Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

The oxidation of fatty acids combined with albumin by isolated rat liver mitochondria

Long chain fatty acids at concentrations inhibiting mitochondrial respiration were, in the presence of serum albumin, found to produce almost as high a rate of oxygen uptake as alpha-ketoglutarate, succinate, or acetate. This oxidation was characterized in terms of its coupling to phosphorylation, need for cofactors, and production of different metabolites during the reactions. Fatty acids were oxidized to carbon dioxide, acetoacetate, beta-hydroxybutyrate, and other water-soluble metabolites, tentatively identified as intermediates of the citric acid cycle. An agent to spark the citric acid cycle and adenosine tri- or monophosphate were necessary for optimal oxidation rate, as described for other fatty acid oxidation systems. Balance experiments with different amounts of malate were performed with incubations lasting as long as oxygen uptake took place. In the presence of 1 mumole of malate, practically all added palmitic acid was used up and found to be converted primarily to carbon dioxide, acetoacetate, and other water-soluble metabolites of which the major part was tentatively identified as succinate. A significant portion was found in mitochondrial phospholipids. With 10 mumoles of malate some palmitic acid remained in the system, while a comparatively small amount was converted to carbon dioxide, and a major part was found as succinate. Here also incorporation into phospholipids occurred. With no malate added, fatty acid oxidation was much smaller than with malate, although significant conversion to carbon dioxide took place. Only a little succinate and phospholipid were found. Oxygen uptake was greater than a theoretical value calculated from radioactive balance experiments. It was concluded that albumin contains oxidizable material even after extraction and dialysis. Albumin at high concentrations inhibited both fatty acid and alpha-ketoglutarate oxidation. The oxidation of long chain fatty acids in high concentrations in the form of albumin-fatty acid complex was coupled to phosphorylation. Thus P:O ratios above 2 were found as well as evidence for respiratory control. It was concluded that oxidation of long chain fatty acids by isolated mitochondria occurs from their albumin complex. This process can also be studied at high concentrations of fatty acids, where high rates of oxygen uptake are obtained from oxidation which is coupled to phosphorylation.     

Genome sequence of the unclassified marine Gammaproteobacterium BDW918

The unclassified marine gammaproteobacterium BDW918, which utilizes volatile fatty acids but not most common carbohydrates and amino acids, was isolated from Dokdo seawater in South Korea. Here we present a draft genome of the strain BDW918, which encodes many putative genes related to fatty acid metabolism and aromatic hydrocarbon degradation.     

Influence of particle size and pH on anaerobic degradation of cellulose by ruminal microbes

Batch experiments were performed to investigate the influence of cellulose particle size and pH on the anaerobic degradation of crystalline cellulose by ruminal microbes. At a particle size of 50 μm there was a higher hydrolysis and acidogenesis rate, and a reduced degradation time, than for 100-μm particles. Reduction in cellulose particle size resulted in decreased methane production, but an increase of soluble products. Cellulose degradation increased with pH from pH 6.0 to 7.5, whereas at pH⩽5.5 there was no degradation. The inhibitory effect of low pH (⩽5.5) on ruminal microbes was not completely remedied even when the pH of the medium was adjusted to a neutral range. In an anaerobic cellulosic waste degrading system inoculated with ruminal microbes the fermentation system should therefore be maintained above pH 6.0. In all cases, volatile fatty acids were the major water-soluble products of cellulose degradation; acetate and propionate accounted for more than 90% of the volatile fatty acid total.     

Naphthalene degradation and incorporation of naphthalene-derived carbon into biomass by the thermophile Bacillus thermoleovorans

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60 degrees C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO(2), was determined by the application of [1-(13)C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2, 3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.     

Aromatic and Volatile Acid Intermediates Observed during Anaerobic Metabolism of Lignin-Derived Oligomers

Anaerobic enrichment cultures acclimated for 2 years to use a ¹⁴C-labeled, lignin-derived substrate with a molecular weight of 600 as a sole source of carbon were characterized by capillary and packed column gas chromatography. After acclimation, several of the active methanogenic consortia were inhibited with 2-bromoethanesulfonic acid, which suppressed methane formation and enhanced accumulation of a series of metabolic intermediates. Volatile fatty acids levels in 2-bromoethanesulfonic acid-amended cultures were 10 times greater than those in the uninhibited, methane-forming consortia with acetate as the predominant component. Furthermore, in the 2-bromoethanesulfonic acid-amended consortia, almost half of the original substrate carbon was metabolized to 10 monoaromatic compounds, with the most appreciable quantities accumulated as cinnamic, benzoic, caffeic, vanillic, and ferulic acids. 2-Bromoethanesulfonic acid seemed to effectively block CH₄ formation in the anaerobic food chain, resulting in the observed buildup of volatile fatty acids and monoaromatic intermediates. Neither fatty acids nor aromatic compounds were detected in the oligolignol substrate before its metabolism, suggesting that these anaerobic consortia have the ability to mediate the cleavage of the β-aryl-ether bond, the most common intermonomeric linkage in lignin, with the subsequent release of the observed constituent aromatic monomers.     

Metabolites and biodegradation pathways of fatty alcohol ethoxylates in microbial biocenoses of sewage treatment plants.

The biodegradation of fatty alcohol polyglycol ethers was studied by analyzing the 14C-labeled intermediates isolated from the effluent of a model continuous-flow sewage treatment plant after dosage of either alkyl- or heptaglycol-labeled stearyl alcohol ethoxylate (SA-7EO). In each case, uncharged and carboxylated (mainly dicarboxylated) polyethylene glycols constituted the most prominent metabolites. The results indicate that there is a faster degradation of the alkyl than the polyethylene glycol moiety and that there are two distinct primary degradation mechanisms acting simultaneously in microbial biocenoses: intramolecular scission of the surfactant as well as omega- and beta-oxidation of the alkyl chain. Characterization of the bulk of 14C-labeled metabolites as a homologous series of neutral and acidic polyglycol units and identification of several C2-fragments accounted for the depolymerization of the hydrophilic part of the surfactant by stepwise cleavage of ether-bound EO units; from additional degradation studies employing either neutral or carboxylated 14C-labeled polyethylene glycols as model metabolites, it was concluded that hydrolytic as well as oxidative cleavage of C2-units is involved. Most of the identified low-molecular-weight 14C-labeled acids suggest an ultimate degradation of EO monomers by the oxidative dicarbonic acid cycle or the glycerate pathway or both. In addition, the finding of considerable amounts of oxalic and formic acids allow consideration of an additional mineralization route via glyoxylic, oxalic, and formic acids. The simultaneous action of different degradation mechanisms indicates the involvement of several distinct bacterial groups in the biodegradation of fatty alcohol ethoxylates under environmental conditions.     

Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates

Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.     

Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.     

Cellular fatty acid uptake: the contribution of metabolism

PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.     

Lipid degradation during manufacture of black tea

About 85% of the fatty acids liberated during the manufacture of black tea can be attributed to autolysis of 4 major polar lipid classes in tea leaf tissue, phosphatidylcholine, monogalactosyldiglyceride, digalactosyldiglyceride and phosphatidylethanolamine. Linolenic, linoleic and palmitic acids are the principal fatty acids released from these lipids and they all undergo further degradation. Linolenic acid (60% of fatty acids released) is derived mainly from galactolipids and thus the upper limit of release is dependent on the chloroplast maturity and content of the leaf tissues. Lipid breakdown is complete after 2 hr fermentation and, as there appears to be no accumulation of long chain fatty acid intermediates, it is probable that volatile production has also ceased at this time.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Novel scheme for biosynthesis of aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with L-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ((14)C- and (13)C-labelled L-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that L-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from L-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via beta-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a beta-oxidation degradation intermediate. To our knowledge, this is the first time that a beta-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from L-phenylalanine is proposed.     

Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.     

Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.     

Naphthalene Degradation and Incorporation of Naphthalene-Derived Carbon into Biomass by the Thermophile Bacillus thermoleovorans

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO₂, was determined by the application of [1-¹³C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.     

Cellular fatty acid patterns inPseudomonas sp. CF600 during catechol and phenol degradation in media supplemented with glucose as an additional carbon source

Fatty acid composition inPseudomonas sp. CF600 during degradation of catechol and phenol individually and their mixture was investigated. Moreover, the influence of glucose as an additional, easily degradable carbon source on fatty acid profiling in bacteria grown on these aromatic substrates was studied. Both catechol and phenol treatments caused in bacterial cells crucial changes in the distribution of tested groups of fatty acids. The major changes included the increase of fatty acid saturation, decrease in the percentage of cyclopropane fatty acid 17:0cy and the appearance of branched and hydroxy fatty acids. Under catechol, phenol and their mixture exposure saturated/unsaturated ratio showed the value 6.5, 5.68 and 6.38 whereas in control cells this ratio reached the value 3.05. As a response to aromatic compounds bacteria formed fatty acids that were not detected in control cells growing on glucose. It has been demonstrated that the supplementation of cultured media containing single aromatic substrates or/and their mixture with glucose resulted in changes in degradation rates of catechol and phenol. It seemed that glucose influenced some metabolic pathways responsible for the assimilation of aromatic compounds. The incubation of cells in the presence of aromatic compounds and glucose rapidly led to alterations of whole-cell derived fatty acid composition. The most important changes were associated with saturation level of fatty acids and cyclopropane fatty acid contents.     

FAME profiles in Pseudomonas vesicularis during catechol and phenol degradation in the presence of glucose as an additional carbon source.

The aim of this study was to evaluate the impact of catechol and phenol added to culture media separately and with glucose as an additional, easily-degradable carbon source on fatty acid methyl ester (FAME) composition in Pseudomonas vesicularis. Simultaneously, the degradation rates of aromatic substrates used were investigated in single and binary substrate systems. Both catechol and phenol treatments caused changes in the distribution of tested groups of fatty acids. The most noticeable changes included an increase in degree of fatty acid saturation, the appearance of branched and disappearance of hydroxy fatty acids as compared to the control sample with glucose. Under catechol or phenol treatment sat/unsat ratio showed the values of 8.63 and 11.38, respectively, whereas in control cells it reached the value of 2.66. The high level of saturation comes from the high content of cyclopropane fatty acids in bacteria under exposure to aromatic substrates, regardless of the presence of glucose. In these treatments their content was more than 3-fold higher compared to the control. It has been demonstrated that glucose supplementation of culture media containing single aromatic substrate extended the degradation rates of catechol and phenol by P. vesicularis, caused an increase in number of cells but did not significantly change the fatty acid profiles in comparison with bacteria growing on catechol and phenol added to the media individually.     

Oxylipins: Structurally diverse metabolites from fatty acid oxidation

Oxylipins are lipophilic signaling molecules derived from the oxidation of polyunsaturated fatty acids. Initial fatty acid oxidation occurs mainly by the enzymatic or chemical formation of fatty acid hydroperoxides. An array of alternative reactions further converting fatty acid hydroperoxides gives rise to a multitude of oxylipin classes, many with reported signaling functions in plants. Oxylipins include the phytohormone, jasmonic acid, and a number of other molecules including hydroxy-, oxo- or keto-fatty acids or volatile aldehydes that may perform various biological roles as second messengers, messengers in inter-organismic signaling, or even as bactericidal agents. The structural diversity of oxylipins is further increased by esterification of the compounds in plastidial glycolipids, for instance the Arabidopsides, or by conjugation of oxylipins to amino acids or other metabolites. The enzymes involved in oxylipin metabolism are diverse and comprise a multitude of examples with interesting and unusual catalytic properties. In addition, the interplay of different subcellular compartments during oxylipin biosynthesis suggests complex mechanisms of regulation that are not well understood. This review aims at giving an overview of plant oxylipins and the multitude of enzymes responsible for their biosynthesis.