Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Continuous fermenter growth of a methionine-overproducing mutant of Candida utilis

Summary An ethionine resistant mutant of Candida utilis was found to maintain an expanded intracellular pool of free l-methionine in batch and continuous cultures. During glucose-limited growth in mineral salts medium in a continuous fermenter, the free l-methionine pool of the mutant was 40–80% higher than in batch cultures, and varied in the range of 25–30 moles/g dry cells (3.7–4.5 mg/g dry cells).     

The production of extracellular thermostable neutral proteinase and α-amylase byBacillus stearothermophilus

Summary Extracellular thermostable neutral proteinase was produced byBacillus stearothermophilus strains NCIB 8924 and NRRL B-3880 growing at 55°C. The formation and stabilization of this proteinase was found to be dependent on the concentration of free calcium ions. Therefore, procedures that removed free calcium ions from the medium, such as the use of phosphate buffer, resulted in a lower production of proteinase. The calcium-deficient proteinase was denaturated or adsorbed by calcium phosphate compounds. During the sterilization procedure of the culture medium, the CaCO₃ precipitation, caused by the removal of CO₂, influenced the amount of proteinase produced in a phosphate buffered medium made with tap water. An improved medium without phosphate buffer was used for 10 and 300 l batch cultivations and the calcium requirement for proteinase formation by the two strains was determined.     

The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10⁻⁶ M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

Enhanced <Emphasis Type="SmallCaps">d</Emphasis>-ribose biosynthesis in batch culture of a transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain by citrate

In this study, the effects of citrate addition on d-ribose production were investigated in batch culture of a transketolase-deficient strain, Bacillus subtilis EC2, in shake flasks and bioreactors. Batch cultures in shake flasks and a 5-l reactor indicated that supplementation with 0.2–0.5 g l⁻¹ of citrate enhanced d-ribose production. When B. subtilis EC2 was cultivated in a 15-l reactor in a complex medium, the d-ribose concentration was 70.9 g l⁻¹ with a ribose yield of 0.497 mol mol⁻¹. When this strain was grown in the same medium supplemented with 0.3 g l⁻¹ of citrate, 83.4 g l⁻¹ of d-ribose were obtained, and the ribose yield was increased to 0.587 mol mol⁻¹. Addition of citrate reduced the activities of pyruvate kinase and phosphofructokinase, while it increased those of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Metabolic flux distribution in the stationary phase indicated that citrate addition resulted in increased fluxes in the pentose phosphate pathway and TCA cycle, and decreased fluxes in the glycolysis and acetate pathways.     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Influence of monosaccharides on aerobactin production byAerobacter aerogenes 62-1

Supplementation of cultures ofAerobacter aerogenes 62-1, 43/4 h after initiation of growth withd-glucose (20 mM), resulted in a threefold increase in the production of aerobactin. Administration ofl-lysine under similar conditions led to a twofold incrasse in the yield of the siderophore. Studies with a cell-free system ofAerobacter aerogenes 62-1 revealed considerable stimulation of lysine-N⁶-hydroxylase activity by glucose and several of its derivatives. Inclusion of ferric chloride (0.1 mM) in the growth medium led to the repression of both lysine-N⁶-hydroxylase and aerobactin synthetase.     

Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection

The extracellular domain of the epidermal growth factor receptor (EGFR) was expressed using the baculovirus expression vector system. The maximum level of the EGFR extracellular domain secreted into the medium in Sf-9 (Spodoptera frugiperda or fall armyworm) cell batch culture was approximately 2.5 g ml^–1. In order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant virus, and replacement of spent medium. By using this process, the specific yield of recombinant protein, which in batch culture drops when infection is carried out at densities greater than 3 × 10⁶ cells ml^–1, can be maintained at a maximum in cultures infected at densities of 10⁷ cells ml^–1 or greater. The process, when applied to 3-1 and 11-1 bioreactor cultures, allowed a maximum volumetric yield of triple the maximum value attainable in batch culture. Spent-medium analysis indicates that medium replacement provides certain nutrients that could otherwise be limiting for recombinant protein production.     

Alkaline serine proteinase and lectin isolation from the culture fluid of Bacillus subtilis

Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipiration. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and d-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mm. *** DIRECT SUPPORT *** AG903053 00002     

Formation of secondary metabolism enzymes in the tylosin producerStreptomyces T59-235

Production of the macrolide antibiotic tylosin byStreptomyces T59-235 was inhibited in cultures containing high phosphate concentrations (30 mM P_i). Vegetative growth (dry weight increase, DNA and RNA synthesis) was hardly affected. Tylosin production began when macromolecule synthesis had slowed down to minimum level; in cultures with 30 mM P_i the onset of antibiotic production was retarded compared to cultures with low phosphate concentration (5 mM). The activities of three enzyme systems involved in tylosin biosynthesis (dTDP-D-glucose-4,6-dehydratase; dTDP-mycarose-forming enzyme system; SAM: macrocin-O-methyl transferase) were measured and found to be significantly lower in cultures with 30 mM P_i than in low phosphate cultures.Chloramphenicol, but not rifampicin, caused a rapid decrease of both tylosin formation rate and dTDP-D-glucose-4,6-dehydratase activity when added to tylosin producing cultures.Abbreviations P_i inorganic phosphate - dTDB 2-deoxythymidine diphosphate - SAM S-adenosyl-L-methionine - LP low phosphate (5 mM) - HP high phosphate (30 mM)     

Composition of the essential oil from cell suspension cultures of Achillea millefolium ssp. millefolium

The composition of the essential oil isolated from Achillea millefolium L. ssp. millefolium cell suspension cultures was analysed by GC and GC-MS. The yield of the oil obtained by hydrodistillation or a simultaneous distillation -extraction of these cultures, harvested at days 8–10 (end of exponential phase), was 0.001 % (w/w). The analysis of the volatiles showed the presence of thirteen components; monoterpenes amounted to 5%, sesquiterpene hydrocarbons attained 40%, while eugenol, demethoxyencecalin and two unidentified compounds amounted to 45% of the total oil. Several methods were tested in an attempt to increase the essential oil production by the cultures: growth on solid medium, growth in light, use of a different culture medium, elicitation with cellulase or yeast extract, and growth in a two-phase system. Of the different methods tested, the growth in B5⁺ medium with Miglyol 812 led to the highest essential oil yield (0.002%, w/w), and resulted in a more diverse oil composition.     

Competition between the green alga Scenedesmus and the cyanobacterium Synechococcus under different modes of inorganic nitrogen supply

In this study, we evaluated growth responses of the green alga Scenedesmus and the cyanobacterium Synechococcus supplied with inorganic nitrogen in different ways. A competitive situation in which nitrogen was limiting was created in mixed cultures as well as in cultures growing in the same vessel but separated by a permeable dialysis membrane. Supplying inorganic nitrogen in small pulses at a high frequency favoured the cyanobacterium Synechococcus, whereas batch additions favoured the green alga Scenedesmus. When using a large-pulse/low-frequency supply mode, the yield of the green alga was higher when ammonium was added as nitrogen source compared to when nitrate was added. By contrast, the yield of the cyanobacterium was higher in the nitrate regime. However, uptake experiments using unialgal cultures showed that both organisms depleted the medium of ammonium more rapidly than they depleted the medium of nitrate; i.e. the higher yield of the cyanobacterium in the nitrate regime than in the ammonium regime can be attributed to the effects of competition with the green alga. Since nitrate assimilation involves the consumption of reductive power, we suggest that the outcome of competition was governed by the fact that green alga was light limited and therefore better able to compete for ammonium than for nitrate. The results from the laboratory studies are discussed in relation to results from an enclosure experiment performed in Lake Erken, Sweden. In that field experiment, in which additions of both phosphate and ammonium were applied every second day to 350-l enclosures, the green algal biomass increased exponentially during an incubation period of 22 days.     

Reduced Growth Yield of Activated Sludge in Organic Protonophore-Containing Batch Culture

Abstract The effects of organic protonophores 2,4-dinitrophenol (dNP) and para-nitrophenol (pNP) on the observed growth yield (Y _obs) was studied using batch cultures of activated sludge microorganisms. A growth yield model was proposed in relation to the ratio of initial protonophore concentration (C _u) to initial biomass concentration (X ₀) and was verified with experimental data. It was found that Y _obs decreased with the increase of the C _u/X ₀ ratio, while the specific rate of glucose consumption was increased. Results showed that the C _u/X ₀ ratio could more reasonably reflect the real strength of organic protonophore exerted to activated sludge than using C _u only. Based on the concept of growth yield, a model describing the uncoupling degree between energy generated via electron transport system and energy used for growth was further developed for protonophore-containing batch culture. It was shown that more than 60% of glucose was consumed through a futile cycle of energy rather than for growth at higher C _u/X ₀ ratios. This research usefully shows that the dissipation of energy via uncoupling biochemical processes can reduce excessive production of activated sludge markedly. Received: 28 April 1999; Accepted: 14 September 1999; Online Publication: 6 March 2000     

Growth of Escherichia coli cells in the presence of cysteine on sulphate-deficient media

Summary The behaviour of E. coli B culture grown on SO₄ ^2--free minimal glucose-salt medium was examined in the presence of exogenous cysteine at various concentrations. This was done by means of using the following parameters: length of lag, growth rate and total population. Up to a concentration of cysteine at 0.2mm the growth sets in without a lag phase, the growth rate is optimal (identical with that of cultures grown on media containing Na₂SO₄ as source of sulphur), only the size of total population being decreased by cysteine. At concentrations of 0.2mm and upwards, after a concentration-dependent lag-period, the cultures were found to increase at various lower growth rates.The toxic effect of cysteine was reduced by leucine itself, as well as by a mixture of leucine, isoleucine, valine and threonine. The anti-cysteine action of these amino acids showed itself in the shortening of the lag period and in the recovery of the growth rate which, however, failed to reach the original level.Cysteamine failed to provide sulphur for cultures of E. coli B grown on the above medium. Neither was the utilization of cysteamine affected by the application of amino acids possessing an anti-cysteine action.We have postulated that beside the inhibition of the biosynthesis of amino acids having an anti-cysteine effect, toxic concentrations of cysteine posses additional sites of action.     

Strategies for the improvement of salidroside production in cell suspension cultures of Rhodiola sachalinensis

Strategies of elicitation and precursor feeding were applied to improve salidroside production in cell suspension cultures of Rhodiola sachalinensis. Of the seven elicitors examined, that extracted from Aspergillus niger was the most effective, increasing the salidroside content by five-fold when added on the day of inoculation 40 mg carbohydrate is medium. Three possible precursors for salidroside synthesis, l-phenylalanine, l-tyrosol and l-tyrosine were added to the cultures. A high content of salidroside (1.440%) was attained with an initial l-tyrosol concentration of 0.5 mm in the medium. Combined application of the two strategies resulted in a significantly high salidroside content of 1.734%, corresponding to a salidroside yield of 200 mg/l. Received: 5 November 1996 / Revision received: 17 February 1997 / Accepted 11 April 1997     

Effect of urea on the production of 2,3-butanediol by K. oxytoca

This paper deals with the production of 2,3- butanediol by K. oxytoca in batch cultures. The effect of urea on various kinetic parameters was studied by replacing the ammonium salts in the medium with the corresponding nitrogen equivalent in the form of urea. The specific growth rate and the product yield in an unacclimatised batch culture were found to be 0.29 h^?1 and 0.26 g·g^?1 respectively. The acclimatised batch cultures on the other hand behaved similar to that grown using the original medium with a specific growth rate of 0.66 h^?1 and the product yield of 0.345 g·g^?1. However the cultures were unable to grow when urea was used both as the carbon and nitrogen source.     

Interpreting the role of phosphorus and growth rate in enhanced fungal induction of sesquiterpenes from Hyoscyamus muticus root cultures

The effect of thiamine limitation in combination with fungal elicitation on sesquiterpene (solavetivone) production was studied in Agrobacterium-transformed hairy-root cultures of Hyoscyamus muticus as a potential means of manipulating the growth rate independent of phosphorus availability. Limiting the initial supply of thiamine did not affect the growth of these cultures compared to growth at the control level of thiamine (0.01 g/l). There was also no enhancement in sesquiterpene production when thiamine supply was limited. Serial culturing in thiamine-free media suggests that these root cultures are not strictly auxotrophic for thiamine, in contrast to previously published results for untransformed root culture. The effect of phosphate limitation combined with elicitation on the production of solavetivone was examined at constant media volume to provide a constant elicitor concentration and to eliminate feedback-inhibition effects. Limiting the initial supply of phosphate to elicited cultures resulted in a twofold increase in solavetivone production as compared to the elicitation at control media phosphate levels (1.1mm). Because growth was attenuated, production per unit cell mass increased 11-fold compared to the control. The effect of phosphate limitation on solavetivone production at constant cell mass and elicitor per root mass was studied. Limiting the initial supply of phosphate to elicited cultures under these conditions did not result in enhanced production of solavetivone. The initially observed enhanced production of solavetivone at limiting initial phosphate concentrations is therefore due to factors other than the growth rate or phosphate involvement in secondary metabolism. Correspondence to: W. R. Curtis