耐氨米根霉菌株ST50-2产L-乳酸发酵条件研究

为研究耐氨米根霉菌株ST50-2产L-乳酸的发酵特性,采用氨水中和剂,进行7L反应器发酵实验。与ST50-2的出发菌株AS3.819相比,发酵周期由原来的72h缩短为56h,L-乳酸产量提高12.30%,乙醇含量降低22.22%,乳酸脱氢酶(LDH)比活力提高83.09%,乙醇脱氢酶(ADH)比活力降低16.06%,对还原糖的利用速率及生物量的积累均高于出发菌株。7L反应器发酵实验表明：CaCO3影响菌体的成球;搅拌转速、氨水体积分数影响菌体的生长、成球大小及L-乳酸产量;pH值影响L-乳酸产量及发酵周期。其优化结果为：在搅拌转速400r/min,中和剂氨水体积分数为15%,pH值控制在6.0时,ST50-2菌球直径为1.0～1.5mm,发酵周期为56h,L-乳酸产量达93.32g/L。     

米根霉L- 乳酸发酵菌丝球形成条件的研究

研究米根霉CS323-9 在摇瓶培养条件下影响菌丝成球及产乳酸的因素。考察(NH4)2SO4 质量浓度、接种量、摇瓶转速及表面活性剂吐温-80 用量4 个因素,再利用L9(34)的正交试验,对菌丝球形成及产酸的条件进行优化。结果表明：(NH4)2SO4 质量浓度4g/L、接种量7%、摇瓶转速200r/min、吐温-80 用量0.5g/L 时,摇瓶发酵菌丝球数目达115 个/mL、菌丝球平均直径为1.2mm、L- 乳酸积累量为78.9g/L。说明米根霉CS323-9 在摇瓶培养条件下菌丝球的形成与乳酸的积累有较大的相关性。     

真菌固定床反应器发酵L(+)-乳酸的初步研究

研究了在5L发酵罐中采用纤维床固定化技术发酵生产乳酸过程不同溶氧量和固定化面积对乳酸发酵过程的影响,测定了不同固定化面积发酵过程中的氧传质系数。在通气率为1.0vvm和固定化面积为400cm2条件下,产酸速率为2.13g/(L·h),发酵液中乳酸最终浓度为73.1g/L,L(+)-乳酸光学纯度为98.9%,证明提高发酵过程中菌体层内部溶氧传质系数有助于增加乳酸产率。乳酸发酵液浊度0.49NTU,显著改善发酵液的流体力学条件,为应用膜生物反应器技术连续发酵生产乳酸奠定了基础。     

耐氨米根霉的分批补料发酵及发酵动力学初探

以氨水为中和剂,替代CaCO3,对耐氨米根霉R.oryzaeJS-N0-2-02进行15L自动发酵罐的分批和分批补料发酵及其发酵动力学的初步研究,结果表明,降低起始糖浓度,产酸期补糖可明显提高菌体L-乳酸比生产速率和耗糖产酸能力,提高L-乳酸产量和纯度,降低残糖。在发酵起始时添加1 g/L CaCO3能进一步提高补糖发酵的L-乳酸比生产速率,增强发酵后期菌体耗糖产酸能力,从而进一步提高L-乳酸产量和纯度,降低残糖。发酵结果:起始糖浓度为120 g/L,25h时补糖使最终发酵总糖浓度达137 g/L,发酵培养60 h,L-乳酸产量可达101.8 g/L,纯度97.3%,菌体耗糖转化率76%,比生产速率0.27 g/g.h,残糖降至3 g/L。     

樟芝真菌深层培养条件研究

溶氧浓度直接影响樟芝真菌菌体生长和产多糖情况.通过调节转速和通气量,将溶氧浓度控制为15%～20%,菌丝体生长较快,还原糖消耗速率和菌体得率明显提高,菌丝体产量和胞外多糖产量分别为7.48g/L和19.20g/L.     

基于CFD模拟的七烯甲萘醌发酵过程优化

七烯甲萘醌(menaquinone-7,MK-7)是维生素K₂的重要组成部分之一,前期研究发现,摇瓶培养条件下MK-7的最高产量远高于搅拌釜反应器培养条件下的最高产量。为缩小从摇瓶放大至3 L搅拌釜反应器带来的差异,使用计算流体力学(CFD)及动态吸收法对不同转速下摇瓶与搅拌釜反应器内的剪切力及氧气传递性能进行比较,并探讨氧气供应及剪切力对枯草芽孢杆菌BS20菌体生长与MK-7合成的影响。结果表明,充足的氧气供应对菌体生长及MK-7合成有促进作用,但过高的剪切力会对菌体产生破坏作用,降低MK-7的产量。基于上述结果,在3 L搅拌釜反应器中,以较低搅拌转速(400 r/min)保证低剪切力,同时采用通富氧空气(氧气体积分数55%)的方式提高供氧水平,MK-7产量达到296.25 mg/L,比对照批次(氧气体积分数21%)提高152%,达到摇瓶培养最高水平的79%。本研究在搅拌釜反应器中所采用的降低搅拌转速、提高进气氧体积分数的方法,能够有效提高对剪切敏感且高好氧微生物的发酵性能。     

液态发酵条件对红曲菌菌体形态及产物Monacolin K的影响

红曲菌是一种重要的食药双用丝状真菌,红曲菌9901种子扩大培养及液态发酵过程中,发酵条件、菌体形态与代谢产物Monacolin K(MK)之间有着紧密的联系。研究发现,斜面菌种的种龄和孢子接种量对红曲菌种子液的菌球直径、菌球外菌丝长度和菌体量等有明显的影响;在30 L机械搅拌发酵罐培养过程中,搅拌转速对红曲菌菌体形态、生物量和代谢产物MK的产量有较明显的影响。当搅拌转速较低时(150~350 r/min),红曲菌菌球直径和菌球比例随着转速的加大而逐渐增加,当搅拌转速为350 r/min时,发酵液的中菌体为均匀的、较大的、表面光滑的菌丝球,其液态发酵合成MK产量较高;当搅拌转速为400 r/min,初始直径为1 500μm的菌球减小至最终大小稳定为900μm的菌丝球,这些分散的、较小的、表面有较长菌丝的菌丝球,液态发酵合成MK产量较低。最后选择种子液初始菌球大小为1 500μm,设搅拌转速为350 r/min时,30 L发酵罐分批发酵384 h时MK的产量为1 308 mg/L,进而为更大规模的发酵罐以至工业化生产的实现奠定基础。     

高山被孢霉产花生四烯酸的发酵和老化工艺

高山被孢霉对于淀粉质原料具有优良的利用能力。采用经淀粉酶液化的玉米粉-豆粕培养基,研究了高山被孢霉在5 L容积机械搅拌罐液态发酵的主要参数,如搅拌转速和通气体积比对于高山被孢霉发酵产花生四烯酸的影响,得到最优的搅拌转速和通气体积比,分别为200 r/min和1∶1。研究发现,采用发酵罐培养5 d的菌丝体,最佳的老化温度和保温时间分别是20℃和5 d。本研究把谷物粗原料成功用于高山被孢霉发酵产花生四烯酸,经发酵罐优化和菌体老化后的花生四烯酸产量达到11.7 g/L,菌体老化可以在保持产量的同时缩短发酵周期,有效降低生产成本,有望实际用于花生四烯酸的工业化生产。     

米曲霉形态对L-苹果酸生产的影响

L-苹果酸是一种重要的C_4化合物,广泛应用于食品、化工和医药行业。本文中以米曲霉为出发菌株,研究氮源种类、CaCO_3质量浓度、搅拌转速和通气量等关键营养条件和环境条件对Aspergillus oryzae形态和产L-苹果酸的影响。最优发酵条件为:胰蛋白胨为氮源、CaCO_3质量浓度为80 g/L、搅拌转速为600 r/min、通气量为2 vvm。进一步分析菌体形态与L-苹果酸产量的关系,得出当单位体积发酵液菌球总体积(V值)为76.4 mm3/mL时,L-苹果酸产量最高达109.9 g/L。     

高产蛹虫草诱变菌株发酵条件优化及放大

考察高产蛹虫草诱变菌株放大发酵条件,为产业化奠定实验基础。以蛹虫草菌丝体干重、多糖、腺苷和虫草酸质量浓度为指标,采用单因素试验的方法优化5 L发酵罐发酵条件:搅拌转速、发酵培养基初始pH值和接种量,并在此条件下重复4次实验,以考察发酵条件的稳定性;进一步考察15 L和100 L发酵罐发酵条件。5 L发酵罐发酵条件为:转速250 r/min、起始pH值7、接种体积分数6%,发酵温度26℃,通气量250 L/h,菌丝体干重、多糖、腺苷和虫草酸质量浓度分别达到23.29 g/L、0.94 g/L、162.84 mg/L和2.06 g/L,4次重复实验发酵条件稳定(P0.01)。15 L发酵罐的最佳发酵时间为72 h,菌丝体干重、多糖、腺苷和虫草酸质量浓度分别达到30.84g/L、1.10 g/L、233.22 mg/L和1.89 g/L;100 L发酵罐的最佳发酵时间为47 h,菌丝体干重、多糖、腺苷和虫草酸质量浓度分别达到32.05 g/L、1.33 g/L、187.20 mg/L和3.51 g/L。5 L发酵罐发酵条件稳定,以菌丝体干重、多糖、腺苷和虫草酸质量浓度为综合考察指标,分别绘制了高产蛹虫草诱变菌株15 L、100 L发酵罐发酵生长曲线,为生产提供了数据依据。     

Mechanism for the effect of agitation on the molecular weight of hyaluronic acid produced by Streptococcus zooepidemicus

Various views persist about the effect of agitation on hyaluronic acid (HA) molecular weight, and the mechanism remains unclear. In this paper, the effect of agitation on HA molecular weight was investigated. The HA molecular weight increased with impeller speed, due to a mass transfer enhancement; however, higher impeller speed caused a decrease in the HA molecular weight. According to the experimental results, it is proposed for the first time that reactive oxygen species (ROS) under higher impeller speed in the bioreactor, which were generated by NADH oxidase, could cause the reduction in the HA molecular weight. ROS generation increased remarkably during the exponential growth phase when the impeller speed increased from 450 rpm to 1000 rpm, and the NADH oxidase activity increased correspondingly. Finally, it was proved that the addition of salicylic acid could increase the HA molecular weight, but inhibit the efficiency of the HA synthesis.     

搅拌转速和通气量对灰树花深层发酵培养的影响

采用15L搅拌式发酵罐研究灰树花的扩大培养条件，主要考察了搅拌转速和通气量对灰树花菌丝量、胞外多糖产率、溶氧、菌丝形态以及发酵液粘度的影响．研究结果表明搅拌转速和通气量能显著影响灰树花的生长，并且通过对灰树花菌丝球的大小、形态以及胞外多糖的分泌影响发酵液的粘度．并得出较为优化的培养条件为：温度25℃，通气量0．75vvm，搅拌转速80r／min，装液量60％；在此条件下，发酵10d后，灰树花菌丝体最大得率为22．95g／L，胞外多糖的得率为1．521g／L。     

振荡条件对发酵生产颗粒状细菌纤维素的影响

为打破静置发酵生产膜状细菌纤维素的传统模式,并开发出新型细菌纤维素,采用摇床振荡培养的方法,发酵生产外观均匀且为颗粒状的细菌纤维素.利用单因素试验,以粒径为0.5cm～0.8cm的细菌纤维素颗粒产量及其占总纤维素比率为考察指标,研究转速、装液量、温度、接种量、种龄及培养时间对形成细菌纤维素颗粒的影响,探索最佳振荡条件.结果表明,转速及装液量对颗粒的形成及粒径影响较大,温度、接种量、发酵时间、种龄主要影响细菌纤维素颗粒产量;最优培养条件为140r/min,30℃、60mL装液量,种龄48h,3％接种量发酵5d,此条件下产物中粒径为0.5cm～0.8cm的颗粒纤维素占有率高,其产量达28.5g/100mL.     

粉被虫草液体培养条件的优化

通过摇瓶液体培养正交试验获得了粉被虫草的液体培养条件,即蔗糖2.5%,马铃薯淀粉2%,黄豆0.5%,牛肉膏0.5%,酵母膏0.1%,K2HPO4 0.1%,KCl 0.02%,MgSO4•7H2O 0.05%,pH5～7,装液量(100ml/250ml摇瓶),加15颗玻璃珠作分散剂,5%接种量,旋转式摇床150r/min,28℃培养7d,达到最大菌丝干重31.86g/L,于9d获得最大胞内产物3.13g/L。同时,对摇瓶液体培养和生物反应器液体深层分批培养的动力学做了较系统的研究,无论生物反应器还是摇瓶,其培养过程中相对应的pH、残糖、氨基氮、菌丝干重、胞内产物等参数变化趋势基本一致,但生物反应器培养时间大大缩短,48h即可达到最大菌丝干重29.97g/L,54h达到4.95g/L的最高胞内产物产率。生物反应器设定培养温度28℃、装液量65%、接种量10%、0.2%的消泡剂、搅拌转速350r/min、通气量(12±3)L/min、罐压1.1MPa、初始pH6.5,培养基配方用食用蔗糖3%、蔗糖糖蜜2%、花生0.5%分别替代蔗糖、马铃薯淀粉、牛肉膏以降低成本。实验表明,粉被虫草的液体培养条件基本能达到工业发酵水平的要求。     

Image analysis of morphological change during arachidonic acid production by Mortierella alpina 1S-4

The changes in mycelial morphology during arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated using an image analysis system. Cultivation was performed in a 10-kl fermentor, and the culture broth was separated into two fractions by sieving (0.5 mm aperture size): the filament fraction (F-fraction, <0.5 mm), and the pellet fraction (P-fraction, >0.5 mm). The effect of the mycelial morphology in each fraction on AA production was analyzed. As a result, a product distribution in the culture broth wherein the AA content in the mycelia of the P-fraction was observed to be higher than that in the mycelia of the F-fraction throughout the cultivation. Morphological analysis of the P-fraction revealed that the hairy pellets became smooth because the mycelia on the pellet surface were shaved off; some pellets were broken and reduced in size. The shaved-off mycelia from the hairy pellets surface moved into the F-fraction and aggregated there. From the above findings, it was likely that the low AA content in the F-fraction was due to mycelial damage during the cultivation. In addition, the morphology of the hairy pellets was found to contribute to an increase in the viscosity of culture broth.     

Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis in a centrifugal impeller bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.     

茶叶对灵芝菌菌丝生长和菌体形态的影响

研究了茶叶对灵芝菌菌丝生长、菌体形态及灵芝多糖合成的影响。平板培养时,普通平板和茶叶平板菌丝增殖速率分别是13.5mm/d和6.4mm/d,表明茶叶可在一定程度上降低菌丝平板增殖速率。研究了摇瓶发酵时灵芝菌丝球的外形和内部结构,结果表明:与普通培养基相比,茶培养基菌丝球直径较大且结构疏松,菌丝球骨架菌丝较少,生殖菌丝和联络菌丝丰富,锁状联合明显,有利于菌丝球供氧平衡和灵芝多糖等代谢产物的合成;茶培养基中灵芝胞外多糖和菌丝体胞内多糖的含量分别比普通培养基的增加34.4%和25.2%。     

茂原链霉菌谷氨酰胺转氨酶合成与菌体形态分化的关系

目的：研究茂原链霉菌（Streptomyces mobaraensis）合成谷氨酰胺转氨酶（transglutaminase,TGase）与菌体分化之间的关系,以及TGase的生理功能。方法：以S. mobaraensis为出发菌株,通过向培养基中添加乙二胺四乙酸（ethylenediaminetetraacetic acid,EDTA）抑制TGase激活关键蛋白酶的活性从而调控成熟TGase的合成,同时利用激光共聚焦显微镜和扫描电镜观察发酵过程中菌体的活力和形态变化。结果：对照组随着TGase产量的升高,菌体生存活力呈先上升后下降的趋势,并且菌体表现出复杂的形态变化;实验组自加入EDTA后TGase产量不再升高,菌体生存活力也逐渐减弱,菌体形态分化滞后。结论：TGase的合成会影响茂原链霉菌生存活力及其形态分化。     

Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor

Submerged cultivation of Podophyllum hexandrum for the production of podophyllotoxin was carried out in a 3l stirred tank bioreactor fitted with a low-shear Setric impeller. The specific requirements of the medium, such as carbon source (sugar) and light, were established for the growth of and podophyllotoxin production by P. hexandrum in suspension cultures. Substitution of sucrose by glucose resulted in higher growth and podophyllotoxin production. The biosynthesis of podophyllotoxin was favored when plant cells were cultivated in the dark. An agitation speed of 100 rpm was sufficient to mix the culture broth in the bioreactor without causing any significant cell damage. Biomass and podophyllotoxin accumulation in 3 l bioreactor under batch growth conditions were 6.5 g/l and 4.26 mg/l, respectively, in 22 d. This resulted in an overall podophyllotoxin productivity of 0.19 mg/(l.d), which represented an increase of 27% in comparison to its productivity in a shake flask. Podophyllotoxin production was found to be a combined growth-associated and non-growth associated process.