Low-nitrogen Medium Research Articles

Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions.

In order to delineate the roles of lignin and manganese peroxidases in the degradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium, the biodegradation of phenanthrene (chosen as a model for polycyclic aromatic hydrocarbons) was investigated. The disappearance of phenanthrene from the extracellular medium and mycelia was determined by using gas chromatography. The disappearance of phenanthrene from cultures of wild-type strains BKM-F1767 (ATCC 24725) and ME446 (ATCC 34541) under ligninolytic (low-nitrogen) as well as nonligninolytic (high-nitrogen) conditions was observed. The study was extended to two homokaryotic (basidiospore-derived) isolates of strain ME446. Both homokaryotic isolates, ME446-B19 (which produces lignin and manganese peroxidases only in low-nitrogen medium) and ME446-B5 (which totally lacks lignin and manganese peroxidase activities), caused the disappearance of phenanthrene when grown in low- as well as high-nitrogen media. Moreover, lignin and manganese peroxidase activities were not detected in any of the cultures incubated in the presence of phenanthrene. Additionally, the mineralization of phenanthrene was observed even under nonligninolytic conditions. The results collectively indicate that lignin and manganese peroxidases are not essential for the degradation of phenanthrene by P. chrysosporium. The observation that phenanthrene degradation occurs under nonligninolytic conditions suggests that the potential of P. chrysosporium for degradation of certain environmental pollutants is not limited to nutrient starvation conditions.

EFFECT OF NITROGEN AND PHOSPHORUS SUPPLY ON GROWTH AND TISSUE COMPOSITION OF ULVA FENESTRATA AND ENTEROMORPHA INTESTINALIS (ULVALES, CHLOROPHYTA)1

The chlorophyte macroalgae Ulva fenestrata (Postels and Ruprecht) and Enteromorpha intestinalis (Linnaeus) Link. were grown under various nutrient regimes in indoor semi‐continuous and batch cultures. Tissue nitrogen contents ranged from 1.3–5.4% N (dry wt), whereas tissue P ranged from 0.21–0.56% P (dry wt). Growth in low nitrogen medium resulted in N:P ratios of 5–8, whereas growth in high nitrogen medium resulted in N:P ratios of 21–44. For U. fenestrata, tissue N:P < 16 was indicative of N‐limitation. Tissue N:P 16–24 was optimal for growth and tissue N:P > 24 was indicative of P‐limitation. Growth of U. fenestrata was hyperbolically related to tissue N but linearly related to tissue P. Phosphorus‐limited U. fenestrata maintained high levels of tissue N, but N‐limited algae became depleted of P. For E. intestinalis, tissue N remained at maximum levels during P‐limitation whereas tissue P decreased to about 85% of maximal levels during N‐limitation. Growth rates for U. fenestrata decreased faster during P‐limitation than during N‐limitation. Simultaneously, tissue P was depleted faster than tissue N. Our results suggest that comparing tissue N and P of macroalage grown in batch cultures is useful for monitoring the nutritional status of macroalgae.

Nitrogen-deregulated mutants of Phanerochaete chrysosporium ?a lignin-degrading basidiomycete

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (14C-synthetic lignin----14CO2), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the other hand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H2O2 in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Medium-induced changes in patterns of free and combined amino acids in mycelium of Serpula lacrimans

Mycelium grown on medium containing either 7·5 or 0·075 m m ammonium sulphate as sole nitrogen source was separated into soluble and insoluble fractions and the amino acid compositions of extracts and hydrolysed residues were determined. Mycelial strands grown from wood were also analysed. Glutamic acid, and possibly glutamine and pyroglutamate, were found to be the most abundant free amino acids, and, together with ornithine, were most affected by the abundance of nitrogen in the medium. The total amino acid content of the hydrolysed residues also varied with the medium but no single amino acid showed a disproportionate change in amount. The amino acid composition of mycelial strands was very similar to that of the mycelium on a low-nitrogen medium. It was concluded that nitrogen can be stored in the form of free amino acid in the mycelium, and that the amino acids most likely to act as such stores are glutamic acid, possibly with glutamine and pyroglutamic acid, and ornithine.

The involvement of glutamate dehydrogenase and glutamine synthetase in ammonia assimilation by the rapidly growing ectomycorrhizal ascomycete, Cenococcum geophilum Fr.

summaryIn order to determine significance of the glutamate dehydrogenase pathway and a glutamine synthetase/glutamate synthase cycle in NH4+ assimilation, we followed a number of different metabolikc parameters (nutrient uptake, free amino acid pools, NH4+‐induced glutamine accumulation) and 15N incorporation into amino acids of rapidly growing Cennoccum geophilum. Arginine was a major free amino acid in C. geophilum during its entire growth period. C. geophilum synthesized and accumulated very large amounts of glutamine at the beginning of the rapid phase of growth in low nitrogen medium, during the whole growth period in high nitrogen medium, and immediately after addition of NH4+. Therefore, the accumulation of a large amount of glutamine tool; place when the external ammonium concentration was high. The current data identify four pathways of N metabolism in rapidly growing C. geophilum: (1) glutamine synthesis, invoking transfer of N to both amino and amino moieties; (2) glutamate formation; (3) transamination with pyruvate to yield alanine; (4) transamination with oxaloacetate to yield asparate. The higher accumulation of glutamate and related amino acids (alanine and aspartate) in the presence of the glutamine synthetase inhibitor methionine sulphoximine indicates that glutamate, the precursor of glutamine, was formed by a pathway insensitive to methionine sulphoximine, the glutamate dehydrogenase pathway. Up to 40% of the assimilated 15N terminated in the amido‐N of glutamine. These data are consistent with a pivotal role for glutamine synthetase activity and indicates that the primary assimilation of NH4+ in rapidly growing C. geophilum is brought about by concurrent activity of the GDH and GS pathways. The pathway of primary assimilation of NH4+ by C. geophilunt in the rapid phase of growth therefore differs from those operating in the stationary phase of growth where N flax through GDH is higher than the flux through the GS pathway.

Nitrate reductase activity in leaves and roots ofAlnus glutinosa (L) Gaertner

Thein vivo nitrate reductase activity (NRA) was determined inAlnus glutinosa plants grown nonsymbiotically on ammonium, nitrate, a combination of both, or symbiotically with atmospheric nitrogen as the only nitrogen source. Root NRA was absent when ammonium or atmospheric nitrogen was the nitrogen source. With nitrate in the culture solution the roots showed a high NRA. However, the leaf NRA behaved quite differently: with negligible activities on all nitrogen sources except atmospheric nitrogen. The foliar NRA measured, however, is likely not due to the activity of the plant but of microbial origin. Methods commonly used to facilitate produced nitrite to leak out of the tissue, such as addition of propanol and cutting the plant material, did not increase the nitrite release from the leaves. A turbidity developed when testing the samples for nitrite which was positively correlated with the NRA. Populations of microorganisms in the phyllosphere did not differ between the nutritional treatments. Bacteria, able to grow on a low-nitrogen medium, were present on the leaves. Nitrifiers could not be detected. The bacteria on the leaves appear to produce nitrite when incubated with leaf material.

Catabolic Fate of Streptomyces viridosporus T7A-Produced, Acid-Precipitable Polymeric Lignin upon Incubation with Ligninolytic Streptomyces Species and Phanerochaete chrysosporium

Degradation of ground and hot-water-extracted corn stover (Zea mays) lignocellulose by Streptomyces viridosporus T7A generates a water-soluble lignin degradation intermediate termed acid-precipitable polymeric lignin (APPL). The further catabolism of T7A-APPL by S. viridosporus T7A, S. badius 252, and S. setonii 75Vi2 was followed for 3 weeks in aerated shake flask cultures at 37 degrees C in a yeast extract-glucose medium containing 0.05% (wt/vol) T7A-APPL. APPL catabolism by Phanerochaete chrysosporium was followed in stationary cultures in a low-nitrogen medium containing 1% (wt/vol) glucose and 0.05% (wt/vol) T7A-APPL. Metabolism of the APPL was followed by turbidometric assay (600 nm) and by direct measurement of APPL recoverable from the medium. Accumulation and disappearance of soluble low-molecular-weight products of APPL catabolism were followed by gas-liquid chromatography and by high-pressure liquid chromatography, utilizing a diode array detector. Identified and quantified compounds present in culture media included p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, protocatechuic acid, vanillic acid, and vanillin. The further catabolism of these APPL-derived aromatic compounds varied with the culture examined, and only S. setonii and P. chrysosporium completely degraded all of them. Some new intermediates of APPL metabolism also appeared in culture media, but the patterns were culture specific. Additional evidence from high-pressure liquid chromatography analyses indicated that one strain, S. badius, converted a water-soluble fraction evident by high-pressure liquid chromatography (7 to 10 min retention time range) into new products appearing at shorter retention times. Mineralization of a [C-lignin]APPL was also followed. The percent C recovered as CO(2), C-APPL, C-labeled water-soluble products, and cell mass-associated radioactivity, were determined for each microorganism after 1 and 3 weeks of incubation in bubbler tube cultures at 37 degrees C. P. chrysosporium evolved the most CO(2) (10%), and S. viridosporus gave the greatest decrease in recoverable C-APPL (23%). The results show that S. badius was not able to significantly degrade the APPL, while the other microorganisms demonstrated various APPL-degrading abilities. The significance of these findings relative to the fate of APPLs in nature was discussed.

Nitrogen utilization in bacterial isolates from the equine cecum.

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.

Factors influencing leakage of intermediates from yeast grown in continuous culture

An investigation into the levels of metabolic intermediates in the medium derived from yeast grown in continuous culture has been carried out. Yeast was grown in continuous culture at different growth rates using three different media; control medium, low nitrogen and low phosphate medium. Samples at each steady state were taken and filtered to produce a cell-free sample which was assayed for glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, 6-phosphogluconate, dihydroxyacetone phosphate, glyceraldehyde phosphate, phosphoenol pyruvate, pyruvate, oxalacetate, citrate and glyoxylate. The levels of ATP, ADP and AMP were also determined. Most of the intermediates were detected in the medium of most of the samples investigated. Citrate (661 μmol l −1 ) was the most abundant intermediate in the medium with ADP (109.4 μmol l −1 ), ATP (70.0 μmol l −1 ) and pyruvate (80.8 μmol l −1 ) the next most abundant. Although no consistent relationship between growth conditions and intermediate levels in the medium was observed, it was proposed that the concentration of extracellular intermediates could be used as markers of the physiological state of a fermentation, since they are more available for analysis than intracellular intermediates.

Induction of proheterocyst-like cells in Oscillatoria princeps by β-2-thienylalanine under microaerobic conditions

Summary When grown microaerobically in a low-nitrogen medium containing β -2-Thienylalanine, certain heterocyst-like cells appeared at irregular intervals in the trichomes of the non-heterocystous cyanobacterium, Oscillatoria princeps .

The involvement of hydroxyl radical derived from hydrogen peroxide in lignin degradation by the white rot fungus Phanerochaete chrysosporium.

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.

Factors affecting microbial growth and polysaccharide production during the fermentation of Xanthomonas campestris cultures

Fermentations of Xanthomonas campestris have been carried out on laboratory and pilot plant scales using various organic nitrogen sources in order to test their effectiveness in polysaccharide (xanthan) production. It was discovered that high nitrogen concentrations give highest yields of crude product and result in a need for only short fermentation times to achieve maximum product formation. These products, however, have inferior solution rheology to those produced from low-nitrogen media due partly to their high concentrations of co-precipitated microbial cells and partly to differences in tertiary molecular structure.

Two new species of Phillipsia from Central America

Phillipsia rugospora and P. guatemalensis are described as new species from Costa Rica and Guatemala, Central America. Phillipsia rugospora is distinguished by its yellow hymenium and wrinkled (under the SEM) ascospores. Phillipsia guatemalensis has a rosaceous tan hymenium, relatively thin flesh, and striate ascospores. Both species have been grown in culture. Phillipsia guatemalensis produces holoblastic, hyaline conidia on a low-nitrogen medium. Phillipsia rugospora does not form conidia in culture. However, small, conidium-like structures are produced by germinating ascospores of the latter species.

Comparative metabolism of dieldrin - 14C in mouse, rat, rabbit, rhesus monkey and chimpanzee

White rot fungi can degrade a wide spectrum of recalcitrant organic pollutants, including polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs). In this experiment, 20 white rot fungi, belonging to genus Phlebia, were investigated for their ability to degrade dieldrin. Based on the screening results, we further investigated Phlebia acanthocystis, Phlebia brevispora, and Phlebia aurea to determine their degradation capacity and metabolic products towards dieldrin and aldrin. The three fungi were able to remove over 50% of dieldrin in a low nitrogen medium, after 42 d of incubation. Three hydroxylated products were detected as metabolites of dieldrin, suggesting that in Phlebia strains, hydroxylation reactions might play an important role in the metabolism of dieldrin. In contrast to dieldrin, aldrin exhibited higher levels of degradation activity. Over 90% of aldrin was removed after 28 d of incubation, and several new metabolites of aldrin in microorganisms, including 9-hydroxyaldrin and two carboxylic acid products, were detected in fungal cultures. These results indicate that the methylene moiety of aldrin and dieldrin molecules might be prone to enzymatic attack by white rot fungi. In this study, we describe for the first time a new metabolic pathway of both compounds by fungi of genus Phlebia.

Nitrogen Metabolism Control of the Acetate — Stimulated Carotenoid Synthesis in Rhodotorula sanneii

The addition of sodium acetate to the culture medium stimulated the formation of carotenoids of Rhodotorula sanneii (CBS 333) on a 3% high nitrogen (C/N ahout 11) glycerol medium. Chloramphenicol treatment intensified the favourable effect of sodium acetate on the formation of carotenoids on high nitrogen media. The concentration of chloramphenicol had a strong effect on the intensity of carotenoid synthesis. On low nitrogen medium (C/N 55) acetate did not stimulate the formation of carotenoids neither in the case of the sole addition nor with chloramphenicol. As the sole additive, acetate stimulated the assimilation of ammonium nitrogen both on high and low nitrogen media. Der Zusatz von Natriumazetat zu einem stickstoffreichen Nährmedium (C:N = 11) mit Glyzerin (3%) als Kohlenstoffquelle stimulierte die Karotinoidbildung in Rhodotorula sanneii -Kulturen (CBS 33). Eine Behandlung mit Chloramphenicol erhöhte den günstigen Einfluß des Azetats. Die Karotinoidsynthese war von der Konzentration des Chloramphenicols abhängig. In einem stickstoffarmen Nährmedium (C:N = 55) stimulierte der Azetatzusatz die Karotinoidbildung weder mit noch ohne Chloramphenicolzugabe. Azetat stimulierte die Ammoniumstickstoffaufnahme aus dem stickstoffreichen sowie aus dem Stickstoffarmen Nährmedium.

Levels of glycogen and trehalose in Mycobacterium smegmatis and the purification and properties of the glycogen synthetase.

The levels of glycogen, free trehalose, and lipid-bound trehalose were compared in Mycobacterium smegmatis grown under various conditions of nitrogen limitation. In a mineral salts medium supplemented with yeast extract and containing fructose as the carbon source, the accumulation of glycogen increased dramatically as the NH(4)Cl content of the medium was lowered. However, levels of free trehalose remained relatively constant. Cells were grown in low nitrogen medium and were then shifted to medium containing high nitrogen. Under these conditions, there was a rapid accumulation of glycogen in low nitrogen, and this glycogen was rapidly depleted when cells were placed in high nitrogen medium. Again the concentration of free trehalose remained fairly constant. However, when cells were grown in low nitrogen medium with [(14)C]fructose and then transferred to high nitrogen medium with unlabeled fructose, the specific radioactivity (counts per minute per micromole) of the free trehalose fell immediately, indicating that it was being synthesized and turned over continually. On the other hand, the specific radioactivity of the glycogen and bound trehalose declined much more slowly, suggesting that these two compounds were not turning over as rapidly or were being synthesized at a much slower rate. Experiments on the incorporation of [(14)C]fructose into glycogen and trehalose indicated that cells in high nitrogen medium synthesized much less glycogen than those in low nitrogen. However, synthesis of both free trehalose and bound trehalose was the same in both cases. The specific enzymatic activities of the glycogen synthetase and the trehalose phosphate synthetase varied somewhat from one growth condition to another, but there was no correlation between enzymatic activity and the amount of glycogen or trehalose, suggesting that changes in glycogen levels were not due to increased synthetic capacity. The glycogen synthetase was purified about 35-fold and its properties were examined. This enzyme was specific for adenosine diphosphate glucose as the glucosyl donor.

Synchronous initiation and maturation of Aspergillus niger conidiophores in culture

Synchronous development of Aspergillus niger conidiophores was obtained by using a system in which complete development was divided into four distinct morphogenetic stages, (i) Conidiophore initiation occurred in a low nitrogen (LN) medium, but not in a medium in which both glucose and nitrogen were exhausted simultaneously and not in LN medium under oxygen-limited conditions, (ii) Conidiophore growth occurred without exogenous nitrogen although exogenous glucose was essential. In addition, development was enhanced by a colonial growth form indicating that translocation from mycelium to the conidiophore was an important feature of this stage. During stages (i) and (ii) there was the abundant production of a carotenoid pigment, (iii) Vesicle and phialide formation did not occur on conidiophores formed in LN medium. Transfer of aborted conidiophores to a medium containing a nitrogen source and a tricarboxylic acid cycle intermediate as a carbon source stimulated vesicle and phialide formation. There was a concurrent stimulation of respiration and an increase in RQ, value. Conidiospore formation was inhibited and this resulted in vesicle and phialide proliferations, (iv) Heavy conidiospore production occurred on a further transfer of conidiophores with vesicles and phialides to a glucose nitrate medium. Conidiophore initiation, vesicle and phialide formation and carotene production were inhibited by reduction of the oxygen supply rate although vegetative growth could continue.

MATING TYPES OF CHLAMYDOMONAS FROM THE COLLECTION OF GILBERT M. SMITH

SUMMARY After the death of Dr. Gilbert M. Smith on July 11, 1959, the strains of Chlamydomonas maintained in his collection were sent to the Culture Collection of Algae at Indiana University. Axenic cultures of 29 strains were investigated to determine the nature of their sexual behavior. Mating types for 6 species (C. elliptica var. britannica, C. frankii, C. minutissima, C. moewusii f. microstigmata, C. reinhardtii, C. smithii) produced zygotes when agar cultures were harvested with sterile distilled water or low nitrogen medium and the complementary mating types for each species were mixed. Three species, C. moewusii f. microstigmata, C. reinhardtii, and C. smithii, produced sufficient zygotes to be of value in experimental work. C. smithii is an undescribed species interfertile with C. reinhardtii. The description of C. smithii will be published in a separate paper. Fourteen mating types of Chlamydomonas originally isolated and maintained by Smith are now maintained in the Culture Collection.

Nutritional control of sexuality in Chlamydomonas reinhardi.

1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated.