Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Live food production using batch culture and chemostat systems in Kuwait

A comparison of live food production using batch andchemostat systems between the facilities initiated in1988 and the re-establishment of new larger chemostatsystems, after Kuwaits liberation, is made. The livefood production dynamics achieved during the 1995–1996 spawning season of Shaem (Acanthopagrus latus)are discussed. Growth rate of the rotifers (Brachionusplicatilis) treated 24 h with a mixture of marinealgal species (Nannochloropsis, Isochrysis,Tetraselmis, Chlorella) plus other enrichment mediasuch as Super Selco, Protein Selco and Sanders priorto being supplied to the marine fish larvae isdiscussed. Rotifer production reached up to 2000 ×10⁶ individuals per day.     

Comparative stability of ethanol production by Escherichia coliKO11 in batch and chemostat culture

Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded. Received 13 January 1999/ Accepted in revised form 23 April 1999     

Stability of recombinant protein production by Penicillium chrysogenum in prolonged chemostat culture

Abstract A strain (WKW2) of Penicillium chrysogenum transformed with heterologous fungal acetamidase ( amd S) and bacterial β-galactosidase ( lac Z) was grown at a dilution rate of 0.17 h⁻¹ (doubling time of approx. 4.1 h) for 1600 h in a glucose-limited culture. By the end of the experiment the original strain had been almost completely replaced by spontaneous, morphological mutants, but the acetamidase and β-galactosidase activities of the culture were essentially unaltered. Furthermore, when WKW2 and the non-transformed parental strain (NRRL1951) were grown together in glucose- or NH₄⁺-limited chemostat cultures, neither strain had a selective advantage over the other. Thus, heterologous gene expression does not result in NRRL1951 having a selective advantage over WKW2. These results suggest that continuous flow culture systems could be used for efficient (and cost effective) production of recombinant proteins.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Amylase production by a Schwanniomyces occidentalis mutant in chemostat culture

The crystalline cell surface layer (S-layer) from Bacillus stearothermophilis PV72 was used as a matrix for reversible immobilization of -d-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2-dipyridyldisulphide, 550 g -d-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one -d-galactosidase molecule [relative molecular mass (M_r), 116000] per two S-layer subunits (M_r, 130 000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies -d-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.Correspondence to: M. Sára     

The Effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 +/- 0.01 h(-1), pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.     

Mass production of thermostable D-hydantoinase by batch culture of recombinant Escherichia coli with a constitutive expression system

D-Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D-amino acids. A gene encoding thermostable D-hydantoinase of Bacillus stearothermophilus SD-1 has previously been cloned and constitutively expressed by its native promoter in Escherichia coli XL1-Blue (Lee et al., 1996b). In this work, we attempted mass production of the D-hydantoinase by batch culture of the recombinant E. coli using glycerol as a carbon source. The plasmid content in cells increased in proportion to the culture temperature, which resulted in a two- or three-fold increase of the specific D-hydantoinase activity at 37 degrees C compared with that at 30 degrees C. The plasmid was stably maintained over 80 generations. When glycerol was initially added to a concentration of 100 g/L, the final biomass concentration reached about 50 g-dry cell weight/L in a 50 L-scale fermentation, resulting in the specific enzyme production of 3.8 x 10(4) unit/g-dry cell weight in a soluble form. Glycerol-using batch cultivation of recombinant E. coli was found to be a cost-effective process for the mass production of industrially useful D-hydantoinase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 449-455, 1997.     

Growth yield determination in a chemostat culture of the marine microalgaIsochrysis galbana

The growth yield of the PUFA-producing marine microalgaIsochrysis galbana ALII-4 grown in a light limited chemostat, was measured under a wide variety of conditions of incident irradiance (I _O ) and dilution rates (D). The experiments were conducted under laboratory conditions at 20 °C under continuous light. D ranged from 0.0024 to 0.0410 h^–1 at three intensities of Io (820, 1620 and 3270 µmol photon m^–2 s^–1) close to those found in outdoor cultures. A maximum efficiency _max = 0.616 g mol photon^–1 was obtained at I _O = 820 µmol photon m^–2 s^–1 and D = 0.030 h^–1 and the maximum capacity of the biomass to metabolize the light harvested was found to be 13.1 µmol photon g^–1 s^–1. Above this value, a significant drop in the system efficiency was observed. A new approach based in the averaged irradiance is used to assess the photon flux absorbed by the biomass.     

Glucose uptake of Cytophaga johnsonae studied in batch and chemostat culture

The influence of different physiological states on the glucose uptake and mineralization by Cytophaga johnsonae, a freshwater isolate, was examined in batch and chemostat cultures. At different growth rates under glucose limitation in chemostat cultures, different uptake patterns for ¹⁴C labeled glucose were observed. In batch culture and at high growth rates the glucose uptake potential showed a higher maximum velocity and a much lower substrate affinity than at lower growth rates. These findings and the results of short-term labeling patterns could be explained by two different glucose uptake mechanisms which enable the strain to grow efficiently both at high and low substrate concentrations. Substrate specificity studies showed that a structural change of the C-2 atom of the glucose molecule was tolerated by both systems. The consequences of these results for the ecophysiological classification of the Cytophaga group and for the operation of continuous cultures are discussed.     

Bacterial bioreactors for high yield production of recombinant protein

We developed a new bacterial expression system that utilizes a combination of attributes (low temperature, induction of an mRNA-specific endoribonuclease causing host cell growth arrest, and culture condensation) to facilitate stable, high level protein expression, almost 30% of total cellular protein, without background protein synthesis. With the use of an optimized vector, exponentially growing cultures could be condensed 40-fold without affecting protein yields, which lowered sample labeling costs to a few percent of the cost of a typical labeling experiment. Because the host cells were completely growth-arrested, toxic amino acids such as selenomethionine and fluorophenylalanine were efficiently incorporated into recombinant proteins in the absence of cytotoxicity. Therefore, this expression system using Escherichia coli as a bioreactor is especially well suited to structural genomics, large-scale protein expressions, and the production of cytotoxic proteins.     

Population dynamics of a recombinant culture in a chemostat under prolonged cultivation

The dynamics of a chemostat culture of Escherichia coli K12 harboring plasmid pBR322 under prolonged cultivation with a nonselective complex medium were studied. The ability of the culture to form colonies on plates supplemented with different ampicillin concentrations was monitored. It was observed that almost all cells sampled were able to grow on a high concentration of ampicillin at the beginning of the experiment. However, a subpopulation which formed colonies on intermediate-concentration (500-1000 mg/L) plates, but not on a high-concentration (2000 mg/L) plate, was detected just before the appearance of the plasmid-free cells. As time progressed, the percentage of this subpopulation increased, reached a maximum, then decreased toward the end of experiment. At this time the culture was dominated by a subpopulation which could not form colonies on the 100 mg/L ampicillin plates. These results indicate that three major processes may occur in the chemostat: a gradual shift of the higher plasmid copy number population toward a relatively lower copy number population; the complete shedding of the plasmid due to faulty segregation of plasmids during cell division; and growth competition among the subpopulations. A previously derived model is extended to account for all subpopulations. The model agrees qualitatively with the experimental results.     

The relationship between inorganic nitrogen oxidation and organic carbon production in batch and chemostat cultures of marine nitrifying bacteria

Rates of nitrification and organic C production were determined in batch and chemostat cultures of marine nitrifying bacteria; two NH ₄ ⁺ -oxidizing species and one NO ₂ ^– -oxidizing spezies. With increasing age in batch cultures and with decreasing flow rates in chemostats, cellular organic C and N concentrations declined while the intracellular ratio of C:N remained constant. With decreasing flow rates in chemostats, there was a reduction in (a) carboxylating enzyme activity per unit of cellular organic C (the potential for chemoautotrophic CO₂ fixation), and (b) the yield of organic C. For both NH ₄ ⁺ and NO ₂ ^– oxidizers, rates of nitrification and C yield were lowest at very slow chemostat growth rates, when compared with optimal growth rates in batch cultures. For both NH ₄ ⁺ and NO ₂ ^– -oxidizing species, the stoichiometric relationship between nitrification and organic C production did not remain constant and appeared to be dependent on the availability of the inorganic N substrate. The organic C yield from NH ₄ ⁺ oxidation and hence the free energy efficiency declined with increasing age in batch cultures and with decreasing flow rates in chemostats. The C yield from NO ₂ ^– oxidation and the free energy efficiency at slow chemostat growth rates was also lower than that at the optimal growth rate in batch culture.     

Perfusion bioreactors for the production of recombinant proteins in insect cells

Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.     

The effect of the dilution rate on CHO cell physiology and recombinant interferon-gamma production in glucose-limited chemostat culture

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (mu) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (mu(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 muM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma. (c) 1993 John Wiley & Sons, Inc.     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

Cell weight kinetics simulation in chemostat and batch culture of the rhodophyte Porphyridium cruentum

Experimental observations of cell size variations in the proliferating rhodophyte Porphyridium cruentum cultured under fully controlled conditions showed significant decreases from inoculation to a steady state in the chemostat with 0.23 d(-1) dilution rate and to a minimum in batch, dropping in size by ratios of over 10. To numerically simulate these variations, we assumed that the cell is made up of two categories of components that behave differently during the interphase and mitosis. These have been called essential (EC) and accessory (AC) components. It is assumed that the cell divides once the EC have doubled in size, regardless of the AC's state. The experimental cell weight time courses were correctly simulated by a model of synchronous cell kinetics based on these assumptions. The EC's specific growth rate was 1.5 times that of the whole cell, when no limitation occurred. The increase in cell weight observed during batch cultures after nutrient exhaustion was suitably simulated by assuming that EC growth stops when a limiting nutrient is exhausted. Several parameters characterizing the cell kinetics were defined, particularly the minimum minimorum EC or cell weight (26 and 15 pg for chemostat and batch, respectively), which was influenced by the cultivation method, and the maximum whole cell weight (224 to 244 pg), which depended on the inoculum's age. The influence of culture conditions on the amount of essential and accessory components contained in a cell was examined. A new approach was developed with respect to these compartments to determine the most suitable strategy and conduct a predictive approach for valuable molecule production.     

分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响

研究了2．5L罐分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响,确定了分批培养时生产谷胱甘肽合成酶系的最佳pH和最佳温度。研究结果表明：在发酵液的pH为7．2和温度为37℃时,谷胱甘肽合成酶系产量和细胞干重达到最大,分别为690．6U／L和3．77g／L。采用变温控制对菌体的生长和谷胱甘肽合成酶系的合成并没有明显的优点。