Formation of vitamin B6 by by the fungus Cladosporium resinae on media with n-dodecane or glucose

Accumulation of vitamin B6 by the fungus Cladosporium resinae str. Moscow University eas investigated on media containing n-dodecane or glucose. Vitamin B6 was built up in the mycelium and the culture liquid filtrate. Under stationary condiditions of growth the maximum amount was found on the 28th day and reached 750/mkg and 925/mkg per 1 of the culture liquid on the glucose and n-dodecane containing medium, respectively.     

The uptake of n-alkanes from alkane mixtures during growth of the hydrocarbon-utilizing fungus Cladosporium resinae

Summary Cladosporium resinae growing on alkane mixtures removed n-alkanes sequentially in order of increasing molecular weight, each at about the same rate as during growth on it as single alkane. This sequence is not in order of the ability of each alkane to support growth. No alkane-specific extracellular solubilizing agent able to affect the order o metabolism could be detected during the growth of C. resinae on mixed n-alkanes, but supplementing the medium with phospholipids like those produced during growth on specific alkanes increased the rate or removal of the respective alkanes. Kinetic analysis indicated that the uptake of dodecane, hexadecane and octadecane from a mixture could be by a common mechanism, the order being determined, through competition, by the affinity of the system for each alkane.     

Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.     

Phospholipids of Cladosporium resinae cultured on glucose and on n-alkanes.

Cladosporium resinae was grown on glucose, on n-dodecane, and on n-hexadecane. Total lipid was greatest in dodecane-grown cells and least in hexadecane-grown cells, while glucose-grown cells contained the most phospholipid and hexadecane-grown cells contained the least. Cells from all three media contained phosphatidylethanolamine and phosphatidylcholine as their major phospholipids, with lesser amounts of phosphatidylserine and traces of a cardiolipin-like compound. The major fatty acids associated with each phospholipid were palmitic acid and one or more 18-carbon unsaturated fatty acids. There was no correlation between n-alkane growth substrate and fatty acyl components of cellular phospholipids.     

Utilization of n-Alkanes by Cladosporium resinae

Four different isolates of Cladosporium resinae from Australian soils were tested for their ability to utilize liquid n-alkanes ranging from n-hexane to n-octadecane under standard conditions. The isolates were unable to make use of n-hexane, n-heptane, and n-octane for growth. In fact, these hydrocarbons, particularly n-hexane, exerted an inhibitory effect on spore germination and mycelial growth. All higher n-alkanes from n-nonane to n-octadecane were assimilated by the fungus, although only limited growth occurred on n-nonane and n-decane. The long chain n-alkanes (C(14) to C(18)) supported good growth of all isolates, but there was no obvious correlation between cell yields and chain lengths of these n-alkanes. Variation in growth responses to individual n-alkane among the different isolates was also observed. The cause of this variation is unknown.     

Production of biosurfactants by Cladosporium resinae

Summary Cladosporium resinae produces extracellular biosurfactants when growing in a hydrocarbon source such as the jet fuel JP8. This production of biosurfactants was observed by the reduction of the surface tension of the aqueous phase of growing medium, and by the increase in emulsion and foaming properties. A partial purification by collapsed foam gave better physical properties by decreasing surface tension and increasing foaming power and stabilization of emulsions. Surface active substances were purified by reversed phase chromatography. Six compounds representing over 75% of fraction containing surface activity were present. This fraction gave an improvement of all surface properties.     

Extracellular lipids of Cladosporium (Amorphotheca) resinae grown on glucose or on n-alkanes.

Cladosporium (Amorphotheca) resinae was grown in shake culture on glucose, n-dodecane, or n-hexadecane. Growth was most rapid on glucose, and more acid accumulated in the medium than in n-alkane-grown cultures. Neutral lipid was the major lipid fraction and triglycerides were the only extracellular neutral lipids detected. Dodecanoic (lauir) acid was the predominant fatty acid (greater than 60%) in neutral lipids from all three media, with lesser amounts of tetradecanoic, hexadecanoic, and octadecanoic acids. Extracellular phospholipids identified were phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and cardiolipin or a cardiolipin-like compound. Phospholipids from all three media contained dodecanoic acid as their principle fatty acid. Dodecanoic acid was the only extracellular free fatty acid detected. Glucose medium contained acetic, glyoxylic, and glycolic acids and an unidentified organic acid which may contribute to the lower pH in cultures after growth on glucose. In all classes of extracellular lipids the fatty acids do not correspond to the fatty acids previously determined to be associated with cellular lipids. Moreover, the fatty acids of extracellular lipids do not reflect the chain length of the n-alkane growth substrate.     

Pathway of n-Alkane Oxidation in Cladosporium resinae

Pathways of initial oxidation of n-alkanes were examined in two strains of Cladosporium resinae. Cells grow on dodecane and hexadecane and their primary alcohol and monoic acid derivatives. The homologous aldehydes do not support growth but are oxidized by intact cells and by cell-free preparations. Hexane and its derivatives support little or no growth, but cell extracts oxidize hexane, hexanol, and hexanal. Alkane oxidation by extracts is stimulated by reduced nicotinamide adenine dinucleotide (phosphate). Alcohol and aldehyde oxidation are stimulated by nicotinamide adenine dinucleotide (phosphate), and reduced coenzymes accumulate in the presence of cyanide or azide. Extracts supplied with (14)C-hexadecane convert it to the alcohol, aldehyde, and acid. Therefore, the major pathway for initial oxidation of n-alkanes is via the primary alcohol, aldehyde, and monoic acid, and the system can act on short-, intermediate-, and long-chain substrates. Thus, filamentous fungi appear to oxidize n-alkanes by pathways similar to those used by bacteria and yeasts.     

Hydrolysis of alpha-D-glucans and alpha-D-gluco-oligosaccharides by Cladosporium resinae glucoamylases

Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain alpha-D-glucosidase, alpha-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1 leads to 6)-alpha-D linkages in pullulan, amylopectin, isomaltose, panose, and 6(3)-alpha-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1 leads to 6)-alpha-D linkage in panose much more readily than that in isomaltose.     

Incorporation of 8-Azaguanine and Guanine by Cladosporium resinae

With (14)C-tagged 8-azaguanine and guanine in a Bushnell-Haas medium with glucose as a carbon source, the rate of incorporation of the two bases was determined in Cladosporium resinae. There was a marked preference for the incorporation of 8-azaguanine over guanine.     

X-ray microanalysis of electron-dense bodies in Cladosporium resinae

Abstract One of the structural changes which occur in Cladosporium resinae during growth on hydrocarbons is the formation of electron-dense bodies. In this paper we report the results of X-ray microanalysis and X-ray mapping, which have shown that these bodies are associated with high concentrations of calcium and phosphorus. Such accumulation of these elements is probably a reflection of the low growth rates which appear to be characteristic of growth of C. resinae on hydrocarbons.     

Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Effects of Poorly Metabolized Hydrocarbons on Substrate Oxidation by Cladosporium resinae

Twelve hydrocarbons which singly support no growth or little growth of Cladosporium resinae were examined for their effects on utilization of four substrates which do support growth of the fungus. Of the 48 combinations of an oxidizable substrate with a potential hydrocarbon substrate, 8 combinations supported increased oxygen consumption above the level obtained with the oxidizable substrate alone. There was no evidence of co-oxidation of the potential co-substrates toluene or p -xylene; their effects on increasing O₂-uptake appear to be due to permeability changes. With hexadecane alone, the ratio hexadecane oxidized to CO₂: hexadecane taken up by cells was 97:3. Addition of p -xylene or toluene decreased that ratio slightly to 96:4 and 89:11, respectively. These high ratios of hydrocarbon oxidized to hydrocarbon taken up may be advantageous during degradation of petroleum in the natural environment, since petroleum components could be degraded without formation of a large biomass.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.