分批发酵动力学模型参数的估算

本文基于通用的发酵动力学数学模型,导出了用于描述分批发酵特征的解析解。藉助于由FORTRAN－77编写的POWELL优化算法,以赖氨酸分批发酵为倒^［5］,一举估算出该解析解中所有的发酵动力学参数;μ_max、K_s、α、β、Y_G、Y_p,及m。结果表明t(1)用模型所得到的计算值与实测值具有较好的一致性,(2)赖氨酸合成速度取决于微生物的生长速度及浓度。     

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。     

聚球藻7002在光生物反应器中的光自养培养

通过对聚球藻7002在光生物反应器中的培养,研究了光强在聚球藻7002培养液中的衰减规律,得到了培养过程光强随藻细胞浓度和光程距离变化的关系式,即I=I₀exp［-(-0.0239+0.0777OD₇₅₀)·L］。并对培养过程特性及培养温度、外加CO₂浓度和光照强度对藻细胞生长的影响进行了较为详细的研究,得到了反应器中较为适宜的聚球藻7002的培养条件,藻细胞培养密度达到3.4g/L(干重),体积产率达到0.57g/(L·d)的较高水平。     

促甲状腺激素单克隆抗体的制备

获得了抗促甲状腺激素(TSH)单克隆抗体杂交瘤细胞20株,其中T₇₄A₁₀小鼠腹水滴度为1:50 000,亲和常数为7.15×10⁹L/mol,T₇₁B₁₁小鼠腹水滴度为1:150000,亲和常数为8.75×10⁹L/mol.两个抗体与人绒毛膜促性腺激素(HCG)、促卵泡激素(FSH)和促黄体生成激素(LH)的交叉反应分别小于1.1×10^-6%、0.01%和0.016%.将T₇₄A₁₀和T₇₁B₁₁应用于TSH免疫放射分析中,得到了满意的结果.     

西藏双须叶须鱼年龄与生长特点研究

双须叶须鱼（Ptychobarbus dipogon Regan）隶属裂腹鱼亚科（Schizothoracinae）叶须鱼属（Ptychobarbus）,是西藏特有经济鱼类,2016年被列入中国脊椎动物红色名录,必须加快推动该鱼的养护工作。2013年2~3月份及2014年2~6月份,在雅鲁藏布江谢通门段与支流拉萨河上游段采集1 030尾双须叶须鱼样本,以脊椎骨作为年龄鉴定材料进行年龄与生长特点研究,以期分析和评价该类群鱼类资源情况。研究结果如下：双须叶须鱼样本中最大年龄为49龄,最小年龄为4龄;体重与体长关系是样本总体W=4.4×10^-5?SL^2.7688、雌鱼W_♀=5.0×10^-4?SL^2.3474、雄鱼W_♂=2.8×10^-5?SL^2.8414;体长生长方程为雌鱼L_t（♀）=431.8［1-e^-0.19（t+1.19）］,渐进体长L∞_（♀）=431.8 mm,拐点年龄为3.3龄,雄鱼L_t（♂）=367.6［1-e^-0.42（t+3.37）］,渐进体长L∞_（♂）=367.7 mm;体重生长方程为雌鱼W_t（♀）=767.40［1-e^-0.19（t+1.19）］^2.3474,雄鱼W_t（♂）=545.02［1-e^-0.42（t+3.37）］^2.8414;群体异速生长指数b=2.768 8与匀速生长指数3存在显著性差异,即P<0.05。研究表明双须叶须鱼体趋向低龄化,应当予以充分关注。     

碳源和氮源对水母雪莲悬浮培养细胞生长和黄酮合成的影响

在MS培养基上进行水母雪莲(Saussurea medusa Maxim)细胞悬浮培养时研究了碳源和氮源的影响。结果表明碳源以蔗糖最适合,蔗糖浓度则以40g/L较好,细胞生长量干重为18.12g/L,总黄酮合成量达到1423.25mg/L。在培养过程中,水母雪莲细胞能够快速将蔗糖水解为葡萄糖和果糖,并首先利用葡萄糖。氮源总浓度(包括NH⁺₄和NO^-₃)为60～120mmol/L,NH⁺₄/NO^-₃比例为20/40有利于雪莲细胞生长和黄酮合成。用HPLC检测显示4′,5,7-三羟基3′,6-二甲氧基黄酮(Jaceosidin)和4′,5,7-三羟基-6-甲氧基黄酮(Hispidulin)2种黄酮的含量分别达到细胞干重的1.46%和0.010%     

葡萄糖氧化酶产生菌Z—l—C的分批发酵与恒化培养

本文对葡萄糖氧化酶产生菌z—I—c的分批发酵与恒化培养进行了研究。实验发现,在以葡萄糖为生长限制性基质的恒化培养中,该产生菌的维持系数为O．04 g葡萄糖／g菌体．H,生长得率系数Y^max_G=O．714 g菌体／g葡萄糖,最大比生长速率μmax=O．385h^-1,饱和常数Ks=4．76g/L,理论最适稀释度Dm=0．260h^-1,最大酶比活(E／x)max为2．16×10³μ／mg,其值较分批发酵的最大酶比活(1．5l×10³μ／mg)提高43％。当向恒化培养的补料培养基中添加0．02％的α-甲基-D-葡萄糖时,由于该葡萄糖结构类似物的诱导作用,可使(E／x)max达3．11×10³μ／mg,较分批发酵之值提高106％。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

锌酵母分批流加发酵动态优化

对锌酵母分批流加发酵过程的控制变量反应温度和pH的动态最优化进行研究。基于酵母流加发酵有抑制的状态模型．通过龙格一库塔法计算微分方程组、单纯形法优化对模型参数进行估计。采用不同的温度和pH控制策略进行研究,由此获得动力学模型参数与温度和pH关系的回归模型。在此基础上,以极大值原理、梯度法优化求解以获得最高锌酵母产量为目标的最优温度和pH分布T*（t）、pH*(t)。实验验证,在T*(t)和pH*(t)下操作,锌酵母产率可提高13.7％。     

硒对NO诱导的内皮细胞内游离钙离子浓度变化的影响

用Fura-2显微荧光测钙技术,研究了用外源性一氧化氮（NO）供体S-亚硝基谷胱甘肽（GSNO）诱导的,人脐静脉内皮细胞系ECV-304细胞胞内游离钙离子浓度（［Ca²⁺］_i ）升高以及硒的抑制效应.结果表明,GSNO作用于ECV-304细胞,短时间内即可导致其胞内游离钙离子浓度升高.胞外液换为无钙液或向胞外液中加入CdCl₂（1 mmol/L）对GSNO引起的［Ca²⁺］_i升高无影响.提示,GSNO刺激主要引起胞内钙库释放.而且,一氧化氮清除剂血红蛋白（Hb）对这一过程有抑制作用,说明GSNO引起的胞内钙库释放由NO介导.经亚硒酸钠（1 μmol/L）处理的细胞,其NO引起的［Ca²⁺］_i升高幅度明显被抑制,说明NO的这种作用可能与细胞的氧化还原状态有关.     

溶氧及pH对产朊假丝酵母分批发酵生产谷胱甘肽的影响

在7 L发酵罐中研究了溶氧和pH对产朊假丝酵母分批发酵生产谷胱甘肽的影响。结果表明,当葡萄糖浓度为30 g/L且通气量控制在5 L/min时,搅拌转速达到300 r/min即可满足细胞生长和谷胱甘肽合成对溶解氧的需求。不同pH控制方式对谷胱甘肽分批发酵的影响有较大差异。不控制pH时,细胞干重和谷胱甘肽产量比控制pH为55的发酵分别低27%和95%,且有50%的谷胱甘肽向胞外渗漏。研究了将pH控制在4.0、4.5、5.0、5.5、6.0和6.5的谷胱甘肽分批发酵过程,发现在pH 5.5时谷胱甘肽总产量最高。用前期研究建立的动力学模型模拟了不同pH (4.0～6.5)下的分批发酵过程,并从动力学角度解释了pH对细胞生长和谷胱甘肽合成的影响。     

Enhanced intracellular glutathione synthesis and excretion capability of Candida utilis by using a low pH-stress strategy

AIMS: To study the effect of low pH stress on glutathione (GSH) synthesis and excretion capability of GSH fermentation production in Candida utilis. METHODS AND RESULTS: When C. utilis WSH 02-08 was cultivated in a glucose-ammonium sulfate medium without pH control, GSH leakage occurred when the pH of the medium decreased to 1.5. However, analysis of the cell viability indicated that the cells were not lysed. To further study the effect of low pH stress on GSH production, pH-controlled batch cultures were conducted, where the pH was switched from 5.5 to 1.2 at 24 h and maintained at 1.2 for 6 h. Nearly all intracellular GSH was leaked into the medium and the cell viability decreased dramatically, conceiving a long-term exposure of strain WSH 02-08 at low pH environment led to a complete cell lysis. A critical point (treated at pH 1.2 for 3 h) was experimentally determined, where most cells were alive but suffering a low pH stress. Low pH-stressed C. utilis cells displayed an increased intracellular GSH synthesis and export capability, which protected the cells against short-term low pH treatment. CONCLUSIONS: Using this knowledge, a low pH-stress strategy was developed and applied in fed-batch production of GSH and 197.3 mg l-1 of GSH was secreted into the medium. The GSH-specific production yield could be increased from 2.11 to 2.67% (w/w), and the total GSH concentration could reach 737.1 mg l-1 and increased by 24.9%. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of GSH secretion of C. utilis at low pH. This study demonstrated the importance of the physiology-based fermentation strategy in the production of useful metabolites.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.     

Fermentation Kinetics and Continuous Process of L-Asparaginase Production

For the purpose of obtaining L-asparaginase in quantities from Erwinia aroideae, cell growth and enzyme formation were investigated in both batch and continuous fermentation. Using yeast extract as a growth-limiting substrate, the relationship between specific growth rate and substrate concentration was found to fit the Monod equation. The optimum temperature for enzyme production was 24 C, although cell growth was higher at 28 C. The enzyme yield reached its maximum of 4 IU/ml during the negative acceleration growth phase which occurs just prior to stationary growth. Compared to batch fermentations, the continuous fermentation process gave a lower enzyme yield except when the fermentation was conducted at a dilution rate of 0.1 hr(-1). The graphical method frequently used for prediction of continuous fermentation does not apply to L-asparaginase production by E. aroideae. The optimum temperature for enzyme production in continuous process was 24 C, which was the same as in batch process. Increasing the temperature from 24 to 28 C resulted in a 20% loss of enzyme yield.     

Kinetics and modeling of temperature effects on batch xanthan gum fermentation

Batch fermentation kinetics of xanthan gum production from glucose by Xanthomonas campestris at temperatures between 22 degrees C and 35 degrees C were studied to evaluate temperature effects on cell growth and xanthan formation. These batch xanthan fermentations were modeled by the logistic equation for cell growth, the Luedeking-Piret equation for xanthan production, and a modified Luedeking-Piret equation for glucose consumption. Temperature dependence of the parameters in this model was evaluated. Growth-associated rate constants increased to a maximum at approximately 30 degrees C and then decreased to zero at approximately 35 degrees C. This temperature effect can be modeled using a square-root model. On the contrary, non-growth-associated rate constants increased with increasing temperature, following the Arrhenius relationship, in the entire temperature range studied. The model developed in this work fits the experimental data very well and can be used in a simulation study. However, due to the empirical nature of the model, the parameter values need to be reevaluated if the model is to be applied to different growth conditions.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

一株嗜热子囊菌产生的碱性耐热过氧化氢酶及其应用潜力

研究了一株嗜热子囊菌产过氧化氢酶的摇瓶发酵条件,并对其在纺织工业中的应用潜力进行了评价。以20 g/L糊精和1%(V/V)乙醇为混合碳源时,过氧化氢酶酶活达到1594 u/Ml,比以糊精和乙醇单独为碳源时过氧化氢酶的活力之和还高23%。改变培养基的初始Ph、提高发酵液中的溶氧水平及添加外源过氧化氢,过氧化氢酶的产量进一步提高到2762 u/Ml,比优化前提高了5.8倍。将嗜热子囊菌的过氧化氢酶同来源于牛肝、黑曲霉的过氧化氢酶进行了热(70℃, 80℃, 90℃)、碱(Ph 9.0, Ph 10.0, Ph 11.0)稳定性的比较。结果显示,产自嗜热子囊菌的过氧化氢酶对高温和强碱性的耐受性能明显优于其它来源的酶,在纺织染整工艺中具有良好的应用潜力。