Hydrogen production from aromatic acids byRhodopseudomonas palustris

The capability of five strains of the phototrophic bacteriumRhodopseudomonas palustris to produce molecular hydrogen (H₂) from the aromatic acids benzoate,p-hydroxybenzoate, cinnamate and D- and L-mandelate was investigated. Optimal H₂ production was achieved when the strains were grown anaerobically in the light at 10,000 lx under nitrogen (N) limitation using 1 mM L-glutamate as an N source. In the presence of 2 mM benzoate or L-mandelate as carbon and electron sources, strain DSM 131 produced 45% H₂ of the maximal theoretical value and strain F2 32%, respectively. Increased H₂ production correlated with increased nitrogenase activities, but H₂ formation was not further stimulated by inhibition of the H₂ uptake (hup) hydrogenase with ethylenediaminetetraacetic acid (EDTA).     

Phenotype Fingerprinting Suggests the Involvement of Single-Genotype Consortia in Degradation of Aromatic Compounds by Rhodopseudomonas palustris

Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms that maintain the redox homeostasis during the degradation of aromatic compounds by one organism are not fully understood. Here we present a hypothesis that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris forms its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. We have revealed the consortia from large-scale measurements of mRNA and protein expressions under p-coumarate, benzoate and succinate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach, marker genes for known R. palustris phenotypes are employed to determine the relative expression levels of genes and proteins in aromatics versus non-aromatics degrading condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading conditions may lead to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO₂ and H₂. Benzoate degrading conditions may also produce at least three subpopulations utilizing benzoate, CO₂ and H₂, and N₂ and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The N₂-fixing subpopulation in the benzoate degrading consortium has preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. This subpopulation in the consortium was confirmed in an independent experiment by consumption of dissolved nitrogen gas under the benzoate degrading conditions.     

Growth performance and pathway flux determine substrate preference of Alcaligenes eutrophus during growth on acetate plus aromatic compound mixtures

During batch growth of Alcaligenes eutrophus on various aromatic compounds in the presence of acetate, several distinct behaviour patterns were observed. The utilization of substrates of the meta pathway (phenol or p-cresol) was inhibited by acetate. When the aromatic was a substrate of the p-hydroxybenzoate branch of the ortho pathway, growth was mixotrophic, i.e. both substrates were consumed simultaneously. For the substrates of the gentisate pathway or the benzoate branch of the ortho pathway, substrate preference was governed by growth performance. Aromatic compounds enabling growth rate and yields higher than those obtained on acetate alone (i.e. benzoate, benzaldehyde, m-hydroxybenzoate and gentisate) inhibited acetate utilization, while acetate was the substrate consumed preferentially in mixtures containing aromatic compounds supporting only slow growth (i.e. benzoyl formate and 4-fluorobenzoate). Received: 18 April 1996 / Received revision: 9 July 1996 / Accepted: 15 July 1996     

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.In the opinion of the authors, D.J. Fairley and G. Wang should be regarded as joint first authors.     

Methane Fermentation of Ferulate and Benzoate: Anaerobic Degradation Pathways

The anaerobic biodegradation of ferulate and benzoate in stabilized methanogenic consortia was examined in detail. Up to 99% of the ferulate and 98% of the benzoate were converted to carbon dioxide and methane. Methanogenesis was inhibited with 2-bromoethanesulfonic acid, which reduced the gas production and enhanced the buildup of intermediates. Use of high-performance liquid chromatography and two gas chromatographic procedures yielded identification of the following compounds: caffeate, p-hydroxycinnamate, cinnamate, phenylpropionate, phenylacetate, benzoate, and toluene during ferulate degradation; and benzene, cyclohexane, methylcyclohexane, cyclohexanecarboxylate, cyclohexanone, 1-methylcyclohexanone, pimelate, adipate, succinate, lactate, heptanoate, caproate, isocaproate, valerate, butyrate, isobutyrate, propionate, and acetate during the degradation of either benzoate or ferulate. Based on the identification of the above compounds, more complete reductive pathways for ferulate and benzoate are proposed.     

Benzoyl Coenzyme A Pathway-Mediated Metabolism of meta-Hydroxy-Aromatic Acids in Rhodopseudomonas palustris

Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.     

Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source

Abstract Enrichment cultures for anoxygenic phototrophs capable of using cinnamic acid as sole organic carbon source consistently yielded the nonsulfur purple bacterium Rhodopseudomonas palustris . Pure cultures of R. palustris obtained from the enrichments grew photoheterotrophically on cinnamate and benzoate as well as on derivatives of these compounds. Photosynthetic growth on cinnamate was greatly stimulated by addition of exogenous CO₂, and resulted in breakage of the aromatic nucleus. Growth yield studies suggested that cinnamate was converted by R. palustris to intermediates that can be quantitatively assimilated into cell material.     

Fermentative degradation of monohydroxybenzoates by defined syntrophic cocultures

From anaerobic freshwater enrichment cultures with 3-hydroxybenzoate as sole substrate, a slightly curved rod-shaped bacterium was isolated in coculture with Desulfovibrio vulgaris as hydrogen scavenger. The new isolate degraded only 3-hydroxybenzoate or benzoate, and depended on syntrophic cooperation with a hydrogenoxidizing methanogen or sulfate reducer. 3-Hydroxybenzoate was degraded via reductive dehydroxylation to benzoate. With 2-hydroxybenzoate (salicylate), short coccoid rods were enriched from anaerobic freshwater mud samples, and were isolated in defined coculture with D. vulgaris. This isolate also fermented 3-hydroxybenzoate or benzoate in obligate syntrophy with a hydrogen-oxidizing anaerobe. The new isolates were both Gram-negative, non-sporeforming strict anaerobes. They fermented hydroxybenzoate or benzoate to acetate, CO₂, and, presumably, hydrogen which was oxidized by the syntrophic partner organism. With hydroxybenzoates, but not with benzoate, Acetobacterium woodii could also serve as syntrophic partner. Other substrates such as sugars, alcohols, fatty or amino acids were not fermented. External electron acceptors such as sulfate, sulfite, nitrate, or fumarate were not reduced. In enrichment cultures with 4-hydroxybenzoate, decarboxylation to phenol was the initial step in degradation which finally led to acetate, methane and CO₂.     

The metabolism of p-hydroxybenzoate by Rhodopseudomonas palustris and its regulation

Summary Rhodopseudomonas palustris metabolizes p-hydroxybenzoate aerobically via a series of specific reactions which are partly described. The pathway similar to that described for aerobic pseudomonads. The requisite enzymes are absent in extracts of cells grown anaerobically under photosynthetic conditions with p-hydroxybenzoate, benzoate and malate, or aerobically with benzoate or malate. An intermediate of the reaction sequence presumably induces in a retrograde fashion.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday. I wish especially to acknowledge a debt to Prof. C. B. van Niel, who offered an assistant so much encouragement and suggested this and so many other problems in his summer Microbiology Course.The early portion of this work was done in collaboration with Dr. E. R. Leadbetter in the laboratory of R. Y. Stanier at Berkeley. Mrs. C. Feist generously provided a sample of -hydroxymuconic semialdehyde. This investigation was supported by the U.S. Public Health Service, Grant HD-02448.     

Cinnamyl alcohol: An attractant of the flower thrips Frankliniella intonsa

Flower-inhabiting thrips find hosts using olfactory and visual cues. In this study, we report the identification of a plant-produced attractant of the flower thrips Frankliniella intonsa (Trybom), an important agricultural pest worldwide. GC–MS analysis of solid-phase microextraction samples from blueberry flowers, Vaccinium corymbosum L., that mediate the attraction of adult F. intonsa revealed that the major component was cinnamyl alcohol, followed by cinnamyl acetate, cinnamaldehyde, germacrene D, β-bourbonene, β-caryophyllene, and benzyl benzoate. The biological activity of the floral compounds was investigated using commercial cinnamaldehyde, cinnamyl alcohol, β-caryophyllene, cinnamyl acetate, and benzyl benzoate in hot pepper (Capsicum annuum L.) fields. Significantly more F. intonsa males and females were caught in red delta traps with cinnamyl alcohol than in all other traps. Cinnamaldehyde and cinnamyl acetate attracted adult F. intonsa but were not as attractive as cinnamyl alcohol. β-Caryophyllene and benzyl benzoate were not attractive. Furthermore, the addition of four minor components to cinnamyl alcohol did not result in increased trap catches relative to cinnamyl alcohol alone, indicating that cinnamyl alcohol is responsible for attracting adult F. intonsa toward blueberry flowers. Therefore, this phenylpropanoid could be used as an effective lure for monitoring and controlling F. intonsa.     

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, -carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.     

Anoxygenic degradation of aromatic substances byRhodopseudomonas palustris

Three strains of the phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris were isolated from different environments and were evaluated for their aromatic degradative potential under phototrophic conditions. All three strains (PFR1, PNR4, and MRL1) utilized benzoate, 4-hydroxybenzoate, 4-aminobenzoate, 4-aminophenol, cinnamate, ferulate, phloroglucinol, and 4-dimethylaminobenzaldehyde in the absence of exogenous CO2. 4-Aminobenzoate and 4-aminophenol served as a carbon and nitrogen source for all the three strains. Utilization of 4-aminophenol was enhanced in the presence of 4-hydroxybenzoate. Salicylate was utilized by PFR1 and MRL1 strains, and phenol was utilized by the MRL1 strain only in the presence of exogenous CO2.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Utilization of aromatic compounds by phototrophic purple nonsulfur bacteria

Biodegradation of aromatic compounds byRhodopseudomonas blastica andRhodospirillum rubrum appears to be lacking in the literature. The above species grew phototrophically (illuminated anaerobic conditions) on a variety of organic compounds. They were found to degrade benzoate, benzyl alcohol, 4-hydroxy-3,5-dimethoxybenzoate (Syringate) and 4-hydroxy-3-methoxybenzoate (vanillate). The ability of the above species to photocatabolize aromatic compounds indicates that these organisms may be ecologically significant as scavengers of aromatic derivatives in illuminated anaerobic habitats in nature.     

New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp.

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds compounds completely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via benzoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aerobically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-hydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoate was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments with whole cells seemingly suggested a sequential induction of enzymes of a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Cells grown aerobically with benzoate contained a benzoate-CoA ligase (AMP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3-hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. Cells grown aerobically with 3-hydroxybenzoate contained a similarly active CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (AMP forming). Extracts from cells grown aerobically with benzoate catalyzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidation of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzyme, benzoyl-CoA 3-monooxygenase, was specifically induced during aerobic growth with benzoate and converted [U-14C]benzoyl-CoA stoichiometrically to [14C]3-hydroxybenzoyl-CoA.     

Optimization of the solvent-tolerant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 as host for the production of <Emphasis Type="Italic">p</Emphasis>-coumarate from glucose

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular l-tyrosine availability, and formation of the by-product cinnamate via l-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l⁻¹) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.     

Isolation and characterization of a new spore-forming sulfate-reducing bacterium growing by complete oxidation of catechol

A new mesophilic sulfate-reducing bacterium, strain Groll, was isolated from a benzoate enrichment culture inoculated with black mud from a freshwater ditch. The isolate was a spore-forming, rod-shaped, motile, gram-positive bacterium. This isolate was able of complete oxidation of several aromatic compounds including phenol, catechol, benzoate, p-and m-cresol, benzyl alcohol and vanillate. With hydrogen and carbon dioxide, formate or O-methylated aromatic compounds, autotrophic growth during sulfate reduction or homoacetogenesis was demonstrated. Lactate was not used as a substrate. SO _inf4 ^sup2- , SO _inf3 ^sup2- , and S₂O _inf3 ^sup2- were utilized as electron acceptors. Although strain Groll originated from a freshwater habitat, salt concentrations of up to 30 g·l^-1 were tolerated. The optimum temperature for growth was 35–37°C. The G+C content of DNA was 42.1 mol%. This isolate is described as a new species of the genus Desulfotomaculum.     

Effect of Fluorination on Skin Sensitization Potential and Fragrant Properties of Cinnamyl Compounds

A series of three α‐ and three β‐fluorinated representatives of the family of cinnamate‐derived odorants (cinnamaldehyde ( 1 ), cinnamyl alcohol ( 2 ), and ethyl cinnamate ( 3 )) as used as fragrance ingredients is described. Olfactive evaluation shows that the fluorinated compounds exhibit a similar odor profile to their parent compounds, but the olfactive detection thresholds are clearly higher. In vitro evaluation of the skin sensitizing properties with three different assays indicates that α‐fluorination of Michael acceptor systems 1 and 3 slightly improves the skin sensitization profile. α‐Fluorocinnamyl alcohol 2b is a weaker skin sensitizer than cinnamyl alcohol 2a by in vitro tests and the fluorinated product drops below the sensitization threshold of the KeratinoSens^® assay. On the other hand, β‐fluorination of compounds 1  –  3 results in highly reactive products which display a worsened in vitro skin sensitization profile.     

Anaerobic degradation of phenol and phenol derivatives by Desulfobacterium phenolicum sp. nov.

A new sulfate-reducing bacterium was enriched and isolated from marine sediment with phenol as sole electron donor and carbon source. Strain Ph01 grew well in defined media without growth factors. Further aromatic compounds oxidized by strain Ph01 were benzoate, phenylacetate, 2-hydroxybenzoate, 4-hydroxybenzoate, 4-hydroxyphenylacetate, p-cresol, indole, anthranilic acid, and phenylalanine. Various fatty acids, alcohols and dicarboxylic acids were also utilized by strain Ph01. Sulfate and thiosulfate served as electron acceptors and were reduced to H₂S. Stoichiometric measurements with strain Ph01 showed complete oxidation of phenol to CO₂. Cytochromes and menaquinone MK-7(H₂) were present; desulfoviridin could not be detected. Strain Ph01 is described as type strain of the new species Desulfobacterium phenolicum.In further marine enrichments with 4-hydroxybenzoate, 4-hydroxyphenylacetate, p-cresol or o-cresol as substrates and sulfate as electron acceptor a variety of morphologically different sulfate-reducing bacteria developed. However, since the new isolate strain Ph01 was able to degrade all these aromatic compounds (except o-cresol) no further studies with the enrichment cultures were carried out.