摇瓶的体积氧传递系数和氧通透率的测定

通过设计特殊摇瓶,用亚硫酸钠法测出摇瓶的体积氧传递系数和氧透过纱布层的通透率。以氧电极测其内外氧的分压降后,可以算出摇瓶表观体积氧传递系数(k_La)app及真实体积氧传递系数k_La,并进一步求出氧通透率。由实验得出：氧分压降低6．1％,氧传递系数增加一倍;在37c、220r／min、500m1摇瓶(内盛液50m1)8层纱布的氧通透率Pm=43．7moI／m²·h·arm;并且关联出摇瓶容积V、装液量VL、转速n、摄氏温度t之间的模型式：(k_La)_app=1．84×10^-7[t]1.8479·[n]2.3906.(VLV]-0.6360(kLa)=2．02×10^-7[t]1.8525·[n]2.39441·[VLV]-0.6370     

假丝酵母99-125脂肪酶的发酵工艺研究

对假丝酵母99-125脂肪酶的发酵工艺条件进行了一系列研究。选择了合适的培养基成分并进行优化 ,获得了最优的摇瓶培养基配方 (% ,W/V) :豆油 4.0 ,全脂豆粉 4.0 ,K₂HPO₄0.1,KH₂PO₄ 0.1。产酶水平能达到 5000IU/mL。在 30L发酵罐上进行初步放大实验 ,其产酶水平能达到 8100IU/mL。在1m³发酵罐上进行中试放大 ,产酶水平可达到 8000IU/mL。     

培养幽门螺旋杆菌的发酵工艺研究

采用摇瓶微需氧培养技术,模拟发酵条件,探讨不同因素（pH、转速、种子菌接种量）对幽门螺旋杆菌（Helicobacter pylori,Hp）生长的影响,优化工艺参数,并级联放大到10L发酵罐发酵,经多次实验,建立了稳定的Hp发酵工艺,24h发酵细菌的最大吸光度A600达3.89,收获菌体湿重达5.2g／L。     

低高径比喷射环流生化反应器流体力学和发酵性能的研究

对高径比s≤2．5喷射环流生化反应器的流体力学和传质特性进行了系统的研究,选出反应器的最佳结构,关联出氧的体积传递系数(k_La)表达式。在此基础上,进行了谷氨酸发酵试验,摸索出用该设备进行各氨酸发酵的最佳工艺条件,使5批一次性投糖发酵的糖酸转化率达到50％以上。     

腐霉素发酵工艺的研究

本文报道吸水链霉菌(Streptomyces hygroscopicus)α-1080菌在小型发酵罐上产生腐霉素(Carriomycin)的研究结果。在发酵罐中进行了通气、搅拌、补加葡萄糖调节碳源浓度及控制溶氧水平等试验,使腐霉素的产量从摇瓶的2000μg/ml提高到3000μg/ml以上,为工业生产打下了基础。     

CellCul－20A反应器的丝网间液体交换速率及氧传递模型

针对笼式通气搅拌生物反应器的特点,本文提出了一种简单可信的测量丝网内外液体交换速率的实验方法,根据此方法,在CellCul-20 A生物反应器中得出了通过丝网的液体交换速率关联式: 单层丝网：Qs=1．93×1O^-5N^1.81+1．13×10^-4UG^0.61(Ⅰ) 双层丝网：Qs=3．42×10^-5N^1.49+1．46×10^-4UG ^0.68 (Ⅱ)并在此基础上,建立了台适的深层通气的氧传递模型,得到： 1- 1- 1- (K_La)d.Vc Qs + Vb.[(K_La)b-(K_La)d](Ⅲ)理论分析和实验结果表明,增大丝网内的液体体积和提高丝网内的氧传递速率,可以有效地提高反应器的体积氧传递系数。     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

透明颤菌血红蛋白在肉桂地链霉菌中的表达对其细胞生长及抗生素合成的影响

肉桂地链霉菌(S.cinnamonensis)是莫能菌素(Monensin)的产生菌。大肠杆菌链霉菌穿梭表达载体pHZ1252中的透明颤菌血红蛋白基因(vhb)位于硫链丝菌素诱导启动子P_tipA之下,它在肉桂地链霉菌中的结构不稳定,发生了重组缺失,缺失的片段包括大肠杆菌质粒部分和vhb基因。但来自阿维链霉菌(S.avermitilis)中缺失了大肠杆菌质粒部分却保留了完整的vhb基因及tipA启动子的pHZ1252,可在肉桂地链霉菌中稳定复制,不再发生缺失,经硫链丝菌素诱导表达出了有生物活性的VHb蛋白。摇瓶发酵实验证明,VHb蛋白在氧限条件下可明显促进肉桂地链霉菌的菌体生长和抗生素合成。     

离子注入花生四烯酸产生菌诱变选育

利用离子束注入生物技术对花生四烯酸产生菌(Mortierella alpina)进行诱变高产菌筛选。筛选到高产菌I₄₉N₁₈,该菌每升培养液可得生物量30.80g(约4%的含水量),干菌体油脂含量为25.8%,其中花生四烯酸的含量占总.脂的45.37%。30L和250L发酵罐发酵试验,该高产菌的花生.四烯酸得率为4.0g/L。     

地衣芽孢杆菌产生碱性蛋白酶的动力学研究

应用自动控制发酵设备,首先进行分批发酵试验摸索了地衣芽孢杆菌2709生长与代谢的基本规律。然后采用补料分批发酵方法限制生长基质浓度,测定了一系列(S_I,μ_I)、(μ_j,q_pj)数据,获得K_S、μ_max、α、β等参数的值,并且推导出了细胞生长与产物合成的动力学公式,从而证明了用Monod方程描述地衣芽孢杆菌2709生长速率与基质浓度关系的合理性和合成碱性蛋白酶的发酵属于生长部分关联型。     

A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

We describe a study of oxygen transfer in shake flasks using a non-invasive optical sensor. This study investigates the effect of different plugs, presence of baffles, and the type of media on the dissolved oxygen profiles during Escherichia coli fermentation. We measured the volumetric mass transfer coefficient (k(L)a) under various conditions and also the resistances of the various plugs. Finally, we compared shake flask k(L)a with that from a stirred tank fermentor. By matching k(L)a's we were able to obtain similar growth and recombinant protein product formation kinetics in both a fermentor and a shake flask. These results provide a quantitative comparison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts.     

Scale-up of biopesticide production processes using wastewater sludge as a raw material

Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e⁺⁰⁸ cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Scale-up of Agrobacterium-mediated transient protein expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture

The reporter gene beta-glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through co-culture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to transient expression including the timing of Agrobacterium inoculation relative to the plant cell growth phase, plant tissue culture hormonal triggers and plant cell cycle synchronization. The co-culture process was simplified to facilitate implementation in a pilot-scale bioreactor. At the shake flask scale it was determined that elevated concentrations of oxygen in the headspace were detrimental to transient expression levels and the addition of acetosyringone to the co-culture had a negligible effect. The bacterial preparation process was also streamlined, permitting the direct transfer of the Agrobacterium culture from a bench-scale fermentor to the pilot-scale plant cell culture bioreactor. Increasing expression levels and overcoming batch-to-batch variability despite extensive procedure systemization remain the major technical hurdles.     

Exopolysaccharide production by Acremonium persicinum in stirred-tank and air-lift fermentors

Summary Exopolysaccharide production by the fungus Acremonium persicinum was affected by the culture system used. The yields achieved in shake flasks were not obtained in a stirred tank reactor, except at very low stirring speeds (100 rpm). However when grown in an air-lift fermentor, exopolysaccharide levels were similar to those found with shake flask cultures. Results suggest that both dissolved oxygen tension and shear rate may determine the ability of this organism to synthesise this exopolysaccharide. Offprint requests to: R. J. Seviour     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Model-based workflow for scale-up of process strategies developed in miniaturized bioreactor systems

Miniaturized bioreactor (MBR) systems are routinely used in the development of mammalian cell culture processes. However, scale-up of process strategies obtained in MBR- to larger scale is challenging due to mainly non-holistic scale-up approaches. In this study, a model-based workflow is introduced to quantify differences in the process dynamics between bioreactor scales and thus enable a more knowledge-driven scale-up. The workflow is applied to two case studies with antibody-producing Chinese hamster ovary cell lines. With the workflow, model parameter distributions are estimated first under consideration of experimental variability for different scales. Second, the obtained individual model parameter distributions are tested for statistical differences. In case of significant differences, model parametric distributions are transferred between the scales. In case study I, a fed-batch process in a microtiter plate (4 ml working volume) and lab-scale bioreactor (3750 ml working volume) was mathematically modeled and evaluated. No significant differences were identified for model parameter distributions reflecting process dynamics. Therefore, the microtiter plate can be applied as scale-down tool for the lab-scale bioreactor. In case study II, a fed-batch process in a 24-Deep-Well-Plate (2 ml working volume) and shake flask (40 ml working volume) with two feed media was investigated. Model parameter distributions showed significant differences. Thus, process strategies were mathematically transferred, and model predictions were simulated for a new shake flask culture setup and confirmed in validation experiments. Overall, the workflow enables a knowledge-driven evaluation of scale-up for a more efficient bioprocess design and optimization.     

Oxygen uptake rate measurements to monitor the activity of terpene transforming fungi

The application of a simple system for measuring the oxygen uptake rate (OUR) to determine the activity of pellet-forming fungi is described. A non-invasive easy-to-use respirometric system was used to measure the OUR as an activity parameter. The model sensor system was optimized in shake flask experiments with Pleurotus sapidus (DSMZ 8266) and Chaetomium globosum (DSMZ 1962). The toxicity of (+)-limonene and (+)-valencene to submerged fungal cultures and the influence of solvents on C. globosum were investigated. Finally, the new respirometric method was used for the determination of oxygen uptake rates in small scale bioreactors to control the activity of terpene transforming fungi.     

Determination of in vivo oxygen uptake and carbon dioxide evolution rates from off-gas measurements under highly dynamic conditions

In vivo kinetics of Saccharomyces cerevisiae are studied, in a time window of 150 s, by analyzing the response of O(2) and CO(2) in the fermentor off-gas after perturbation of chemostat cultures by metabolite pulses. Here, a new mathematical method is presented for the estimation of the in vivo oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) directly from the off-gas data in such perturbation experiments. The mathematical construction allows effective elimination of delay and distortion in the off-gas measurement signal under highly dynamic conditions. A black box model for the fermentor off-gas system is first obtained by system identification, followed by the construction of an optimal linear filter, based on the identified off-gas model. The method is applied to glucose and ethanol pulses performed on chemostat cultures of S. cerevisiae. The estimated OUR is shown to be consistent with the independent dissolved oxygen measurement. The estimated in vivo OUR and CER provide valuable insights into the complex dynamic behavior of yeast and are essential for the establishment and validation of in vivo kinetic models of primary metabolism.