【作者】 王蓉；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 生物医学工程， 2005， 硕士

【摘要】 本文以资源丰富的玉米淀粉为原料,以米根霉为菌种,进行液体发酵研究获得高品质L-乳酸的最适发酵条件。研究了碳源浓度、氮源种类及其浓度、装液量、温度和pH 等因素对发酵过程菌体生长及产酸的影响,确定了发酵的最适碳源、氮源、装液量、温度及pH 等参数,同时利用固定化技术,采用摇瓶发酵试验确定了最佳固定化载体材料——丝瓜布,研究了载体的边长大小以及用量等方面对固定化发酵产酸的影响,同时比较了固定化细胞与游离细胞的批次发酵能力,为L-乳酸规模化生产提供依据,为聚L-乳酸的批量生产提供可靠的原料来源。本次用米根霉As3.3462,经过研究的主要结论如下: 1、种子培养的最佳培养基为(L):葡萄糖60g;KH2PO4 0.3g;MgSO4 .7H2O 0.25g;ZnSO4 .7H2O 0.08g;尿素2g。最佳培养条件为:250mL 的三角瓶中装入50mL 种子培养基,接种量10%,一次性添加CaCO3 10g/L 用于调节pH 值,置于30℃,转速180 r/min 震荡培养箱,培养20h。2、摇瓶发酵最佳培养基(L):玉米淀粉120.0g, KH2PO4 0.3g,MgSO4.7H2O 0.25g,ZnSO4.7H2O 0.08g,尿素2.0g。最适摇瓶发酵条件:500mL 三角瓶中转入100mL 的发酵培养基,接种量10%,置于30℃,转速180r/min 震荡培养箱中,培养72h。发酵12h 后一次性加入CaCO3 60.0g/L。在该最适发酵条件下,该米根霉菌株的摇瓶发酵L-乳酸积累量可达86.80g/L,对原淀粉的转化率可达65.17%,最高生物量可达8.8g/L。3、利用米根霉固定化发酵玉米淀粉制备L-乳酸,试验利用丝瓜布作为固定化载体,发酵的适宜条件为:载体边长在8～10mm,载体用量选择15mL/50mL 培养基较为适宜。固定化发酵时间比游离发酵时间缩短12h,即固定化发酵产率(1.37g乳酸/L*h)相对游离发酵的产率(1.15g 乳酸/L*h)有所提高。而且固定化发酵有较强的批次重复发酵能力,可连续发酵六批次以上。而游离细胞在发酵第二批时产酸能力显著下降,在第三批时已经难以维持。4、固定化技术的长处在于它是一种良好发酵和分离耦合的生物反应器,能提高微生物的耐性和利用率,能减少后期分离的工艺过程。丝瓜布的吸附固定法简单易行,所制得的固定化细胞稳定性强,可反复发酵,有利于提高设备和菌株的使用效率。更多还原

【Abstract】 Fermentation of high optical purity of L-lactic acid from corn starch was conducted by Rhizopus oryzae As3.3462 .The effects of carbon resources and its concentration,nitrogen and its concentration,air supply,temperature and pH value on cell growth and L-lactic acid fermentation were studied in shake flask.At the same time a method for immobilization of R. oryzae As3.3462 in loofa (Loofa cylindrica) sponge ,cotton cloth and PVA —poly(vinyl alcohol) in shake flask was investigated.Some importants results are as the follows: 1. Loofa sponge as immobilization carrier was better to produce L-lactic acid by R.oryzae As3.3462 compared with PVA and cotton cloth. 2. The optimal compositions of seed medium were as follows(L): glucose 60g;KH2PO4 0.3g; MgSO4.7H2O 0.25g; ZnSO4.7H2O 0.08g; Urea 2g. The number of spores in the suspension was adjusted to 107 per milliliter by dilution with water and the spore suspension was inoculated into a 250ml flask containing 50ml medium and were cultivated at 30℃with shaking at 180r/min for 20h.10g/L CaCO3 autoclaved previously was added to regulate pH.The optimal compositions of fermentation medium were as follows (L): corn starch 120g; Urea 2.0g; KH2PO4 0.3g; MgSO4.7H2O 0.25g; ZnSO4.7H2O 0.08g.The corn starch was liquefied by addition of α-amylase at 85~90 ℃for about 30 min in advance.The fermentation was carried out at 30℃in a 500mL flask containing 100mL medium for 72h.The pH was controlled by adding 60g/L sterile CaCO3,agitation was at 180r/min. On the conditions above,L-lactic acid productivity and the crude corn starch conversion rate were 86.80g/L and 65.17% ,respectively.The biomass concentration was 8.8g/L. 3. Fermentation with loofa (Loofa cylindrica) sponge ,cotton cloth and PVA to be immobilized cell was also studied in shake flask.The immobilization capacity of loofa sponge was found to be preferable to that of PVA and cotton cloth regardless of the cultivation time and L-lactic acid productivity.The productivity of L-lactic aicd was high when the carrier size was about 8～10mm,the concentration of the carrier was 15mL loofa sponge per 50mL seed medium.Results showed that the immobilized cell fermentation circulating six times with loofa sponge was still feasible while the free cell fermentation just two times. The L-lactic acid production velocity had been increased from 1.15 to 1.37g L-lactic acid per litre per hour.It is expected that loofa sponge may be used as an excellent carrier for the immobilization of Rhizopus oryzae.更多还原