Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Neuromedin U causes biphasic cardiovascular effects and impairs baroreflex function in rostral ventrolateral medulla of spontaneously hypertensive rat

Neuromedin U (NMU) causes biphasic cardiovascular and sympathetic responses and attenuates adaptive reflexes in the rostral ventrolateral medulla (RVLM) and spinal cord in normotensive animal. However, the role of NMU in the pathogenesis of hypertension is unknown. The effect of NMU on baseline cardiorespiratory variables in the RVLM and spinal cord were investigated in urethane-anaesthetized, vagotomized and artificially ventilated male spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats (WKY). Experiments were also conducted to determine the effects of NMU on somatosympathetic and baroreceptor reflexes in the RVLM of SHR and WKY. NMU injected into the RVLM and spinal cord elicited biphasic response, a brief pressor and sympathoexcitatory response followed by a prolonged depressor and sympathoinhibitory response in both hypertensive and normotensive rat models. The pressor, sympathoexcitatory and sympathoinhibitory responses evoked by NMU were exaggerated in SHR. Phrenic nerve amplitude was also increased following intrathecal or microinjection of NMU into the RVLM of both strains. NMU injection into the RVLM attenuated the somatosympathetic reflex in both SHR and WKY. Baroreflex sensitivity was impaired in SHR at baseline and further impaired following NMU injection into the RVLM. NMU did not affect baroreflex activity in WKY. The present study provides functional evidence that NMU can have an important effect on the cardiovascular and reflex responses that are integrated in the RVLM and spinal cord. A role for NMU in the development and maintenance of essential hypertension remains to be determined.     

The effect of oscillating dissolved oxygen concentrations on the metabolism of a Spodoptera frugiperda IPLB-Sf21-AE clonal isolate

The effect of oscillating dissolved oxygen (DO) concentration on the metabolism of a clonal isolate of the Spodoptera frugiperda IPLB-Sf21-AE insect cell line was investigated. Specifically, the effect on cell growth, re- combinant protein synthesis, glucose and glutamine consumption, and lactate accumulation was determined. Prior to conducting the oscillating DO experiments, it was found that the DO concentration could be reduced to 15% air saturation without adversely affecting the growth rate. Under these conditions, glucose and glutamine became depleted as the maximum cell density was reached. The introduction of DO oscillations, that is, cycles consisting of 30 min at 15% DO followed by 30 min of anoxia, significantly altered cell metabolism, including inhibition of cell growth and recombinant protein synthesis. The effect of DO oscillations on glucose consumption was dependent on the experimental conditions. Glucose exhaustion occurred when the DO oscillations contained either an "apparent" anoxia period (nitrogen sparging discontinued upon reaching 0% DO) without pH control or a "true" anoxia period (nitrogen sparging continued throughout anoxia period) with pH control. Glucose consumption was significantly decreased, however, when the cells were exposed to a "true" anoxia period without pH control, that is, low pH inhibited glucose utilization. Glutamine uptake was not significantly affected by DO oscillations. Lactate only accumulated in the oscillating DO runs, a finding consistent with previous results demonstrating that significant lactate accumulation only occurs under DO-limited conditions. (c) 1995 John Wiley & Sons, Inc.     

Novel role of the anorexigenic peptide neuromedin U in the control of LH secretion and its regulation by gonadal hormones and photoperiod

Neuromedin U (NMU) is a widely spread neuropeptide, with predominant expression at the gastrointestinal tract and brain, putatively involved in the regulation of a diversity of biological functions, including food intake, energy balance and circadian rhythms; all closely related to reproduction. Yet, the implication of NMU in the control of the gonadotropic axis remains scarcely studied. We report herein analyses on the hypothalamic expression and function of NMU in different physiological and experimental states of the rat reproductive system. Expression of NMU mRNA at the hypothalamus was persistently detected along female postnatal development, with maximum levels in adulthood that fluctuated across the cycle and were modulated by ovarian steroids. Acute central administration of NMU evoked increases of serum LH levels in pubertal female rats, while repeated injection of NMU tended to advance vaginal opening. Likewise, central injection of NMU increased serum LH concentrations in cycling female rats, with peak responses in estrus. In contrast, NMU significantly inhibited preelevated LH secretion in gonadectomized and kisspeptin-treated rats. Finally, in noncycling females due to photoperiodic manipulation (constant light), hypothalamic NMU mRNA levels were markedly depressed, but relative LH responses to exogenous NMU were significantly augmented. All together, our present data support a predominant stimulatory role of NMU in the control of the female gonadotropic axis, which appears under the influence of developmental, hormonal, and photoperiodic cues, and might contribute to the joint regulation of energy balance, biological rhythms, and reproduction.     

An approach for prediction of optimum reaction conditions for laccase-catalyzed bio-transformation of 1-naphthol by response surface methodology (RSM)

Response surface methodology (RSM) was successfully applied to enzymatic bio-transformation of 1-naphthol. The experiments were conducted in a closed system containing acetone and sodium acetate buffer, with laccase enzyme. Laccase enzyme used as catalyst was derived from Trametes versicolor (ATCC 200801). The enzymatic bio-transformation rate of 1-naphthol, based on measurements of initial dissolved oxygen (DO) consumption rate in the closed system, was optimized by the application of RSM. The independent variables, which had been found as the most effective variables on the initial DO consumption rate by screening experiments, were determined as medium temperature, pH and acetone content. A quadratic model was developed through RSM in terms of related independent variables to describe the DO consumption rate as the response. Based on contour plots and variance analysis, optimum operational conditions for maximizing initial DO consumption rate, while keeping acetone content at its minimum value, were 301 K of temperature, pH 6 and acetone content of 7% to obtain 9.17 x 10(-3) mM DO/min for initial oxidation rate.     

Short-cut nitrification of domestic wastewater in a pilot-scale A/O nitrogen removal plant

The feasibility of nitrite accumulation in a pilot-scale A/O (anoxic/oxic) nitrogen removal plant treating domestic wastewater was investigated at various dissolved oxygen (DO) concentrations and pH levels. The results showed that the pH was not a useful operational parameter to realize nitrite accumulation. Significant nitrite accumulation was observed at the low DO concentration range of 0.3–0.8 mg/l and the maximum nitrite accumulation ratio of about 90% occurred at a DO concentration of 0.6 mg/l. This suggests a reduction of 22% in the oxygen consumption, and therefore a considerable saving in aeration. However, the nitrite accumulation was destroyed at the high DO concentration and the resumption was very slow. In addition, the average ammonia removal efficiency reached as high as 93% at the low DO level. Moreover, experimental results indicated that nitrogen could be removed by simultaneous nitrification and denitrification (SND) via nitrite in the aerobic zones at the low DO concentration, with the efficiency of 6–12%.     

DO,pH, and Eh microprofiles in cyanobacterial granules from Lake Taihu under different environmental conditions

To understand characteristics of cyanobacterial granules from Lake Taihu, dissolved oxygen (DO), pH, and redox potential (Eh) microelectrodes were used to investigate physiological responses within these granules under different irradiance, temperature, and external pH levels. DO and pH levels increased with rising irradiance, while the Eh had an opposite trend. High light combined with high temperature decreased photosynthesis of the cyanobacterial granules. DO diffused from the surrounding water to the granules at low irradiance; however, DO began to diffuse from the granules to the water at high irradiance owing to increased photosynthesis. Dynamic changes of DO, pH, and Eh levels existed within the cyanobacterial granules under light–dark cycles. High DO levels within intercellular space of the cyanobacterial granules are another important buoyancy regulation mechanism. An external initial pH affected photosynthesis of the cyanobacteria in the granules. DO and pH levels of the granules in slightly alkaline solution (pH 8–9) were higher than those in highly alkaline solution (pH 10). Such physical and chemical characteristics within cyanobacterial granules in eutrophic water allowed them to outcompete other aquatic algae. The characterization of the physiological microenvironment within these cyanobacterial granules provides a new research approach to a better bloom management.     

Neuromedin U and Neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status

Neuromedin U (NMU) has been associated with the regulation of food-intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor-2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter-mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.     

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here.     

Effect of N-nitroso-N-methylurea on viability and mutagenic response of repair-deficient strains of Escherichia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N-nitroso-N-methylurea (NMU).Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA^? strains was not affected by NMU concentrations up to 3.0 mM. Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA^? while strains carrying uvrA^?polA^? or recA^? were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks.Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 mM the numbers of revertants induced in exrA^? carrying strains, prossessing accurate rec-dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.     

Distribution of neuromedin U receptor subtype 2 mRNA in the rat brain

Neuromedin U (NMU) is a family of peptides found in the gut and the central nervous system [Neuroscience 25 (1988) 797; Biochem. Biophys. Res. Commun. 130 (1985) 1078]. While several peripheral activities such as uterus stimulating and hypertensive effects have been described for NMU [Biochem. Biophys. Res. Commun. 130 (1985) 1078], its role in the CNS remains poorly understood. Recently, we reported the identification of two receptors for NMU (NMU1R and NMU2R), and demonstrated that NMU may play a role in regulating feeding behavior. The central effect of NMU is likely mediated primarily via NMU2R, since NMU1R is detectable only in the periphery, but not in the brain [Nature 406 (2000) 70]. In this report, we describe detailed mapping of NMU2R mRNA expression in the rat brain by in situ hybridization. The most intense signals were observed in the ependymal cell layer along the wall of the third ventricle in the hypothalamus, CA1 region of the hippocampus, indusium griseum and septohippocampal nucleus. Moderate expression was detected in the hypothalamic paraventricular nucleus, dorsal raphe nucleus as well as a number of other brain structures. The presence of NMU2R in the hypothalamus is consistent with its role in energy balance. Significant levels of expression of NMU2R elsewhere in the brain may suggest additional physiological functions for this neuropeptide.     

Operational parameters affecting the performannce of a mediator-less microbial fuel cell

A mediator-less microbial fuel cell was optimized in terms of various operating conditions. Current generation was dependent on several factors such as pH, resistance, electrolyte used, and dissolved oxygen concentration in the cathode compartment. The highest current was generated at pH 7. Under the operating conditions, the resistance was the rate-determining factor at over 500 omega. With resistance lower than 500 omega, proton transfer and dissolved oxygen (DO) supply limited the cathode reaction. A high strength buffer reduced the proton limitation to some extent. The DO concentration was around 6 mg l(-1) at the DO limited condition. The fact that oxygen limitation was observed at high DO concentration is believed to be due to the poor oxygen reducing activity of the electrode used, graphite. The current showed linear relationship with the fuel added at low concentration, and the electronic charge was well correlated with substrate concentration from up to 400 mg l(-1) of COD(cr). The microbial fuel cell might be used as a biochemical oxygen demand (BOD) sensor.     

On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.     

pH, Redox, and oxygen microprofiles in rhizosphere of bulrush (Scirpus validus) in a constructed wetland treating municipal wastewater

Microenvironmental studies regarding plant oxygen release in a wastewater environment are important to understand the principles of constructed wetlands for wastewater treatment. pH, oxidation reduction potential (ORP), and dissolved oxygen (DO) microprofiles for the lateral and main roots of the bulrush (Scirpus validus) in a vertical flow constructed wetland fed with municipal wastewater were measured using microelectrode techniques. pH was found to be low (6.91-6.98) near the lateral root surface, indicating possible nitrification or H(+) extrusion. The ORP at the lateral root surface was between +250 and +317 mV and gradually reached the bulk solution ORP (+14 to -54 mV) at a radial distance of approximately 4,750 microm. DO values at the lateral root surface varied from 0.64-2.04 mg L(-1) as bulk biochemical oxygen demand (BOD) changed from 24 to 1,267 mg L(-1). DO at the lateral root surface and the thickness of the oxygen layer around the root marginally increased with an increase in bulk BOD, while ORP at the lateral and main root surface decreased. pH and DO values did not change near the main root and had the bulk solution values. The results of this study provide insights into root-induced microenvironments and would be helpful for the quantification of the total amount of oxygen contributed by plants in constructed wetlands.     

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。     

Fumaric acid fermentation with immobilised Rhizopus oryzae: Quantifying time-dependent variations in catabolic flux

A novel fermenter system utilising immobilised Rhizopus oryzae is presented. The impact of dissolved oxygen (20%, 60% and 80%) and pH (4 and 5) was investigated. All fermentations exhibited three distinct phases. Phase A, at the start, was associated with no fumarate production, minimal respiration and ethanol as the major product. Phase B was characterised by the onset of fumarate production and significant ethanol and respiration fluxes. Phase C was associated with zero ethanol flux. Inhibition of fumarate production was more severe at low pH. The DO 20% fermentation (pH 5) had a low respiration flux which resulted in excessive ethanol production. Higher DO levels resulted in less inhibition of fumarate production during phase C. Instantaneous fumarate yields on glucose were at a maximum at the start of phase C, with values in excess of 0.75 g g⁻¹ achieved for the DO 60% and 80% fermentations.     

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (q(O2) and q(CO2), respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate q(O2), q(CO2) and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), q(O2) and q(CO2) ranges were 5.14-5.77 and 5.31-6.36 pmol/cell day, respectively, resulting in RQ values of 0.98-1.14. Changes to DO had a slight reducing effect on respiration rates with q(O2) and q(CO2) values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with q(O2) and q(CO2) values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective q(O2) and q(CO2) values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in q(O2) and q(CO2) values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real-time q(O2) and q(CO2) estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing.     

Staphylococcal Enterotoxin B and Nuclease Production Under Controlled Dissolved Oxygen Conditions

Enterotoxin B, nuclease, and total exoprotein production by Staphylococcus aureus strain S-6 was studied in a 0.5-liter fermentor system. While these extracellular products were elaborated over a wide range of aeration rates, maximal production occurred within the very narrow range of 125 to 150 cm(3) of air per min. The levels attained at the optimal aeration rate were not increased by maintaining a constant pH, although yield of enterotoxin:cell mass was highest at a constant pH of 7.0. During the growth cycle of the cultures, when aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen (DO) levels, initially set at 100% of saturation, decreased to 5 to 10% 4 to 5 h after inoculation. The oxygen demand of the culture then maintained this level for an additional 4 to 6 h. This interval of low DO was characterized by maximal growth and exoprotein production. When the DO was controlled at a constant value throughout growth (by increasing or decreasing the airflow rate as appropriate), the culture demonstrated different optima for maximal growth and exoprotein production. A constant DO of 100% stimulated growth to extremely high densities, but the accumulation of toxin and nuclease was not observed. On the other hand, maintaining constant DO levels at 50 or 10% raised exoprotein levels higher than those achieved in a culture grown at the optimal aeration rate. Compared to the optimal aeration rate culture, the 10% DO culture yielded 20% more nuclease, 25% more toxin, and 40 to 50% more total exoprotein. These results indicate that it is the DO and not the aeration rate, per se, that is influential in controlling growth, toxin, nuclease, and total exoprotein production.     

Combined dissolved oxygen and pH control strategy to improve the fermentative production of l-isoleucine by Brevibacterium lactofermentum

The effect of both dissolved oxygen (DO) and pH on l-isoleucine production by batch culture of Brevibacterium lactofermentum was investigated. A two-stage agitation speed control strategy was developed, and the isoleucine production reached 23.3 g L⁻¹ in a relative short time (52 h), increased by 11.6% compared to the results obtained in the single agitation speed control process. In order to make sure whether the combination of DO and pH control can boost the production by a mutual effect, different control modes were conducted, based on the data obtained from the two-stage agitation speed control strategy and the analysis of kinetics parameters at different pH values. The results showed that the mode of combining two-stage DO with two-stage pH control strategy was the optimal for isoleucine production. The isoleucine production can reach 26.6 g L⁻¹ at 56 h, increased by 14.3% comparing to that obtained by the single two-stage DO control strategy.