Vol. 54, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1988, p. 712-7170099-2240/88/030712-06$02.00/0Copyright © 1988, American Society for Microbiology

Anaerobic and Aerobic Metabolism of Diverse AromaticCompounds by the Photosynthetic Bacterium

Rhodopseudomonas palustrisCAROLINE S. HARWOOD'* AND JANE GIBSON2

Department of Microbiology, New York State College ofAgriculture and Life Sciences,' and Section ofBiochemistry,Molecular and Cell Biology, Division of Biological Sciences,2 Cornell University, Ithaca, New York 14853-7201

Received 2 September 1987/Accepted 19 December 1987

The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic com-pounds for growth under anaerobic and aerobic conditions. Many phenolic, dihydroxylated, and methoxylatedaromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001in both the presence and absence of oxygen. Some compounds were metabolized under only aerobic or underonly anaerobic conditions. Two other strains, CGCO23 and CGDO52, had similar anaerobic substrateutilization patterns, but CGDO52 was able to use a slightly larger number of compounds for growth. Theseresults show that R. palustris is far more versatile in terms of aromatic degradation than had been previouslydemonstrated. A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respectto anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobicbreakdown of diverse aromatics. Another mutant (CGA047) was unable to grow anaerobically on eitherbenzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown onmore complex aromatic compounds. This indicates that R. palustris has two major anaerobic routes foraromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate.

Aerobic pathways of aromatic metabolism have beenstudied extensively in bacteria and show an almost universalrequirement for molecular oxygen (7, 11). The biochemicalstrategies involved in anaerobic aromatic degradation, bycontrast, are fundamentally different and are still incom-pletely understood. The earliest and most thorough studieson this topic were carried out with the photosyntheticbacterium Rhodopseudomonas palustris. Nearly 20 yearshave elapsed since Dutton and Evans (9) proposed a novelring reduction mechanism leading to anaerobic cleavage ofthe aromatic ring of benzoate. These studies have beensubstantiated and elaborated on (14, 15, 17, 30), and workwith additional anaerobes, including fermentative anaerobesand nitrate reducers, indicates that the reductive ring cleav-age pathway for benzoate degradation is widespread amongmicroorganisms (2, 18, 24, 31).Recent concern about the environmental fate of industri-

ally produced organic compounds has prompted a resur-gence of interest in the anaerobic degradation of aromaticcompounds. Numerous studies have shown that substitutedbenzoates, including chlorinated, nitro-, and aminoaroma-tics and also aromatic hydrocarbons and phenolic com-pounds, can be broken down under anaerobic conditions bybacteria (3, 5, 6, 13, 19, 25, 32, 33). However, in most casesthe metabolic fates of these structurally diverse aromaticcompounds have not been delineated. One might ask, forexample, whether diverse aromatic compounds are metabo-lized via totally independent catabolic pathways or whethergroups of related compounds are degraded to form commonintermediates which then undergo ring fission reactions.To begin to address this question, we surveyed several R.

palustris strains for the ability to grow anaerobically on arange of aromatic compounds and we tested a mutant that isblocked in anaerobic benzoate and 4-hydroxybenzoate utili-

* Corresponding author.

zation to see whether it could degrade aromatic compoundswhich are more complex structurally. The aerobic growthcapabilities of R. palustris were also examined. The resultsreported in this paper show that R. palustris is far morenutritionally versatile with respect to aromatic utilizationthan had been previously reported. Furthermore, variouscomplex aromatic compounds are metabolized to form eitherbenzoate or 4-hydroxybenzoate before anaerobic cleavageof the aromatic ring. This indicates that the pathways forbenzoate and 4-hydroxybenzoate metabolism play a generalrole as central anaerobic ring fission pathways in R. palus-tris.

MATERIALS AND METHODS

Bacterial strains. Strain CGCO23 is the type strain of R.palustris (ATCC 11168). Strain CGA001 was originally iso-lated by S. Taniguchi and has been maintained in the culturecollection of R. K. Clayton at Cornell University, Ithaca,N.Y. Strains CGA033 and CGA047 are derivatives ofCGA001 that were obtained after NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis. CGA033 is unable to growaerobically with 4-hydroxybenzoate as the sole carbonsource but is wild type with respect to anaerobic growth.Strain CGA047 is unable to utilize either benzoate or 4-hydroxybenzoate under anaerobic conditions but is unim-paired in aerobic growth capabilities. Strain CGDO52 wasrecently isolated by us from liquid enrichment cultures (4)that had been supplemented with benzoate, inoculated withsewage sludge, and incubated anaerobically in light.Media and growth conditions. Unless otherwise noted, all

strains were grown at 30°C in defined basal medium (22)supplemented with 0.15 mM 4-aminobenzoic acid and 0.1mM sodium thiosulfate (PM medium). Carbon sources wereadded to autoclaved medium at the time of inoculation fromsterile solutions that had been adjusted to pH 6.8 to 7.0.Anaerobic medium was prepared by bubbling nitrogen gas

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through PM liquid medium for 1 h, transferring the mediumto an anaerobic glove box (Coy Laboratory Products, AnnArbor, Mich.), and then dispensing 10-ml aliquots of themedium into culture tubes (Bellco Glass, Inc., Vineland,N.J.) in the glove box. The tubes, containing a nitrogenatmosphere, were sealed with butyl rubber stoppers andautoclaved. Sodium bicarbonate (final concentration, 10mM) was added to anaerobic growth medium as a sterilesolution after autoclaving. Anaerobic cultures were illumi-nated with 40-W incandescent lamps at a distance of 20 to 40cm. Cells were grown aerobically in liquid medium providedwith constant aeration by a gyratory environmental shaker(New Brunswick Scientific Co., Inc., Edison, N.J.).Auxanography was used to screen strains for the ability to

utilize aromatic and hydroaromatic compounds as growthsubstrates (23). Cells grown aerobically at 37°C in liquid PMmedium supplemented with 10 mM succinate were harvestedby centrifugation, washed once, and suspended in basalmedium to a final density of approximately 5 x 108 cells perml. Suspended cells were diluted 20-fold into PM mediumcontaining 0.5% (wt/vol) melted Gelrite (Kelco, San Diego,Calif.), and the medium was then immediately poured intopetri dishes. Each carbon compound to be tested as a growthsubstrate was applied to the surface of the solidified mediumnear the periphery of the petri dish. One compound wastested per plate, and each compound was applied as aspatula-pointful of solid chemical. Auxanographic plateswere incubated aerobically in the dark or anaerobically inlight in polycarbonate jars to which GasPak hydrogen pluscarbon dioxide generator envelopes (BBL MicrobiologySystems, Cockeysville, Md.) had been added.Growth in liquid cultures was monitored by measuring the

increase in A660 in a Spectronic 21 (Bausch & Lomb, Inc.,Rochester, N.Y.).

Substrate conversion. The absorption spectra of samples ofculture fluid that had been centrifuged and diluted 10-foldinto 50 mM P04 buffer (pH 7.0) were determined by scanningbetween 200 and 400 nm with a Beckman DU-7 spectropho-tometer (Beckman Instruments, Palo Alto, Calif.). The me-tabolism of various aromatic substrates during anaerobicgrowth was also followed by reversed-phase high-perfor-mance liquid chromatography. A Beckman binary gradientmodel 344 system with a 254-nm fixed-wavelength UVdetector was used. The supernatant liquid of centrifugedsamples (20 ixl) was injected into a Ultrasphere C-18 column(Beckman Instruments). The mobile phase consisted ofacetonitrile and 0.01 N perchloric acid in a linear gradient of0.6 to 60% acetonitrile. The flow rate was 1.0 ml/min, and therun time was 20 min.

RESULTS

Growth patterns of strain CGA001 in auxanographic plates.To explore the full potential of R. palustris CGA001 forutilizing diverse aromatic compounds, we exposed cells toconcentration gradients of potential substrates by the tech-nique of auxanography. Diffusion of a given carbon com-pound from its site of addition effectively exposes cellsseeded in plates of agar medium to a range of substrateconcentrations, and growth is reflected as the formation ofan arc of turbidity (Fig. 1). This approach allowed rapidscreening of potential growth substrates, and it also revealedthe ability of cells to grow with compounds that are toxic athigh concentrations. Cinnamaldehyde, for example, stimu-lated the growth of a narrow arc of cells some distance fromits origin of application to the petri dish (Fig. la). Growth

FIG. 1. Growth patterns of R. palustris on auxanographic plates.(a) Growth with cinnamaldehyde, a toxic substrate. (b) Arc of turbidgrowth observed with the substrate, 4-hydroxycinnamate. (c) Pat-tern of growth with the relatively nontoxic substrate, 4-hydroxyben-zoate.

with more moderately toxic substrates, such as 4-hydroxy-cinnamate and 4-hydroxybenzoate, was reflected in theformation of bands at intermediate distances from the site ofsubstrate addition (Fig. lb and c). Approximately 50 aro-matic and hydroaromatic compounds were screened for theability to serve as carbon sources for CGA001 cells grownunder both phototrophic (anaerobic in light) and heterotro-phic (aerobic) conditions.The following substrates supported both aerobic and an-

aerobic growth: caffeate, cinnamaldehyde, cinnamate, cy-clohexanecarboxylate, cyclohexanepropionate, A-1-cyclo-hexenecarboxylate, A-3-cyclohexenecarboxylate, ferulate,

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714 HARWOOD AND GIBSON

TABLE 1. Anaerobic transformations of aromatic acids by R. palustris strains'

Results in CGAOO1 Results in CGDO52Growth substrate (mM) Generation R f Generation

time (h) Ring fission time (h) Ring fission

Benzoate (2) 16 + 12 +Benzoylformate (1) 40 + NT NACaffeate (1.5) 90 100 +Cinnamate (1) 12 + 10 +Ferulate (1.5) 35 35 +Hydrocaffeate (1) 100 60 +3-Hydroxybenzoate (2) 110 + 75 +4-Hydroxybenzoate (1) 16 + 12 +4-Hydroxybenzoylformate (1) 60 + NT NA4-Hydroxycinnamate (0.75) 22 + 25 +DL-Mandelate (1) 55 + 30 +4-Phenylbutyrate (1) 30 163-Phenylpropionate (1) 12 + 17 +5-Phenylvalerate (0.6) 18 + 13 +Protocatechuate (1) NG NA 170 +Vanillate (1) NG NA 170 +

a NT, Not tested; NA, not applicable; NG; no growth. Symbols indicate presence (+) or absence (-) of ring fission.

hydrocaffeate, hydrocinnamaldehyde, 4-hydroxybenzal-dehyde, 4-hydroxybenzoate, 4-hydroxybenzoylformate, 4-hydroxycinnamate, 4-phenylbutyrate, 3-phenylpropionate(hydrocinnamate), and 5-phenylvalerate. The following sub-strates supported aerobic but not anaerobic growth: homo-gentisate, isovanillate, phenylacetate, protocatechuate, andvanillate. The following substrates supported anaerobic butnot aerobic growth: benzaldehyde, benzoate, benzoylfor-mate, 3-hydroxybenzoate, and DL-mandelate. The followingsubstrates did not support growth: 4-aminobenzoate, anthra-nilate (2-aminobenzoate), catechol, 3-chlorobenzoate, coni-feryl alcohol, 4-cresol, cyclohexanol, cyclohexanone, ethyl-vanillate, 2-fluorobenzoate, gallate (trihydroxybenzoate),gentisate, nicotinate, phenol, phenoxyacetate, 3-phenyl-butyrate, quinate, resorcinol, salicylate (2-hydroxyben-zoate), shikimate, syringate, trimethoxybenzoate, trime-thoxycinnamate, 3-toluate, 4-toluate, and vanillin. As can beseen from the above list, 14 aromatic acids were anaerobicgrowth substrates and several hydroaromatic compounds, aswell as several aromatic aldehydes, also supported growth.Most of these compounds were also utilized by aerobicallygrown cells. Growth on a few of the compounds tested wasrestricted to only heterotrophically grown cells or onlyphototrophically grown cells.The generation times of CGA001 on those aromatic acids

that supported growth were determined in liquid cultures(Table 1).

Utilization of aromatic compounds by strains CGCO23 andCGDO52. Two additional strains of R. palustris were testedfor anaerobic growth in liquid medium on the aromatic acidslisted in Table 1. Strain CGCO23 had substrate utilizationpatterns that were indistinguishable from those of CGA001.Strain CGDO52 had somewhat broader anaerobic growthcapabilities and was able to utilize vanillate and protocatech-uate under phototrophic conditions (Table 1).

Anaerobic, aromatic ring fission. A review of the structuralfeatures of the aromatic compounds used by R. palustrisstrains shows that these organisms are particularly wellsuited for degrading compounds with carbon side chains. Toassess this metabolic potential in greater detail, cells weregrown anaerobically in liquid with various substrates andspent growth media were analyzed for accumulation ofpartial degradation products. Spectrophotometric scans and

high-performance liquid chromatographic analyses revealedthat only aromatic acids with no ring substitutions or thosewith a single hydroxyl group in either position 3 or position4 were fully degraded under anaerobic conditions byCGA001 (Table 1). Compounds such as ferulate, caffeate,and hydrocaffeate that have multiple ring substitutions wereonly partially degraded. Cells used the side chain as a carbonsource and then excreted the remaining aromatic moiety intothe external medium. In these cases, ferulate was degradedto give vanillate, and protocatechuate was formed fromcaffeate and hydrocaffeate.

Strain CGDO52 had more extensive anaerobic ring fissioncapabilities and was able to fully degrade 4-hydroxylatedaromatic compounds with a hydroxyl or methoxyl group inposition 3 (i.e., ferulate, caffeate, hydrocaffeate, vanillate,and protocatechuate). Both CGA001 and CGDO52 metabo-lized 4-phenylbutyrate to form phenylacetate under anaero-bic conditions.Growth of a mutant strain blocked in anaerobic benzoate

and 4-hydroxybenzoate degradation. Mutant strain CGA047is unable to grow on either benzoate or 4-hydroxybenzoateanaerobically, but it has the same aerobic growth capabilitiesas its parental strain, CGA001. This mutant strain was alsounable to grow with mandelate, benzoylformate, 3-hydroxy-benzoate, or 4-hydroxybenzoylformate under anaerobicconditions. CGA047 retained the ability to grow anaerobi-cally with aromatic compounds that have carbon side chains,but the cell yields on many of these compounds weresubstantially lower than those obtained with the wild type.Analyses of spent growth media showed that none of thearomatic acids tested was fully degraded by CGA047. Cin-namate, 3-phenylpropionate, and 5-phenylvalerate were me-tabolized to form benzoate, and 4-hydroxycinnamate wasconverted to 4-hydroxybenzoate. Ferulate, caffeate, andhydrocaffeate were metabolized by CGA047 to yield thesame products as were observed with strain CGA001.Growth of a mutant blocked in aerobic 4-hydroxybenzoate

metabolism. Mutant strain CGA033 is unable to grow with4-hydroxybenzoate under aerobic conditions. It was alsoincapable of aerobic growth on several other aromatic com-pounds (i.e., 4-hydroxybenzoylformate, protocatechuate,vanillate, and 4-hydroxycinnamate) that have a hydroxylgroup in position 4. However, phenylacetate, 3-phenylpro-

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COOHI

HCI ICH

L%UY OCH3OH

Feulic Acid

COOH

OCH3

OHVanillic Acid

I?COOH

v OH

OHProlcatechuic Acid

4?COOH

A.-

OH

4-Hydroxybezoic Acid

Central

COOH MetabolitesCOOHA

Benzoic Acid

FIG. 2. Aromatic compounds utilized by R. palustris as anaerobic growth substrates. Proposed major routes of aromatic breakdown areindicated by the arrows.

pionate, cinnamate, 4-phenylbutyrate, 5-phenylvalerate, hy-drocaffeate, caffeate, and ferulate all supported aerobicgrowth of this mutant. Strain CGA033 behaved identically tothe wild type with respect to its ability to utilize 4-hydroxy-benzoate and other aromatic acids under anaerobic condi-tions. This suggests that a number of aromatic compoundswith hydroxyl groups in position 4 probably follow a com-mon aerobic degradative route and that distinct metabolicpathways are used under anaerobic and aerobic conditions inthe catabolism of other aromatic compounds in addition to4-hydroxybenzoate.

DISCUSSION

The results presented here show that R. palustris canutilize a much larger number of aromatic compounds underanaerobic conditions than had been previously reported (8,16). It is not restricted to growth on only very simplearomatic acids but can also utilize dihydroxylated and me-thoxylated compounds, as well as aromatic acids with car-bon side chains. Several aromatic aldehydes and hydroaro-matic acids also supported growth. These diverse metaboliccapabilities make R. palustris one of the most versatile of theanaerobic bacteria described to date with respect to aromaticdegradation.

Furthermore, recent work, showing that R. palustris canbe selectively enriched from a variety of habitats withcinnamate as the sole carbon source, suggests that aromaticacid-degrading strains of R. palustris are widespread and

may make a significant contribution to anaerobic aromaticbreakdown in natural environments (M. Madigan and H.Gest, FEMS Microbiol. Ecol., in press).Many aerobic pathways for the metabolism of diverse

aromatic compounds converge to a small number of com-pounds which serve as the starting substrates for subsequentring cleavage reactions (7). Although the pathways of anaer-obic aromatic degradation differ radically from those takenunder aerobic conditions, our data suggest that R. palustrisuses an analogous metabolic strategy when degrading aro-matic compounds anaerobically and converts diverse com-pounds to two major key substrates before initiating ringfission pathways. The probable degradative routes taken inthe anaerobic metabolism of aromatic compounds by R.palustris are summarized in Fig. 2. These were assessed byanalyzing growth patterns and the accumulation of partialdegradation products by wild-type strains and by a mutantstrain (CGA047) blocked in the anaerobic degradation ofbenzoate and 4-hydroxybenzoate. The main conclusion to bedrawn is that many aromatic compounds are metabolizedanaerobically to form either benzoate or 4-hydroxybenzoatebefore cleavage of the aromatic ring. This indicates that R.palustris uses two major routes for aromatic degradation,one that passes through benzoate and one that passesthrough 4-hydroxybenzoate. This conclusion may also applyto other microorganisms and consortia of microorganismsthat have similar substrate utilization patterns (3, 25, 32) andto organisms that can degrade aromatic hydrocarbons (5), aswell as phenol and substituted phenols (19, 28).

COOH COOHI I

HC Cu211 ICH CH2

U OH JOHOH OH

Caffec aid Hydnxffeic AcidCOOH

HC COOHIICH C'O

OH OH

I4-Hydn,xycinmnic Acid 4-Hydroxybeazoylimnic AcidI

COOH COOH COOHI ~~~~II COICH ] HC COOH 2 COOH

I CI CHOH CHC

5-Phenylvalcric Acid Cinnamic Acid Maidelic Acid 3-Phenylpropionic Acid Benzoylfornic Acid

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716 HARWOOD AND GIBSON

The degradative fates of vanillate and protocatchuate are

uncertain. These compounds were metabolized by just one

of the strains that we examined (CGDO52), and no degrada-tive intermediates appeared even transiently in liquid culturemedia during growth. Anaerobic demethoxylation and re-

ductive dehydroxylation of aromatic compounds has beenobserved in other bacteria, however (1, 10, 12, 20, 21, 26, 27,29), so it seems reasonable to suggest that vanillate andprotocatechuate are converted to 4-hydroxybenzoate. Theisolation of a mutant derivative of strain CGDO52 that isblocked in 4-hydroxybenzoate utilization should clarify thispoint.

R. palustris also has considerable aerobic growth capabil-ities on aromatic compounds. That the pathways followedare distinct from those used for anaerobic degradation was

shown by the behavior of two mutants, one blocked in theanaerobic degradation of selected aromatics (CGA047) andthe second deficient in aerobic aromatic breakdown(CGA033). Strain CGA047 was unimpaired in aerobicgrowth on aromatic compounds, whereas CGA033 behavedidentically to its wild-type parental strain when grown on

aromatic compounds under anaerobic conditions.These studies were facilitated considerably by the use of

auxanography to screen a large number of potential growthsubstrates. Since this technique exposes cells to a gradient ofsubstrate concentrations, we could exclude the possibilitythat failure to grow on a given substrate was due to toxicity.An additional advantage of auxanography is that it providesa way to rapidly screen the substrate utilization patterns ofslow-growing organisms. Since each petri dish typicallyreceives 5 x 108 cells, even a small amount of growthquickly becomes apparent. Thus, growth of R. palustris,which has a generation time of greater than 20 h on manyaromatic compounds, could be detected on auxanographicplates after just an overnight incubation. This techniqueshould therefore be widely applicable and useful for testingthe substrate utilization patterns of many kinds of anae-robes.

ACKNOWLEDGMENTS

We thank Johanna Geissler for helpful discussions and for per-forming some of the initial experiments and Constance Thomas forisolating the mutants used in this study.

This work was supported in part by grant DE-FG02-86ER13495from the Department of Energy and by a grant from the CornellBiotechnology Program, which is sponsored by the New York StateScience and Technology Foundation and a consortium of industries.

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