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Consumption of Tropospheric Levels of Methyl Bromide by C1 Compound-Utilizing Bacteria and Comparison to Saturation Kinetics

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Consumption of Tropospheric Levels of Methyl Bromide by C1 Compound-Utilizing Bacteria and Comparison to Saturation Kinetics
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    10.1128/AEM.67.12.5437-5443.2001. 2001, 67(12):5437. DOI: Appl. Environ. Microbiol. Ronald S. OremlandKelly D. Goodwin, Ruth K. Varner, Patrick M. Crill and  KineticsBacteria and Comparison to Saturation Compound-Utilizing1Methyl Bromide by CConsumption of Tropospheric Levels of http://aem.asm.org/content/67/12/5437Updated information and services can be found at: These include:  REFERENCES http://aem.asm.org/content/67/12/5437#ref-list-1at: This article cites 48 articles, 22 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  onN  ov  em b  er 1 7  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om  onN  ov  em b  er 1 7  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om    A  PPLIED AND  E NVIRONMENTAL   M ICROBIOLOGY ,0099-2240/01/$04.00  0 DOI: 10.1128/AEM.67.12.5437–5443.2001Dec. 2001, p. 5437–5443 Vol. 67, No. 12Copyright © 2001, American Society for Microbiology. All Rights Reserved. Consumption of Tropospheric Levels of Methyl Bromide by C 1 Compound-Utilizing Bacteria and Comparison toSaturation Kinetics KELLY D. GOODWIN, 1 * RUTH K. VARNER, 2 PATRICK M. CRILL, 2  AND  RONALD S. OREMLAND 3 Cooperative Institute for Marine and Atmospheric Studies, Rosenstiel School of Marine and Atmospheric Sciences,University of Miami, Miami, Florida 33149 1  ; Complex Systems Research Center, University of New Hampshire, Durham, New Hampshire 03824 2  ; and U.S. Geological Survey, Menlo Park, California 94025 3 Received 9 July 2001/Accepted 1 October 2001 Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however,their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested.Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropo-spheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters (  2 pM). Kinetic investigationsusing picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed usingstrain IMB-1 and  Leisingeria methylohalidivorans  strain MB2 T — terrestrial and marine methylotrophs capableof halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains.Strain MB2 T displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, withan apparent  K   s  of 2.4  M MeBr and a  V  max  of 1.6 nmol h  1 (10 6 cells)  1 . Apparent first-order degradation rateconstants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1  10 7 -fold-higher MeBr concentrations.  Ruegeria algicola  (a phylogenetic relative of strainMB2 T ), the common heterotrophs  Escherichia coli  and  Bacillus pumilus , and a toluene oxidizer,  Pseudomonas mendocina  KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, theability to consume ambient mixing ratios of MeBr was limited to C 1  compound-oxidizing bacteria in this study. Aerobic C 1  bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soilsand waters. Methyl bromide (MeBr; CH 3 Br) is the major source of in-organic bromine in the stratosphere, making it an importantcontributor to stratospheric ozone depletion. MeBr accountsfor 5 to 10% of stratospheric ozone destruction on a globalbasis (48). Use of MeBr as a fumigant is being phased outunder amendments to the Montreal Protocol (49), but unlikemany of the other compounds scheduled for phase out (e.g.,chlorinated fluorocarbons), most of the MeBr released to theatmosphere is derived from natural sources.The global MeBr budget as currently understood is out of balance because known sources do not balance identified sinks(3). Natural sources of MeBr include macroalgae, phytoplank-ton, fungi, higher plants (11, 20, 29, 37, 38, 53), and varioustypes of wetlands (25, 36, 51). Sinks for MeBr are abiotic(hydrolysis and halide substitution) (21, 28) and biotic. In theoceans, microbial degradation of MeBr is widespread (45, 46).The processes of production and degradation occur simulta-neously in the oceans, the balance of which results in a net sinkfor atmospheric MeBr (27). The biological mechanisms thatcontrol net flux are not well understood, but biotic sinks are of sufficient global magnitude to affect the atmospheric burdenand lifetime of MeBr (41, 57).Mechanistic studies with elevated MeBr concentrations(parts per million by volume [ppmv]) have demonstrated bio-degradation in a variety of environments, including anaerobicsediments (35), fumigated agricultural soils (32), and a numberof water types (5, 14). Experiments with soils have demon-strated that unidentified bacteria consume MeBr at ambienttropospheric mixing ratios (pptv) (16, 41, 52). Experiments with seawater have indicated that unidentified microbes areresponsible for degradation at relatively low MeBr concentra-tions (  100-fold above ambient) (28, 46).Cometabolism of MeBr has been well documented in cul-ture studies. Bacteria able to cooxidize MeBr include meth-anotrophs (13, 43), nitrifiers (10, 24), and certain marinemethylotrophs that grow on dimethylsulfide or methanesulfo-nate (18; J. C. Murrell, personal communication). Investiga-tions with specific inhibitors applied to environmental sampleshave shown that methanotroph/nitrifier cooxidation of ppmvMeBr was 50 to 72% in certain soils (34) and 82% in a fresh- water lake (14). Methanotroph/nitrifier involvement was notdetected in compost (34), agricultural soils (16, 32) or in ma-rine and estuarine waters (14). The ability of methanotrophs inculture to consume pptv levels of MeBr has not been testedpreviously.Certain facultative methylotrophs isolated from soil (strainsIMB-1 and CC495) (6, 8) and from seawater (  Leisingeria methylohalidivorans  strain MB2 T ) (39) can grow with MeBrprovided as the sole source of carbon and energy. Addingstrain IMB-1 to soil increased the uptake of MeBr supplied atppmv levels (6), and strains IMB-1 and MB2 T fractionatedstable carbon during MeBr oxidation (up to 72‰) (33). Such * Corresponding author. Mailing address: Cooperative Institute forMarine and Atmospheric Studies, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4301 RickenbackerCauseway, Miami, FL 33149. Phone: (305) 361-4384. Fax: (305) 361-4392. E-mail: kelly.goodwin@noaa.gov.5437   onN  ov  em b  er 1 7  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   fi ndings highlight MeBr-metabolizing bacteria as possiblemodels for bacterial uptake of tropospheric MeBr. However,threshold concentrations for both growth and substrate degra-dation may exist (7), and the ability of such strains to consumetropospheric mixing ratios of MeBr (  12 pptv) has not beentested directly. The ability of such bacteria to metabolize pptvmixing ratios of MeBr would corroborate degradation experi-ments conducted with marine waters and soils (16, 28, 41, 45,46, 52), and it would support using these isolates for mecha-nistic studies of MeBr oxidation. In this work, we directlytested whether a number of bacteria, including methanotrophsand MeBr-metabolizing methylotrophs, could oxidize MeBrsupplied at ambient MeBr mixing ratios. MATERIALS AND METHODSGrowth of organisms.  Strain MB2 T is a marine methylotroph recently identi- fi ed as  Leisingeria methylohalidivorans  (ATCC BAA-92) (39). It was grown onmarine broth (Difco 2216) either with or without MeBr or on a mineral saltsmedium (MAMS) supplied with MeBr as the sole carbon and energy source. TheMAMS medium was adapted from that of Thompson et al. (44) and contained(in grams per liter): NaCl, 16; (NH 4 ) 2 SO 4,  1.0; MgSO 4    7H 2 O, 1.0; CaCl 2   2H 2 O, 0.2; FeSO 4    7H 2 O, 0.002; Na 2 MoO 4    2H 2 O, 0.002; Na 2 WO 4,  0.003;KH 2 PO 4,  0.36; K  2 HPO 4,  2.34; and 1.0 ml of SL-10 trace metals (55). Thephosphates were added after autoclaving from sterile stock solutions. The  fi nalpH of the medium was 6.9 to 7.1.  Ruegeria algicola  (ATCC 51440) (47), a marine bacterium closely related to  L. methylohalidivorans  (97.5% identity) (39), was also grown on marine broth.  R. algicola  and  L. methylohalidivorans  are members of the  Roseobacter   group (alsoknown as the marine alpha bacteria), a numerically dominant group of marinebacteria (12). Strain IMB-1 is a terrestrial methylotroph closely related to thegenus  Aminobacter   (8, 23). It was grown on Luria-Bertani (LB) broth (Lennox;Difco) or on a de fi ned mineral salts medium (Doronina medium [DM]) (40).When grown on DM, carbon and energy sources were supplied as MeBr (  188  M), glucose (15 mM), or both glucose and MeBr.  Pseudomonas mendocina KR1, a toluene-oxidizing bacterium containing toluene-4-monooxygenase(T4MO), was grown with 100  M toluene either on a mineral salt medium (31)or on LB broth.  Bacillus pumilus  and  Escherichia coli  were also grown on LBbroth.  Methylomonas rubra , a type 1 methanotroph, was grown on nitrate mineralsalts medium (NMS) (54). Strain BB5.1, an estuarine methanotroph (42), wasgrown on NMS supplemented with 1.5% salt.The methanotrophs were grown under a 50 – 50 methane-air atmosphere. Cells were grown using laboratory air; thus, they were exposed to ambient laboratorylevels of MeBr during growth. Bacterial density was determined using acridineorange direct counts (AODC) (17). Three separate samples with a minimum of 8 grids per sample were counted for each experiment. The average percentcoef  fi cient of variation for AODC measurements was 17%. Supplying pptv MeBr.  A three-stage dynamic dilution system supplied samples with a steady stream of an experimental atmosphere consisting of precise, near-ambient mixing ratios of MeBr (26). In brief, ultra-high-purity (UHP) air(99.999%; NE Air Gas)  fl owed through an oven held at 30 ° C  0.1 ° C, where itmixed with MeBr emitted from a gravimetrically calibrated permeation tube(KIN-TEK). The air  fl owed through a three-stage dilution box, where it wassubsequently diluted with UHP air and/or bled from the system using mass  fl owcontrollers. The mass  fl ow controllers were manually adjusted to produce thedesired mixing ratio of MeBr. The permeation tube was weighed periodicallyover 5 years and determined to produce 16.8  0.4 ng of MeBr min  1 . The MeBremitted at this permeation rate in conjunction with the supplied dilution airproduced calibrated mixing ratios ranging from 6.4 to 2,000 pptv MeBr. MeBr was normally supplied to bacterial cell suspensions at a mixing ratio of 12.03 pptvto mimic the tropospheric mixing ratio of MeBr, which is 11.9 pptv in thenorthern hemisphere (27). A sample pump and mass  fl ow controller delivered the experimental air to thesamples at a measured  fl ow rate of 80 ml min  1 . The experimental air wasbubbled through 15 ml of culture placed in a gas washing tube  fi tted with a 19/22frit (Corning; ML-1490-702). Equilibrium dissolved concentrations were calcu-lated using the equations of King and Saltzman (28). The dissolved concentra-tions were 2.2 pM and 2.0 pM MeBr at 23 ° C for medium with no added salt and16% salt, respectively. To verify that MeBr supersaturation did not occur, 15 mlof distilled (DI) water was  fl ushed with experimental air for 20 min, and subse-quently the sample was stripped for 30 min with UHP nitrogen to  fl ush all theMeBr from the sample into the GC Cryotrap. The mass of MeBr recovered wasin agreement with equilibrium calculations, indicating that supersaturation didnot occur (data not shown).Samples were equilibrated with experimental air for 20 min. A known volumeof gas  fl owing from the fritted device was cryogenically trapped at  70 ° C usinga GC Cryotrap (model 951; Scienti fi c Instrument Services, Inc.) in conjunction with a totalizer/mass  fl ow meter (MFM) (Brooks Instruments). The total volumeof air was typically 400 ml, sampled over 20 min. The GC Cryotrap was heatedto 120 ° C to volatilize the MeBr into the carrier gas stream of a Shimadzu GC-8A gas chromatograph equipped with an electron capture detector (GC-ECD). Thecarrier gas was oxygen-doped UHP nitrogen  fl owing at 12 ml min  1 .MeBr was separated using a precolumn (1 m by 0.16 cm outer diameter [o.d.])packed with PoropakQ 100/120 mesh and an analytical column (2 m by 0.16 cmo.d.) packed with 80/100 mesh HayeSepQ (Alltech). Column and injector/detec-tor temperatures were 140 ° C and 290 ° C, respectively. Standards from a gascylinder were analyzed daily to verify the accuracy of the dilution system mixingratios. The daily standard curves included replicates of the following  fi  ve stan-dards: 0.05, 0.25, 0.5, and 1.0 ml of 270 ppbv MeBr, corresponding to a range of 0.06 to 12 pmol of MeBr, which bracketed the range of masses delivered to theGC during experiments. The  r  2 of the linear regression  fi t of 6 months of GCresponse versus nanomoles of MeBr standard was 0.9998. The detection limit was ca. 0.04 pptv MeBr. Measurement precision typically was 5% (coef  fi cient of  variation [CV]). Calculations for experiments supplied with pptv MeBr.  Consumption of MeBr was measured on duplicate live and control samples. Autoclaved cell suspensionsand sterile medium behaved similarly (data not shown); thus, sterile mediumtypically was used as a control. Live cultures in late exponential phase were used,and uptake rates by live samples were corrected for any uptake observed incontrols.The experimental system functioned as a steady-state, continuously stirredtank reactor (CSTR). The mass balance equation (15) for the concentration of MeBr (nanomolar) in the in fl uent ( C in ) and ef  fl uent ( C out ) gas is given inequation 1: QC in  QC out  Vr   (1) where  Q  is the gas  fl ow rate (liters per hour),  V   is the liquid volume (0.015 L),and  r   is the rate of MeBr consumption which occurs in the liquid phase (nano-molar). By rearrangement, the rate of MeBr consumption can be expressed interms of the gas phase concentrations:  r   QV    C in  C out   (2)For a reaction  fi rst-order in substrate and cell concentration, the rate of MeBrconsumption can be expressed as follows (4):  r    kXC  L  (3) where  X   is the cell density (cells per liter),  C  L  is the concentration of MeBr in theliquid phase (nanomolar), and  k  is the apparent  fi rst-order reaction rate constant(per hour liter per cell). The exiting gas is in equilibrium with the liquid phase;therefore,  C  L    C out  /   H  , where  H   is the dimensionless Henry ’ s constant forMeBr. The Henry ’ s constant was calculated from the equations of De Bruyn andSaltzman (9); e.g.,  H     0.22 at 23 ° C for medium without salt and  H     0.25 at23 ° C for medium with 16 g of NaCl per liter. Combining equations 2 and 3 resultsin the following expression for the uptake rate of MeBr:  r  X     kC  L  QVX   C in  C out  . (4)Equation 4 illustrates that a plot of the MeBr uptake rate,  r   /   X  , versus theliquid-phase MeBr concentration ( C  L ) will be a straight line with a slope equalto the apparent  fi rst-order reaction rate constant (  k ). This relationship is illus-trated in Fig. 1 and 2. Inhibition of protein synthesis.  Chloramphenicol (20   g ml  1 ) was used toassess the speed of turnover for MeBr-degrading enzymes. Strain IMB-1 wasgrown on glucose and 188   M MeBr. The uptake rate of MeBr supplied at 12pptv was measured for IMB-1 samples in the absence of chloramphenicol and forsamples that were exposed to chloramphenicol 90 min prior to being placed intothe gas washing tube.  L. methylohalidivorans  was grown on MAMS mediumsupplemented with MeBr (50  M) as the sole carbon and energy source, or it wasgrown on marine broth with no additional MeBr. Chloramphenicol was added to  L. methylohalidivorans  in a fashion similar to that for strain IMB-1; however, no 5438 GOODWIN ET AL. A  PPL  . E NVIRON . M ICROBIOL  .   onN  ov  em b  er 1 7  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   uptake of MeBr was observed under any conditions after 2 h of incubation (datanot shown).To better equalize the conditions between the control and treated samples andto normalize for possible general metabolic inhibition, chloramphenicol wasadded simultaneously to pairs of samples and for short durations. One samplereceived chloramphenicol soon after it was equilibrated with experimental airand just as the sample was being cryotrapped; thus, it was exposed to chloram-phenicol during the 20 min it took to cryotrap the gas sample. The second samplereceived chloramphenicol at the same time, which was about 20 min before it wasequilibrated with experimental air, and thus 40 min of cumulative exposureoccurred before analysis. Consumption of micromolar MeBr.  Strains IMB-1 and MB2 T have beenshown to readily consume MeBr provided at micromolar concentrations (39, 40).Other cultures tested here for uptake of picomolar MeBr (pptv) were also testedfor their ability to consume micromolar levels of MeBr (ppmv). Cultures weregrown in batch, and 10 ml of culture was added aseptically to 58-ml serum vials which were crimp sealed with blue butyl stoppers. MeBr was added to achieve a fi nal concentration of 5  M in the liquid phase for the methanotrophs  M. rubra and strain BB5.1.  R. algicola  was tested for degradation using concentrations of 282, 141, 10, and 1  M MeBr.  P. mendocina  KR1 was tested using concentrationsof 5 and 1  M MeBr.Degradation of MeBr was monitored by injecting vial headspace (100  l) intoa Hewlett-Packard 5890 Series II GC-ECD. Experiments were performed usinga Restek RTX-624 wide-bore capillary column (30 m, 0.53  m i.d.; 3.0  m depthof   fi lm). The oven, injector, and detector temperatures were 50 ° C, 240 ° C, and300 ° C, respectively. Batch kinetics of   L. methylohalidivorans  at micromolar concentrations.  StrainMB2 T  was grown on 50  M MeBr in MAMS medium. The culture was dilutedthreefold with sterile medium, and 20 ml of diluted culture was added to 160-mlserum vials which were crimp sealed with blue butyl stoppers. MeBr was addedto achieve  fi nal concentrations ranging from 0.18 to 29   M and analyzed byGC-ECD as described above. Rates of MeBr degradation were determined bylinear least-squares regression and normalized by cell density. The cell densityranged from 3.3    10 6 to 4.9    10 6 cells ml  1 for three separate experiments.Two replicate bottles were used for each concentration, and each bottle wassampled twice per time point. The data from three experiments (22 data points) were used to determine kinetic parameters by nonlinear least-squares regressionto the Michaelis-Menten equation (Origin 6.0 software). RESULTS Ability of bacteria to consume 12 pptv MeBr.  A marinemethylotroph (strain MB2 T ) and a terrestrial methylotroph(strain IMB-1) consumed MeBr supplied at a mixing ratio of 12 pptv (  2 pM equilibrium dissolved concentration). Con-sumption of MeBr was  fi rst order in concentration for bothorganisms (Fig. 1 and 2). Neither a threshold for MeBr uptakenor saturation was observed for the concentrations appliedhere. The apparent degradation rate constants,  k  (equation 4),for strains MB2 T and IMB-1 and the range of applied MeBrconcentrations are given in Table 1. A terrestrial methanotroph (  M. rubra ) and an estuarinemethanotroph (strain BB5.1) also consumed MeBr supplied at12 pptv (Table 2). This appears to be the  fi rst direct demon-stration that methanotrophic isolates can consume tropo-spheric levels of MeBr. These two methanotrophs were alsoable to completely remove 5   M MeBr within 24 h (data notshown).No consumption of 12 pptv MeBr was observed for theheterotrophs  B. pumilus  and  E. coli  (Table 2).  R. algicola , aphylogenetic relative of   L. methylohalidivorans  strain MB2 T ,also did not consume MeBr whether it was supplied at micro-molar or picomolar levels. Similarly,  P. mendocina  KR1, whichcan cooxidize a variety of halogenated compounds (31), did notconsume 12 pptv MeBr (Table 2), nor did it consume 1 or 5  M MeBr even after 1 week of incubation (data not shown). Although the survey was not exhaustive, biodegradation of MeBr did not appear to be a universal trait of oxygenase-containing bacteria or a general trait of some common hetero-trophs; in this study, the ability was limited to methylotrophsand methanotrophs. Effect of growth conditions on MeBr uptake by strainsMB2 T and IMB-1.  Strains MB2 T and IMB-1 consumed 12 pptvMeBr under most growth conditions. The presence of alterna-tive growth substrates such as glucose did not appear to inhibitMeBr uptake, and both strains could consume 12 pptv MeBr whether or not they had been previously exposed to high levelsof MeBr during growth (Table 3). On a per-cell basis, degra-dation rates were highest for cells grown on mineral medium with MeBr added as the sole source of carbon and energy(Table 3). Doubling the MeBr concentration from 154 to 330  M during growth of strain IMB-1 had no adverse effect onuptake of near-ambient mixing ratios of MeBr, but increasingthe growth concentration to 500   M MeBr caused a markeddecline in the consumption of 12 pptv MeBr (Table 3). Al-though strain IMB-1 can tolerate fairly high concentrations of MeBr (6, 40), doses in the 500  M range appeared to be toxic. FIG. 1. Uptake rate of MeBr by  L. methylohalidivorans  strainMB2 T (  r   /   X  ) versus the dissolved equilibrium concentration ( C  L ). Con-centrations correspond to supplied mixing ratios of 14 to 9,559 pptvMeBr. The slope of the line (1.4  10  9 h  1 liter cell  1 ) is equal to theapparent  fi rst-order rate constant,  k , for this experiment (see equation4).FIG. 2. Uptake rate of MeBr by strain IMB-1 (  r   /   X  ) versus thedissolved equilibrium concentration ( C  L ). Concentrations correspondto supplied mixing ratios of 12 to 512 pptv MeBr. The slope of the line(3.9    10  9 h  1 liter cell  1 ) is equal to the apparent  fi rst-order rateconstant,  k,  for this experiment (see equation 4).V OL  . 67, 2001 BACTERIAL UPTAKE OF 12-PPTV METHYL BROMIDE 5439   onN  ov  em b  er 1 7  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   Inhibition of protein synthesis.  Consumption of 12 pptvMeBr by strain IMB-1 was not signi fi cantly affected by theaddition of chloramphenicol (Table 4). Conversely, chloram-phenicol did affect MeBr consumption by strain MB2 T . Al-though uptake of MeBr was observed with a 20-min exposureof chloramphenicol, a 40-min exposure essentially inhibiteduptake by cells grown on marine broth (Table 4). In a separateexperiment, strain MB2 T  was actively growing on MeBr; thus,an ample supply of MeBr-degrading enzymes should have beenavailable. Nonetheless, a 40-min exposure to chloramphenicolcaused a 73% reduction in the MeBr uptake rate (Table 4). Batch kinetics for  L. methylohalidivorans.  Saturation kinetics were observed for strain MB2 T (Fig. 3). Nonlinear regressionto the Michaelis-Menten equation produced values of   V  max   1.6  0.1 nmol h  1 (10 6 cells)  1 and  K   s   2.4  0.6  M. DISCUSSION The role of microorganisms in controlling the biogeochem-istry of trace gases such as methane (CH 4 ) and MeBr appearsto be pronounced (7, 16, 41). However, the biological oxidationof tropospheric CH 4  (  1.75 ppmv) by soil methanotrophsposes a physiological paradox because more energy is ex-pended in the production and maintenance of enzymes likeCH 4  monooxygenase (MMO) than can be recovered from theavailable substrate (7). Thresholds for methane (CH 4 ) degra-dation and a biphasic pattern have been reported for CH 4 oxidation in soils (1), raising questions about how bacteriaconsume trace gases from the atmosphere. Tropospheric mix-ing ratios of MeBr are about  fi  ve orders of magnitude lowerthan those of CH 4 , so MeBr poses an even greater physiolog-ical paradox from the standpoint of energy yields. It was thusquestionable whether previously characterized CH 4 - or MeBr-oxidizing bacteria would have any detectable af  fi nity for 12pptv levels of MeBr.To directly address this issue, we measured the kinetics of MeBr degradation starting with near-ambient mixing ratios of MeBr. The methanotrophs and methylotrophs tested wereable to consume MeBr supplied at 12 pptv (Table 2). Further-more, the methylotrophic strains MB2 T and IMB-1 showedclear  fi rst-order kinetics with no apparent threshold or biphasicpattern (Fig. 1 and 2).Kinetic saturation was not reached in these experiments(Fig. 1 and 2), precluding the calculation of   K   s  and  V  max  .However, these parameters were measured in batch experi-ments for  L. methylohalidivorans  strain MB2 T (Fig. 3). Theapparent  fi rst-order reaction rate constant (  k ) can be estimatedas  V  max   /   K   s  for  S   K   s . This value, 0.67  10  9 h  1 liter cell  1 ,is equivalent to the rate constant obtained from the CSTRexperiments (Table 1). Kinetic parameters previously weremeasured in batch experiments for strain IMB-1 by Schaeferand Oremland (40). They reported values of   K   s  190 nM and V  max     210 pmol h  1 (10 6 cells)  1 . The resulting value for V  max   /   K   s , 1.1    10  9 h  1 liter cell  1 , is equivalent to the rateconstant obtained from the CSTR experiments for strainIMB-1 (Table 1). Note that the reported rate constant of 0.056h  1 for a cell density of 10 6 cells was actually for a cell densityof 10 6 cells per 20 ml of cell suspension (J. K. Schaefer, per-sonal communication).The CSTR employed near-ambient concentrations, whereasthe batch experiments used MeBr concentrations that were 35to 1  10 7 times higher. Nonetheless, the values of   K   s  and  V  max  determined in batch experiments adequately described the ap-parent rate constants measured using the CSTR. For strainsMB2 T and IMB-1,  K   s  values were 10 2 to 10 3 times higher thanconcentrations of MeBr that could be supplied from the tro-posphere. Even so, both strains were able to consume environ-mentally relevant levels of MeBr.Known methanotrophs and soil bacteria consuming ambient TABLE 1. Average apparent  fi rst-order rate constants,  k , for MeBr uptake Strain MeBr applied, pptv (pM)  a Mean cell density (cells ml  1 )  SD Mean  k  b (h  1 liter cell)  SE IMB-1 7 – 2,970 (1.3 – 584) (  n  5) 3.4  10 7  3.8  10 7 3.0  10  9  2.1  10  9 MB2 T 14 – 9,559 pptv (2.8 – 1,886) (  n  2) 4.1  10 7  3.0  10 7 6.9  10  10 – 1.4  10  9  a Range of applied gas phase mixing ratios and calculated equilibrium liquid concentrations for  n  experiments.  b Mean  SE or range (for  n  2). Each experiment utilized 5 to 13 separate MeBr concentrations, as demonstrated in Fig. 1 and 2. TABLE 2. Uptake rate of MeBr supplied at 12 pptv fordifferent bacteria  a SampleMean uptake rate(10  8 pmol day  1 cell  1 )  SD Strain MB2 T .......................................................... 6.0  1.8 (  n  7)Strain IMB-1 ......................................................... 4.5  1.1 (  n  5)  Methylomonas rubra .............................................. 8.1  1.0 (  n  2)  Methylobacter   sp. strain BB5.1 ............................ 2.9  1.5 (  n  3)  Pseudomonas mendocina  KR1 ............................ Not detected (  n  2)  Ruegeria algicola .................................................... Not detected (  n  4)  Escherichia coli ...................................................... Not detected (  n  2)  Bacillus pumilus ..................................................... Not detected (  n  2)  a Strains MB2 T and IMB-1 were grown with MeBr as the sole source of carbonand energy. SD includes cumulative error for GC and AODC measurements. TABLE 3. Uptake rate versus growth condition for MeBr suppliedat 12 pptv (  2 pM) for terrestrial and marine methylotrophs Strain Growth substrate and MeBrconcn (  M)Mean uptake rate  a (10  8 pmol day  1 cell  1 )  SD MB2 T MeBr (50) 7.6  3.8Marine broth/MeBr (50) 0.92  0.10Marine broth 0.47  0.07IMB-1 MeBr330 2.7  0.4500 0.096  0.001Glucose/MeBr154 0.20  0.02188 0.22  0.02330 0.27  0.07500 0.0043  0.0032Methylamine/MeBr (330) 0.44  0.07LB broth 0.36  0.16  a SD includes cumulative error for GC and AODC measurements. 5440 GOODWIN ET AL. A  PPL  . E NVIRON . M ICROBIOL  .   onN  ov  em b  er 1 7  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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