High-yield growth of E. coli at different temperatures in a bench scale fermentor

E. coli wax grown exponentially at different temperatures in a bench scale fermentor. pH was maintained at 6.8 by ammonia which served also as the nitrogen source. Glucose was introduced semi-continuously at a predetermined rate which ensured a glucose concentration of 25–50 g/liter during growth. The culture was sparged with pure oxygen. Yield constants for glucose, nitrogen, phosphorus, and oxygen were determined at the different temperatures of propagation. When all growth conditions, except temperature, were kept constant, the maximal possible yield of exponentially grown cell mass was found to be directly proportional to the doubling time. Concentrations of up to 55 g dry cells/liter culture were achieved.     

Growth of E. Coli W to high cell concentration by oxygen level linked control of carbon source concentration

The growth of E. coli W in a bench scale fermentor to high cell concentration is described. The method involves growth-linked introduction of ammonia to the culture, sparging the culture with oxygen, and maintenance of aerobic conditions during the final growth phase by gradually and automatically decreasing the concentration of the carbon source, sucrose, in the culture. Thus, the oxygen demand is kept within the limits of the supply capacity, and a linear growth rate during the final phase of growth is obtained. A concentration of 42 g dry cell per liter was obtained. The yield constants for nitrogen and phosphorous were determined and were compared with those obtained using the temperature variation method.     

Dense growth of aerobic bacteria in a bench-scale fermentor

Escherichia coli B, Escherichia coli MRE 600, Escherichia coli K 12-3300, Pseudomonas fluorescens, and Aerobacter aerogenes were grown exponentially in a bench-scale fermentor to cell concentrations in the range of 20 to 41 g dry cells/liter at 30°C and 30 to 55 g dry cells/liter at 25°C. The high cell concentrations were achieved in a growth system previously described for growth of Escherichia coli W (Biotechnol. Bioeng., 16 , 933 (1974); ibid. 17 , 227 (1975)). Various enzyme activity levels in the high-concentration cells were compared to those in cells grown in conventional low-density cultures. No significant differences were found. The culture supernatants were found to be essentially free of high-molecular weight metabolic or cell lysis products. Yield constants for glucose, nitrogen, oxygen, and phosphorus were also determined in the dense cultures and some of their relations to the growth conditions are discussed.     

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

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.     

Acetate metabolism by Escherichia coli in high-cell-density fermentation.

Little is known about the cellular physiology of Escherichia coli at high cell densities (e.g., greater than 50 g [dry cell weight] per liter), particularly in relation to the cellular response to different growth conditions. E. coli W3100 cultures were grown under identical physical and nutritional conditions, by using a computer-controlled fermentation system which maintains the glucose concentration at 0.5 g/liter, to high cell densities at pH values of 6.0, 6.5, 7.0, and 7.5. The data suggest a relationship between the pH of the environment and the amount of acetate excreted by the organism during growth. At pH values of 6.0 and 6.5, the acetate reached a concentration of 6 g/liter, whereas at pH 7.5, the acetate reached a concentration of 12 g/liter. Furthermore, at pH values of 6.0 to 7.0, the E. coli culture undergoes a dramatic metabolic switch in which oxygen and glucose consumption and CO2 evolution all temporarily decreased by 50 to 80%, with a concomitant initiation of acetate utilization. After a 30-min pause in which approximately 50% of the available acetate is consumed, the culture recovers and resumes consuming glucose and oxygen and producing acetate and CO2 at preswitch levels. During the switch period, the specific activity of isocitrate lyase typically increases approximately fourfold.     

Effect of interstage mixing in multistage culture systems on continuous biomass production

The significance of the interstage mixing on important process parameters of biomass production was studied. The experiments were performed in a multistage tower fermentor and in fermentors in series. The interstage mixing effect can be evaluated under conditions of geometrical similarity, identity of oxygen transfer rate, and identity of dilution rate per stage in the individual stages of both culture systems. Candida utilis was cultivated on a synthetic medium with ethanol as the sole carbon and energy source in the concentration range 10–100 g/liter. Dilution rate, temperature, and pH in each stage of both culture systems were kept constant. It was demonstrated that in the multistage tower fermentor the definite backflow which ensures the permanent reinoculation by adapted cells significantly decreases the inhibitory effect of higher ethanol concentrations on the cell growth and on the rate of ethanol utilization.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Growth and cell wall changes in rice coleoptiles growing under different conditions I. Changes in turgor pressure and cell wall polysaccharides during intact growth

The effect of the oxygen supply on growth, water absorptionof cells and cell wall changes was studied in coleoptiles ofrice seedlings growing under three different conditions: underwater, under water with constant air bubbling and in air. Coleoptilegrowth was larger when they were grown under water than in waterwith air bubbling and in air. Coleoptile growth under waterwas limited by the suction force of their cells rather thanby mechanical properties of the cell wall, while that of thecoleoptiles growing under the other two conditions was limitedby the cell wall rigidity. A decrease in the relative amountof noncellulosic glucose of the cell wall, and an increase inthe noncellulosic xylose during coleoptile growth were foundfor all three culture conditions. ¹ Present address: Departamento de Fisiologia Vegetal, Facultadde Ciencias, Universidad, Salamanca, Spain. (Received May 21, 1979; )     

Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli

A novel air separation system based on permeable membrane gas separation technology was used to cultivate Escherichia coli. The system fulfilled the dissolved oxygen requirements of a culture of E. coli grown on a glucose synthetic medium at a high and constant growth rate of 0.55 h-1. A biomass yield of 45 g (dry weight) per liter was achieved, and no by-product inhibition by acetate or CO2 was observed.     

Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli.

A novel air separation system based on permeable membrane gas separation technology was used to cultivate Escherichia coli. The system fulfilled the dissolved oxygen requirements of a culture of E. coli grown on a glucose synthetic medium at a high and constant growth rate of 0.55 h-1. A biomass yield of 45 g (dry weight) per liter was achieved, and no by-product inhibition by acetate or CO2 was observed.     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. I. Saccharomyces spp.

Saccharomyces cerevisiae and Saccharomyces carlsbergensis were grown in batch culture with and without oxygen control. The concentrations of A-, B- and C-type cytochromes of both yeasts were dependent on the oxygen concentration during growth as well as on the initial glucose concentration of the growth medium. S. cerevisiae cytochromes were maximal after growth in low glucose and low oxygen; S. carlsbergensis cytochromes were maximal after growth in low glucose and high oxygen. Except when glucose was in very low concentration, its catabolism by S. carlsbergensis was directed predominantly towards ethanolic fermentation regardless of the oxygen concentration. Growth rate, total cell mass and yield were maximal, and anabolism was closely balanced with catabolism, when glucose and oxygen of S. carlsbergensis cultures were both high. Under these conditions neither catabolism, respiratory or ethanolic, nor glucose uptake were maximal.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

An evaluation of suspension culture systems for the growth of murine lymphoblastoid lines expressing TL and Thy-1 alloantigens

As a prelude to our studies on TL and Thy-1 differentiation alloantigens, three murine lymphobhastoid cell lines were examined for expression of these components. Optimal conditions for their mass culture were also determined. Several suspension culture systems were evaluated: (a) 50 ml through 500 ml Wheaton and Bellco spinner flasks as well as 1, 4, and 8 liter Wheaton flasks modified for semicontinuous culture conditions, (b) 3 liter Chemapec Vibrofermentor, and (c) 14 liter New Brunswick fermentor. Utilizing these types of vessels the optimal culture conditions were evaluated as to the effect of: (1) pH, (2) initial concentration of cell inoculum, (3) types of media, and (4) methods of gassing and gas mixtures on the rate of growth and alloantigen expression. This study demonstrated that cells could be cultured on a semicontinuous basis up to densities of 2–4 × 10⁶ cells/ml if a vessel of appropriate dimensions was utilized, the appropriate medium selected, and the pH controlled by CO₂ and air overlay. Once these parameters were established the growth of a given cell line was highly reproducible: Under optimal culture conditions the expression of Thy-1 was maximum while the cells were in the exponential stage of growth and reduced during the lag and stationary phases of growth. The expression of TL did not vary as significantly during the various stages of growth. One cell line grown in medium supplemented with 10% horse serum expressed lass Thy-1 than those grown in medium containing 10% fetal calf serum. The factors affecting cell growth and alloantigen expression have been considered in the design of a large-scale suspension culture facility for culturing 1000 liters of cells per week.     

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758–766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 452–462, 2013     

Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Technological problems in cultivation of plant cells at high density

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technology problems in the cultivation of the plant cells at high density were investigated. Using “shake” flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen on order to obtain high cell densities in shaken cultures, particles breakdown and damage to the largest cell aggregate group (above 1981 μm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K)for a model solid–liquid system (β-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in subsequent cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 × 10^?3 cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble desperation). The most suitable bioreactor for culturing plant cells at high density was ajar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

A CORRELATION BETWEEN THE APICAL CELL AND THE HETEROBLASTIC LEAF SERIES IN MARSILEA

Plants of three species of Marsilea (M. vestita, M. villosa, M. drummondii) were grown in sterile culture under controlled conditions, and stem apices were sampled at one of the three heteroblastic leaf forms typical of this plant: spatulate, bifid, or quadrifid leaves. Statistical analyses were made of the relationship between the area of the apical cell and the leaf form which the plant produces under varied growth conditions. For all three species there is a statistically significant correlation (1% level) between apical cell area and leaf form. The analysis indicates that 83% of the variation in apical cell area in Marsilea vestita, 52% in M. villosa, and 54% in M. drummondii can be related to the change in leaf form. An increase in the glucose concentration of the culture medium increases the average apical cell area, and the addition of the protein synthesis inhibitor 2-thiouracil at concentrations of 10 mg/liter and 25 mg/liter decreases the average apical cell area. A certain average apical cell area is necessary for the production of a particular leaf form in the series. The area is a relative rather than an absolute size under any one particular growth condition. Any growth condition which inhibits or reverses the increase in apical cell size (which is typical of the normal growth pattern) will inhibit or reverse the normal heteroblastic series. Under constant conditions, the average size at which particular leaf forms develop appears to be species specific. These results confirm a generally held idea that the apex size is related to the heteroblastic leaf series, and they indicate that the area of the apical cell is the key factor rather than the volume or the number of cells of the apex. Undoubtedly the area of the apical cell is only a reflection of the physiological or morphological characteristics of the apical meristem that underlie the heteroblastic leaf series and which currently do not appear to lend themselves to more direct quantitative analysis.     

Use of continuous-culture techniques for determining the growth kinetics of a celluloytic Thermoactinomyces sp.

In order to better understand the kinetics of cellulose degradation by Thermoactinomyces sp., continuous-culture experiments were performed utilizing the various intermediates of cellulose degradation as the feed substrates. Steady-state data from the glucose runs suggest that this organism has a growth yield of 0.42 g cell/g glucose, and a specific maintenance of 0.24 g glucose/g cell/hr. The Monod equation did not seen to model the growth well, since a plot of 1/D vs. 1/S gave a maximum specific growth rate that was even lower than one of the steady-state dilution rates. A dynamic washout experiment suggested a maximum specific specific growth rate of 0.36 hr^?1 and indicated that glucose is only slightly growth inhibitory as the inhibition constant, K_i, is 19 g glucose/liter. An equation for substrate concentration for washout conditions was derived. This equation predicted the transient glucose concentration relatively well. A fill-and-draw technique was investigated for determination of the growth parameters. It was not successful because of difficulties in contamination and accurately monitoring the dissolved oxygen in the small highly agitated vessel. However, the technique could be useful in studying the growth characteristics of sludge in a waste treatment system where contamination is not a worry. One could cover the medium surface and use a nonsterilizable dissolved oxygen probe of high sensitivity membrane to overcome these difficulties.     

Lipid accumulation inRhodotorula glutinis on sugar cane molasses in single-stage continuous culture

Microbial lipids produced byRhodotorula glutinis grown in continuous culture with molasses under nitrogen-limiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h^–1. The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.     

Growth and composition of Escherichia coli subjected to square-wave perturbations in nutrient supply: Effect of varying amplitudes

The effect of regular variations in the input limiting nutrient concentration at different cycle amplitudes on the growth and composition (elemental and macromolecular) of Escherichia coli ML30 in chemostat culture is described. The cycle time was maintained constant at 2.0 hr. The basal level of nutrient fed to cultures was also maintained constant but the concentration amplitude was varied over a range from 0.2 to 5.0 g/liter. A lag between the stimulus and organisms response was observed and found to be independent of cycle amplitude over most of the range examined. Increasing the cycle amplitude did not effect the cell yield from glucose or the elemental composition of the organisms. However, large variations in the macromolecular composition of the cells (on a dry weight basis) were found; at 4.0 g/liter amplitude the cells contained some 82% protein. The relevance of this type of reactor operation in large-scale fermentations is discussed.