【作者】 王珍；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 生物医学工程， 2010， 博士

【摘要】 L-乳酸是一种重要的天然有机酸,在食品、医药、生物降解塑料的制造上有广泛应用。特别地,应用L-乳酸生产的聚乳酸是一种具有多种良好医学性能的生物高分子材料,在整形外科、骨修复、生物手术缝合线、药物缓释系统等方面的应用不断增加。米根霉( Rhizopus oryzae)由于具备好氧发酵、发酵产物L-乳酸光学纯度高、易于分离等特点,而成为目前制备高光学纯度L-乳酸的主要菌种。当前米根霉发酵生产L-乳酸的工业化过程中的最大困难是:细胞形态的控制,底物成本和反应器的放大可行性。本文主要从一种新型棉布载体的设计为出发点,进行了米根霉固定化发酵生产L-乳酸的研究。该载体以棉布为固定化材料,不锈钢网为支撑,组成六枝星状,具有高表面积而有利于米根霉生长和产酸。为了证明该固定化载体的可行性,放大潜力,长期稳定性和广泛适用性,研究内容包括了米根霉的固定化和发酵动力学,固定化发酵条件的优化,该棉布载体与其他载体方式的优劣比较,低成本底物的利用和生物反应器的放大验证五方面。首先,在棉布载体对米根霉固定过程的研究中,通过对孢子萌芽过程的研究,确定了可以量化米根霉孢子萌芽的停滞期tβ和50%吸附时间λ,并将转速对tβ和λ的影响进行了比较;通过对不同接种浓度下孢子固定化曲线的数学拟合,建立了孢子固定过程的一级反应方程ln( Ct /Co)= ?kt,C代表孢子浓度,Co代表初始孢子浓度,Ct代表时间t时的孢子浓度。吸附速率常数k由C-t的半对数曲线斜率计算得出。其中,前4小时内,k为0.12±0.04 h-1;孢子萌发后,k为0.50±0.07 h-1。其次,在固定化发酵条件的研究中,通过研究固定化载体参数,得到了最佳发酵环境参数:摇床转速180r/min,载体直径2cm,每一摇瓶3个载体,接种孢子浓度1×106/ml。在此条件下,获得乳酸终浓度为49.5 g/L,底物转化率为68.7%,产率为1.03 g/L·h。此外,通过单因素实验方法和均匀设计的结合运用,对固定化米根霉的发酵培养基成分及其影响进行了研究。单因素实验确定了最佳葡萄糖浓度80g/L,氮源为尿素,碳氮比为100:1。均匀设计方法的使用,确定了各无机盐的显著性和交互作用,确定了最佳的无机盐浓度组合:0.5g/L KH2PO4,0.2g/L MgSO4.7H2O,0.04 g/L ZnSO4.7H2O,0.006 g/L FeSO4.5H2O。通过对CaCO3添加浓度和添加方式的研究,从pH控制,乳酸生产和生物量控制三方面进行评价,确定了最佳的CaCO3添加浓度(40g/L)和添加方式(间隔12小时添加一次)。发酵结果证明了该新型棉布载体在米根霉的固定化及乳酸生产中的可行性。随后,通过棉布载体与海藻酸钙凝胶,聚乙烯醇(PVA)凝胶和丝瓜布三种固定化方式以及游离发酵的比较,证明了新型棉布载体固定化米根霉的发酵效果优于其他各组实验。单批发酵中,新型棉布载体固定化米根霉的乳酸产量,得率和产率分别为48.50g/L,60.12%,1.01g/L.h,比游离发酵分别提高了~60%,~70%和~150%。副产物乙醇浓度为游离发酵生成的乙醇浓度的~5%。在8批重复发酵中,新型棉布载体固定化米根霉的平均乳酸产量,平均得率和平均产率分别为50.13g/L,62.66%,1.04g/L.h,比游离发酵分别提高了~110%,~120%和~200%。副产物乙醇的浓度分别为游离发酵和丝瓜布固定化发酵生成的乙醇浓度的~16%和~60%。单批发酵结束,蓬松菌丝层均匀包裹载体,形成星状载体单元。重复发酵结束时,载体形状维持较好,无结团或破裂现象,生物量为游离发酵的~60%。为了进一步实现降低发酵中的底物成本的目的,选取了葡萄糖,玉米淀粉(可溶和不可溶),玉米秸秆水解物,木糖四种具有代表性的碳源,通过单批和半连续发酵,探究了以新型棉布载体固定米根霉发酵中的碳源利用度和偏好性。实验结果表明,玉米淀粉能够达到与葡萄糖相似的乳酸生产效果,有助于降低发酵成本。而玉米秸秆水解物由于纤维二糖和木糖的累积,乳酸产率和得率较低。同时,通过对木糖的发酵动力学研究,表明在纤维类材料中,木糖的底物抑制对米根霉细胞活性具有较大影响,明确了米根霉在戊糖利用上的局限性。通过进一步研究葡萄糖与木糖混合物的长期发酵试验,为未来提高戊糖在发酵中的利用率指明了潜在努力方向。最后,为了验证新型棉布载体在生物反应器中的放大潜力,采用350ml和3.5L的生物反应柱进行了的相关研究。单因素实验确定了350ml生物反应柱中的最佳反应条件:葡萄糖浓度120g/L,孢子浓度为106个/ml,10 M NaOH维持pH为6.0。对两种型号的生物反应柱中的溶氧传递速率常数与空气线速度的关系的研究,确定了kLa与Vs的线性关系。通过350ml和3.5L生物反应柱中的发酵结果与溶氧浓度、溶氧传递速率常数和溶氧传递速率三个重要参数之间关系的研究,确定了溶氧传递速率作为放大原则的可行性和相关性。经过长达27天,共9批次的重复发酵实验,探索了生物反应柱中的发酵变化趋势,证明了新型固定化载体在放大实验中的长期稳定性。更多还原

【Abstract】 Recently, L-lactic acid has attracted a lot of attentions for its use as the starting material in the synthesis of polylactic acid, which can be used for manufacture of biodegradable materials. Current industrial lactic acid fermentations use homolactic acid bacteria, which have high lactic acid yield and productivity but are fastidious and require complex nutrients for fermentation. In contrast, some filamentous fungi such as Rhizopus oryzae and Rhizopus arrhizus can grow in simple media with minimal nutrients to produce L-lactic acid from various carbohydrates, including glucose, xylose and starch. Besides the simple medium cost, the fungal fermentation also has the advantage of producing L-lactic acid with a high optical purity of greater than 99% that is easier to purify from the simple medium used in the fermentation. However, filamentous fungi are difficult to use in large-scale industrial fermentation because of their complex morphology, substrate cost and process engineering instability in scaling-up.A new support matrix inspired by honeycomb was developed for cell immobilization to control fungal morphology and enhance mass transfer in bioreactor for lactic acid production by Rhizopus oryzae. The immobilization matrix composed of asterisk-shaped fibrous matrices in a honeycomb configuration provided high surface areas for cell attachment and biofilm growth. The feasibility, scaling-up potential, stability and scope of application of the matrix were all investigated.First, in immobilization and fermentation kinetics study, lag phase of germination process (tβ) and 50% immobilization time (λ) were determined by germination plot. Compared to to other environmental factors such as temperature, pH, and nutrients, the rotational speed did not strongly influence R. oryzae spore germination. The cell adsorption kinetics was found to follow the first-order reaction kinetics and can be represented by ln( Ct /Co)= ?kt, The adsorption rate constant k can be estimated from the slope of the semi-logarithmic plot of C vs. t and was found to be 0.12±0.04 h-1 for the initial 4-h period and 0.50±0.07 h-1 for the subsequent period after spore had germinated.Furthermore, the immobilized fermentation using the novel matrix was optimized both in environmental factors and medium compositions. The optimal culture setting was as following: rotational speed 180 r/min, matrix diameter 2 cm and three matrices in each flask, initial spore concentration 1×106/ml. Uniform design was carried out in medium optimization. The highest L-lactic acid production was achieved when the medium consisted of : 80g/L glucose, 0.15g/L urea, 0.5g/L KH2PO4 , 0.2g/L MgSO4.7H2O,0.04 g/L ZnSO4.7H2O,0.006 g/L FeSO4.5H2O,40g/L CaCO3 added every 12 hours.Then, the novel matrix was compared to calcium alginate gel, polyvinyl alcohol gel and loofa sponge in immobilized fermentation by R.oryzae. In batch fermentation, novel matrix obtained better morphological control and L-lactic acid production and other methods. Moreover, it showed better stability and producing ability in long-term fermentation, while both calcium alginate gel and polyvinyl alcohol gel tended to abrupt after relatively short period.In study on preference of carbon assimilation of R.oryzae using this matrix, glucose, corn starch (soluble and in soluble), corn cob hydrolysate and xylose were investigated. Corn starch was proved to be similar with glucose in L-lactic acid production, while corn cob hydrolysate was not favorable because cellobiose and xylose accumulation in fermentation limited cell activity of R.oryzae. The long-term fermentation using glucose/xylose proved that R.oryzae preferred glucose to xylose and the potential of xylose could be explored by assistant of glucose.At last, matrices were united in 350ml and 3.5 L column bioreactors to testify its scaling-up feasability and process parameters. In 350 ml column bioreactor, the best culture conditon was: 120g/L glucose, 106 spore /ml, pH 6.0 by adding 10 M NaOH solution. Volumetric oxygen transfer rate coefficient (kLa) was found to be linearly related with superficial air velocity (Vs). Compared to oxygen transfer rate coefficient, oxygen transfer rate was more reliable as the scaling-up key parameter since it included liquid height and air pressure in the process.更多还原