【作者】 任慧卿；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 生物医学工程， 2010， 硕士

【摘要】 乳酸是生物医学工程材料聚乳酸的生产原料,而且随着生物可降解材料-聚乳酸研究的迅速发展,乳酸的生产越来越受到人们的高度关注。目前乳酸生产的方法大部分采用微生物发酵法,常用的菌种有米根霉和乳酸菌。米根霉的营养需求低,产出的乳酸光学纯度高。但是由于真菌的菌丝形态难控制,游离发酵易形成凝团和结块,降低了生产效率。采用固定化方法,可以控制菌丝形态,缩短发酵时间,且固定化细胞可重复稳定使用。此外,米根霉可以利用的碳源有葡萄糖和淀粉,但是随着能源和粮食问题的加重,需要找到廉价且易得的原料。目前研究较多的是利用纤维素原料—农业废弃物如玉米秸秆进行发酵,本文对此也进行了探讨。本文分为两部分进行了研究:采用聚乙烯醇丝作为吸附材料,制成十字形立体载体,研究了该载体的参数、发酵条件及载体的长期稳定性;在此基础上,初步研究了以秸秆酶解液为碳源进行发酵的产乳酸（游离与固定化）。首先,以葡萄糖作为碳源,确定载体的形状为十字形,载体长度2.5cm、PVA丝质量0.12g/个、孢子浓度0.5×10⁶个/ml、1个载体进行发酵,乳酸浓度可达到51.13g/L,转化率为63.90%。与游离发酵相比,摇瓶固定化发酵乳酸浓度提高了25%,发酵时间缩短了33%。其次,确定了载体的摇瓶发酵条件:在种子培养的开始就加入载体固定化培养24h、转速180 r/min、发酵开始一次性添加20g/L的CaCO3,乳酸浓度54.32g/L。摇瓶和1L的鼓泡式生物反应器的半连续发酵研究表明载体固定化发酵可保持长期稳定性。摇瓶可稳定发酵10批次,平均转化率65.00%,平均产率稳定1.10 g/L·h;1L反应器乳酸浓度最高达到55.68 g/L,转化率69.60%。最后,以玉米秸秆作的酶解液作为碳源,初步探讨了酶解液的发酵。选择蒸汽爆破和物理化学法相结合对秸秆进行了预处理,降低了木质素的含量,提高了纤维素酶对原料的敏感性。通过均匀设计得到最佳的纤维素酶浓度35FPU/g和底物浓度50g/L,酶解率可达到94.03%。采用酶解液进行发酵的研究中,优化培养基为:MgSO₄·7H₂O 0.35 g/L,ZnSO₄·7H₂O 0.20 g/L,KH₂PO₄ 0.15 g/L,尿素1g/L。游离发酵中,48小时乳酸转化率52.36%;采用PVA丝载体固定化发酵,发酵周期缩短为30小时,乳酸转化率30.19%。更多还原

【Abstract】 Lactic acid is precursor of poly lactic acid which is a kind of biomedical engineering materials.Recently, with the rapid development of biodegradable materials-poly lactic acid, there has been an high interest in lactic acid production. The main lactic acid production method is microorganism fermentation in which lactic acid bacteria and Rhizopus oryzae are commonly used. The nutritional requirement of Rhizopus oryzae is simple and its output is in high optical purity. However, it is difficult to control the fungal cell morphology and the dispersed mycelia would easily form clumps which will impose diffusion limitation and reduce lactic acid production. The immobilized-cell fermentation can easily control the mycelium morphology and shorten fermentation time, besides the immobilized-cell can be reused stably. Glucose and starch are often used as the carbon source of Rhizopus oryzae, but as the energy and food problem becoming more and more serious, it is urgent to find new raw materials which are inexpensive and easy to get. Nowadys, the agriculture waste such as corn straw are extensively studied as cellulosic raw material in lactic acid fermentation which was also investigated in this paper.This paper was divided into two parts. Firstly, polyving achohol fiber as absorbing material for cruciform matrix production was adopted, and the parameters, fermentation conditions and long-term stability of matrix were researched. At the same time, the fermentation production of lactic acid （free and immobilized cell） by using straw hydrolyzate as carbon source was studied.Firstly, using glucose as carbon source and determining the shape of matrix to be cruciform in the fermentation, with the matrix inner diameter of 2.5 cm, PVA quality of 0.12g/per matrix, spores concentration of 0.5×10⁶/ml and one matrix, as a result, the lactic acid concentration achieved 51.13 g/Lwith the yield of 63.90%. Compared with free fermentation, using immobilized fermentation in shake-flasks, the final lactic acid concentration was increased by 25% and the fermentation time was shortened by 33%.Secondly, the matrix conditions of shake-flasks fermentation were determined: matrix was put in at the beginning and culturing for 24h, with a rotation speed of 180r/min and adding 20g/L CaCO3 at the beginning of the fermentation at once, lactic acid concentration reached 54.32 g/L. The performance in both of shake-flasks and 1L bubble-column bioreactor operated in a repeated batch mode was indicated the long-term reusability. The overall yield of first 10 cycles in shake-flasks was 65%, corresponding to a volumetric productivity of 1.1g/L·h. Moreover, the highest lactic acid concentration in the bubble-column bioreactor was 55.68% with a yield of 69.60%.Finally, the fermentation with hydrolyzate of corn straw as carbon source was primary investigated. The pretreatment of corn straw adopted the method of combined steam explosion and physical-chemical process. The content of lignin was reduced; meanwhile the sensitivity of cellulase enzyme to corn straw was increased. The optimal condition for hydrolyze was developed by using Uniform Design theory. When the concentration of cellulose enzyme was 35FPU/g and substrate was 50g/L, the hydrolysis achieved 94.03%. During the study of hydrolyzate fermentation, an optimal medium of MgSO₄·7H₂O 0.35 g/L, ZnSO₄·7H₂O 0.20 g/L, KH₂PO₄ 0.15 g/L and urea 1g/L was used. The lactic acid yield was 52.36% in 48h by using free cell while the lactic acid yield was 30.19% by using immobilized cell and the fermentation time was shortened to 30h.更多还原