Carbon and Nitrogen Source Nutrition of Fumagillin Biosynthesis by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L⁻¹ xylan and 50 g L⁻¹ mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L⁻¹ was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings. Received: 8 April 2002 / Accepted: 24 June 2002     

Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica (Bacillariophyceae): effect of nitrogen source and concentration

To investigate the nutritional value of the diatom Cyclotella cryptica Reimann, Lewin, and Guillard as an alternative feed for the use in the aquaculture industry, the heterotrophic growth characteristics, total fatty acids, and the resultant fatty acid profile of the microalga were studied when cultivated with sodium nitrate, ammonium chloride, or urea. All three nitrogen sources supported growth under heterotrophic conditions, and their uptake affected the pH of the cultivation medium, even when buffered. The use of sodium nitrate or urea resulted in a significant increase in the pH of the cultivation media, whereas the use of ammonium chloride caused a minor decrease in the pH of the cultivation media. The maximum specific growth rate was highest when urea and ammonium chloride were supplied at a low concentration; however, the total fatty acid content was not significantly affected (P = 0.101) by the nitrogen source when supplied at 10.7 mM nitrogen. The total fatty acid content and fatty acid profile of C. cryptica was more affected by the growth phase (predominately influenced by the initial nitrogen concentration) than by the source of nitrogen.     

Inorganic nitrogen source for autotrophic growth of Euglena mutabilis Schmitz

The effect of inorganic nitrogen source on population growth of Euglena mutabilis, an acidophillic benthic protozoa colonizing on the sediment of acid mine drainage, was investigated. Sodium nitrate, ammonium chloride, and ammonium sulfate were tested as nitrogen sources. The population density of E. mutabilis at equilibrium density cultivated in ammonium chloride‐ and ammonium sulfate‐containing media was 9–11 times higher than that in sodium nitrate‐containing medium at the optimal salt medium concentration. The population growth of E. mutabilis in ammonium sulfate‐containing medium was rapid and reached half of the equilibrium density after ca. 228 h, which was ca. 77 h earlier than that in ammonium chloride‐containing medium. Culture medium with ammonium sulfate as the nitrogen source achieved the highest maximum population density and the fastest growth rate among the three nitrogen salts used as nitrogen sources.     

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media. Received: 4 October 1996 / Accepted: 25 October 1996     

Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities

A morpholinepropanesulfonic acid (MOPS)-buffered rich defined medium (RDM) was optimized to support a reproducible 2.6-h doubling time at 35 °C for Deinococcus radiodurans R1 and used to gain insight into vitamin and carbon metabolism. D. radiodurans was shown to require biotin and niacin for growth in this medium. A glutamine–serine simple defined medium (SDM) was developed that supported a 4-h doubling time, and this medium was used to probe sulfur and methionine metabolism. Vitamin B₁₂ was shown to alleviate methionine auxotrophy, and under these conditions, sulfate was used as the sole sulfur source. Phenotypic characterization of a methionine synthase deletion mutant demonstrated that the B₁₂ alleviation of methionine auxotrophy was due to the necessity of the B₁₂-dependent methionine synthase in methionine biosynthesis. Growth on ammonium as the sole nitrogen source in the presence of vitamin B₁₂ was demonstrated, but it was not possible to achieve reproducibly good growth in the absence of at least one amino acid as a nitrogen source. Growth on sulfate, cysteine, and methionine as sulfur sources demonstrated the function of a complete sulfur recycling pathway in this strain. These studies have demonstrated that rapid growth of D. radiodurans R1 can be achieved in a MOPS-based medium solely containing a carbon source, salts, four vitamins, and two amino acids.     

Carbon and nitrogen source nutrition of fumagillin biosynthesis by Aspergillus fumigatus

The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L(-1) xylan and 50 g L(-1) mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L(-1) was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings.     

Tetrahydrofolate and other growth requirements of certain strains of Ruminococcus flavefaciens.

Two strains of Ruminococcus flavefaciens were studied. Each grew in a chemically defined minimal medium containing: minerals; ammonium sulfate as a nitrogen source; amino acids as a nitrogen source, a growth promotant(s) or as both; cellobiose as an energy and carbon source; isobutyric acid, isovaleric acid, carbonic acid, and bicarbonate as additional carbon sources; and biotin, thiamine, and tetrahydrofolic acid as vitamins. Tetrahydrofolic acid (5 ng/ml) served as a replacement for rumen fluid that was required in previous media tested for the growth of these bacteria. The present bacteria differ from many of the ruminococci previously studied in that they do not require either p-amino-benzoic acid or folic acid but do require tetrahydrofolic acid for maximum growth. Dihydrofolic acid and 5-methyltetrahydrofolic acid can substitute for tetrahydrofolic acid in minimal chemically defined medium. Thus, there must be extensive metabolic interaction between the microbes inhabitating the rumen, because the R. flavefaciens isolated had complex requirements for growth and yet was among the predominant bacteria in the rumen of cattle fed a simple vitamin B-deficient, nonprotein nitrogen, high-fiber, purified diet.     

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation

Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation. Received: 30 March 2000 / Accepted: 19 May 2000     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

Plant-growth-promoting compounds produced by two agronomically important strains of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>, and implications for inoculant formulation

We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA₃, ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA₃ were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 μg ml⁻¹). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 μg ml⁻¹). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus l-methionine (3.94 ng ml⁻¹ h⁻¹) and Az39 cultured in NFb plus l-lysine (36.55 ng ml⁻¹ h⁻¹). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.     

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.     

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans

Two protein bands with strong esterase activity are present in broths of Nocardia lactamdurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed K _m values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C. Received: 17 December 1999 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

白葡萄酒活性干酵母对不同氮源利用的研究

选用5种不同的白葡萄酒活性干酵母,以硫酸铵、氯化铵、硝酸铵、尿素、酵母粉等5种物质为氮源,观察其生长量并称量菌体重,以此分析其对氮源利用情况及不同氮源对酵母生长的影响。研究表明:供试菌系在不同氮源中均能生长。不同氮源对酵母的生长速度和生长量有不同影响;不同酵母菌种对不同氮源的利用也有差异。在以酵母粉为氮源的培养基中生长最好。在实验提供的氮源中,酵母粉为供试菌最优氮源,其次是硫酸铵,氯化铵与硫酸铵基本相当,而硝酸铵最差。8#菌种对各种氮源的利用能力相对较强。17#菌种对各种氮源的利用能力最弱。     

Differential growth response of <Emphasis Type="Italic">Ulva lactuca</Emphasis> to ammonium and nitrate assimilation

Controlled cultivation of marine macroalgal biomass such as Ulva species, notably Ulva lactuca, is currently studied for production of biofuels or functional food ingredients. In a eutrophic environment, this macrophyte is exposed to varying types of nutrient supply, including different and fluctuating levels of nitrogen sources. Our understanding of the influences of this varying condition on the uptake and growth responses of U. lactuca is limited. In this present work, we examined the growth response of U. lactuca exposed to different sources of nitrogen (NH₄⁺; NO₃⁻; and the combination NH₄NO₃) by using photo-scanning technology for monitoring the growth kinetics of U. lactuca. The images revealed differential increases of the surface area of U. lactuca disks with time in response to different N-nutrient enrichments. The results showed a favorable growth response to ammonium as the nitrogen source. The NH₄Cl and NaNO₃ rich media (50 μM of N) accelerated U. lactuca growth to a maximum specific growth rate of 16.4 ± 0.18% day⁻¹ and 9.4 ± 0.72% day⁻¹, respectively. The highest biomass production rate obtained was 22.5 ± 0.24 mg DW m⁻²·day⁻¹. The presence of ammonium apparently discriminated the nitrate uptake by U. lactuca when exposed to NH₄NO₃. Apart from showing the significant differential growth response of U. lactuca to different nitrogen sources, the work exhibits the applicability of a photo-scanning approach for acquiring precise quantitative growth data for U. lactuca as exemplified by assessment of the growth response to two different N-sources.     

Glucose repression of anthracycline formation in Streptomyces peucetius var. caesius

The effect of glucose on growth and anthracycline production by Streptomyces peucetius var. caesius was examined in a chemically defined medium. Glucose concentrations above 100 mM inhibited anthracycline synthesis in the original strain without causing significant change in growth and final pH values. This effect was observed when the carbohydrate was added initially or after 24 h fermentation, but not when added during the stationary growth phase. When the microorganism was pregrown in 100 mM glucose and then transferred to a resting cell system with 444 mM glucose, no significant differences in antibiotic production were observed compared to the control without glucose. The negative effect of glucose on antibiotic synthesis was not observed in a mutant (2-dog^R–21) resistant to growth inhibition by 2-deoxyglucose. Glucose consumption by this mutant was approximately 30% of that utilized by the original strain. Compared to the original strain, the mutant 2-dog^R–21 exhibited a reduction of 50% in glucose transport and an 85% decrease in glucose kinase activity. The experimental evidence obtained suggests that glucose represses anthracycline formation in a transitory manner and that this effect is related to glucose transport and phosphorylation. Received: 15 January 1999 / Received revision: 7 April 1999 / Accepted: 1 May 1999     

Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Streptoverticillium mobaraense

In the later stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense the availability of a nitrogen source accessible to the microorganism becomes critical. Fed-batch fermentation is investigated with the aim of avoiding this substrate limitation. When peptone is used as a nitrogen source in the feed, no significant improvement of growth and transglutaminase production is observed. This is probably due to crosslinking of the nitrogen source by the transglutaminase produced. Using an inorganic nitrogen source alone does not give satisfactory growth and production. A fed-batch fermentation method has thus been developed to deal with this problem. In the batch phase of the fermentation, an initial medium containing peptone, designed on the basis of the stoichiometric requirements of the microorganism, is used to ensure optimal growth. In the feeding phase, ammonium sulphate is used instead to avoid the crosslinking effect. The feed composition, mainly the amount of nitrogen and carbon source, is also based on the stoichiometric requirements of the organism, taking into account the replacement of peptone by ammonium sulphate. By using this fed-batch fermentation technique, cell-mass dry weight and transglutaminase production could be increased by 33% and 80% respectively, compared to those in a batch fermentation. Received: 10 July 1997 / Received revision: 24 October 1997 / Accepted: 24 October 1997     

NUTRITIONAL REQUIREMENTS FOR OVULE FORMATION IN EXCISED PISTILS OF NIGELLA

The nutritional requirements for ovule formation in Nigella saliva L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mil) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mM nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of α-alanine (1 mM) and γ-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high nitrate medium.     

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Received: 11 April 1997 / Received revision: 19 June 1997 / Accepted: 23 June 1997     

Nutritional requirements ofLysobacter lactamgenus for the production of cephabacins

Summary To optimize the fermentation medium for the production of new cephem compounds, cephabacins, by an eubacteriaLysobacter lactamgenus IFO 14,288, the effects of medium components on cephabacin production were investigated. Supplementation of glucose as a sole carbon source in liquid media was the best for the antibiotic production as well as for the cell growth. Casamino acid was the best nitrogen source for antibiotic biosynthesis and cell growth among nitrogen sources tested, and this strain could utilize sulfate or thiosulfate as a sulfur source. No significant effects of growth factors (vitamins) on the antibiotic production and cell growth were observed, but ferrous, magnesium and nickel ions slightly enhanced the cephabacin production.     

Influence of carbon and nitrogen sources on Flavobacteriumgrowth and zeaxanthin biosynthesis

Growth and production of zeaxanthin by Flavobacterium sp were studied using different carbon and nitrogen sources in a chemically defined medium. The best growth was supported by sucrose, but glucose yielded similar carotenoid concentrations. Both asparagine and glutamine stimulated growth and pigment formation. Carotenoid production and glucose consumption increased as a function of asparagine concentration. In the presence of asparagine, high glucose concentrations decreased pigment production without affecting biomass formation. In the absence of glucose, asparagine could not support growth and zeaxanthin production. When compared to the effect of 55 mM glucose, 10 mM oxaloacetate increased growth and carotenoid production. Pyruvate and other intermediates of the citric acid cycle showed a similar stimulatory effect. The intermediates of glycolysis: glucose 6-phosphate and fructose 1,6-diphosphate did not support growth. These results suggest that Flavobacterium sp utilizes asparagine primarily as a nitrogen source for growth and production of zeaxanthin. Received 29 September 1998/ Accepted in revised form 23 April 1999