest lactic acid productivity was achieved with brown macroalgae (Sargassum cristaefolium) using L. plantarum 23, which produced maximum lactic acid concentration and productivity of 30.91±0.45 g/L and 7.09±0.64 g/L/h, respectively.

摘要 ............... 1
ABSTRACT .............. I
ACKNOWLEDGEMENT .......... III
CONTENT.............IV
List of Tables ........... VIII
LIST OF FIGURES ...........XI
CHAPTER 1 INTRODUCTION .......... 1
1.1 Motivation and Purpose .......... 1
1.2 Research Scheme .......... 2
CHAPTER 2 LITERATURE REVIEW ......... 4
2.1 Lactic acid ............ 4
2.1.1 General Description .......... 5
2.1.2 Lactic Acid Fermentation ......... 7
2.1.3 Metabolic pathways for lactic acid production.... 7
2.1.4 Lactic acid bacteria (LAB) ....... 10
2.2 Lactic Acid Fermentation .......... 12
2.2.1 Fermentation Cell System ........ 12
2.2.2 Fermentation Modes ......... 14
2.2.3 Factor-factors Affecting LA production ...... 16
2.3 Macroalgae ........... 17
2.3.1 Green Algae (Ulva sp.) ........ 18
2.3.2 Red Algae (Gracillaria sp.)........ 20
2.3.3 Brown Algae (Sargassum cristaefolium) ..... 22
2.3.4 Pretreatment of Macroalgae Biomass for Microbial Fermentation
............. 23
2.3.5 Macroalgae as Renewable Source ...... 25
CHAPTER 3 MATERIALS AND METHODS ........ 27
3.1 Chemicals and Materials ......... 27
3.2 Equipment ............ 28
3.3 Microorganism and Mediums ......... 29
3.3.1 Bacteria strain .......... 29
3.3.2 Medium composition and Cultivation Condition .... 30
3.4 Feedstock ........... 31
3.4.1 Ulva sp. ............ 31
3.4.2 Gracillaria. sp. ......... 32
3.4.3 Sargassum cristaefolium ......... 34
3.5 Experimental methods .......... 35
3.5.1 Preculture.......... 35
3.5.2 Lactic Acid fermentation using Suspended Cells .... 35
3.5.3 Immobilization of Lactic Acid Bacteria (LAB) using PVA.. 35
3.5.4 Preparation of Macroalgae Hydrolysate ...... 36
3.5.5 Concentration of Macroalgae Hydrolysate using Vacuum
Evaporation ........... 36
3.5.6 Batch fermentation using Green Macroalgae Hydrolysate as
Carbon Source......... 36
3.5.7 Batch fermentation using Red Macroalgae Hydrolysate as Carbon
Source ............ 36
3.5.8 Batch fermentation using Brown Macroalgae Hydrolysate as
Carbon Source......... 37
3.6 Measurement Method ......... 37
3.6.1 Measurement of cell concentration ...... 37
3.6.2 Measurement of reducing sugar and soluble metabolites
concentration by high performance liquid chromatography (HPLC)
............. 37
3.6.3 Measurement of carbohydrate content ...... 38
3.6.4 Measurement of lipid content....... 38
3.6.5 Measurement of amino acid ....... 39
3.6.6 Measurement of ionic compound content ...... 39
3.6.7 Measurement of hydroxymethylfurfural (HMF) and furfural . 39
3.6.8 Measurement of ash content ........ 40
3.6.9 Analysis of transient behavior by modified Gompertz equation .. 40
3.6.10 Measurement of lactic acid yield, productivity and sugar
consumption .......... 41
3.6.11 Calculation of Biomass Hydrolysis Efficiency and Reducing Sugar
Yield ........... 42
CHAPTER 4 RESULTS AND DISCUSSION........ 43
4.1Screening of LA bacteria and carbon source for LA production .... 43
4.1.1 Effect of various lactic acid bacteria on LA fermentation .. 43
4.1.2 Effect of carbon source on LA fermentation ..... 45
4.2 Using green macroalgae hydrolysate as renewable feedstock for LA
production ........... 46
4.2.1 Green Macroalgae Pretreatment...... 46
4.2.2 Lactic Acid Bacteria (LAB) Screening ..... 48
4.2.3 Batch Fermentation using Macroalgae Powder .... 50
4.2.4 Batch Fermentation using Suspended Cell and Green Macroalgae
Hydrolysate as Feedstock ........ 52
4.2.5 Batch fermentation using PVA-immobilized cell and green
macroalgae hydrolysate as feedstock ...... 56
4.3 Using Red macroalgae hydrolysate as renewable feedstock for LA production
.............. 58
4.3.1 Red Macroalgae Pretreatment ....... 60
4.3.2 Lactic Acid Bacteria (LAB) Screening ..... 62
4.3.3 Batch fermentation using Suspended Cell and Red Macroalgae
Hydrolysate as feedstock ......... 68
4.3.4 Batch fermentation using PVA-immobilized Cell and Red
Macroalgae Hydrolysate as Feedstock ..... 70
4.4 Using Brown macroalgal hydrolysate as renewable feedstock on LA
production ........... 72
4.4.1 Brown Macroalgae Pretreatment ....... 72
4.4.2 Lactic Acid Bacteria (LAB) Screening ..... 75
4.4.3 Batch fermentation using Suspended Cell and Brown Macroalgae
as Carbon Source .......... 81
4.4.4 Batch fermentation using PVA-Immobilized Cell and Brown
Macroalgae as Carbon Source ........ 82
CHAPTER 5 CONCLUSIONS .......... 85
APPENDIX ............. 91
                                    

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