摘要：该研究以Yarrowia lipolytica SWJ-1b为发酵菌株，以地沟油为原料，进行柠檬酸的发酵生产。对发酵培养基的组成成分进行了组合优化，结果发现，适度添加氮和镁使得柠檬酸发酵过程中副产物油脂和异柠檬酸产量显著下降，柠檬酸发酵产率提高；而维生素B1和磷酸盐的添加则导致副产物的累积而对柠檬酸生成产生不利影响。同时，发酵培养基最佳初始地沟油浓度为80.0 g/l，最佳接种量为1×107个细胞/l培养基。在此发酵条件下，副产物的生成得到有效抑制，达到较高的柠檬酸产量。在5-l发酵体系中进行发酵，经过336个小时，柠檬酸产量达到31.7 g/l，生物量为5.9 g/l，异柠檬酸浓度6.5 g/l，胞内油脂为42.1 g/100g细胞干重。发酵结束时，有94.6%的地沟油被酵母菌利用，柠檬酸产率达到了0.4 g/g地沟油。这表明Y. lipolytica SWJ-1b利用地沟油发酵柠檬酸时, 通过培养基成分优化能有效抑制发酵过程中副产物生成，从而显著提高该菌的柠檬酸发酵产率。60162

毕业论文关键词：柠檬酸，解脂耶罗维亚酵母菌，地沟油，异柠檬酸，胞内油脂

Abstract In this study, citric acid producing from illegal cooking oil by Yarrowia lipolytica SWJ-1b was investigated. Here the compositions of the medium for citric acid producing with illegal cooking oil were optimized, and the results showed that extra nitrogen and magnesium rather than vitaminB1 and phosphate were needed for CA producing. To get the maximal CA yield, the optimal concentration of the illegal cooking oil in the medium was 80.0 g/l, and the suitable inoculation size was 1*107 cells/l medium. We also reported that during 10-l fermentation, 31.7 g/l of citric acid, 6.5 g/l of isocitric acid, 5.9 g/l of biomass and 42.1 g/100.0 g cell dry weight of lipid were attained from 80.0 g/l of illegal cooking oil within 336 h. At the end of the fermentation, 94.6 % of the illegal cooking oil was used by cells of Y. lipolytica SWJ-1b, and the yield of citric acid was 0.4 g/g illegal cooking oil, which suggested that illegal cooking oil is a suitable carbon resource for citric acid production.

 Key words: citric acid, Yarrowia lipolytica, illegal cooking oil, isocitric acid, intracellular lipid 

1 前言 3

2 材料与方法 5

2.1 材料 5

2.2 方法 6

3 结果与讨论 8

3.1 (NH4)2SO4浓度对产物产量的影响 8

3.2 KH2PO4浓度对产物产量的影响 9

3.3 Mg2+浓度对产物产量的影响 10

3.4 维生素B1浓度对产物产量的影响 11

3.5 接种量对产物产量的影响 11

3.6 地沟油含量对产物产量的影响 12

3.7 发酵过程产物含量变化趋势 13

结论 14

参考文献 15

致谢 17

1 前言

柠檬酸（Citric acid）又名2-羟基丙烷-1, 2, 3-三羧酸，是三羧酸循环的中间代谢物，也是一种重要的有机酸，由于其特殊性质能够作为酸化剂、调剂和抗氧化剂，因此被广泛应用于食品业、制药业和农业等众多领域。据报道，世界柠檬酸发酵年产量已达170万吨，其中我国年产量达到94万吨，占世界柠檬酸产业的50%以上[1]。目前工业柠檬酸主要采用黑曲霉（Aspergillus niger）发酵淀粉质原料的方式获得，然而A. niger及其孢子具有极大的致病隐患（常导致操作工人罹患肺曲霉肿等职业疾病），其发酵过程也往往伴随大量固液体废弃物产生，给社会及环境造成很大负担。此外，A. niger对发酵环境敏感，很难利用低成本的粗原料，在加剧粮食危机的同时，也使得发酵成本不断增加[2]。近年来，在激烈的行业竞争下，柠檬酸产业利润空间被大幅压缩而陷入空前的尴尬境地，亟需寻找一种环境友好、经济可行的新型发酵模式。