Effect of carbon source on aroma production byTrichoderma viride

Summary The effect of carbon source on aroma production by a strain ofTrichoderma viride was studied in static and agitated cultures. Different carbon sources affected the quantity but not the nature of the aroma chemical produced. The formation of the aroma lactone (6-pentyl--pyrone) was also affected by the method of cultivation.

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Resumen Se estudió el efecto de la fuente de carbono en la producción de aromas porTrichoderma viride en cultivos estáticos y en agitación. Las distintas fuentes de carbono utilizadas influyeron en la cantidad pero no en la naturaleza del aroma químico producido. En la formación de un aroma de tipo lactona (6-pentil--pirona) también influyó el método de cultivo.

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Résumé L'effet de la source de carbone sur la production d'arome par une souche deTrichoderma viride a été étudiée en cultures statitiques et agitées. Les différentes sources de carbone modifient la quantité, mais pas la nature chimique, de l'arome produit. La production de l'arome lactonique (6-pentyl--pyrone) est affectée aussi par le mode culture.

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Paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–18 August 1985     

Pyrimidine base catabolism in Pseudomonas putida biotype B

Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl--alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source.     

Formation of 2-(5-Hyclroxymethyl-2-formylpyrrol-1-yl)alkyl Acid Lactones on Roasting Alkyl-α-amino Acid with d-Glucose

d-Glucose and several alkyl-α-amino acids (glycine, dl-α-alanine, dl-α-amino-n-butyric acid, l-valine, l-leucine and dl-α-amino-n-caproic acid) were roasted at 200°C or 250°C in a simple two components system. From the roasting products were newly isolated a series of 2-(5-hydroxymethyl-2-formylpyrrol-1-yl)alkyl acid lactones which were characterized by elementary analysis, UV, IR, MS (GC-MS) and NMR spectra.

These lactones have characteristic aroma which may contribute to the flavor produced by sugar-amino acid reaction. The subjective evaluation of aroma of the lactones obtained wrere as follows: 2-(5-hydroxymethyl-2-formyipyrrol-1-yl)propionic acid lactone, caramel and a little scorching; -n-butyric acid lactone, maple and strong sweet; isovaleric acid lactone and isocaproic acid lactone, miso, soy sauce and a little chocolate-like.     

Production of tobacco aroma from lutein. Specific role of the microorganisms involved in the process

A mixed culture formed by Bacillus sp. and Geotrichum sp. produced tobacco aroma compounds from the carotenoid lutein through the formation of the intermediate -ionone. Both microorganisms can grow independently in a medium supplemented with lutein, but only Geotrichum produces -ionone. This intermediate was incorporated by the bacilli, converted to aroma and this product excreted to the culture medium. Bacillus sp. did not utilize -ionone for growth but modified it. We conclude that, in the bioconversion of lutein to products with tobacco aroma, Geotrichum sp. is involved in carotenoid oxidation to produce -ionone and Bacillus sp. is responsible for the norisoprenoid reduction to produce 7,8-dihydro--ionone and 7,8-dihydro--ionol.     

Kinetics and effect of nitrogen source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture

Summary A kinetic study of the production of poly--hydroxybutyric acid (PHB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the PHB production phase. The effect of ammonia feeding on PHB production was consequently investigated. The nitrogen source (ammonia water) was supplied at a low constant feeding rate after the growth phase in which cell mass concentration reached 60 g/l. Feeding with a small quantity of ammonia resulted in a more rapid increase in intracellular PHB content than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated PHB but also reduction of microbial PHB synthetic activity.     

Maturation of black spruce somatic embryos. Part I. Effect of l-glutamine on the number and germinability of somatic embryos

Different concentrations of l-glutamine and different nitrogen sources in the medium were compared during maturation of black spruce (Picea mariana (Mill.) B.S.P.) somatic embryos. l-glutamine can be used as the sole nitrogen source for the maturation of Picea mariana somatic embryos at 2 to 3 gl^-1. A significantly lower number of somatic embryos was obtained on a medium prepared with only inorganic nitrogen. Compared with a medium supplement to inorganic nitrogen resulted in a twofold increase in the number of embryos for six genotypes. The nitrogen source and concentration in the maturation medium significantly affected the germination sensus stricto of somatic embryos (radicle appearance), but not their development into plantlets; at the time of epicotyl appearance, an effect of the nitrogen source was no longer found. A comparison of the development of somatic embryos into plantlets from seven genotypes showed that the genotype had more effect in terms of epicotyl appearance and in conversion rate than the nitrogen source present in the maturation medium.Abbreviations HLM-1 half-Litvays's medium with 10 M 2,4-D and 5 M BA - i only inorganic nitrogen in the medium - i+1 gG inorganic nitrogen plus 1 g l^-1 glutamine in the medium - SMM standard maturation medium - 2.5gG only 2.5 g l^-1 glutamine in the medium     

Urea as sole source of nitrogen for plant growth

Summary Spirodela oligorrhiza grown in sterile culture was able to use urea as sole source of nitrogen but only when the pH of the culture medium was below 4.3. Plants inoculated into urea media at pH 6.4 initially made little growth and became nitrogen-deficient in appearance and composition although they contained about 100 grams of urea per gram fresh weight of tissue. After a period the pH of the medium usually fell below 4.3 and growth commenced. Growth with other compounds, e.g. ammonium, nitrate or allantoin, as sources of nitrogen was not similarly affected by the pH of the culture medium.Urease activity could always be detected in the tissues of Spirodela oligorrhiza growing on urea. Plants with little or no urease activity soon developed significant activity when inoculated into urea media at pH 4.0. When the pH of the medium was higher there was no increase in urease activity and no growth ensued. Plants growing on urea possessed an activity of about 50 milliunits per gram fresh weight of tissue, but if the pH of the medium fell to 3.5 or lower, the activity present rose to 10 times this level.Urease activity also appeared, in the absence of supplied urea, as plants became increasingly nitrogen-deficient.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Interaction of odorous lactones with phospholipids: implications in toxicity towards producing yeast cells

Some lactonic aroma compounds, that can be produced industrially by microorganisms, become toxic towards the producing cells as these compounds reach high concentrations in the culture medium. To determine the manner by which these metabolites may influence yeast physiology, the effects of four lactones (concentration range of 100 to 300 mg l^–1) on the growth of Yarrowia lipolytica and on the phase behaviour of deuterated dimyristoylphosphatidylcholine (DMPC-d27) were studied. The results showed that the hydrophobic lactones decrease the phase transition temperature (Tm) of DMPC-d27 bilayers and that Tm decrease (Tm) was related to the inhibitory action of the lactones on yeast growth (evaluated by the lag time). These results suggest that whatever the lactone, a Tm of at least 2.5 °C resulted in a total growth inhibition: this implicates the lactone-phospholipids interaction in the mechanism of yeast growth inhibition. The test used in the present study may be a predicting method to assess the in vivo action of potential membrane active compounds.     

Bioconversion of lutein using a microbial mixture—maximizing the production of tobacco aroma compounds by manipulation of culture medium

The generation of aroma compounds by carotenoid cleavage in the 9–10 position was studied, due to the importance of these compounds in the flavor and fragrance industry. The bioconversion of the carotenoid lutein to C₁₃ norisoprenoids utilizing a microbial mixture composed of Trichosporon asahii and Paenibacillus amylolyticus was carried out by a fermentation process. Applying an experimental design methodology, the effects of nutritional factors on the production of aroma compounds present in the tobacco profile were studied. After an assessment of the significance of each nutritional factor, the levels of the variables yielding the maximum response were calculated. Glucose, tryptone, and yeast extract exerted a strong negative effect over the objective function, with glucose being the strongest. Lutein possessed a positive effect over the tobacco aroma production, while sodium chloride and trace elements showed no influence over the process. The yield attained after culture medium manipulation was almost ten-fold higher, compared with the base medium; and the aroma mixture was characterized as: 7,8-dihydro--ionol (95.2%), 7,8-dihydro--ionone (3.7%), and -ionone (1.1%).     

Production of 6-pentyl-α-pyrone byTrichoderma harzianum from 18∶n fatty acid methyl esters

Biosynthesis of 6-pentyl--pyrone byTrichoderma harzianum in two different media was evaluated. Best yields were found in nitrogen deficient medium (C/N=60). Limited growth seems to favour the production of this lactone. When fungal cells, precultured in low nitrogen medium, were incubated on methyl ricinoleate (10 g/l, C/N=60) an increase in 6-pentyl--pyrone production was observed in comparison with the media containing methyl oleate or methyl linoleate.     

Nitrogen source effects on the growth and toxicity of two strains of the ciguatera-causing dinoflagellate Gambierdiscus toxicus

Two Caribbean strains (1651 and 1655) of the ciguatera-causing dinoflagellate Gambierdiscus toxicus were grown in xenic, batch culture under defined, measured nutrient conditions with nitrate, ammonium, urea, a mix of free amino acids (FAA), or putrescine as the nitrogen source. Cultures were maintained at 27 °C, salinity 35, 110 μmol m⁻² s⁻¹ (12 h:12 h light:dark cycle) on L2 medium at an initial nitrogen concentration of 50 μM N. Toxicity was determined using a ouabain/veratridine-dependent cytotoxicity assay (N2A assay) standardized to a ciguatoxin standard. Nitrate, ammonium, FAA, and putrescine supported growth, but urea did not. The appearance of ammonium in the organic nitrogen cultures indicated that G. toxicus and/or associated bacteria remineralized the available organic nitrogen. Both strains were exposed to nitrogen-limiting conditions as evidenced by chlorophyll a content per cell, nitrogen content, and nitrogen (N) to phosphorus (P) (N:P) ratio significantly declining once nitrogen was no longer available in the medium and cells entered stationary phase. Strain 1651 grew significantly faster than strain 1655 when nitrate, FAA, and putrescine was the nitrogen source, but not ammonium. Nitrogen source had no effect on growth rate (0.14 d⁻¹) in strain 1651. The growth rate of strain 1655 (0.10–0.13 d⁻¹) was significantly faster on ammonium than the other nitrogen sources. Strain 1655 was significantly more toxic (10-fold) than strain 1651 except when growing on ammonium at exponential phase. Toxicity ranged from 1.3 to 8.7 fg C-CTX1-Eq cell⁻¹ in strain 1651 and from 30.7 to 54.3 fg C-CTX1-Eq cell⁻¹ in strain 1655. Nitrogen source had no significant affect on toxicity. Toxicity was greater in stationary versus exponential phase cells for strain 1651 when grown on nitrate and strain 1655 regardless of nitrogen source. The difference in toxicity between growth phases may result from an increase in ciguatoxin and/or maitotoxin. Our results suggest that some strains of G. toxicus when associated with bacteria are able to take advantage of organic as well as inorganic nitrogen sources on short time scales to support future growth. The uncoupling of total nitrogen and phosphorus pools from conditions in the water column suggest that instantaneous growth rates can be supported by nutrients acquired hours to days earlier.     

Metabolism of twelve herbicides by Streptomyces

Experiments were conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize twelve herbicides representing several different classes including: acetanilides, triazines, ureas, uracils, and imidazoles. Incubations in aqueous culture with dextrin as carbon source and either ammonium or Casamino acids as nitrogen source resulted in transformations (>50%) of eight of the herbicides tested: alachlor, metolachlor, atrazine, prometryne, ametryne, linuron, tebuthiuron, and bromacil; the remaining four herbicides (cyanazine, diuron, metribuzin, and imazapyr) were also transformed, but to a lesser extent. In most instances, biotransformations occurred concurrently with growth and results were consistent regardless of the nitrogen source (ammonium vs. Casamino acids). However, in some instances there were differences in rates of biotransformation as a consequence of the nitrogen source (e.g. alachlor, metribuzin), suggesting the selective induction of certain metabolic enzymes; in other instances biotransformations were not associated with growth, suggesting secondary metabolism. An experiment was also conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize atrazine contaminated soil. Inoculation of soil amended with 20 g/g of atrazine and 5% chitin as carbon source resulted in ca. 78% removal of atrazine within 28 days. These data suggest that Streptomyces species may be potential candidates for soil inoculation to bioremediate herbicide contaminated soils.The U.S. Government's right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri

A bacterium tentatively classified as Arthrobacter strain Py1 being capable to degrade pyrrole-2-carboxylate as only source of carbon, nitrogen, and energy was isolated from soil. In contrast to many other N-heterocyclic compounds, growth of the isolate on pyrrole-2-carboxylate was not affected by molybdate or its specific inhibitor tungstate, indicating a molybdoenzyme-independent breakdown. The latter was initiated by a hydroxylation reaction catalyzed by a pyrrole-2-carboxylate oxygenase, which also exhibited an NADH-cytochrome c reductase activity. The pyrrole-2-carboxylate oxygenase reaction as examined in cell extracts depended on NADH, FAD, and pyrrole-2-carboxylate; the apparent K _m values were 44, 6, and 43 M, respectively. A degradation pathway for pyrrole-2-carboxylate is proposed which involves 5-hydroxy-pyrrole-2-carboxylate and 2-oxoglutarate.     

Growth and β-Glucan 10C3K Production by a Mutant of Alcaligenes faecalis var. myxogenes in Defined Medium

A defined medium was developed in which Alcaligenes faecalis var. myxogenes 10C3 mutant K produced a large quantity of β-glucan 10C3K. The medium contained 4% glucose together with 0.1% citrate, succinate or fumarate as the carbon source, 0.15% (NH₄)₂HPO₄ as the nitrogen source and mineral salts. When NaNH₄HPO₄, KNO₃ or urea was used at a concentration of 0.03% nitrogen as the sole nitrogen source, salts of organic acid were not needed in addition to glucose.

In culture medium containing phosphate buffer (M/15, pH 6.5~8.0) large amounts of polysaccharide were formed and its yield from the 4% glucose added was about 50%. Thus, it was shown that polysaccharide production is enhanced greatly if a suitable pH for polysaccharide production is maintained during incubation.     

Lysine catabolism inStreptomyces ambofaciens producer of macrolide antibiotic,spiramycin

Spiramycin production was highly stimulated when lysine was used as the sole nitrogen source. This amino acid was catabolized by the -transaminase pathway characterized by dosage of cadaverine aminotransferase (CAT) enzyme. The Km_cadaverine was of 57mM. CAT was highly induced by lysine (634% in comparison with ammonium). Addition of 40mm of ammonium in a culture begun with 20mm of lysine as the sole initial nitrogen source repressed CAT biosynthesis by 24% but did not affect spiramycin production seriously. Addition of 20mm of lysine in a culture started with 40mm ammonium induced CAT biosynthesis of 425%, but did not allow spiramycin production. In these two cases, spiramycin production seems to be conditioned by the nitrogen source initially present in the culture medium. CAT activity was inhibited by ammonium ions (33% at 20mm), whereas lysine had no effects.     

Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium

In Phanerochaete chrysosporium fruit body formations is subject to strong catabolite repression by glucose in the presence of physiological levels of nitrogen. Walseth cellulose was found to be the best source of carbon for the induction of fruit body and consequent basidiospore synthesis. Ejected basidiospores collected from cultures grown under these conditions for two weeks are contaminated with neither conidia nor mycelial fragments and are therefore suitable for genetic analysis of recombination. Under conditions of nitrogen limitation, the glucose catabolite repression of fruit body synthesis was relieved. Exogenous adenosine 3,5-monophosphate but not other related nucleotides, also relieved glucose catabolite repression of fruit body formation.     

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of -keto--methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 °C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.     

α-Amylase production in batch and continuous cultures by Bacillus caldolyticus

Summary The concentration and productivity of -amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture.It was suggested that the higher production of -amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.