Showing papers in "Biotechnology and Bioengineering in 1969"

Dynamic behavior of the chemostat subject to substrate inhibition

Abstract: A theoretical study was made on the dynamic behavior of a single-vessel continuous fermentation subject to a growth inhibition at, high concentration of the rate limiting substrate. Phase plane plotting and stability analysis showed three steady states to exist; namely, a “washout”; state, a nontrivial stable state, and an unstable state. Whether the system attains a nontrivial steady state or is to be washed-out depends upon the initial values of the cell and/or substrate concentration(s). Since this property is a characteristic feature of the present system, an experimental procedure was suggested to insure a stable operation in practice.

The kinetics of β‐galactosidase attached to porous cellulose sheets

Abstract: The properties of β-galactosidase attached to cellulose and DEAE-cellulose sheets arc described. Those insoluble enzyme derivatives obey the Michael-Menten relationship but, the measured kinetic parameters are very dependent on the flow conditions. The results of long-term stability tests are given.

Microprobe techniques for determining diffusivities and respiration rates in microbial slime systems

Abstract: A microprobe electrode was used to determine dissolved oxygen concentrations near the surface and within a bacterial slime mass supplied with a continuous flow of nutrient solution. With dilute medium, the oxygen profile became level at high concentrations within the film, indicating substrate-limited respiration. More concentrated medium caused the profile to fall to low oxygen concentrations characteristic of oxygen-limited respiration. Oxygen responses to sudden changes in concentration of nutrient medium were measured. Estimates of microbial respiration rate and of diffusivity of oxygen were based on well-known diffusion equations.

Studies of fungal growth and intermediary carbon metabolism under steady and non‐steady state conditions

Abstract: The physiology of Aspergillus nidulans strain 224 has been studied under conditions of batch‐ and glucose‐limited chemostat‐culture and the effect of different steady state growth rates and dissolved oxygen tensions (DOT) examined. Measurements of the specific activities of selected glucose enzymes, the extent of oxygen uptake inhibition by glycolytic inhibitors, and radiorespirometric analyses were made in order to follow the variations in glucose catabolism, which occurred under these conditions. Greatly increased activity of the hexosemonophosphate (HMP) pathway was found during: (i) exponential growth of batch cultures; (ii) at near maximum specific growth rates (μ = 0.072 hr−1) (DOT = 156 mm Hg); and (iii) at low DOT levels (<30 mm Hg) (μ = 0.050 hr−1) in chemostat cultures. These changes in glucose eatabolism have been discussed in terms of the biosynthetic demands of the fungus under the influence of changing growth pressures. Preliminary studies also have been made of transition state behavior following stepwise alteration of the DOT. A new steady state was established after 4–5 culture doublings during which period an “overshoot” in HMP pathway activity occurred; these kinetics are indicative of a derepression of certain glucose enzymes. Low molecular weight phenols are synthesized during the exponential phase in batch cultures and these are further metabliized to a major secondary metabolite, melanin, at the onset of stationary phase conditions. The kinetics of tyrosinase production in steady state chemostats differs from those that might be predicted for an enzyme associated solely with secondary metabolism. A primary physiological role for this oxidase in Aspergillus nidulans has been postulated.

The conversion of benzyl penicillin to 6-aminopenieillanie acid using an insoluble derivative of penieillin amidase

Abstract: Penicillin amidase was extracted from Escherichia coli ATCC 9637, grown on phenylacetic, acid and glutamate, and purified by fractional ion with streptomycin sulphate, ammonium sulphate and polyethylene glycol, followed by chromatography on DEAE–cellulose. The purification factor was 100–200 × and the overall yield was about 115%. The enzyme was chemically attached to derivatives of cellulose and the kinetics of these insolubilized penicillin amidase preparations was investigated.

Homogeneous cultivation of animal cells for the production of virus and virus products

Abstract: Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 107 plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.

Investigation of some methods for increasing the digestibility in vitro of microalgae.

Abstract: In order to increase the availability of the cell bound protein in Scenedesmus algae, mechanical, enzymatic, and chemical methods of degrading the cell wall structure were investigated. Mechanical treatment involved the use of a ball-mill. The algae suspension together with glass beads was milled in a water-cooled chamber equipped with rotating disks. The enzyme tested was a cellulolytic enzyme (Meicelase) and the chemical employed was hydrogen peroxide. In the ball-mill experiments a complete disintegration was achieved ina disintegrator, working with batches. Trails wwere also performed with a continuous disintegrator and the depedence of disintegration on bead size and flow rate was studied. The disintegration determined by microscropic cell count was compared to the increase of the pepsin digestibility. The meicelase treatment caused a slight increase of the pepsin digestibility, as measured after 3 hr pepsin incubation. No increase of the pepsin disgestibility could be detected with hydrogen peroxide treatment. After the ball-mill disintegration 95% of contaminating bacteria were killed and yields of extractable proteins were higher. The capacity of availble continuous ball-mills is such that they could be used on a pilot-plant scale and the energy cost of disintegration would be of the same magnitude as that of separation.

Continuous enzymatic saccharification of cellulose with culture filtrates of trichoderma viride QM 6a.

Abstract: Continuous saccharification of Solka Floc (cellulose pulp) in single and four-vessel stirred-tank reactor systems has been possible employing enzymes obtained directly from submerged fermentation of Trichoderma viride QM 6a. Studies on the effect of modification of the solid substrate, enzyme stability, substrate concentration, and the influence of reducing sugar concentration on the rate of hydrolysis are reported. While susceptibility of substrate to digestion is not affected by heating alone, it is strikingly increased by heating plus grinding, or by grinding following heating. Batch and steady state continuous saccharification experiments have yielded more than 5% reducing sugar in the effluent with a dilution rate of 0.025 hr−1 at 50°C, at a substrate level of 10%. An average glucose concentration of 3.4% has been obtained in the effluent of a continuous saccharification using 5% substrate at the same dilution rate and temperature.

Preparation and use of insolubilized amyloglucosidase for the production of sweet glucose liquors

Abstract: Amyloglucosidase (EC. 3.2.1.3), partially purified from an Aspergillus species, was chemically attached to DEAE cellulose using the bifunctional reagent 2-amino-4,6-dichloro-s-triazine. The action of the insolubilized enzyme derivative on dilute maltose and dextrin solutions was studied in a packed bed. A second and deeper bed was used to demonstrate the possibility of a continuous process for raising the dextrose; equivalents of “glucose” liquors of high concentration formed by acid hydrolysis of maize starch.

Kinetics of fermentation processes.

Abstract: Kinetic studies on fermentation processes were made and a general equation of production rate was newly presented applying the kinetic theory on mierobial cell growth which was reported previously by the authors.l,2 Equations for product concentration in fermentation time courses were derived by developing mathematically the general equation of production rate, and characteristic properties of fermentation processes were clarified. Some examples of fermentations were analyzed kinetically using the new kinetic theory. The calculated values of product, and cell concentrations were in good agreement with the observed values.

The effect of Eh on regulatory processes in facultative anaerobes

Abstract: On increasing aeration rate in chemostat cultures of E. coli the redox potential (Eh) of the culture changes from −l50 mv to +250 mv. During this period only a slight increase in measured dissolved oxygen concentration is observed. It is suggested that Eh be used as a control variable for aeration in the oxygen limited growth condition, whilst dissolved oxygen concentration be used as control variable in the presence of excess oxygen. Change in cytochrome b1, three tricarboxylic acid cycle enzymes and hydrogenase can be related to culture Eh. These changes are discussed.

Predator-prey relationships in a model for the activated process

Abstract: Summary The primary objective of this paper was to develop a mathematical description for the food chain,

A laboratory‐scale pulper for leafy plant material

Abstract: A machine is described that, makes, from 2 to 3 kg samples of leaf, a pulp comparable to that made by the large-scale equipment used in leaf protein extraction. It is therefore suitable for use in agronomic experiments on leaf protein yield.

The response by microorganisms to steady state growth in controlled concentrations of oxygen and glucose. I. Candida utilis.

Abstract: Dissolved oxygen and glucose concentrations have been independently maintained at various concentrations for extended periods during growth of Candida utilis in continuous culture. Simultaneous observations of cytochrome concentration, growth rate, rates of uptake of oxygen and glucose and rates of production of CO2, ethanol and acid have been made during steady states at various levels of oxygen and glucose. There is an inverse relationship between dissolved oxygen and cytochrome and between glucose cocentration and cytochrome. Studies of the transient state following a step change from high to low dissolved oxygen show that there is a lag of about 10 hr during which there is no change in the above parameters. This is followed by rapid oscillatory changes in cytochrome content and a change to a more fermentative metabolism.

Design and physical characteristics of a multistage, continuous tower fermentor

Abstract: A multistage tower laboratory fermentor has been constructed consisting of eight compartments separated by sieve plates. Flow of substrate and air is concurrent from the bottom to the top of the column. It, was hoped that this system could be used to reproduce, simultaneously on a continuous basis, eight distinct phases of a batch growth curve. It was believed that the extent of batch curve simulation would depend upon the character of hydraulic mean residence time of broth in the column and in the individual compartments. The expected relationship did not occur. Rather it was found that growth in the column involved residence time characteristics not only for the fluid but also for the microorganisms, and for the growth limiting substrate. Depending upon the column operation, these could be distinct and different. The purpose of this investigation was to study the residence time distribution (RTD) of the continous (fluid) and dispersed (microorganisms) phases for model systems as well as for a yeast fermentation. Various degrees of flow nonideality, i.e., fluid blackflow and dispersed phase sedimentation, were noticed. The former seems to be due to interaction of the concurrent gas and liquid flow; it is particularly dependent upon void area of the sieve plate holes. Sedimentation is probably a function of plate design as well as cell size and density. It wa concluded that for a particular plate design the gas hold-up wass controlled by superficial air velocity and was the main parameter governing the differences between dispersed and continous phase(Rt1). This conclusion was supported by a computeraided styudy utilizing a mathematical model of fluid flow to fit the growth kinetics and cell distribution observed experimentally throughout the fermentor. Some advantages of foam control in the tower fermentor by surface active compounds are mentioned. Also, suggestions are made for carrying out fermentations that have two liquid phases, such as a hydrocarbon fermentation. The possibility of closely approximating plug-flow conditions in the multistage tower fermentor, a necessary condition for batch growth simulation, is discussed from a practical point of view.

Activation of the biosynthesis of carotenoids by Blakeslea trispora

Abstract: The research carried out by several scientists has made possible the industrial preparation of β-carotene by fermentation. A fungus, Blakeslea trispora, abundantly synthesizes carotenoids when its two opposite forms are cultivated together in a special fatty medium. When ionones or other natural substances are introduced into the culture, a very obvious increase in the biosynthesis of carotenoids, more specifically of β-carotene, is obtained. Our own work has shown that; (1) several synthetic products chemically related to β-ionone, such as 2,6,6-trimethyl-l-acetyleyelohexene, can advantageously replace either partially or totally the ionones as inductors of the biosysnthesis of β-carotene; (2) various nitrogen-containing substances when added to the culture medium can considerably enhance the biosysnthesis of carotenoids while sometimes very specically orienting it. Their action comes on top of that of the ionones or their substitutes; actually this action is unexplained. Thus certain amides, imides, lactams, hydrazides, or substituted pyradines, and in particular succinimide and isonicotinoylhydrazine, have produced a two or threefold increase in the quantity of β-carotene present in the culture media of Blackeslea trispora. Conversely some heterocyclic substances such as pyridine itself or imidazole totally inhibit the biosysnthesis of β-carotene but induce the production of very important quantities of lycopene.

Effects of oxygen and glucose levels on lipid composition of yeast candida utilis grown in continuous culture

Abstract: Total lipids were extracted from the cells of Candida utilis grown at a constant population density in continuous culture. At different steady states, the environment was controlled with respect to dissolved oxygen and glucose concentrations, pH and temperature. Gas liquid chromatography was used to follow quantitative and qualitative changes in the fatty acid composition of the cells. Increasing glucose concentration resulted in higher lipid content; high oxygen concentrations increased the level of polyunsaturated fatty acids. The most significant changes in fatty acid composition took place when both glucose and oxygen concentrations were high, and under these conditions the amount, of linolenic acid was at its highest value.

Factors affecting the flocculation of bacteria by chemical additives.

Abstract: Pure bacterial cultures can be flocculated by a variety of chemical flocculants. Flocculation of bacteria will assist in their recovery, especially where the cells themselves are of interest, as in microbial protein production. Studies with several genera of bacteria indicate that the mechanism of flocculation is highly complex. Such interacting variables as temperature, ionic environment, physiological age, flocculant, bacterial genus, and surface shear have been observed. Jar test experiments with washed cells indicate that many of the variables are related to the release by the cell of proteins, nucleic acids, or polysaccharides. When released, these polymers may increase the required dosage of flocculant for recovery as in the case of E. coli, or the dosage may decrease as it does for Lactobacillus.

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity

Abstract: In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0−5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.

Growth models of cultures with two liquid phases. I. Substrate dissolved in dispersed phase

Abstract: Mathematical models which can be used to describe batch growth in fermentations with two liquid phases are developed for systems in which the growth limiting substrate is dissolved the dispersed liquid phase. In the models, the possibilities of growth occurring at the surface of the dispersed phase and in the continuous phase are considered. It is assumed that the composition of the dispersed phase is such that substrate utilization from it causes little of no change, in interfacial area. Three special cases are examined. In the first, it is assumed that all growth occurs at the surface of the dispersed phase. In the second and third, it is assumed that growth occurs both at the interface and in the continuous phase. The second case assumes that substrate equilibrium is continuously established between the two phases while the third assumes substrate consumption in the continuous phase is limited by rate of transport of substrate to that phase. Comparison of the first model with available experimental data shows good agreement between model and data.

Studies on methanol oxidizing bacteria

Abstract: A mixed culture of methanol oxidizing bacteria has been cultivated on simple inorganic salts medium supplemented with methanol. Optimal growth occurred at 31°C, pH 6.0–6.3, and a methanol concentration between 1 and 2 ml/1, of medium. The maximum yield was 4.5 g dw/I and the mean generation time 3.2 hr. It was estimated that 41% of methanol carbon was converted into cell-carbon, and that 73% of the inorganic nitrogen was converted to organic nitrogen.

Effect of high substrate concentration and cell feedback on kinetic behavior of heterogeneous populations in completely mixed systems

Abstract: Growth kinetics of heterogeneous populations of sewage origin were studied in completely mixed reactors of the once-through type at a high concentration of incoming substrate, 3000 mg/l glucose, and in systems employing cell feedback or sludge recycle at an incoming substrate concentration of 1000 mg/1 glucose. The recycle flow rate employed was 25% of the incoming feed flow, and the concentration of cells in the recycle was maintained as closely as possible at 150% of the cell concentration in the reactor. Studies were made at various dilution rates. Throughout these studies, batch experiments using cells grown at the various dilution rates were made to determine ks and μm values. As in previous studios using heterogeneous populations, the relationship between specific growth rates μ and substrate concentration S was represented better by the Monod equation than by any other which was tested. The growth “constants” μm, ks, and Y were found to fall in the same general range as those determined in previous studies in once-through systems operated at 1000 mg/l glucose. It was observed that cell recycle, even at the relatively low concentration factor employed in these studies, greatly enhanced the flocculating and settling characteristics of the cells.

Animal cell cultures as a source of hormones.

Abstract: Current methodology now makes it possible to establish in culture a variety of mammalian cells which perform organ-specific functions during serial propagation for periods of months or years. This report describes the results of experiments with animal and human cells that produce growth hormone, adrenocortical steroid hormones, thyrocalcitonin, and parathyroid hormone. Within the next decade it should be possible to use cell culture methods for manufacturing purposes to produce hormones and other valuable cellular products which are difficult to obtain in other ways. At first it may be necessary to use neoplastic cells for this purpose; but evidence is accumulating to suggest that it may eventually be possible to establish in culture normal, functional animal and human cells.

Microbial transformation of hydrocarbons. II. Growth constants and cell composition of microbial cells derived from n‐alkanes

Abstract: Cultivation of Norcardia sp., Mycobacterium phlei, and Candida lipolytica in inorganic salt solution containing n-alkanes C10–C20 as solo carbon and energy source was investigated. Generation times of 0.5–7.0 hr were typical during the exponential growth phase. The final cell concentrations (dry weight) were usually 9–26 g/l with n-alkane mixtures ranging from n-decane through n-eicosane. A linear dependence was found between the production of cell mass and the consumption of n-alkanes. The rest concentration of n-alkanes in the cell mass is in all experiments smaller than 0.5% (w/w). Cell yields were Ysub 60–142% and for Ye 50–97% based on n-alkane utilization. In one case, with the Nocardia NBZ 23, the substrate specifity on hydrocarbons and on a n-alkane mixture C10-C20 was studied. The cell mass recovered from the fermentations contained 47.8–57.7% carbon, 5.6–9.95% nitrogen, 7.2–9.4% hydrogen, 35–62% crude protein, and 6–36% lipid. Cellular protein and lipid synthesized by an organism is influenced by the type of nitrogen source. The amino acid, glucosamine, muramic acid, 2,6-diaminopimelinic acid, and fatty acid distribution in organisms grown on n-alkanes compared with a corresponding fermentation on glucose as sole carbon source were also estimated.

Clutivation of yeast on gas oil

Abstract: The results achieved by the cultivation of the yeast. Candida lipolytica on gas oil are referred. By using a distillation fraction of gas oil distilling between 180–400°C, containing 10–20% of n-alkanes, the optimal condition for biomass production and deparaffination were estimated for various dilution rates and various amounts of gas oil in the medium. The main factor, which influences the yield coefficient by hydrocarbon fermentation is the polyauxie of the hydrocarbon substrate. The penetration of dispersed hydrocarbons into the yeast cell is demonstrated on electron micrographs and the velocity and reversibility of this process is estimated by using tritium-traced hexadecane.

Use of the glucose oxidase system to measure oxygen transfer rates

Abstract: This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D‐glucono‐δ‐lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, Km(O2), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench‐scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.

Biosynthesis of chloramphenicol

Abstract: The current knowledge concerning the biosynthesis of chloramphenicol is discussed. Cultures of Streptomyces sp. 3022a fed 14C-shikimie acid incorporated the label to the same extent into phenylalanine, tyrosine, and chloramphenicol. Of possible precursors of the phenylpropanoid nucleus of this antibiotic only p-aminophenylalanine and DL-threo-p-amino phenylserine specifically labeled chloramphenicol. On the basis of these results a pathway for the biosynthesis of chloramphenicol is presented. The lack of specific incorporation of 15N-nitrogen from a competitive feeding experiment in which both l5N-nitrate and 14N-DL-serine were fed to growing cultures suggests that both the amido- and the nitro-nitrogen atom present in this antibiotic are derived from a common pool. Studies on the enzyme, DAHP synthetase, show that in streptomyces sp. 3022a it is not subject to feed back inhibition by either phenylalanine, tyrosine, or chloramphenicol.

A continuous, multistage tower fermentor. I. Design and performance tests

Abstract: The design of a continuous column fermentor with a multiple staging effect is described. The column is divided into four compartments by horizontal perforated plates and is provided with a central agitator shaft driving an impeller in each compartment. A tube at the center of each plate forms a liquid seal around the shaft and also acts as a “downcomer.” The fermentor is normally operated with counter-current flow of gas and medium. Fresh medium is added to the top stage and product is withdrawn from the bottom. The effect of plate and agitator design on fermentor performance was studied in terms of factor such as oxygen transfer rate, gas holdup, and interstage mixing. By proper choice of the design parameters, the fermentor was made to approximate a perfect four-stage cascade in terms of reactor performance. Preliminary experiments were performed with air-water systems, but a more realistic picture of fermentor performance was obtained in experience involving propagation of Escherichia coli. Data for business and substrate concentrations in each stage confirmed the staging effect of the apparatus. The fermentor operated in a stable manner for periods of more than two weeks.

Increased oxygen mass transfer rates from single bubbles in microbial systems at low reynolds numbers.

Abstract: The presence of microorganisms have been shown to increase by over 40% the mass transfer rates from small oxygen bubbles at low Reynolds number flow. This increase was found to be due only to the microbe cells as inert particles disrupting the quasi-static liquid surface film surrounding the gas bubble and thus decreasing the gas-liquid interfacial resistance. The observed increase in oxygen mass transfer rates was not dependent on cell viability, no effect was noted due to cell-liquid interfacial resistance, nor was the phenomenon due to altering the physical properties of the liquid during cell propagation. These results were obtained in a unique plexiglass apparatus designed for observing under a microscope a small (0.4 mm dia.) stationary oxygen bubble collapsing into a flowing fluid. The oxygen bubble was injected by a small hypodermic needle and the fluid was suspensions of the yeast Candida intermedia, the bacterium Pseudomonas ovalis, 0.3μ alumina, as well as base points of cell free broth and pure water. Several well-known chemical inhibitors of oxidative phosphorylation were used to limit cell oxygen uptake. Calculations of oxygen mass transfer rates were compared with the semi-empirical model of Frossling, the circulating sphere model of Levich, and the rigid sphere concentration boundary layer model of Fried-lander, the latter two showing strong Reynolds number dependence that may be due to radial fluid motion.