Aerobic and anaerobic catabolism of phthalic acid by a nitrate-respiring bacterium

A Bacillus sp., isolated by anaerobic enrichment on a o-phthalic acid-nitrate medium, grew either aerobically or anaerobically on phthalic acid. Cells grown anaerobically on phthalate immediately oxidized phthalate and benzoate with nitrate, whereas aerobic oxidation only occurred after a lag period and was inhibited by chloramphenicol. 2-Fluoro-and 3-fluorobenzoate were formed from 3-fluorophthalate by cells grown anaerobically on phthalate. Aerobically grown cells immediately oxidized phthalate, benzoate, 3-hydroxybenzoate and gentisate with oxygen. The aerobic and anaerobic route of catabolism of phthalate may thus share an initial decarboxylation to benzoate. This is the first report of the anaerobic dissimilation of phthalic acid by a pure bacterial culture.     

Anaerobic Degradation of the Benzene Nucleus by a Facultatively Anaerobic Microorganism

A bacterium was isolated by elective culture with p-hydroxybenzoate as substrate and nitrate as electron acceptor. It grew either aerobically or anaerobically, by nitrate respiration, on a range of aromatic compounds. The organism was identified as a pseudomonad and was given the trivial name Pseudomonas PN-1. Benzoate and p-hydroxybenzoate were metabolized aerobically via protocatechuate, followed by meta cleavage catalyzed by protocatechuic acid-4,5-oxygenase, to yield alpha-hydroxy-gamma-carboxymuconic semialdehyde. Pseudomonas PN-1 grew rapidly on p-hydroxybenzoate under strictly anaerobic conditions, provided nitrate was present, even though protocatechuic acid-4,5-oxygenase was repressed. Suspensions of cells grown anaerobically on p-hydroxybenzoate oxidized benzoate with nitrate and produced 4 to 5 mumoles of CO(2) per mumole of benzoate added; these cells did not oxidize benzoate aerobically. The patterns of the oxidation of aromatic substrates with oxygen or nitrate by cells grown aerobically or anaerobically on different aromatic compounds indicated that benzoate rather than protocatechuate was a key intermediate in the early stages of anaerobic metabolism. It was concluded that the pathway for the anaerobic breakdown of the aromatic ring is different and quite distinct from the aerobic pathway. Mechanisms for the anaerobic degradation of the benzene nucleus by Pseudomonas PN-1 are discussed.     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Anaerobic degradation of 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria.

Three strains of anaerobically benzoate-degrading, denitrifying bacteria of the genus Pseudomonas were able to grow on 2-fluorobenzoate as the sole carbon and energy source. Fluoride ion release was stoichiometric, and the reduction of dissolved organic carbon indicated total degradation. Cells grown anaerobically with benzoate were adapted for immediate growth with 2-fluorobenzoate, and both compounds were substrates for an inducible benzoyl-coenzyme A synthetase, the initial enzyme of anaerobic degradation. It is proposed that fluoride is eliminated gratuitously by a regioselective reaction in a sequence common to both carbon sources. Benzoate, but not 2-fluorobenzoate, was oxidized by aerobically grown cells.     

Activation of aromatic acids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomonas strain

The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K _m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K _m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K _m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.     

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1

Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O₂ or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O₂. The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.     

Degradation of 2-fluorobenzoate by a pseudomonad

Pseudomonas (spp), isolated from a complex petrochemical sludge, was able to utilize 2-fluorobenzoate as its sole source of carbon and energy. At the end of the growth phase, about 42% of the organically bound fluoride was released. Catechol, 3-fluorocatechol, and 6-fluorodihydrodihydroxybenzoate were confirmed as intermediates by chromatographic and spectral analyses. During 2-fluorobenzoate metabolism, fluoride is eliminated before the aromaticity of the ring is lost. Twofold higher levels of catechol 1,2-oxygenase were detected in 2-fluorobenzoate-grown cells compared with cells grown on benzoate. When used as assay substrates, 3-chlorocatechol showed less catechol 1,2-oxygenase activity than catechol or 4-chlorocatechol. The ability to degrade 4-fluorobenzoate could be transferred toPseudomonas (spp) by the conjugal transfer of plasmid pWR1 fromPseudomonas sp. B13.     

Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate

Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).     

Two-dimensional gel electrophoretic analysis of Escherichia coli proteins: influence of various anaerobic growth conditions and the fnr gene product on cellular protein composition

Two-dimensional gel electrophoresis was used to examine the response of the cellular proteins of Escherichia coli to various anaerobic growth conditions and to the presence or absence of a functional Fnr protein. The steady-state levels of 125 polypeptides were found to vary in either a positive or negative manner, with many polypeptides being affected under a number of conditions. A large number (21) of the anaerobically inducible polypeptides were shown to be totally independent of the presence of Fnr while 22 were shown to be reduced in a fnr mutant under all anaerobic growth conditions tested. A total of 8 proteins were shown to be reduced in a fnr mutant only in aerobically grown cells indicating that the Fnr protein has a function in the presence of oxygen. This was further confirmed by the observation that 15 anaerobically inducible polypeptides were also found to show an increase in aerobically grown cells, however, only in a fnr strain. This latter finding implies that Fnr may also exhibit repressor function. This effect of Fnr-dependent repression was also observed with several polypeptides in anaerobically grown cells.Abbreviation CRP cyclic AMP receptor protein     

Effect of fluorinated analogues of phenol and hydroxybenzoates on the anaerobic transformation of phenol to benzoate

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At 250 M 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of 250 M 3-fluorophenol. In contrast, 4-fluorophenol at 2 mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2 : 1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para-carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.Abbreviations 3F4HB 3-fluoro-4-hydroxybenzoate - 3F2HB 3-fluoro-2-hydroxybenzoate (3-fluorosalicylate) Contribution No. 692, Environmental Research Laboratory, U.S. EPA, Gulf Breeze, FL. 32561, USA     

Production of cis,cis-muconate from benzoate and 2-fluoro-cis,cis-muconate from 3-fluorobenzoate by 3-chlorobenzoate degrading bacteria

Summary 3-Chlorobenzoate grown cells of Pseudomonas sp. strain B13 or Alcaligenes sp. strain A7-2 converted 3-fluorobenzoate to 2-fluoro-cis,cis-muconate with 87% yield. The latter strain produced 1.6 g/l. The type II muconate cycloisomerases of neither strain exhibit acitivity for 2-fluoro-cis,cis-muconate. Succinate grown cells of Pseudomonas sp. strain B13 converted benzoate to cis,cis-muconate (91% yield; 7.4 g/l). Enzyme tests confirmed that no muconate cycloisomerising enzyme was induced within 24 h.     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

The anaerobic biodegradation ofo-, m- andp-cresol by sulfate-reducing bacterial enrichment cultures obtained from a shallow anoxic aquifer

Summary Sulfate-reducing bacterial enrichments were obtained from a shallow anoxic aquifer for their ability to metabolize eithero-, m-, orp-cresol. GC/MS and simultaneous adaptation experiments suggested that the anaerobic decomposition ofp-cresol proceeds by the initial oxidation of the aryl methyl group to formp-hydroxybenzoic acid. This intermediate was then converted to benzoic acid. Benzoic acid and a hydroxybenzaldehyde were also found in spent culture fluids from ano-cresol-degrading enrichment culture. This result, in addition to others, suggested thato-cresol may also be anaerobically degraded by the oxidation of the methyl substituent. An alternate pathway for anaerobicm-cresol decomposition might exist. Enrichment cultures obtained with eitherp- oro-cresol degraded both of these substrates but notm-cresol. In contrast, am-cresol enrichment culture did not metabolize theortho orpara isomers. Anaerobic biodegradation in all enrichment cultures was inhibited by molybdate and oxygen, and was dependent on the presence of sulfate as a terminal electron acceptor. The stoichiometry of sulfate-reduction and substrate depletion by the various enrichment cultures indicated that the parent cresol isomers were completely mineralized. This result was confirmed by the conversion of¹⁴C-labeledp-cresol to¹⁴CO₂. These results help clarify the fate of alkylated aromatic chemicals in anoxic aquifers.     

Identification and properties of yeasts associated with the aerobic deterioration of wheat and alfalfa silages

Populations of fungi in aerobically deteriorating wheat and alfalfa silages were identified as: Endomycopsis burtonii, E. selenospora, Hansenula canadensis, Candida tenuis and C. silvicola. The yeasts recovered were similar for both silages, but H. canadensis was recovered only in wheat silages. All of these yeasts could utilize lactic acid aerobically, but not anaerobically. Only Endomycopsis spp. could utilize propionic acid aerobically and none of the yeasts utilized this acid anaerobically. However, all yeasts grew in complete media supplemented with propionate. Therefore, while lactic and propionic acids may contribute to stability under anaerobic conditions, they are much less less effective after the silage is exposed to air.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Effect of pH and Oxygen Tension on Staphylococcal Growth and Enterotoxin Formation in Fermented Sausage

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 × 10⁷ cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.     

Critical Reactions in Fluorobenzoic Acid Degradation by Pseudomonas sp. B13

3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3-chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cis-muconic acid was identified as a dead-end metabolite from 2- and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.     

Changes in protein composition of facultatively aerobic sulfur-dependent archaebacteria depending on growth conditions

Cell-free extracts of facultatively anaerobic, sulfur-dependent archaebacteria Acidianus infernus (DSM 3191), and Acidianus brierleyi (DSM 1651) were examine by two-dimensional gel electrophoresis. 56 out of 250 protein spots were induced in anaerobically grown cells of A. infernus compared with 57 out of 251 in aerobically grown cells. In aerobically grown cells of A. brierleyi 62 out of 160 spots were induced, compared with 84 out of 182 in anaerobically grown cells. Changes in the protein patterns of both species were not comparable.     

Metabolism of Hydroxylated and Fluorinated Benzoates by Syntrophus aciditrophicus and Detection of a Fluorodiene Metabolite

Transformations of 2-hydroxybenzoate and fluorobenzoate isomers were investigated in the strictly anaerobic Syntrophus aciditrophicus to gain insight into the initial steps of the metabolism of aromatic acids. 2-Hydroxybenzoate was metabolized to methane and acetate by S. aciditrophicus and Methanospirillum hungatei cocultures and reduced to cyclohexane carboxylate by pure cultures of S. aciditrophicus when grown in the presence of crotonate. Under both conditions, transient accumulation of benzoate but not phenol was observed, indicating that dehydroxylation occurred prior to ring reduction. Pure cultures of S. aciditrophicus reductively dehalogenated 3-fluorobenzoate with the stoichiometric accumulation of benzoate and fluorine. 3-Fluorobenzoate-degrading cultures produced a metabolite that had a fragmentation pattern almost identical to that of the trimethylsilyl (TMS) derivative of 3-fluorobenzoate but with a mass increase of 2 units. When cells were incubated with deuterated water, this metabolite had a mass increase of 3 or 4 units relative to the TMS derivative of 3-fluorobenzoate. ¹⁹F nuclear magnetic resonance spectroscopy (¹⁹F NMR) detected a metabolite in fluorobenzoate-degrading cultures with two double bonds, either 1-carboxyl-3-fluoro-2,6-cyclohexadiene or 1-carboxyl-3-fluoro-3,6-cyclohexadiene. The mass spectral and NMR data are consistent with the addition of two hydrogen or deuterium atoms to 3-fluorobenzoate, forming a 3-fluorocyclohexadiene metabolite. The production of a diene metabolite provides evidence that S. aciditrophicus contains dearomatizing reductase that uses two electrons to dearomatize the aromatic ring.