Causes of conductance change in yeast cultures

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rabra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds.
Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

The culture of plant cells with ammonium salts as the sole nitrogen source

Soybean cell suspension cultures grew on defined media with ammonium as the sole nitrogen source if Krebs cycle acids were added. Satisfactory growth was obtained with ammonium salts of citrate, malate, fumarate, or succinate, when compared with the regular medium containing nitrate and ammonium. Little or no growth occurred when ammonium salts of shikimate, tartrate, acetate, carbonate, or sulfate were used. The cells also grew well with l-glutamine as nitrogen source. The specific activities of glutamine synthetase and isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate) were lower than in cells grown on a nitrate medium, but ammonium enhanced the activity of glutamate dehydrogenase. Cells of soybean, wheat, and flax have been cultured for an extended period on the ammonium citrate medium.     

Effect of nitrogen source on pullulan production by Aureobasidium pullulans grown in a batch bioreactor

Pullulan production by Aureobasidium pullulans ATCC 201253 using selected nitrogen sources was studied in a medium using corn syrup as a carbon source. Independent of the corn syrup concentration present, the use of corn steep liquor or hydrolysed soy protein as a nitrogen source instead of ammonium sulphate did not elevate polysaccharide production by ATCC 201253 cells grown in an aerated, batch bioreactor containing 4 litres of medium. Pullulan production on corn steep liquor or hydrolysed soy protein as a nitrogen source became more comparable as the concentration of corn syrup was increased. Cell weights after 7 days of growth on any of the nitrogen sources were similar. The viscosity of the polysaccharide on day 7 was highest for cells grown on ammonium sulphate and 12.5% corn syrup. The pullulan content of the polysaccharide elaborated by ammonium sulphate-grown cells on day 7 decreased as the corn syrup level rose in the medium while the pullulan content of polysaccharide produced by cells grown on corn steep liquor or soytone generally increased.     

The route of lysine breakdown in Candida tropicalis

Candida tropicalis was found to contain high levels of the following enzymes after growth in defined medium on L-lysine as sole nitrogen source: L-lysine N6-acetyltransferase, N6-acetyl-lysine aminotransferase, and aminotransferase activity for 5-aminovalerate and 4-aminobutyrate. Extracts were also capable of converting 5-acetamidovalerate (and 4-acetamidobutyrate) to acetate. N6-Acetyllysine however, only gave rise to acetate in the presence of 2-oxoglutarate, NAD+ and thiamine pyrophosphate. These activities were undetectable or present in much lower concentrations in cells that had been grown on ammonium sulphate as sole nitrogen source. It is concluded that L-lysine is degraded in this organism via N6-acetyllysine, 5-acetamidovalerate and 5-aminovalerate, both nitrogen atoms being removed by transamination.     

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.     

Flower Induction by Nitrogen Deficiency in Lemna paucicostata 6746

Lemna paucicostata 6746, a short-day plant, produced flowerbuds even under continuous light when cultured in nitrogen-deficientmodified Hoagland medium with 1% sucrose for 3 days or morefollowed by culture on nitrogen-rich medium (either nitrateor ammonium). Flowering was also induced by culture on mediumcontaining 20–100 µM nitrate as the sole nitrogensource for 10 days or more, but not on medium with a low ammoniumconcentration. However, if plants cultured on medium containing5–20 µM ammonium as the sole nitrogen source for10 days were grown in a nitrogen-rich medium for a further 4days, they produced flower buds. Thus, nitrogen deficiency caninduce day length-independent flowering in Lemna paucicoslata6746, but nitrogen is required for the manifestation of flowering. (Received January 31, 1986; Accepted April 24, 1986)     

An organic solvent-tolerant protease from Pseudomonas aeruginosa strain K: Nutritional factors affecting protease production

An organic solvent-tolerant bacterium producing an organic solvent-stable protease was isolated from soil and identified as Pseudomonas aeruginosa strain K. Nutritional requirements for optimized protease production by this strain were investigated. Maximum protease activity was achieved with sorbitol as the sole carbon source, followed by starch and lactose at pH 7.0 and 37 °C. Dextrose, sucrose and glycerol greatly reduced the protease production. The best organic nitrogen source was casamino acid. Tryptone, soytone and yeast extract supported protease production while corn steep liquor and beef extract inhibited the protease activity. Significant protease production was observed with sodium nitrate as a sole nitrogen source however, ammonium nitrate completely inhibit it. More than 62% drop in production occurred in the presence of amino acids. Addition of metal ions such as K⁺, Mg²⁺ and Ca²⁺ maximized the enzyme production.     

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adenine-dependent growth of marine yeast,Rhodotorula glutinis var.salinaria under sodium chloride-hypertonic condition

Nitrate and ammonium were shown to alter the growth ofRhodotorula glutinis var.salinaria in saline and non-saline media. In saline medium in which ammonium was the sole nitrogen source, ammonium inhibited growth in the presence of molybdate ions. Detailed comparisons of the growth in saline and non-saline media when nitrate was supplemented in the presence of molybdate ions showed that differences in utilizability of purine bases of nucleic acid were responsible for the differences in growth,i.e. adenine increased the growth in such saline medium, but decreased it in non-saline medium. There was not such a specific requirement for adenine in saline medium in the presence of molybdate ions when nitrate was substituted for ammonium as the sole nitrogen source. It was suggested that adenine might provide the necessary skeleton of nucleic acid for serving nitrate reduction in saline medium.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Inhibition of tobacco nitrite reductase activity by expression of antisense RNA

A tobacco nitrite reductase (NiR) cDNA and its corresponding gene were isolated from cDNA and genomic libraries. An NiR antisense mRNA was expressed in transgenic tobacco under the control of a double 35S promoter. Transformants were obtained on a medium containing ammonium as the sole source of nitrogen. One plant growing normally on ammonium but displaying drastically reduced development and chlorotic leaves when grown on nitrate as the sole source of nitrogen was studied further. This plant accumulated nitrite fivefold over wild-type level and showed reduced amounts of ammonium (11% wild-type level), glutamine (19%), and total protein (8%). NiR mRNA and activity were below detectable levels. Under these conditions, nitrate reductase (NR) activity and mRNA were overexpressed, suggesting that N-metabolites resulting from nitrate reduction are responsible for the repression of the expression of the NR gene, independently from the presence or absence of a functional NR protein.     

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentrationon the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) wascultured in media with different nitrogen sources and concentrations.During the pre-logarithmic phase, the alga grew faster with ammoniumas N source than with nitrate, but the reverse applied during thepost-logarithmic phase. The alga grew poorly in N-free mediumor medium with urea as the sole N source. In the same growth phase,ammonium medium resulted in higher yield of total lipid, but the EPA yielddid not differ significantly different from that using nitrate medium. Themaximum growth rate occurred in medium containing 1.28 mmolL^-1 sodium nitrate, while maximum EPA and total lipid contents werereached at 1.92 mmol L^-1, when EPA accounted for 27.9% totalfatty acids. The growth rate kept stable when NH₄Cl ranged from0.64 to 2.56 mmol L^-1, and the maximum content of total lipidand EPA occurred in the medium with 2.56 mmol L^-1NH₄Cl. The EPA content was higher in the pre- thanpost-logarithmic phase, though the total lipid content was lower. Thehighest EPA content expressed as percent total fatty acid was 27.9% innitrate medium and and 39.0% in ammonium medium.     

Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization

In the present work, erythromycin production was carried out in submerged culture using Saccharopolyspora erythraea. Different experiments were conducted to optimize the cultivation medium through the change of carbon and nitrogen sources to cheaper one in order to reduce the cost of medium and to utilize sugar cane molasses as one of major sugar industry by-products in Egypt. It was found that the addition of sugar cane molasses a sole carbon source at a concentration of 60 g/l accompanied by corn steep liquor (as organic N-source) in combination with ammonium sulphate (as inorganic N-source) gave the maximal erythromycin production. The antibiotic production in this medium reached about 600 mg/l which is about 33% higher than the value obtained in glucose based medium. On the other hand, the addition of n-propanol in concentration of 1% (v/v) increased the antibiotic production reaching about 720 mg/l after 144 h. Concluding, the new medium formulation based on cheap carbon source, sugar cane molasses, was a good alternative solution for the production of erythromycin economically.     

Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F

Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.     

Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs

The role of nitrate, ammonium, and culture medium pH on shoot organogenesis in Nicotiana tabacum zz100 leaf discs was examined. The nitrogen composition of a basal liquid shoot induction medium (SIM) containing 39.4 mM and 20.6 mM was altered whilst maintaining the overall ionic balance with Na(+) and Cl(-) ions. Omission of total nitrogen and nitrate, but not ammonium, from SIM prevented the initiation and formation of shoots. When nitrate was used as the sole source of nitrogen, a high frequency of explants initiated and produced leafy shoots. However, the numbers of shoots produced were significantly fewer than the control SIM. Buffering nitrate-only media with the organic acid 2[N-morpholino]ethanesulphonic acid (MES) could not compensate for the omission of ammonium. Ammonium used as the sole source of nitrogen appeared to have a negative effect on explant growth and morphogenesis, with a significant lowering of media pH. Buffering ammonium-only media with MES stabilized pH and allowed a low frequency of explants to initiate shoot meristems. However, no further differentiation into leafy shoots was observed. The amount of available nitrogen appears to be less important than the ratio between nitrate and ammonium. Shoot formation was achieved with a wide range of ratios, but media containing 40 mM nitrate and 20 mM ammonium (70:30) produced the greatest number of shoots per explant. Results from this study indicate a synergistic effect between ammonium and nitrate on shoot organogenesis independent of culture medium pH.     

Induction,maintenance, and differentiation of rice callus cultures on ammonium as sole nitrogen source

Rice callus was initiated from cultured anthers and was maintained on medium containing ammonium as sole nitrogen source and supplemented with either a buffer or an organic acid. Plant regeneration also was obtained on this medium, but the morphogenetic capacity of the callus culture declined as rapidly as on a medium containing a mixture of ammonium and nitrate. The results of the studies suggest that organic acids provide an additional function beyond that of buffering the medium during growth on ammonium.     

Enhancement and Cultural Characteristics of Leucomycin Production by Streptomyces kitasatoensis in the Presence of Magnesium Phosphate

Stimulation of leucomycin production with Streptomyces kitasatoensis by magnesium phosphate (MgP), and a concomitant decrease of ammonium ion concentration in the culture supernatant have been reported from this laboratory. Cultural characteristics of the MgP-supplemented culture were further examined in relation to the stimulation of leucomycin production. When the leucomycin titer increased 4- to 5-fold in the presence of MgP, mycelial growth level and assimilation rate of a major carbon source (glycerol) increased about 2-fold. MgP-grown mycelia were not appreciably different in shape or in carbon and nitrogen contents from the control mycelia. Magnesium and phosphate ions were released from MgP into the medium, while the addition of magnesium or phosphate salts to the control medium was rather inhibitory for leucomycin production. The pH values of MgP-supplemented cultures were lower than those of the control culture. When modifications of the basal media and culture conditions were done so as to permit the pH-change almost identical with that of the control media, the leucomycin titer was still higher in the presence of MgP than in its absence. When uric acid, an insoluble compound, was used as the sole nitrogen source, the leucomycin production increased to a high level even in the absence of MgP, which was comparable to that seen when MgP was added to the ammonium lactate-containing medium. The ammonium ion concentration in the culture supernatant of the uric acid-containing medium was maintained at a considerably low level. While in the ammonium lactate-containing medium, leucomycin production was low and ammonium ion concentration remained at a high level. Thus, it was demonstrated that the stimulation of leucomycin production by MgP is related to the lowering of ammonium ion level, but not to the other factors examined such as growth level, carbon or phosphate regulation and pH-effect.     

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Summary Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.