J. B. DAVIS, H. H. CHASE, AND R. L. RAYMOND

SUMMARYA study was made of the factors affecting a routine

novobiocin cylinder-plate assay. Combining the ad-dition of 0.25 per cent glucose to the basic assay me-dium, the use of pH 6.0 phosphate buffer 10 per centand 7.5 ml-medium volume resulted in a 6-fold increasein assay sensitivity and good zone clarity.

REFERENCESCHRISTIANSEN, E. H. AND ZYGMUNT, W. A. 1952 A cylinder

dispenser for use in agar diffusion assays. J. Am. Pharm.Assoc., Sci. Ed., 41, 131-132.

DYE, W. E., LYNCH, H. P., CULLEN, T. M., AND SPRAGUE, E. M.1952 Use of synergism to increase the sensitivity of amicrobiological method for the assay of viomycin. Anti-biotics & Chemotherapy, 2, 610-614.

PINZEL1K, J., NISONGER, L. L., AND MURRAY, F. J. 1953Some variables in the assay of bacitracin. Appl. Micro-biol., 1, 293-296.

REILLY, H. C. AND SOBERS, H. 0. 1952 The use of plain agaras a base layer in the paper disc assay. Antibiotics &Chemotherapy, 2, 469-471.

WALLICK, H., HARRIS, D. A., REAGAN, M. A., RUGER, M., ANDWOODRUFF, H. B. 1956 In: Antibiotics Annual 1955-1956. Medical Encyclopedia, Inc., New York, p. 909.

Mycobacterium paraffinicum n. sp., a Bacterium Isolated from SoilJ. B. DAVIS, H. H. CHASE AND R. L. RAYMOND

Magnolia Petroleum Company, Field Research Laboratories, Dallas, Texas

Received for publication June 18, 1956

The utilization of hydrocarbons by bacteria wasdescribed by Stone et al. (1940, 1942) and Bushnelland Haas (1941). These workers presented their ownobservations and reviewed observations of previousworkers. The large variety of microorganisms capableof oxidizing hydrocarbons include the genera Methano-monas, Mycobacterium, Pseudomonas, and Coryne-bacterium, as well as other bacteria, certain actino-mycetes, and filamentous fungi. Sohngen (1906) isolatedfrom methane-enrichment culture a bacterium which hecalled Bacillus methanicus, a short motile rod, laterrenamed Methanomonas methanica by Orla-Jensen(1909). Tausz and Peter (1919) isolated with n-heptaneand cyclohexane enrichment, respectively, two soilbacteria which they named Bacterium aliphaticum andBacterium aliphaticum liquefaciens, both of which prob-ably belong to the genus Pseudomonas. These workersalso isolated with mineral oil enrichment a "Paraffinbacterium" described as a gram positive, sporeformingrod, not since classified. Corynebacteria were observedby Haag (1926), Jensen (1934), and Bushnell and Haas(1941) to utilize paraffin hydrocarbons, and S6hngen(1913), Buttner (1926), Haag (1927), Jensen (1934),and Bushnell and Haas (1941) observed the utilizationof paraffin by certain mycobacteria. Nechaeva (1949)described two methane-utilizing mycobacteria, Myco-bacterium flavum var. methanicum and Mycobacteriummethanicum n. sp. Both of these bacteria were reportedto utilize a wide range of complex organic media.Nechaeva pointed out that M. methanicum n. sp. alsoutilized propane as a sole carbon source and thatMycobacterium flavum var. methanicum utilized propaneand hexane. These were the only hydrocarbons beside

methane that he tested. Bokova (1954) reported ob-servations on two mycobacteria: Mycobacterium per-rugosum var. ethanicum which utilized ethane andhigher paraffinic gaseous hydrocarbons, but notmethane; and Mycobacterium rubrum var. propanicumwhich utilized propane and higher paraffinic gaseoushydrocarbons, but not methane or ethane. Both ofthese mycobacteria were reported to utilize a widerange of complex organic media. The observations ofNechaeva and of Bokova came to the attention of theauthors following the completion of work describedherein.

It is not our purpose to discuss the numerous observa-tions made regarding hydrocarbon assimilation bybacteria. For an extensive review see that of ZoBell(1950). In the present paper, a description is given of amycobacterium which utilizes for growth paraffinicgaseous hydrocarbons, with the exception of methane,and is further differentiated from common soil formssuch as Mycobacterium lacticola and Mycobacteriumphlei by its inability to utilize common bacteriologicalorganic media.

EXPERIMENTAL METHODS AND RESULTS

Isolation methods. Soil was obtained from below thegrass roots level. Ten-g aliquots of the soil were placedin 60-ml bottles, to which were added 10 ml of a mineral-salts solution of the following per cent composition:(NH4)2SO4, 0.1; Na2CO3, 0.01; KH2PO4, 0.05;MgSO4.7H20, 0.02; CaCl2, 0.001; FeSO147H20, 0.0005;and MnSO4, 0.0002. The bottles were fitted with capil-lary glass manometers (figure 1), and ethane was added

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FIG. 1. Manometer system, assembled and disassembled.a, 2-oz bottle; b, metal cap; c, neoprene gasket; d, capillarytubing manometer with flared end.

FIG. 2. Colonies of Mycobacterium paraffinicum n. sp. onmineral-salts agar plate. Sprinkled with soil, incubated under40 per cent ethane, 20 per cent oxygen, and 40 per cent nitrogen.

through the manometers after evacuation of the bottles.The ethane (99.9 mol per cent)' was used ordinarily ina concentration of 40 per cent mixed with 20 per centoxygen and 40 per cent nitrogen. Mercury was used asthe manometer fluid.Where growth of the bacteria occurred, usually after

10 to 20 days of incubation at 30 C, gas uptake resultedwith the manometer registering negative pressure withinthe bottles. Negative pressure gradually reached a maxi-mum of about 150 mm of mercury as the result ofoxygen depletion. A pellicle formed over the surfaceof the supernatant mineral-salts medium concurrentlywith progressive gas uptake. This pellicle, at firstwhite, later became yellow and flaky in appearance. A

'Research Grade ethane, purchased from Phillips Petro-leum Company, Bartlesville, Oklahoma.

FIG. 3. Stock slants with copious growth of the purifiedbacterial cultures upon mineral-salts agar in a gas mixturesimilar to that used in isolation.

loop of the pellicle was streaked upon plates of the min-eral salts medium containing 1.5 per cent washed agar.Colonies which developed were typical of mycobacteria,being waxy, wrinkled, and yellow pigmented.An even simpler means of isolation was accomplished

bv directly applying air-dried, powdered soil upon a.mineral-salts agar plate. After incubation of the platefor about 20 days in a desiccator containing the ethanegas mixture, colonies of the bacteria ranging in diameterfrom 1 to 5 mm were observed upon the soil and agarsurfaces (figure 2). These colonies had a similar appear-ance to the colonies which developed from the pelliclegrowth previously described. Purification of the colonieswas accomplished in either case, ordinarily after 2 or 3transfers. Stock slants with copious growth of thepurified bacterial cultures upon mineral-salts agarmay be maintained in screw-cap tubes (figure 3).These were prepared by inoculating the slants andincubating the open tubes in desiccators for 15 days in agas mixture similar to that used in isolation.No growth of the bacterium has been obtained in gas

uptake systems with methane (at least 99 mol per cent)serving as the carbon source in a 40 per cent concentra-tion in air. Ethane-grown bacterial cells were tested inrespirometer experiments for their ability to oxidizemethane. The methane used contained 0.5 per centethane as an impurity. Forty per cent methane in airwas used. Oxidation occurred, but subsequent experi-ments using (C14) radiomethane and (C14) radioethaneshowed that the apparent oxidation of methane (see

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200200

2 3 4TIME, HOURS

FIG. 4. Oxidation of gaseous paraffinic hydrocarbons by cellsuspensions of ethane-grown Mycobacterium paraffinicumn. sp. All the hydrocarbons were used at a 40 per cent concen-tration in air.

FIG. 6. Electron photomicrograph of Mycobacterium paraf-finicum n. sp.

FIG. 5. Mycobacterium paraifinicum n. sp., phase contrastphotomicrograph.

figure 4) was actually due to oxidation of the ethaneimpurity. A bacterial-cell suspension was incubatedwith radiomethane and the carbon dioxide producedwas recovered as barium carbonate which had a specificactivity of only 4 cpm. The use of radioethane (of thesame activity as the radiomethane) resulted in a bariumcarbonate specific activity of 234 cpm.Ethane-grown bacterial cells readily oxidize propane

(99.99 mol per cent) and n-butane (99.78 mol per cent),both of which are ethane free (figure 4), and likewiseutilize these gaseous hydrocarbons for growth. In thegrowth experiments each of these gases was usedarbitrarily in a 40 per cent concentration in air.

Description of the bacterium. The bacterium is a

slender rod about 0.5 to 0.7 ,u wide and 3 to 7 A long(see figure 5). On microscopic examination of the bac-terial growth, the cells have a characteristic tendency

to appear in cords or show what could be termed alog-jam arrangement. This is particularly pronouncedin pellicular growth taken from liquid mineral saltsmedium. Under phase contrast examination, the cellsare observed to have granules, ordinarily one at eachterminal. These are particularly pronounced in anelectron photomicrograph (figure 6). The bacterium isnonmotile, gram positive, acid-fast, and forms yellow,waxy, wrinkled colonies on mineral salts agar medium.These colonies are similar in appearance to those ofM. phlei. A distinctive characteristic which separatesthe bacterium from M. phlei is the inability of theformer to grow on bacteriological media such as pep-tone, yeast extract, or glucose. Attempts to adapt theorganism to grow on these media have failed, althoughthe bacterium will grow readily upon these media inthe presence of ethane. Transfers of growth from thesemedia to fresh media in the absence of the hydrocarbonconsistently results in no growth of the bacterium. Thebacterium will grow in the presence of n-pentane, n-hexane, n-heptane, n-octane, and n-decane, and possiblymany other paraffinic hydrocarbons.

It is proposed that the bacterium herein describedbe given the name Mycobacterium paraffinicum. Thisproposal is based upon the following three observations:(1) the morphological, staining, and cultural charac-teristics of the bacterium place it in the genus Myco-bacterium; (2) the bacterium can be distinguished

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MYCOBACTERIUM PARAFFINICUM N. SP.

readily from other known mycobacteria by its inabilityto utilize common bacteriological media; and (3) thebacterium utilizes paraffinic hydrocarbons (other thanmethane).

Physiologic characteristics. Glucose, peptone, yeastextract, glycerol, ethanol, acetate, and acetaldehydeare among the nonhydrocarbon carbon sources used inattempts to grow M. paraffinicum. Only ethanol andacetate supported the growth of the bacterium. Growthupon these substrates was meager, but sufficient for theobtention of cells for respirometer experiments.

Stanier (1947) pointed out that bacteria grown upona particular substrate develop enzymes capable ofoxidizing compounds intermediate between the par-ticular substrate and the final products. Their abilityor inability to oxidize certain hypothetical intermediatesmay thus serve as criteria of whether or not the testedcompounds are true intermediates. Incorporated in thisphilosophy is the theory that bacterial growth on aparticular intermediate will result in bacterial enzymesadapted to oxidize intermediates principally in thedirection of the more oxidized intermediates orproducts, and not in the reverse direction. Bacteriaordinarily do not develop enzymes capable of oxidizingintermediates that occur in the biochemical chain pre-ceding the intermediate (substrate) upon which thebacteria are grown.A study of the oxidation of ethane and its hypo-

thetical oxidative intermediates has been approachedwith the above considerations in mind. M. paraffinicum,grown on mineral-salts agar with ethane as the onlycarbon source, was tested for its ability to oxidizeethane, ethylene, ethanol, acetaldehyde, and acetate.The cells were harvested, suspended in 0.85 per centsodium chloride solution, centrifuged, and the bacterialcells resuspended in the saline solution with 0.1 percent phosphate buffer added. The suspensions wererecentrifuged and the cells resuspended in the abovesolution. The final suspension contained about 20 mg(dry wt) of bacterial cells per ml. The above procedurewas followed in subsequent experiments. Ethane,ethylene, ethanol, acetaldehyde, and acetate wereoxidized by these suspensions (see figure 7). The oxida-tion of the above hypothetical intermediates of ethaneoxidation by M. paraffinicum indicates only that thesecompounds could be true intermediates. It does not,of course, prove that they are intermediates. A possiblebiochemical route of ethane oxidation in its simplestform would be:

CH3-CH3 -*CH2-=CH2 -+CH3CH20H>CH3CHO -÷ CH3COOH -+ bacterial cells,

C02 and H20

Experiments were performed in which M.paraffinicum was grown upon 1 per cent ethanol and 1per cent potassium acetate, respectively, and cell

O/

soox0

50~~~~~~~~~

50 V~~~~~~~~

o 0 3~

o ~~~~~0

I0 20 30 40TIME, MINUTES

FIG. 7. Oxidation of ethane and its hypothetical oxidativeintermediates by ethane-grown cells of Mycobacterium paraf-finicum n. sp. Ethane, 20 per cent; ethylene, 1 per cent inair; all others 0.1 mM.

suspensions tested for their ability to oxidize ethane.2In these experiments, another bacterium, isolate A,was also employed. Isolate A, isolated from soil usingethane-enrichment culture, is a rod-shaped, acid-fast,yellow-pigmented, nonsporeforming bacterium whichreadily utilizes common bacteriologic media such aspeptone, yeast extract, and glucose, in addition toethane. This bacterium was grown upon ethanol andacetate, respectively, and cell suspensions were testedfor their ability to oxidize ethane, alongside M.paraftinicum. M. paraffinicum cells, grown on ethanol,readily oxidized ethane, whereas acetate-grown cellsdid not (see figures 8 and 9). Both ethanol-grown andacetate-grown isolate A cells failed to oxidize ethane.The point of most interest is that cells of M.paraffinicum, when grown upon the hypothetical inter-mediate, ethanol, still retain the enzymic mechanismfor oxidizing ethane, which, of course, precedes ethanolin the oxidative chain. This observation points up theconstitutiveness of the ethane-oxidizing enzymes inM. paraffinicum as against the lack of this constitutiveproperty in isolate A.

Carbon balance. Attempts have been made to detectintermediates in ethane-growth cultures of M.paraffinicum. While traces of ethylene and an un-identified alcohol have been indicated, the onlydefinitely detectable products of ethane oxidation arebacterial cells and carbon dioxide. The equation forcomplete oxidation of ethane is as follows:

C2H6 + 3.502 -- 2CO2 + 3H20 (1)2 No growth of the bacteria occurred with ethylene or, as

mentioned above, acetaldehyde.

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J. B. DAVIS, H. H. CHASE, AND R. L. RAYMOND

100

y4

a.

n

(I)

4D

I~-

0

50

IKt~~~4.1~

4,00 t

V_I It0 20 30

TIME, MINUTES40

FIG. 8. Oxidation of ethane by ethanol-grown cells of Myco-bacterium paraffinicum n. sp. Ethane, 20 per cent in air; ethanol0.1 mM.

100

o/101

M 50

0~~~~

Io 20 30 40TIME, MINUTES

FIG. 9. No oxidation of ethane by acetate-grown cells ofMycobacterium paraffinicum n. sp. Ethane, 20 per cent in air;sodium acetate, 0.1 mM.

However, during growth about one-half of the carbonis incorporated into cell material, thus:

C2H6 + 2.502 -> (CH20) + C02 + 2H20 (2)

bacterial cells

A respirometer determination of ethane and oxygenuptake, in the absence of growth of M. paraffinicum,shows that equation (1) obtains. In growth culturesequation (2) obtains, with slight variation. Very littleof the oxidative intermediates between ethane and theabove-mentioned products accumulate, and presumablythe intermediates of ethane oxidation never get outsidethe bacterial cell before they are further metabolized.

DISCUSSION

The most interesting characteristic of M. paraffinicumn. sp. is its inability to assimilate methane and complexorganic media. The organism has been isolated fromnumerous soil samples from widely distributed oilfields. Currently, we have 19 purified isolates whichconform rigidly to the following characteristics: long,slender, strongly acid-fast rods showing Much's granuleswith Ziehl-Neelsen stain; yellow, waxy, wrinkledcolonies; no growth on nutrient-agar media, glycerol,or methane.

In contrast of this type of ethane utilizer, we haveisolated five other distinct ethane-utilizing micro-organisms on which we plan to report in detail in thefuture. These, tentatively, may be described as follows:(1) Two acid-fast rod-shaped bacteria distinguishedonly by pigmentation; both (yellow form, 12 isolates;white form, 10 isolates) form fairly smooth, softcolonies, grow readily on a wide range of organic media,but do not attack methane. (2) Nocardia (16 isolates),which form salmon or pink colonies, definitely are notacid-fast, grow readily on complex organic media, donot attack methane, and their mycelia fragment toirregularly shaped rods and coccoids after a few dayswith ethane media, or 12 hr on nutrient agar. Coloniesnever form aerial mycelia. (3) Streptomyces (only 2isolates on hand), which readily attack complex organicmedia and methane, form white, powdery colonies withcharacteristic odor of actinomycetes. (4) A nonseptate,sporogenous, filamentous fungus, which utilizes com-plex organic media but not methane, forms copiouswhite, aerial mycelia.M. paraffinicum n. sp. is distinguished from all the

other types by its inability to grow on nonhydro-carbon media (with the exception of ethanol and acetate-see above). The other types attack ethane only aftera lag period if the organisms are grown on nutrient agaror glucose media, for example. Ethanol-grown cells ofM. paraffinicum n. sp. attack ethane immediatelywhereas ethanol-grown cells of isolate A, a representa-tive of the 12 yellow, soft, bacterial cultures describedabove, do not.Of the 19 isolates classified as M. paraJnicum n. sp.

there is a degree of variation in the rapidity with whichpropane and n-butane are utilized for growth. Highernormal paraffinic hydrocarbons are attacked morereadily and consistently by the 19 isolates. The speciesterm "paraffinictim" is meant to connote the generalutilization of gaseous and liquid paraffinic hydro-carbons, although methane is not utilized. Whilemethane is a paraffinic hydrocarbon, its distinction ofnot being oxidized by the organism should not preventthe use of the term paraffinicum. Furthermore, the useof ethane as a selective substrate for isolation purposesshould not rule the selection of a species name, since

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1MYCOBACTERIUM PARAFFINICUM N. SP.

other paraffinic hydrocarbons are utilized by the iso-lates. The creation of a new species of mycobacteriawhich utilizes paraffinic hydrocarbons almost ex-clusively for growth appears warranted, since formerworkers have consistently reported that the paraffinichydrocarbon-utilizing mycobacteria with which theyworked grew on nutrient agar, glucose, glycerol, and/orother nonhydrocarbon media. Furthermore, besidesthe reports of Nechaeva (1949) and Bokova (1954), nomycobacteria capable of utilizing gaseous, paraffinichydrocarbons have been adequately described. It isworthwhile to discuss, briefly, two points pertinent toobservations concerning hydrocarbon-utilizing bacteria.Firstly, in the course of work with M. paraffinicum n. sp.we observed some growth of the bacterium uponmineral agar plates incubated with pure grade methane.3This methane contained about 0.5 per cent ethane.Using small amounts of radioactive (C14) methane andradioactive ethane alternately as supplements to thepure grade methane, it was determined that only ethanecarbon is assimilated-that is, the formed cell materialwas radioactive only when radioactive ethane was usedas the supplement. It is apparent that the bacteriagrew solely on the ethane present as an impurity in themethane. The purity of the hydrocarbon used is of theutmost importance in establishing whether or not acertain microorganism is capable of utilizing it. It ispreferable to use research grade hydrocarbons with apurity approaching, as much as possible, 100 mol percent.

Secondly, one must be careful in interpreting resultsbased upon testing the hydrocarbon-utilizing ability ofcolonies which develop upon nutrient-agar plates as aresult of streaked inoculum from hydrocarbon enrich-ment cultures. The colonies which develop and appearto be isolated may be contaminated with hydrocarbon-oxidizing bacterial cells which do not develop uponnutrient agar, and hence are not noticed. Therefore,when one tests the developed colony for its hydro-carbon-utilizing ability and reports positive results, itis often the unseen contaminant, and not the observedcolony on nutrient agar, that is responsible. This maylead further to the false conclusion that the hydro-carbon utilizer grows upon nutrient agar. Culturaltechnique in going from hydrocarbon-enrichment cul-tures to pure cultures of hydrocarbon-oxidizing bacteriais not always a simple, straightforward process, par-ticularly when complex nutrient media are introducedbefore complete isolation of the hydrocarbon-utilizingbacterium is effected.

ACKNOWLEDGMENTS

The authors are indebted to Mr. R. M. Squires fortesting the purity of certain gaseous hydrocarbons and

3 99 mol per cent methane, purchased from Phillips Petro-leum Company, Bartlesville, Oklahoma.

to Mr. J. P. Stanley for his assistance in the performanceof experiments.

SUMMARY

A mycobacterium of the proposed name Mycobac-terium paraffinicum n. sp. was isolated from the soilusing ethane-enrichment culture. This bacteriumutilizes ethane and other gaseous and liquid paraffinichydrocarbons for growth, but not methane. Complexorganic media are not utilized by the bacterium; ofthose tested the only nonhydrocarbon substrates uti-lized for growth were ethanol and acetate. Evidence ispresented relative to the constitutiveness of the hydro-carbon-oxidizing enzyme system of the bacterium.

REFERENCES

BOKOVA, Ek. N. 1954 Oxidation of ethane and propane bycertain species of mycobacteria. Mikrobiologiya, 23,No. 1, 15-21. English translation from Russian by Asso-ciated Technical Services, East Orange, N. J.

BUSHNELL, L. D. AND HAAS, H. F. 1941 The utilization ofcertain hydrocarbons by microorganisms. J. Bacteriol.,41, 653-673.

BUTTNER, H. 1926 Zur Kenntnis der Mykobacterien, Ins-besondere ihres quantitative Stoffwechsel auf Paraffin-nahrboden. Arch. Hyg. u. Bakteriol., 97, 12-27.

HAAG, F. E. 1926 Uber die Bedeutung von Doppelbindun-gen im Paraffin des Handels fur des Wachstum von Bak-terien. Arch. Hyg. u. Bakteriol., 97, 28-46.

HAAG, F. E. 1927 Die Saprophytischen Mykobakterien.Zentr. Bakt. Parasitenk., Abt. II, 71, 1-45.

JENSEN, H. L. 1934 Studies on saprophytic mycobacteriaand corynebacteria. Proc. Linnean. Soc. N. S. Wales,59, 19-61.

NECHAEVA, N. B. 1949 Two species of methane-oxidizingmycobacteria. Mikrobiologiya, 18, No. 4, 310-317. Eng-lish translation from Russian by Associated TechnicalServices, East Orange, N. J.

ORLA-JENSEN, S. 1909 Die Hauptlinien des natiirlichen Bak-terien systems. Zentr. Bakt. Parasitenk., Abt. II, 22,305-346.

SOHNGEN, N. L. 1906 tUber Bakterien, welche methan alsKohlenstoffnahrung Energiequelle gebrauchen. Zentr.Bakt. Parasitenk., Abt. II, 15, 513-517.

S6HNGEN, N. L. 1913 Benzin, Petroleum, Paraffinol undParaffin als Kohlenstoff und Energiequelle fur Mikroben.Zentr. Bakt. Parasitenk., Abt. II, 37, 595-609.

STANIER, R. Y. 1947 Simultaneous adaptation: a newtechnique for the study of metabolic pathways. J.Bacteriol., 54, 339-348.

STONE, R. W., FENSKE, M. R., AND WHITE, A. G. C. 1942Bacteria attacking petroleum and oil fractions. J.Bacteriol., 44, 169-178.

STONE, R. W., WHITE, A. G. C., AND FENSKE, M. R. 1940Microorganisms attacking petroleum and petroleumfractions. J. Bacteriol., 39, 91-92.

TAUSZ, J. AND PETER, M. 1919 Neue methode der Kohlen-wasserstoffanalyses mit Hilfe von Bakterien. Zentr.Bakt. Parasitenk., Abt. II, 49, 497-554.

ZoBELL, C. E. 1950 Assimilation of hydrocarbons by micro-organisms. Advances in Enzymol., 10, 443-486.

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