On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Utility of culture redox potential for identifying metabolic state changes in amino acid fermentation

We investigated the relationship of dissolved oxygen and culture redox potential (CRP) on amino acid production. Corynebacterium glutamicum ATCC 14296 was used for all experiments. The fermentation can be divided into a growth phase and a production phase. Our results indicate that in order to get higher amino acid production, a lower oxygen supply during the exponential phase is favored. A higher oxygen supply rate appears to be necessary during the production phase. Culture redox potential (CRP) was used to monitor the fermentation. CRP readings were observed to drop to a characteristic minimum value as the metabolic state changed from a growth to production phase. This was evidenced by the commencement of amino acid production and a simultaneous uptake of lactate. Upon lactate exhaustion, the CRP increased abruptly. At the same time, maximal amino acid yields were observed. By the use of minimum CRP as an indication of metabolic phase changes, the agitation rate was changed to increase oxygen supply during the production phase. This significantly increased amino acid production. These results show that culture redox potential measurements can be used to monitor and optimize amino acid production by process manipulation.     

Studies on the effect of changing the environments of paddy roots on the uptake of zinc,manganese and iron

Summary The results of the present investigation revealed that the magnitude of the potential is high in the case of oxygen gas treatment and low potential has been observed under hydrogen gas treatment. The uptake of Zn is reduced under hydrogen and hydrogen plus nitrogen gas treatments. The plants growing under reduced condition take up less Zn than the plants growing under drought or upland condition. The uptake of Fe and Mn fall gradually as we proceed from hydrogen to oxygen gas treatment in the nutrient solution. The lowest uptake of Fe and Mn have been observed under oxygen gas treatment for all the varieties. re]19760102     

Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration

Cells of Escherichia coli O157:H7 were heat-treated at 59 °C and enumerated in (i) anaerobic medium with a low redox potential, (ii) anaerobic media with the oxidizing agents potassium ferricyanide or 2,6-dichloroindophenol (DPIP) added to raise the redox potential, (iii) aerobic medium with a high redox potential and (iv) aerobic medium with the reducing agent dithiothreitol added to lower the redox potential. The measured heat-resistance was greatest when the enumeration medium was highly anaerobic due to the absence of oxygen and the presence of hydrogen and cysteine HCl. Measured heat resistance was influenced by the redox potential of the enumeration medium independently of the chemical used to adjust it and therefore, independently of the presence of oxygen. Sub-lethally heat-damaged cells regained their ability to grow in media of high redox potential at a similar rate whether the redox potential was increased by the addition of potassium ferricyanide, DPIP or oxygen.     

Photoproduction of hydrogen by adapted cells of Chlorella pyrenoidosa

Photoproduction of hydrogen gas by the green alga Chlorella pyrenoidosa was studied in a large scale culture of 2.1. Hydrogen was produced by adding sodium hydrosulfite directly to an algal suspension after anaerobiosis in darkness for activation of hydrogenase. The hydrogen production rate showed a characteristic course of an initial burst of gas then steady production, and this course appeared most clearly at cell concentrations around 0.6–0.7 kg/m³. In the final third phase, the hydrogen production rate gradually decreased until evolution ceased. The steady hydrogen evolution was inhibited 75% by a herbicide, DCMU, which blocks electron flow through photosystem II, indicating that the electron donor for hydrogen production was mainly water. The average light intensity within the culture vessel was measured with a diffusing sphere photoprobe. The rate of hydrogen evolution increased hyperbolically with the average light intensity. The duration of hydrogen photoproduction was shorter at higher light intensity due to the inhibition of hydrogenase by concomitantly released oxygen. The duration was shorter also at higher concentrations of algal suspension. It was foudd that the optimum concentration of algae, about 0.7 kg/m³ in this system, must be selected to maximize the yield of hydrogen.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

Effect of hydrogen acceptors on D-xylose fermentation by anaerobic culture of immobilized Pachysolen tannophilus cells

The effect of hydrogen acceptors on the kinetic parameters of D-xylose fermentation under anaerobic conditions was studied in a transient culture of immobilized Pachysolen tannophilus cells. Addition of oxygen to a steady-state culture resulted in a rapid increase (up to fivefold) in the rates of ethanol production and D-xylose uptake, but the rate of xylitol production was unaffected. Furthermore, the molar ethanol yield increased from 0.97 to 1.43 in the presence of oxygen. The moles of ethanol produced per moles of oxygen utilized were considerably greater than would be predicted from the stoichiometry of D-xylose fermentation, which suggests that the organism required oxygen for other functions in addition to its role as a hydrogen acceptor in D-xylose metabolism. When the artificial hydrogen acceptors acetone, acetaldehyde, and acetoin were added to the culture, the rate of ethanol production increased while the xylitol production rate decreased but the rate of xylose uptake was unaffected. The molar ethanol yields increased from 1.03 to 1.63, 1.43, and 1.24 upon addition of acetaldehyde, acetone, and acetoin, respectively, at the expense of the molar xylitol yields. The hydrogen acceptors sodium acetate, methylene blue, benzyl viologen, phenazine methosulfate, indigo carmine, and tetrazolium chloride had no effect on ethanol production.     

Oxygen Uptake In Cysts and Trophozoites of Giardia Lamblia

ABSTRACT. Oxygen uptake in cysts and trophozoites of the parasitic protozoan Giardia lamblia was examined. Both showed oxygen uptake activity, but that of cysts was only 10% to 20% that of trophozoites. Oxygen dependence of oxygen uptake in cysts and trophozoites showed oxygen maxima above which oxygen uptake decreased. the oxygen concentration at which the oxygen uptake rate was greatest was higher for trophozoites than for cysts. the effect of various inhibitors on cyst and trophozoithe oxygen uptake suggested that flavoproteins and quinones play some role in oxygen uptake. the substrate specificities and the effect of inhibitors on G. lamblia trophozoites were similar to those observed for G. muris. Metronidazole, the drug most commonly used in treatment of giardiasis, inhibited oxygen uptake and motility in trophozoites; however, it had no obvious effect on either oxygen uptake or excystation in cysts. Menadione, a redox cycling naphthaquinone, first stimulated, then completely inhibited, oxygen uptake in cysts and trophozoites; a complete loss of cyst viability and trophozoite motility was also observed. the effect of menadione on G. Iamblia may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.     

Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

固氮蓝藻高光放氢突变种的筛选和放氢特点

作者以前报道过几种快速生长的固氮蓝藻在某种条件下能好气光放氢,其速度可以达到光合放氧速度的10—15%,但这种活性只有在不积累氢气的流动气相下或在短时间内发生。本文报道用亚硝基胍诱变所得到的Anabaena spp。Strain CA的高光放氢突变种——N9A和18A——的筛选和氢代谢特点。在达生长饱和光照以后,野生型的光放氢活性与光照强度的增加成正相关,而其吸氢活性则与之成负相关,显示高光照强度可能抑制吸氢酶的活性。无论在什么光强下,均测不到两个突变种的吸氢活性,暗示在突变种中,吸氢酶或有关系统受损伤。把细胞固相化在琼脂上,在密闭系统中,高光强下培养50个小时,两个突变种光释放和积累的氢分别为野生型的2倍(N9A)和6倍(18A),后者等于氢占气相(1%CO_2的空气)的1.8%。两个突变种在生长速度、叶绿素含量、乙炔还原活性以及光合放氧方面与野生型无明显不同。当以含50—100nM的镍离子的培养基培养时,野生型的好气净产氢活性完全丢失,其吸氢活性却增加约10倍。培养基中镍离子的存在,对两个突变种的高光放氢活性则毫无影响,而且在此情况下,仍测不出其吸氢活性。实验结果表明,这两个突变种系吸氢酶缺陷型突变种。     

Efficient Hydrogen photoproduction by synchronously grown cells of a marine cyanobacterium, Synechococcus sp. Miami BG 043511, under high cell density conditions

The capability of hydrogen photoproduction under high cell density conditions was examined using synchronously grown cells of nitrogen-fixing Synechococcus sp. Miami BG 043511. Optimum hydrogen yield was obtained when vessels (25 ml) contained 0.2 to 0.3 mg chlorophyll (a) in 3-mL cell suspension. During a 24-h incubation period, an initial phase of hydrogen and carbon dioxide production and a subsequent phase of carbon dioxide uptake and oxygen accumulated as major products after 24 h. after the initial 24-h. After the initial 24-h incubation, as high as 7.4 and 3.7 L (at standard condition) of hydrogen and oxygen, respectively, accumulated in vessels with 22-ml gas phase. This indicated that the pressure in the flask increased to 1.5 atmosphere. Energy conversion efficiency based on photosynthetically active radiation (25 W/m(2)) was about 2.6%. However, increased pressure somehow reduced the duration of hydrogen production. Duration of hydrogen and oxygen production was prolonged by periodical (24-h interval) gas replacement during incubation. (c) 1994 John Wiley & Sons, Inc.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough)

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.     

Influence of redox potential on the glucose catabolism of chemostat grownBacillus licheniformis

Summary The effect of the redox potential on the by-production of acetic acid byBacillus licheniformis grown in a chemostat has been studied. It was demonstrated thatB. licheniformis produced acetic acid when it was grown at a low growth rate, low oxygen supplies, and low redox potentials.Furthermore, it was shown that the acetic acid productivity was strongly dependent on redox potential, giving a maximum productivity at redox potentials about 110 mV. The observed effect was correlated to the established theory of the dependence of intracellular enzymes on oxygen and redox potential. It is proposed that the redox potential can be a variable that can be used in a regulatory procedure.     

Physico-chemical properties of aqueous solutions, prepared in a membrane electrolyzer

The physicochemical properties of aqueous solutions resulting from membrane electrolysis were studied. It was shown that the catholyte contains hydrogen peroxide at a concentration of 10(-7), which is formed during the reduction of soluble oxygen. It was found that the relaxation of the catholyte redox potential is caused by the transition of the reducing agent to the gaseous phase. The relaxation characteristics of the redox potentials of the catholyte and molecular hydrogen solution were compared. The similarity of the relaxation characteristics of the catholyte and the hydrogen solution as well as the fact that the catholyte, despite its low redox potential, does not reduce either potassium ferricyanide or 5-5'-dithiobis(2-nitrobenzoic acid) support the suggestion that the redox potential of the catholyte is due to molecular hydrogen. However, based on this suggestion, it is impossible to explain the increase in the relaxation time of the catholyte with increasing ionic strength and the fact that, as the redox potential of the catholyte decreases, the concentration of other gases dissolved in the catholyte remains unchanged. Thus, the question regarding the nature of the reducing agent remains open.     

Whole-cell fluorescence for observing reductive metabolism from carbon compounds and hydrogen inAzotobacter vinelandii

The change in fluorescence of intactAzotobacter vinelandii was observed to study the oxidation and reduction of flavin and pyridine nucleotides resulting from carbon and hydrogen metabolism. Metronidazole, acetaldehyde, and oxygen each oxidized flavin. Flavin oxidized by metronidazole or acetaldehyde was reduced by addition of mannitol or ethanol, but not by acetate or hydrogen. The fluorescence induced by oxygen was transient. Mannitol, ethanol, acetate, acetaldehyde, and hydrogen shortened the duration of the oxygen-dependent fluorescence and supported respiration. The changes in redox state of pyridine nucleotides corresponded to the changes in flavin redox state. This indicates that the use of reducing equivalents from uptake hydrogenase is limited to the respiratory electron transport system inAzotobacter vinelandii.     

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.     

Inhibition of astrocyte glutamate uptake by reactive oxygen species: role of antioxidant enzymes.

BACKGROUND: The recent literature suggests that free radicals and reactive oxygen species may account for many pathologies, including those of the nervous system. MATERIALS AND METHODS: The influence of various reactive oxygen species on the rate of glutamate uptake by astrocytes was investigated on monolayers of primary cultures of mouse cortical astrocytes. RESULTS: Hydrogen peroxide and peroxynitrite inhibited glutamate uptake in a concentration-dependent manner. Addition of copper ions and ascorbate increased the potency and the efficacy of the hydrogen peroxide effect, supporting the potential neurotoxicity of the hydroxyl radical. The free radical scavenger dimethylthiourea effectively eliminated the inhibitory potential of a mixture containing hydrogen peroxide, copper sulphate, and ascorbate on the rate of glutamate transport into astrocytes. The inhibitory effect of hydrogen peroxide on glutamate uptake was not altered by the inhibition of glutathione peroxidase, whereas the inhibition of catalase by sodium azide clearly potentiated this effect. Superoxide and nitric oxide had no effect by themselves on the rate of glutamate uptake by astrocytes. The absence of an effect of nitric oxide is not due to an inability of astrocytes to respond to this substance, since the same cultures did respond to nitric oxide with a sustained increase in cytoplasmic free calcium. CONCLUSION: These results confirm that reactive oxygen species have a potential neurotoxicity by means of impairing glutamate transport into astrocytes, and they suggest that preventing the accumulation of hydrogen peroxide in the extracellular space of the brain, especially during conditions that favor hydroxyl radical formation, could be therapeutic.     

Influence of some aliphatic alcohols on the metabolism of rat liver slices

The influence of some aliphatic alcohols on oxygen uptake, carbon dioxide production, acid formation and lactate and pyruvate concentrations of rat liver slices was studied. At the concentrations used, none of the alcohols was found to influence oxygen uptake. Of the alcohols that are not oxidized by liver alcohol dehydrogenase, methanol increased carbon dioxide production, propan-2-ol decreased it and 2-methylpropan-2-ol was without influence. All the alcohols that are oxidized by the enzyme strongly decreased carbon dioxide production. The alcohols that are not oxidized had no marked effect on the lactate/pyruvate concentration ratio, whereas the other alcohols strongly increased the ratio. A highly significant correlation was found between the effects of the alcohol on pyruvate concentration and carbon dioxide production. It is assumed that the shift in the redox potential inhibits the function of the tricarboxylic acid cycle of the liver.     

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.)

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H₂O₂. The cells recover and start producing ATP immediately upon the cessation of H₂O₂ production. This indicates that the increased O₂ uptake is primarily incorporated into active O₂ species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M_r 40 000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.