Continuous cultivation of Hydrogenobacter thermophilus at low partial pressures of H

Growth of the thermophilic H-oxidizing bacterium Hydrogenobacter thermophilus (ITI 553) was achieved in continuous culture using a gas-lift bioreactor. Although the highest equilibrium cell density was obtained at a dilution rate of 0.05 h, the maximum productivity was obtained at 0.1h. At 0.05 h, the maintainable cell density was 7.10 cells/mL at 2-4% oxygen with a gas flow rate of 0.1vvm. The steady-state cell density increased further with increasing gas flow rates. Maximum specific activity of the peroxidase was obtained at 8% oxygen. The total productivity of the enzyme had its maximum at 6-8% oxygen. Increasing gas flow rates had hardly any effect upon peroxidase specific activity, however, productivity was increased due to the increased cell densities obtained.     

Studies on a gram-positive hydrogen bacterium,Nocardia opaca strain 1b

Summary A new hydrogen bacterium has been isolated by enrichment culture on propane. It is a strictly aerobic, Gram-positive, non acid-fast bacterium, characterized by filamentous growth, and has been tentatively assigned to Nocardia opaca (strain 1 b).It grows heterotrophically, on many organic compounds (71 out of 138 tested substrates including organic acids and sugars), on hydrocarbons (C₁₁–C₁₈) as well as under autotrophic conditions (under an atmosphere of hydrogen, oxygen, and carbon dioxide=8:1:1) In the absence of a nitrogen source storage materials, mainly carbohydrates, are accumulated.Hydrogenase is an inducible enzyme. Under appropriate growth conditions the specific hydrogenase activity reaches high values: 2700 enzyme units/g cell protein. The formation of hydrogenase is repressed by fructose. With increasing oxygen concentrations during growth the specific hydrogenase activity decreases. In resting cell oxygen progressively inhibits the oxyhydrogen reaction.Cell-free extracts of autotrophically grown cells are able to reduce oxygen benzyl-and methyl viologen, dichlorphenolindophenol, methylene blue and nicotinamide adeninedinucleotide with hydrogen.     

Determination of maintenance coefficients of Saccharomyces cerevisiae cultures with cell recycle by cross-flow membrane filtration

The fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single-stage process with recycle of the cells via cross-flow micro-filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.     

Growth kinetics and cellulase biosynthesis in the continuous culture of Trichoderma viride.

Continuous culture studies have been carried out growing Trichoderma viride QM 9123 in a 10 liter stirred fermentor on a medium containing commercial glucose as the carbon source. Experiments were carried out at 30 degrees C and at three controlled pH values of 2.5, 3.0, and 4.0 over a range of dilution rates from 0.01 to 0.11 hr-1. Steady-state values of cell, glucose, and cellulase concentration oxygen tension, and outlet gas oxygen partial pressure were recorded. Values of maximum specific growth rate, endogenous metabolism coefficient, Michaelis-Menten coefficient, yield and maintenance coefficient for glucose were derived and correlated the effect of the hydrogen ion concentration. Specific oxygen uptake rates were correlated with specific growth rates and absorption coefficients were shown to be a function of dilution rate independent of pH. Some data on cellulase biosynthesis were examined and correlated in terms of a maturation time model.     

Growth kinetics of glucose-limited petunia hybrida cells in chemostat cultures: Determination of experimental values for growth and maintenance parameters

With glucose-limited continuous cultures of Petunia hybrida six steady states were obtained at specific growth rates varying from 0.0035 to 0.012 h(-1) (corresponding with culture residence times varying from 285 to 85 h). The macromolecular and the elemental biomass composition which were determined in four steady states showed no major differences over the range of growth rates examined. During all six steady states specific subtrate and oxygen consumption as well as biomass and extracellular product formation rates were monitored. Moreover the specific activities of the mitochondrial cytochrome and alternative pathway were determined and used to estimate specific adenosine triphosphate (ATP) production rates. Data thus obtained were used in the determination of maintenance and true growth yield parameters. For the maintenance on glucose and ATP values of 0.0070 C-mol/C-mol/h and 0.034 mol/C-mol/h were obtained, respectively. True yields of biomass on glucose and ATP were 0.50 C-mol/C-mol and 0.28 C-mol/mol, respectively. (c) 1995 John Wiley & Sons, Inc.     

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.     

Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.     

Monitoring Growth of Microorganisms using the Methods of Mass-Energy Balance and Utilization of Computer on Line with Fermenter in Real Time Processing (in Russian)

The effective means of microbial culture monitoring is the measurement of low-inertial parameters (respiration rate, rates of supply of alkali for pH maintenance and the limiting substrate) and utilization of computer on line with fermenter for recalculation of these rates into the instant values of mass and energy cell yields, specific rates of cell growth and substrate and oxygen consumption, using the method of mass-energy balance. In this paper, the equations of mass-energy balance are presented both in general form and in the form of numerical algorythms for computer programming. The installation for automation of microbial cultivation experiment is described. Experimental data are presented which indicate the effectiveness of the method of indirect measurement of cell biomass yield and specific rates of physiological processes.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.     

Acclimation of the photosynthetic response of Chromatium vinosum to light-limiting conditions

The photosynthetic response of the purple sulfur bacterium Chromatium vinosum DSM 185 to different degrees of illumination was analyzed. The microorganism was grown in continuous culture, and samples were taken from the effluent of the culture and incubated at different irradiances to determine the specific rate of sulfur oxidation as a measure of the photosynthetic activity of the organism. The activities obtained were plotted as a function of the specific rate of light uptake, and for each set of data a photosynthesis equation was fitted, which allowed the estimation of P_max (photosynthetic capacity), q_k (the threshold irradiance for light limitation), and m (maintenance coefficient). The results indicated that cells grown under light limitation are able to achieve higher photosynthetic activities than cells grown under light saturation. The photosynthetic capacity (P_max) remained constant under all the conditions of illumination tested, while the maintenance expenses (m) were higher under light limitation. The parameter q_k, on the contrary, decreased considerably at limiting irradiances. Received: 16 January 1998 / Accepted: 7 September 1998     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH. Reprinted from Biotechnology and Bioengineering, Vol. 32, Pp 947-965 (1988)

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli.

Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation.     

Microaerobic hydrogen production by photosynthetic bacteria in a double-phase photobioreactor

The rate of hydrogen production by the marine nonsulfur photosynthetic bacterium, Rhodovulum sp., increased with increasing light intensity. A light intensity of 1800 W/m(2) hydrogen production rate was achieved at the rate of 9.4 micromol/mg dry weight/h. The hydrogen production of this strain was enhanced by the addition of a small amount of oxygen (12 micromol O(2)/reactor). Intracellular ATP content was most efficiently accumulated under microaerobic, dark conditions. Hydrogen production rate by Rhodovulum sp. was investigated using a double-phase photobioreactor consisting of light and dark compartments. This rate was compared with data obtained using a conventional photobioreactor. Rhodovulum sp. produced hydrogen at a rate of 0.38+/-0.03 micromol/mg dry weight/h under microaerobic conditions using the double-phase photobioreactor. The hydrogen production rate was four times greater under microaerobic conditions, as compared with anaerobic conditions using either type of photobioreactor. Hydrogen production using a double-phase photobioreactor was demonstrated continuously at the same rate for 150 h.     

Fed-batch culture: Modeling and application in the study of microbiol energetics

Microbial growth in the fed-batch mode is described by a simple unstructured model. The model is found to be in good agreement with agreement with the experimental observation, except under highly transient conditions. Extensive experimental data were collected and the energetics of the bacterium Klebsiella pneumoniae is evaluated. It is shown that the fed-batch culti vation is a powerful experimental tool in the study of microbial kinetics and energetics simultaneously. Methods for determining the maintenance requirements are shown and evaluated. The maintenance coefficients determined from fed-batch data are systematically smaller than those reported for continuous culture systems. Results suggest a decrease in maintenance demands at low specific growth rates.     

Monoclonal antibody productivity and the metabolic pattern of perfusion cultures under varying oxygen tensions

The metabolic pattern and cell culture kinetics of high-cell-density perfusion cultures were compared under two different oxygen transfer conditions: oxygen limiting and not limiting. When oxygen was a limiting factor during perfusion culture, both specific glucose uptake and lactate production rates increased, compared to non-oxygen-limited condition, by about 60% and 30%, respectively. The specific glutamine uptake rate under oxygen-limited conditions was almost 4.0 times higher than that under non-oxygen-limited conditions. The activity of lactate dehydrogenase (LDH) released into the medium by the dead cells can be used as an indicator for the metabolic and physiological conditions related to oxygen limitation. There was a 3.2 times higher specific rate of LDH activity released by dead cells in oxygen-limited cultures than those in non-oxygen-limited cultures. The specific production rate of monoclonal antibody was not significantly affected by the oxygen transfer conditions during the rapid cell growth period, but it rapidly increased toward the end of perfusion cultures. The higher perfusion rate may have limited further cell growth during high-cell-density perfusion culture, because cell damage was caused by the hydrodynamic shear within a hollow fiber microfiltration cartridge installed to withdraw the spent medium and the waste metabolites. (c) 1993 John Wiley & Sons, Inc.     

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.