Oxidation of aromatic compounds in soil

The oxidation ofp-hydroxybenzoic acid, quinic acid, vanillin and coumarin in soil was studied. With vanillin, and particularly with coumarin, the lag phase for oxygen consumption was longer and the rate of oxygen consumption attained more than one peak. In soil preincubated with the relevant substrate, the second dose of the same substrate was oxidized more rapidly. If the soil was preincubated with glucose, the lag phase was also shortened and oxygen consumption was raised with all aromatic substrates.     

Decomposition of vanillin by soil microorganisms

In chernozem soil, vanillin was decomposed via vanillic and protocatechuic acid before the aromatic ring opened. The rate curves of oxygen consumption for the oxidation of vanillin were seen to have more than one maximum. During incubation of the soil with vanillin, the number of bacteria increased, especially those capable of utilizing vanillin as the sole carbon source. Of the 21 such strains isolated, 15 were identified asPseudomonas sp., five asCellulomonas sp. and one asAchromobacter sp. It was found that the course of the oxidation of vanillin varied at different p.H values and in different strains was found that the course of the oxidation of vanillin varied at different p.H values and in different strains of bacteria. In some cases, the phase of the oxidation of vanillin to vanillic acid was clearly differentiated from the subsequent decomposition of vanillic acid.     

The oxidation of pipecolic acid in preincubated soils

Chernozem soil was preincubated with 0.1% glucose, glucose plus ammonium nitrate, hydrolyzed casein or amino acids for five days and with 1% wheat straw, pectin, peptone or cellulose for ten days. The soil was modified by this treatment so that the pipecolic acid was oxidized in two steps, as was shown by two peaks on the graphic plot of the oxygen uptake. In the control soil sample preincubated with water, the oxygen consumption curve formed one peak. In brown soil it was necessary to increase the concentration of glucose and hydrolyzed casein to 1% to obtain the oxidation of pipecolic acid in two phasos. Approximately a third of the oxygen for the complete oxidation of pipecolic acid was consumed in the first phase of oxidation, another third in the second phase of oxidation. A relation was found between the occurrence of the second period of oxidation of pipecolic acid and the amount of organic carbon in the soil.     

Oxidation of vanillin by Serratia marcescens

A Serratia marcescens strain quantitatively converted vanillin (0·1%, w/v) to vanillic acid, which exerted an inhibitory effect on bacterial growth. Vanillic acid producing activity was found in cell-free extracts of cultures grown in media with and without vanillin, when glucose was the substrate.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.     

Effect of dexamethasone on neutrophil metabolism

The effect of dexamethasone on glucose and glutamine metabolism was investigated. The consumption and oxidation of glucose and glutamine, and the production of glutamate and lactate were determined in neutrophils cultured for 3 h in the presence of dexamethasone. The activities and expression of glucose-6-phosphate dehydrogenase (G6PDH) and phosphate-dependent glutaminase were also determined under the same conditions. Addition of dexamethasone to the culture medium caused a significant increase of glucose consumption at 0.5 microm (123.9%) and 1.0 microm (78.3%) concentrations. In spite of this, however, glucose oxidation remained unchanged. The glucocorticoid did not change glutamine consumption but caused a significant increase of glutamate production and did not alter glutamine oxidation. Dexamethasone-treated neutrophils had a significant decrease of G6PDH activity and expression in particular at 1.0 microm concentration. Phosphate- dependent glutaminase activity was also decreased (about 34%) by dexamethasone treatment. A similar effect was observed on glutaminase expression as indicated by RT-PCR analysis. Thus, the effect of dexamethasone on neutrophil metabolism was particularly noticeable with respect to G6PDH and glutaminase activities where a decrease in the respective mRNA levels was demonstrated.     

Effect of bioconversion conditions on vanillin production by Amycolatopsis sp. ATCC 39116 through an analysis of competing by-product formation

This study investigated the effects of transformation conditions such as initial pH, the initial concentration of glucose and yeast extract in the medium, and the separate addition of ferulic acid and vanillic acid, on the production of vanillin through an analysis of competing by-product formation by Amycolatopsis sp. ATCC 39116. The extent and nature of by-product formation and vanillin yield were affected by initial pH and different initial concentrations of glucose and yeast extract in the medium, with a high yield of vanillin and high cell density obtained at pH 8.0, 10 g/l glucose, and 8 g/l yeast extract. High concentrations of ferulic acid were found to negatively affect cell density. Additional supplementation of 100 mg/l vanillic acid, a metabolically linked by-product, was found to result in a high concentration of vanillin and guaiacol, an intermediate of vanillin. Via an analysis of the effect of these transformation conditions on competing by-product formation, high concentrations of ferulic acid were transformed with a molar yield to vanillin of 96.1 and 95.2 %, by Amycolatopsis sp. ATCC 39116 and Streptomyces V1, respectively, together with a minor accumulation of by-products. These are among the highest performance values reported in the literature to date for Streptomyces in batch cultures.     

Enzymatic oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared Guinea pig liver slices.

BACKGROUND/AIMS: The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared liver slices has not been previously reported. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activities in the oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared liver slices. METHODS: Vanillin, isovanillin or protocatechuic aldehyde was incubated with liver slices in the presence/absence of specific inhibitors of each enzyme, followed by HPLC. RESULTS: Vanillin was rapidly converted to vanillic acid. Vanillic acid formation was completely inhibited by isovanillin (aldehyde oxidase inhibitor), whereas disulfiram (aldehyde dehydrogenase inhibitor) inhibited acid formation by 16% and allopurinol (xanthine oxidase inhibitor) had no effect. Isovanillin was rapidly converted to isovanillic acid. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. Protocatechuic aldehyde was converted to protocatechuic acid at a lower rate than that of vanillin or isovanillin. Allopurinol only slightly inhibited protocatechuic aldehyde oxidation, isovanillin had little effect, whereas disulfiram inhibited protocatechuic acid formation by 50%. CONCLUSIONS: In freshly prepared liver slices, vanillin is rapidly oxidized by aldehyde oxidase with little contribution from xanthine oxidase or aldehyde dehydrogenase. Isovanillin is not a substrate for aldehyde oxidase and therefore it is metabolized to isovanillic acid predominantly by aldehyde dehydrogenase. All three enzymes contribute to the oxidation of protocatechuic aldehyde to its acid.     

Effect of drying and rewetting on bacterial growth rates in soil

The effect of soil moisture on bacterial growth was investigated, and the effects of rewetting were compared with glucose addition because both treatments increase substrate availability. Bacterial growth was estimated as thymidine and leucine incorporation, and was compared with respiration. Low growth rates were found in air-dried soil, increasing rapidly to high stable values in moist soils. Respiration and bacterial growth at different soil moisture contents were correlated. Rewetting air-dried soil resulted in a linear increase in bacterial growth with time, reaching the levels in moist soil (10 times higher) after about 7 h. Respiration rates increased within 1 h to a level >10 times higher than that in moist soil. After the initial flush, there was a gradual decrease in respiration rate, while bacterial growth increased to levels twice that of moist soil 24 h after rewetting, and decreased to levels similar to those in moist soil after 2 days. Adding glucose resulted in no positive effect on bacterial growth during the first 9 h, despite resulting in more than five times higher respiration. This indicated that the initial increase in bacterial growth after rewetting was not due to increased substrate availability.     

Dual effect of glucose on LDL oxidation: dependence on vitamin E

The aim of the present study was to determine the direct effect of glucose on LDL oxidation, a key step in the development of atherosclerosis. Purified human LDL were incubated with glucose (500 mg/dl) and LDL oxidation was started by adding CuCl(2) to the media. Glucose delayed the vitamin E consumption, but accelerated the formation of conjugated dienes and increased both the formation of thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility. When LDL were incubated with increasing concentrations of glucose and submitted to oxidation, the formation of conjugated dienes, TBARS, and the electrophoretic mobility increased in a concentration-dependent manner. When LDL was enriched with vitamin E, it showed a delay in the formation of conjugated dienes, even in the presence of glucose. To determine whether glucose had any effect on LDL oxidation, once the process was started and vitamin E consumed, LDL were submitted to oxidation and, at different times thereafter, glucose was added into the media. Under these conditions glucose also accelerated the LDL oxidation. In summary, present results show that in LDL submitted to oxidation, glucose delays the early phases of the oxidation, slowing the vitamin E consumption, but it accelerates the rate of LDL oxidation once LDL vitamin E has been consumed; the effect being concentration-dependent.     

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.     

Stimulation of lactobacilli during alcoholic fermentation: action of sucrose hydrolysis by yeast

Summary Behaviour of lactic acid bacteria especially their stimulation in mixed culture with yeast was studied. In alcoholic fermentation of molasses worts, bacterial growth was stimulated as the yeast inoculum size increase. The consumption of monosaccharides (glucose and fructose) liberated during hydrolysis of sucrose by yeast is proposed as a major factor accounting for this phenomenon.     

Presence and oxidation of amino acids in rhizosphere and non-rhizosphere soil

Manometric studies were carried out on the respiratory activity of different rhizosphere and non-rhizosphere soils to follow quantitatively the over-all microbial activity in the rhizosphere soil as affected by the species and growth phase of plant. Oxygen consumption was distinctly greater in rhizosphere soils as compared to that in non-rhizosphere soils. The difference between oxygen consumption by rhizosphere and non-rhizosphere soils changed with the course of plant growth and it was not the same in different plants. This difference in oxygen consumption might be a measure of the amount of available oxidizable substrate in the rhizosphere. A rhizosphere sample had greater diversity as well as higher concentration of free amino acids than a non-rhizosphere sample of the same soil. Bacterial counts pointed to preferential stimulation in the rhizosphere of bacteria requiring individual amino acids. amino acids, such as glycine, alanine, asparsic acid of tyrosine were oxidized more rapidly in rhizosphere than in non-rhizosphere soil, but the extsent of oxidation for each of the amino acids studied did not differ. The amount of oxygen consumed during oxidation of alanine, aspartic acid or tyrosine was about one-half of the total amount necessary for complete oxidation. With glycine a higher extent of oxidation (60%) was observed. These extents of oxidation of glycine and aspartic acid did not change on investigation at two different phases of plant growth.     

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

Effect of several environmental parameters on carbon metabolism in histosols

High specific activity¹⁴C-labeled glucose, succinate, acetate, salicylate, and amino acids were used to examine carbon metabolism by the microbial community of Pahokee muck (aLithic medisaprist), a drained, cultivated soil of the Florida Everglades. Variations in carbon oxidation were observed from the end of the wet season through the dry season in a fallow (bare) field. Evolution of¹⁴CO₂ varied with the substrate added and time. Calculation of¹⁴CO₂ evolution for each substrate as a proportion of total respiration of the microbial community which was measured by succinate oxidation (relative oxidation) allowed for determination of the proportion of metabolic activity contributed by the oxidation of each carbon source. Except for the May sample when an approximate 30% decline in relative salicylate oxidation activity was observed, the proportion of total catabolic activity contributed by salicylate oxidation and acetate degradation was constant with time. Relative oxidation of glucose and amino acids ranged from 0.12 to 0.52 and 0.10 to 0.23, respectively. At two times during the dry season, the effect of depth of soil and crop on the carbon oxidation was examined. Relative acetate and amino acid oxidation were constant with depth whereas statistically significant variation was observed in glucose and salicylate oxidation. Generally, with the latter substrates, the activity declined with increased soil depth. Greatest effect of crop on these metabolic activities was noted with oxidation of salicylate in soils from a St. Augustinegrass [Stenatophrum secundatum (Walt.) Kuntz] pasture. In these soils, oxidation of salicylate was nearly double that of the fallow field or of soil planted with sugarcane (Saccharum sp.).     

Evidence for mutualism between a plant growing in a phosphate-limited desert environment and a mineral phosphate solubilizing (MPS) rhizobacterium

Alkaline desert soils are high in insoluble calcium phosphates but deficient in soluble orthophosphate (Pi) essential for plant growth. In this extreme environment, one adaptive strategy could involve specific associations between plant roots and mineral phosphate solubilizing (MPS) bacteria. The most efficient MPS phenotype in Gram-negative bacteria results from extracellular oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase. A unique bacterial population isolated from the roots of Helianthus annus jaegeri growing at the edge of an alkaline dry lake in the Mojave Desert showed no MPS activity and no gluconic acid production. Addition of a concentrated solution containing material washed from the roots to these bacteria in culture resulted in production of high levels of gluconic acid. This effect was mimicked by addition of the essential glucose dehydrogenase redox cofactor 2,7,9-tricarboxyl-1H-pyrrolo[2,3]-quinoline-4,5-dione (PQQ) but the bioactive component was not PQQ. DNA hybridization data confirmed that this soil bacterium carried a gene with homology to the Escherichia coli quinoprotein glucose dehydrogenase. These data suggest that expression of the direct oxidation pathway in this bacterium may be regulated by signaling between the bacteria and the plant root. The resultant acidification of the rhizosphere may play a role in nutrient availability and/or other ecophysiological parameters essential for the survival of this desert plant.     

Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis

Leptin regulates fatty acid metabolism in liver, skeletal muscle, and pancreas by partitioning fatty acids into oxidation rather than triacylglycerol (TG) storage. Although leptin receptors are present in the heart, it is not known whether leptin also regulates cardiac fatty acid metabolism. To determine whether leptin directly regulates cardiac fatty acid metabolism, isolated working rat hearts were perfused with 0.8 mm [9,10-(3)H]palmitate and 5 mm [1-(14)C]glucose to measure palmitate and glucose oxidation rates. Leptin (60 ng/ml) significantly increased palmitate oxidation rates 60% above control hearts (p < 0.05) and decreased TG content by 33% (p < 0.05) over the 60-min perfusion period. In contrast, there was no difference in glucose oxidation rates between leptin-treated and control hearts. Although leptin did not affect cardiac work, oxygen consumption increased by 30% (p < 0.05) and cardiac efficiency was decreased by 42% (p < 0.05). AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels. Leptin has also been shown to increase fatty acid oxidation in skeletal muscle through the activation of AMPK. However, we demonstrate that leptin had no significant effect on AMPK activity, AMPK phosphorylation state, ACC activity, or malonyl-CoA levels. AMPK activity and its phosphorylation state were also unaffected after 5 and 10 min of perfusion in the presence of leptin. The addition of insulin (100 microunits/ml) to the perfusate reduced the ability of leptin to increase fatty acid oxidation and decrease cardiac TG content. These data demonstrate for the first time that leptin activates fatty acid oxidation and decreases TG content in the heart. We also show that the effects of leptin in the heart are independent of changes in the AMPK-ACC-malonyl-CoA axis.     

Effect of Four Nematocides on Activities of Microorganisms in Soil

Tests were conducted to determine the effects of four nematocides, Dasanit, carbofuran, D-D, and Vorlex on microbial activities in a loamy sand. The results indicated that bacterial and fungal populations initially decreased with some nematocide treatments but recovered rapidly to levels similar to those in the controls. In some instances, ammonium production from added peptone increased in the nematocide-treated soils, whereas mineralization of soil organic nitrogen and nitrification and oxidation of elemental sulfur were depressed. Oxygen consumption generally increased in proportion to the concentration of nematocide in the soil. However, with Vorlex, an increase in respiration was evident at the lower concentration, whereas an inhibitory effect occurred at the higher concentration. The study indicated that indigenous soil microorganisms can tolerate these chemicals used for control of nematodes in soil.     

Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.