Brief report

Nitrogen fixation by reductively dechlorinating bacteria

Xiongfei Ju, Liping Zhao and Baolin Sun*Hefei National Laboratory for Physical Sciences atMicroscale and School of Life Sciences, University ofScience and Technology of China, Hefei, Anhui 230027,China.

Summary

Significant advances in the ecology, physiology andgenetics of reductively dechlorinating bacteria haverevealed their important environmental roles in biore-mediation and in the global chlorine cycle. N2 fixationhas been widely observed in symbiotic, associativeand free-living bacteria. Here we show physiologicaland molecular evidence that reductively dechlorinat-ing bacteria are capable of fixing atmosphericnitrogen. Furthermore, N2 fixation in some of thesedechlorinating bacteria stimulated reductive dechlo-rination, which should help predict and regulate theenvironmental function of dechlorinating bacteria inin situ bioremediation of chlorinated pollutants.These results imply that N2 fixation in dechlorinatingbacteria interacts with other biogeochemical cyclesto control the nitrogen status of the anaerobicecosystem.

Introduction

A wide range of man-made and naturally producedchlorinated hydrocarbons has been released into theenvironment. These compounds include many of the mosttoxic and environmentally persistent pollutants. Their widedistribution causes a significant risk to public health andthe environment. Fortunately, reductively dechlorinatingbacteria are capable of transforming chlorinated hydro-carbons through reductive dechlorination in anaerobicenvironments such as anoxic soils, groundwaters andsediments (Mohn and Tiedje, 1992; Fetzner, 1998), in anenergy-generating process termed halorespiration. Bac-terial culture, carbon isotope fractionation and molecularbiology studies have showed that halorespiring anaer-

obes are widely distributed in nature, implying their impor-tant environmental roles in bioremediation and in theglobal chlorine cycle (Drenzek et al., 2001; SherwoodLollar et al., 2001; Smidt and de Vos, 2004; Watts et al.,2005). Some reductively dechlorinating bacteria alsoconduct alternative physiological activities such as nitrateand sulfate reduction, depending on environmentalconditions. In addition, reductive dechlorination may becompatible with other important physiological featuressuch as nitrogen fixation through long-term evolutionaryadaptation.

N2 fixation is an energy-consuming process and hasbeen widely observed in symbiotic, associative and free-living bacteria. This bacterial physiological feature plays atremendous role in ecological sustenance, world agricul-ture and global nitrogen cycle. Nitrogen availability canlimit microbial growth and affect ecosystem activity (Dixonand Kahn, 2004). Although N2 fixation is widely distributedamong Bacteria and Archaea (Raymond et al., 2004), thissignificant physiological feature has not been revealedin reductively dechlorinating bacteria. Results fromsequencing of three whole genomes of the halorespiringbacteria Desulfitobacterium hafniense and Dehalococ-coides indicated that nitrogen fixation-related genes arefound in D. hafniense strain Y51 (Nonaka et al., 2006) andDehalococcoides strain 195 (Seshadri et al., 2005), butare missing in Dehalococcoides strain CBDB1 (Kubeet al., 2005). In this research we provide experimentalevidence that reductively dechlorinating bacteria arecapable of fixing atmospheric nitrogen.

Results and discussion

nifH cloning and phylogenetic analysis

We detected N2 fixation capability in halorespiring anaer-obes Desulfomonile tiedjei (Shelton and Tiedje, 1984),Sulphurospirillum multivorans (Scholz-Muramatsu et al.,1995), Desulfovibrio dechloracetivorans (Sun et al., 2000)and Desulfitobacterium dehalogenans (Utkin et al., 1994),which were isolated from distinct habitats and representGram-negative and Gram-positive bacteria. Partial nifHfragments (approximate 390 bp) were amplified usingdegenerate primers (Mehta et al., 2003) corresponding tothe conserved regions of nifH. All these dechlorinating

Received 20 July, 2006; accepted 30 October, 2006. *Forcorrespondence. E-mail sunb@ustc.edu.cn; Tel. (+86) 551 360 6748;Fax (+86) 551 360 7438.

Environmental Microbiology (2007) 9(4), 1078–1083 doi:10.1111/j.1462-2920.2006.01199.x

© 2006 The AuthorsJournal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd

bacteria possessed nifH homologues. InD. dechloracetivorans, two divergent nifH homologues(nifH-1 and nifH-2) were found. nifH-2 grouped withincluster I of the NifH phylogenetic tree (Fig. 1), whichincludes a-, b- and g-proteobacterial nitrogenases; nifH-1fell into cluster III, which includes diverse anaerobic bac-teria like sulfate reducers and reductive dechlorinators.

Single nifH homologues were found in three other bacte-ria and grouped into different clusters: S. multivorans intocluster I, D. tiedjei into cluster III and D. dehalogenansinto cluster IV respectively. The distribution of nifH homo-logues in these dechlorinating bacteria coincides withconventional opinion that nifH was phylogeneticallydiverse in nature (Ueda et al., 1995; Zehr et al., 1998;

Fig. 1. Unrooted Neighbour-Joining phylogenetic tree of deduced NifH sequences of diazotrophic and reductively dechlorinating bacteria.Polymerase chain reaction products (approximate 390 bp) were cloned with TOPO TA Cloning Kit (Invitrogen) and 10 colonies were randomlyselected for sequencing. The obtained nifH partial sequences have been submitted to GenBank databases under Accession numberDQ337203 for nifH-1 of D. dechloracetivorans (ATCC700912); DQ337204 for nifH-2 of D. dechloracetivorans; DQ337205 for nifH of D. tiedjei(ATCC49306); DQ337206 for nifH of S. multivorans (DSMZ12446) and DQ337207 for nifH of D. dehalogenans (DSMZ9161). The previouslydetermined nifH gene sequences used for comparisons in this study were selected from other representative diazotrophs that fell into fourclusters and retrieved from the GenBank database at the National Center for Biotechnology Information. Deduced NifH amino acid sequenceswere aligned using ClustalW version 1.81, and Unrooted Neighbour-Joining phylogenetic tree was created with the program MEGA version 3.1.Analysis was bootstrapped and bootstrap values greater than 60 are indicated at respective nodes (1000 replications). The scale indicates thenumber of amino acid substitutions per site.

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2003; Mehta et al., 2003), suggesting that lateral transferof nifH genes may have occurred in diverseenvironments. Previous studies have shown that somesymbiotic and free-living bacteria harbour multiple nifHhomologues (Lilburn et al., 2001; Choo et al., 2003), buthow these nifH homologues respond to environmentalcues and are expressed remains unknown. The diver-gence of nifH genes in these bacteria may proffer themecological advantages in natural environments.

N2-dependent growth and acetylene reduction activity

The nitrogenase activity in D. dechloracetivorans andS. multivorans was demonstrated by their N2-dependentgrowth and their NH4

+-repressible acetylene reductionactivity (Fig. 2). Dechlorination products such as phenoland benzoate were analysed by high-performance liquid

chromatography (Agilent 1100 Series) with reverse C18

column (Phenomenex). Dichloroethene (DCE) was moni-tored by gas chromatography (Agilent 6890 N) equippedwith a flame ionization detector with a DB-1 column(30 m ¥ 250 mm ¥ 1 mm). For acetylene reduction assay,6 ml of acetylene was injected into headspace of eachbottle when bacterial growth entered exponential phase.Ethylene was analysed by gas chromatography (Agilent6890 N) equipped with a HP-PLOT/AL203 ‘S’ Deactivatedcolumn (50 m ¥ 530 mm ¥ 15 mm). Ethylene productionrate was determined from the slope of the linear regres-sion of time versus ethylene concentration using softwareOrigin version 7.5.

N2 fixation in D. tiedjei was evidenced by bacterialgrowth and reductive dechlorination when N2 was the solenitrogen source, although acetylene reduction activity wasnot observed in this organism. No growth or reductive

Fig. 2. Growth optical density (OD600) or reductive dechlorinating product concentration and rate of acetylene reduction to ethylene.A. Growth of D. dechloracetivorans under sulfate reduction conditions (32 mM sulfate + 10 mM lactate).B. Reductive dechlorination of 2-chlorophenol (2-CP) to phenol with pyruvate (10 mM) as electron donor by D. dechloracetivorans.C. Fumarate respiration by S. multivorans (10 mM fumarate + 10 mM pyruvate).D. Reductive dechlorination of tetrachloroethene (PCE) to DCE with pyruvate (10 mM) as electron donor by S. multivorans. Addition of 5.6 mMNH4Cl into culture resulted in complete suppression of acetylene reduction (data not shown). The error bars indicate the standard errors of themeasurements. Symbols: �, nitrogenase activity determined by the acetylene reduction with N2 as the sole nitrogen source; �, addition ofammonia as nitrogen source; �, bacterial growth with N2 as the sole nitrogen source; �, addition of Ar instead of N2 as negative control.

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dechlorination was observed in D. dehalogenans underany growth conditions when ammonia was omitted, indi-cating that nifH is not functional in N2 fixation in thebacterium. This result is consistent with the previousobservation that diverse nifH homologues in cluster IV arenot involved in N2 fixation (Raymond et al., 2004; Mehtaet al., 2005).

It is interesting to note that N2 fixation gave riseto higher reductive dechlorination rates inD. dechloracetivorans and D. tiedjei, which were sus-tained over repeated feedings of chlorinated compoundsas terminal electron acceptors. When N2 was provided asthe sole nitrogen source and pyruvate was used aselectron donor in D. dechloracetivorans, dechlorinationrate (4.24 mmol l-1 h-1) was the same as with ammonia(Fig. 2B). On the other hand, when N2 was provided asthe sole nitrogen source and acetate was used aselectron donor, reductive dechlorination rate(3.94 mmol l-1 h-1) was higher than that (3.26 mmol l-1 h-1)of ammonia (Fig. 3A); under both growth conditions,biomass production was about 3 g of cells (dry weight) permol of 2-chlorophenol dechlorinated. This suggests that amore efficient halorespiration occurred when acetateserved as electron donor, considering that N2 fixation is anenergy-consuming process. Similar dechlorination patternwas observed in D. tiedjei (Fig. 3B) and this phenomenonwas only observed under dechlorination conditions. Com-paring with acetate, pyruvate oxidation produces reducingequivalents with much more negative redox potential,which are preferred by both reductive dechlorination andN2 fixation. The competition for some components of theelectron transport chain may affect dechlorination activity.Other possible explanations for this phenomenon include

that acetate oxidation provides a more efficient and bal-anced carbon and nitrogen flux, and/or ammonia assimi-lation affects reductive dechlorination. Although we do nothave convincing data to explain this phenomenon, it hassignificant implications in in situ bioremediation. In somecases, nitrogen input is necessary in bioremediation ofhaloorganics-contaminated sites, where nitrogen sourceis limited. Nitrogen input may lead to the dominance ofbacterial populations other than dechlorinators, therebydecreasing their environmental function. In addition,excess nitrogen input may cause nitrate contamination inthese sites. This finding not only may provide clues of howreductively dechlorinating bacteria take advantage of theirdiverse physiological features in natural environment, butalso help us optimize nitrogen input and regulate theenvironmental function of dechlorinating bacteria in con-taminated sites.

Reverse transcription polymerase chain reaction(RT-PCR)

Reverse transcription polymerase chain reaction wasexecuted to further detect the capability of N2 fixation indechlorinating bacteria. Anticipated RT-PCR productswere amplified from D. tiedjei and S. multivorans cultures(Fig. 4C and D), further demonstrating the nifH expres-sion and N2 fixation in these bacteria. As predicted, noRT-PCR product was observed from D. dehalogenanscultures (data not shown). Interestingly, inD. dechloracetivorans an expression discrepancy ofnifH-1 and nifH-2 was observed (Fig. 4A and B). OnlynifH-1 was expressed under sulfate reduction conditions;both nifH-1 and nifH-2 were expressed under pyruvate

Fig. 3. Reductive dechlorination and growth under various culture conditions.A. Reductive dechlorination of 2-CP to phenol with acetate (2.5 mM) as electron donor by D. dechloracetivorans.B. Reductive dechlorination of 3-chlorobenzoate (3-CB) to benzoate with lactate (10 mM) as electron donor by D. tiedjei. The error barsindicate the standard errors of the measurements. Symbols: �, addition of ammonia as nitrogen source; �, N2 as the sole nitrogen source; �,addition of Ar instead of N2 as negative control.

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fermentation and reductive dechlorination conditions.Further elucidation of the nifH expression pattern in thisorganism should aid in our understanding of genetic regu-lation of N2 fixation in reductively dechlorinating bacteria.

The diversity and global distribution of reductivelydechlorinating bacteria have been revealed in recentyears, suggesting the significant role they play in thebiogeochemical transformations of elements and eco-system sustenance (Smidt and de Vos, 2004). Thisstudy reveals a heretofore unrecognized role for reduc-tively dechlorinating bacteria in the anoxic ecosystemand in the global nitrogen cycle. Our results may alsolead to a better understanding of the dynamics, the spe-cific activities and the conditions required for environ-mental function of dechlorinating populations in in situbioremediation.

Acknowledgements

We thank Zhang J. and Wang J. for technical assistance. Thiswork was supported by the One Hundred Talent Project ofChinese Academy of Sciences.

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