氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

植物乳杆菌Lp-2的高密度发酵

高密度培养植物乳杆菌是制作其发酵剂的重要环节。首先,研究了不同的溶氧和pH对植物乳杆菌的分批发酵的影响。在分批发酵的基础上,为进一步提高发酵液中的菌体浓度,进行了补料分批发酵实验。最终通过对蔗糖反馈补料发酵试验对比改造获得了pH反馈补料发酵工艺。此发酵补料工艺可以控制蔗糖残糖量始终处于较低的水平,因此获得了最高的菌体产量。菌体干重达到13.56g/L,较分批培养提高90.05%。     

氮源种类及溶氧水平对锁掷酵母产类胡萝卜素组分的影响

在锁掷酵母（Sporidioboluspararoseus）发酵产类胡萝卜紊的过程中,发酵产物中类胡萝卜紊种类繁多,而且性质相似,加大了不同色素分离纯化的难度。为定向积累不同种类的类胡萝卜素,以本实验室保藏锁挪酵母JD-2为出发菌,研究了氮源种类和浓度及溶氧对锁掷酵母产类胡萝卜素的影响,并在7L发酵罐中进行了补料分批发酵试验。发现培养基中同时添加有机氮源和无机氮源且溶氧控制较低（5％）时有利于β-胡萝卜素的大量积累,最佳有机氮源和无机氮源分别为玉米浆（20g／L）、硫酸铵（5g／L）。补料分批发酵时β-胡萝卜素产量达到31．28mg／L,红酵母烯12．38mg／L。培养基中只添加有机氮源且相对溶氧控制相对较高（30％）时有利于红酵母烯的大量积累,最佳有机氮源为酵母膏（20g／L）。补料分批发酵时红酵母烯产量达到38．96mg／L,8．胡萝卜素12．36mg／L。     

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。     

L-缬氨酸的发酵工艺研究

目的:以黄色短杆菌XV0505为生产菌株,探讨发酵培养基和发酵控制条件对L-缬氨酸的产量和糖酸转化率的影响。方法:应用单因素实验确定发酵的工艺条件;利用纸层析-色斑洗脱比色法测定发酵液中L-缬氨酸的含量。结果:在最优发酵条件下,通过10L罐流加发酵72h,产酸量可达53.4g/L,糖酸转化率为26.7%,分别比补料分批发酵提高11.9%和3.5%。结论:环境因子和发酵控制工艺对发酵生产L-缬氨酸具有重要影响。     

环境因素对L-异亮氨酸发酵的影响

通过 5L自控发酵罐发酵实验 ,结合发酵过程中菌生长形态的变化 ,对L -异亮氨酸补料分批发酵进行研究 ,研究了环境因素对黄色短杆菌 (Brevibacteriumflavum)TJCN - 1的影响 ,优化出发酵最佳控制条件 ,提出分阶段发酵控制模式 ,对L -异亮氨酸生产有指导意义。     

L-谷氨酸补料分批发酵的研究

对谷氨酸棒杆菌(Corynebacteriuin glutamicum)HCJ46产L-谷氨酸的补料分批发酵条件进行研究.结果表明:最适初糖质量浓度和最佳残糖维持质量浓度分别为100和(10～20)g/L;对发酵控温方式进行研究,确定了最佳温度控制策略为0～8h维持32℃,8～16h维持34℃、16～32h维持36℃,同时发现相对溶氧控制在30%左右时产酸最高.在以上的优化条件下,L-谷氨酸产量从72g/L提高到95g/L,提高了31.9%.     

赤霉素发酵溶氧优化调控研究

为了解溶氧对赤霉素发酵过程影响以及相应工艺优化,采用不同溶氧条件下藤仓赤霉菌Gibberella fujikuroi分批发酵生产赤霉素的过程进行菌丝浓度、残糖浓度和GA3产物浓度检测,并微分运算得出比生长速率与比产物合成速率随发酵时间变化,分析了溶氧对比生长速率与比产物合成速率以及得率的影响,进而提出Gibberella fujikuroi发酵高产的溶氧控制策略:在发酵初始阶段(0–50h)控制溶氧30%左右,以维持较高的菌体生长速率;发酵中后期(50–184h),溶氧控制在15%,以获取菌丝持续较高的GA3合成速率能力。采用这一优化溶氧控制策略,发酵过程中最大菌丝浓度19.24g/L、最终赤霉素浓度2 180mg/L和平均比产物合成速率0.616mg/(g·h),比未优化前发酵分别提高了8.33%、13.25%和4.58%,表明所采取的分阶段溶氧控制策略对促进GA3生产有效。     

基于途径分析的L-异亮氨酸发酵溶氧控制研究

利用途径分析方法对黄色短杆菌（Brevibacterium flavum）TC-21 生产L-异亮氨酸的途径进行了分析,确定了黄色短杆菌TC-21生产L-异亮氨酸的最佳途径的通量分布,根据途径分析的结果,TCA循环的代谢流量对L-异亮氨酸产量有明显影响,而TCA循环与发酵过程中的溶氧密切相关,因此可以通过控制溶氧来提高L-异亮氨酸产量。在发酵过程的不同阶段,根据菌体生长和产酸的需求,改变TCA代谢流量,可以有效提高产酸率。实验证明,通过溶氧分阶段控制发酵生产L-异亮氨酸,比溶氧恒定控制方式发酵产率提高了15.77％。实验结果说明,用途径分析的结果指导发酵过程中的溶氧可以大幅度提高L-异亮氨酸的产量。     

裂殖壶菌发酵产DHA过程呼吸参数的在线检测分析

利用尾气分析仪对发酵过程的尾气中的O2、CO2含量进行实时检测,建立了裂殖弧菌发酵生产DHA过程中的呼吸参数在线检测方法,实现了裂殖壶菌补料分批发酵过程及双阶段供氧控制发酵过程中的呼吸参数在线检测分析。通过呼吸参数在线检测分析,从氧消耗机制方面解释了双阶段氧传递控制工艺能获得较高生物量、油脂和DHA含量的原因,从而为该工艺过程提供了理论指导。根据发酵过程中菌体生长不同时期的呼吸参数的变化情况,建立了基于呼吸商变化的在线补料控制方法,设计了一种基于RQ-Stat的补料工艺。RQ-Stat补料方式最终获得的油脂含量、DHA产量和产率比间歇式补料工艺分别提高了11.58%、12.19%和11.40%。     

不同溶氧条件下L-苏氨酸生物合成菌株的代谢流量分析

[目的]探索L-苏氨酸生物合成机理及影响因素.[方法]建立了大肠杆菌L-苏氨酸的代谢流平衡模型,应用MATLAB软件计算出不同溶氧条件下发酵中后期代谢网络的代谢流分布及理想代谢流分布.[结果]5%溶氧条件下,25.5%碳架进入HMP途径,74.5%碳架进入糖酵解途径,获得33.9%质量转化率;20%溶氧条件下,58.08%碳架进入HMP途径,41.92%碳架进入糖酵解途径,获得46.5%质量转化率;[结论]与理想代谢流(88.23%质量转化率)相比,应从菌种改造和发酵控制方面通过改变6-磷酸葡萄糖异构酶借以增加HMP途径代谢流量,通过增加磷酸烯醇式丙酮酸羧化反应代谢流提高天冬氨酸族合成代谢流,减少TCA循环代谢流量,从而达到减少副产物生成,增加L-苏氨酸生物合成的目的.     

L-苏氨酸工业生产发酵条件优化研究

发酵法生产L-苏氨酸是目前广泛采用的方法,因此研究工业生产发酵条件优化具有重要意义。试验以高产L-苏氨酸菌作为出发菌株,结合本公司的实际工业生产条件对发酵各条件进行了一系列优化研究,结果表明：添加0.2%的工业级生长促进剂,以复合糖代替葡萄糖为初糖,并控制初糖浓度在60g/L,除生长高峰期外,发酵过程中溶解氧（DO）控制在10%~20%之间;最终发酵放罐湿菌体在45g/L左右,L-苏氨酸含量可达110g/L左右。     

Rebalancing microbial carbon distribution for L-threonine maximization using a thermal switch system

In metabolic engineering, unbalanced microbial carbon distribution has long blocked the further improvement in yield and productivity of high-volume natural metabolites. Current studies mostly focus on regulating desired biosynthetic pathways, whereas few strategies are available to maximize L-threonine efficiently. Here, we present a strategy to guarantee the supply of reduced cofactors and actualize L-threonine maximization by regulating cellular carbon distribution in central metabolic pathways. A thermal switch system was designed and applied to divide the whole fermentation process into two stages: growth and production. This system could rebalance carbon substrates between pyruvate and oxaloacetate by controlling the heterogenous expression of pyruvate carboxylase and oxaloacetate decarboxylation that responds to temperature. The system was tested in an L-threonine producer Escherichia coli TWF001, and the resulting strain TWF106/pFT24rp overproduced L-threonine from glucose with 111.78% molar yield. The thermal switch system was then employed to switch off the L-alanine synthesis pathway, resulting in the highest L-threonine yield of 124.03%, which exceeds the best reported yield (87.88%) and the maximum available theoretical value of L-threonine production (122.47%). This inducer-free genetic circuit design can be also developed for other biosynthetic pathways to increase product conversion rates and shorten production cycles.     

Requirements for induction of the biodegradative threonine dehydratase in Escherichia coli.

Synthesis of the biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, was prevented by dissolved oxygen concentrations of 6 micrometer or greater. This effect was shown to be exerted solely on synthesis, rather than being the result of enzyme inactivation in vivo. In addition to an anaerobic environment, maximum enzyme synthesis was dependent upon the presence of a complete complement of amino acids, with omission of L-threonine, L-valine, or L-leucine producing the largest decreases in enzyme formation. L-Threonine, the most essential of the amino acid requirements, could be partially replaced by DL-allothreonine or alpha-ketobutyrate. Half-maximal stimulation of enzyme synthesis occurred with 0.4 mM threonine in the medium. The roles of anaerobiosis and amino acids are interpreted as being in accord with the concept that threonine dehydratase functions in anaerobic energy production under conditions of amino acid sufficiency.     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

Effect of culture conditions on synthesis of L-asparaginase by Escherichia coli A-1.

The nutritional requirements and culture conditions affecting biosynthesis of L-asparaginase in a mutant of Escherichia coli HAP designated strain A-1 were studied. Asparaginase activity was increased by the addition of L-glutamic acid, L-glutamine, or commercial-grade monosodium glutamate. The rate of enzyme synthesis was dependent on the interaction between the pH of the culture and the amount of oxygen dissolved in the medium. A critical oxygen transfer rate essential for asparaginase formation was identified, and a fermentation procedure is described in which enzyme synthesis is controlled by aeration rate. Enhancement of L-asparaginase activity by monosodium glutamate was inhibited by the presence of glucose, culture pH, chloramphenicol, and oxygen dissolved in the fermentation medium.     

Effect of culture conditions on synthesis of L-asparaginase by Escherichia coli A-1.

The nutritional requirements and culture conditions affecting biosynthesis of L-asparaginase in a mutant of Escherichia coli HAP designated strain A-1 were studied. Asparaginase activity was increased by the addition of L-glutamic acid, L-glutamine, or commercial-grade monosodium glutamate. The rate of enzyme synthesis was dependent on the interaction between the pH of the culture and the amount of oxygen dissolved in the medium. A critical oxygen transfer rate essential for asparaginase formation was identified, and a fermentation procedure is described in which enzyme synthesis is controlled by aeration rate. Enhancement of L-asparaginase activity by monosodium glutamate was inhibited by the presence of glucose, culture pH, chloramphenicol, and oxygen dissolved in the fermentation medium.     

Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.     

D-核糖发酵过程参数的研究与控制

以７Ｌ自控发酵罐进行Ｄ－核糖发酵实验,对Ｄ－核糖发酵过程参数进行优化,发现对数生长后期接种,溶氧控制在４０％左右,以葡萄糖酸补料并调节ｐＨ值在７．２左右,可使罐的发酵单位由４５ｇ／Ｌ提高至６５ｇ／Ｌ左右。     

溶氧对L-缬氨酸发酵过程的影响

目的:以黄色短杆菌XV0505为供试菌,探索溶氧对L-缬氨酸发酵过程的影响及其控制策略。方法:利用5L发酵罐,考察了不同溶氧浓度对L-缬氨酸发酵的影响,并采用代谢流分析对其结果进行阐述,提出分阶段溶氧控制策略。结果:采用分阶段溶氧控制策略,即在0～24h溶氧浓度为20%,24～60h溶氧浓度为5%,发酵60h,L-缬氨酸可达到58.36g/L,比5%和20%溶氧浓度下分别提高了18.95%和13.56%。结论:溶氧浓度对L-缬氨酸发酵有重要影响。