Showing papers in "Biotechnology and Bioengineering in 1968"

A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates

Abstract: A mathematical model is presented for both batch and continuous cultures of microorganisms utilizing inhibitory substrates. The key feature of the model is the use of a inhibition function to relate substrate concentration and specific growth rate. Simulation studies show that the primary result of inhibition by substrate in a batch culture is an increase in the lag time whereas in continuous culture inhibition by substrate may result in process instability. The model should be of value in investigations of the stability of biological processes used for the treatment of certain industrial wastes such as those containing phenols, thiocyanates, nitrates, ammonia, volatile acids, etc., which are known to be inhibitory to many of the organisms metabolizing them.

Response of biological waste treatment systems to changes in salt concentrations

Abstract: The effect of NaCl on the yield of biological solids and on the ability of continuously cultured heterogeneous microbial populations to remove substrate was assessed by changing the salt concentration in the inflowing synthetic waste. During the period of increasing the salt concentration to 30,000 mg/l the system could not maintain a high substrate removal efficiency. However, after an acclimation period the system regained its former efficiency. Upon diluting the salt out of the system, a significant rise in cell yield was noted as the salt level passed through the range 8,000–10,000 mg/l. It was found that steady operation at a salt level of 8,000 mg/l sustained the cell yield at a high level.

Automatic analysis of gas exchange in microbial systems

Abstract: An automatically working test arrangement for the permanent analysis of O2 and CO2 in microbiological cultures is described. The measuring principle is based on the paramagnetic properties of oxygen and on the absorption of infrared by carbon dioxide. The preparation of the gas for measuring and the correction of the recording are indicated. The formula of correction was programmed and the values were calculated for a range of 3%. The routine correction of analysis values is done with a nomogram established on the basis of these individual values. The advantages of the described test arrangement are illustrated by two examples of growth experiments on Saccharomyces cerevisiae.

Kinetics of gas diffusion in mammalian cell culture systems. II. Theory

Abstract: Mathematical models have been constructed which relate the depth of the culture fluid overlay to the oxygen available to mammalian cells cultured under static conditions. These models suggest that the maintenance of a given rate of oxygen utilization by some culture systems may be critically depended on this fluid depth and on the solubility and rate of diffusion of oxygen in the culture fluid. The importance of these concepts as applied to the isolation and growth of differentiated cells representative of the tissue of origin are noted.

The role of aeration and agitation in the production of glucose oxidase in submerged culture. II

Abstract: The main purpose of the work reported here was to establish the effectiveness of aeration and agitation, and to determine the best conditions of aeration for the growth and production of glucose oxidase of Aspergillus niger, on a semi-industrial scale. Concentration of dissolved O2, O2 consumption and CO2 production were measured. It was found that the rate of growth and the activity of glucose oxidase per gram mycelium increased with the increase of speed of agitation. The concentration of dissolved oxygen of the fermentation broth, as well as the rate of respiration (O2 consumption and CO2 production) increased in direct proportion to the increase of speed of agitation, while assimilation of sugars was accelerated. The values of the respiratory ratio showed a fluctuation according to the presence or absence of sugar in the medium.

Kinetics of microbial cell growth

Abstract: As a rate equation of microbial cell growth, the Monod equation is widely used. However, this equation cannot fully correspond to real courses of microbial cell growth in many batch cultivations. Especially, predicted values based on this equation do not agree with observed values in many continuous cultivations. In this paper, which introduces new concepts of critical concentration and coefficient of consumption activity, the growth rate equation which corresponds to the whole period including lag period is newly derived and characteristics of microbial cell growth in batch cultivation are clarified. Further, applying the new rate equation to continuous cultivation, a general equation with which to calculate cell concentration is derived and characteristics of microbial cell growth in continuous cultivation are clarified. The calculated values of cell concentration based on the new theory showed quite good agreement with the observed values in both batch and continuous cultivation.

Mathematical representation of batch culture data

Abstract: A generalized logistic equation is proposed for the mathematical representation of batch culture kinetic data. Properties of the equation are discussed. A computer program is used to fit the generalized equation to both artificial and actual batch culture data. The equation is shown to be capable of fitting data exhibiting lag, exponential, deceleration, stationary, and death phases, as well as diauxic growth. The fitted equation is useful for differentiation, interpolation, and other manipulations of the data, and it is a convenient means of data storage.

Kinetics of gas diffusion in mammalian cell culture systems. I. Experimental

Abstract: It has been demonstrated experimentally that the thickness of fluid overlay in conventional tissue culture systems limits the oxygen available to mammalian cells growing as a submerged monolayer. A rocker culture system is described which circumvents critical problems associated with thin film culture while permitting nearly unlimited access of oxygen to the cell monolayer. Good growth of primary hepatic cells as isolated sheets has been obtained.

Toxic spiroepoxy compounds from Fusaria and other hyphomycetes

Abstract: Many hyphomyceteous fungi have been found to produce closely related toxic metabolites which form the class of compounds called scirpenes. The structures of those compounds are reviewed and current studies on their biological activity and possible implication in moldy corn poisoning are discussed.

Continuous cultivation of Saccharomyces cerevisiae. I. Growth on ethanol under steady‐state conditions

Abstract: Growth of Saccharomyces cerevisiae LBG H 1022 on ethanol under steady-state conditions was studied. As a cultivation device, an aerated Chemap fermentor combined with continuously working gas analyzers for oxygen and carbon dioxide was used. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release, and respiration quotient, as well as nitrogen, carbon, phosphorus, hydrogen, and protein content of the cells were measured in dependence on the dilution rate. Cell size distribution, as a function of the specific growth rate, was determined with the aid of a Celloscope 202. A fair agreement with the theory of continuous culture for all metabolic curves could be established. An increased turnover rate resulted from the addition of glutamic acid to the synthetic growth medium. The primary effect of this supplement could be a rise in the flow rate of the tricarboxylic acid cycle.

A multisurface tissue propagator for the mass‐scale growth of cell monolayers

Abstract: Various multisurface mass-scale tissue culture propagators ranging in capacity up to 200 liters have been developed at Abbott Laboratories. These patented units consist, of an enclosed vessel containing a multiplicity of separated glass plates or disks on which cells may attach and proliferate. Means for mixing and aeration of the medium are provided. Sample ports facilitate the addition of cultures and media, the withdrawal of samples, the washing of cell monolayers, and the harvesting of cells and cell products. The large cell growth surface per occupied volume, the provision for separating tissue cells from media simply and easily, and the minimization of the amount of labor required per cell growth area are some of the many advantages of the multisurface tissue propagator that are described.

Oxygen diffusion through zoogloeal flocs

Abstract: The mechanism of oxygen transfer through a pure culture floc of Zoogloea ramigera I-16M has been described quantitatively. Oxygen uptake rates for both blended and nonblended floc particles indicated that, at a certain dissolved oxygen concentration, diffusion of oxygen through the floc matrix was the mechanism controlling the rate of oxygen utilization by the floc. This mechanism was quantitatively described by determining the oxygen diffusivity values for the floc. The diffusional distances of the floc particles along with the oxygen utilization rates of the floc were measured on floc grown under various conditions. Anoxic core equations were then used to calculate the oxygen diffusivity values for each experiment. These diffusivity values were then used to estimate the oxygen concentrations necessary in activated sludge plants.

Simultaneous gas–liquid interfacial oxygen absorption and biochemical oxidation

Abstract: Two experimental methods were used in measuring oxygen-transfer rates. The results indicate that two possible mechanisms are operating in simultaneous gas–liquid interfacial oxygen absorption and biochemical oxidation. One of the mechanisms, the direct absorption mechanism, has not been much studied in bioengineering. Mathematical equations are derived to describe the simultaneous mechanisms.

Production of fatty acids and lipid by a Candida sp. growing on a fraction of n‐alkanes predominating in tridecane

Abstract: A Candida sp. was grown on a fraction of n-alkanes (dodecane 22%, tridecane 48%, tetradecane 28%) as sole carbon source. The growth rate was increased most markedly by using high concentrations of n-alkanes (16.7% v/v). When grown in a 5 liter fermentor, the yeast reached its highest yield (60 g. of cell dry wt/l) with a concomitant high yield of fatty acids (21 g of fatty acids/l), by using a nitrogen-deficient medium. To achieve good growth, it was essential to use an inoculum (1 part into 10) of rapidly growing cells and beneficial to increase the agitation rate gradually once growth had begun. After 108 hr maximum conversions of substrate to product were: 71.5% (w/w) for alkanes into cells and 24.8% (w/w) for alkanes into fatty acids. Of the, total fatty acids at the end of the fat-accumulating phase of growth 54% were shorter in chain length than palmitic acid (C16H32O2). When grown on glucose, as sole carbon source, less than 2% of the total fatty acids were shorter than palmitic acid. When n-alkanes were added to cells growing on glucose, short-chain fatty acids (C10 to C14) were synthesized immediately, indicating a derepressed enzyme system for hydrocarbon assimilation and the absence of diauxie. The production of these acids was at the apparent sacrifice of linoleic acid synthesis. In spite of the high conversion ratios, it is concluded that it would be uneconomical to produce fatty acids, even expensive ones such as lauric acid, by microbial transformation of n-alkanes.

Virus interactions with membrane filters

Abstract: A survey of interactions of membrane filters with viruses has included 28 types of membranes, 4 types of enteroviruses, and 1 reovirus. Losses of these viruses in filtration, due to adsorption to the filter membranes, appear to be governed by three factors: the chemical composition of the filter membrane, the ratio of pore diameter to the diameter of the virus particle, and the presence of substances, such as those occurring in serum, which interfere with adsorption. Membranes of cellulose triacetate and of certain other materials have a very low affinity for these viruses. Cellulose triacetate filters adsorb virtually none when the pore size exceeds the virus diameter by a factor of more than 3. At porosities nearer the virus diameter, even low‐affinity membranes adsorb large quantities of virus unless serum or some other additive interferes. Cellulose nitrate membranes, in the absence of interfering substances, adsorb enterovirus significantly at a pore size 285 times the virus diameter.

Sterilization with gaseous ethylene oxide: A review of chemical and physical factors

Abstract: Although the basic parameters for ethylene oxide sterilization are established, it is sometimes difficult to attain in practice where the principal limiting factor is moisture availability. There are situations which can limit or enhance the dynamics of sterilization. Such factors, if overlooked, could upset experiments and lead to erroneous conclusions, or defeat the sterilization process entirety. Such are, namely: stratification effects, diffusion barriers, moisture-reducing effects, polymerization, and temperature distribution gradients.

A pilot plant for mammalian cell culture

Abstract: Studies of the possible viral etiology of human leukemia have required large quantities of cultured cells derived from human hematopoietic tissues. Since cultures sufficiently large and free from contamination could not readily be produced according to existing methods, a pilot, cell culture plant has been constructed for the production of mammalian cells in mass quantity. 500‐ml to 20‐liter trophocell units have already proved to be scientifically and economically practical, as they provide good reliability, excellent growth rates, and sustained yield of human cells. 200‐liter stainless steel culture units have now been added to the trophocell system. Five complete 200 liter units are now in operation. The design of the original stainless steel unit was based on that of a stainless steel, jacketed soup kettle. There are no openings in the vessel other than those in the lid, which provide convenient access points for sampling, sensor probes, etc. Environmental parameters, e.g., liquid level, temperature, and pH, are monitored and controlled with commercially available apparatus. Many initial problems connected with the new 200 liter units have been resolved, but operational and design problems remain in the areas of stable instrumentation, cell harvesting, salvaging and reuse of unspent media components, establishment of physiologic steady stale, recovery of virus‐containing cells with reculture of the remaining unaffected cells, and the recovery and separation of cell components and special products such as immunoglobulins, interferons, and hormones. A definitive cell plant with culture units of 20, 50, 250, and 1250 liters is now being constructed.

Transient response of continuous cultures to changes in temperature

Abstract: The purpose of this experimental study was to examine the transient response of a chemostat-type continuous culture of Escherichia coli B to step changes in temperature by following transient limiting substrate concentration and calculating from it the transient growth rate. The transient response to step changes of temperature was tested for four different situations. In the first two cases, temperature was shifted down from 37 to 27°C., and 37 to 32°C. In the last two, it was shifted up from 32 to 37°C., and 27 to 37°C. When the temperature was shifted up, the growth rate increased rather rapidly to its transient maximum value and then decreased slowly until it, settled back into the steady-state value. On the other hand, when the temperature was shifted down, the growth rate decreased relatively rapidly to its transient minimum and then it slowly increased and returned gradually to the steady-state value. The magnitude of the transients was less than would be expected if the transient growth rates followed an Arrhenius function.

Microflotation: Coagulation and foam separation of aerobacter aerogenes

Abstract: The foam separation technique of microflotation was studied using A. aerogenes as the test organisms. Gas flow rate, collector and alum (when used) concentrations, and frother dose were held constant. In contrast to E. coli, previously reported, A. aerogenes are removed using both lauric acid and laurylamine as the collectors without prior coagulation with alum. The removals are improved after coagulation, with laurylamine being the more efficient collector. In all cases the removals decrease upon increasing the pH above about 8.

Cultivation of the yeast candida lipolytica on hydrocarbons. I. Degradation of n‐alkanes in batch fermentation of gas oil

Abstract: The growth of batch‐cultivated yeast Candida lipolytica on three kinds of gas oil using mineral medium was studied. A linear dependence was found between the production of yeast biomass and the consumption of n‐alkanes, while the decrease of freezing point of gas oil during cultivation had a distinct course. This disproportion was explained by different degradation of individual n‐alkanes contained in gas oil. The rate of degradation of pentadecane, hexadecane, and heptadecane was the same during the entire cultivation. On the contrary, in the first phase the utilization of shorter chain n‐alkanes, nonane to tetradecane, was more rapid while that of longer chain homologs, octadecane to pentacosane, lagged. Rapid utilization of longer chain n‐alkanes did not occur before the concentration of the other n‐alkanes decreased. Only then the rapid decrease of freezing point appeared.

Continuous cultivation of Saccharomyces cerevisiae. II. Growth on ethanol under transient-state conditions

Abstract: Growth of Saccharomyces cerevisiae LBG H 1022 on ethanol under transient-state conditions was studied. As a cultivation device, an aerated Chemap fermentor combined with continuously working gas analyzers for oxygen and carbon dioxide was used. Yeast cell dry matter, substrate concentration, specific oxygen uptake, specific carbon dioxide release, and respiration quotient were measured during the different transient states. Depending on which range of the dilution rate the initial steady state was found, we obtain different responses to the shift experiment. For the lower range, up to D = 0.07, we deal with damped oscillations ranging above and below the steady-state values. For the higher specific growth rates, the rate of damping is strongly enhanced and the shape of the curves becomes an asymptotic approach to the final steady states.

The influence of dissolved oxygen partial pressure on the level of various enzymes in mouse LS cells

Abstract: The intracellular levels of seven enzymes in mouse LS cells growing in suspension culture at controlled dissolved oxygen partial pressures (pO2) have been measured. During the growth of each culture large fluctuations were observed in the levels of some enzymes, particularly aldolase and cytochrome oxidase. Mean values for the concentration of each enzyme during the growth phase have been calculated. These results are discussed in relation to previous observations made on the growth of mouse LS cells.

Application of a multiple surface tissue culture propagator for the production of cell monolayers, virus, and biochemicals

Abstract: A new concept of tissue culture equipment and procedures was developed for the mass-scale growth of several types of animal tissue cells in monolayers on multiple glass surfaces. Continuous, cell lines, primary and diploid cell strains were grown in this equipment. Cells studied include primary bovine kidney, human diploid WI-38, human foreskin, and mouse CCL1 cells. Photomicrographic comparisons of cells grown by these techniques indicate they are morphologically identical to tissue culture cells grown in glass bottles or tubes. The growth of the tissue culture cells in the propagator was monitored by carbohydrate Utilization and acid production. Large-scale production of viruses and biochemicals on cells grown in the multiple-plate tissue culture propagator was accomplished. Virus titers were equal to those obtained from conventional bottle or tube cultures for several strains of influenza, parainfluenza, and respiratory syneytial viruses. High-titred mouse interferon was also produced in this system. In addition to tissue culture cell production, Eaton agent, Mycoplasma pneumoniae was grown on the multiple glass surfaces on a mass scale.

Emetic material associated with Fusarium species in cereal grains and artificial media

Abstract: Substances which cause emesis in pigeons were extracted from corn (Zea mays) artificially inoculated with Fusarium graminearum and from liquid culture medium inoculated with F. moniliforme, F. roseum, F. poae, F. culmorum, and F. nivale. Emetic preparations were obtained also from infected wheat (Triticum aestivum L. em. Thell), (Hordeum vulgare L. em. Lam), and durum (Triticum durum Desf). Partial purification resulted from chromatography with columns of cellulose and DEAE cellulose and with thin layers of silica gel. Two active materials were obtained from liquid culture of F. moniliforme but only one from infected cereals. Emetic preparations from F. moniliforme and infected cereals contained a polypeptide as a minor component. Ultraviolet and infrared spectrums, elemental analyses, refractive indices, and amino acid composition of the emetic from corn and one of the emeties from liquid culture of F. moniliforme were similar but not identical. Attempts to crystalline these emetics and to characterize them were unsuccessful.

A mathematical model for the batch reactor kinetics of algae growth

Abstract: A mathematical model based on the Einstein law of photochemical equivalence is proposed to describe the batch growth of unicellular algae. The model was applied in an integrated form to cell concentration versus growth time data taken over an extended range of cell concentrations which include both the regions of “exponential” and “linear” growth. It is shown that a certain function of cell concentration contained in the integrated form of the model is linearly dependent on the growth time over both the “exponential” and “linear” growth regions.

Studies on ventilation of culture broths. I. Behavior of CO2 in model systems

Abstract: The rate of dissolution and dehydration of CO2 in a liquid model system was investigated. Components in the model system established the main conditions which may exist, in the extracellular space of a microbiological culture liquid. The charge in voltage of a glass electrode was measured which indicated the formation of H+ ions in the H2CO3 ⇌ HCO 3− H+ reaction. The rate of CO2 hydration increased with the increase of temperature from 0 to 40°C. Likewise the equilibrium of the reaction was shifted towards the forward reaction. Similar results were observed when the tip velocity of the impeller was increased. Data suggest that agitation promotes the dissolution of CO2 in the culture liquid through the reduction of gas‐liquid film resistance in the diffusion of this gas. The rate of hydration of CO2 into the bulk of the liquid was independent of pCO2 above the surface of the liquid but depended on pCO2 in the gas bubble within the liquid. The concentration of HCO 3− was, furthermore, influenced by the buffer components, buffer capacity, and the viscosity of the system. Since pCO2 and the HCO 3− concentration in the extracellular space depend on both physical and chemical factors, the ventilation of a culture liquid necessitates both exhaust of CO2 from the gas bubbles of the culture broth and shift of the H2CO3 ⇌ HCO 3− + H+ reaction towards the backward direction.