高密度发酵生产突变型重组人肿瘤坏死因子rhTNFα-DK2的研究

通过对发酵基质和发酵关键参数的优化,确定了发酵培养基的磷酸盐浓度为０．１５Ｍ,甘油浓度为１．２ｍｌ／Ｌ,补料中甘油浓度为２０ｍｌ／Ｌ,发酵过程中溶解氧控制在３０％～６０％,ｐＨ控制在６．８５左右。在５Ｌ在ＮＢＳ－Ｂｉｏｆｌｏ３０００型自动控制发酵罐中采取加速补料的补料分批培养,重组大肠杆菌ＹＫ５３７／ｐＳＢ－ＴＫ经１０ｈ３０°Ｃ培养和５ｈ４２°Ｃ诱导培养,最终密度达到６０ＯＤ６００,ｒｈＴＮＦα－ＤＫ２的表达量占菌体总蛋白的５０％以上,每升发酵液纯化可得到近２ｇ的ｒｈＴＮＦα－ＤＫ２。     

补料速度对工程菌生长和产物表达的影响研究

通过研究各培养阶段补料速度对温敏启动子控制的rIL-2工程菌E.coliK802(pLY-4)培养密度和rIL-2表达的影响,发现在各培养阶段控制不同的补料速度有利于提高菌密度和rIL-2表达,缩短培养周期。确定了rIL-2工程菌高密度培养方案,三批重复实验,平均菌密度为58OD_(600);菌干重18.0g/L,rIL-2表达水平为42.4％。     

重组巴斯德毕赤酵母高密度发酵表达植酸酶

对巴斯德毕赤酵母的高密度发酵条件进行了试验,并根据摇瓶发酵的优化结果进行了补料方式的研究。在摇瓶发酵时,最佳种龄为16h,接种量为3％,甲醇的诱导浓度为15g／L,生长阶段最适pH为5．0,诱导阶段最适pH为5．5。在间歇补料、恒速补料、变速补料三种补料方式中以变速流加最优。     

酵母菌HL的批次发酵及发酵动力学初探

在摇瓶培养的基础上,对酵母菌Ｌｉｐｏｍｙｃｅｓ ｓｔａｒｋｅｙｉ ＨＬ进行了小型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,通过后期补料既可明显地延长菌体脂类合成期,减缓油脂比合成速率的降低,又可增加菌液的细胞密度,最终提高了整个发酵罐的油脂产量和平均容量产率。发酵结果如下：发酵时间１２０ｈ;油脂产量１１.０ｇ／Ｌ;菌体生物量１９．４ｇ／Ｌ。油脂百分含量 ５６．５％,显然比分批培养８４ｈ所得的１１．２ｇ／Ｌ细胞生物量和 ６.１ｇ／Ｌ油脂产量分别增长了７３％和８０％。此外,通     

葡萄糖流加策略对基因工程FR-008／杀念菌素衍生物CS103补料分批发酵过程的影响

以组合生物合成技术得到的链霉菌FR-008突变株CS103为研究对象,研究了3．7L发酵罐上维持一定葡萄糖浓度对其次级代谢产物脱羧FR-008／candicidin衍生聚酮抗生素CS103生物合成的影响。当初始葡萄糖浓度20g／L,发酵过程还原糖浓度维持在10g/L时,抗生素CS103最高产量较分批发酵最高产量相比提高30％。研究了3．7L罐上补料分批发酵生产CS103的工艺,主要考察了脉冲补料、间歇流加补料和连续流加补料三种补料分批发酵工艺,并与分批发酵进行了比较。连续流加补料维持糖浓度的效果明显,最高产量达到126．9μg/mL,与分批发酵相比提高了44％左右。     

枯草芽孢杆菌β-甘露聚糖酶补料发酵及其特性研究

采用正交实验设计对枯草芽孢杆菌产β－甘露聚糖酶的培养基组成和培养条件进行优化,在此基础上进行摇瓶补料发酵,酶活达223．47U／mL,较未补料的酶活提高了32．09％。酶反应最适pH为6．5,最适温度为70℃,该酶在pH5.0-10.0和70摄氏度以下稳定,水解魔芋胶产物主要为二糖以上低聚糖。     

温度对大肠杆菌L-色氨酸发酵过程的影响

目的：研究变温控制对大肠杆菌TRTH L-色氨酸补料分批发酵过程中生物量、色氨酸产量、比生长速率及质粒稳定性的影响。方法：利用5L自控发酵罐对L-色氨酸补料分批发酵过程进行温度控制,对不同温度下相关参数进行分析比较,确定优化的温度控制方案。结果：以30-36％顺序升温的工艺进行发酵得到理想结果,与单一温度控制策略相比,L-色氨酸产量提高了15．4％;色氨酸的比合成速率提高了21．6％;质粒稳定性增加,未出现质粒丢失现象,质粒拷贝数保持在恒定水平。结论：温度对大肠杆菌L-色氨酸发酵有重要影响。     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。     

补料培养与溶氧控制联合应用对红豆杉细胞培养的影响

为进一步提高红豆杉 (Taxuschinensis (Pilg.)Rehd .)细胞培养过程中紫杉醇的产量 ,采用细胞悬浮培养方法研究了补料培养与溶氧控制联合应用对紫杉醇产量的影响。 5L反应器中补料培养研究表明 ,培养过程中第 16天添加含 2 0g/L蔗糖的补料培养液有利于细胞的生长及紫杉醇的合成。 2 0L反应器中补料培养的研究结果表明 :2 0 %饱和度培养时紫杉醇含量最高 (0 .98mg/gDW) ,但 4 0 %～ 6 0 %溶氧饱和度能提高紫杉醇的产量。进一步研究表明 ,细胞在 6 0 %溶氧饱和度培养 2 0d后转入 2 0 %溶氧饱和度继续培养 12d ,能显著提高紫杉醇产量。补料培养与溶氧控制联合应用时 ,2 0L反应器中红豆杉细胞培养紫杉醇产量可达 18.7mg/L。     

补料培养与溶氧控制联合应用对红豆杉细胞培养的影响

为进一步提高红豆杉(Taxus chinensis (Pilg.) Rehd.)细胞培养过程中紫杉醇的产量,采用细胞悬浮培养方法研究了补料培养与溶氧控制联合应用对紫杉醇产量的影响.5 L反应器中补料培养研究表明,培养过程中第16天添加含20 g/L蔗糖的补料培养液有利于细胞的生长及紫杉醇的合成.20 L反应器中补料培养的研究结果表明:20%饱和度培养时紫杉醇含量最高(0.98 mg/g DW),但40%～60%溶氧饱和度能提高紫杉醇的产量.进一步研究表明,细胞在60%溶氧饱和度培养20 d后转入20%溶氧饱和度继续培养12 d,能显著提高紫杉醇产量.补料培养与溶氧控制联合应用时,20 L反应器中红豆杉细胞培养紫杉醇产量可达18.7 mg/L.     

Production of thuringiensin by fed-batch culture of Bacillus thuringiensis subsp. darmstadiensis 032 with an improved pH-control glucose feeding strategy

A improved pH-control fed-batch strategy for Bacillus thuringiensis subsp. darmstadiensis 032 producing thuringiensin was developed based on the analysis of the batch culture, constant rate fed-batch cultures and the original pH-control fed-batch. Having considered the pH variation and the glucose consumption status, the pH was adjusted from 6.5 to 7.0 by adding base in the late cultivation period of batch culture, and then the pH was kept at 7.0 by glucose feeding. The feeding was terminated when the pH could not be controlled by glucose feeding anymore. The proposed fed-batch strategy effectively avoided underfeeding or overfeeding, and it increased the thuringiensin yield and Y_P/X by 89.51% and 103.2% compared to that of the batch culture, respectively.     

Nisin Production by Lactococcus Lactis Subsp. lactis under Nutritional Limitation in Fed-Batch Culture

Nisin production was improved under nutritional limitation in fed-batch culture of Lactococcus lactis. Nisin titre reached 3887 IU ml(-1) by a slow feeding of sucrose and 4131 IU ml(-1) by slow feeding of nitrogen source, which were, respectively, 64% and 74% above the values in batch culture.     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

法夫酵母高密度培养及虾青素的高产研究*

本文对法夫酵母Phaffia rhodozyma的不同流加培养模式进行了研究。实验结果表明,采用指数流加,虾青素产率和细胞干重具有较大值,分别达到14.52mg/l和32.56g/l;其次是恒pO2流加和恒速流加培养,虾青素产率分别达到8.89mg/l和6.70mg/l; 恒pH流加方式更有利于法夫酵母细胞的生长(14.62g/l DCW)。但是,不同流加培养模式所得的μmax和qasta具有较大的差距。恒pH、恒pO2流加培养及间歇培养有较大值,分别为0.0613 h-1、0.056 h-1、0.053 h-1;指数流加的μmax较小。间歇培养中虾青素生成比率最大,qasta=0.048×10-3h-1。     

Feed development for fed‐batch CHO production process by semisteady state analysis

Semisteady state cultures are useful for studying cell physiology and facilitating media development. Two semisteady states with a viable cell density of 5.5 million cells/mL were obtained in CHO cell cultures and compared with a fed‐batch mode control. In the first semisteady state, the culture was maintained at 5 mM glucose and 0.5 mM glutamine. The second condition had threefold higher concentrations of both nutrients, which led to a 10% increase in lactate production, a 78% increase in ammonia production, and a 30% reduction in cell growth rate. The differences between the two semisteady states indicate that maintaining relatively low levels of glucose and glutamine can reduce the production of lactate and ammonia. Specific amino acid production and consumption indicated further metabolic differences between the two semisteady states and fed‐batch mode. The results from this experiment shed light in the feeding strategy for a fed‐batch process and feed medium enhancement. The fed‐batch process utilizes a feeding strategy whereby the feed added was based on glucose levels in the bioreactor. To evaluate if a fixed feed strategy would improve robustness and process consistency, two alternative feeding strategies were implemented. A constant volume feed of 30% or 40% of the initial culture volume fed over the course of cell culture was evaluated. The results indicate that a constant volumetric‐based feed can be more beneficial than a glucose‐based feeding strategy. This study demonstrated the applicability of analyzing CHO cultures in semisteady state for feed enhancement and continuous process improvement. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Distribution of substrates carbon in sophorose lipid production by Torulopsis bombicola

Summary Torulopsis bombicola (ATCC 22214) produced sophorose lipid to 80 g/l in batch culture containing 11% glucose and 10% soybean oil as carbon and energy sources. According to the carbon mass balance analysis, 13% and 37% of input carbon were channeled to cells and to products, respectively, and 50% of the total input carbon was channeled to CO₂ gas in batch culture. In fed-batch culture with intermittent oil feeding, however, the carbon fractions incorporated into sophorose lipid and cells were 60% and 12%, respectively, and the carbon fraction evolved as CO₂ gas was 30%. In conclusion, yield of sophorose lipid based on total input carbon substrates was increased from 0.37 g/g-substrate in batch culture to 0.6 g/ g-substrate by employing a fed-batch culture.     

Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies

In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end-products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL-1606) was cultivated in T-flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed-batch culture and by 26-fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed-batch culture and by 50-fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five- and tenfold respectively, compared with conventional batch culture. (c) 1994 John Wiley & Sons, Inc.     

Patterns of RNA synthesis in batch and cyclic fed-batch cultures of tylosin-producingStreptomyces fradiae

Ribonucleic acid (RNA) synthesis was studied in batch and cyclic fed-batch cultures of tylosin-producingStreptomyces fradiae. In batch culture, net RNA synthesis increased with increasing growth rate then decreased as the fermentation progressed. A cyclic response pattern of net RNA synthesis was observed when glucose and glutamate solutions were cyclically fed to the batch culture; RNA synthesis rate increased sharply to a maximum level following the feeding cycles then decreased to lower values between the maxima. Data obtained from an extended cyclic fed-batch culture showed that cyclic feeding of the key nutrients allowed RNA and tylosin syntheses to continue during a period when such activities had ceased in the control batch culture.