厨余垃圾发酵L-乳酸的光学研究

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厨余垃圾发酵

L-乳酸的光学研究

摘要

本论文对米根霉 AS3.819 发酵开放式厨余垃圾生产 L-乳酸，进而合成聚乳

酸进行研究。利用分光光度法测定 L-乳酸含量，优化了摇瓶游离发酵培养条件，

并进行了优化条件下的放大实验；同时采用了固定化技术，对海藻酸钙固定化

米根霉作了初步的探讨，在本论文中，主要研究结论有以下几点：

1)以开放式厨余垃圾为原料，以米根霉 AS3.819 为菌种进行的摇瓶游离优

化实验表明：最佳发酵条件为：PH 为8，种龄 16 小时，接种量 10%，温度为

34℃ 。在该最适发酵条件下该米根霉菌株的摇瓶发酵 L- 乳酸积累量达到

63.2g/L，发酵时间为 80 小时，L-乳酸生产率为 0.79g/(L·h)。在该最适条件下，

发酵罐发酵 L-乳酸积累量达到 63.1g/L，发酵时间为 120h，L-乳酸生产率为 0.53

g/(L·h)。

2)以海藻酸钙为载体固定化细胞，进行固定化细胞优化工艺的研究，实验

表明：最佳发酵条件为：海藻酸钙凝珠直径 1.8mm，凝珠增值时间为 20 小时，

载体添加量为 35%。在此条件下进行的半连续发酵结果为：用 1.8mm 直径凝珠

半连续发酵六次，初始发酵时 L-乳酸产量保持基本稳定，后五个批次中随着批

次的增加，L-乳酸的产量也有小幅提高。固定化发酵周期比摇瓶游离发酵周期要

短得多，第一批次发酵用时 50 小时，发酵产酸 66.05g/L，产酸率 1.32g/(L·h)。后

五个批次发酵周期为 24 小时，发酵产酸 67.76-67.78g/L，产酸率大约为 2.82g/

(L·h)。可见，固定化发酵较游离发酵有较大优势。

关键词：厨余垃圾 L-乳酸米根霉 AS3.819 聚 L-乳酸分光光度法

ABSTRACT

In this paper, spectrophotometry is used to calculate L-lactic acid concentration. The

shake-flask suspension fermentation conditions of L-lactic acid production by rhizopus

oryzae AS3.819 from nonautoclaved minced kitchen waste is investigated, and the

amplification experiment under the optimized conditions is also conducted; at the same

time this paper also optimized the immobilized fermentation conditions in shake

flasks, which is immobilized by the calcium alginate. Important results are as

followings:

1)Fermentation conditions in shake-flask suspension fermentation by Rhizopus

oryzae AS3.819 were carried out. The most suitable fermentation conditions:

temperature of 34℃, seed age of 16 hours and 10% for inoculum size, PH8. Under

such fermentation conditions, the final concentration of the L-lactic acid of shake-flask

experiments arrived 63.2g/L and the fermentation productivity was 0.79g/(L·h) for

80h. Under such fermentation conditions, the final concentration of the L-lactic acid of

amplification experiment arrived 63.1g/L and the fermentation productivity was 0.53

g/(L·h) for 120h.

2)The L-lactic acid fermentation conditions by immobilized Rhizopus oryzae

AS3.819 was investigated. Favorite productivity of L-lactic acid was induced when the

carrier size was about 1.8mm, culture time of the carrier was 20h, concentration of the

carrier was 20%. Under above fermentation conditions, this paper conducted six

times’s emicontinuous fermentations, the L-lactic acid concentration of the first cycle

was 66.05 g/L, with production rate of 1.32 g/(L. h), the later five cycles had a little

increase one by one. The fermentation time of first cycle was 50h, and the later five

cycles was 24h, the production rate of the later five cycles were above 2.82g/L, which

was much higher than suspension fermentation.

Key Words：Kitchen waste, L-lactic acid, Rhizopus oryzaeAS3.819,

poly-L-lactic acid, spectrophotometry

中文摘要

ABSTRACT

第一章绪论..............................................................................................................1

1.1 课题研究背景..................................................................................................1

1.2 厨余垃圾简介..................................................................................................1

1.2.1 厨余垃圾特性...........................................................................................1

1.2.2 厨余垃圾资源化技术探讨.......................................................................2

1.3 乳酸及 L-乳酸.................................................................................................3

1.3.1 乳酸的性质...............................................................................................3

1.3.2 乳酸及 L-乳酸的应用...............................................................................4

1.3.3 发酵法生产 L-乳酸的代谢途径...............................................................5

1.3.3.1 同型乳酸发酵.........................................................................................5

1.3.3.2 异型乳酸发酵.........................................................................................5

1.3.3.3 混合酸发酵.............................................................................................6

1.3.4 米根霉发酵产 L-乳酸...............................................................................6

1.3.4.1 菌种选育.....................................................................................................6

1.3.4.2 发酵生产工艺条件.....................................................................................7

1.4 厨余垃圾发酵产乳酸现状..............................................................................7

1.5 本课题研究意义及主要内容..........................................................................8

1.5.1 本课题的研究意义...................................................................................8

1.5.2 本课题研究的主要内容...........................................................................9

第二章分光光度法测定 L-乳酸及葡萄糖含量....................................................10

2.1 光的吸收定律................................................................................................10

2.1.1 液层厚度与光吸收关系.........................................................................10

2.1.2 溶液的浓度和光吸收的关系.................................................................11

2.1.3 吸收定律.................................................................................................12

2.2 分光光度法....................................................................................................12

2.3 分光光度法测定 L-乳酸及葡萄糖含量.......................................................14

2.3.1 L-乳酸测定.............................................................................................14

2.3.1.1 材料....................................................................................................14

2.3.1.2 分光光度法测定乳酸含量...................................................................14

2.3.1.3 发酵液样品预处理...............................................................................14

2.3.1.4 标准曲线的制定...................................................................................14

2.3.1.5 发酵液 L-乳酸的测定..........................................................................15

2.3.2 还原糖的测定.........................................................................................15

2.3.2.1 DNS 试剂的配置..................................................................................15

2.3.2.2 原理.......................................................................................................16

2.3.2.3 标准曲线的制定...................................................................................16

2.3.2.4 发酵液中残糖的测定...........................................................................18

第三章发酵设计、材料与方法.............................................................................19

3.1 光的吸收定律..................................................................................................19

3.1.1 菌种.........................................................................................................19

3.1.2 试剂.........................................................................................................19

3.1.3 仪器.........................................................................................................20

3.2 分析方法........................................................................................................21

3.2.1 菌体干重测定.........................................................................................21

3.2.2 PH 值的测定...........................................................................................21

3.2.3 溶氧的测定.............................................................................................21

3.2.4 孢子浓度计数.........................................................................................21

3.3 发酵实验系统................................................................................................21

3.4 实验控制方法................................................................................................23

3.5 乳酸产生速率................................................................................................25

第四章米根霉游离发酵工艺条件的优化.............................................................26

4.1 引言................................................................................................................26

4.2 菌种................................................................................................................26

4.3 培养基............................................................................................................26

4.3.1 PDA 培养基............................................................................................26

4.3.2 液体种子培养基.....................................................................................26

4.3.3 发酵培养基.............................................................................................26

4.4 培养方法........................................................................................................27

4.4.1 米根霉孢子悬液的制备.........................................................................27

4.4.2 液体种子增殖培养.................................................................................27

4.4.3 摇瓶悬浮发酵培养.................................................................................27

4.5 结果与讨论....................................................................................................27

4.5.1 PH 对发酵的影响...................................................................................27

4.5.2 种龄对发酵的影响.................................................................................29

4.5.3 接种量对发酵的影响.............................................................................30

4.5.4 温度对发酵的影响.................................................................................31

4.5.5 优化条件下的发酵罐发酵.....................................................................32

4.6 小结................................................................................................................34

第五章米根霉固定化发酵工艺条件的优化.........................................................35

5.1 菌种................................................................................................................35

5.2 培养基............................................................................................................35

5.3 培养方法........................................................................................................35

5.3.1 米根霉孢子悬液的制备.........................................................................35

5.3.2 细胞固定方法.........................................................................................35

5.3.2.1 海藻酸钙凝珠制备.................................................................................35

5.3.2.2 固定化细胞的增殖培养.........................................................................35

5.3.2.3 发酵实验.................................................................................................35

5.4 结果与讨论....................................................................................................35

5.4.1 凝珠增殖时间对发酵的影响.................................................................35

5.4.2 凝珠直径大小对发酵的影响.................................................................36

5.4.3 凝珠添加量对发酵的影响.....................................................................36

5.4.4 优化条件下的摇瓶半连续发酵实验.....................................................37

5.5 小结................................................................................................................38

第六章结论与展望.................................................................................................39

6.1 结论................................................................................................................39

6.2 展望................................................................................................................39

参考文献..................................................................................................................41

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摘要：

厨余垃圾发酵L-乳酸的光学研究摘要本论文对米根霉AS3.819发酵开放式厨余垃圾生产L-乳酸，进而合成聚乳酸进行研究。利用分光光度法测定L-乳酸含量，优化了摇瓶游离发酵培养条件，并进行了优化条件下的放大实验；同时采用了固定化技术，对海藻酸钙固定化米根霉作了初步的探讨，在本论文中，主要研究结论有以下几点：1)以开放式厨余垃圾为原料，以米根霉AS3.819为菌种进行的摇瓶游离优化实验表明：最佳发酵条件为：PH为8，种龄16小时，接种量10%，温度为34℃。在该最适发酵条件下该米根霉菌株的摇瓶发酵L-乳酸积累量达到63.2g/L，发酵时间为80小时，L-乳酸生产率为0.79g/(L·h)。在该最适条...

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