Effect of amino acids on the heat production and growth efficiency of Streptococcus bovis: balance of anabolic and catabolic rates.

Streptococcus bovis JB1 grew nearly twice as fast (0.9 versus 1.6 h-1) and had a 40% greater growth yield (18 versus 12.5 mg of protein per mmol of glucose) when an ammonia-based medium was supplemented with amino acids, but the glucose consumption rate (88 mumol mg of protein-1 h-1) and specific rate of heat production (2.1 mW/mg of protein) were unaffected. Amino acid availability had little effect on the catabolic rate, but the specific heat decreased 40% (8.8 to 5.2 J/mg of protein). These growth rate-dependent changes in metabolic efficiency were fivefold greater than the maintenance energy. Chloramphenicol (100 mg/l), an inhibitor of protein synthesis, caused a gradual decrease in anabolic (growth) rate, but there was little change in the rate of glucose consumption and the specific heat increased. When growth was inhibited by iodoacetate, the catabolic and anabolic rates both declined and there was not increase in specific heat. On the basis of these results, the benefit of amino acid supplementation was largely explained by the balance of anabolic and catabolic rates. When amino acids were available, the anabolic and catabolic rates were more closely matched and less energy was spilled as heat.     

Specific heat flow rate: an on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy

One of the requirements for enhanced productivity by the animal culture systems used in biotechnology is the direct assessment of the metabolic rate by on-line biosensors. Based on the fact that cell growth is associated with an enthalpy change, it is shown that the specific heat flow rate is stoichiometrically related to the net specific rates of substrates, products, and indeed to specific growth rate, and therefore a direct reflection of metabolic rate. Heat flow rate measured by conduction calorimetry has a technical advantage over estimates for many material flows which require assays at a minimum of two discrete times to give the rate. In order to make heat flow rate specific to the amount of the living cellular system, it would be advantageous to divide it by viable biomass. This requirement has been fulfilled by combining a continuous flow microcalorimeter ex situ with a dielectric spectroscope in situ, the latter measuring the viable cell mass volume fraction. The quality of the resulting biosensor for specific heat flow rate was illustrated using batch cultures of Chinese hamster ovary cells (CHO 320) producing recombinant human interferon-gamma (IFN-gamma) during growth in a stirred tank bioreactor under fully aerobic conditions. The measuring scatter of the probe was decreased significantly by applying the moving average technique to the two participant signals. It was demonstrated that the total metabolic rate of the cells, as indicated by the specific heat flow rate sensor, decreased with increasing time in batch culture, coincident with the decline in the two major substrates, glucose and glutamine, and the accumulation of the by-products, ammonia and lactate. Furthermore, the specific heat flow rate was an earlier indicator of substrate depletion than the flow rate alone. The calorimetric-respirometric ratio showed the intensive participation of anaerobic processes during growth and the related IFN-gamma production. Specific heat flow rate was monotonically related to specific cell growth rate and associated with specific IFN-gamma production. Specific heat flow rate is potentially a valid control variable for the growth of genetically engineered cell lines producing target proteins.     

Kinetic study on pH dependence of growth and death of Streptococcus faecalis

A chemostat culture was used for lactic acid fermentation with Streptococcus faecalis at various pH values (8.0, 7.0, 6.0, 5.5, 5.0) and glucose concentrations (10, 20, 30 g/l). At every pH value, the reciprocals of the specific consumption rate of glucose and the specific production rate of lactic acid were linearly correlated to the reciprocal of the specific growth rate. The product, lactic acid, caused non-competitive inhibition of the specific growth rate at every pH value. Moreover, it was found that the cell death rate was dependent on pH and lactic acid. The death rate was smallest at pH 7.0 and increased with increasing lactic acid concentration. The kinetic equations of growth and death are proposed in a broader pH range. Correspondence to: H. Ohara     

Monitoring of morphological development of the arachidonic-acid-producing filamentous microorganism Mortierella alpina

Morphological parameters, such as hyphal growth rate, tip formation rate, tip extension rate and branch formation rate, of Mortierella alpina have been measured using a flow-through chamber under 25 different combinations of carbon and nitrogen concentrations. Morphological parameters were influenced not by C/N ratio but by carbon concentration in the medium. Specific rates of hyphal growth and tip formation both remained constant at a low carbon concentration of 5 g/l. Tip extension rate from one tip was 60 microm tip(-1) h(-1) at a carbon concentration below 15 g/l, and the branching formation rate was independent of carbon concentration. Tip extension rate was a function of specific hyphal growth rate, which in turn was linearly proportional to the specific tip formation rate, demonstrating that tip extension rate was exponentially proportional to the specific tip formation rate. Branch formation rate per hyphal element remained unchanged even at tip extension rates lower than 60 microm tip(-1) h(-1) and at specific hyphal growth rates lower than 0.83 h(-1), but decreased drastically at higher rates of tip extension and hyphal growth.     

Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.     

The heat generated by yeast cultures with a mixed metabolism in the transition between respiration and fermentation

The heat generated by both batch and continuous cultures of the yeast K. fragilis was studied using a modified Bench Scale Calorimeter. Batch cultures were used to measure the heat dissipation rates and the heat yields during fully aerobic and completely anaerobic growth, whereas continuous cultures enabled, in addition, a quantitative study of heat dissipation rates during growth on mixed metabolism. In this case, the extent of fermentation versus respiration could be specified and controlled by varying the degree of oxygen limitation. The heat dissipated per unit biomass formed was highest for fully respirative catabolism and fell continuously to a much lower value typical of anaerobic cultures as the catabolism was shifted increasingly to the fermentative mode. The heat generated per mole of oxygen taken up stayed quite close to the fully aerobic value of 506 kJ mol(-1) even when a sizable fraction of the substrate available to catabolism was fermented. If the fraction of respiration in the metabolism is lowered beyond a certain threshold, the ratio of the heat generation to oxygen consumption starts to increase dramatically and finally tends to infinity for fully anaerobic growth. All experimental results were quantitatively analyzed and explained on the basis of a simple model which formally describes the cultures in terms of two parallel "chemical" reactions. In simple cases such as the one presented here, the model enables calculation of the whole stoichiometry of the culture from a single measured heat yield.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Calorimetric analysis of Escherichia coli in continuous culture

The heat evolution rate was measured in continuous culture of Escherichia coli k-12 on glucose-limiting medium. The specific heat production rate based on the dry cell mass increased in proportion to increase of dilution rate. The specific heat production rate based on a viable cell increased nonlinearly because the cell size varied in each dilution rate. Several thermochemical data were calculated for each dilution rate from the stoichiometric equation which was derived from elementary analysis of the cells. Growth yields based on energy measured directly was found to be less influenced by dilution rate than the growth yield based on consumed glucose. This suggested that the energy efficiency of utilization of catabolic energy for anabolism was almost constant.     

Effect of specific production rate of recombinant protein on multimerization of plasmid vector and gene expression level

In fed-batch cultures of recombinant Escherichia coli BL21(DE3)[pT7-G3IL2] at high cell concentration, the post-induction specific growth rate was carefully regulated by controlled medium feed to maximize the synthesis level of recombinant fusion interleukin-2, G3.IL-2. A maximum concentration of G3.IL-2 (11.25 g l(-1)) was achieved in the induced recombinant culture growing at the rate of 0.056 h(-1). A steep decrease in the expression level of G3.IL-2 was observed at the post-induction specific growth rates higher than its optimal value (0.056 h(-1)). In the induced recombinant cultures, plasmid multimerization was observed and highly dependent on specific growth and production rate: a higher post-induction specific growth rate and an increased specific production rate tended to significantly promote it much further. Moreover, plasmid stability was found to decrease rapidly in a faster growing culture.     

An algorithmic approach to constructing the on-line estimation system for the specific growth rate

The objective of this article is to propose an algorithm for the on-line estimation of the specific growth rate in a batch or a fed-batch fermentation process. The algorithm shows the practical procedure for the estimation method utilizing the macroscopic balance and the extended Kalman filter. A number of studies of the on line estimation have been presented. However, there are few studies discussing about the selection of the observed variables and for the tuning of some parameters of the extended Kalman filter, such as covariance matrix and initial values of the state.The beginning of this article is devoted to explain the selection of the observed variable. This information is very important in terms of the practical know-how for using technique. It is discovered that the condition number is a practically useful and valid criterion for number is a practically useful and valid criterion for choosing the variable to be observed.Next, when the extended Kalman filter in applied to the online estimation of the specific growth rate, which is directly unmeasurable, criteria for judging the validity of the estimated value from the observed data are proposed. Based on the proposed criterial, the system equation of the specific growth rate is selected and initial value of the state variable and covariance matrix of the system noises are adjusted. From many experiments, it is certified that the specific growth rate in the batch or fed -batch fermentation can be estimated accurately by means of the algorithm proposed here. In these experiments, that is, when the cell concentration is measured directly, the extended Kalman filter using the convariance matrix with a constant element can estimate more accurately values of the specific growth rate than the adaptive extended Kalman filter does.     

Dependence of penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity

The influence of the agitation conditions on the growth, morphology, vacuolation, and productivity of Penicillium chrysogenum has been examined in 6 L fed-batch fermentations. A standard Rushton turbine, a four-bladed paddle, and a six-bladed pitched blade impeller were compared. Power inputs per unit volume of liquid, P/VL, ranged from 0.35 to 7.4 kW/m3. The same fermentation protocol was used in each fermentation, including holding the dissolved oxygen concentration above 40% air saturation by gas blending. The mean projected area (for all dispersed types, including clumps) and the clump roughness were used to characterize the morphology. Consideration of clumps was vital as these were the predominant morphological form. For a given impeller, the batch-phase specific growth rates and the overall biomass concentrations increased with agitation intensity. Higher fragmentation at higher speeds was assumed to have promoted growth through increased formation of new growing tips. The mean projected area increased during the rapid growth phase followed by a sharp decrease to a relatively constant value dependent on the agitation conditions. The higher the speed, the lower the projected area for a given impeller type. The proportion by volume of hyphal vacuoles and empty regions decreased with speed, possibly due to fragmentation in the vacuolated regions. The specific penicillin production rate was generally higher with lower impeller speed for a given impeller type. The highest value of penicillin production as well as its rate was obtained using the Rushton turbine impeller at the lowest speed. At given P/VL, changes in morphology, specific growth rate, and specific penicillin production rate depended on impeller geometry. The morphological data could be correlated with either tip speed or the "energy dissipation/circulation function," but a reasonable correlation of the specific growth rate and specific production rate was only possible with the latter. Copyright 1998 John Wiley & Sons, Inc.     

Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2

Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications.     

氮、磷营养盐对底栖硅藻的生长及生化组成影响

目的:研究了从福建连江海域分离得到的一株野生底栖硅藻(Nitzschia.constricta)的生长和生理特性.方法:测定了在不同氮磷浓度条件下比增长速率、色素,以及不同时期底栖硅藻蛋白和胞外多糖的积累情况.结果:当硝酸氮浓度为900mg/l时,获得最大比增长速率0.21和叶绿素a含量4.31mg/l、类胡萝卜素含量3.44mg/l、胞外多糖产率90.57μg/ml.当氮浓度为75mg/l时,在第10d得到最大蛋白产率28.90μg/ml.当磷浓度为4.4mg/l时,可得到最大比增长速率0.13、叶绿素a含量3.42mg/l、类胡萝卜素2.82mg/l、蛋白含量23.16μg/ml,无磷培养基中第5d的胞外多糖产率最高13.51μg/ml.结论:氮磷营养盐浓度的增加促进了此种底栖硅藻的生长,但不一定会促进蛋白及胞外多糖的产生.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg).

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.     

Inhibition of Candida valida growth by copper ions]

The high toxicity of copper ions for Candida valida growth was established at pH-auxostat regime. The value of mu max decreased even at the residual Cu2+ concentration 1.0 mg/l. The inhibition constant (Ki) that characterized a copper ion concentration at which yeast specific growth rate was halved was equal to 7.7 mg/l. A linear dependence of 1/mu max on a residual concentration of copper ions indicates that yeast growth inhibition is due to inhibition of one enzymic reaction which is the most sensitive to copper. Yeast growth inhibition by copper was accompanied by accumulation of Cu2+ ions in biomass, a decrease in nucleic acid and true protein contents, and changes in amino acid composition of protein. The amounts of cystine and cysteine in protein increased and tryptophane content decreased with inhibition of yeast growth. Yeast growth inhibition by copper did not affect the lipid content but significantly reduced the degree of unsaturation due to a decrease in the amounts of polyunsaturated linoleic and alpha-linolenic acids.     

Growth kinetics of gel-immobilized yeast cells studied by on-line microscopy

A microscope reactor was used to study online the dynamics of gel immobilized cell systems. The applicability of the reactor is demonstrated by a study of the growth kinetics of Saccharomyces cerevisiae entrapped in 2% calcium alginate. The specific growth rates of single immobilized cells and free cells were measured. The growth of a microcolony in Ca-alginate was followed and the specific growth rate of the cells in the microcolony determined. A simple growth model was used to estimate the cell volume fraction of the yeast cells in the microcolony. As internal and external mass transfer limitations can be neglected and immobilized cell growth rates were found to be identical to those of free cells, one may conclude that immobilization does not influence cell growth under our experimental conditions.     

Erythritol production by controlling osmotic pressure in Trigonopsis variabilis

Specific erythritol production rate by Trigonopsis variabilis increased from 0.09 to 0.19 g/g-day by varying the osmotic pressure from 1.3 to 3.9 kPa in glucose medium, but the specific growth rate decreased from 0.28 to 0.13 h -1 . Therefore, osmotic pressure was adjusted to 1.4 kPa during growth phase and to 3.7 kPa during production phase in a two-stage fermentation. Erythritol reached 46 g/ l in such a system and was twice that obtained in one-stage fermentation.     

Kinetic analysis of the growth of Chlorella vulgaris

The energy of the total transmitted light was subtracted from that of the incident light in a culture vessel and the difference was divided by the weight of cells. The value thus obtained was defined as the amount, E(x), of light energy absorbed per unit cell weight per unit time.Batch and continuous cultures of Chlorella vulgaris were carried out at 30 degrees C in the pH range of 6.4-6.7 while restricting illumination. Next the specific growth rate, mu, in the batch culture and the fixed dilution rate, D, in the continuous culture were plotted against E(x). The results showed that the relation between D and E(x) can be expressed in a Michaelis-Menten equation, where the maximal specific growth rate is 0.24 h (-1) and the saturation constant is 6.58 kcal/g . h.Cell concentration calculated by substituting the apparent concentration, X(e), of incubated cells and the apparent maintenance constant, M(e), for this equation agreed with that observed in almost all growth phases. Furthermore, from the change of chlorophyll productivity and the relationship between D and E(x) expressed in this equation, it is assumed that E(x) involves the light energy directly utilized in photosynthesis in the cells and that which is converted into, e.g., heat. This equation also indicated that a maximum in the growth yield existed. Then the growth yield of 0.029 g/kcal obtained at the incident light of 1.46 or 2.63 cal/cm(2) . h was maximum (maximal conversion efficiency of light energy, 15.6%).These results indicate that this method of deriving the equation for the growth rate from this study is a useful procedure for obtaining bioengineering findings.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.