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1.
根据重组人碱性成纤维细胞生长因子基因工程菌的生长特点,对其高密度发酵工艺条件进行研究和改进,采用“平衡DO-State”控制策略进行分批补料培养中的葡萄糖流加,有效地控制了培养过程中代谢副产物-乙酸的产生及其对工程菌生长的抑制作用,使发酵终了时乙酸浓度由15.6g/L下降为2.6g/L,而菌体密度则由15.2gDCW/L提高到30.2gDCW/L。  相似文献   

2.
The efficiency of the fed-batch cultivation of recombinant Escherichia coli AT2471 for phenylalanine production is highly dependent on the distribution of the carbon flow between the main process products — biomass, phenylalanine, acetic acid and carbon dioxide. In order to enhance the process performance, the effects of several factors, namely glucose feeding, tyrosine feeding and oxygen supply, were investigated experimentally. As a result, a set of control strategies was developed, designed to tolerate phenylalanine synthesis at the expense of the remaining products. The DO was controlled to prevent acetic acid excretion due to oxygen limitation. The total amount of tyrosine fed was used to provide an optimal balance between biomass synthesis and that of phenylalanine. Special algorithms for control of the glucose feed rate were applied to eliminate the threat of acetic acid excretion due to overfeeding, and at the same time, to reduce excessive CO2 evolution caused by unnecessarily severe glucose limitation. The joint application of these strategies resulted in greatly improved efficiency in the phenylalanine production process: the final phenylalanine concentration reached 46 g/l, the yield was above 17%, and the productivity-0.85 g/l·h. In combination, these data exceed the results reported by others, and are much higher than those obtained by use before the implementation of the proposed complex of techniques.  相似文献   

3.
可溶性TRAIL蛋白的高密度培养及补料策略研究   总被引:3,自引:0,他引:3  
采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略:间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L(WCW)以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。  相似文献   

4.
This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.  相似文献   

5.
乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。  相似文献   

6.
Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more beta-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h(-1), the methionine addition enhanced beta-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h (-1), the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. (c) 1993 John Wiley & Sons, Inc.  相似文献   

7.
本研究旨在优化重组大肠杆菌Escherichia coli BL21 (DE3) harboring pRSF-aad-ldh10-fdh菌株的培养条件,获得高密的供生物转化苯丙氨酸为苯乳酸的细胞。实验考察了摇瓶发酵培养基碳源、氮源种类和浓度,3 L发酵罐中转速和通气量及恒速补料、DO-stat和pH-stat等不同分批补料策略对菌体密度的影响。结果表明,当碳源为4 g/L葡萄糖,氮源为24 g/L安琪酵母浸粉FM802,细胞干重最大可达9.24 g/L;当转速为400 r/min和通气量为1.5 vvm时,细胞干重最大可达10.18 g/L;以4 g/(L·h)恒速流加葡萄糖时,细胞干重最大可达13.71 g/L。本研究还对工程菌酶表达的诱导条件进行了优化,菌体培养2 h后,添加终浓度为0.08 mmol/L IPTG诱导剂,在25℃下诱导培养14 h所得细胞有利于生物转化。底物苯丙氨酸浓度为60 g/L,转化为苯丙酮酸的转化率为50.2%,转化为苯乳酸的转化率为35.2%。  相似文献   

8.
玉米秸秆酸解副产物对重组酿酒酵母6508-127发酵的影响   总被引:10,自引:0,他引:10  
将木质纤维素类生物质如玉米秸秆等用稀酸水解预处理,在半纤维素水解为单糖的同时,水解液中还会产生一些可能对后续发酵有影响的副产物。本实验分别考查了在玉米秸秆稀酸水解液中检测出的乙酸、甲酸、香草醛、糠醛和羟甲基糠醛对重组木糖发酵菌株S. cerevisiae 6508-127生长和发酵的影响。结果表明,甲酸和乙酸对菌体生长的抑制强于乙醇生成,且甲酸的抑制程度远大于乙酸;2g/L香草醛可使菌体生长延滞期明显延长,而在较低浓度(≤1.2g/L)此现象不明显。糠醛在0.5-1.5g/L范围内对菌体生长有抑制作用,但使乙醇得率提高;羟甲基糠醛在0.2g/L浓度存在就使乙醇得率有明显降低,但使生物量得率提高;研究中还发现,糠醛、羟甲基糠醛和香草醛可被S. cerevisiae 6508-127代谢。  相似文献   

9.
代谢副产物乙酸对L-色氨酸发酵的影响   总被引:5,自引:0,他引:5  
分析了重组大肠杆菌(E.coli TRTH/pSV-709)发酵生产L-色氨酸的发酵过程,检测结果表明发酵液中有大量代谢副产物乙酸的积累。利用外源添加试验研究了乙酸对L-色氨酸发酵的影响,结果表明乙酸浓度高于2g/L时对L-色氨酸生产菌的生长和产酸均有抑制作用。分析了乙酸的产生机制,并采取了调节溶氧水平、确定合适初始葡萄糖浓度、限制葡萄糖流加及控制菌体比生长速率等措施来减少乙酸的生成。在优化条件下,乙酸含量与原工艺相比降低了51.35%,菌体生物量和L-色氨酸产量分别提高了51.07%和46.54%,实现了高密度发酵培养的目的。  相似文献   

10.
Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two evolutionary engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L(-1) : (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L(-1) acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g(-1) biomass h(-1) . After storage of samples from both selection experiments at -80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L(-1) acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol.  相似文献   

11.
Efforts in optimizing reducing agents, cysteine-HCl.H2O and sodium sulfide in order to attain satisfactory responses during acetic acid fermentation have been carried out in this study. Cysteine-HCl.H2O each with five concentrations (0.00-0.50 g/L) was optimized one at a time and followed by sodium sulfide component (0.00-0.50 g/L). Response surface methodology (RSM) was used to determine the optimum concentrations of cysteine-HCl.H2O and sodium sulfide. The statistical analysis showed that the amount of cells produced and efficiency in CO conversion were not affected by sodium sulfide concentration. However, sodium sulfide is required as it does influence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl.H2O and sodium sulfide respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% CO conversion.  相似文献   

12.
为了提高重组大肠杆菌FB讲/pSV-04发酵生产L-色氨酸的产量,减少代谢副产物乙酸的生成,考察了比生长速率和无机盐对重组大肠杆菌发酵生产L.色氨酸的影响。在确定了合适的比生长速率和无机盐浓度之后,乙酸积累很少,L-色氨酸的产量为53.4g/L,比优化前提高了141.6%。经30L发酵罐初步放大,L-色氨酸的产量达53.6g/L,发酵结果稳定,具有工业化应用前景。  相似文献   

13.
Acetate is a primary inhibitory metabolite in Escherichia coli cultivation which is detrimental to bacterial growth and the formation of desired products. It can be derived from acetyl coenzyme A by the phosphotransacetylase (Pta)–acetate kinase (AckA) pathway. In this study, the fermentation characteristics of Pta mutant strain E. coli TRTHΔpta were compared with those of the control strain E. coli TRTH in a 30-L fermentor. The effects of glucose concentration and dissolved oxygen (DO) level were investigated, and the results suggest that DO and glucose concentration are vital influencing parameters for the production of L-tryptophan. Based on our experimental results, we then tested a DO-stat fed-batch fermentation strategy. When DO was controlled at about 20 % during L-tryptophan fermentation in the DO-stat fed-batch system, the pta mutant was able to maintain a higher growth rate at the exponential phase, and the final biomass and L-tryptophan production were increased to 55.3 g/L and 35.2 g/L, respectively. Concomitantly, as the concentration of acetate decreased to 0.7 g/L, the accumulation of pyruvate and lactate increased in the mutant strain as compared with the control strain. This characterization of the recombinant mutant strain provides useful information for the rational modification of metabolic fluxes to improve tryptophan production.  相似文献   

14.
乙酸渗漏型丙酮酸高产菌的选育   总被引:7,自引:0,他引:7  
对Torulopsis glabrata WSH-IP303进行NTG诱变,挑选以乙酸为补充碳源的平板上透明圈较大的菌落,经初筛和复筛,发现T.glabrataWSH-LQ307生产丙酮酸能力强且稳定。以乙酸为补充碳源摇瓶培养48h,其丙酮酸产量(46.2g/L)比出发菌株(38.3g/L)提高21%,采用该菌株在5L发酵罐上进行4批发酵实验,丙酮酸产量在64h最高可达68.7g/L,对葡萄糖的转化率为0.651g/g。  相似文献   

15.
In this study, a dissolved oxygen (DO)-stat fed-batch process was conducted in a pressurized 75-L bioreactor, resulting in the production of the short version of human leukotactin-1 (shLkn-1) using Pichia pastoris as the host, with control of the DO-stat profile and an extension of the recombinant shLkn-1 production phase. By regulation of the exhaust-gas valve, we were able to maintain the vessel pressure at up to 120 kPa, in order to overcome DO limitations associated with the use of the DO-stat. The lowest DO value was adjusted by varying the feed pump speed, allowing us to control the DO-stat profile. This principle was successfully applied to both glycerol feeding during the growth phase and methanol feeding for the induction of shLkn-1. The extension of the methanol induction phase to a total of 192 h of culture time resulted in a shLkn-1 concentration of 2.5 g/L, and a total of 102 g of cumulative production. During this extended induction period, the C-terminal residue of shLkn-1 was truncated and this was confirmed by both reverse-phase HPLC and mass spectrometry.  相似文献   

16.
A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.  相似文献   

17.
Summary The acetic acid concentration in a batch culture of Acetobacter aceti M23 increased up to 90 g/l by adding ethanol intermittently. Although the bacterial cells ceased growth at about 60 g acetic acid/l, non-viable cells still preserved ethanol oxidation activity. Cell recycling by filtration in a repeated fed-batch culture increased the overall acetic acid production rate 2.84-fold compared to that without cell recycling for the purpose of obtaining an acetic acid concentration of 80.8 g/l. Repeated fed-batch cultivation with cell recycle was effective for increasing the production rate of acetic acid and obtaining high amounts close to a lethal concentration (90 g/l).Offprint requests to: Kiyoshi Toda  相似文献   

18.
添加有机酸对Clostridium acetobutylicum合成丙酮和丁醇的影响   总被引:2,自引:0,他引:2  
为提高丙酮-丁醇梭菌厌氧发酵生产丙酮和丁醇的能力,在发酵过程中添加有机酸(乙酸和丁酸),考察其对菌体生长、溶剂合成影响。实验表明:当添加1.5 g/L乙酸时能够促进菌体的生长,促进丙酮的合成,在600 nm处的最大OD值比参照值高出18.4%,丙酮的最终质量分数提高了21.05%,但不能促进丁醇的合成;当添加1.0g/L丁酸时能够促进菌体生长,促进丁醇的合成,在600 nm处的最大OD比参照值高22.29%,丁醇的最终质量分数比对照组提高了24.32%,但不能促进丙酮的合成。  相似文献   

19.
Of various carbon sources, examined for the cultivation of Phaffia rhodozyma, ethanol enhanced the astaxanthin content but severely decreased growth. Therefore, high cell mass was obtained by glucose fed-batch culture with pH-stat, and the ethanol feeding was performed based on DO-stat. As a result of this two-stage fed-batch cultivation, 30 g dry cells per liter were obtained, and the astaxanthin content reached 0.72 mg/g, which was 2.2-fold higher than that without ethanol feeding.  相似文献   

20.
The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (r(DHA)) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The r(DHA) was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring.  相似文献   

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