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Journal of General Microbiology (1993), 139, 1331-1336. Printed in Great Britain 1331

Microbial transformation of nitroaromatic compounds under anaerobicconditionsTHOMASORONTZY,ANKUVER nd KARL-HEINZLOTEVOGEL*Fachbereich Biologie, AG Mikrobiologie, Universitat Oldenburg, Postfach 25 03, W-2900 Oldenburg,Federal Republic of Germany(Received 5 October 199 2; revised 14 January 19 93; accepted 20 January 19 93)

The transformation of several mono- and dinitroaromatic compounds (tested at 50-200 p ~ )y methanogenicbacteria, sulphate-reducingbacteria and clostridia was studied. Some of the nitroaromatics tested were transformedchemically by 1.5 mM qua ntities of culture media reducing agents, like cysteine or sulphide. This abiotic reductio noccurred a t the o-nitro-grou ps preferentially. Nitrophenols, p-nitroaniline and p-nitrobe nzoic acid were completelytransform ed biologically into the corresponding amino derivatives. The nitro arom atics were transform ed by all ofthe bacterial strains tested. While growing cells of sulphate-reducing bacteria and Clostridium spp. carried outnitroreduction, methanogen cells lysed in the presence of nitroaromatics. Nevertheless these culture suspensionsconverted nitroaromatics to the corresponding amino derivatives. This was also confirmed by crude cell extractsof methanogenic bacteria. The rate of nitroreduction by sulphate-reducing bacteria depended on the electrondonors supplied and the ce ll density, with molecu lar hydrogen being the m ost e ffective donor of red ucingequivalents. The toxicity of p-nitrophenol to some of the organisms tested depended on the concentration of thenitroaromatic compound and the type of organism.

IntroductionNitroaromatic compounds are released into the en-vironment in large quantities, since they are widely usednot only as pesticides, but also as explosives andprecursors for dyes, pharmaceuticals and plastics. Theyare also formed during incineration of organic com-pounds and synthesized photochemically in the at-mosphere (Gibson, 1982). Some nitroaromatic com-pounds, such as 1aitropyrene, are highly mutagenicand/or carcinogenic (Thiem et al., 1979), and severalnitroaromatics are potent uncouplers of oxidative andphotosynthetic phosphorylation (Terada, 198 1; Shea etal., 1983). Once released into the environment, nitro-aromatics underlie complex physical, chemical, andbiological processes. Depending on the prevailing con-ditions they may be transformed photochemically(Nakagawa & Crosby, 1974), or reduced electro-chemically (Macalady et al., 1986). Nitroaromatics canalso be transformed by micro-organisms under aerobic

* Author for correspondence. Tel. 441 7980; fax 441 798 3250.Abbreviations: p-NP, p-nitrophenol ; p-AP, p-aminophenol; m-NP,

m-nitrophenol; p-NB, p-nitrobenzoic acid.

conditions (Germanier & Wuhrmann, 1963; Zeyer &Kearney, 1984; Schackmann & Muller, 1991; Spain &Gibson, 1991). Little is known about the transformationof nitroaromatics by growing cultures of anaerobicbacteria. Transformation reactions have been examinedmostly in mixed cultures and enzyme preparations (Boydet al., 1983;Hallas & Alexander, 1983;Zeyer & Kearney,1984; Blotevogel& Butte, 1989). Specific nitroreductasescatalysing the reduction of nitro-groups of aromatics toamino-groups have been isolated and characterized(Kinoushi & Ohnishi, 1983; Bryant & DeLuca, 1991;Rafii et al., 1991). One of these enzymes acts as anelectron carrier protein for the artificial electron-acceptormenadione, indicating quinone reductase activity(Bryant & DeLuca, 1991).The present study was performed to investigate thebehaviour of nitroaromatics in the presence of purecultures of sulphate-reducing bacteria, methanogenicbacteria, and Clostridium spp., as well as the effect ofnitroaromatics on these bacteria.

MethodsStrains of bacteria. The following strains of bacteria were used :

Methanobacterium ormicicu m (DSM 1535),Methanobacterium thermo-0001-7876 0 1993 SGM

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1332 T. Gorontzy, J . Kiiver and K.-H. Blotevogelautotrophicum (DSM 1053), Methanosarcina barkeri (DSM 800),Methanosarcina frisia (DSM 33 18), Methanosarcina sp. KS2002(isolated from sewage sludge), Methanospirillum hungatei (DSM 864),Methanogenium tationis (DSM 2702) and Methanoculleus oldenbur-gensis (DSM 62 16); Desulfovibrio desulfuricans (DSM 642), Desulfo-vibrio gigas (DSM 496), Desulfovibrio sp. AS (isolated from sedimentfrom the river Amazon, Brazil), Desulfovibrio sp. HB (isolated fromsewage sludge), Desulfococcus multivorans (DSM 2059), Desulfoto-maculum orientis (DSM 765), Desulfotomaculum sp. GROL (isolatedfrom a ditch sediment at Bremen, FRG) ; Clostridium pasteurianum(DSM 525) and Clostridium sp. W1 (isolated from soil).

Chemical transformation. An appropriate amount of each nitro-aromatic compound was placed into serum flasks containing 50mlpotassium phosphate buffer (50mM; pH 7.0) to give a final con-centration of 0.2 mM. Then the reducing agents Ti(II1) nitrilotriacetate,sodium dithionite, sodium sulphide, sodium sulphite, and cysteine.HClwere added to give a final concentration of between 1.5 and 10.0 mM.Before analysis the reaction mixtures were incubated at 37 "C for oneweek.Media and culture conditions. The methanogenic bacteria werecultivated and maintained as previously described (Balch et al., 1979;Blotevogel et al., 1986), with molecular hydrogen and carbon dioxideas energy and carbon sources. The sulphate-reducing bacteria werecultivated and maintained as described by Widdel& Bak (1992). Unlessotherwise indicated, the sulphate-reducing bacteria were grown withsulphate (20mM) and lactate (20 mM) except for Desulfotomaculum sp .GROL, which was grown with butanol(20 mM) as energy and carbonsource. The clostridia were grown in the medium described by Widdel

& Bak (1992), with 10mwfructose as energy and carbon source.Experiments with added nitroaromatics were also performed underthese conditions, while transformation experiments with p-NP werecarried out in fed-batch culture. Samples were withdrawn every 24 hand the losses of p-NP, the production of p-AP, and the growthparameters were measured. All experiments were carried out at 37 "Cin triplicate.Analytical procedures. The optical density was measured spectro-photometrically at 580 nm. The protein content was determined asdescribed by Bradford (1976) with bovine serum albumin as standard.Methane was analysed by gas chromatography with a Varian model6000 gas chromatograph equipped with a thermal conductivitydetector. The injector and the detector were heated to 70C and

180"C , respectively; the oven temperature was 50 "C. The gases wereseparated from each other in a 2 m glass column packed with PorapacQ (Supelco) with 30 ml N, min-' as a carrier gas.The nitroaromatic and aminoaromatic compounds were measuredby means of UV/VIS-spectrophotometry based on their specificabsorbance at a certain wavelength (Alexander & Lustigman, 1966;Hallas& Alexander, 1983). Absorption spectra were taken from 200 to500 nm after precipitating the hydrogen sulphide in the 1 ml samplewith 0.1 ml 2% (w/v) zinc acetate solution. Reference spectra of thenitro- and aminoaromatics (20-1000 p ~ ) ere determined in phos-phate-buffered standard solutions (10 mM; pH 7.2). Disappearance ofnitrophenols and production of aminophenols was determined byreverse-phase HPLC [LiChrosorb C18 (10 pm); eluent H,O/methanol/glacial acetic acid (65: 30:5 by vol.), pH 3.0; flow rate 2.0 ml min-';detection UV 254 nm].

Chemicals. All chemicals were of reagent grade and were obtainedfrom Serva, Fluka or Janssen. For the preparation of 1 0 0 m ~tocksolutions the nitroaromatics and aminoaromatics were dissolved indeionized distilled water with the exception of 2,4-dinitrophenol, whichwas dissolved in methanol (97%, v/v).

ResultsChemical reduction of nitroaromatics by m ediumconstituents and possible metabolic productsIn order to differentiate chemical from biologicaltransformation reactions it was first necessary to studythe behaviour of the nitroaromatics in the presence oftypical reducing agents which are used for the prep-aration of media for anaerobic bacteria (Table 1). 0-Nitrophenol (0-NP) was completely reduced chemicallyto its corresponding amino derivative by the reducingagents sulphide and/or cysteine. Therefore it was notused to study biological transformation. The trans-formation of 2,4-dinitrophenol (2,4-DNP) by sulphideand cysteine resulted in the formation of 2-amino-4-nitrophenol ;p-nitroaniline (p-NA) was transformed toan unidentified product under the same conditions.Ti(II1) nitrilotriacetate (Ti-NTA) and sodium dithionitecaused a complete transformation of every nitroaromaticcompound tested into the corresponding amino- ordiamino-derivative herefore these compounds were notused in biological transformation experiments.

Reduction of nitroaromatics by growing cultures ofbacteriaUninoculated and autoclaved controls showed no trans-formation of the nitroaromatics tested. Only 2,4-dinitrophenol and p-nitroaniline were partially trans-formed by media reducing agents. All of the organismsstudied were able to perform the transformation ofnitroaromatic compounds and to complete the partialtransformation of 2,4-dinitrophenol and p-nitroaniline(Table 2). Desulfococcus multivorans and Desulfoto-macu lum orientis only transformed p-nitrophenol. Trans-formation always resulted in the formation of amino-

Table 1. Transformation of several nitroaromaticcompounds by reducing agentsreducing agent was tested at 1-5-10 mM.

Experiments were carried out at pH 7.2 and 37 "C for 7 d. Each

Nitroaromatic Reducing agent*compound(0.5 mM) Ti-NTA Dithionite Sulphite Sulphide Cysteine- - --NP + +m-NP + +0-NP + + + +2,4-DNP + + * *p-NB + +p-NA + + k IfI- - ---- - --

* Ti-NTA, Ti(II1) nitrilotriacetate ; + , complete transformation intothe corresponding amino derivative; f partial transformation; -, notransformation

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Microbial transformation o nitroaromatics 1333Table 2 . Transform ation of several nitroaromatic compounds b y growingcultures of methanogenic bacteria, sulphate-reducing bacteria and Clostridium

The compounds were used at 50 PM , 100 PM and 100PM for the three groups of bacteriarespectively. Experiments were carried out in typical media for 7 d (except M s. barkeri andMethanosarcina sp. KS2002, which were incubated for 14 d at pH 7.0-7.2 and 30 "C/37 "C.Complete transformation of the nitroaromatics was ascertained by UV/VIS and HPLC. Theabiotically transformed nitroaromatics 2,4-dinitrophenol and p-nitroaniline were investigatedfor complete transformation by the listed organisms.Nitroaromatic compound*

Organism p-NP m-NP 2,4-DNP p-NB p-NAMethanobacteriumformicicum + + + + +thermoautotrophicum + + + + +

hungatei + + + + +barkeri + + + + +frisia + + + + +sp. KS2002 + + + + +tationis + + + + +

MethanospirillumMethanosarcina

MethanogeniumMethanoculleusDesulfovibrio

oldenburgensis + + + + +desulfur cans + + + + +gigas + + + + +sp. AS + + + + +sp. HB + + + + +multivorans +orientis +

DesulfococcusDesulfo tomacu lum

- - - -- - - -

sp. GROL + + + + +pasteurianum + + + + +sp. W I + + + + +

Clostridium

* +, 100% transformation into the corresponding amino derivative; - no transformation.aromatics as ascertained by UV/VIS-spectrophotometryand HPLC.As a typical representative of the sulphate-reducingbacteria, Desulfovibrio gigas was studied more ex-tensively. The nitro-group reduction (0.1 mM each maxi-mally) by growing cultures of Desulfovibrio gigas wasstrongly correlated with the growth phase (Fig. 1).Controls without added nitroaromatics showed the samecourse of growth. The maximum rate of p-NP trans-formation during the exponential phase of growth wasaround 0.28 pmol (mg protein)-' h-'.The two Clostridium species studied were also able tocompletely transform all the nitroaromatics tested totheir corresponding amino derivatives (Table 2). TheClostridium spp. showed the same transformation be-haviour as Desulfovibrio gigas. Again the course ofgrowth was not affected by the nitroaromatic compound.The maximum rate of p-NP transformation during theexponential phase of growth was about 0-42pmol (mgprotein)-' h-I.

The transformation of nitroaromatic compounds bymethanogenic bacteria only occurred while cells lysed.Therefore it was necessary to pregrow the cells to anoptical density of about 0-15 before adding the nitro-aromatics. As shown in Table 2, all of the methanogenstested were able to perform a complete transformation ofthe nitroaromatics. Methanosarcina frisia was investi-gated in more detail. The results in Fig. 2 indicate thatp-NP was transformed by growing cultures of Methano-sarcina fris ia (OD,,, 0.15) accompanied by a decrease inthe optical density. As long as p-NP was present, themethane production ceased entirely. When transform-ation had been completed, growth and methane pro-duction of Methanosarcina frisia recovered.Eflect o diflerent electron donors on the rate onitroaromatic reduction by Desulfovibrio gigasFurther experiments with Desulfovibrio gigas showedthat different electron donors affected the p-NP trans-

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1334 T. Gorontzy, J . Kiiver and K.-H. Blotevogel

100hs 5

502

25,

4I I 0.75

0.50

0-25

1 2 3 4 5 6Time (d)Fig. 1. Repeated transformation of p-NP (100 p ~ )y cultures ofDesulfovibrio gigas with lactate and sulphate (each 20 mM) as carbonand energy sources. 0,D,,,; 4, p-NP recovery (YO)+ addition ofp-NP (100 pM).

1.254

1.oo0.75 a"00.50

- 0.251 12 4 6 8 10

Time (d)Fig. 2. Repeated transformation of p-NP (50 VM) by cultures ofMethanosarcina frisia with molecular hydrogen and carbon dioxideas carbon and energy sources. 0, Dsso; U, p-NP recovery (%);+ addition of p-NP (50 p~).formation differently (Fig. 3a). The following sequenceof transformation velocity could be observed :molecularhydrogen > pyruvate > lactate > ethanol. Except in thecase of molecular hydrogen the transformation velocitywas correlated with the specific growth rates and thebiomass yields (Fig. 3b ) . With molecular hydrogen thetransformation was completed within the early stages ofthe exponential-phase of growth.E f e c t o nitro- and aminoaromatics on growthConcentrations of p-NP higher than 0.2 mM impaired thegrowth of Desutfovibrio gigas, which became apparent byan extended lag phase. A concentration of 1.0 mM ledto complete inhibition of growth. The transformation

100

75nsW

pc5 251 A I M I

5 10 15 20

0.50

0.25

Time (d)Fig. 3. ( a ) Influence of different carbon and energy sources on thereduction of p-NP (200 p ~ )y growing cultures of Desulfouibrio gigas.(b )Growth of Dv. gigaswith different energy and carbo n sources in thepresence of 200 p~ p-NP. 0,ontrol; a, thanol (30mM); 0,actate(20 mM); B, pyruvate (20 mM); and V, H2/C02 plus acetate (20 mM).product p-aminophenol (p-AP; 0.1 to 1 0 m ~ )id notinfluence growth. Similar results were obtained withClostridium pasteurianum, but this organism toleratedhigher concentrations of p-NP than did Desulfovibriogigas. Complete inhibition of growth of Clostridiumpasteurianum was observed in the presence of 5 0 m ~ -p-NP. The transformation product p-AP did notinfluence the growth of this organism nor the growth ofMethanosarcina frisia .

DiscussionOur results conclusively show that under anaerobicconditions nitroaromatics were converted into the corre-sponding amino compounds by living bacteria as wellas abiotically. Chemical transformation occurred in thepresence of media-reducing compounds like sulphideor Ti(II1) nitrilotriacetate (Table 1). In the case ofo-nitrophenol and 2,4-dinitrophenol, nitro-group trans-formation occurred only in the ortho-position; thetransformation product of p-nitroaniline is not known.

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Microbial transformation of nitroaromatics 1335In contrast to recent findings (Macalady et al., 1986), ou rresults clearly dem onstrate tha t nitroarom atics (0-2 mM)can be reduced directly by certain reducta nts (1.5 mM).A chemical transformation of 2,4,6-trinitrotoluene to2,4-diamino-6-nitrotoluene by sulphide ions was alsoobserved (Preuss et al., 1990).

Biological transform ation of certain nitroarom atics bycell-free extracts of anaer obic bacteria h as been observedin Clostridium spp. (OBrien & Morris, 1971;Angermaier& Simon, 1983; Rafii et al., 1991), Bacteroides fra gilis(Kinoushi & Ohnishi, 1983), Veillonella alcalescens(McCormick et al., 1976) and Enterobacter cloacae(Bryant & DeLuca, 1991). Our results extend thisrange of species for which transformation has beendemonstrated to some strains of sulphate-reducing andmethanogenic bacteria (Table 2). According to otherauthors (Boyd et al., 1983; Battersby & Wilson, 1989)the reductive transformation of nitroaromatic com-pounds leads to a detoxification of the substance.Methanogenic bacteria lysed in the presence ofnitroarom atics. This was correlate d with the reduction ofthe nitro-substituent to the corresponding amino-group.In Fig. 2 the behaviour of Methanosarcina fris ia in thepresence of 50 VM p-NP is shown. Cell lysis does notdepend on the type of nitroaromatic compound. Thismay be the reason why methanogenesis is inhibited bynitroaromatics during anaerobic sewage treatment(Horowitz et al., 1982; Boyd et al., 1983; Johnson &Young, 1983). However, Fedorak et al. (1990) found noevidence that anilines negatively influenced methano-genesis. This was also confirmed in our experiments:after the reduction of the nitro-substituent, the resultingamin oarom atic molecule no longer hindered cell grow th.Additionally, other aminoaromatics did not cause celllysis. Therefore we assumed that nitroaromatics orintermediates of the reduction process like nitroso- orhydroxylamines are the real toxicants, since they mayreact with the unique cell membrane components of themethanogens.A second toxic function of a n itroaroma tic compoundmight be determined by the fact that it acts as anelectron trap. This has already been assumed for theoxygen sensitivity of methan ogens (Jarrell, 1985), conse-quently leading to the breakdown of ATP synthesis. Athird possibility might be the uncoupling activity ofnitroaromatics on electron trans port chains thus inhibit-ing ATP synthesis, followed by cell lysis. Althoughinformation about the biochemistry of nitro-groupreduction under methanogenic conditions is rather po or,it cannot be excluded that the coenzyme F420s involvedin such a reaction due to its low redox potential(Eh = -340 mV). Apparently the methanogens weremore sensitive to the presence of nitroaromatics thanwere the sulphate-reducing bacteria and clostridia. This

is probably du e to the destruction of the twofold functionof the cell membranes of most methanogens as aphysiological barrier an d as the cell skeleton. In sulpha te-reducing bacteria and clostridia such a destructiveeffect of nitroaromatics might be prevented by thepresence of a murein-containing cell wall and a differentcomposition of the cell membrane.In contrast to the methanogens, Clostridium spp. andsulphate-reducing bacteria reduced the nitro-substituentswithout cell lysis (Table 2) . It is not known whyDesulfococcus multivorans and Desulfotoma culum orientisonly transformed p-nitrophenol. As shown in Fig. 1,there is a strong correlation between the growth phaseand the reduction process. In addition, p-NP reductionby Desulfovibrio gigas is also dependent on the kind ofelectron donor. Due to the electron donor applied andthe bioma ss yielded the following sequence of the ra te oftransformation of p-NP could be measured: ethanol

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