Pergamon

Geochimica et Cosmochimica Acta, Vol. 68, No. 15, pp. 3271–3283, 2004Copyright © 2004 Elsevier Ltd

Printed in the USA. All rights reserved0016-7037/04 $30.00� .00

doi:10.1016/j.gca.2003.11.028

Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable Cisotope fractionation and stable isotope probing to identify reactions

and the responsible microorganisms

LAURENCE G. MILLER,1,* K AREN L. WARNER,2 SHAUN M. BAESMAN,1 RONALD S. OREMLAND,1 IAN R. MCDONALD,2

STEFAN RADAJEWSKI,2 and J. COLIN MURRELL2

1U.S. Geological Survey, Menlo Park, CA 94025, USA2Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

(Received May 28, 2003;accepted in revised form November 25, 2003)

Abstract—Bacteria in soil microcosm experiments oxidized elevated levels of methyl chloride (MeCl) andmethyl bromide (MeBr), the former compound more rapidly than the latter. MeBr was also removed bychemical reactions while MeCl was not. Chemical degradation dominated the early removal of MeBr andaccounted for more than half of its total loss. Fractionation of stable carbon isotopes during chemicaldegradation of MeBr resulted in a kinetic isotope effect (KIE) of 59� 7‰. Soil bacterial oxidation dominatedthe later removal of MeBr and MeCl and was characterized by different KIEs for each compound. The KIEfor MeBr oxidation was 69� 9‰ and the KIE for MeCl oxidation was 49� 3‰. Stable isotope probingrevealed that different populations of soil bacteria assimilated added13C-labeled MeBr and MeCl. The identityof the active MeBr and MeCl degrading bacteria in soil was determined by analysis of 16S rRNA genesequences amplified from13C-DNA fractions, which identified a number of sequences from organisms notpreviously thought to be involved in methyl halide degradation. These includedBurkholderia, the major clonetype in the13C-MeBr fraction, andRhodobacter, Lysobacter andNocardioides the major clone types in the13C-MeCl fraction. None of the 16S rRNA gene sequences for methyl halide oxidizing bacteria currently inculture (includingAminobacter strain IMB-1 isolated from fumigated soil) were identified. Functional geneclone types closely related toAminobacter spp. were identified in libraries containing the sequences for thecmuA gene, which codes for the enzyme known to catalyze the initial step in the oxidation of MeBr and MeCl.The cmuA gene was limited to members of the alpha-Proteobacteria whereas the greater diversity demon-strated by the 16S rRNA gene may indicate that other enzymes catalyze methyl halide oxidation in different

groups of bacteria. Copyright © 2004 Elsevier Ltd

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1. INTRODUCTION

Methyl bromide (MeBr) and methyl chloride (MeCl) aimportant atmospheric trace gases (tropospheric mixing r� 12 and 600 pptv, respectively) that contribute directlstratospheric ozone depletion. Both compounds have nand anthropogenic sources and sinks. The significant soand sinks contribute to the balance of MeBr or MeCl inatmosphere as well as the stable C isotope ratios of amMeBr or MeCl in air (McCauley et al., 1999; Bill et al., 20022002b). Soils comprise the largest known sink for MeBr onEarth’s surface (Kurylo et al., 1999) and are also a large sifor MeCl (Harper, 2000). However, the processes that influethe flux of these compounds from air to soil or soil to airpoorly understood at present.

Degradation of MeBr has been studied at low (ambconcentrations as well as at high concentrations achieveding agricultural soil fumigations (up to 30,000 ppmv). Suptake of tropospheric mixing ratios of MeBr was attributebacteria (Shorter et al., 1995; Hines at al., 1998) and purecultures of methanotrophs and methylotrophs have been sto oxidize these low mixing ratios characteristic of troposphMeBr (Goodwin et al., 2001). High levels of MeBr are toxic tmost organisms, hence the widespread commercial applic

* Author to whom correspondence should be addressed (lgmi

usgs.gov).

3271

of MeBr as a preplant soil fumigant. However, certainbacteria, such asAminobacter strain IMB-1, can grow boxidizing high levels of MeBr (Miller et al., 1997; ConnelHancock et al., 1998). Chemical losses (including hydrolyand reaction with organic matter) may also contribute todegradation of MeBr observed in soils (Gan et al., 1996Papiernik et al., 2000; Tao and Maciel, 2002).

The soil sink for MeCl is less well studied, in part becauis less significant to the global budget (Khalil and Rasmusse1999). However, because the atmospheric concentratioMeCl is higher than MeBr, MeCl appears to be more dynaRhew et al. (2000)reported that soils take up low concentions of MeCl�40 times more rapidly than they do MeBr.with MeBr, the MeCl sink can be separated into microbioical and chemical components (Harper, 2000). Chemical uptakis probably much less significant than for MeBr becausedrolysis occurs slowly if at all (Elliott and Rowland, 1995).Other chemical losses, including reaction with soil orgmatter may be important.

It is not known to what extent microbiological or chemiprocesses affect stable isotope fractionation of methyl hain soil. Previous studies of13C/12C ratios of MeBr and MeCemissions from salt marshes (Bill et al., 2002a) and of MeBrefflux from fumigated soil (Miller et al., 2001; Bill et al.2002b) showed the importance of degradation in determithe isotopic composition of gases leaving the soil, but w

unable to evaluate the in situ removal mechanisms. Determi-

3272 L. G. Miller et al.

nation of the relative contribution of chemical vs. microbiolog-ical uptake of MeBr in agricultural soil was one of the goals ofthis study.

Methylotrophic bacteria capable of oxidizing MeBr or MeCl(or both) have been isolated from soils. The described bacterialcultures are taxonomically diverse within the �-Proteobacteria,and include representatives of several different genera includ-ing Methylobacterium, Hyphomicrobium and Aminobacter spp.(McDonald et al., 2002), yet they have common characteristicswith respect to their enzymes and metabolic pathways formethyl halide oxidation. All of the soil methylotrophs testedthus far contain a gene (cmuA) that codes for the methyltransferase (CmuA) responsible for methyl halide degradation(Woodall et al., 2001; McDonald et al., 2002). The enzyme hasbeen purified (Coulter et al., 1999) and contains both methyl-transferase and corrinoid binding domains (McAnulla et al.,2001a).

Similarities among methylotrophs extend to their kineticisotope effects or KIEs. The three strains tested by Miller et al.(2001), including two isolated from soils, fractionated stablecarbon isotopes of MeCl to the same degree. However, largedifferences were observed in the stable isotope fractionationassociated with the oxidation of MeBr by these organisms.Such differences might allow the use of carbon isotope frac-tionation studies to determine which bacteria are responsiblefor degrading MeBr in soil. Other soil bacteria, includingmethanotrophs and nitrifiers, degrade MeBr in soils and inculture (Oremland et al., 1994). It is not know to what degreemethanotrophs or nitrifiers fractionate carbon during co-oxida-tion of MeBr.

Further insights into the pathways of microbial oxidation aregained from identifying and sequencing specific genes of mi-croorganisms that are responsible for MeBr and MeCl oxida-tion in soil and comparing these sequences with those fromknown methylotrophs. This was accomplished here by follow-ing the uptake of added 13C-labeled MeBr or MeCl throughstable isotope probing (SIP) (Radajewski et al., 2000) to deter-mine which bacteria accumulated the label and hence wereactively involved in oxidizing MeBr and MeCl. The organismsactively involved in oxidizing MeBr and MeCl were deter-mined by analyzing the diversity of sequences of the phyloge-netic 16S rRNA gene and the functional cmuA gene, whichcodes for the enzyme responsible for oxidizing MeBr and MeClin this soil (functional gene probing).

2. MATERIALS AND METHODS

2.1. Measuring Chemical vs. Biological Degradation

Headspace concentrations of MeBr and MeCl were measured inbottles with soil (Elder sandy loam; organic carbon �1%, pH � 6.8)collected on 11/6/02 from Monterey Bay Academy, Watsonville, CA(Bill et al., 2002b). Soil was stored in sealed glass jars at 4°C and fieldwater content (�15% w/w) for 1 to 2 months before use. Soil was airdried for 1 d at room temperature (22°C) and sieved through a 2-mmscreen. Soil water content was determined in order that adjustments toconstant water content (10%) could be made later by adding water tobottles. Soil (50 g oven dried weight) was added to 58-mL amber glassserum bottles, sealed with butyl rubber stoppers and crimped withaluminum seals (Bellco Glass Inc.). Triplicate bottles of live soil wereprepared for each of six concentrations of MeBr or MeCl applied to thesoils. A single control bottle for each concentration of MeBr or MeCl

was autoclaved (121°C, 100 kPa) for 1 h. This step was repeated after

24 h to ensure that spores were also killed. Controls were flushed withfilter-sterilized (0.2 �m) air after cooling following the second heattreatment. Various amounts (1.1–22 �mol) of MeBr or MeCl (Mathe-son Gas Products) were added to bottles via gas-tight syringes using25-g needles. Headspace gas samples (0.1 mL) were collected severaltimes during each experiment for analysis by GC-FID (Miller et al.,1997) and results reported in micromoles per liter of gas phase. Preci-sion of FID analyses was 5% of the reported concentration for bothmethyl halides.

2.2. Determining Kinetic Isotope Effects

2.2.1. Experiments and measurements

Microcosm experiments with soil were conducted to determine theseparate kinetic isotope effects (KIEs) of carbon resulting from chem-ical and biologic uptake of MeBr and MeCl. Soils were treated asabove, with the difference that duplicate heat-killed controls wereprepared. A single level of MeBr or MeCl (6.7 �mol) was added at thestart and headspace samples were collected over time for both GC-FIDanalysis of concentration (0.1 mL), as above, and for simultaneousGC-combustion-mass spectrometry (irmGCMS) determination of the�13C of the reactant MeBr or MeCl (1.0 mL) (Miller et al., 2001; Billet al., 2002b). The large sample volumes required for the combinedanalyses (1.1 mL) necessitated correction of the total pressure in thebottles, resulting in an upward adjustment of 2.0 and 2.3% (v/v) in theheadspace concentration of MeBr and MeCl, respectively, followingeach sampling. Stable carbon isotope ratios of samples and standardswere determined by comparison with CO2 of known isotopic compo-sition and reported in delta notation as per mil vs. the Vienna PDBstandard.

2.2.2. Calculation of KIE

The isotope fractionation associated with degradation was deter-mined from the slope of the regression of �13C values of the reactantMeBr or MeCl against the logarithm of the fraction (C/Co) of reactantremaining (Miller et al., 2001). Fractionation factors were positive (i.e.,reaction rates with the lighter isotope were greater than rates with theheavier isotope) and are reported as �, the deviation from unity times103. The chemical KIE was derived using the heat-killed controls.Separate determinations of KIE resulting from biologic uptake weremade by correcting the measured concentrations and �13C values forlive soil at each sampling time by the linear changes over time ofheadspace concentration and �13C in heat-killed soil to obtain theresulting values due solely to bacterial oxidation at that time (Kalin etal., 2001). Data from live soils (three bottles) or heat-killed soils (twobottles) for each methyl halide were combined to provide greaternumbers of samples for statistical analysis and graphical representation.Standard errors (1 S.E.) are reported for the determination of each KIE.

2.3. Stable Isotope Probing

2.3.1. Extraction of 13C-Enriched DNA

DNA of microorganisms that assimilated MeBr or MeCl was en-riched in13C by adding pulses (22–180 �mol per pulse) of 13C-labeledMeBr or MeCl (99 atom % 13C; Isotec Inc.) to 150 g of live soil in159-mL glass serum bottles. Soil was collected from the same site atMonterey Bay Academy, 1 month after the most recent MeBr fumiga-tion. Headspace MeBr and MeCl concentrations were monitored usingGC-FID and additions were only made following complete degradationof the previous pulse. Up to eight pulses were added for a total of 600�mol MeBr and MeCl.

DNA was extracted from eight 5-g samples (40 g total) of each soilby a method based on Zhou et al. (1996). Soil (5 g) was mixed with13.5 mL DNA extraction buffer (100 mmol/L Tris-HCl pH 8.0, 100mmol/L NaEDTA pH 8.0, 100 mmol/L sodium phosphate pH 8.0, 1.5mol/L NaCl) and 1% (w/v) cetyl trimethylammonium bromide (CTAB)and 100 �L proteinase K (10 mg/mL) in Oakridge tubes and shakenhorizontally at 37°C for 30 min. Then 1.5 mL of 20% (w/v) sodiumdodecyl sulfate (SDS) was added and the solution incubated at 65°C for

2 h with gentle shaking every 20 min. The solution was centrifuged at

3273Methyl halide degradation by soils and bacteria

6000g for 10 min at 22°C and the supernatant transferred into sterileOakridge tubes. The soil pellets were extracted twice more by re-suspending in 4.5 mL of extraction buffer and 0.5 mL 20% (w/v) SDSby vortexing for 10 s, incubated at 65°C for 10 min, then centrifugedas before. The supernatants were combined and cleaned by mixing withan equal volume of chloroform:isoamyl alcohol (24:1). Purified DNAobtained after each extraction was precipitated with isopropyl alcohol,pelleted, washed in 70% (v/v) ethanol and re-suspended in 0.5 mLH2O. CsCl (4 g) was dissolved in the total DNA (4 mL) and centrifuged(265,000g) for 19 h in a CsCl-ethidium bromide density-gradient asdescribed by Radajewski et al. (2002). Following ultra-centrifugation,two distinct DNA fractions were visualized in each gradient with UVlight (365 nm). A “ light” (12C) DNA fraction was prominent in eachgradient, which represented the total microbial community that grew onnatural, unlabelled carbon substrates in the soil. A faint “heavy” (13C)DNA fraction was also visible �5 to 10 mm below the 12C-DNA,which represented the microbial community that had assimilated theadded 13C-labeled MeCl or MeBr. The 13C- and 12C-DNA fractionswere collected separately from each gradient (13C-DNA collected first)as described previously (Radajewski et al., 2002). Each fraction wasadded to a fresh CsCl gradient and re-centrifuged (140,000g) for 66 h(Radajewski and Murrell, 2002), after which only single discrete DNAfractions were visible in each gradient. The resulting fractions werecollected and ethidium bromide was removed from the DNA by ex-traction in 1-butanol saturated with TE buffer (10 mmol/L Tris HCl, 1mmol/L EDTA, pH 8.0). Following dialysis against TE buffer, the 13C-and 12C-DNA fractions were ethanol precipitated (overnight) and dis-solved in 0.1 mL of TE buffer.

2.3.2. PCR Characterization of 13C- and 12C-DNA fractions

The 12C- and 13C-DNA fractions from MeCl and MeBr were used astemplates for PCR. The primers Eubac 27F and 1492R (Lane, 1991)were used to amplify the bacterial small subunit rRNA gene. Themethyl halide utilization gene cmuA was also targeted using the cmuAprimers cmuA802f (5�-TTC AAC GGC GAY ATG TAT CCY GG-3�)and cmuA1609r (5�-TCT CGA TGA ACT GCT CRG GCT-3�), re-cently designed from the four cmuA sequences currently available.Amplification reactions were performed using the reagents supplied inthe GIBCO-BRL (Paisley, Scotland) Taq polymerase kit in a 0.5-mLmicrocentrifuge tube in 50-�L reaction mixtures containing 1� PCRamplification buffer (20 mmol/L Tris-HCl, pH8.4, 50 mmol/L KCl),1.5 mmol/L Mg2�, 0.2 mmol/L of each deoxynucleoside triphosphate,20 �g bovine serum albumin (Boehringer Mannheim, Germany), 100pmol of each primer set and 2.5 U of Taq polymerase. The reactionswere carried out in a DNA thermocycler (Touchdown model; Hybaid,Teddington, Middlesex, United Kingdom) with initial denaturation at94°C for 5 min, followed by addition of Taq polymerase and 30 cyclesof 94°C for 1 min, annealing (60°C with 27f/1492r and 55°C withcmuA802f/cmuA1609r) for 1 min and 72°C for 1 min, followed by afinal extension at 72°C for 10 min. Reaction products were checked forsize and purity on 1% (w/v) agarose gels (Sambrook et al., 1989). PCRproducts of the correct size (�1450 bp for 16S rRNA and 807 bp forcmuA) were cloned using the TOPO TA cloning kit (Invitrogen) andlibraries of 100 clones (for bacterial 16S rRNA) and 50 clones (forcmuA) from both the 13C-MeBr and 13C-MeCl DNA fractions werescreened by Restriction Fragment Length Polymorphism (RFLP) anal-ysis. Smaller 16S rRNA gene libraries (25 clones each) were preparedto analyze representatives in the 12C-DNA fractions. EcoRI and RsaIwere used in the restriction digests for the 16S rRNA gene libraries andEcoRI and DdeI were used for the cmuA libraries. The clones from eachlibrary were grouped into operational taxonomic units (OTUs) usingthe restriction patterns and representative clones from the OTUs weresequenced. Complete sequence data (between the amplification prim-ers) were obtained for one clone from each OTU in the 13C-16S rRNAgene libraries that contained more than two clones, and for one clonefrom each OTU in the cmuA libraries with more than one clone. Partial16S rRNA gene sequences were obtained using primers 357f and 1100r(Lane, 1991). For all libraries, at least 10% of clones within each OTUwere partially sequenced (�500 bp) to verify that the restriction patternrepresented a single sequence type: DNA sequences of multiple clonesfrom each OTU were �99% identical. The sequences have been

deposited in GenBank AY439185 to AY439212.

2.3.3. Denaturing gradient gel electrophoresis (DGGE)

DGGE was used to compare the 13C- and 12C- fractions of DNAobtained for each methyl halide and to contrast the 13C-MeBr fractionwith the 13C-MeCl fraction. The PCR primers 341F-GC and 907R(Muyzer et al., 1993) were used to obtain 16S rRNA gene amplificationproducts from the MeBr and MeCl 12C- and 13C-DNA fractions.Amplification reactions were performed using the reagents supplied inthe GIBCO-BRL (Paisley, Scotland) Taq polymerase kit in a 0.5-mLmicrocentrifuge tube in 50-�L reaction mixtures containing 1� PCRamplification buffer (20 mmol/L Tris-HCl, pH8.4, 50 mmol/L KCl),1.5 mmol/L Mg2�, 0.2 mmol/L of each deoxynucleoside triphosphate,20 �g bovine serum albumin (Boehringer Mannheim, Germany), 1.3%(v/v) DMSO/1.3 mol/L glycine betaine, 100 pmol of each primer and2.5 U of Taq polymerase. The reactions were carried out with initialdenaturation at 94°C for 5 min, followed by addition of Taq polymer-ase and 20 cycles of 94°C for 1 min, 65°C. . .55°C (Touchdown, 0.5°Cper cycle) for 1 min and 72°C for 3 min, followed by 15 cycles of 94°Cfor 1 min, 55°C for 1 min and 72°C for 3 min, followed by a finalextension at 72°C for 7 min. These were separated on a 6% (w/v)DGGE gel with an 80 to 20% gradient using the methods of Schaferand Muyzer (2001).

2.3.4. Phylogenetic analysis and construction of dendrograms

The ARB program (http://www.mikro.biologie.tu-muenchen.de) wasused for sequence alignments and phylogenetic analyses. 16S rRNAgene sequences were aligned using the automatic alignment tool ofARB (ALIGNER, version 2.0), and the alignments were correctedaccording to secondary structural constraints of the 16S rRNA. Toidentify close relatives of the 16S rRNA gene clone sequences in theARB database, the aligned sequences were added to a phylogenetic treeof sequences (�1400 bp) by using a maximum-parsimony tool withinARB. 16S rRNA gene sequences from other closely related bacteriawere identified by BLAST searches of GenBank and aligned to theARB database. A filter was generated that omitted alignment positionsof missing data or sequence ambiguity (N) for sequences of �1300 bp.

Phylogenies were reconstructed using evolutionary distance (Jukesand Cantor model with FITCH) analysis of the aligned near full length(�1300 bp) 16S rRNA gene sequences. Partial 16S rRNA gene se-quences were added to the final tree using a maximum parsimony toolwithin ARB. The functional gene sequences (cmuA) were alignedmanually to related sequences from GenBank. A filter was generatedfrom the derived amino acid sequences that excluded alignment posi-tions containing amino-acid ambiguity (X) and missing sequence data.Phylogenetic trees were constructed using the PROTDIST (PAM-Dayhoff matrix with FITCH). The confidence of branch points wasdetermined for each analysis by bootstrapping (100 replicates) with thesame evolutionary distance parameters.

3. RESULTS

3.1. Different Uptake Patterns for MeBr and MeCl

MeBr or MeCl introduced into the headspace of vials con-taining soil was rapidly distributed between gas and liquidphases such that 60% of the MeBr or 70% of the MeCl was inthe headspace after 1 h equilibration. The remaining 40% of theMeBr or 30% of the MeCl was reversibly dissolved in the liquidphase. These estimates of the amount dissolved agree with calcu-lations of the fraction in the liquid phase using the water content(5 mL) and dimensionless Henry’s Law constants for MeBr(0.24) and MeCl (0.35), suggesting that little physical adsorp-tion of the methyl halides to the soil surfaces had occurred.

MeBr was consumed by live soil within 4 to 19 d, with lowerquantities of MeBr degraded in less time and greater quantitiesin more time (Fig. 1a) MeBr concentrations in heat-killedcontrols decreased linearly (see example of the highest concen-tration), suggesting a constant rate of its removal from the

headspace by chemical processes. There were two phases of

3274 L. G. Miller et al.

MeBr uptake by live soils; a chemically dominated early phasewhere MeBr decreased linearly, tracking the heat-killed con-trols for a period of time ranging from 3 to 13 d, followed bya biologically dominated phase during which the rate of uptakein live soil was greater than the rate in heat-killed controls.

MeCl was removed more rapidly than equivalent amounts ofMeBr (Fig. 1b), being completely degraded by live soil within3 to 6 d. Concentrations of MeCl in heat-killed controls re-mained constant over this time (see example of the highestconcentration), indicating no chemical loss. As with MeBr,MeCl in live soil exhibited two phases of uptake; a lag phase inwhich MeCl remained constant for an initial period after thestart and a biologically dominated phase in which MeCl was

Fig. 1. Mean (n � 3) headspace concentrations of MeBr (a) andMeCl (b) in 58-mL serum bottles containing 50 g (dry weight) soil withvarying amounts of MeBr or MeCl added. Errors bars represent �1S.D. Also shown are headspace concentrations of MeBr and MeCl (�)in heat-killed controls (n � 1) for the 22-�mol additions and linearregressions of these data. The arrows show the end of the lag period forthe 22-�mol additions.

rapidly degraded. The length of the lag phase increased with

greater amounts of added MeCl, from 2 d at the lowest levelsto 4 d at the highest levels. MeCl was biologically degraded inall live samples within 2 d following the lag.

Chemical degradation rates of MeBr or MeCl were calcu-lated from the linear regression of headspace concentrations inheat-killed controls over time. MeBr chemical degradationrates increased proportionally with the amount of MeBr added(Fig. 2a) The slope of the line created by the regression of therates vs. the amount added (0.03 d1) is the chemical degra-dation rate constant for MeBr. Chemical degradation rates ofMeCl were near zero, regardless of the amount of MeCl added.

Total (chemical � bacterial) degradation rates of MeBr orMeCl were determined for the biologically dominant phase ofuptake using linear regressions of headspace concentrations inlive soils over time, following the lag period. The end of the lagperiod was defined as the time when the uptake curves for livesoils diverged by �2 S.D. from the controls. Degradation ratesof MeCl following the lag increased with greater amounts of

Fig. 2. Rate of degradation of MeBr (O) and MeCl (�) as a functionof the amount added in heat-killed soils (a) and in live soils following

the lag period (b). Error bars represent �1 S.E.

ar.

3275Methyl halide degradation by soils and bacteria

added MeCl (Fig. 2b) Degradation rates of MeBr following thelag increased less rapidly than for MeCl and reached a maxi-mum level (�2 �mol d1) beyond which no further oxidationwas achieved with increased MeBr, suggesting that microbialoxidation was inhibited at the higher levels of MeBr.

3.2. Kinetic Isotope Effects in Live and Killed Soils

MeBr was degraded by live soils over the 9 d of the KIEexperiment (Fig. 3a) Loss of MeBr in heat-killed controls waslinear over this time but slower than the loss measured in livesoils. During the first 6 d of degradation, coinciding with thelag period, �75% of the total loss of MeBr was because ofchemical reactions, whereas �25% of the loss was because ofmicrobial activity. By day 9, the fraction of MeBr lost bybacterial oxidation increased to half of the total degradation. Incontrast, there was no significant loss of MeCl in heat-killedcontrols (Fig. 3b) The lag period for MeCl degradation inbottles containing live soil was only 3 d, and was followed by2 d of rapid biologic uptake during which microorganismsoxidized the remaining MeCl.

Starting �13C values for MeBr and MeCl (–70 and –60‰,respectively) were similar to values reported in previous studies

Fig. 3. Changes in headspace concentrations of MeBr(d) with time during isotope fractionation experiments. Clines are linear regressions of concentration (a, b) and mChanges in bottles containing live soil (�) were not line

(Miller et al., 2001; Bill et al., 2002b). Degradation resulted in

increased �13C of the methyl halides with time. Changes in�13C-MeBr measured during the first 6 d of degradation in livesoil were similar to changes in heat-killed controls (Fig. 3c).However, the effect of bacterial oxidation upon �13C-MeBrwas pronounced following the lag period. By day 9, values of�13C-MeBr in live samples were enriched over 80‰ from theirstarting values and were, on average, 50‰ heavier than corre-sponding heat-killed controls. There was insufficient MeBrpresent after day 9 to measure �13C. In the case of MeBr, thetotal effect was 63 � 3‰ of which about half was chemical andhalf was bacterial. This made partitioning of the KIE intochemical and bacterial components difficult. Scatter in the datacontributed to uncertainty in the slopes of the lines, but it seemsclear that the slope of the bacterial fractionation line wasgreater than the slope of the chemical fractionation line (Fig.4a). This is consistent with the calculated KIE values of 69 �9‰ and 59 � 7‰, respectively. However, it should be cau-tioned that the compounded error in estimating the bacterialKIE by difference is large and that the measured chemical KIEand the calculated bacterial KIE are not significantly different.By contrast with MeBr, there was a large increase in �13C-MeCl values with time in bottles containing live soil but nocorresponding change in �13C-MeCl of heat-killed controls

MeCl (b) and in headspace �13C of MeBr (c) and MeClation is reported as a fraction of the initial amount. Solidd �13C (c, d) in bottles containing heat-killed soil (�).

(a) andoncentr

easure

(Fig. 3d). In this case, degradation of MeCl was exclusively

3276 L. G. Miller et al.

biologic, and therefore the measured total KIE of 49 � 3‰ wasthe same as the biologic KIE (Fig. 4b).

3.3. Stable Isotope Probing of Bacterial Communities

13C-DNA was collected from soils incubated with 13C-la-beled substrates, which represented the microbial communitythat had actively assimilated MeBr or MeCl and incorporatedthe 13C into their DNA as the primary carbon source. The12C-DNA fraction was also collected as it represented themicrobial community that did not metabolize the methyl ha-lides. A comparison of the profiles produced from the 12C- and13C-DNA fractions for each substrate (MeBr or MeCl) indi-cated that the 13C-DNA fraction was different from that of thenon-methyl halide degrading bacterial population (12C-DNA)(Fig. 5). The 12C-MeCl fraction was a smear suggesting thatthere were a large number of different organisms present in thefraction. This corresponded with results from the 12C-MeCl16S rRNA gene library (where 22 OTUs were detected from 25clones). Furthermore, the 16S rRNA gene profiles producedfrom the MeBr and MeCl 13C-DNA fractions were differentfrom each other, and may reflect the incorporation of 13C-carbon from each substrate into the DNA of different bacteria.The profiles of four MeBr or MeCl utilizing isolates currentlyin culture also differed from those of the 13C-DNA fractions,indicating that as yet uncultured bacteria were important MeBrand MeCl oxidizers in these soils. The prominent bands wereexcised from the DGGE gel, re-amplified by PCR, run onanother DGGE gel to check their purity and were then se-quenced. However, it proved difficult to get good sequencesfrom the re-amplified bands. Sequences from three of the13C-MeBr fraction bands were most similar to a Burkholderia16S rRNA gene sequence, which corresponded with resultsfrom the 13C-MeBr 16S rRNA gene library (OTU MeBr-1).The DGGE bands sequenced from both the 12C- and 13C-MeClfractions were not good, except for one band from the 13C-MeCl fraction which was most similar to the 16S rRNA genesequence from Nocardioides.

Fig. 4. KIE plots for MeBr (a) and MeCl (b) during isotoof measured �13C vs. –ln (C/C0) in live soil (�); solid l(C/C0) in heat-killed soil (�); and dotted line in (a) is a lidue solely to bacterial oxidation (‚).

Cloned 16S rRNA gene PCR products amplified from the

separate 13C-DNA fractions were screened to identify the bac-teria that had actively assimilated MeBr or MeCl (Fig. 6). Atotal of five OTUs were identified in the 13C-MeBr library, anda representative 16S rRNA gene clone of each was sequenced.Twenty-five OTUs were obtained from the 13C-MeCl library.Ten of these OTUs contained more than one clone and arepresentative 16S rRNA gene clone of each was sequenced.Two of the 13C-MeBr clones (MeBr 1 and MeBr 20), repre-senting separate OTUs (containing 85 and 2 clones respec-tively), contained 16S rRNA gene sequences which groupedclosely with the 16S rRNA gene sequence from a �-proteobac-terial soil isolate, Burkholderia caribiensis (97% identity).Three other 13C-MeBr clones (MeBr 10, MeBr 23 and MeBr14; containing 6, 1 and 1 clones respectively), were mostrelated to 16S rRNA gene sequences from uncultured pro-teobacteria and actinobacteria. The 13C-MeCl clones were dis-tributed among a greater number of genera. A clone represent-ing one OTU (MeCl 28, 9 clones) aligned with the 16S rRNA

Fig. 5. DGGE gel of 16S rRNA gene fragments amplified from 12C-and 13C-DNA fractions extracted from soils exposed to 13C-MeBr or13C-MeCl. Also shown are DGGE gel positive controls with PCRproducts of 16S rRNA genes from known MeBr and MeCl degradersHyphomicrobium chloromethanicum CM2, Methylobacterium chlo-

tionation experiments. Dashed lines are linear regressionsa) is a linear regression of measured �13C-MeBr vs. –lnression of calculated values of �13C-MeBr vs. –ln (C/C0)

pe fracine in (near reg

romethanicum CM4 and Aminobacter spp. strains IMB-1 and CC495.

3277Methyl halide degradation by soils and bacteria

gene sequences of Mesorhizobium loti and M. plurifarium(98% identity), which are closely related to two methyl halide

Fig. 6. Phylogenetic tree of 16S rRNA gene sequence13C-MeBr and 13C-MeCl supplemented soils. The 16S rRFull-length sequence (�1450 bp) was obtained for all cconfidence of branch points was determined by bootstrapare indicated at branch points. Sequences labeled MeBr anfrom MeBr and MeCl supplemented soils, respectively. Nfrom methyl halide SIP experiments. Scale bar, 10 inferr

degrading Aminobacter species (strains IMB-1 and C495). A

clone representing another OTU (MeCl 87, 14 clones) groupedwith the 16S rRNA gene sequence of Nocardiodes spp., a

ved from the 13C-labeled DNA fractions extracted fromuence of Thermotoga maritima was used as an outgroup.xcept those marked with a broken line (�500 bp). Thes and values �95% (�) and in the range 70 to 95% (�)were derived from cloned PCR products using 13C-DNAbold are known methyl halide utilizers or clones derived

titutions/100 nucleotide positions.

s retrieNA seqlones eanalysid MeClames ined subs

genus of actinobacteria from which one MeCl utilizer (SAC-4)

3278 L. G. Miller et al.

has been isolated previously (McAnulla et al., 2001a), butsubsequently lost from culture. The 16S rRNA gene sequencesof the remaining OTUs aligned with the 16S rRNA genesequences of various �-, �- and �-Proteobacteria that have notbeen previously reported to grow on methyl halides. 16S rRNAgene sequences from OTUs derived from the MeBr or MeCl12C-DNA fractions were widely distributed and reflected thebroad bacterial diversity that is present in soil (not shown).

Development of cmuA-specific PCR primers (Warner, 2003)led to the application of functional gene probing of the soilDNA. The functional gene cmuA, encoding the enzyme CmuA,was PCR amplified from 13C-DNA extracted from soils incu-bated with 13C-MeBr or 13C-MeCl as the substrate. A total of13 OTUs were identified from both the MeBr and MeCl clonelibraries. Representative clones from each OTU were se-quenced and their similarity to CmuA sequences from knownmethyl halide degrading organisms was determined (Fig. 7). Incontrast to results from 16S rRNA gene sequences, the func-tional phylogenetic tree identified groups of CmuA sequencesthat were highly similar to those of Aminobacter sp. CC495(MeCl 26, MeCl 47, MeBr 2, MeBr 18 and MeBr 19; contain-

Fig. 7. Unrooted phylogenetic tree of deduced amino-acid sequencesof the functional gene cmuA retrieved from the 13C-labeled DNAfractions extracted from 13C-MeBr and 13C-MeCl supplemented soils.The confidence of branch points was determined by bootstrap analysisand values �95% (�) and in the range 70 to 95% (�) are indicated atbranch points. Sequences labeled MeBr and MeCl were derived fromcloned cmuA PCR products using 13C-DNA from MeBr and MeClsupplemented soils, respectively. Scale bar, 10 inferred substitutions/100 amino-acid positions.

ing 11, 4, 17, 6, and 1 clones, respectively) and Hyphomicro-

bium chloromethanicum CM2 (MeCl 41, 2 clones), whichsuggests that similar bacteria to the known species activelyassimilated the added 13C-MeCl and 13C-MeBr. CmuA se-quences (MeCl 5, MeCl 44, MeBr 12; containing 1, 2 and 5clones respectively) that were closely related to, but distinctfrom, Aminobacter sp. IMB-1 are likely to be present in bac-teria which are closely related to this genus. The distinct groupof sequences (MeCl 1, MeCl 20, MeCl 28 and MeBr 26;containing 14, 6, 2 and 1 clones, respectively) that were notclosely related to the CmuA sequences of the strains currentlyin culture suggests that as yet uncultured methyl halide degrad-ers were active in the soil. The cmuA gene was also success-fully PCR amplified from the 12C-DNA fractions, but cloneswere not analyzed further.

To estimate whether the gene sequence diversity within theclone libraries generated in this study approaches the diversitywithin a sample of infinite size, the coverage was calculated(Ravenschlag et al., 1999). The statistical coverage value forthe 16S rRNA gene libraries was 98% for 13C-MeBr, 75% for12C-MeBr, 86% for 13C-MeCl and 12% for 12C-MeCl. For thecmuA libraries the coverage values were 93% for 13C-MeBrand 98% for 13C-MeCl. These values indicate that most of thediversity within the 13C-DNA fractions had been identified inboth the 16S rRNA gene and cmuA libraries. The differencebetween the coverage of the 12C-DNA libraries of the MeBrand MeCl fractions, was probably due to the toxicity of MeBrreducing the populations of bacteria remaining in the soil thatwere available for survey.

4. DISCUSSION

4.1. Chemical Degradation of MeBr

Chemical reactions with MeBr occur in soil where Br ex-changes with other nucleophiles via SN2-type chemical reac-tions. Suggested nucleophilic sites include –OH, -COO, -Cl,-SH, and -NH2 (Swain and Scott, 1953; Papiernik, et al., 2000).Hydrolysis and methylation of organic matter are the mostlikely removal mechanisms for MeBr in soil (Gan et al., 1994;Tao and Maciel, 2002). The MeBr losses we observed inheat-killed soil followed pseudo first order kinetics over therange of MeBr concentrations studied (Figs. 1a and 2a) Uptakewas linear and the loss rate increased with the amount added.We calculated a net removal rate constant of 0.03 d1, whichis 50% greater than the hydrolysis rate constant for pure waterat 22°C using the equation in Elliott and Rowland (1995). Thissuggests that an additional reaction, namely methylation oforganic matter, was also significant in the chemical removal ofMeBr in this soil.

In contrast to MeBr, there was no chemical loss of MeCl inheat-killed soil. Headspace concentrations of MeCl remainedconstant in controls measured 9 d beyond the end of the periodof uptake by live soil (Fig. 1b) The mean slope of linearregressions of concentration over this time was zero and theuncertainty (1 S.D.) was �3 �mol L1. MeCl is hydrolyzed toa lesser degree than is MeBr (Elliott and Rowland, 1995). Wecan add here that reaction with organic matter was, likewise,

not a significant sink for MeCl in this soil.

3279Methyl halide degradation by soils and bacteria

4.2. Patterns of Bacterial Oxidation of MeBr and MeCl

There were pronounced differences with respect to uptake ofMeBr and MeCl by live soil. More time was required forcomplete degradation of MeBr than of equivalent amounts ofMeCl (Fig. 1). The lag or delay in uptake of MeBr (4–13 d)was also longer than the lag in uptake of MeCl (2–4 d).However, the duration of the lag increased with increasedconcentration of both substrates, suggesting that the two methylhalides may have broadly similar effects on microbial activityin soil. Toxic effects of higher MeBr concentrations likelycontributed to the lag, hence the lag in MeCl degradation waslikely shorter because that compound was less toxic to theorganisms involved in its degradation. The lag period also maybe a time in which bacteria are growing or enzymes needed foroxidizing elevated concentrations of methyl halides are induced(Schaefer and Oremland, 1999). Following the lag period,MeCl degradation rates increased more rapidly with increasedconcentration than did MeBr degradation rates (Fig. 2b) Therewas partial inhibition of bacterial MeBr oxidation activity atelevated concentrations (�180 �mol L1 MeBr in the head-space). Partial inhibition was previously observed in pure cul-tures of Aminobacter strain IMB-1 at similar MeBr levels(Miller et al., 2003).

A bacterium previously isolated from fumigated soil, Ami-nobacter strain IMB-1, was able to oxidize and grow on MeBr,MeCl and other C1 substrates (Miller et al., 1997; Connell-Hancock et al., 1998). Schaefer and Oremland (1999) proposedthat Aminobacter strain IMB-1 possessed separate constitutiveand inducible enzymes, capable of degrading low and highconcentrations of MeBr, respectively. They also determinedthat strain IMB-1 expressed enzymes with a higher affinity forMeBr than for MeCl (Ks � 0.19 and 1.60 �mol/L, respec-tively). Other bacteria noted for the ability to degrade bothMeBr and MeCl in culture include Aminobacter strain CC495(Coulter et al., 1999) and the marine methylotroph, Leisingeramethylohalidovorans strain MB2 (Schaeffer et al., 2002).Given the disparity observed here in the patterns of MeBr andMeCl oxidation, it is possible that different microorganisms areresponsible for oxidizing each compound in soils. However,different oxidation rates could also result from expression ofseparate enzymes for oxidation of MeBr and MeCl by a singleorganism. Different oxidation rates are not sufficient evidenceby themselves to determine whether one or more organisms areactively degrading MeBr and MeCl. Ultimately, DNA patterns(see below) showed that several different bacteria were activelyoxidizing each methyl halide.

4.3. Stable Isotope Fractionation by Soils and Bacteria

Stable isotope mass balances are useful in constraining theatmospheric budgets of MeBr and MeCl, especially if the majorsources are isotopically distinct (McCauley et al., 1999).Changes in isotopic composition resulting from removal can beimportant if the sink is large, as is the case with soils, or ifsufficient fractionation occurs such that the remaining inven-tory is affected. However, quantification of isotopic fraction-ation resulting from the soil sink at ambient tropospheric mix-ing ratios remains to be done.

In studies of natural emission of methyl halides from salt

marshes, production of MeBr and MeCl by plants dominatedthe flux from soils (Rhew et al., 2000). Measurements of�13C-MeBr and �13C-MeCl efflux from salt marsh soilsshowed a large range and strong diurnal variability with thelightest (most negative) values associated with the middaymaximum in emission (Bill et al., 2002a). Daily weighted meanemissions were more positive for MeBr than for MeCl (43 vs.62‰, respectively) and heavier values were associated withthe night-time minimum in emission. This pattern suggestedenrichment in the heavier isotope by bacterial oxidation, espe-cially for MeBr. These authors proposed that oxidation by soilbacteria attenuated the flux and that the balance between pro-duction by plants and consumption by bacteria in the soildetermined the isotopic composition of the emitted methylhalide. From the results of our study, we would add thatchemical degradation can also impact the flux and the isotopiccomposition of MeBr.

During agricultural soil fumigations, fractionation resultingfrom degradation within the soil produced an enrichment incarbon isotope ratios of the residual MeBr and shifted theisotopic composition of the MeBr emitted from that applied(Miller et al., 2001; Bill et al., 2002b), thus affecting the sourceto the atmosphere. It was not determined whether chemical orbiologic reactions (or both) controlled the carbon isotope frac-tionation during degradation of MeBr in fumigated soils. In thisstudy we demonstrate that large shifts of the carbon isotopes ofMeBr are associated with both chemical and microbial losses insoil. This is the first direct determination of stable isotopefractionation during chemical degradation of MeBr in soil.Previously, Kalin et al. (2001) reported a KIE of � � 42‰ forchemical transhalogenation of MeBr to MeCl in a chloride-richsolution. Chemical reactions in the soil studied here result inloss of MeBr with time constants of weeks (half-life for abioticdegradation of MeBr � 6–12 d). The large isotope fraction-ation concurrent with the chemical loss of MeBr results in acalculated KIE of � � 59 � 7‰. At the present time, we cannotsay what amount of this fractionation is because of hydrolysisvs. reaction with soil organic matter or why this fractionation isso large. We found a similarly large KIE (�60‰) for chemicaldegradation of MeBr in a study using soil collected at MontereyBay Academy from a nearby organic plot of the same soil type(data not shown).

The KIE for oxidation of MeBr by microorganisms in soil (�� 69 � 9‰) is similar to values previously determined forpure cultures of the methylotrophs Leisingera methyloha-lidovorans (a marine bacterium) and Aminobacter strain IMB-1(isolated from agricultural soil previously fumigated withMeBr) (Miller et al., 2001). These strains exhibited high KIEs(57‰ � � � 72‰) while Aminobacter strain CC495 (isolatedfrom pristine forest soil) produced almost no fractionation(� � 4 � 2‰) during oxidation of MeBr. This suggests that themicroorganisms active in degrading elevated concentrations ofMeBr in the soil used in this study were predominantly thosethat exhibit high KIEs. It may be that these KIEs do notrepresent the in situ fractionation, as the population of soilbacteria may have changed in the month between sampling andthe experiments. This study represents a first attempt to sepa-rate soil carbon isotope fractionation into chemical and biologiccomponents and, therefore, should be repeated with fresh soils

to see if these results are reproducible.

3280 L. G. Miller et al.

The KIE for total (chemical � biological) degradation ofMeBr was � � 63 � 3‰. This KIE was fivefold greater thanthe net fractionation determined for a system open to theatmosphere based on changes in 13C-MeBr of soil gas duringfield fumigation at the same site (� � 12 � 2‰; Bill et al.,2002b). These authors suggested that abiotic losses with lowKIE could influence the effective fractionation, resulting in alower value for the effective KIE. Our data indicate, however,that chemical losses are associated with high KIEs and canresult in significant shifts in the carbon isotopic ratio of theMeBr emitted. Typical field fumigations consist of a tarpedperiod of 5 to 6 d during which chemical degradation isdominant, followed by a fallow period of several days duringwhich bacterial degradation is dominant. Throughout the entirefumigation much of the MeBr (up to 86%) is lost by emissionand escapes to the atmosphere (Yagi et al., 1995). Much of thisemission takes place during the early days of fumigation beforesignificant chemical or bacterial modification of 13C/12C ratiosof the MeBr has occurred. The result of loss of MeBr withoutcommensurate change in �13C is that a lower effective frac-tionation is determined for open-system (field) vs. closed-system (bottle) experiments. This distinction was used byMiller et al. (2001) to interpret �13C measurements of open-system flux chamber experiments that resulted in an effectivefractionation of � � 17‰ in soil at another agricultural site inCalifornia and is a likely explanation of the low effectivefractionation observed by Bill et al. (2002b).

Large fractionation of the carbon isotopes of MeCl was onlyassociated with microbial oxidation in soil. There was nochemical loss and no significant change in the isotopic com-position of MeCl in heat-killed controls. Therefore, the mea-sured total fractionation (� � 49 � 3‰) was due entirely tomicrobial activity in the soil. This value is within the range ofvalues reported in Miller et al. (2001) for fractionation of MeClby the three methylotrophs Leisingera methylohalidovoransstrain MB2, Aminobacter strain IMB-1, and Aminobacter strainCC495 (42‰ � � � 50‰). Since all three of these bacteriawere shown to oxidize MeCl and fractionate carbon isotopes tothe same extent, it is not possible to discriminate among thespecies oxidizing MeCl in soils using stable carbon isotopes.Molecular techniques (see below) allowed us to determine ifmore than one type of MeCl oxidizing bacterium was active inthe soil and whether the bacteria with the capacity to oxidizeMeCl were the same as those that oxidize MeBr. The utility ofmeasuring isotope fractionation of MeCl in the soil was thatany change in the �13C MeCl signature provided unequivocalevidence of microbial oxidation, since there was no chemicaldegradation of this compound.

4.4. Different Populations of MeBr and MeCl OxidizingBacteria

Stable isotope probing revealed that different populations ofsoil bacteria assimilated the carbon from MeBr and MeCl astheir primary carbon source. If MeBr and MeCl were incorpo-rated into the DNA of the same bacterial population, the DGGEpatterns of PCR products for 16S rRNA genes would have beensimilar for the 13C-DNA fractions from the MeCl and MeBrtreated soils. Different DNA profiles (Fig. 5) indicate that the

metabolism of MeBr and MeCl was carried out by several

different bacteria in these soil samples. It is important to notehowever, that the 13C-DNA fractions only represent microbesthat assimilated methyl halides as a primary carbon source.Therefore bacteria which co-oxidize methyl halides, such asmethanotrophs and nitrifiers, or bacteria which only use thelabeled methyl halide for a small portion of their carbon growthsubstrate would not be detected with this technique. Incubationof the soils with the high levels of methyl halide that werenecessary to obtain a 13C-DNA fraction might have also shiftedthe population of bacteria from that which oxidizes tropo-spheric mixing ratios of MeCl and MeBr (Shorter et al., 1995).

The phylogeny of 16S rRNA gene sequences retrieved fromthe 13C-DNA fractions (Fig. 6) indicated that a diverse range ofbacteria in this soil are capable of degrading and assimilatingMeBr or MeCl. The statistical coverage value (Ravenschlag etal., 1999) for the 16S rRNA gene clone libraries was 98% for13C-MeBr and 86% for 13C-MeCl, indicating that most of thediversity within these 13C-DNA fractions had been identified.Although there were somewhat fewer MeBr oxidizing bacteriaidentified than MeCl oxidizers, the capacity for MeBr and/orMeCl oxidation was implied for a broad range of bacteria,which was much more diverse than that of described methylhalide-degrading bacteria in pure culture at the present time. Inthe 16S rRNA 13C-MeBr gene library, 87% of clones wereclosely related to Burkholderia sp., which are not known todegrade methyl halides. However, this genus is known for itsnutritional versatility and its ability to degrade a wide range oforganic compounds (Gillis et al., 1995), and is an importantcomponent of soil microbial communities (Salles et al., 2002).The nutritional versatility of Burkholderia has led to its use inthe biodegradation of environmental pollutants (Fain and Had-dock, 2001). Burkholderia Strain LB400, isolated from soilcontaminated with polychlorinated biphenyls (PCBs), grows onbiphenyl as a sole source of carbon and energy and can co-oxidize a range of polychlorinated biphenyls containing one tosix chlorine substitutes. This strain is closely related to Burk-holderia caribiensis and Burkholderia graminis which have16S rRNA gene sequences that are closely related to the Burk-holderia sequences in the 13C-MeBr library. Another clone inthe 13C-MeBr library had 97% sequence identity with the 16SrRNA sequence from an environmental clone, WCHB1-08,retrieved from soil that had been contaminated with hydrocar-bons and chlorinated solvents including dichloroethane, chlo-roethane and vinyl chloride (Dojka et al., 1998). Other OTUshad sequences that were closely related to 16S rRNA genesequences from Sphingomonas sp., Rhodobacter sp., Mesorhi-zobium sp., Rhodanobacter sp., and several other species ofbacteria, all of which are commonly found in soils suggestingthe diversity of MeBr- and MeCl-oxidizing bacteria extendsbeyond the known cultures of �-Proteobacteria.

The 16S rRNA 13C-MeCl library was more diverse than the13C-MeBr library, perhaps due to the lesser toxicity of MeCl orto more cross feeding of metabolites produced from 13C-MeClby bacteria in the soil microcosm. Clones in this library had16S rRNA gene sequences closely related to sequences fromknown methylotrophs, including Methylophilus methylotro-phus and Methylotrophus leisingeri which are both capable ofutilizing dichloromethane (Leisinger and Braus-Stromeyer,1995). Fourteen percent of the clone library had sequences

closely related to the 16S rRNA gene sequences from Nocar-

3281Methyl halide degradation by soils and bacteria

dioides and in particular Nocardioides strain SAC-4 (McAnullaet al., 2001b), which was capable of growth on MeCl as a solesource of carbon and energy, and is of particular interestbecause it is the only gram-positive strain isolated to date thatis capable of growth on methyl halides. This suggests that thediversity of bacteria capable of methyl halide degradation ex-tends beyond the Proteobacteria.

Interestingly, no 16S rRNA gene sequences from strains ofmethyl halide utilizers presently in culture were found in any ofthe libraries. This could be because they are not present in largeenough numbers to be detected by PCR, or they do not play animportant role in methyl halide degradation in this environmentbut are merely more easily isolated than other strains or theycan grow on MeBr and/or MeCl and other (12C) carbon sourcessimultaneously, thereby reducing their 13C-labelling.

Several sequences found in the 13C-DNA libraries were alsofound in the 12C-DNA libraries. This may be because not allcells of a particular strain capable of utilizing methyl halidesare active at the same time, with the inactive cells present in the12C-fraction, or because the ability of one member of a genusto degrade methyl halides does not mean that all species in thegenus are capable of methyl halide degradation. AlthoughHyphomicrobium chloromethanicum CM2 is able to utilizemethyl halides, other Hyphomicrobium strains are not(McAnulla et al., 2001b). Therefore, although similar se-quences were found in both 12C- and 13C-DNA libraries, theymay differ at the species or strain level and the sequences in the12C-DNA libraries may represent organisms that are unable todegrade methyl halides.

When looking at 16S rRNA gene libraries, PCR bias has tobe taken into account (Polz et al., 1999). However resultssuggest there is little PCR bias in this study since the PCRreactions for the 16S rRNA gene DGGE study were with adifferent set of primers to those used to make the libraries, andthe results show that similar sequences are dominant from bothexperiments. The results of the DGGE analyses show thatoverall there is a difference between the 12C and 13C-DNAfractions, more notably in the MeBr fractions. There is moresmearing in the 12C-MeCl fraction and more defined banding inthe 13C-fraction. This correlates with the observed greaternumber of OTUs in the 12C-MeCl library. As with the libraries,the sequences of the major bands from the 13C-MeBr DGGEanalysis are similar to 16S rRNA gene sequences from Burk-holderia sp., which do not appear in the 12C-lane. Althoughsequencing of bands from the DGGE gel was difficult, it can beseen that the results from DGGE correspond favorably withthose of the libraries.

Since the 13C-DNA fraction contained the combined genomeof the microbial population that assimilated the labeled methylhalides, it was possible to target the functional gene (cmuA) thatencodes CmuA, the enzyme required for initiation of the pri-mary degradation pathway for both MeBr and MeCl in knownmethyl halide degraders. The diversity of cmuA sequencesrecovered from the active bacterial population (Fig. 7) supportsthe 16S rRNA analysis in suggesting that only a subset of themethyl halide oxidizing bacteria present in soils has beenisolated in pure culture thus far from the environment. Also,significant differences in diversity were found between the13C-MeCl and the 13C-MeBr 16S rRNA gene libraries (Fig. 6),

but very similar representation was seen in the diversity of

cmuA genes. This may be because the cmuA primers weredesigned from the four sequences available from extant methylhalide utilizers (Methylobacterium chloromethanicum CM4,Hyphomicrobium chloromethanicum CM2, Aminobacter sp.strain IMB-1 and Aminobacter sp. strain CC495, all membersof the �-Proteobacteria), and may not detect more novel cmuAsequences from organisms outside of the �-Proteobacteria, or itmay be that some of the organisms identified in the 16S rRNAgene libraries utilize methyl halides via an alternative mecha-nism. However, phylogenies derived from both the 16S rRNAgene and the functional gene cmuA are not necessarily indica-tive of the uptake kinetics or isotope fractionation effectsexhibited by the methyl halide degrading bacteria. For instance,it is somewhat surprising that closely related organisms (e.g.,Aminobacter strains IMB-1 and CC495) can achieve very dif-ferent C isotopic fractionations of MeBr (Miller et al., 2001).Therefore, further isolates need to be obtained to understandmore fully the physiology, biochemistry and genetics of MeBrand MeCl degrading bacteria. Applying multiple tools to thestudy of bacteria in soil, as we have done here, has providedinsight into the taxonomic and physiologic diversity of themethylotrophs present in soil that can account for much of thebehavior with respect to oxidation of MeBr and MeCl.

5. CONCLUSIONS

Degradation of elevated concentrations of MeBr in soilsproceeded by both chemical and biologic processes. Chemicaldegradation was more important during the initial lag period,after which bacterial oxidation removed MeBr more rapidly.The lag period increased with greater MeBr concentration. Onthe other hand, degradation of MeCl was by microbial oxida-tion only. There was no chemical degradation of MeCl.

Different patterns of stable carbon isotope fractionation wereobserved for MeBr and MeCl. The KIE for chemical degrada-tion of MeBr was 59 � 7‰. Oxidation of MeBr by microor-ganisms in soil resulted in a similarly high KIE (69 � 9‰).Bacterial fractionation was at the high end of the rangeachieved by methylotrophs in culture (4‰ � � � 72‰).Oxidation of MeCl by microorganisms in soil produced simi-lar fractionation (49 � 3‰) to cultures of methylotrophs (42‰� � � 50‰).

Stable isotope probing was successfully applied to identifybacteria in soil that actively degraded MeBr and MeCl. DGGEpatterns of amplified 16S rRNA genes revealed that differentbacterial populations degraded each substrate. Representativesof the major groups of clones from 16S rRNA gene librarieswere sequenced to identify the bacteria that degraded MeBr orMeCl in the soil. A parallel survey of cmuA gene sequencesamplified from the 13C-DNA fractions was also undertaken.The combined phylogenetic analyses indicated that the range ofbacteria with the capacity to degrade methyl halides was morediverse than expected, that the methyl halide degraders alreadyin culture represent only a subset of this diversity, and thatenzymes different from CmuA in known cultures may be in-volved in methyl halide degradation.

Acknowledgments—This paper is dedicated to the memory of DougWhite. The California Strawberry Commission (CSC) provided accessto the Monterey Bay Academy. We appreciate the advice and encour-

agement of CSC staff and fellow researchers at the site. We thank C.

3282 L. G. Miller et al.

Kendall, S. Silva, and L. D. White for making the USGS mass spec-trometry facility available and for technical assistance and H. Schaferfor assistance with running DGGE. We are grateful to S. L. Shaw andK. D Goodwin for their thoughtful reviews of an earlier version of thismanuscript as well as to K. Hinrichs and three anonymous reviewersfor their comments. Financial support was from USGS, National Re-search Program, NASA Upper Atmosphere Research Program (1995-AU-0006), U.K. Natural Environment Research Council (NERC)grants (NER/T/S/2000/00618 and NER/A/S/2000/00423) and anNERC studentship to K. Warner.

Associate editor: R. Summons

REFERENCES

Bill M., Rhew R. C., Weiss R. F., and Goldstein A. H. (2002a) Carbonisotope ratios of methyl bromide and methyl chloride emitted from acoastal salt marsh. Geophys. Res. Lett. 29, 4.1–4.4.

Bill M., Miller L. G., and Goldstein A. G. (2002b) Carbon isotopefractionation of methyl bromide during agricultural soil fumigations.Biogeochemistry 60, 181–190.

Connell-Hancock T. L., Costello A. M., Lidstrom M. E., and OremlandR. S. (1998) Strain IMB-1, a novel bacterium for removal of methylbromide in fumigated agricultural soils. Appl. Environ. Microbiol.64, 2899–2905.

Coulter C., Hamilton J. T. G., McRoberts W. C., Kulakov L., LarkinM. J., and Harper D. B. (1999) Halomethane: bisulfide/halide ionmethyltransferase, an unusual corrinoid enzyme of environmentalsignificance isolated from an aerobic methylotroph using chlo-romethane as the sole carbon source. Appl. Environ. Microbiol. 65,4301–4312.

Dojka M. A., Hugenholtz P., Haack S. K., and Pace N. R. (1998)Microbial diversity in a hydrocarbon- and chlorinated-solvent-con-taminated aquifer undergoing intrinsic bioremediation. Appl. Envi-ron. Microbiol. 64, 3869–3877.

Elliott S. M. and Rowland F. S. (1995) Methyl halide hydrolysis ratesin natural waters. J. Atmos. Chem. 20, 229–236.

Fain M. G. and Haddock J. D. (2001) Phenotypic and phylogeneticcharacterization of Burkholderia (Pseudomonas) sp. strain LB400.Curr. Microbiol. 42, 269–275.

Gan J., Yates S. R., Anderson M. A., Spencer W. F., and Yates M. V.(1994) Effect of soil properties and sorption of methyl bromide insoil. Chemosphere 29, 2658–2700.

Gan J., Yates S. R., Wang D., and Spencer W. F. (1996) Effect of soilfactors on methyl bromide volatilization after soil application. En-viron. Sci. Technol. 30, 1629–1636.

Gillis M., Van T. V., Bardin R., Goor M., Hebbar P., Willems A.,Segers P., Kerstens K., Heulin T., and Fernadez M. P. (1995)Polyphasic taxonomy in the genus Burkholderia leading to anamended description of the genus and proposition of Burkholderiavietnamiensis sp. nov. for N2-fixing isolates from rice in Vietnam.Int. J. Syst. Bacteriol. 45, 274–289.

Goodwin K. D., Varner R. K., Crill P. M., and Oremland R. S. (2001)Consumption of tropospheric levels of methyl bromide by C1 com-pound-utilizing bacteria and comparison to saturation kinetics. Appl.Environ. Microbiol. 67, 5437–5443.

Harper D. B. (2000) The global chloromethane cycle: biosynthesis,biodegradation, and metabolic role. Nat. Prod. Rep. 17, 337–348.

Hines M. E., Crill P. M., Varner R. K., Talbot R. W., Shorter J. H.,Kolb C. E., and Harriss R. C. (1998) Rapid consumption of lowconcentrations of methyl bromide by soil bacteria. Appl. Environ.Microbiol. 64, 1864–1870.

Kalin R. M., Hamilton J. T. G., Harper D. B., Miller L. G., Lamb C.,Kennedy J. T., Downey A., McCauley S., and Goldstein A. H.(2001) Continuous flow stable isotope methods for study of �13Cfractionation during halomethane production and degradation. RapidCommun. Mass Spectrom. 15, 357–363.

Khalil M. A. K. and Rasmussen R. A. (1999) Atmospheric methylchloride. Atmos. Environ. 33, 1305–1321.

Kurylo M. J., Rodriguez J. M., Andrea M. O., Atlas E. L., Blake D. R.,Butler J. H., Lal S., Lary D. J., Midgley P. M., Montzka S. A.,Novelli P. C., Reeves C. E., Simmonds P. G., Steele L. P., Sturges

W. T., Weiss R. F. and Yokouchi Y. (1999) Short-lived ozone

related compounds. In Scientific Assessment of Ozone Depletion:1998 World Meteorological Organization (eds. G. M. Albritton,R. T. Watson and P. J. Aucamp), pp 2.1–2.37.

Lane D. J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Tech-niques in Bacterial Systematics (eds. E. Stackebrandt and M. Good-fellow), pp. 115–175. John Wiley, Chichester, UK.

Leisinger T. and Braus-Stromeyer S. A. (1995) Bacterial growth withchlorinated methanes. Environ. Health Perspect. 103, 33–36.

McAnulla C., Woodall C. A., McDonald I. R., Studer A., VuilleumierS., Leisinger T., and Murrell J. C. (2001a) Chloromethane utilizationgene cluster from Hyphomicrobium chloromethanicum strain CM2T

and development of functional gene probes to detect halomethane-degrading bacteria. Appl. Environ. Microbiol. 67, 307–316.

McAnulla C., McDonald I. R., and Murrell J. C. (2001b) Methylchloride utilizing bacteria are ubiquitous in the natural environment.FEMS Microbiol. Lett. 201, 151–155.

McCauley S. E., Goldstein A. H., and DePaulo D. J. (1999) An isotopicapproach for understanding the CH3Br budget of the atmosphere.Proc. Natl. Acad. Sci. 96, 10006–10009.

McDonald I. R., Warner K. L., McAnulla C., Woodall C. A., OremlandR. S., and Murrell J. C. (2002) A review of bacterial methyl halidedegradation: Biochemistry, genetics and molecular ecology. Envi-ron. Microbiol. 4, 193–203.

Miller L. G., Connell T. L., Guidetti J. R., and Oremland R. S. (1997)Bacterial oxidation of methyl bromide in fumigated agriculturalsoils. Appl. Environ. Microbiol. 63, 4346–4354.

Miller L. G., Kalin R. M., McCauley S. E., Hamilton J. T. G., HarperD. B., Millet D. B., Oremland R. S., and Goldstein A. H. (2001)Large carbon isotope fractionation associated with oxidation ofmethyl halides by methylotrophic bacteria. Proc. Natl. Acad. Sci. 98,5833–5837.

Miller L. G., Baesman S. M., and Oremland R. S. (2003) Bioreactorsfor removing methyl bromide following contained fumigations. En-viron. Sci. Technol. 37, 603–607.

Muyzer G., De Waal E. C., and Uitterlinden A. G. (1993) Profiling ofcomplex microbial populations by denaturing gradient gel electro-phoresis analysis of polymerase chain reaction-amplified genes cod-ing for 16S rRNA. Appl. Environ. Microbiol. 59, 695–700.

Oremland R. S., Miller L. G., Culbertson C. W., Connell T. L., andJahnke L. (1994) Degradation of methyl bromide by methanotrophicbacteria in cell suspensions and soils. Appl. Environ. Microbiol. 60,3640–3646.

Papiernik S. K., Gan J., and Yates S. R. (2000) Mechanism of degra-dation of methyl bromide and propargyl bromide in soil. J. Environ.Qual. 29, 1322–1328.

Polz M. F., Harbison C., and Cavanaugh C. M. (1999) Diversity andheterogeneity of epibiotic bacterial communities on the marine nema-tode Eubostrichus dianae. Appl. Environ. Microbiol. 65, 4271–4275.

Radajewski S. and Murrell J. C. (2002) Stable isotope probing fordetection of methanotrophs after enrichment with13CH4. In GeneProbes: Principles and Protocols (eds. M. Aquino de Muro and R.Rapley), pp. 149–157. Humana Press, Totawa, NJ.

Radajewski S., Ineson P., Parekh N. R., and Murrell J. C. (2000) Stableisotope probing as a tool in microbial ecology. Nature 403, 646–649.

Radajewski S., Webster G., Reay D., Morris S. A., Ineson P. NedwellD. B., Prosser J. I., and Murrell J. C. (2002) Identification of activemethylotroph populations in an acidic forest soil by stable isotopeprobing. Microbiology 148, 2331–2342.

Ravenschlag K., Sahm K., Pernthaler J., and Amman R. (1999) Highbacterial diversity in permanently cold marine sediments. Appl.Environ. Microbiol. 65, 3982–3989.

Rhew R. C., Miller B. R., and Weiss R. F. (2000) Natural methylbromide and methyl chloride emissions from coastal salt marshes.Nature 403, 292–295.

Salles J. F., De Souza F. A., and Van Elsas J. D.. (2002) Molecularmethod to assess the diversity of Burkholderia species in environ-mental samples. Appl. Environ. Microbiol. 68, 1595–1603.

Sambrook J., Fritsch E. F., and Maniatis T. (1989) Molecular Cloning:A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.

Schafer H. and Muyzer G. (2001) Denaturing gradient gel electrophoresis

in marine microbial ecology. Meth. Microbiol. 30, 425–468.

3283Methyl halide degradation by soils and bacteria

Schaeffer J. K. and Oremland R. S. (1999) Oxidation of methyl halidesby the facultative methylotroph strain IMB-1. Appl. Environ. Micro-biol. 65, 5035–5041.

Schaefer J. K., Goodwin K. D., McDonald I. R., Murrell J. C., andOremland R. S. (2002) Leisingera methylohalidovorans gen. Nov.,sp. nov., a marine methylotrophs that grows on methyl bromide. Int.J. Syst. Evol. Microbiol. 52, 851–859.

Shorter J. H., Kolb C. E., Crill P. M., Kerwin R. A., Talbot R. W.,Hines M. E., and Harriss R. C. (1995) Rapid degradation of atmo-spheric methyl bromide in soils. Nature 377, 717–719.

Swain C. G. and Scott C. B. (1953) Quantitative correlation of relativerates. Comparison of hydroxide ion with other nucleophilic reagentstoward alkyl halides, esters, epoxides and acyl halides. J. Am. Chem.Soc. 75, 141–147.

Tao T. and Maciel G. E. (2002) Interaction of methyl bromide withsoil. Environ. Sci. Technol. 36, 603–607.

Warner K. L. (2003) Molecular Biology and Molecular Ecology ofBacterial Methyl Halide Degradation. Ph.D. dissertation, Universityof Warwick, England.

Woodall C. A., Warner K. L., Oremland R. S., Murrell J. C., andMcDonald I. R. (2001) Identification of methyl halide-utilizinggenes in the methyl bromide-utilizing bacterial strain IMB-1suggest a high degree of conservation of methyl halide-specificgenes in gram-negative bacteria. Appl. Environ. Microbiol. 67,1959 –1963.

Yagi K., Williams J., Wang N.-Y., and Cicerone R. J. (1995) Atmo-spheric methyl bromide (CH3Br) from agricultural soil fumigations.Nature 267, 1979–1981.

Zhou J., Bruns M. A., and Tiedje J. M. (1996) DNA recovery fromsoils of diverse composition. Appl. Environ. Microbiol. 62, 316 –322.