Effects of Arene-type Hydrocarbon Air Pollutants onBacillus megateriumr1' 2

WILLIAM D. WON AND JEROME F. THOMAS

Sanitary Engineering Research Laboratory, University of California, Berkeley, California

Received for publication November 13, 1961

ABSTRACTW/ON, WILLIAM D. (University of California, Berkeley),

AND JEROME F. THOMAS. Effects of arene-type hydrocarbonair pollutants on Bacillus megaterium. Appl. Microbiol.10:217-222.-Effects of certain common carcinogenic andnoncarcinogenic polycyclic aromatic hydrocarbon organicair pollutants on Bacillus megaterium cultures were noted.Depending on the medium used, either growth suppressionor induction of atypical cell forms was observed in cul-tures grown in the presence of a carcinogen. By contrast,no such alterations were apparent in cultures grown inmedia supplemented with a noncarcinogen. Both carcino-genic and noncarcinogenic hydrocarbons exerted anenhancing influence, of varying degree, on lipogenesis,glycolysis, and methylene blue reductase activity. Ahigher than normal level of these reactions, however, wasassociated with cultures exposed to a carcinogen. In addi-tion, infrared examination of lipids revealed unique spec-tral characteristics for materials extracted from carci-nogen-treated cultures. No difference was noted betweenmaterials derived from noncarcinogen-treated cultures andfrom control cultures.

Although a large number of condensed polycyclic aro-matic hydrocarbons and their derivatives have been sepa-rated from polluted atmospheres, only a small portion hasbeen identified and classified. Included in the latter groupare some of the well established, powerful chemical car-cinogens of which benz(a)pyrene is an example. Because ofthe very low concentrations at which these substancesgenerally occur, conventional laboratory and animal pro-cedures are inadequate for their characterization. Conse-quently, a more sensitive method was sought to investigatethe properties of some of these materials. The develop-ment of a microbiological assay system seemed to be areasonable approach to the problem. However, growthinhibition as well as growth stimulation and induction ofabnormal forms in microbial cultures resulting from theinfluence of chemical carcinogens have been reported inthe literature (Goldstein, 1937; Hopper and Clapp, 1939;

1 Presented in part before the American Society for Micro-biology, 61st Annual Meeting, Chicago, Ill., April 23 to 27, 1961.

2 The investigation is supported by research grant RG 7692from the National Institutes of Health, U. S. Public HealthService, Bethesda, Md.

Melroy and Spencer, 1940; Novelli, 1942; Miller, Kingsley,and Miller, 1947).

Interest in this subject was stimulated by the observa-tion that a variety of abnormal morphological forms ofseveral microorganisms occurred in cultures grown in twodifferent media containing a carcinogenic hydrocarbon.The present report is concerned with the growth, theintracellular reactions, and the biochemical alterations ofBacillus megaterium grown in the presence of hydrocarbons,including both the so-called noncarcinogens, phenanthrene,chrysene, and anthracene; and the established powerfulcarcinogens, benz(a)pyrene and 20-methylcholanthrene.

MATERIALS AND METHODS

B. megaterium, lysogenic strain 899, obtained from theDepartment of Bacteriology, University of California,Berkeley, was employed. Stock slants, subcultured at2-month intervals, were maintaied at 4 C on blood-agarbase (Difco).3Media used in the study were Difco heart infusion broth

(HIB) and a chemically defined medium (CD) of thefollowing composition (per liter): Na2HPO4 12H20,15.0 g; KH2PO4, 3.0 g; NaCl, 1.0 g; NH4Cl, 1.0 g; andMgSO4.7H20, 0.2 g. Media were dispensed in 200-ml quan-tities into 500-ml Erlenmeyer flasks. Sterilization waseffected by saturated steam at 15 psi for 15 min. Aftercooling, sterile glucose was added aseptically to give a

final concentration of 1.0 %.Media to be supplemented with a hydrocarbon were

prepared by adding 0.20 ml (40 ,ug) of stock solution of thehydrocarbon to each 200-ml portion of medium beforeautoclaving. Stock solutions of the arenes consisted of20 mg of reagent dissolved in 100 ml of methylene chlorideand were kept in amber bottles at room temperature.Cultures were prepared by inoculating media with 1.0 mlof a 24-hr HIB growth of B. megaterium. Incubation was

carried out at 35 C on a Brunswick4 gyratory shakeroperating at 100 cycles per min. At 24-hr intervals,samples were removed for viable determinations and formorphological and biochemical analyses.

Examination for morphological variations was accom-

plished by making standard smears at specified intervalsand staining them with Giemsa stain. Concomitantly, a

3 Difco Laboratories, Inc., Detroit, Mich.4Brunswick Scientific Company, New Brunswick, N. J.

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W. D. WON AND J. F. THOMAS

parallel series of smears was prepared for the examinationof internal structures according to the technique suggestedby Robinow (Dubos, 1949).

Lactic acid concentration was determined colorimetri-

loll I I I

~10' ~ a HIB

lb,0

6

l4

HOURS

FIG. 1. Growth of Bacillus megateriu?n in HIB and CD mediasupplemented with various polycyclic aromatic hydrocarbons.

cally at 0560 m,u on a Beckman5 model B spectrophotometeraccording to the method of Barker (Colowick and Kaplan1957).

Lipid materials were extracted from 250-ml aliquots ofcultures previously adjusted to pH 12.0 with saturatedNaOH, and subsequently concentrated to a syrupy con-sistency (about 20 ml) in a rotating vacuum evaporatorimmersed in a 40 C water bath. The residue was acidifiedwith 10 to 20 ml of 6 N HCl and refluxed at 60 C for60 min with 100 ml of methanol-diethyl ether (1:1); asecond reflux was carried out under similar conditionswith a 1:3 mixture of methanol-diethyl ether. A finalextraction was made with diethyl ether after an additional60 min of reflux. The extracts were combined and trans-ferred to a separatory funnel of adequate size. Sufficientvolume (150 ml) of distilled water was then introduced tofacilitate the separation of ether, methanol, and waterphases. The ether layer was collected and the process wasrepeated once with 50 ml and twice with 25 ml of distilledwater. The pooled ether fraction was returned to theseparatory funnel and washed once with 25 ml of distilledwater and once with 25 ml of saturated NaCl. Then it wasdried over anhydrous sodium sulfate and the solvent re-moved by distillation at 45 C. The extracts were dissolvedin CCl4 and samples were injected into a sodium chloridecell for infrared examination. The spectra were recordedautomatically by a Beckman model IR-5 double-beaminfrared spectrophotometer equipped with NaCl optics.An alternate technique was to smear and air dry a sam-

ple on the surface of KBr discs that were slightly tilted soas to produce wedgeshaped films of variable thicknessSuch a procedure permitted structural analysis with verysmall samples, and the variable thickness of the film per-mitted the regulation of the sample beam to give identical

I Beckman Instruments, Inc., Fullerton, Calif.

FIG. 2. Cells of Bacillus megaterium, 24-hr cultures grown in HIB medium. Stained by Rubinow's method; approximately X 1,000. A)normal cells; B) atypical large, granutlated cells induced by benz(a)pyrene; C) abnormal cells induced by 20-methylcholanthrene surroundedby unaffected cells.

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ARENE-TYPE HYDROCARBON AIR POLLUTANTS

transmission with a common band absorption at a specifiedwavelength.

For the methylene blue reduction test, the cultures were

washed twice with normal saline. The cells were resus-

pended in saline and standardized to approximately1.0 X 1010 viable cells per ml as determined by optical

density on a Beckman model B spectrophotometer at

640 m,u. Nine-milliliter aliquots of cell suspension were

then transferred to Pyrex tubes (150 by 8 mm), and 1.0 ml

of 1: 20,000 methylene blue solution was added. The tubeswere closed with rubber stoppers, mixed gently, andincubated at 37 C. Complete reduction of the dye as deter-mined visually was taken as the end point.

RESULTS

Growth and morphology. Quantitative data on the growthof B. megaterium in HIB and CD media containing eithera carcinogenic or a noncarcinogenic polycyclic aromatichydrocarbon are summarized in Fig. 1. Viability was deter-mined on triplicate plates of blood agar base medium(Difco). This figure shows that, with respect to the con-

trol culture, there was no alteration in the growth patternof the organism grown in HIB supplemented with a

hydrocarbon. Inhibition of growth was evidenced earlyin the CD cultures grown in the presence of a carcino-genic hydrocarbon. The inhibitory effect disappearedabruptly, however, and was not discernible at the 24-hror other assay periods. Thus, with the exception of thetemporary inhibition in the CD medium, the growth pat-terns appeared virtually normal.Another interesting feature observed in these experi-

ments was the appearance of atypical cells in culturesgrown in HIB supplemented with a carcinogen. Mostreadily detectable, among a considerable proportion ofapparently normal cells, were giant cells containingnumerous vacuoles as well as giant cells characterized byextensive granulations (Fig. 2B, 2C). Other variationsoccasionally observed were cells showing reticular cyto-plasm and cells exhibiting a ballooning of the cytoplasm.However, these changes were not found in smears of sub-

cultures made in carcinogen-free HIB media. Similarly,there was no evidence of cytological changes in cultures

grown in media supplemented with a noncarcinogen. The

cytological characteristics of such cultures appeared to be

as normal as those of the control cultures (Fig. 2A).Occasionally, however, atypical cytological and morpho-

TABLE 1. Effects of arenes on methylene blue reductase activity ofBacillus megaterium of various age at 37 C

Reduction time (min)Supplement

24 hr 48 hr 72 hr

None (control) ....................... 50 9065

Phenanthrene ........................ 40 4540

Anthracene ........................... 35 45 40

Benz (a)pyrene ........................ 25 40 35

Methylcholanthrene .................. 20 35 25

logical cells appeared in cultures grown in the presence ofthe so-called noncarcinogenic anthracene.

Methylene blue reduction. The effects of growing B. mega-terium in the presence of carcinogenic and noncarcinogenicpolycyclic aromatic hydrocarbons on the rate of methyleneblue reductions are indicated by the data listed in Table 1.In general, cells harvested from cultures grown in mediasupplemented with a hydrocarbon uniformly manifestedan accelerated reduction rate. Moreover, quantitativedifferences in the reduction reactions were demonstrable.As is shown in Table 1, reduction time for the dye wasconsiderably lower for cells exposed to a carcinogen thanfor those in contact with a noncarcinogen.

Correspondingly, the reduction rate for cells obtainedfrom preparations containing a noncarcinogenic hydro-carbon was more rapid than that of the controls.

Lipid and lactate levels. Only the CD culture mediumwas used in the study of the influence of carcinogenic andnoncarcinogenic hydrocarbons on the lipid and lactateproductions of the organism. Data obtained from thisstudy are presented in Table 2. According to the results,the average of the results of three experiments, the yieldof lipids is greater from cultures grown in media containingcarcinogens than from those containing noncarcinogenichydrocarbons. In turn, the yield from cultures grownin the presence of noncarcinogenic hydrocarbons wasgreater than that from the controls.On infrared examination, these lipid materials showed

absorption characteristics that might be of diagnosticvalue. Examples of such characteristics are illustrated bythe series of typical spectra presented in Fig. 3. As isshown in the figure, curve C, representing the spectrum oflipids derived from cultures grown in the presence of thepowerful carcinogen 20-methylcholanthrene, differs instructure significantly from curves A and B. It has pro-nounced absorption peaks indicated at approximately5.1 A (1,950 cm-l), at 7.7,u (1,300 cm-'), and at 10.6,(942 cm-l) that are not demonstrable in the curves forlipids obtained from the culture extracts of growths grownin the presence of phenanthrene (noncarcinogen) andfrom those of the controls. The spectra of the latter twowere remarkably similar.

Quantitative differences in lactate production amongthe cultures were suggested by the variation in intensityof the characteristic odor of lactic acid. The results of the

TABLE 2. Effects of arenes on lactate formation and lipid synthesis byBacillus megaterium grown in defined medium

Total lipid (mg/loo ml) Lactic acid (Ag/ml)Supplement

24 hr 48 hr 72 hr 24 hr 48 hr 72 hr

None (control).7.3 17.4 19.3 15.0 32.0 60.0Chrysene ............... 8.0 21.0 23.6 17.5 31.0 67.0Phenanthrene ........... 8.6 20.3 25.0 16.0 32.6 64.0Anthracene ............. 9.7 23.3 26.2 15.8 34.0 66.0Benz(a)pyrene .......... 10.1 31.0 29.4 16.6 39.0 73.0

Methylcholanthrene . ... 10.5 26.0 27.8 16.4 38.0 70.0

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Z80B

z 40

j20

80C

60

40

20

C2~~~~ ~ WAELEGT (M1ICRONS)'.FREQUENCY (CM-)

40

20

0

p

w 8

S 60

Z240

j20

40

80

60

2 3 4 '5 6 7 8a 9 10 1~I 2 I1iI4 15 6WAVELENGTH (MICRONS)

FIG. 3. Infrared absorption spectra of lipid preparations of 24-hr (top) and of 48-hr (bottom) defined medium cultures of Bacillus mega-terium. A) control; B) phenanthrene added; C) 20-methylcholanthrene added.

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experiments on lactate production again showed a familiarpattern of differential enhancing influence exerted by thehydrocarbons. The level of enhancing activity character-istically was highest in cultures supplemented with acarcinogenic-type hydrocarbon, whereas that in culturesconitaining noncarcinogenic hydrocarbons was higher thanthat of the controls.

DiscussIONWith respect to the contradictions and inconsistencies

found in earlier studies concerning the effects of carci-dogenic hydrocarbons on bacterial growth and morphology,Brown (1951) attributes such discrepancies to the use ofunstable suspensions or of solutions of uncertain conceni-tration. In his investigation, he prepared and employedwater-soluble derivatives of hydrocarbons. He observedthat the carcinogenic derivatives, although weaker incarcinogenicity than the parent compounds, were decidedlygrowth inhibitory, whereas a noncarcinogenic derivativewas without effect; and that marked inhibition in onemedium may be consistently absent in another.The demonstration in the present study of a temporary

growth suppression in a mineral medium containing acarcinogen, but not in the carcinogen-containing heartinfusion broth (HIB) medium, was in agreement withBrown's findings. Similarly, the induction of abnormalcytological and morphological forms in cultures exposedto carcinogens was consistent with previous observationsmade in mold (Reese and Reese, 1945), yeast (Dodge andDodge, 1937), and paramecium cultures (Mottram, 1940).However, in contrast to the stable "polyploidal giants"of Pasteurella pestis (Won, 1950) and Escherichia coli(Ogg and Zelle, 19,57) produced by exposure to the oxy-genated derivatives of the terpene-type hydrocarbon(camphor), the atypical B. megaterium cells were capableof reverting readily to normal appearance when subcul-tured in carcinogen-free media.

It is disconcerting to note, however, that at no time dida cell population of the affected cultures appear to be com-prised homogeneously of atypical or abnormal forms. Per-haps these seemingly normal or "unaltered" cells mayhave had, or have developed, resistance to the carcinogens;or there may have been a very low "mutation" rate inthese cultures.

Results of experiments involving determination of meth-ylene blue reductase activity, a qualitative and anempirical method of measurement, indicate that anincreased level of enzyme activity occurred in culturesgrown in the presence of a polycyclic aromatic hydrocar-bon. It is particularly noteworthv that although the over-all reaction appeared to be nonspecific with respect tocarcinogenicity or noncarcinogenicity, the quantitativedifferences in the reduction rate varied according to thedegree of carcinogenic potency of the compound used.A similar pattern of differential, quantitative enhance-

ment was also found to be true with respect to lipid syn-

thesis and lactic formation by the organism. Higher yieldsof either lipid or lactic compounds were obtained fromculture materials initially supplemented with a carcino-gen. Similarly, higher yields were obtained from culturematerials exposed to a noncarcinogen than were obtainedfrom the controls. Apparently, the over-all enhancinginfluence is a characteristic common to the various hydro-carbons studied. M\iost notable in this connection, however,was the association of higher levels of enhancing activitieswith the carcinogenic hydrocarbons.

Preliminary evidence obtained in the present studyindicates a parallelism to a phenomenon that Goerner(1938) observed in rabbits injected with a carcinogen. Henoted a remarkable increase of total lipids in the hepaticmitochondria of the animals after they were injected witha dose of 1,2,5, 6-dibenzanthracene. Similarly, Boyd,MIcEwen, and Mlurdock (19-6) observed a significantincrease in levels of neutral fat, total lipids, and totalfatty acids in the malignant spleen of rats.

Of additional interest was the demonstration of uniqueinfrared absorption characteristics in the lipid materialsextracted from cultures grown in the presence of a car-cinogen. Although the results indicate a feasible micro-biological procedure in classifying certain air pollutants,the data cannot be considered conclusive. Further investi-gation is needed to determine the extent of the implica-tions involved in the occasional induction of atypical cellsin cultures supplemented with anthracene, a noncarci-nogen. The relationship between analogues of the hydro-carbons used in the study and their effects on cell activitiesshould also receive attention.

ACKNOWLEDGMENT

The technical assistance of Hugh Hildebrant is grate-fully acknowledged.

LITERATURE CITED

BOYD, E. M., D. MCEWEN, AND M. E. MURDOCK. 1956. Usualhydrolipotropic shifts in the spleen of albino rats bearingWalker carcinosarcoma. J. Natl. Cancer Inst. 16:913-925.

BROWN, A. 1951. Action of carcinogens on bacteria. J. InfectiousDiseases 89:59-75.

COILOWICK, S. P., AND N. 0. KAPLAN (Editors). 1957. Methods inenzymology, p. 241-246. vol. 3. Academic Press, Inc., NewYork.

DODGE, C. W., AND B. S. DODGE. 1937. Some effects of methyl-cholanthrene on morphological growth of yeasts. Ann. Mis-souri Botan. Garden 24:583-590.

DUBOS, R. J. 1949. The bacterial cell, p. 365. Harvard UniversityPress, Cambridge, Mass.

GOERNER, A. 1938. Effect of dibenzanthracene on vitamin A andtotal lipid of mitochondria. J. Biol. Chem. 122:529-537.

GOLDSTEIN, S. 1937. A microbiological test for carcinogenic hydro-carbons. Science 86:176-177.

HOPPER, S. H., AND D. B. CLAPP. 1939. Effect of carcinogenic andother hydrocarbons on growth of Escherichia comnmnunior. J.Bacteriol. 38:13-23.

MEIROY, M. B., AND R. R. SPENCER. 1940. Effect of carcinogens on

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small free living organisms. I. Eberthella typhi. J. Natl. Can-cer Inst. 1:129-134.

MILLER, E. C., H. N. KINGSLEY, AND J. A. MILLER. 1947. Theinhibition of growth of Lactobacillus casei by p-monomethyl-aminoazobenzene and its reversal by riboflavin. Cancer Re-search 7:730.

MOTTRAM, J. C. 1940. 3,4-benzpyrene, Paramecium and the pro-duction of tumors. Nature 145:184-185.

NOVELLI, G. D. 1942. The effect of certain carcinogenic hydro-

carbons on the growth rate of Escherichia coli and Staphylo-coccus aureus. J. Bacteriol. 44:394.

OGG, J. E., AND M. R. ZELLE. 1957. Isolation and characterizationof a large cell possibly polyploid strain of Escherichia coli.J. Bacteriol. 74:477-484.

REESE, E., AND L. REESE. 1945. The effects of carcinogens ongrowth of fungus hyphae. Growth 9:177-187.

WON, W. D. 1950. The production of giant cells of Pasteurella pestisby treatment with camphor. J. Bacteriol. 60:102-104.

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