Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture

Oxygen transport is a major limitation in large-scale mammalian cell culture. The effects of the dissolved oxygen concentration (DO; from 0.1 to 100% saturation with air) on Sp2/0-derived mouse hybridomas were investigated using continuous culture. The steady-state concentration of viable cells increased with decreasing DO until a critical dissolved oxygen concentration of 0.5% of air saturation was reached. The cell concentration declined at lower DO because of incomplete glutamine oxidation, and the specific lactate production from glucose increased to offset the reduced energy production from glutamine. Cell viability increased as the DO was decreased; the viability continued to increase even when the DO was reduced below 0.5%. The specific oxygen uptake rate was essentially constant for DO greater than or equal to 10% of air saturation and then decreased with decreasing DO. The P/O ratio (ATP molecules produced per O atom consumed) appears to change from 2 to 3 between 10 and 0.5% DO. The specific ATP production rate calculated using this assumption decreases only slightly with decreasing DO. The optimum DO of 50% for antibody production is different than the optimum (approximately 0.5% DO) for cell growth.     

Photobioreactor design: Mixing, carbon utilization, and oxygen accumulation

Photobioreactor design and operation are discussed in terms of mixing, carbon utilization, and the accumulation of photosynthetically produced oxygen. The open raceway pond is the primary type of reactor considered; however small diameter (1-5 cm) horizontal glass tubular reactors are compared to ponds in several respects. These are representative of the diversity in photobioreactor design: low capital cost, open systems and high capital cost, closed systems. Two 100-m(2) raceways were operated to provide input data and to validate analytical results. With a planktonic Chlorella sp., no significant difference in productivity was noted between one pond mixed at 30 cm/s and another mixed from 1 to 30 cm/s. Thus, power consumption or CO(2) outgassing limits maximal mixing velocities. Mixing power inputs measured in 100-m(2) ponds agreed fairly well with those calculated by the use of Manning's equation. A typically configured tubular reactor flowing full (1 cm diameter, 30 cm/s) consumes 10 times as much energy as a typical pond (20 cm deep flowing at 20 cm/s). Tubular reactors that flow only partially full would be limited by large hydraulic head losses to very short sections (as little as 2 m length at 30 cm/s flow) or very low flow velocities. Open ponds have greater CO(2) storage capacity than tubular reactors because of their greater culture volume per square meter (100-300 L/m(2) vs. 8-40 L/m(2) for 1-5-cm tubes). However, after recarbonation, open ponds tend to desorb CO(2) to the atmosphere. Thus ponds must be operated at higher pH and lower alkalinity than would be possible with tubular reactors if cost of carbon is a constraint. The mass transfer coefficient, K(L), for CO(2) release through the surface of a 100-m(2) pond was determined to be 0.10 m/h. Oxygen buildup would be a serious problem with any enclosed reactor, especially small-diameter tubes. At maximal rates of photosynthesis, a 1-cm tubular reactor would accumulate 8-10 mg O(2)/L/min. This may result in concentrations of oxygen reaching 100 mg/L, even with very frequent gas exchange. In an open pond, dissolved oxygen rises much more slowly as a consequence of the much greater volume per unit surface area and the outgassing of oxygen to the atmosphere. The maximum concentration of dissolved oxygen is typically 25-40 mg/L. The major advantage of enclosed reactors lies in the potential for aseptic operation, a product value which justifies the expense. For most products of algal mass cultivation, open ponds are the only feasible photobioreactor design capable of meeting the economic and operating requirements of such systems, provided desirable species can be maintained.     

Effect of dissolved oxygen control on growth and antibiotic production in Streptomyces clavuligerus fermentations.

A proportional-integral control system was used to control dissolved oxygen in a fermentor at constant shear and mass transfer conditions. Growth and antibiotic production in Streptomyces clavuligerus were studied at different dissolved oxygen levels during the fermentation. Three protocols were employed: no-oxygen control to provide a base case, oxygen controlled to a preset saturation level throughout the fermentation, and oxygen controlled at a high level only during the growth phase. The last protocol was aimed at optimizing the consumption of oxygen. Lower specific growth rates and cephamycin C yields were obtained when dissolved oxygen was controlled at 50% throughout the fermentation, compared to the base case. A 2.4-fold increase in the final cephamycin yield was observed when dissolved oxygen was controlled at saturation levels during the growth phase, compared to the experiments without dissolved oxygen control. This enhancement in yield was independent of the dissolved oxygen (DO) level after exponential growth, in the range of 50-100% saturation. The most effective control strategy, therefore, was to control DO only during active growth when the biosynthetic enzymes were probably synthesized.     

Modeling of the mixed culture and periodic control for PHB production

The unstructured mathematical model was developed in the present investigation for the mixed culture, where the metabolites produced by one microorganism is assimilated by the other microorganism. For this, we specifically employed such model system in which sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted in turn to poly-β-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Several batch and fed-batch culture experiments were conducted using each microorganism at different dissolved oxygen (DO) concentrations. Those experimental data were then fitted to the mathematical model, which can describe the dynamics of a mixed culture. Some of the model parameters were expressed as functions of DO concentrations, and some of the other model parameters were tuned based on the mixed culture experiments. The model developed describes the effects of such concentrations of glucose, lactate, DO, and NH₃ on the dynamic behavior of such concentrations as both microorganisms, glucose, lactate, and PHB. Optimal operating condition was then investigated using the model developed. It was found that the periodic change in DO concentration improved such performance as PHB yield, and it was verified by experiments. The optimal NH₃ concentration profile was also obtained for the efficient PHB production by the application of the maximum principle.     

Measurement of hydrodynamic shear by using a dissolved oxygen probe

When a dissolved oxygen (DO) probe is submerged in an air-saturated cell culture medium the thickness of the liquid film that exists outside the membrane of a DO probe changes with hydrodynamic shear. The response of the DO probe thus varies with the hydrodynamic shear environment near the DO probe in cell culture reactors. The thickness of the liquid film was estimated by using a three-layer model, which describes the flow of DO molecules through the liquid layer, the membrane, and the electrolyte, to the cathode of a DO probe. According to the three-layer model, the current output of the DO probe was a strong function of thickness of the liquid film outside the membrane of the DO probe. A correlation between shear rates on the surface of the probe and the DO saturation reading was obtained by using two concentric cylinders with a rotating inner cylinder. This correlation was then used to characterize the local hydrodynamic shear environment in a cell culture reactor. (c) 1993 John Wiley & Sons, Inc.     

Effect of low dissolved oxygen on simultaneous nitrification and denitrification in a membrane bioreactor treating black water

Effect of low dissolved oxygen on simultaneous nitrification and denitrification was evaluated in a membrane bioreactor treating black water. A fully aerobic membrane bioreactor was operated at a sludge age of 60 days under three low dissolved oxygen (DO) levels below 0.5mg/L. It sustained effective simultaneous nitrification/denitrification for the entire observation period. Nitrification was incomplete due to adverse effects of a number of factors such as low DO level, SMPs inhibition, alkalinity limitation, etc. DO impact was more significant on denitrification: Nitrate was fully removed at low DO level but the removal was gradually reduced as DO was increased to 0.5mg/L. Nitrogen removal remained optimal within the DO range of 0.15-0.35 mg/L. Experimental results were calibrated and simulated by model evaluation with the same model coefficients. The model defined improved mass transfer with lower affinity coefficients for oxygen and nitrate as compared to conventional activated sludge.     

Scale-down model to simulate spatial pH variations in large-scale bioreactors

For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.     

Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture

Baker's yeast, Saccharomyces cerevisiae, was investigated for the combined influence of dissolved oxygen and glucose concentration in continuous culture. A reactor was operated at a range of dilution rates (0.1, 0.2, 0.25, 0.27, and 3.0 h(-1)), above and below the critical value that separates the oxidative and fermentation regions. For each dilution rate (D), steady states were established at each of five to ten different dissolved oxygen concentrations (DO) in the range of 0.01-5 mg/L. The use of on-line mass spectrometry facilitated the measurement of gaseous and dissolved O(2), CO(2), and ethanol. Intracellular carbohydrate, protein, RNA, DNA, lipid, and cytochrome concentrations were measured. Cell size measurements were reduced to specific surface areas. Cytochrome content showed up to 100% variation during a 20-day period of adaptation at D = 0.2 h(-1) to low DO. Eventually, the culture behaved the same at DO = 0.05 mg/L as it did initially at 3 mg/L. At D = 0.2, 0.25, and 0.27 h(-1), the transition between oxidation and fermentation was characterized by a critical DO which decreased with decreasing D. The X-D curves were shifted such that the critical D value was reduced with decreasing DO. Specific oxygen update rates varied with DO according to the saturation kinetics. Specific cell surface areas increased with decreasing DO. Cytochrome content generally decreased with decreasing DO, and Q(O(2) ) could be linearly related to the total cytochrome content, which exhibited a maximum at D = 0.27 h(-1).     

Influence of constant and oscillating dissolved oxygen concentrations on keto acid production by Gluconobacter oxydans subsps. melanogenum.

Gluconobacter species are known to oxidise glucose via a direct oxidation pathway which is distinct from the pentose phosphate pathway. In the present communication results of an investigation on the influence of different dissolved oxygen concentrations (DO) on the production of 2,5-diketogluconic acid in batch and chemostat cultures are given. DO of 30% relative to air at 1 bar was found as a threshold level for optimum productivity. The positive influence of continuous availability of dissolved oxygen on the process of rapid glucose oxidation was unambiguously shown as the result of induction of membrane bound dehydrogenases involved in direct glucose oxidation. Furthermore data of scale-down experiments in which the organism was cultivated under oscillations of dissolved oxygen, are given. The influences of such oscillations of DO in the region of the established threshold (30% saturation) were found to result in a prolonged lag phase for growth and product formation. The data obtained in this study revealed critical residence times at low DO that could be employed as a criterion for scale up of this aerobic process.     

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

In large-scale bioreactors, there is often insufficient mixing and as a consequence, cells experience uneven substrate and oxygen levels that influence product formation. In this study, the influence of dissolved oxygen (DO) gradients on the primary and secondary metabolism of a high producing industrial strain of Penicillium chrysogenum was investigated. Within a wide range of DO concentrations, obtained under chemostat conditions, we observed different responses from P. chrysogenum: (i) no influence on growth or penicillin production (>0.025 mmol L⁻¹); (ii) reduced penicillin production, but no growth limitation (0.013–0.025 mmol L⁻¹); and (iii) growth and penicillin production limitations (<0.013 mmol L⁻¹). In addition, scale down experiments were performed by oscillating the DO concentration in the bioreactor. We found that during DO oscillation, the penicillin production rate decreased below the value observed when a constant DO equal to the average oscillating DO value was used. To understand and predict the influence of oxygen levels on primary metabolism and penicillin production, we developed a black box model that was linked to a detailed kinetic model of the penicillin pathway. The model simulations represented the experimental data during the step experiments; however, during the oscillation experiments the predictions deviated, indicating the involvement of the central metabolism in penicillin production.     

Lake Number,a Quantitative Indicator of Mixing Used to Estimate Changes in Dissolved Oxygen

Lake Number, L_N, values are shown to be quantitative indicators of deep mixing in lakes and reservoirs that can be used to estimate changes in deep water dissolved oxygen (DO) concentrations. L_N is a dimensionless parameter defined as the ratio of the moments about the center of volume of the water body, of the stabilizing force of gravity associated with density stratification to the destabilizing forces supplied by wind, cooling, inflow, outflow, and other artificial mixing devices. To demonstrate the universality of this parameter, L_N values are used to describe the extent of deep mixing and are compared with changes in DO concentrations in three reservoirs in Australia and four lakes in the U.S.A., which vary in productivity and mixing regimes. A simple model is developed which relates changes in L_N values, i.e., the extent of mixing, to changes in near bottom DO concentrations. After calibrating the model for a specific system, it is possible to use real-time L_N values, calculated using water temperature profiles and surface wind velocities, to estimate changes in DO concentrations (assuming unchanged trophic conditions).     

Influence of dissolved oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor

The biofilm airlift suspension (BAS) reactor can treat wastewater at a high volumetric loading rate combined with a low sludge loading. Two BAS reactors were operated, with an ammonium load of 5 kg N/(m(3) d), in order to study the influence of biomass and oxygen concentration on the nitrification process. After start-up the nitrifying biomass in the reactors gradually increased up to 30 g VSS/L. Due to this increased biomass concentration the gas-liquid mass transfer coefficient was negatively influenced. The resulting gradual decrease in dissolved oxygen concentration (over a 2-month period) was associated with a concomitantly nitrite build-up. Short term experiments showed a similar relation between dissolved oxygen concentration (DO) and nitrite accumulation. It was possible to obtain full ammonium conversion with approximately 50% nitrate and 50% nitrite in the effluent. The facts that (i) nitrite build up occurred only when DO dropped, (ii) the nitrite formation was stable over long periods, and (iii) fully depending on DO levels in short term experiments, led to the conclusion that it was not affected by microbial adaptations but associated with intrinsic characteristics of the microbial growth system. A simple biofilm model based on the often reported difference of oxygen affinity between ammonium and nitrite oxydizers was capable of adequately describing the phenomena.Measurements of biomass density and concentration are critical for the interpretation of the results, but highly sensitive to sampling procedures. Therefore we have developed an independent method, based on the residence time of Dextran Blue, to check the experimental methods. There was a good agreement between procedures.The relation between biomass concentration, oxygen mass transfer rate and nitrification in a BAS reactor is discussed. (c) 1997 John Wiley & Sons, Inc.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

溶氧水平对红豆杉细胞悬浮培养的影响研究

紫杉醇 (Ｔａｘｏｌ)是源自红豆杉提取物的一种高度衍生化的二萜类化合物 ,临床实验结果表明紫杉醇对于卵巢癌、乳腺癌、胃肠道癌等具有明显的抗肿瘤活性[1] ,因而受到世界各国的广泛关注 ,并已被美国食品与药品管理局 (ＦＤＡ)批准用于卵巢癌与乳腺癌的治疗[2 ] 。到目前为止紫杉醇仍然主要从树皮中提取 ,但由于红豆杉生长缓慢 ,天然资源非常有限 ,加快其替代来源的研究势在必行。利用植物细胞悬浮培养生产紫杉醇作为一种可行的选择 ,近年来取得了较大的进展[3 ,4 ] 。本文研究了摇瓶及 2 0 Ｌ反应器培养过程的溶氧水平对细胞生长及紫杉醇…     

Kinetics of benzene biotransformation under microaerophilic and oxygen-limited conditions

A special microbial consortium adapted to degrade petroleum hydrocarbons at limited availability of oxygen, transformed benzene, a highly toxic and carcinogenic contaminant of groundwater and soil, at low initial dissolved oxygen (DO) concentrations of 0.05-2 mg/L. The employed initial concentrations of dissolved oxygen were considerably lower than the previously reported values. Under these conditions, the overall transformation of benzene ranged from 34% +/- 1.7% to 100%, considerably higher than the theoretical predictions for complete mineralization of benzene based on the requirement of 3.08 mg oxygen/mg benzene. Unlike biotransformation that proceeded at the lowest examined DO concentration of 0.05 mg/L, the mineralization of benzene, defined by its conversion to CO(2) and water, required a minimum DO concentration of 0.2 mg/L. The mineralization of benzene under microaerophilic conditions (DO < 2 mg/L), ranged from 0.83% +/- 0.06% to 89% +/- 1.3%, which was less than the theoretical predictions at any given initial DO concentration. The regulatory effects of dissolved oxygen concentration or its partial pressure on the activities of enzymes catalyzing the biotransformation of aromatic hydrocarbons was postulated to account for the reduced mineralization of benzene. The ratio between the transformed benzene and the consumed oxygen increased with the decrease of initial DO concentration, reaching a value of 2.8, considerably higher than the theoretical value of 0.33 obtained for a complete aerobic oxidation of benzene. Phenol was the major and the most stable intermediate metabolite during the biotransformation of benzene at low concentrations of DO. While benzene transformation stopped after the depletion of oxygen in the experimental system, phenol continued to accumulate under strictly anaerobic conditions, indicating its formation from an alternative carbon source, possibly biomass.     

Generalised additive modelling approach to the fermentation process of glutamate

In this work, generalised additive models (GAMs) were used for the first time to model the fermentation of glutamate (Glu). It was found that three fermentation parameters fermentation time (T), dissolved oxygen (DO) and oxygen uptake rate (OUR) could capture 97% variance of the production of Glu during the fermentation process through a GAM model calibrated using online data from 15 fermentation experiments. This model was applied to investigate the individual and combined effects of T, DO and OUR on the production of Glu. The conditions to optimize the fermentation process were proposed based on the simulation study from this model. Results suggested that the production of Glu can reach a high level by controlling concentration levels of DO and OUR to the proposed optimization conditions during the fermentation process. The GAM approach therefore provides an alternative way to model and optimize the fermentation process of Glu.     

The half-saturation coefficient for dissolved oxygen: A dynamic method for its determination and its effect on dual species competition

A simple approach was developed to determine the half-saturation coefficient for dissolved oxygen (K(DO)) for three bacteria by maintaining a constant oxygen concentration in continuous culture, and employing a dynamic method to obtain the specific growth rate (mu) for each species. Measurement of mu at selected dissolved oxygen concentrations (DO) resulted in a typical Monod curve for a plot of mu vs. DO. Values for K(DO) and mu(max) were obtained from the Lineweaver-Burk reciprocal plot. The bacteria studied included representative strains of three microorganisms isolated in pure culture from poorly settling activated sludge: two filamentous microorganisms, Sphaerotilus natans and a second Sphaerotilus sp., and an unidentified floc-forming microorganism. The K(DO) values obtained for Sphaerotilus sp., S. natans, and the floc former were 0.014, 0.033, and 0.073 mg/L, respectively. Dual species competition experiments were conducted in continuous culture under low and high DO conditions. Successful growth competition by these microorganisms under DO-limiting conditions was consistent with experimentally determined K(DO) values. The finding of lower K(DO) values for the two Sphaerotilus species, compared to the floc former, confirmed the hypothesis that these filamentous microorganisms can outgrow floc-forming microorganisms in activated sludge when DO in the aeration basin is low.     

不同溶氧对谷氨酸棒杆菌代谢的影响

【目的】以谷氨酸棒杆菌为研究对象,分别控制在0、30%、50%3种溶氧水平下进行发酵,分析不同溶氧水平下代谢的变化。【方法】通过检测发酵代谢物中有机酸、氨基酸的含量,以及测定代谢途径中关键酶活性及其编码基因的表达情况来考察不同溶氧水平下物质代谢发生的变化。通过检测胞内还原力和ATP的含量来分析不同溶氧水平对能量代谢产生的影响。【结果】谷氨酸棒杆菌代谢支路受溶氧的影响而发生改变,氨基酸、有机酸的产量也随之改变。特别是在低溶氧(0)情况下,细胞内氧化磷酸化减弱,导致维持生命活动所必需的ATP供应减少,因此细胞通过增强底物水平磷酸化来产生ATP以满足生命活动的需求。在此情况下,胞内NADH得到较多积累,TCA循环代谢流量减小,而转向糖酵解、乙醛酸循环等,并且这个过程伴随多种杂酸包括乳酸、缬氨酸、亮氨酸等的产生,必将影响目的产物的产量。【结论】研究结果对于进一步采取措施优化溶氧的控制策略,提高目的产物的产量具有指导意义。     

Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae are known to exhibit oscillatory behavior in the oxidative region. Important findings of a series of experiments conducted to identify the causes for initiation of and the means for elimination of oscillations in these cultures are reported in this paper. These oscillations are seen to be connected to the growth kinetics of the microorganism and are induced at very low glucose concentrations and at dissolved oxygen (DO) levels that are neither high nor low (DO values between 20 and 78% air saturation at a dilution rate of 0.2 h(-1) and pH of 5.5 at 30 degrees C). The oscillatory behavior is encountered over a range of dilution rates (0.09-0.25 h(-1) at 30 degrees C for pH = 5.5 and DO = 50% air saturation). The oscillations can be eliminated by raising the DO level above a critical value or by lowering the DO level below a critical value.     

Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.