Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric,and ferulic acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60 degrees C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The beta-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-beta-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-beta-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

Catabolism of Substituted Benzoic Acids by Streptomyces Species

Four thermotolerant actinomycetes from soil, identified as Streptomyces albulus 321, Streptomyces sioyaensis P5, Streptomyces viridosporus T7A, and Streptomyces sp. V7, were grown at 45°C in media containing either benzoic acid or hydroxyl- and methoxyl-substituted benzoic acids as the principal carbon sources. Benzoic acid was converted to catechol; p-hydroxybenzoic, vanillic, and veratric acids were converted to protocatechuic acid; and m-hydroxybenzoic acid was converted to gentisic acid. Catechol, protocatechuic acid, and gentisic acid were cleaved by catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, and gentisate 1,2-dioxygenase, respectively. Dioxygenases appeared only in induced cultures. m-Hydroxybenzoic, m-anisic, and p-anisic acids were gratuitous inducers of dioxygenases in some strains. One strain converted vanillic acid to guaiacol.     

Possible pathways for destruction of polyaromatic hydrocarbons by some oil-degrading bacteria isolated from plant endosphere and rhizosphere

Six strains of oil-degrading bacteria isolated from the endosphere and rhizosphere of plants growing on oil polluted soils of the Irkutsk region were studied to determine the pathways for biodestruction of polyaromatic oil hydrocarbons. All strains were able to efficiently degrade polyaromatic hydrocarbons with the formation of pyrocatechin as a final product; strains 90, 108, and 112 additionally formed protocathechuic acid. The culture broth of the studied strains contained ferulic, n-coumaric, n-oxybenzoic, vanillic, and lilac acids, which probably represent metabolites of cinnamic alcohol, cinnamic aldehyde, and benzoic acid presenting in oil and metabolized by bacteria.     

Metabolism of Aromatic Amino Acids in Microorganisms

It was found that when Rhodotorula rubra IFO 0911 was grown in a phenylalanine medium, benzoic acid and p-hydroxybenzoic acid besides cinnamic acid were formed in the cultured both. The conversions of cinnamic acid into benzoic acid and of benzoic acid into p-hydroxybenzoic acid, and the degradation of p-hydroxybenzoic acid were demonstrated in intact cells of Rhodotorula rubra. These activities were observed in the cells grown on various media, including the medium containing no phenylalanine, and were found to be distributed widely in Rhodotorula. The cells of Rhodotorula rubra were also able to degrade p-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-hydroxyphenyl-acetic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). From these results, the metabolic pathways for phenylalanine and tyrosine in Rhodotorula were discussed.     

Isolation of Enterobacteria able to degrade simple aromatic compounds from the wastewater from olive oil extraction

Summary Enterobacteria growing on wastewater from olive oil extraction were selected. Among this microflora, strains of Klebsiella oxytoca and Citrobacter diversus able to degrade simple monomeric aromatic compounds were isolated by enrichment culture of the effluent lacking simple sugars. In this preliminary investigation, the phenolic acids tested on solid and liquid media were gentisic, protocatechuic, p-hydroxybenzoic, benzoic, vanillic and ferulic. It was shown that the biodegradation of an aromatic acid is tightly dependent on both the type and the position of the radical substituted on the aromatic ring. Citrobacter was the most efficient strain in metabolizing ferulic acid in liquid medium at a concentration of 1.5 g/l. The substrate biodegradation yield achieved exceeded 86%.     

Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent,Beta-Oxidative Deacetylation Pathway

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)–CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.     

Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers

To alleviate the stress of continuous cropping for cucumber continuous cropping (CCC) system, a beneficial fungus Trichoderma harzianum SQR-T037 (SQR-T037) was isolated and applied to soil to degrade allelochemicals exuded from cucumber plants in a Rhizobox experiment. The following phenolic acids (PAs), classified as allelochemicals, were isolated and identified from cucumber rhizospheres: 4-hydroxybenzoic acid, vanillic acid, ferulic acid, benzoic acid, 3-phenylpropionic acid, and cinnamic acid. Mixed PAs added in potato dextrose broth, each with 0.2 gram per liter, were completely degraded by SQR-T037 after 170 h of incubation. In Rhizobox experiments, inoculation of SQR-T037 in the CCC soil also degraded the PAs exuded from cucumber plant roots. This degradation was 88.8% for 4-hydroxybenzoic acid, 90% for vanillic acid, 95% for benzoic acid, and 100% for ferulic acid, 3-phenylpropionic acid, and cinnamic acid at 45 days after plantation. Simultaneously, a significant (p ≥ 0.05) decrease in the disease index of Fusarium wilt and an increase in dry weights of cucumber plants were obtained in pot experiments by application of SQR-T037. This was mostly attributed to degradation of PAs exuded from cucumber roots in CCC soil by SQR-T037 and alleviation of the allelopathic stress. Application of beneficial microorganisms, such as SQR-T037 that biodegrades allelochemicals, is a highly efficient way to resolve the problems associated with continuous cropping system.     

Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.     

The properties of syringyl, guaiacyl and p-hydroxyphenyl artificial lignins

1. Artificial lignins have been produced on potato parenchyma. 2. The methoxyl-free lignin and 4-hydroxy-3-methoxy (guaiacyl) lignins could be estimated by the sulphuric acid method but the 4-hydroxy-3,5-dimethoxy (syringyl) lignins could not. 3. Permanganate oxidation of isolated p-coumaric lignin gave 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and small amounts of hydroxytrimesic acid and 4-hydroxyphthalic acid. Ferulic lignin gave vanillic acid and 5-carboxyvanillic acid and also small amounts of 4-hydroxybenzoic acid and dehydrodivanillic acid. The sinapic lignin gave traces of syringic acid and of 4-hydroxybenzoic acid. 4. The p-coumaric lignin is a highly condensed polymer. The ferulic lignin is partly uncondensed and partly condensed through the 5-position like gymnosperm lignin. The sinapic lignin shows no evidence of condensation and is probably an ether-linked polymer.     

Studies on Flavor Components in Soybean

Ethanol (1:1) extract of defatted soybean flour was fractionated systematically and the resulting phonolic acid fraction was investigated. This fraction had strong phenol-like flavor and contained at least seven phenolic acids including syringic, vanillic, ferulic, gentisic, salicylic, p-coumaric, and p-hydroxybenzoic acids. The main component among these was syringic acid, which was isolated as 3,5-dinitrobenzoate.

In addition, two isomers of chlorogenic acids, presumably isochlorogenic and chlorogenic acids approximately in a ratio of 1 : 10, were found in this extract. These substances have sour, bitter and astringent flavors.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : II. Effects of Precursor Feeding and Metabolic Inhibitors

Feeding of cinnamic acid and ferulic acid to non-treated and chitosan-treated cell suspension cultures of Vanilla planifolia resulted in the formation of trace amounts of p-hydroxy benzoic acid (5.2 micrograms per gram fresh weight of cells) and vanillic acid (6.4 micrograms per gram fresh weight of cells), respectively. Addition of a 4-hydroxycinnamate: CoA-ligase inhibitor, 3,4-(methylenedioxy)-cinnamic acid (MDCA), resulted in a reduced biosynthesis of ligneous material with a simultaneous significant increased vanillic acid formation (around 75 micrograms per gram fresh weight of cells). A K₁ of 100 micromolar for 4-hydroxycinnamate: CoA-ligase in a crude preparation was estimated for this inhibitor. It is suggested that the conversion of cinnamic acids into benzoic acids does not involve cinnamoyl CoA esters as intermediates. Feeding of ¹⁴C-cinnamic acid and ¹⁴C-ferulic acid to cells treated with MDCA indicate that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in these cultivated cells of V. planifolia.     

Labotory studies on retting of coconut husk

Laboratory rets set by soaking the coconut husk in water were monitored by physico-chemical analysis of leachate and husk. Increasing amounts of pectins and phenols were leached in ret liquor during a period of 9 days of soaking. A GLC analysis revealed the presence of various aromatic p-hydroxy-phenolic and methoxy compounds, such as benzoic, p-hydroxybenzoic, veratric, gentisic, syringic, p-coumaric, vanillic and cinnamic acids in ret liquor, ret liquor supplemented with Fusarium solani and controls. Aeration turned the leachate and the fibre brown in colour. Pectins, phenols, Klason and milled wood lignin (MWL) and cellulose contents of soaked husk varied disproportionately. Tensile strength and the diameters of the fibres varied widely; the soaked husk gave softer fibre than the unsoaked ones.     

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml⁻¹ for two of the three test organisms).     

Anaerobic Biodegradation of Eleven Aromatic Compounds to Methane

A range of 11 simple aromatic lignin derivatives are biodegradable to methane and carbon dioxide under strict anaerobic conditions. A serum-bottle modification of the Hungate technique for growing anaerobes was used for methanogenic enrichments on vanillin, vanillic acid, ferulic acid, cinnamic acid, benzoic acid, catechol, protocatechuic acid, phenol, p-hydroxybenzoic acid, syringic acid, and syringaldehyde. Microbial populations acclimated to a particular aromatic substrate can be simultaneously acclimated to other selected aromatic substrates. Carbon balance measurements made on vanillic and ferulic acids indicate that the aromatic ring was cleaved and that the amount of methane produced from these substrates closely agrees with calculated stoichiometric values. These data suggest that more than half of the organic carbon of these aromatic compounds potentially can be converted to methane gas and that this type of methanogenic conversion of simple aromatics may not be uncommon.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : III. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids

Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-¹⁴C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia.     

Composes phenoliques du latex d'Hevea brasiliensis: aglycones

The latex of Hevea brasiliensis; clone PR 107, contains from 160 to 1100 μg total phenolics per ml. This wide variability is associated with season, tapping system and application or not of a stimulant (Ethrel). The following aglycones have been identified in hydrolysed extracts: vanillic, salicyclic, syringic, gentisic, p- and m-hydroxybenzoic and protocatechuic acids; scopoletin, aesculetin and coumarin; ferulic, sinapic, caffeic, o- and p-coumaric acids; quercetin and kaempferol; tyrosine and dihydroxyphenylalanine. Flavans and condensed tannins are also present in latex.     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

Ueber den abbau von polyphenolen durch pilze der gattung Fusarium

The ability of 16 Fusarium species to degrade polyphenols was investigated. Phenols, benzoic acids, cinnamic acids, flavonoids and isoflavones are efficiently catabolized by all strains investigated. o-coumaric acid is transformed into 4-hydroxycoumarin by 7 species. A pronounced capability for methyl ether cleavage is demonstrated by stepwise o-demethylation of veratric acid and 5,7,4′-trimethoxyisoflavone. The latter compound is degraded via the sequence: 5,7,4′-trimethoxyisoflavone → 5,4′-dimethoxy-7-hydroxyisoflavone → biochanin A → genistein → orobol → ring fission products.     

Biodegradation of aromatic compounds byRhodopseudomonas gelatinosa

A few photosynthetic bacteria have been isolated from a biological treatment system treating producer gas plant effluent. One of them was identified asRhodopseudomonas gelatinosa on the basis of physiological and morphological characteristics. Biodegradation of aromatic compounds byR. gelatinosa appears not to have been reported in the literature. The culture was found to degrade benzoic acid,p-hydroxybenzoic acid, cinnamic acid, andp-coumaric acid. The doubling time for this culture was found to be 18, 19, 23, and 31 h for benzoic acid, p-hydroxybenzoic acid, cinnamic acid, andp-coumaric acid respectively.     

Involvement of phenolic acids in disease resistance of potato tubers from CEPA-treated plants

Treatment of vegetative parts of potato plants two weeks before the harvest with 0.2% 2-chloroethylphosphonic acid (CEPA) delayed the sprouting of tubers and increased the resistance of tubers to infections caused byPhytophthora infestans, Erwinia carotovora andFusarium spp. during the storage period. Levels of free, soluble ester- and glycoside-bound phenolic acids and cell wall-bound phenolics were determined in cortical parenchyma of tubers (periderm). The enhancement of phenolic acids in tubers from treated plants was caused primarily by the increase in the contents of free vanillic, caffeic andp-hydroxybenzoic acids and cell wall-bound ferulic, vanillic andp-coumaric acids.