C群流行性脑膜炎球菌多糖分批发酵动力学模型的建立

目的建立C群流行性脑膜炎球菌多糖50 L罐分批发酵动力学模型,为实现对C群流行性脑膜炎球菌多糖发酵过程的工艺控制、优化及其放大提供理论依据。方法利用分批发酵的数据,以Logistic方程建立菌体生长动力学模型,以Leudedeking-Piret微分方程建立产物合成动力学模型,以底物消耗的物料平衡方程建立底物消耗的动力学模型,并应用Origin软件进行模型拟合,获得3个模型的参数。结果菌体的最大比生长速率(μmax)为0.731 2 h-1,最大菌体浓度(Xmax)为3.949 5 g/L,与菌体生长速率关联的产物合成常数(a)为0.004 97 g/(L·h),与菌体浓度关联的产物合成浓度(β)为0.015 44 g/(L·h),菌体对底物的得率(Yx)为1.905 2 g/g,产物对底物的得率(Yp)为0.130 1 g/g,菌体的维持因子(m)为1.070 1。结论建立的动力学模型能较好地反映C群流行性脑膜炎球菌多糖分批发酵过程中菌体生长、C群多糖合成和基质消耗的的变化规律。     

全细胞催化合成医用纺织新材料聚γ-谷氨酸

在3.7L生物反应器中研究了反应条件对固定化地衣芽孢杆菌催化合成聚γ-谷氨酸的影响.结果表明,添加赖氨酸与谷氨酰胺都可加强产物的合成,反应体系温度37℃及pH值7.0、催化剂用量4%(ω)、通气量4L/min时,搅拌转速达300r/min即可满足细胞的基础代谢和聚γ-谷氨酸合成对溶解氧的需求.以溶氧水平作为L-谷氨酸代谢指标控制L-谷氨酸限制性流加,既可维持一定的固定化菌体的基础代谢,又不会发生反应体系中残余谷氨酸及有害代谢产物阻遏作用,聚γ-谷氨酸转化得率最高可达92.74%.全细胞生物催化剂反应5次后聚合得率可保持在81%以上.     

全细胞催化合成医用纺织新材料聚g-谷氨酸

在3.7 L生物反应器中研究了反应条件对固定化地衣芽孢杆菌催化合成聚g-谷氨酸的影响. 结果表明,添加赖氨酸与谷氨酰胺都可加强产物的合成,反应体系温度37℃及pH值7.0、催化剂用量4%(w)、通气量4 L/min时,搅拌转速达300 r/min即可满足细胞的基础代谢和聚g-谷氨酸合成对溶解氧的需求. 以溶氧水平作为L-谷氨酸代谢指标控制L-谷氨酸限制性流加,既可维持一定的固定化菌体的基础代谢,又不会发生反应体系中残余谷氨酸及有害代谢产物阻遏作用,聚g-谷氨酸转化得率最高可达92.74%. 全细胞生物催化剂反应5次后聚合得率可保持在81%以上.     

碱性纤维素酶基因在巴氏毕赤酵母中的表达及重组酵母菌发酵工艺的优化

目的克隆碱性纤维素酶基因,构建酵母整合型表达质粒,在巴氏毕赤酵母中表达,并对重组菌的发酵工艺进行优化。方法应用PCR技术从嗜碱性芽孢杆菌ATCC21833中扩增碱性纤维素酶基因,克隆至酵母整合型表达载体pGAPZαA中,构建重组表达质粒pGAPZαA-ATCC21833,并转化至巴氏毕赤酵母GS115。通过单因素实验及正交实验,确定重组酵母的最佳发酵培养基。在20L发酵罐中进行高密度发酵,观察碳源对批式发酵的影响,并检测在4种流加方式(连续恒速流加、间歇匀速流加、间歇递减流加、维持底物浓度流加)下的菌体干重及发酵液中的酶活性。结果重组表达质粒pGAPZαA-ATCC21833经酶切及DNA测序证明构建正确,其基因序列与嗜碱性芽孢杆菌KSM-635的碱性纤维素酶基因序列一致。最佳发酵培养基组成为6%葡萄糖、2%硫酸铵、12g/L磷酸二氢钾。碳源浓度对于重组酵母菌体生长及产酶至关重要。SDS-PAGE表明表达产物的相对分子质量约为103000。维持底物浓度的流加方式可获得最高的菌体干重(29.8g/L)及酶活力(24U/ml)。结论已成功构建了表达碱性纤维素酶的巴氏毕赤酵母工程菌,并确定了维持底物浓度的流加方式为最佳发酵方式。     

葡萄糖作辅助碳源对klebsiella pneumoniae发酵生产1,3-丙二醇的影响

在Klebsiella pneumoniae发酵甘油生产1,3 丙二醇的种子培养及发酵实验中,考察了葡萄糖作辅助碳源对菌体生长及1,3 丙二醇生成的影响. 结果表明,在种子培养期,以葡萄糖和甘油为混合碳源可缩短种子培养周期;在批次发酵和流加发酵中,葡萄糖作辅助碳源可使1,3 丙二醇产率及得率明显提高,但不同的葡萄糖加入方式对产率及得率促进的效果不同. 在初始发酵培养基中添加5 g/L葡萄糖、并在4 40 h进行葡萄糖与甘油混合液连续流加的条件下,1,3 丙二醇浓度、产率及得率较单一甘油为底物的流加发酵结果分别提高41.2 , 38.6 和8.3 .     

黄原胶发酵的动力学模型及过程模拟

在5L 通气式搅拌发酵罐中,非溶氧限制的条件下,实验测定了间歇黄原胶发酵的过程数据,并建立了描述这一过程的动力学方程。菌体生长动力学方程(dC_b)/(dt)=0.2675C_b(1(-C_b/1.75))底物(碳源)消耗的动力学方程-(dC_s)/(dt)=0.14C_b(1-(C_b/1.75))+0.5806C_b~2/(1+0.06326C_p)产物(黄原胶)生成动力学方程(dC_p)/(dt)=0.4452C_b~2/(1+0.06326C_p)利用上述动力学方程能较好地描述生长期和稳定期的黄原胶发酵行为。     

红平红球菌LSSE8–1的培养及其对二苯并噻吩中硫元素脱除的影响

红平红球菌(Rhodococcus erythropolis)LSSE8-1是一株新分离的专一性脱硫菌，HPLC分析表明该菌能选择性地脱除二苯并噻吩(DBT)中的硫，最终代谢产物是2-羟基联苯(2-HBP). 适宜的初始pH为6.5~9；能利用多种碳源，其中以甘油为最佳，适宜的甘油浓度为8 g/L；2 g/L的乙酸铵是菌体生长和脱硫的合适氮源；二甲基亚砜是提高该菌细胞收率和脱硫比活性的有效硫源.     

溶氧对Candida glycerinogenes产甘油发酵代谢流分布的影响

分析了耐高渗高产甘油工业菌株产甘油假丝酵母(Candida glycerinogenes)的摇瓶发酵过程,发现溶氧水平是影响C.glycerinogenes 生长、代谢产物生成的重要因素。在此基础上,以5 L发酵罐研究了不同恒溶氧水平甘油的合成过程,通过代谢流分析表明,低溶氧环境下乙醇碳流量增加,导致糖耗快而甘油合成相对较少;碳流分布表明HMP途径在甘油过量合成中起着重要的作用。分析C.glycerinogenes发酵过程中能量代谢和物质代谢（氧化磷酸化和底物水平磷酸化）的变化发现高溶氧环境下ATP合成水平高,一定程度抑制底物水平磷酸化从而使碳流更多地流向了甘油合成;C.glycerinogenes胞内氧化还原平衡(NADH/NAD+比率)的研究进一步解释了产甘油假丝酵母中甘油合成和乙醇生成的竞争关系。     

基于消耗碳氮比进行底物分配和油脂得率预测

微生物油脂作为胞内代谢产物,其脂肪酸组成和植物油类似,是生物柴油和油脂工业理想替代原料。为了进一步了解底物中碳氮比对油脂合成的影响,通过恒化培养的方法,研究了圆红冬孢酵母在不同稀释率条件下,消耗碳氮比和底物在油脂和非油生物量之间的分配以及和油脂得率之间的关系。通过碳、氢、氧和氮的化学反应计量学分析,并根据不同稀释率稳态时所消耗C/N比,构建了底物碳在油脂和非油生物量之间的分配模型。利用实验数据确定了模型参数:最大油脂碳得率Y_L^max为0.51 mol C·(mol C)^-1,最大菌体碳得率Y_L^max为0.52 mol C·(mol C)^-1,促使油脂合成的临界C/N比为12.1 mol C·(mol C)^-1。利用该分配模型预测不同消耗C/N比的油脂得率,预测值为实验值的 95.2%～116.7%,表明模型可靠性较好。     

多拷贝毕赤酵母表达猪胰岛素前体的动力学模型

以携带多拷贝猪胰岛素前体(PIP)基因的毕赤酵母(Pichia pastoris)为供试菌株,对分批补料发酵PIP合成动力学模型进行研究。建立了不同拷贝数毕赤酵母发酵合成PIP的菌体生长、产物合成和底物消耗的动力学模型,并通过Origin8.0软件对模型参数进行了非线性拟合。根据动力学模型结果发现,随着拷贝数的增加与菌体生长相关的产物系数 和细胞生长代谢系数k₁绝对值不断增加,12拷贝时PIP表达量达到最高,说明只有进一步促进高拷贝菌株细胞生长并减少其代谢负担才能更有效地提高产物的生成速率。同时表明:预测值与实验值有良好的拟合性,所建模型能较好地反映分批补料发酵过程PIP的合成。     

Stoichiometric analysis of Kluyveromyces fragilis growth on lactose

This paper describes results of the application of simple metabolic model principles to aerobic growth of Kluyveromyces fragilis during the utilisation of a semi‐synthetic medium simulating dairy waste (whey). The theoretical yield coefficients (maximum, true and real) of biomass on lactose and oxygen, available electrons and ATP were determined and compared with experimental data. The experimental runs were performed in a B Brown fermenter (15 dm³ of operating capacity). The yeast K fragilis was cultured in semi‐synthetic medium containing lactose (40 g dm⁻³) as a main carbon and energy source and ammonia salts as a main nitrogen source (C/N = 8.74). The yeast growth is closely related to the concentration of oxygen in the media. The experimental yields of biomass on lactose and oxygen reached 94% and 88% of real maximum theoretical values, respectively. Calculated true biomass yield coefficients were closely correlated with the values resulting from the balance analysis of stoichiometric equations. The specific maintenance requirement (m_ATP) was 14.7 mmole ATP(g DM h)^‐1. Determination of the elemental composition of the biomass during the course of fermentation resulted in observations of the cellular C/N ratio changes. The conversion of lactose carbon to biomass and CO₂ was 74.3%–81.2%. The recovery of ATP reached 79%, and the recovery of oxygen 96.2%. © 2000 Society of Chemical Industry     

通过通量平衡分析计算经验和真实维持系数

The stoichiometric matrix of a simplified metabolic network in Bacillus Subtillis was contructed from the flux balance equations, which were used for reconciliation of the measured rates and determination of the inner metabolic rates. Thus more reliable results of the true and empirical maintenance coefficients were obtained. The true maintenance coefficient is linearly related to the specific growth rate and changes with the P/O ratiol. The neasured biomass yield of adenosine triphosphate (ATP) is also linearly related to the P/O ratio.     

金色链霉菌碳代谢流量变化特性的研究

建立简化的金色链霉菌代谢网络模型,根据其在复合培养基上的代谢特性,用优化手段求解细胞内碳代谢网络流量。根据不同时期金色链霉菌细胞内碳代谢流的分布,分析金色链霉菌生长代谢过程的碳代谢特性。研究表明, 在对数生长期,碳在EMP途径的通量为0.701mmolg-1h-1,占66.6%,进入HMS途径的流量为0.353mmolg-1h-1,占总碳消耗量的33.4%;在产物合成期,进入EMP途径的代谢流量降至0.382mmolg-1h-1,占进入细胞总碳流量的的51.6%;进入HMS途径的碳为0.358mmolg-1h-1,占48.4%。磷酸己糖旁路HMS通量对金霉素的合成起重要的调节作用, 添加磷酸盐和芳香性氨基酸抑制HMS途径的碳代谢通量,金霉素合成量分别降低50%以上。     

Steady-state behaviour of an aerobic mixed culture growing on phenol in a continuous stirred reactor

The kinetic behaviour of an aerobic mixed culture was studied in a well-stirred reactor at various dilution rates with phenol as the sole carbon source and limiting factor. In the range of dilution rates investigated (up to 0.51 h⁻¹), biomass washout from the reactor was not observed. Analysis of steady-state data using the inhibitory kinetic parameters obtained in a previous batch growth study gave a very poor prediction of the reactor's performance. Since microbial growth occurred on the reactor walls, the experimental data were analysed using two steady-state equations (one with Monod, the other with Haldane growth kinetics) together with the Topiwala-Hamer model to describe the wall attachment. The mathematical model using Monod kinetics gave a good fit to the experimental data and the values of the kinetic parameters obtained using this model were as follows: maximum specific growth rate, 0.340 h⁻¹; saturation constant, 1.61 mg l⁻¹; wall growth constant, 2.84 mg l⁻¹. Furthermore, the yield and maintenance coefficients were calculated to be 0.47 mg mg⁻¹ and 0.007 mg mg⁻¹ h⁻¹ respectively.     

Yield and maintenance coefficients in S. cerevisiae cultures

Precise determination of yields deduced from measurements of metabolic rates needs an understanding of the physiology and energetics of growth of microorganisms. The linear growth equation derived from the simplified picture of microbial metabolism is generally accepted. However, the significance of the yields it gives is less well understood. The analysis of the theoretical and real observed and true yield coefficients for partly aerobic and anaerobic growth of S. cerevisiae is presented in this study. In the calculations of yields and maintenance coefficients, the pseudoconstants Y, P/O were used. A set of limitations imposed on true yields in the linear growth equation is established and the significance of the yields is also discussed. The experimental runs were performed in a laboratory-scale bioreactor (1·5 dm³ of operating capacity). The yeast Saccharomyces cerevisiae hybrid G-67 was cultured in synthetic Reader medium containing sucrose as the main carbon and energy source. The comparison between experimentally obtained yield coefficients relative to cell growth and ethanol formation and the real theoretical values is presented. The maintenance coefficients calculated for anaerobic and respiro-fermentative processes are 0·14 C-mole ATP (C-mole DM h)⁻¹. © 1998 SCI     

Formulation and optimization of two culture media for the production of tumour necrosis factor-β in Escherichia coli

Two culture media were designed and optimized by statistical techniques for the growth of Escherichia coli K12 HB101 (pCG402) which expressed human tumour necrosis factor-β (TNF-β). Common compounds, such as MgSO₄ and KH₂PO₄, were used to provide the bacteria with the necessary elements for biomass synthesis. For compounds not required for biomass synthesis, such as thiamine, or compounds used for both biomass synthesis and as an energy source, such as glucose, yield coefficients were used. In formulating the composition of the nutrients, carbon was chosen as the limiting substrate in both media. In the complex medium (CM), casein hydrolysate was selected to supply the two auxotrophic amino acids, proline and leucine. The optimal ratio of glucose to casein hydrolysate was determined to be 1:0·6 by using a centre composite design experiment. In the defined medium (DM), the concentrations of the three carbon sources, glucose, proline and leucine were based on their respective yield coefficients (Y_{biomass/glucose}: 0·5, Y_{biomass/proline}: 7, Y_{biomass/leucine}: 13·5). Shake flask experiments based on a fractional design were used to confirm that the two media were glucose limiting. Bacterial growth was improved in CM whereas DM gave higher TNF-β expression. A 2-dm³ fed-batch fermentation using CM was performed and a dry biomass concentration of 20 g dm^?3 was obtained with the expression of soluble TNF-β being 20 mg g^?1 dry biomass. With this fed-batch system, a high biomass concentration and high expression of TNF-β were achieved concomitantly.     

Media Optimization for Poly(β-hydroxybutyrate) Production Using Azotobacter Beijerinckii

In the present study, efforts were made to optimize the growth of Azotobacter beijerinckii in the media with essential nutrients for production of PHB. The effects of temperature, agitation rate, carbon and nitrogen source concentrations on microbial grows and PHB production were investigated. Glucose and ammonium chloride were used as carbon and nitrogen source, respectively. The optimal temperature for the growth and PHB synthesis appeared to be 30°C. However, over the range of 25–38°C, the effect of temperature was not very significant. Fermentation under controlled conditions such as agitation rate of 250 rpm, incubated inoculums for 15 h, fermentation temperature at 30°C, and fermentation time of 72 h was performed. Glucose concentration of 30 to 60 g/l with increment of 10 g/l and ammonium chloride in the range of 0.5 to 2 g/l with increment of 0.5 g/l were utilized as carbon and nitrogen sources, respectively. The PHB production was maximized at the desired concentration of glucose (50 g/l) and ammonium chloride (1.5) for carbon and nitrogen sources, respectively. A. beijerinckii exhibited maximum biomass of 17.31 g/l with a PHB concentration of 5.84 g/l. Under the best conditions, PHB constituted up to 34% of dry cell mass after 64 h of culture. The average biomass yield coefficient on glucose was about 0.57 kg/kg.     

氨基酸促进甘油合成的动力学分析

在产甘油假丝酵母分批发酵生产甘油的过程中,考察了添加甘氨酸、谷氨酸、天冬酰胺、丙酮酸和a-酮戊二酸等物质对菌体生长、维持代谢、甘油生成及副产物形成的影响. 结果表明,在发酵全过程中,它们对菌体的生长基本没有影响,但能加快葡萄糖的消耗速率、缩短发酵周期、提高甘油得率,从而使甘油的生产强度分别达到1.66, 1.59, 1.61, 1.54和1.68 g/(L×h),比对照分别提高了25.18%, 19.21%, 20.77%, 15.92%和26.32 %;副产物的形成主要发生在快速生长阶段,而此阶段用于菌体生长、维持代谢和甘油合成所消耗的葡萄糖基本没有变化;在稳定生长期,它们可以减少用于菌体生长、维持代谢和形成副产物的葡萄糖消耗量,导致流向甘油合成途径的葡萄糖代谢流增加.     

噻呋酰胺与吡唑醚菌酯复配对马铃薯黑痣病联合作用及其田间效果

采用菌丝生长速率法测定噻呋酰胺与吡唑醚菌酯不同配比复配对马铃薯黑痣病菌的毒力,评价其联合作用,并选择最佳配比加工成制剂进行田间小区药效试验。试验结果表明:噻呋酰胺与吡唑醚菌酯以质量比3︰1、2︰1、1︰1、1︰2、1︰3复配,增效作用显著,其中以质量比1︰2复配,增效作用最佳,共毒系数为181.42。按此比例加工30%噻呋·吡唑酯悬浮剂,在有效成分用量为200~300 g/hm~2时地面喷雾,开花期、收获时对植株黑痣病和薯块黑痣病的防效分别为72.09%~75.58%和77.08%~80.80%。因此,30%噻呋·吡唑酯悬浮剂防治马铃薯黑痣病效果良好,且对马铃薯安全,有较高的推广应用价值。     

A mathematical model for the production of biosurfactants by Pseudomonas aeuginosa CFTR-6: Production of biomass

The fermentation reaction producing biosurfactant using Pseudomonas aeruginosa CFTR-6 was studied. The progress of the bioreaction was monitored in terms of biomass production, product formation and substrate consumption in shake flask experiments using different initial sugar concentrations. A logistic model for biomass growth was found to be satisfactory. Average estimates of the specific growth rate, biomass yield coefficient and maintenance coefficients were obtained.