Biological degradation of tannins in sericea lespedeza (Lespedeza cuneata) by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus analyzed by solid-state 13C nuclear magnetic resonance spectroscopy.

Leaves of sericea lespedeza exhibit a high proportion of condensed tannin, resulting in poor forage quality. The white rot fungi Ceriporiopsis subvermispora and Cyathus sterocoreus are known to preferentially degrade lignin in a variety of plants and were evaluated for their ability to degrade condensed tannin from sericea leaves with the aim of improving digestibility. Relative levels of condensed tannin, cutin, pectin, and cellulose were monitored as a function of fungal treatment by solid-state cross-polarization and magic angle spinning 13C nuclear magnetic resonance spectroscopy. Total soluble phenolics, soluble tannins, and soluble and insoluble proanthocyanidin levels in fungus-treated and control samples were measured by established chemical techniques. Results indicate that both species of fungus preferentially degrade condensed tannin and that C. subvermispora is markedly superior to C. stercoreus in this capacity.     

Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white rot fungi.

Eight rapid Poly R-478 dye-decolorizing isolates from The Netherlands were screened in this study for the biodegradation of polycyclic aromatic hydrocarbons (PAH) supplied at 10 mg liter(-1). Several well-known ligninolytic culture collection strains, Phanerochaete chrysosporium BKM-F-1767, Trametes versicolor Paprican 52, and Bjerkandera adusta CBS 595.78 were tested in parallel. All of the strains significantly removed anthracene, and nine of the strains significantly removed benzo(a)pyrene beyond the limited losses observed in sterile liquid and HgCl2-poisoned fungus controls. One of the new isolates, Bjerkandera sp. strain Bos 55, was the best degrader of both anthracene and benzo(a)pyrene, removing 99.2 and 83.1% of these compounds after 28 days, respectively. Half of the strains, exemplified by strains of the genera Bjerkandera and Phanerochaete, converted anthracene to anthraquinone, which was found to be a dead-end metabolite, in high yields. The extracellular fluids of selected strains were shown to be implicated in this conversion. In contrast, four Trametes strains removed anthracene without significant accumulation of the quinone. The ability of Trametes strains to degrade anthraquinone was confirmed in this study. None of the strains accumulated PAH quinones during benzo(a)pyrene degradation. Biodegradation of PAH by the various strains was highly correlated to the rate by which they decolorized Poly R-478 dye, demonstrating that ligninolytic indicators are useful in screening for promising PAH-degrading white rot fungal strains.     

Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white rot fungi.

Eight rapid Poly R-478 dye-decolorizing isolates from The Netherlands were screened in this study for the biodegradation of polycyclic aromatic hydrocarbons (PAH) supplied at 10 mg liter(-1). Several well-known ligninolytic culture collection strains, Phanerochaete chrysosporium BKM-F-1767, Trametes versicolor Paprican 52, and Bjerkandera adusta CBS 595.78 were tested in parallel. All of the strains significantly removed anthracene, and nine of the strains significantly removed benzo(a)pyrene beyond the limited losses observed in sterile liquid and HgCl2-poisoned fungus controls. One of the new isolates, Bjerkandera sp. strain Bos 55, was the best degrader of both anthracene and benzo(a)pyrene, removing 99.2 and 83.1% of these compounds after 28 days, respectively. Half of the strains, exemplified by strains of the genera Bjerkandera and Phanerochaete, converted anthracene to anthraquinone, which was found to be a dead-end metabolite, in high yields. The extracellular fluids of selected strains were shown to be implicated in this conversion. In contrast, four Trametes strains removed anthracene without significant accumulation of the quinone. The ability of Trametes strains to degrade anthraquinone was confirmed in this study. None of the strains accumulated PAH quinones during benzo(a)pyrene degradation. Biodegradation of PAH by the various strains was highly correlated to the rate by which they decolorized Poly R-478 dye, demonstrating that ligninolytic indicators are useful in screening for promising PAH-degrading white rot fungal strains.     

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

Structural characterization of ficaprenol-11 by 13C nuclear magnetic resonance.

The location of the internal trans and cis isoprene units in ficaprenol-11 isolated from Ficus elastica was determined by 13C nuclear magnetic resonance. The alignment of the isoprene units was estimated to be in the order: omega-terminal unit, three trans units, six cis units and alpha-terminal cis alcohol unit.     

13C nuclear magnetic resonance studies of egg phosphatidylcholine.

Spin lattice relaxation times (T1) and apparent spin-spin relaxation times (T2) derived from linewidth have been used to investigate model membranes composed of egg yolk phosphatidylcholine. T1 measurements appear to be largely dominated by segmental motion and as a consequence are not very sensitive to small changes in membrane structure. On the contrary, apparent T2 times are shown to be sensitive to such changes in the membrane and are thus suggested as a useful tool for further investigation of membrane structure.     

Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dipalmitoylphosphatidylcholine-palmitic acid phase diagram studied by 13C nuclear magnetic resonance

The phase diagram of dipalmitoylphosphatidylcholine (DPPC) and palmitic acid mixtures in excess D2O was studied by 13C-NMR. Phase boundaries were determined from plots of apparent spin-spin relaxation time T2 (for both choline methyl and fatty acid chain carbons) versus temperature. A peritectic transition in the 1-10 mol% region, whose existence has been theoretically inferred from the Gibbs phase rule but which was undetectable by differential thermal analysis (DTA) (S.E. Schullery et al. Biochemistry, 20 (1981) 6818-6824), was located by NMR at 41.6 degrees C. A second, nearby peritectic line at 44 degrees C, which had been shown by DTA to extend from about 3-25 mol% palmitic acid, was seen by NMR only above 10 mol%. The palmitic acid/DPPC complex (2:1), with a sharp melting point at 64 degrees C, reported in earlier studies, was also seen by NMR. A phase diagram including both NMR and DTA results is presented. Important general conclusions from this study are: (i) NMR and scanning thermal analysis are complementary techniques for phase studies; each can see transitions that are invisible to the other. (ii) The case for the applicability of the Gibbs phase rule to lipid bilayer systems has been strengthened by the observance of two predicted, close-spaced boundaries. (iii) Low concentrations of fatty acids and related molecules can not be assumed to disperse as simple ideal solutes in the bilayer matrix.     

High-resolution solid-state 13C nuclear magnetic resonance of bacterial spores: identification of the alpha-carbon signal of dipicolinic acid.

Natural-abundance solid-state 13C nuclear magnetic resonance spectra were obtained for bacterial spores for the first time by using the technique of cross-polarization magic-angle-spinning nuclear magnetic resonance spectroscopy. A resonance at about 150 ppm, detectable in spore samples having a Mn content of less than 0.05%, was consistent with an identification as the alpha-carbon signal of calcium dipicolinate; this signal was missing from a spore sample treated with acid to release dipicolinate and from a spore coat preparation. Carbohydrate peaks were particularly intense in spores and coat preparations of Bacillus macerans. Signals ascribable to beta-hydroxybutyrate were prominent in a B. cereus sample.     

Mobility of elastin chains as determined by 13C nuclear magnetic resonance

Relaxation times and integrated intensities of ¹³C have been obtained from nuclear magnetic resonance spectra of elastin in unstretched calf ligamentum nuchae and indicate that about 80% of the backbone carbonyl carbons have short rotational correlation times, τ_R ～ 40 nanoseconds. τ_R is reduced by only a factor of two when the ligament is in contact with 2 m-KCNS, a strong denaturant. By contrast, the highly ordered chains of collagen in insoluble calf achilles tendon give no spectrum until denatured in 2 m-KCNS, when t_R decreases by many orders of magnitude. These results show that elastin is composed largely of highly mobile chains under physiological conditions, suggesting that configurational entropy has an important role in its elastic properties.     

Pyruvate metabolism in Halobacterium salinarium studied by intracellular 13C nuclear magnetic resonance spectroscopy.

13C nuclear magnetic resonance spectroscopy was used to study the metabolism of [2-13C]pyruvate in intact cells of Halobacterium salinarium. The spectra of these cells show that pyruvate is reduced to lactic acid and transaminated to alanine. The intensity of C-2 lactate is higher under anaerobic conditions than under aerobic conditions. When cells are grown in the absence of glucose, the level of C-2 lactate intensity is lower. In extracts of these cells, the level of NADH-dependent lactate dehydrogenase activity is lower than that of cells grown in the presence of glucose. A C-5 glutamate resonance suggests the entry of pyruvate into the tricarboxylic acid cycle through acetyl-coenzyme A. In addition, the label is also observed at C-3 and C-4 of glutamate, signifying a pyruvate carboxylase-type reaction and scrambling of label at the fumarate-succinate stage plus malic enzyme operation, respectively. Citrate synthase and malic enzyme activity appear to be controlled by the growth conditions of H. salinarium.     

13C nuclear magnetic resonance spectra of 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphorylcholines.

The 13C NMR spectra of all sixteen 1,2-dioctade-cis-enoyl-sn-glycero-3-phosphorylcholines have been obtained. Resonance lines of the olefinic, methylene, methyl and carboxyl carbon nuclei are sufficiently characteristic to permit unequivocal designation of double bond position for each isomer. Two resonances of the sn-glycero-3-phosphorylcholine structure have been reassigned.     

13C nuclear magnetic resonance study of trehalose mobilization in yeast spores.

Using high-resolution 13C nuclear magnetic resonance, we examined the mobilization of endogenous trehalose in suspensions of yeast asci. Sporulation of yeast cells in [1-13C]acetate resulted in incorporation of label into the C-3 and C-4 positions of trehalose within the asci. During germination of these asci with [1-13C]glucose, the consumption of both endogenous trehalose and exogenous glucose were followed simultaneously by 13C nuclear magnetic resonance, as was the formation of glycerol and ethanol, their glycolytic and products. Time courses for carbohydrate consumption indicated that trehalose, although it decreased to 25% of its initial value upon germination, was not preferentially catabolized and did not provide the primary energy supply for germination with glucose. The ratio of trehalose to glucose catabolized was 0.09. Exogenous glucose levels appeared to regulate trehalose mobilization since trehalose was only consumed when sufficiently high levels (more than 2 mM) of glucose were present. Upon glucose depletion newly synthesized [1-13C]trehalose was observed. Nuclear magnetic resonance spectra of extracts confirmed the trehalose peak assignments and showed products of [1-13C]glucose catabolism. In addition by quantitating trehalose consumption and 2-deoxyglucose incorporation in dormant yeast asci, we found that 3.8 +/- 0.l4 molecules of 2-deoxyglucose were incorporated for each trehalose molecule consumed. Trehalose can therefore function as a carbohydrate source for ATP formation during dormancy.     

Biodegradation and bioconversion of endrin by white rot fungi,Phlebia acanthocystis and Phlebia brevispora

Endrin is persistent organic pollutants that contaminate soil in many parts of the world. In this study, endrin was used as the substrate for a degradation experiment with the white rot fungi of the genus Phlebia. The results of tolerance test showed that the tolerance level of Phlebia acanthocystis and Phlebia brevispora to endrin was higher than that of other fungi, and the tolerance coefficient of both strains to 1.0 mg/L endrin exceeded 0.9 in solid PDA medium. P. acanthocystis and P. brevispora could degrade endrin efficiently in pure culture, especially P. acanthocystis had the highest degradability of more than 80% after 20 d incubation. Compared with low-nitrogen medium, PDB medium is more suitable for the biodegradability of two fungi. Several hydroxylated products such as 8-hydroxyendrin and two monohydroxyendrin were detected, indicating that endrin was initially branched to different monohydroxylated products in fungal degradation. Moreover, a carboxylic acid product was obtained from P. acanthocystis culture, suggesting that the carboxylation reaction occurred in bioconversion of endrin. The fungal cytochrome P450 enzymes play significant role in the in the initial hydroxylation process on endrin degradation. This is the first report that endrin is converted to hydroxylated and carboxylated metabolites by microorganisms.     

Biosynthetic pathways in Methanospirillum hungatei as determined by 13C nuclear magnetic resonance

The main metabolic pathways in Methanospirillum hungatei GP1 were followed by using 13C nuclear magnetic resonance, with 13C-labeled acetate and CO2 as carbon sources. The labeling patterns found in carbohydrates, amino acids, lipids, and nucleosides were consistent with the formation of pyruvate from acetate and CO2 as the first step in biosynthesis. Carbohydrates are formed by the glucogenic pathway, and no scrambling of label was observed, indicating that the oxidative or reductive pentose phosphate pathways are not functioning at significant rates. The pathways for amino acid biosynthesis are the usual ones, with the exception of that for isoleucine. The tricarboxylic acid pathway is incomplete and operates in a reductive direction to form alpha-ketoglutarate. The phytanyl chains of lipids are synthesized from acetate via mevalonic acid.