Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

A Chitinolytic Actinomycete Complex in Chernozem Soil

A chitinolytic actinomycete complex in chernozem soil has a specific taxonomic composition, which differs from that of the actinomycete complex typically isolated on standard nutrient media containing sugars and organic acids as carbon sources. The actinomycete complex that was isolated by using nutrient media with chitin as the source of carbon and nitrogen was dominated by representatives of the genus Streptosporangium, and the actinomycete complex that was isolated by using nutrient media with sugars and organic acids as the carbon sources was dominated by representatives of the genus Streptomyces. The confirmation of the ability of actinomycetes to utilize chitin as a sole source of carbon and nitrogen came from the augmented length and biomass of the mycelium, the increased number and biomass of the actinomycete spores, the production of carbon dioxide, and the accumulation of NH₄ ⁺ ions in the culture liquid of the actinomycetes grown in the nutrient media with chitin.     

A novel finding that Streptomyces clavuligerus can produce the antibiotic clavulanic acid using olive oil as a sole carbon source

Aims: This study aims to establish whether commercially available food oils can be used by Streptomyces clavuligerus as sole carbon sources for growth and clavulanic acid production. Methods and results: Batch cultures in bioreactors showed that Strep. clavuligerus growth and clavulanic acid yields in a P‐limited medium containing 0.6% (v/v) olive oil were respectively 2.5‐ and 2.6‐fold higher than in a glycerol‐containing medium used as control. Glycerol‐ and olive oil‐grown cells present different macromolecular composition, particularly lipid and protein content. Conclusions: Streptomyces clavuligerus uses olive oil as the sole carbon and energy source for growth and clavulanic acid production. Yields and production rates in olive oil are comparable to those reported for oil‐containing complex media. Differences in yields and in the macromolecular composition indicate that different metabolic pathways convert substrate into product. Significance and impact of the study: This is the first report of oils being used as the sole carbon source by Strep. clavuligerus. Apart from economic benefits, interesting questions are raised about Strep. clavuligerus physiology. Defined culture media allow physiological studies to be performed in the absence of interference by other compounds. Understanding how Strep. clavuligerus catabolises oils may have an economic impact in clavulanic acid production.     

l-proline as a nitrogen source increases the susceptibility ofSaccharomyces cerevisiae S288c to fluconazole

Fluconazole inhibition ofSaccharomyces cerevisiae S288c growth was evaluated in media containing ammonia,l-proline orl-leucine as a nitrogen source. Growth inhibition by fluconazole was maximum whenl-proline was used as a nitrogen source, while rhodamine 6G accumulation and fluconazole resistance were the highest when ammonia was the sole nitrogen source.     

The role of root-associatedKlebsiella pneumoniae in the nitrogen nutrition ofPoa pratensis andTriticum aestivum as estimated by the method of15N isotope dilution

Summary The technique of¹⁵N isotope dilution was used to verify that nitrogen was fixed and transferred to the plant byKlebsiella pneumoniae strain Pp in association withPoa pratensis orTriticum aestivum. Surface sterilized, sprouting seeds were inoculated withK. pneumoniae and grown in sand in modified Leonard jars. Potassium nitrate enriched with¹⁵N was used to provide N concentrations ranging from 10–40 mg Nl^–1 nutrient solution. After 10–18 weeks the shoots and roots were analyzed separately for dry matter, N content, total N, and atom %¹⁵N excess. The acetylene reduction technique was used to test for the presence of N₂-fixing organisms on the roots. The data from¹⁵N isotope dilution demonstrated that up to 33.8% of N in the shoots ofP. pratensis and 15.9% in those ofT. aestivum were derived from associative N₂ fixation byK. pneumoniae. In most experiments the dry matter yield, N content, and total N yield of the shoots ofP. pratensis were increased byK. pneumoniae inoculation, whereas inoculation had no significant effect on the dry matter yield, N content or total N of the shoots ofT. aestivum.     

Effect of plant growth regulators and medium composition on cell growth and saponin production during cell-suspension culture of mountain ginseng (Panax ginseng C. A. mayer)

We have established cell-suspension cultures of mountain ginseng (Panax ginseng G A. Mayer), and have attempted to increase the yield of saponin by manipulating our processing method and culturing factors (e.g., media strengths; the presence of plant growth regulators or sucrose; ratios of NO⁺ ₃/ NH^- ₄). Maximum biomass yield was obtained in media containing 2,4-D. However, saponin productivity was much higher in a medium comprising either IBA or NAA; 7.0 mg/L IBA was optimal for promoting both cell growth (10.0 g/L dry weight) and saponin production (7.29 mg/g DW total ginsenoside). Although the addition of cytokinins (BA and kinetin) did not affect cell growth, the level of saponin (particularly in the Rb group) was enhanced when the media were supplemented with either 0.5 mg/L BA or 0.5 mg/L kinetin. Half- and full-strength MS media were equally suitable for inducing both biomass as well as saponin production. We also investigated the effect of various concentrations of sucrose and nitrogen, and found that 30 g/L sucrose enhanced biomass yield as well as saponin content However, further increases (i.e., up to 70 g/L) led to a decrease in saponin accumulation and biomass production. Maximum growth and saponin productivity were reported from treatments with an initial nitrogen concentration of 30 mM. In general, the amount of saponin increased when the test media had high NO⁺ ₃/ NH^- ₄ ratios; in fact, saponin production was greatest when nitrate was the sole nitrogen source.     

History of yield improvements in the production of asperlicin byAspergillus alliaceus

Summary The natural product asperlicin is the first nonpeptide antagonist of cholecystokinin isolated from a microbial source. At discovery, production of asperlicin by the original soil isolate ofAspergillus alliaceus was between 15 and 30 mg/l. Selection of natural variants ofA. alliaceus, use of Plackett & Burman and Simplex experimental designs; formulation of synthetic media; amino acid supplementation of production media; analysis of complex nitrogen sources for their amino acid content; evaluation of promising media in fermentors; substitution of glycerol for glucose as a carbon source and rational mutant selection all contributed to titer increases to >900 mg/l.     

Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Is birch a suitable reference in estimating dinitrogen fixation in alders by isotope dilution?

Seedlings ofAlnus incana (nodulated and non-nodulated) andBetula papyrifera were fertilized with varying amounts (0, 10, 25, 50, 100, 250 and 500 g N g^–1 soil) of labelled ammonium-N or nitrate-N ( 5.2 A% excess¹⁵N as ammonium sulphate or potassium nitrate). After 4 months in the greenhouse,¹⁵N excess in the plants were determined and an isotope dilution equation was applied to determine the percent of biomass N fixed by theA. incana/Frankia system. When ammonium was used as the sole N source and birch as the non-fixing reference, N-fixation accounted for 95%, 87% and 60% of the plant nitrogen yields with 10, 25 and 50 g N g^–1 rates, additions respectively. At the 100 g N g^–1 fertilization and above N-fixation accounted for less than 10% of the N yield. Similar results were obtained when non-nodulatedA. incana was used as non-fixing reference. With nitrate as the sole N source, N-fixation accounted for 98%, 97%, 97%, 86%, 56% and 12% of N yield with 10, 25, 50, 100, 250 and 500 g N g^–1 additions respectively. These values were similar for both types of reference plants. The direct isotope dilution method was compared to that of the total nitrogen difference method. There was good agreement between the two methods up to 50 g N g^–1 for ammonium and up to 100 g N g^–1 for nitrate. The difference method produced negative values at high concentrations of nitrogen fertilization. Again similar results were obtained by the two reference plants. The results indicate that birch can be used as a non-fixing control in isotope dilution studies but that care must be exercised in selecting the type and quantity of labelled nitrogen fertilizer.     

Evaluation of the availability ofAzolla-N and urea-N to rice using15N

Summary The symbiotic association of the water fernAzolla with the blue-green algaAnabaena azollae can fix 30–60 kg N ha^–1 per rice cropping season. The value of this fixed N for rice production, however, is only realized once the N is released from theAzolla biomass and taken up by the rice plants. The availability of N applied asAzolla or as urea was measured in field experiments by two¹⁵N methods. In the first,Azolla caroliniana (Willd.) was labelled with¹⁵N in nutrient solution and incorporated into the soil at a rate of 144 kg N ha^–1. The recovery ofAzolla-N in the above ground parts of rice [Oryza sativa (L) cv. Nucleoryza] was found to be 32% vs. 26% for urea applied at a rate of 100 kg N/ha; there was no significant difference in recovery. In the second, 100 kg N/ha of¹⁵N-urea was applied separately or in combination with either 250 or 330 kg N ha^–1 of unlabelledAzolla. At the higher rate, the recovery ofAzolla-N was significantly greater than that of urea. There was a significant interaction when both N sources were applied together, which resulted in a greater recovery of N from each source in comparison to that source applied separately. Increasing the combined urea andAzolla application rate from 350 kg N ha^–1 to 430 kg N ha^–1 increased the N yield but had no effect on the dry matter yield of rice plants. The additional N taken up at the higher level of N application accumulated to a greater extent in the straw compared to the panicles. Since no assumptions need to be made about the contribution of soil N in the method using¹⁵N-labelledAzolla, this method is preferable to the¹⁵N dilution technique for assessing the availability ofAzolla-N to rice. Pot trials usingAzolla stored at –20°C or following oven-drying showed that both treatments decreased the recovery of N by one third in comparison to freshAzolla.     

Studies on the microbial production of theobromine and heteroxanthine from caffeine

Summary From soil a caffeine degrading bacterium was isolated which is able to grow on media containing up to 2% caffeine as the sole source of carbon and nitrogen. The organism was identified as Pseudomonas putida and referred to as Pseudomonas putida WS. Mutants of this strain converted caffeine and were shown to accumulate a mixture of theobromine and heteroxanthine during resting cells experiments.The highest yield in accumulation products was obtained with the mutant strain H8, however the production rate with resting cells was too small for commercial purposes. The yield was significantly increased by growth of the mutant on diluted complex media. With this technique a yield of 50% based on the amount of caffeine could be obtained for heteroxanthine. The concentration maximum is reached when caffeine is completely converted and only traces of theobromine are present.Dedicated to Professor G. Braunitzer on the occasion of his 65th birthday     

On mycological fat production from iraqi date extract: The dibis

Three strong fat-forming fungi, namely,Penicillium lilacinum, Penicillium soppi andAspergillus nidulans were grown on Iraqi date extract: the dibis supplemented with an external source of nitrogen in the form of asparagine, ammonium carbonate, or sodium nitrate. Penicillium lilacinum was the slowest in growth and fat formation, but later it exceeded the two other fungi. Media supplemented with ammonium carbonate were the least conducive to growth and fat formation. Asparagine was most favorable for fat formation in case ofPenicillium lilacinum, sodium nitrate in case ofAspergillusn idulans, but in case ofPenicillium soppi, the two sources were equally good.The use of different levels of nitrogen has shown that in case ofPenicillium lilacinum increase in nitrogen within the experimental limits was accompanied by an increase in fat yield. In case ofPenicillium soppi andAspergillus nidulans, the fat yield increased with decrease of nitrate nitrogen (increase in C:N ratio), but with asparagine, the medium concentration gave the heaviest yield.The highest fat percentages or fat coefficients did not, however, coincide with the highest total fat yield nor with the complete exhaustion of the sugar content. It is concluded that addition of nitrogen at the proper concentration to dibis media can bring up growth and fat formation to a reasonable level. But, still, dibis as substrate for fat formation seems to be yet inferior to synthetic media favourable for fat formation.     

Quinoline biodegradation and its nitrogen transformation pathway by a <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain

A Pseudomonas sp. strain, which can utilize quinoline as its sole carbon, nitrogen and energy source, was isolated from activated sludge in a coking wastewater treatment plant. Quinoline can be degraded via the 8-hydroxycoumarin pathway. We quantified the first two organic intermediates of the biodegradation, 2-hydroxyquinoline and 2,8-dihydroxyquinoline. We tracked the transformation of the nitrogen in quinoline in two media containing different C/N ratios. At least 40.4% of the nitrogen was finally transformed into ammonium when quinoline was the sole C and N source. But addition of an external carbon source like glucose promoted the transformation of N from NH₃ into NO₃ ⁻, NO₂ ⁻, and then to N₂. The product analysis and gene characteristics indicated that the isolate accomplished heterotrophic nitrification and aerobic denitrification simultaneously. The study also demonstrated that quinoline and its metabolic products can be eliminated if the C/N ratio is properly controlled in the treatment of quinoline-containing wastewater.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Enzymatic digestion of spent yeast cells for nutrient recycling in inulase production

Summary An enzyme complex capable of lysing yeast cells was produced byArthrobacter sp. in a medium containing live cells ofKluyveromyces fragilis as the sole source of nutrients. The enzyme complex caused a 90% reduction in the optical density of viable yeast cells in 6 h at 25°C. This yeast cell hydrolysate can be used as a source of nitrogen, vitamins and minerals for subsequent growth of yeast cells (8.3 mg/ml) and further production of inulase (167 U/ml) representing 88 and 87% yield respectively, compared to cells grown on a standard yeast extract (1%) and sucrose (2%) medium.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).     

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.     

Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy

NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in ¹⁵N, ¹³C, or ²H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched ¹⁵N or ¹⁵N/¹³C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the ¹⁵N- and ¹⁵N/¹³C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including ¹H-¹⁵N and ¹H-¹³C HSQCs, ¹⁵N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy.     

An inexpensive medium for production of arachidonic acid by <Emphasis Type="Italic">Mortierella alpina</Emphasis>

The production of arachidonic acid was studied in the fungus Mortierella alpina using an inexpensive medium. Glucose derived from maize starch hydrolysate was the sole carbon source and defatted soybean meal and sodium nitrate were the nitrogen sources. Optimal arachidonic acid yield (1.47 g l^-1) was observed at a glucose concentration of 100 g l^-1. Various treatments of defatted soybean meal to extract soluble nitrogen nutrients were evaluated. Alkali extract was the most effective for arachidonic acid production. A mixture of soybean alkali-extract protein and sodium nitrate was an excellent nitrogen source for fungal growth, lipid accumulation, and arachidonic acid production. A maximum yield of 1.87 g arachidonic acid l^-1 was obtained with a soybean protein concentration of 4.6 g l^-1 and a sodium nitrate concentration of 2.3 g l^-1. Electronic Publication