Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

Growth and phycocyanin formation of Spirulina platensis in photoheterotrophic culture

Summary Glucose and acetate enhanced cell growth and phycocyanin production of S. platensis. The highest specific growth rate, cell concentration and phycocyanin production were respectively 0.62 d^-1, 2.66 g/l and 322 mg/l on glucose and 0.52 d^-1, 1.81 g/l and 246 mg/l on acetate. The specific growth rate of the alga on 2.5 g glucose/l was markedly increased with increasing light intensity up to 2 klux. Further increasing light intensity to 4 klux only resulted in a very slight increase in specific growth rate. At a light intensity above 4 klux, photoinhibition occurred. Light favoured phycocyanin formation. The highest phycocyanin content was obtained at a light intensity of 4 klux. When the light intensity decreased to 2 klux or less, the optimal glucose concentration for biomass production shifted from 2.5 g/l to 5.0 g/l.     

绿色微囊藻的混合营养生长

研究绿色微囊藻(Microcystis viridis)在混合条件下的生长特性,以及葡萄糖,光照强度和pH对绿色微囊藻生长的影响。结果表明:绿色微囊藻混合营养生长与光能自养生长相比,生长速率明显提高,对数期延长,生物量显著提高;随着初始葡萄糖浓度在0~18.0g/l范围内增加,同一光照条件下明显提高了藻细胞的生长速率,但在初始葡萄糖浓度18.0~36.0g/l范围内,同一光照条件下葡萄糖浓度的高低对藻细胞的生长速率不再有更大的影响;在光照强度24~112μE·m^-2·s^-1范围内,初始葡萄糖浓度相同条件下藻细胞的生长速率及对葡萄糖的藻体得率都随光照强度的增强而增大,但当光照强度在112~200μE·m^-2·s^-1时,绿色微囊藻的生长速率增加幅度较小,出现了光饱和现象;当pH处于8.0~10.0间,明显促进了绿色微囊藻的生长,偏离该范围越大,越抑制绿色微囊藻的生长,甚至导致死亡。     

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。     

Intensification of β-poly(l-malic acid) production by Aureobasidium pullulans ipe-1 in the late exponential growth phase

β-Poly(malic acid) (PMLA) has attracted industrial interest because this polyester can be used as a prodrug or for drug delivery systems. In PMLA production by Aureobasidium pullulans ipe-1, it was found that PLMA production was associated with cell growth in the early exponential growth phase and dissociated from cell growth in the late exponential growth phase. To enhance PMLA production in the late phase, different fermentation modes and strategies for controlling culture redox potential (CRP) were studied. The results showed that high concentrations of produced PMLA (above 40 g/l) not only inhibited PMLA production, but also was detrimental to cell growth. Moreover, when CRP increased from 57 to 100 mV in the late exponential growth phase, the lack of reducing power in the broth also decreased PMLA productivity. PMLA productivity could be enhanced by repeated-batch culture to maintain cell growth in the exponential growth phase, or by cell-recycle culture with membrane to remove the produced PMLA, or by maintaining CRP below 70 mV no matter which kind of fermentation mode was adopted. Repeated-batch culture afforded a high PMLA concentration (up to 63.2 g/l) with a productivity of 1.15 g l(-1) h(-1). Cell-recycle culture also confirmed that PMLA production by the strain ipe-1 was associated with cell growth.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

Characterization of factors influencing the growth of Anabaena variabilis in a bubble column reactor

The combined effect of superficial gas velocity, pH, initial phosphate concentration, and light intensity on cell growth was investigated for the mass production of cyanobacterial cells. The light intensity was manipulated to maintain a specific irradiation rate (q(i)) at a constant level for high cell density culture. The optimum condition for the batch culture was achieved at a superficial gas velocity of 2.0 cm/s, pH 7.0, and an initial phosphate concentration of 55 mg/l when the specific irradiation rate was controlled above 11.5 micromol/s/g dry cell. In this condition, the specific growth rate and cell productivity were 1.47 day(-1) and 0.98 g dry cell/l/day, respectively.     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

雨生红球藻在红光下的生长及营养盐消耗特征

研究了在红光下雨生红球藻Haematcoccus pluvialis的细胞增殖过程中, 叶绿素荧光参数的变化以及氮磷消耗特征, 并且研究了在不同初始氮磷浓度下, 细胞增殖周期、色素变化以及氮磷消耗速率, 以期为雨生红球藻的培养工艺提供参考。结果显示: (1)在细胞增殖过程中, 光合荧光参数PSⅡ、qP、ETR与细胞增殖周期一致, 没有显著差异。Fv/Fm、NPQ则随着细胞密度的上升而下降, 随着细胞密度下降而上升, 且发生变化的拐点与营养盐胁迫造成细胞数量下降的时间点基本一致。(2)以不同初始氮磷浓度(氮磷比10:1, 重量比)的培养液接种细胞, 较高浓度的氮磷条件(45/4.5130/13.0 mg/L), 比较低浓度的氮磷条件(15/1.5 20/2.0 mg/L)有利于提高批次培养后的细胞终产量。但是, 在初始高浓度氮磷下, 接种初期细胞受到胁迫, 相对增长速率小; 氮磷消耗率低, 后期浓度维持高位; 藻细胞指数生长延迟, 培养周期变长。在红光下, 最优化的氮磷接种浓度为45/4.5 mg/L。研究结果对半连续培养雨生红球藻的应用进行了讨论。       

PHYSIOLOGICAL AND ULTRASTRUCTURAL RESPONSES OF CYCLOTELLA MENEGHINIANA (BACILLARIOPHYTA) TO LIGHT INTENSITY AND NUTRIENT LIMITATION1

Photosynthetic characteristics and chloroplast ultrastructure of Cyclotella meneghiniana Kütz. were quantified while the organism was simultaneously adjusting to light and nutrient stress. Cells were grown in batch culture at either low or high light intensity on medium with a nitrogen/phosphorus molar ratio of 2:1 as a control, or with nitrogen or phosphorus deleted from the medium to create nutrient deficiencies. Analysis of variance indicated that light intensity, nutrient deficiency and duration of nutrient deficiency all had significant effects on cell growth, chlorophyll (Chl) concentration/cell, cellular fluorescence capacity (CFC), chloroplast volume and thylakoid surface density. Because interactions existed among nutrient deficiency, extent of nutrient deficiency, and light intensity, all three must be considered together in order to describe accurately the physiology and chloroplast ultrastructure of the diatom. Significant correlations were found between the Chl/cell or CFC/cell and chloroplast volume and thylakoid surface density. Through an increase in Chi concentration, chloroplast volume and thylakoid surface density, the cells successfully adapted to the conditions of low light intensity even while under nutrient stress. In contrast, less Chl/cell, smaller chloroplast volume and less thylakoid surface density were found at high light intensity.     

Optimization of a cultivation process for recombinant protein production by Escherichia coli.

A single-stage fed-batch bioprocess for the production of a recombinant protein beta-galactosidase, by E. coli has been developed. The XL1-blue strain of E. coli which harbors a multi-number foreign plasmid PT was cultured in a reformulated medium. Critical medium components were selected and their respective concentrations were optimized with the Orthogonal Table method. An exponential substrate feeding schedule was used to maintain optimum conditions. Inhibition of growth and protein expression caused by excessive concentrations of glucose and acetate was investigated and subsequently minimized with an incremental nutrient feeding schedule which limited the specific growth rate of a culture. The program necessary to facilitate the control of substrate addition is fully described. This program has been used with a 2.5 l bioreactor and a commercially available software package for optimization without on-line or off-line measurement of optical density (OD), CO2, glucose or acetate. The optimized fed-batch process limited the acetate concentration to less than 20 mM; maintained an exponential growth phase for 50 h; and produced a cell density of 51 g l-1 dry cell weight (DCW) or 154 OD600 with a beta-galactosidase activity of 990 U ml-1.     

Energy--dependent bacterivory in Ochromonas minima--a strategy promoting the use of substitutable resources and survival at insufficient light supply

Phagotrophy and competitive ability of the mixotrophic Ochromonas minima were investigated in a three-factorial experiment where light intensity (low: 1.0 micromol m(-2)s(-1) and high: 60 micromol m(-2)s(-1) PPFD), nutrient concentration (ambient: 7.0 micromolNl(-1), 0.11 micromol P l(-1) and enriched: 88 micromol N l(-1), 6.3 micro mol P l(-1)) and DOC supply (without and with enrichment, 250 micromol C l(-1)) were manipulated. Ochromonas minima and bacterial abundance were monitored for 12 days. We found significant and interacting effects of light and nutrients on Ochromonas minima growth rate and abundance. At high light intensity, nutrient enrichment resulted in increased growth rates and population sizes. In contrast, reduced growth rates and population sizes were observed for nutrient enrichment when light intensity was low. Although, Ochromonas minima was able to ingest bacteria under both high and low light conditions, it grew only when light intensity was high. At high light intensity, Ochromonas minima grew exponentially under nutrient conditions that would have been limiting for photoautotrophic microalgae. In non-enriched low light treatments, Ochromonas minima populations survived, probably by using background DOC as an energy source, indicating that this ability can be of relevance for natural systems even when DOC concentrations are relatively low. When competing with photoautotrophic microalgae, the ability to grow under severe nutrient limitation and to survive under light limitation should be advantageous for Ochromonas minima.     

Semi-continuous monitoring of the capacity of sea water for supporting growth of phytoplankton

Overlapping series of dialysis cultures of three marine planktonic algae were grown in running sea water for several months and responded systematically to the variations in the nutrient level of the water. Under the right conditions the algal cultures showed three growth phases, namely, exponential, linear, and asymptotic as predicted for the dialysis system used. The transition point between exponential and linear growth was well correlated with the concentration of the limiting nutrient in the sea water. Furthermore, linear line correlation between growth rate in the linear phase and the nitrate content of the sea water was obtained for the three of the species involved. It was also found that the final cell density of the asymptotic growth phase was determined by the nitrate content of the sea water. The yield coefficients for conversion of nitrate to cells calculated for linear growth agreed well with the protein content of the various algae.For several cultures of one of the algal species the “maintenance requirement” for nitrate was estimated on the basis of the linear correlation obtained between final cell yield and the nitrate content of the sea water.     

小球藻纯种室内模拟培养初探

实验以小球藻为藻种, 构建室内模拟培养装置, 在选定的一组优化的光照、温度和营养盐浓度条件下,采用间歇培养+连续培养的方式对其进行单种群培养, 模拟藻类生物量积累的过程。将培养期划分为5个阶段, 通过分析各阶段的光合荧光参数以及藻细胞内C、N、P含量, 初探生物量积累过程中藻生理参数的变化特征, 为研究水华的发生、维持和消退提供一定的参考。结果显示: 调整期, 藻生长不受生境光热、营养盐限制, 小球藻光合活性较强; 对数生长期, 藻类开始出现光抑制, 热耗散增加, 胞内N、P含量及N/P值较高; 对数生长期和稳定期, 藻同时受光抑制和营养盐抑制, N和P含量、N/P值均下降; 连续培养期, 营养盐限制得到缓解, 小球藻光合活性开始升高, 且藻细胞对N、P营养物吸收能力增强。监控藻类生物量积累过程中细胞内氮磷下降的拐点和细胞内元素比例变化可为预测水华的消退提供一定的参考依据。     

NH+/4-N assimilation by Rhodobacter capsulatus ATCC 23782 grown axenically and non-axenically in N and C rich media

Assimilation of NH+/4-N and formation of cell biomass in Rhodobacter capsulatus ATCC 23782 were studied in batch cultures as a function of N and C concentration and light intensity. Growth occurred satisfactorily up to N and C levels of 1.2 and 6.0g/1, respectively. The maximum biomass density achieved was 2.3 g biomass-C/l at 0.8 g N/l and 4.0g C/l. Media containing initial C/N ratios of 5 provided good growth and almost complete assimilation and recovery of NH+/4-N and lactate-C, respectively. A light intensity of about 120 μE/m²/s was adequate for efficient growth. At low levels of NH+/4-N (<0.05 g N/l), the photobacterium could not maintain dominance under non-axenic growth conditions. Chloroxuron was necessary to prevent algal overgrowth. At concentrations of 0.2 and 0.4 g NH+/4-N/l, the photo-bacterium maintained dominance over several months under the appropriate conditions of temperature (30°C), light intensity (120μE/m²/s), carbon supply (C/N = 5) and cell residence time (5.5d). The protein of Rhb. capsulatus ATCC 23782 was rich in essential amino acids.     

The regulation of nitrogen accumulation in the grain of wheat plants (Triticum aestivum)

Nitrogen accumulation in the ear of wheat plants ( Triticum aestivum L. cv. Klein Chamaco) during ear growth was studied under 4 experimental conditions. Plants were grown in pots with Perlite or soil, and fertilized with nutrient solutions. In one experiment the plants were grown in a greenhouse and supplied with high (16m M ) or low (1.6 m M ) N in the nutrient solutions until anthesis, and then with or without nitrogen supply until ripening. In a second experiment the plants were grown with high N supply until anthesis, and then for half of the plants light intensity was decreased by 50%, and at the same time. N supply was terminated for half of the plants within each light treatment. A third experiment was similar to the previous one, but was carried out in a growth cabinet under 20% of the maximal irradiance in the greenhouse. In a fourth experiment half the ear was excised at anthesis in half of the plants, and these plants were then supplied with or without nitrogen.
In all experiments there was a linear relation between the rate of N accumulation and the rate of ear growth. A wide range of final individual grain weights and N concentration was observed among the experiments. The same maximum N concentration was observed for all grain sizes, although the N concentration could be different between grains of the same size. The grain N concentration correlated with the rate of N accumulation per unit of ear weight increase during ear growth. It is suggested that in wheat plants there is a dependence of nitrogen transport on carbon transport to the ear, and to the ear, and that the final grain N concentration is determined by the N/C ratio exported from the vegetative tissues.     

稀土元素对红豆杉细胞悬浮培养及紫杉醇合成的影响

研究了在250mL摇瓶中,不同浓度的硝酸镧、硫酸铈铵、硝酸亚铈3种稀土化合物对细胞生长及紫杉醇分泌和释放的影响。结果表明,在培养初期加入稀土元素。3种不同稀土化合物对细胞生长影响强弱不同,但趋势相似,均使细胞的延迟期缩短。1ppm的Ce^4 促进细胞生长的效果最明显。细胞干重第17d达到10.9g/L。在指数期加入稀土元素。10ppmCe^3 刺激细胞生长的效果最明显,细胞干重最高值达到11.5g/dL,比对照高1.5g/L,而10ppm的La^3 抑制细胞的生长。经稀土元素处理后,细胞胞内和胞外紫杉醇含量都有大幅度的提高,其中以10ppmCe^3 处理,胞外紫杉醇释放率最大,达37.7%。     

Fed-batch bioconversion of indene to cis-indandiol

The bioconversion of indene to cis-(1S,2R) indandiol, a key intermediate in the synthesis of Merck’s HIV protease inhibitor, CRIXIVAN™ can be achieved during the growth of a Rhodococcus strain. In a previous study, we reported on the application of multi-parameter flow cytometry for the measurement of indene toxicity to the strain, and found that concentrations up to 0.25 g/l of indene (0.037 g indene/g dry cell weight) in batch bioconversions did not influence cell physiology. Using this information, this study reports on the implementation of a single phase indene fed-batch bioconversion. Cytoplasmic membrane (membrane) integrity and membrane polarisation of a large number of cells were measured during such bioconversions using multi-parameter flow cytometry and compared to a control in order to assess any toxic effects of indene feeding. The results indicate that indene supply at a rate of 0.1 g/l/h is feasible without any deleterious effects on cell physiology. The delay in indene metabolism was significantly shorter, with lower concentrations of by-product formation, when it was added to the culture in the stationary phase than when it was added at the beginning of the exponential phase of the fermentation. cis-Indandiol production rates could be enhanced from 20 mg/l/h, in a previously reported silicone oil two-liquid phase system, up to 200 mg/l/h by a combination of suitable indene feeding rates in the stationary phase and operating with a high biomass concentration to limit the effects of toxicity. In addition, the yield of cis-indandiol on indene (g/g) was higher at 0.48 in the single phase system compared to 0.20 in the two-liquid phase system. However, the final concentration of cis-indandiol was considerably lower, possibly as a result of higher dehydrogenase activity resulting in an increased transformation of cis-indandiol to 1-keto-2-hydroxy indan. This study has demonstrated that it is feasible to feed indene directly in the stationary phase of the bioconversion using high biomass concentrations to obtain enhanced cis-indandiol formation rates as well as yields based on indene utilisation compared to a two-phase silicone oil system.     

The regulation of pyocyanin production in Pseudomonas aeruginosa

Summary Pyocyanin was produced only after the exponential phase of growth on all media examined. Pyocyanin was also found to be produced in response to some nutrient limitation (for example, carbon or oxygen). Furthermore, by controlling the growth rate at less than approximately 0.1 h^–1 the repression of pyocyanin production could be overcome to a large degree. An inverse relationship existed at low growth rates between growth rate and pyocyanin production, with a decrease in growth rate resulting in increased pyocyanin levels.Therefore, pyocyanin production appeared to be regulated by the energy status of the cell which would be lowered under conditions of low nutrient concentration, resulting in a decrease in growth rate and an increase in the level of pyocyanin produced. Under conditions of readily available nutrients the energy generating capacity of the cell was increased resulting in an increased growth rate and repression of pyocyanin.The ability of uncouplers of oxidative phosphorylation (e.g. CCCP and FCCP) to induce pyocyanin production, and of inhibitors of the membrane-bound ATPase (e.g. DCCD and sodium azide) to repress pyocyanin production, confirmed the existance of an energy mediated regulatory mechanism. Indeed, the evidence presented here along with the reported regulatory role of inorganic phosphate in pyocyanin production, suggests that production of this antibiotic may be regulated by intracellular ATP levels.