JOURNAL OF BACTERIOLOGY, Nov. 1974, p. 955-964Copyright 0 1974 American Society for Microbiology

Vol. 120, No. 2Printed in U.SA.

Isolation and Characterization of Bacteria That Grow onMethane and Organic Compounds as Sole Sources of Carbon

and EnergyTOM E. PATT, GLORIA C. COLE, JUDITH BLAND, AND R. S. HANSONDepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 5 August 1974

Bacteria capable of growth on methane and a variety of complex organicsubstrates as sole sources of carbon and energy have been isolated. Conditionsused to rigorously establish the purity of the cultures are described. Onefacultative methylotroph has been studied in detail. This organism has peripher-ally arranged pairs of intracytoplasmic membranes characteristic of obligatemethylotrophs. This isolate apparently utilizes the serine pathway of formalde-hyde fixation. The location of methane oxidizers in a dimictic lake indicates thatthese organisms prefer less than saturating levels of dissolved oxygen. Laboratoryexperiments confirmed the preference of these organisms for atmospherescontaining less oxygen than air.

Bacteria with the ability to utilize methane asthe sole source of carbon and energy have beenknown since the early 1900's (21, 25). Untilrecently, there have been only three species inpure culture (22, 31, 32). The three well-docu-mented species are Pseudomonas methanica(8), Methanomonas methanooxidans (2), andMethylococcus capsulatus (9). Whittenburyand co-workers (31) were able to improve thetechnique of enrichment and isolation of me-thane-utilizing bacteria to the point where theyhave isolated over 100 strains, which they classi-fied into 15 species. Some of the strains theyconsidered identical to previously isolated me-thane-utilizing bacteria.Microorganisms are considered methylo-

trophs if they can grow non-autotrophicallyusing carbon compounds containing one or morecarbon atoms but no carbon-carbon bonds (3).Interestingly, all of the isolates with the abilityto utilize methane have been obligate methylo-trophs, that is, organisms that can only usemethane, methanol, or dimethyl ether (32) astheir sole carbon and energy source. Organismsclassified as obligate methylotrophs encompasstype I and type II organisms (31), a classifica-tion by membrane morphology that is paral-leled by the pathway used by the organisms forthe incorporation of reduced Cl compounds(17).The facultative methylotrophs isolated previ-

ously grow on methanol, methylamines, andformate and can also utilize more complexorganic compounds. None of the facultative

methylotrophs studied are able to utilize meth-ane as a sole source of carbon and energy (32).Most of the facultative methylotrophic bacteriacharacterized utilize the serine pathway forformaldehyde incorporation (12-15, 22). Thepresence of internal membrane structure hasnot been established except for Hyphomicro-bium strain H-526 that possesses type II mem-branes (4). Stieglitz and Mateles (29) wereunable to demonstrate internal membrane struc-tures in Pseudomonas C. This organism pos-sessed hexose phosphate synthase activity.This paper reports on the isolation of orga-

nisms that will utilize methane as well as themore complex organic carbon and energysources. They are much like the facultativemethylotrophs previously reported in their abil-ity to utilize C, compounds in addition to themore complex carbon and energy sources, butthey differ in that they also possess the abilityto utilize methane as a sole carbon and energysource.One of these isolates has been more fully

characterized and is described.(The work described in this paper was sub-

mitted to the University of Wisconsin by G. C.C. and T. E. P. in partial fulfillment of therequirements for the M.S. degree.)

MATERIALS AND METHODSOrganisms. Methylosinus sporium 12 and Meth-

ylosinus trichosporium PG were used in some exper-iments for comparison with our isolates (31). Theseorganisms were kindly provided by R. Whittenbury,

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University of Warwick, England. Escherichia coli UWwas obtained from the University of Wisconsin, De-partment of Bacteriology, collection. MicrococcusIysodeikticus was obtained in the form of dried cellsfrom Sigma Chemical Co., St. Louis, Mo. Isolate XXhas been deposited with the American Type CultureCollection, Rockville, Md. (accession number 27886).Media and growth conditions. The two basal

media.of Whittenbury et al. (31) were used with amodification. The AMS basal salts medium con-tained (per liter of distilled water): K,PO4, 0.70 g;KH,PO4, 0.54 g; MgSO4.7H2O, 1 g; CaCl2.2H2O, 0.2g; FeSO4.7H2O, 4 mg; NH4Cl, 0.5 g; ZnSO4.7H20,100 Mg; MnCl2.4H20, 30 Mtg; HSBO4, 300 Mg;CoCl2.6H2O, 200 Mg; CuCl2.2H2O, 10 Mg;NiCl2.6H2O, 20 Mg; and Na2MoO4.2H2O, 30 Mg. TheNMS basal salts substituted 1 g of KNO, for theNH4Cl. FeSO4.7H20, 4 mg/liter, replaced the seques-trene iron complex. When agar (Difco purified orOxoid no. 2 Ionagar) was present, it was added to givea 1.2% (wt/vol) concentration. At other times washedmembrane filters (HAWG, 0.45-am pore size; Mil-lipore Corp.) were placed on glass-fiber filters (Mil-lipore AP 2504700) wetted with the liquid NMSmedium to give a solid support for growth. Bacteriawere streaked on the surfaces with inoculating loopsto obtain clones.Methane as a carbon and energy source was pro-

vided by filtering (through a glass-wool filter) anatmosphere of methane-oxygen (4:1) or methane-air(1:4) into a closed vessel containing the growth media.Methanol, ethanol, and propanol were added directlyto the media and were also provided by incorporationinto separate uninoculated agar slants to provide acontinuous release of vapors at a low level. Ethanewas provided in the same way as methane. All othercarbon sources were sterilized separately in water andwere added directly to the growth medium at a finalconcentration of 0.1% (wt/vol).

Agar cultures were incubated under an atmosphereof methane-oxygen in Brewer jars (BBL) and desicca-tors. Liquid cultures were gassed directly throughports that could be sealed or through rubber serumstoppers by means of a syringe needle.

Antibiotic gradient plates were prepared as de-scribed by Szybalski and Bryson (30), using 100 Mg ofthe antibiotics streptomycin, staphcillin, or gramici-din per ml in the bottom layer of agar.

Liquid cultures were incubated on rotary shakers at30 C unless otherwise stated.

Large batches of cells for enzyme assay were grownin a New Brunswick Microferm 14 L fermenter. NMSmedium (9 liters) was inoculated with 1,200 ml of thecells grown on methane. The fermenter was continu-ously gassed with a mixture of 70 ml of methane and 1liter of air per min by using a gas proportionater(Matheson). The temperature was maintained at30 C. The growth was followed by measuring theabsorbance at 525 nm in a Beckman model BDspectrophotometer. At the end of exponential growth,the cells were harvested in a Sharples centrifuge,washed once with 50 mM potassium phosphate buffer(pH 6.8), and stored frozen at -20 C.

In order to determine if growth would occur on

various carbon and energy sources, the inocula weretransferred several times before inoculation into thetest flasks. A loopful of cells scraped from an AMSagar plate incubated under methane-oxygen was usedfor the initial inoculum into nutrient broth. Aftergrowth had occurred, 5 ml was pipetted into 50 ml ofAMS basal salts and incubated under methane-air(1:4). This culture was transferred once more throughmethane. A 1-ml sample of this culture was used toinoculate 100 ml of AMS and was incubated undermethane-air. These cells were centrifuged and resus-pended before use as an inoculum. The substratetested was added at a concentration of 0.1% (wt/vol)to NMS basal salts.

Single cell isolations. Individual cells from cellpopulations grown in a NMS basal salts mediumunder an atmosphere of methane-air (1:4) and inNMS basal salts supplemented with 0.1% glucosewere isolated by the method of DeVay and Schna-thorst (7).

Labeling of deoxyribonucleic acid (DNA) fordensity centrifugation. Bacteria were grown at 30 Cin NMS medium under an atmosphere of CH4-02(4:1). The atmosphere included 100 MCi of "CH4.Incubation was continued with the addition of air atintervals to keep a pressure of one atmosphere insidethe culture vessel. Cells were also labeled in NMSmedium supplemented with glucose and 100 MCi of[3H Jthymidine.DNA isolation and equilibrium CsCl density

gradients. M. lysodeikticus DNA was isolated by themethod of Marmur (20) and isolate XX DNA wasisolated by the method of Schilperoot (R. A. Schil-peroot, Ph.D. thesis, University of Leiden, The Neth-erlands, 1969).The Schilperoot method consists of destroying the

deoxyribonuclease activity in situ by a pretreatmentat 70 C. Sodium dodecyl sulfate (1.6%) and PronaseB-1 M NaCl (0.6 volume, 1 mg/ml) were added to thesuspension, and the suspension was incubated for 3 hat 60 C. The resulting viscous suspension was depro-teinized by adding successively 1% sodium dodecylsulfate, 6% sodium p(4-)amino-salicylate, and 3%NaCl. Phenol containing 0.1% 8-hydroxyquinoline,saturated with 3 SSC (1 standard saline citrate buffercontained 0.15 M NaCl, 0.15 M trisodium citrate, pH7.3), was then added, and the mixture was incubatedwith gentle shaking. The addition of 1% sodiumdodecyl sulfate, 6% sodium p(4)amino-salicylate, and3% NaCl prevented the loss of DNA in the interphaseafter centrifugation of the phenol-water emulsion.Most of the dissolved phenol was extracted with

chloroform, and the remaining phenol plus chloroformwas removed by dialysis against 0.1 SSC. Ribonucleicacid was removed by the addition of ribonuclease Aand T, (1/20 volume of a stock solution containing 2mg of type A and 600 U of type T1 per ml). A secondPronase treatment was followed by two additionalphenol deproteinizations. The phenol was extractedwith chloroform again. The DNA was precipitatedwith cold absolute alcohol and wound out of solutionon a glass rod. The purified DNA was dissolved in 0.1SSC buffer.A DNA mixture of labeled isolate XX DNA plus

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marker DNA was made up to 4 ml of 0.1 SSC. ThisDNA solution was added to 5.35 g of CsCl (opticalgrade, Schwartz/Mann). The solution was added topolyallomer tubes, and mineral oil was layered on topto fill the tubes. For alkaline CsCl gradients, 5.6 g ofCsCl per 4 ml of DNA solution at pH 12 in 0.1 SSCwas used.The DNA solution was centrifuged to equilibrium

with a Beckman model L-2 centrifuge at 34,000 rpm ina type SW65L Titanium rotor for 60 h at 25 C. Thetubes were punctured with an 18-gauge needle, and12-drop fractions were collected under constant pres-

sure. A 10-Mliter sample from each fraction was placedon a Gelman GF/A filter and dried under a red-heatlamp. The filters, when dry, were placed in 10 ml ofscintillation fluid; 2,5-diphenyloxazole, 4 g, and 1,4-bis-2-(5-phenyloxazolyl)-benzene, 0.05 g in 1 liter oftoluene. The radioactivity was monitored in a Pac-kard Tri-Carb model 3375 liquid scintillation counter.The remaining part of each fraction was diluted to 1

ml with distilled water, and the absorbance (at 260nm) was measured on a Bausch and Lomb spectronic600 spectrophotometer.The guanine plus cytosine content of isolate XX

DNA was calculated from the buoyant density usingthe relationships published by Schildkraut et al. (24).M. lysodeikticus DNA was used as an internal marker(p = 1.731 g/cm3 in neutral CsCl and 1.793 g/cm3 inpH 12 CsCl 124]). The refractive index of the CsCl wasread on a Bausch and Lomb refractometer at 25 C,and the solution density was determined (25).

Preparation of extracts. Cells were harvested andwashed with 50 mM potassium phosphate buffer (pH6.8). Cell extracts were prepared by suspending 1.0 g(wet weight) of cells in 4.0 ml of the lysis buffercontaining 10 mM tris(hydroxymethyl)-amino-methane, 1 mM ethylenediaminetetraacetic acid tet-rasodium salt, 1 mM trisodium-citrate, and 100 mMKCl (pH adjusted to 7.5 with acetic acid). Thesuspended cells were broken by two passes through a

French pressure cell at 6,000 lb/in2. Ribonuclease anddeoxyribonuclease, 25 Mg each, were added to the cellpaste, and it was incubated at 37 C for 10 min. Theextract was centrifuged at 5,900 x g for 15 min toremove intact cells and debris. The following enzymeswere assayed in this crude extract: hydroxypyruvatereductase (EC 1.1.1.29, D-glycerate: nicotinamideadenine dinucleotide oxidoreductase); hexose phos-phate synthase (no EC number); ribulose diphos-phate carboxylase (EC 4.1.1.39, 3-phospho-D-glycer-ate carboxylase [dinierizing]); and phosphoenolpyru-vate-glucose phosphotransferase (no EC number).The supernatant extract was fractionated by cen-

trifugation at 38,000 x g for 1 h to obtain soluble andparticulate fractions. The particulate fraction was

resuspended in one-fifth volume of 50 mM potassiumphosphate buffer (pH 7.5) and was then used for theassay of succinate dehydrogenase (EC 1.3.99.1, succi-nate: [acceptor] oxidoreductase). The soluble fractionwas used to assay citrate synthase (EC 4.1.3.7, citrateoxaloacetate-lyase [coenzyme A-acetylating]); isocit-rate dehydrogenase (nicotinamide adenine dinucleo-tide phosphate [NADP], EC 1.1.1.42, threo-D.-iso-citrate: NADP oxidoreductase [decarboxylating]);

)N AND ENERGY SOURCE 957

malate dehydrogenase (EC 1.1.1.37, L-malate; nico-tinamide adenine dinucleotide oxidoreductase);aconitase (EC 4.2.1.3, citrate [isocitrate] hydro-lyase);fumarase (EC 4.2.1.2, L-malate hydro-lyase); and glu-cose-6-phosphate dehydrogenase (EC 1.1.1.49, D-glu-cose-6-phosphate:NADP oxidoreductase).A portion of the soluble fraction was centrifuged at

133,000 x g for 3 h in order to remove reducednicotinamide adenine dinucleotide oxidase (no ECnumber) activity. The particulate fraction from thishigh-speed centrifugation was resuspended in one-fifth volume of 50 mM potassium phosphate buffer(pH 7.5) and used for the assay of a-ketogluteratedehydrogenase (EC 1.2.4.2, 2-oxoglutarate: lipoateoxidoreductase [acceptor-acylating]).

All operations were carried out at 4 C. The proteincontent of the extracts was determined by the methodof Lowry et al. (18) using crystalline bovine serumalbumin (Sigma, fraction V) as a standard.Enzyme assays. The following enzymes were as-

sayed as previously described (1, 10, 11, 16, 19):citrate synthase, aconitase, isocitrate dehydrogenase(NADP), a-ketoglutarate dehydrogenase, succinic de-hydrogenase, fumarase, malate dehydrogenase, glu-cose-6-phosphate dehydrogenase, hydroxypyruvatereductase, hexose-phosphate synthase, ribulose di-phosphate carboxylase, and phosphoenolpyruvate-glucose phosphotransferase.The changes in absorbance were monitored with a

Gilford model 2000 recording spectrophotometer at30 C. Specific activities were calculated as mi-cromoles per minute per milligram of protein.

Microscopy. For thin sections, preparations werefixed according to the method of Smith and Ribbons(27). Alternatively, the Ryter-Kellenberger procedure(23) was employed. After dehydration, all prepara-tions were embedded in Spurr epoxy resin (28). Thinsections were cut with a diamond knife on a MT-2Sorvall microtome, placed on Parlodion and carbon-coated grids, and stained with uranyl acetate and leadcitrate. An angle of 280 was used for shadowing driedcell preparations with platinum and carbon. Speci-mens were examined and photographed in a Zeiss EM9S electron microscope.

Incorporation of labeled C1 compounds. Cellswere harvested, washed in NMS basal medium, andresuspended in NMS basal medium to give a finalconcentration of 40 to 45 Ag of protein/ml. One-halfmilliliter of the cell suspension was placed in a 12-mlvial sealed with a rubber septum. The labeled com-pounds were added by injection into the vial. Incuba-tion was at 30 C with shaking for the time specified.Ice-cold trichloroacetic acid was added to the cells togive a final concentration of 10%. The samples werestored on ice for 30 min before filtering the samplethrough Gelman GF/A filter pads. The filter padswere then washed twice with 10 ml of ice-cold 95%ethanol to remove the trichloroacetic acid. Afterdrying, the pads were added to scintillation vialscontaining 10 ml of scintillation counting fluid: 2,5-diphenyloxazole, 4 g, and 1,4-bis-2-(5-phenyloxazo-lyl)-benzene, 0.05 g in 1 liter of toluene.The incorporation of labeled one-carbon compound

was used as an indicator of the biological activity of

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methane utilizers in lakewater samples. A measuredvolume of water was incubated for 4 h as describedabove.

Analytical methods. Protein was determined bythe Folin phenol reagent method (18) with crystallinebovine serum albumin (Sigma, fraction V) as astandard. Cells were treated with 1 N NaOH to solu-bilize the protein. Bovine serum albumin standardswere treated identically to the samples. Absorbancewas measured at 500 nm on a Beckman model DBspectrophotometer.Methane and ethane were analyzed on a Beckman

gas chromatograph model GC-4 equipped with ahydrogen-flame ionization detector and a Beckman10-in (ca. 25.4-cm) recorder. The column was 6 ft by'/8 in (ca. 182.8 by 0.32 cm) packed with Poropak R(Waters Association, Inc., Framingham, Mass.) andwas maintained isothermally at 50 C. The carrier gaswas helium at a flow of 50 ml/min.

Lakewater samples were obtained using a VanDoren water sampler (Hydro Products, DillinghamCorp., San Diego, Calif.). The lakewater temperatureand oxygen concentration were measured at variousdepths with a model 54 oxygen meter and tempera-ture thermistor (Yellow Springs Instrument Co., Inc.,Yellow Springs, Ohio). Methane concentration wasdetermined in lakewater samples by injecting 0.5 mlof the water into evacuated serum bottles (8 ml). Themethane partial pressure was determined in the gaschromatograph and compared with a standard meth-ane sample.

RESULTSEnrichment of isolation. Enrichment and

isolation techniques were similar to those ofWhittenbury et al. (31). Lake bottom mud andlakewater samples were used as inocula. Thesample size was approximately 5 to 10 g per 100ml of NMS or AMS containing 10 mg of ferrousammonium citrate. Some of the samples wereheated at 80 C for 15 min prior to their use asinocula. The enrichment flasks were incubatedat 30 C without shaking under an atmosphere ofmethane-oxygen (4:1). Within 7 to 14 days theculture medium usually became turbid, pre-sumably due to the growth of CH4-oxidizingbacteria.

Serial dilutions were made of the enrichmentculture medium and spread onto AMS agarplates. The plates were incubated in Brewer jars(BBL) under methane-oxygen (4:1) at 30 C.Isolated colonies were picked and restreaked.

It was discovered that many of these single-colony isolates would grow on nutrient agarplates as well as on methane. Presumptivegrowth on methane was indicated by methane-dependent growth of colonies on membranefilters incubated with and without a methaneatmosphere. We proceeded to rigorously estab-lish purity of the cultures.

Criteria of purity. Antibiotic gradient platescontaining streptomycin, staphcillin, or grami-cidin were streaked from isolated colonies grow-ing upon methane, beginning at the end of theplate with the lowest concentration of the anti-biotic. The plates were incubated at 30 C underan atmosphere of methane-oxygen (4:1). Thosecolonies growing in the region of highest antibi-otic concentration were tested for growth afterstreaking on nutrient agar. All colonies werefound to grow. This method should be useful forpurification if organisms growing together donot have the same resistance to antibiotics. Theorganisms from colonies on each set of plates,when examined under the phase contrast micro-scope, were found to be indistinguishable.

Further purification was carried out on iso-lates from the antibiotic plates. Single-cellisolations (7) were used to demonstrate theidentity of the organisms growing at the expenseof methane with those growing on glucose.Droplets of medium under the sterile mineral oilthat contained a single cell were selected. Thedroplets were diluted and plated in 20 spotseither on mineral salts medium, incubatedunder an atmosphere of methane-oxygen (4:1),or on nutrient agar. Fifty clones growing onmethane were replicated onto nutrient agar,and an equal number of clones from nutrientagar were replicated onto mineral salts mediumand incubated in an atmosphere of methane-oxygen. All the clones picked were able to growwhen replicated. The cells from the differentmedia were indistinguishable when examinedunder the phase contrast microscope. There-fore, these experiments lend supporting evi-dence that a clone from a single cell could growon nutrient agar as well as on AMS agar under amethane atmosphere. Based on the results ofthis and the preceding experiment, the isolateswere presumed to represent pure cultures offacultative methylotrophs capable of utilizingmethane.

Isolate XX was obtained from a heat-shockedprimary enrichment culture, so it was believedthat cultures of this organism contained aheat-stable form. If the isolate was a mixedculture, it would be unusual for both forms tosurvive high temperatures. Therefore, a culturewas heated until there were few survivors. Thesurvivors were plated on two media. Clones ofthese survivors should constitute a pure culture.Table 1 describes an experiment which mea-sured the survivors from a 2-day-old nutrientbroth culture heated in sterile test tubes at80 C. Samples were taken at 0, 5, 10, 20, 30, and60 min of incubation at 80 C and each was

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METHANE AS SOLE CARBON AND ENERGY SOURCE

TABLE 1. Survival of organisms from a culture ofisolate XX grown on methane as a source of carbon

and energy

Survivors/mlTime in min

(at 80 C)Methane-AMS Nutrient agar

agar

0 8 x 106 7 x 1065 1.8 x 103 1.9 x 10210 300 29520 300 20030 300 20060 70 60

serially diluted. Dilutions were spread on nutri-ent agar plates and on AMS agar plates incu-bated under an atmosphere of methane-oxygen(4:1). It can be seen from the data in this tablethat the number of survivors on the two types ofmedia were similar in each heated sample.Thus, there was only one organism in theoriginal culture, or different organisms hadidentical death rates at 80 C.

Colonies that grew on nutrient agar fromsamples heated 30 and 60 min were picked andtransferred to AMS agar plates which wereincubated under methane, and colonies fromthe same heated samples originally growing onmethane were transferred to nutrient agar. Allthe colonies grew on both sets of plates, andunder phase contrast microscopy they wereindistinguishable.

Finally, because obligate methylotrophs showno growth on complex nutrient solutions (31), itshould be possible to eliminate obligate methyl-lotrophs by transferring our isolates on nutrientagar a number of times. The remaining popula-tion would be heterotrophs. The new isolatesreported here retained their ability to grown onmethane after many single-colony isolations onnutrient agar. This is also shown in the incorp-oration experiments described later.DNA density. Figure 1 shows the density of

isolate XX DNA relative to M. lysodeikticusDNA in a neutral and alkaline CsCl densitygradient. The densities are very similar. Thedensity of XX DNA, determined by the refrac-tive indexes of the gradient fractions, was 1.725g/cm3 in neutral CsCl and 1.787 g/cm3 inalkaline CsCl. The identical density shifts of thetwo DNAs in the alkaline CsCl (0.062 g/cm3)confirm the density of 1.725 obtained in theneutral CsCl gradient. The shift of density ofthe DNA in alkaline CsCl ruled out the possibil-ity that the density near the value for M.

0aC4El

z44

0

49

Ia

0

z

0 10 20 30FRACTION NUMBER

FIG. 1. CsCI density gradients of M. lysodeikticusDNA and isolate XX DNA. (A) Neutral and (B)alkaline gradients. Symbols: 0, absorbance (260 nm)of M. lysodeikticus DNA; 0, dpm of ['H]thymidine-labeled DNA from isolate XX.

lysodeikticus is due to an artifact, such asbinding of the DNA to other materials.

It was also of interest to determine if the DNAfrom isolate XX growing on methane had thesame density and guanine plus cytosine contentas the cells growing on glucose. If the densitieswere different, it would indicate that two differ-ent organisms were responsible for growth onorganic substrates and methane. Figure 2 showsthat both the cells growing on glucose( [3H ]thymidine labeled) and those cells growingon methane (14CH, labeled) had DNA with adensity of 1.725 g/cm3. This density correspondsto a guanine plus cytosine content of 66%.Morphological characterization. Cell mor-

phology varied greatly among our isolates. Mor-phology ranged from long, slender rods (isolatePG-2) to short, fat rods (isolate XX). Manyisolates had pleomorphic forms, some wereclub-shaped, and many formed rosettes (includ-ing isolate XX).

Colonial morphology varied greatly amongthe isolates. The color of colonies ranges fromcolorless, yellow, orange, to white. Isolate XX

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Ia

z

0 5 10 15 20FRACTION NUMBER

FIG. 2. Neutral CsCI density gradient of isolateXX DNA. Symbols: *, [9H]thymidine label in glu-cose-grown cells; 0, ['4C]methane label in methane-grown cells.

formed white colonies, which turned orange onaging.The cells of isolate XX are gram negative,

flagellated, motile rods with a diameter of 1 ,umand a length of about 1.5 um (Fig. 3A). We seeat least one polar flagellum with an averagelength of 5 to 6 gm.The fine structure of XX was studied in more

detail than the other isolates. The two fixationprocedures described in Materials and Methodswere used on methane-grown and on glucose-grown cells. Cells grown on methane as the solesource of carbon and energy had extensiveintracytoplasmic pairs of membranes arrangedat the periphery of the cell (Fig. 3B). Thismembrane type has previously been designatedtype II (6). Thin sections of XX growing onglucose have not shown observable intracyto-plasmic membranes (Fig. 3C).Carbon and energy sources utilized. Table

2 shows the substrates tested as the sole sourceof carbon and energy for our isolates and the twoisolates provided by Professor Whittenbury.Methylosinus sporium and Methylosinustrichosporium grew on methane, but on none ofthe other substrates except methanol. Our iso-lates were able to use metabolically diversecompounds, including tricarboxylic acid-cycleintermediates, sugars, complex media, andmethane. Initial attempts failed to demonstrategrowth of all isolates on methanol. Recently wehave succeeded in obtaining growth of isolate

XX on NMS media with 0.5% (wt/vol) metha-nol. Other isolates have not been retested.The ability to utilize methane as a sole

carbon and energy source was demonstrated inseveral ways. The requirements for methane forgrowth in the absence of other added carbonsources was shown by incubating duplicatecultures, one with methane and one without. Inbroth cultures growth was demonstrated todepend on methane. The isolates would grow onagar with or without methane. The use ofmembrane filters pinned to glass-fiber filterssoaked in the AMS medium eliminated growthin the absence of methane. In the presence ofmethane, visible colonies that turned orangewith age appeared on the membrane filters.The dependence on methane for growth was

also demonstrated using a gas chromatograph.Measurements made at various intervalsshowed that methane disappeared as cell massincreased. The incorporation of ["4C]methaneinto cold trichloroacetic acid-insoluble materialalso showed that these cultures would usemethane as a carbon source.Biochemical characterization. Table 3

shows the results of enzymatic assays of isolateXX grown on NMS under an atmosphere ofmethane-oxygen (4:1). Extracts of E. coli strainUW were also used to indicate that the assaysused would detect all the enzymes assayed.

All enzymes of the tricarboxylic acid cyclewere present at detectable levels. Of the en-zymes that play a key role in Cl incorporation,the only one detected was hydroxypyruvatereductase, an enzyme in the serine pathway forformaldehyde assimilation. Hydroxypyruvatereductase was found in high levels in themethane-grown XX cells. The specific activityranged from 0.28 to 0.89 gmol/min per mg ofprotein.Table 4 shows the results for two enzymes

concerned with the intermediary metabolism ofcarbohydrates. Glucose-6-phosphate dehydro-genase activity can be seen to vary with carbonand energy source used to grow isolate XX. Thenicotinamide adenine dinucleotide-specific en-zyme is not detectable in methane-grown cells,but is present at the specific activity of 0.080Aumol/min per mg of protein in glucose-growncells. The same is true for NADP+-specificenzyme, except it is present at a specific activ-ity of 0.406. Phosphoenolpyruvate-glucose

FIG. 3. Electron micrographs of isolate XX. (A) Negative stain using platinum and carbon; (B) methane-grown cell fixed by the method of Smith and Ribbons (27); (C) glucose-grown cell fixed by the method of Ryterand Kellenberger (23). The bar represents 0.5 tsm in (A) and 0.2 ,m in (B) and (C).

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TAmZ 2. Utilization of carbon and energy sources by methylotrophic bacteria

Carbon and Isolate no. M. tricho-energy sources 7 4 1 14 12-7 9 XM. sporum sporium

Malate + + + ± + + + + _Succinate + + + + + + + + _Acetate ± + + ++ ++ + + + -

Fumarate ±4 4 + + ± + + + - -

Glucose + + + + + + + + - -

Galactose + + + ± ++ + + + - -

Sucrose + + + ± ++ + ++ + - -

Ribose 4_ _ -X _ - -

Lactose + + + ± + + + + _Propanol _ _ _ - _ _ _ _ _Ethanol _ _ _ - -_ _ _ _Methanol ? ? ? ? ± ? + ? + +oNone _ _ _- - - - -

Methane + + + + + + + + + +Nutrient agar + + + + + + + +

a +, Growth; -, no growth; ±, slight growth.* Results of R. Whittenbury.

TABLE 3. Enzymatic activities in extracts ofisolate XX

Sp act (pmol/min per

Enzyme mg of protein)

E. coli Isolate XX

Citrate synthase 0.890 0.160Aconitase 0.074 0.084Isocitrate dehydrogenase 1.83 0.196a-Ketoglutarate dehydrogenase 0.465 0.005Succinate dehydrogenase 0.217 0.053Fumarase 0.586 0.431Malate dehydrogenase 11.60 1.41Glucose-6-phosphate dehydrog- 0.242 NDa

enaseHydroxpyruvate reductase ND 0.28-0.89Ribulose diphosphate carbox- ND ND

ylaseHexosephosphate synthetase ND

a ND, Not detectable.

phosphate transferase was present in isolateXX under both growth conditions at approxi-mately the same level.Location of methylotrophs in a dimictic

lakewater column. Because we isolated most ofour methylotrophs from lakewater samples, wewere interested in their location in the lake.Figure 4 shows the results of a study to deter-mine where methane oxidation occurs in thelake. These results show that the most rapidincorporation of [140 ]methane into trichloroace-tic acid-insoluble material occurs in water sam-ples from the thermocline in Lake Mendota,Madison, Wis. The methane concentration is

TaE 4. Specific activity of glucose-6-phosphatedehydrogenase and phosphoenolpyruvate-glucosephosphate transferase in isolate XX grown on two

different carbon and energy sources

Carbon and energysource (;pmol/min

Enzyme per mg of protein)

CH4 Glucose

Glucose-6-phosphate dehydrog-enase:

NAD+ a ND" 0.080NADP+ ND 0.406

Phosphoenolpyruvate-glucose 0.006 0.005phosphate transferase

a NAD, nicotinamide adenine dinucleotide.° ND, Not detectable.

highest at the sediment-water interface anddecreases at the thermocline.Incorporation of radioactive C1 compounds

by methane- and glucose-grown cells. Table 5shows the results of C, incorporation experi-ments from cells grown on two different carbonand energy sources: methane and glucose. Thecells grown on methane incorporate more "CH4in the presence of glucose than in the presenceof CH4 alone. The incorporation of 14CO, wasshown to be energy dependent, with the greatestincorporation occurring when the same energysource is present during incorporation as wasused for growth of the cells.Methane- and glucose-grown cells incorpo-

rated H14CHO.

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METHANE AS SOLE CARBON AND ENERGY SOURCE

T1MP C

MIT.A..I .OI) SIl

FIG. 4. Physical characteristics and the location ofmethylotrophs in a dimictic lakewater column. Sym-bols: 0, temperature; *, oxygen concentration; 0,1umoles of methane incorporated; *, methane.

TABLE 5. Cl incorporation of one-carbon substratesinto cell material by isolate XX

Growth substrate for isolate XX(dpm incorporated/20 Ag of cell

Substrates testeda b proteinc

CH, Glucose

14CH4 64,500 (5.86) 3,600 (0.33)14CH4 + glucosed 71,000 (6.45) 3,220 (0.29)14CO2 6,200 (0.56) 11,020 (1.02)14CO2 + CH4 98,900 (9.00) 13,580 (1.23)14CO2 + glucosed 8,450 (0.77) 21,969 (1.92)H14CHO 49,700 (4.52) 63,000 (5.72)

a 14C (0.5 uCi) (specific activity: 14CH4, 0.022mCi/mmol; 14CO2, 3 mCi/mmol; H14CHO, 0.15 mCi/mmol).

b Incubation (4.5 h) at 30 C in 0.5 ml of NMS basalsalts.

c Corrected for heat-killed control cells which incor-porate 90 dpm per 20 jig of protein; numbers inparentheses indicate percent incorporated.

d Glucose (6 mM).

DISCUSSIONThis paper describes the isolation of methylo-

trophs that are capable of utilizing, in additionto methane, many common organic carbon andenergy sources. Included is a description of therigorous experiments, which demonstrate thatone organism is responsible for growth on the Clcompounds as well as on the more complexorganic compounds. The methods to demon-strate that a, single species grows on the variouscarbon and energy sources include: single-cellisolations, antibiotic sensitivities, heat toler-

ance, subculturing on rich media for manygenerations to eliminate obligate methylo-trophs, ["4C]methane incorporation, and DNAdensity determinations. In the DNA densitystudies it is important to note that the DNAdensities are identical and relatively high (adensity corresponding to a guanine plus cyto-sine content of 66%).The Cl incorporation results showed that

glucose-grown cells were able to incorporatemethane into cell carbon. On culturing the cellson glucose for several transfers, obligate meth-ylotrophs would be lost. The ability of our cul-tures to continue to incorporate methane wouldindicate that the glucose-grown cells possessed,or could synthesize, the enzyme systems for theincorporation of methane. If the culture weremixed, the obligate methylotrophs should havebeen eliminated in this experiment. The in-corporation data also provides evidence thatmethane-grown cells will incorporate moremethane in the presence of glucose than withoutit. This indicates that isolate XX can usemethane and glucose simultaneously.Methylotrophs have been classified as be-

longing to either type I or type II (31). Thisdivision was initially made on the basis ofmembrane ultrastructure (6), but it has sincebeen shown that the pathway of reduced C1assimilation follows the same division (17).Type I organisms possess parallel, closely

packed bundles of membranes consisting of anumber of disk-shaped vesicles found through-out the cell and use the ribulose-5-phosphatepathway of formaldehyde incorporation. Type IIorganisms possess a system of paired mem-branes running throughout the organism oraggregated at the periphery and use the serinepathway to incorporate C1 units.The enzymatic data and the electron micro-

graphs place isolate XX in the type II category.Isolate XX has a complete tricarboxylic acidcycle, as has been shown in the other type IIorganisms (5); the key enzyme of the serinepathway, hydroxypyruvate reductase, is pres-ent, whereas other C l-assimilating enzymes arenot. Thin sections show the characteristic mem-branes of type II.Within the group of organisms that utilize the

serine pathway there is a further division: theobligate methylotrophs which can only usemethane, methanol, or other Cl compounds;and the facultative methylotrophs which usemethanol, formate, methylamines, and morecomplex organic compounds, but are unable toutilize methane (32). The organisms reported inthis paper would be considered facultative

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PAFT ET AL.

methylotrophs, differing from previous faculta-tive organisms in the ability to utilize methane.The lake study experiments indicate that the

methane-utilization activity is most active atthe thermocline. At the thermocline the dis-solved 0, concentration decreases. In the labo-ratory we have found that isolate XX grows bestin atmos'pheres with less than 20% oxygen

(unpublished data). We also observed that"4CH4 incorporation occurs in the lake bottomwater samples from the water-sediment inter-face. We have tested for anaerobic methaneincorporation and find that bottom sedimentsfrom anaerobic layers of a eutrophic pondincorporate methane in the absence of oxygen

(unpublished data).

ACKNOWLEDGMENTSThis research was supported by the College of Agricultural

and Life Sciences, Madison, Wis. Tom E. Patt and JudithBland were supported by Public Health Service traininggrant 5 TO1-GM00686-13 from the National Institute ofGeneral Medical Sciences.We are grateful to R. Whittenbury for his confirmation of

the facultative nature of the isolate XX and for his many

helpful comments.

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