利用克雷伯肺炎杆菌限制性底物发酵生产1，3-丙二醇

研究了克雷伯肺炎杆菌（Klebsiella pneumoniae）批式流加发酵生产1,3-丙二醇的发酵工艺,根据1,3-丙二醇的生产和菌体生长相关的特点,采用营养基质限制性流加的发酵工艺,通过控制氮源氯化铵以保持细胞稳定生长。结果表明：过低的氮源浓度,细胞生长受到限制,影响产物1,3-PD的合成;过高的氮源浓度,细胞比生长速率增加,但1,3-PD关于消耗甘油的得率降低,用于生长和维持代谢所消耗的甘油量增加。以0.41 g/(L·h)的氮源流加速率,残余氯化铵浓度在0.1 g/L时,转化率和生产强度最高。发酵25 h～28 h后,1,3-丙二醇最终浓度达到52.03 g/L,生产强度为2.04 g/(L·h),相对于甘油的摩尔转化率为0.66,分别比氮源限制前提高了28.0 ％、35.1 ％及29.4 ％。通过限制性流加氯化铵,控制细胞的比生长速率,使底物甘油有效转变为发酵的目标产物1,3-PD,有效实现产物1,3-PD的高生产强度以及对甘油的高转化率。     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

补料分批发酵过程中两阶段发酵生产1,3-丙二醇

在补料分批发酵过程中提高比生长速率不仅减少乙醇、甲酸的生成,而且提高1,3-丙二醇的得率和比生产速率.发酵后期甘油的浓度在15～26 g/L时有利于提高1,3-丙二醇的生产.采取在发酵前期控制菌体较高比生长速率和发酵后期控制适宜甘油浓度相结合的策略,有效地提高了1,3-丙二醇的生产,降低副产物乳酸和乙醇的生成.     

Effects of different pH regulators on 1,3-propanediol production by Klebsiella pneumoniae in fed-batch culture

对肺炎克雷伯氏菌(Klebsiella pneumoniae)发酵生产1,3-丙二醇(1,3-Propanediol,1,3-PD)的补碱策略进行了研究.分别利用NaOH、氨水、KOH三种溶液作为pH调节剂,优化三种pH调节剂并得到按一定比例混合的混合碱.当采用混合碱调控发酵pH值为7.0时,1,3-丙二醇的产量达到了55 g/L,比无pH调控(对照)发酵过程发酵水平提高了10.6倍.     

丙酮酸对肺炎克雷伯氏菌微氧发酵甘油产1,3-丙二醇的影响

微氧条件下,考察肺炎克雷伯氏菌发酵生产1,3-丙二醇过程中柠檬酸和丙酮酸对发酵过程的影响。摇瓶实验结果表明:添加柠檬酸能抑制菌体生长和1,3-丙二醇合成;丙酮酸对菌体生长和1,3-丙二醇合成有一定的促进作用。5 L发酵罐批式发酵表明:补料培养基中加入8 g/L丙酮酸,1,3-丙二醇的产量提高了约10.8%,转化率提高了约4.4%,比生长速率提高了约10.8%。上述结果初步表明,强化能量的产生能够有效促进1,3-丙二醇的合成,可以利用分子生物学手段强化丙酮酸的产生以促进1,3-丙二醇的合成。     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。     

甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析

在甘油厌氧发酵生产1,3-丙二醇的过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-丙二醇的产量和得率。采用PCR方法从Candidaboidinii基因组中克隆编码甲酸脱氢酶基因fdh,将fdh基因片段插入载体pMALTM-p2X中,构建表达载体pMALTM-p2X-fdh,并转入1,3-丙二醇生产菌Klebsiella pneumoniae YMU2,获得重组菌Klebsiella pneumoniae F-1。研究了重组质粒的稳定性和IPTG诱导fdh基因过量表达的条件。结果表明,重组质粒具有良好的稳定性;fdh基因表达的蛋白分子量为40.2kDa;IPTG诱导表达研究表明,在IPTG浓度为0.5mmol/L时,诱导4h后甲酸脱氢酶表达明显;发酵过程中甲酸脱氢酶比酶活达到5.47U/mg;与出发菌株K.pneumoniae YMU2相比,重组菌F-1合成1,3-丙二醇的浓度提高了12.5%。     

pH两阶段控制策略发酵生产重组角质酶

为提高基因工程菌Bacillus subtilis WSHB06-07发酵生产角质酶的产量和生产强度,考察了pH（5.5~8.0）对菌体生长和产酶的影响。基于不同pH发酵过程中菌体比生长速率及比产物合成速率的变化,确定了pH两阶段控制策略,即0~4 h时控制pH 7.5,4 h后将pH调至6.5。通过采用这一优化策略,角质酶酶活有了较大的提高,达170 U/mL,生产强度为16.9 kU/(L·h),比恒定pH 7.5控制模式下分别提高了122.6%和123.2%。     

在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH再生系统

生物法生产1,3-丙二醇（1,3-Propanediol,1,3-PD）是当前工业生物技术研究的热点之一,生产过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-PD的产量和得率。本文采用PCR的方法从Candida boidinii基因组中克隆编码fdh的基因,将该基因片段插入载体pMALTM-p2X,构建表达载体pMALTM -p2X-fdh,并转入醛脱氢酶失活菌Klebsiella pneumoniae DA-1HB,获得重组菌Klebsiella pneumoniae DAF-1。在IPTG浓度0.5 mmol/L时,诱导3 h后甲酸脱氢酶表达明显;发酵过程中甲酸脱氢酶比酶活达到4.82 U/mg;与出发菌株K. pneumoniae DA-1HB相比,重组菌DAF-1合成1,3-丙二醇的浓度提高了19.2%?。     

论文中阿拉伯数字的使用克雷伯氏肺炎杆菌HR526快速合成1,3-丙二醇发酵特性研究

研究了实验室筛选的一株高产1,3-丙二醇(PDO)菌株克雷伯氏肺炎杆菌HR526(Klebsiella pneumoniae HR526),在5 L B.Braun发酵罐进行甘油补料流加发酵30 h,PDO达到91.47 g/L,胞外代谢通量分析显示,PDO在对数中期通量达到最大,而乳酸在稳定期通量达到最大.结合酶学检测分析了PDO合成关键酶PDO氧化还原酶(PDOR)、甘油脱水酶(GDHt)和甘油脱氢酶(GDH)酶活的变化,PDO氧化还原酶活性在对数中期达到最高,甘油脱水酶/甘油脱氢酶在对数期远大于稳定期、衰退期,与代谢通量变化一致甘油脱水酶/甘油脱氢酶活性比例不均衡是3-HPA对数期积累的原因,PDO合成主要集中在对数期,是生长偶联的代谢产物.     

The effect of specific growth rates on the recovery of amino acids

Three specific growth rates, 0.23, 0.45 and 0.51 h^–1, were used to cultivate Corynebacterium glutamicum in a pH-auxostat. The specific formation rates of most amino acids increased by raising the specific growth rates. The highest specific growth rate, 0.51 h^–1, favors the production of LEU; whereas the highest production yield for ALA and GLU were at = 0.23 h^–1. A correlation among specific growth rates, glucose consumption rate, and production yields of amino acids was obtained.     

CIRCADIAN RHYTHM IN GROWTH AND DEATH OF ANABAENA FLOS-AQUAE (CYANOBACTERIA)

Circadian periodicity in cell division and death was investigated in the cyanobacterium Anabaena flos-aquae (Lyngb.) Bréb in a phosphorus (P)-limited, N₂-fixing chemostat culture. When entrained under 12:12 h LD cycles, not only cell division but also cell death showed a clear circadian rhythm in this filamentous cyanobacterium. The rhythm persisted under continuous light and was temperature compensated. Circadian rhythm was clearly observed in the steady-state cell number and instantaneous growth rate, μ(t), which reached a maximum at about 2 h before sunset and a minimum at about 2 h before sunrise. The number of dead cells and the instantaneous death rate γ(t) also showed a circadian periodicity; the peak of γ(t) occurred approximately 8 h before that of μ(t). Therefore, cell growth and death in A. flos-aquae appear to be under the control of circadian clocks, and thus it seems that their death is programmed cell death.     

Nitrate uptake and respiration in roots and shoots: A model

Respiration in plants is often divided into growth and maintenance components. From the growth respiration it is possible to estimate the efficiency of conversion of substrate to plant material. Analysis of recent experimental data on this basis suggests that the conversion efficiency is considerably lower in roots than in shoots, which conflicts with biochemical analysis. The conventional method for describing respiration data is developed to incorporate root activity and is applied to a set of experimental data. The model provides a means for estimating the respiratory cost of nitrate uptake and also a possible explanation for the inconsistency between experimental observations and theoretical analysis.     

Long-term responses of Melilotus segetalis to salinity. II. Nutrient absorption and utilization

Specific absorption rates (SAR) and specific utilization rates (SUR) of sodium, chloride, potassium, calcium, magnesium and phosphate ions were determined for Melilotus segetalis (Brot.) Ser. (annual sweetclover) grown under both control and salinized conditions (NaCl treatment of CE=15 dS m⁻¹) for a complete life cycle with sequential harvests. The behaviour over time of the SARs and SURs of the mineral elements was in general correlated with relative growth rate (RGR) kinetics, with a parabolic trend during the vegetative phase and a progressive linear decrease during the reproductive stage. Salinity significantly reduced the SARs of K and Mg but did not affect the SARs of Ca and P during the vegetative phase. During the reproductive stage, however, the SARs of K, Ca and P of salt-stressed plants were higher than in control plants. The similar SARs of total cations (TC) found in control and salt-stressed plants may indicate compensatory mechanisms to maintain a constant total cation content. Salt-stressed plants showed lower SURs of K, Ca and P during the vegetative phase, and lower SURs of K and P but a higher SUR of Mg during the reproductive stage. A nutrient imbalance, caused by a lower root efficiency in absorbing K and Mg and a lower leaf efficiency in producing biomass per unit of K, Ca and P, apparently contributed to the salt-induced reduction in growth during the vegetative phase of M. segetalis. The switch to non-reduced, compensated growth during the reproductive phase may have been caused by a higher nutrient demand which increased the root efficiency in absorbing K, Ca and P and the leaf efficiency in utilizing Mg.     

Nitrifying bacterial growth inhibition in the presence of algae and cyanobacteria

Nitrifying bacteria, cyanobacteria, and algae are important microorganisms in open pond wastewater treatment systems. Nitrification involving the sequential oxidation of ammonia to nitrite and nitrate, mainly due to autotrophic nitrifying bacteria, is essential to biological nitrogen removal in wastewater and global nitrogen cycling. A continuous flow autotrophic bioreactor was initially designed for nitrifying bacterial growth only. In the presence of cyanobacteria and algae, we monitored both the microbial activity by measuring specific oxygen production rate (SOPR) for microalgae and cyanobacteria and specific oxygen uptake rate (SOUR) for nitrifying bacteria. The growth of cyanobacteria and algae inhibited the maximum nitrification rate by a factor of 4 although the ammonium nitrogen fed to the reactor was almost completely removed. Terminal restriction fragment length polymorphism (T‐RFLP) analysis indicated that the community structures of nitrifying bacteria remained unchanged, containing the dominant Nitrosospira, Nitrospira, and Nitrobacter species. PCR amplification coupled with cloning and sequencing analysis resulted in identifying Chlorella emersonii and an uncultured cyanobacterium as the dominant species in the autotrophic bioreactor. Notwithstanding their fast growth rate and their toxicity to nitrifiers, microalgae and cyanobacteria were more easily lost in effluent than nitrifying bacteria because of their poor settling characteristics. The microorganisms were able to grow together in the bioreactor with constant individual biomass fractions because of the uncoupled solids retention times for algae/cyanobacteria and nitrifiers. The results indicate that compared to conventional wastewater treatment systems, longer solids retention times (e.g., by a factor of 4) should be considered in phototrophic bioreactors for complete nitrification and nitrogen removal. Biotechnol. Bioeng. 2010;107: 1004–1011. © 2010 Wiley Periodicals, Inc.     

Productivity of outdoor algal cultures in enclosed tubular photobioreactor

At quasi-steady-state outdoor cyclic fed batch cultures of Chlorella pyrenoidosa, the growth irradiance incidented on the tubular photobioreactor increased about fivefold between 9:00 a.m. and noon. Overheating of the cultures was observed, resulting in decreasing biomass output rate when culture temperature went above 40 degrees C. In cultures with temperature control, the quasi-steady-state output rate of all cultures increased throughout the day and leveled off in the late afternoon in high-density cultures. The daily area output rate was proportional to the density of the cultures. The specific growth rate of the light-limited cultures increased only marginally (20%) in the morning. (c) 1992 John Wiley & Sons, Inc.     

A general equation for the evaluation of the error that affects the value of the maximum specific growth rate

A general equation is proposed to evaluate the absolute error that affects the maximum specific growth rate calculated from batch or continuous experiments. This error depends on the relative errors of the cell concentration measurements and on the duration of the test.Nomenclature X ₁ Cell concentration at the beginning of the exponential growth phase or of the washing-out period - X ₁ Lowest value of X ₁ due to experimental errors - X ₁ Highest value of X ₁ due to experimental errors - X ₂ Cell concentration at the end of the exponential growth phse or of the washing-out period - X ₂ Lowest value of X ₂ due to experimental errors - X ₂ Highest value of X ₂ due to experiment errors - Relative error that affects X ₁ - Relative error that affects X ₂ - t Exponential growth stage or washing-out duration - Asolute error that affects - Maximum specific growth rate - ₁ Lowest value of due to experimental errors - ₂ Highest value of due to experimental errors The author is with the Instituto Mauá de Technologia, Estrada das Lágrimas 2035, 09580-900, São Caetano do Sul, SP, Brazil     

Comparative study of shortwave heating patterns in phantoms with polyethylene and silk partitions

Specific absorption rate (SAR) and effective depths of heating patterns induced by a shortwave, pancake diathermy applicator in fat-muscle phantom are measured. Midplane partitions of polyethylene and silk screen with and without contact chemicals are used. Thermographically obtained SAR data show nearly the same value for silk-screen partitions with and without contact chemicals and slightly lower values with polyethylene partitions, provided that the partition midplanes are tightly pressed against each other. Thermometry data indicate that for low-power exposures the major error in thermographic measurements obtained after termination of heating is due to thermal diffusion and not evaporative cooling in the opened midplane of the phantom.     

Evaluation of the maximum specific growth rate of a yeast indicating non-linear growth trends in batch culture

The maximum specific growth rate (μ_max) of an ethanolic D-xylose-fermenting yeast, Pichia stipitis, showing non-linear growth trends in batch culture, was calculated using the rate equation μ₂ = (1/Δt) ln(x ₂/x ₁). The absolute error Δμ, affecting μ₂, was derived using an equation given by Borzani (1994). Based on the assumption of linearity of growth curves between two closest time points, the relation between the two rate formulae, μ₁ = (1/xˉ)dx _t /dt and μ₂ = (1/Δt) ln(x ₂/x ₁) was established. In a particular condition, when μ₁ = μ₂, an equation has been developed, the roots of which are the specific growth rates at different time points. This revised version was published online in July 2006 with corrections to the Cover Date.