Bacterial Growth Kinetics on Diphenylmethane andNaphthalene-Heptamethylnonane Mixtures
RICHARD S. WODZINSKI* AND DAVID LAROCCABiology Department, Ithaca College, Ithaca, New York 14850
Received for publication 8 September 1976
Experiments were carried out to determine ifdiphenylmethane is utilized by aspecies ofPseudomonas (Hydrogenomonas) in the dissolved state regardless ofthe physical state (liquid or solid) of the undissolved diphenylmethane sus-pended in the medium. Bacterial growth rates in the presence of variousamounts of solid or liquid diphenylmethane indicate that liquid diphenylme-thane is utilized at the aqueous-diphenylmethane interface but that solid di-phenylmethane is not. A Pseudomonas sp. that was isolated on naphthalene(solid), but could not utilize heptamethylnonane, was grown in the presence ofvarious amounts of a naphthalene-heptamethylnonane mixture (liquid). Thegrowth rates indicate that the bacterium could utilize naphthalene at theaqueous-hydrocarbon interface, which is not the case in the absence of theheptamethylnonane.
For bacteria able to utilize naphthalene,phenanthrene, and anthracene, there is an ap-parent relationship between the water solubil-ity of the aromatic hydrocarbon and the bacte-rial growth rate (8). The bacteria studied grewfaster on the more soluble hydrocarbons. Theseresults suggest that the bacteria utilize the aro-matic hydrocarbons in the dissolved state. Ifthis hypothesis is true, insoluble aromatic hy-drocarbons would not be degraded rapidly bybacteria because the substrate would be lessavailable to the cells due to its low concentra-tion in the medium.
If the solubility of the aromatic hydrocarbondirectly influences the rate of growth on thearomatic hydrocarbon, the bacteria must uti-lize the dissolved hydrocarbon rather than uti-lize directly the hydrocarbon by interactingwith solid particles suspended in the aqueousmedium. Soil pseudomonads can absorb naph-thalene and phenanthrene from aqueous solu-tions saturated with these hydrocarbons (5),and there is direct evidence that some bacteriautilize naphthalene, bibenzyl (1,2-diphenyle-thane), and phenanthrene in the dissolved statefor growth (6, 7).This investigation was designed to determine
if the hypothesis that aromatic hydrocarbonsare utilized in the dissolved state could be ex-tended to include a liquid aromatic hydrocar-bon (diphenylmethane) and a solid aromatichydrocarbon dissolved in a liquid hydrocarbon(naphthalene dissolved in 2,2,4,4,6,8,8-hepta-methylnonane). Diphenylmethane was chosenfor study because it melts at 24°C. Thus, the
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growth of bacteria could be studied in the pres-ence of liquid or solid diphenylmethane byvarying the temperature of the system. Hepta-methylnonane was chosen because it was notoxidized by bacteria used in this study.
MATERIALS AND METHODSOrganisms. The organism used in the studies of
diphenylmethane was a Pseudomonas sp. (Hydro-genomonas) (3) obtained from Martin Alexander ofCornell University. The organism used in the stud-ies of napthalene was a Pseudomonas sp. describedpreviously (6, 8).
Media. A buffered mineral salts (BMS) solution(8) at pH 7.0 was the base of all media. BMS solutioncontaining dissolved diphenylmethane was pre-pared by adding aseptically 0.5 g of sterile diphenyl-methane to 1 liter of sterile BMS solution. The mix-ture was allowed to stand in a 3-liter flask for aminimum of 1 week at 20°C to become saturatedwith air. Since diphenylmethane has a meltingpoint of 24°C, the diphenylmethane is a solid at20°C. Before the medium was used, solid diphenyl-methane was removed aseptically at 20°C by filter-ing the medium through Schleicher and Schuell no.588 filter paper. If the BMS solution containing dis-solved diphenylmethane was to be used at 30°C, thefiltered solution was placed in a sealed flask andkept in an incubator at 30°C until the mediumreached 300C. Medium containing excess liquid orsolid diphenylmethane was prepared by adding theproper amount of liquid diphenylmethane to BMSsolution that had been saturated with diphenylme-thane at 20°C. When medium containing liquid di-phenylmethane was used, the medium was kept at30°C. When medium containing solid diphenylme-thane was used, the diphenylmethane was solidifiedwhile the medium was agitated at 20°C. To prepare
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medium saturated with diphenylmethane in whichthe oxygen concentration was one-half air satura-tion, 0.5 g of sterile diphenylmethane was asepti-cally added to 1 liter of sterile BMS solution in a 2-liter flask. After the solution was in an incubator at200C for 24 h, it was sparged at 200C with a gasmixture of 50% air and 50% nitrogen for 30 min at arate of 500 ml/min. The gas mixture was sterilizedby passing it through a glass wool filter. The flaskwas sealed and placed in an incubator at 200C for atleast 1 week prior to use. This solution was thenfiltered directly into a fermenter by use of a continu-ous-pipetting syringe fitted with a Swinny filter.While the fermenter was being filled, the air spacewas gassed with a gas mixture containing 50% airand 50% nitrogen.The solubility of diphenylmethane at 20°C was
determined to be 0.065 g/liter.Naphthalene-saturated medium was prepared by
adding 1.5 g of naphthalene to 1 liter of BMS solu-tion. The solution was autoclaved and allowed tostand for at least 1 week at 30°C. Before the mediumwas used, solid naphthalene was removed by filter-ing it through Schleicher and Schuell no. 588 filterpaper. Two media that contained heptamethylnon-ane, a hydrocarbon not degraded by the bacteria,were used. One medium was a filtered, saturatednaphthalene-BMS solution with added heptame-thylnonane. The other medium was prepared byfirst dissolving 0.015 g of sterile naphthalene in eachmilliliter of sterile heptamethylnonane and thenadding this mixture to sterile BMS solution.
Inocula. The inoculum for experiments with di-phenylmethane at 200C was grown in 500-ml Erlen-meyer flasks containing 80 ml of BMS solution and0.5 g of diphenylmethane. After the cultures wereincubated for approximately 20 h on a shaker at300C, they were cooled to solidify the diphenylme-thane. Solid diphenylmethane was removed by fil-tration at 200C through Schleicher and Schuell no.588 filter paper. The filtered solution was diluted 20-fold with sterile water. The absorbancy at 620 nm(A620) of the diluted, filtered solution was approxi-mately 0.02. The inoculum for experiments at 300Cwith diphenylmethane was prepared by plating theorganism on BMS agar in inverted petri dishes. Thecarbon source was diphenylmethane vapor derivedfrom diphenylmethane placed on the inside cover. Adilute suspension of cells (A620 of 0.02) was preparedby mixing cells from the petri dish with sterile wa-ter.The inoculum for experiments with naphthalene
was grown in 500-ml Erlenmeyer flasks containing0.5 g of naphthalene in 100 ml of BMS solution.After the cultures were incubated at 300C for 18 to 24h with constant agitation, the medium containingthe inoculum was filtered through Schleicher andSchuell no. 588 filter paper to remove undissolvednaphthalene. The filtered solution was diluted 100-fold with sterile water. In all experiments 0.1 ml ofdiluted, filtered solution was used to inoculate thefermenters.
Fermenter. For experiments with diphenylme-thane, a 500-ml, gas-tight fermenter was con-structed from a 500-ml Erlenmeyer flask fitted with
BACTERIAL GROWTH KINETICS 661
a silicone rubber stopper. The silicone rubber stop-per held an oxygen probe (1) and an exit port, whichconsisted of 6-mm-ID glass tubing. After the fermen-ter was completely filled with medium, the siliconerubber stopper was inserted. After excess mediumleft the fermenter through the exit port, the portwas sealed with a cork covered with Teflon tape.Thus, there was no free gas space in the fermenter.For fermentations with diphenylmethane at 300C,agitation was provided by a Teflon-coated stirringbar (1 by 5 cm) rotating at 180 rpm. For fermenta-tions with diphenylmethane at 200C, agitation wasprovided by a glass-coated stirring bar (1 by 3 cm)rotating at 600 rpm. This degree of agitation dis-persed solid diphenylmethane into particles with adiameter in the range of 0.2 mm.
For experiments with naphthalene, the same fer-menter was used, but the internal end of the exit-port tube was bent upward to prevent droplets ofheptamethylnonane from accumulating in the exitport. The heptamethylnonane was added to the fer-menter via the exit port with a syringe and needlefitted with a piece of 0.025-inch (ca. 0.06 cm)-IDSilastic brand medical grade tubing. For all growthexperiments with naphthalene, the fermenters wereincubated at 300C with agitation provided by a mag-netic stirring bar (1 by 5 cm) rotating at 240 rpm.Measurement of dissolved oxygen. An oxygen
probe (1) was used to measure changes in dissolvedoxygen during fermentations. The oxygen probecurrent was observed on a 10-mV recorder as a po-tential across an external resistance. With air-satu-rated medium and the conditions employed, the po-tential across an external resistance of less than1,500 ohms is proportional to the dissolved oxygenconcentration (6). For experiments done at 200Cwith medium that had an initial oxygen concentra-tion of one-half air saturation, external resistancesofup to 2,100 ohms were used. By methods describedpreviously (6), the potential across the higher exter-nal resistances was found to be proportional to dis-solved oxygen concentration.
Generation times were calculated from thestraight line obtained by plotting the logarithm ofthe percentage of decrease in dissolved oxygen con-centration versus time.
Distribution of naphthalene in media containingheptamethylnonane. After heptamethylnonane wasadded to naphthalene-BMS medium, the distribu-tion of naphthalene between the aqueous BMSphase and the nonpolar heptamethylnonane phasewas determined. Heptamethylnonane was added to500-ml Erlenmeyer flasks that were filled withnaphthalene-BMS solution and to flasks containingBMS solution. All solutions were equilibrated insealed flasks by stirring for at least 3 h with amagnetic stirring bar (1 by 5 cm) rotating at 240rpm. Samples of approximately 35 ml were takenfrom each flask and centrifuged in capped tubes at10,000 x g and 280C for 10 min. The denser aqueousphase was removed by puncturing the bottom of thecentrifuge tubes and collecting the aqueous samplesin test tubes, which were then immediately sealedwith foil-covered stoppers. The A275 of the aqueousphase was measured to determine the concentration
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of naphthalene in that phase. The approximate ab-sorbance for naphthalene in the heptamethylnonanephase was calculated from the difference betweenthe initial (before adding heptamethylnonane) andfinal (after adding heptamethylnonane) absorb-ances in the aqueous phase. It was assumed that theextinction coefficient of naphthalene is the same inwater and heptamethylnonane.A second group of experiments was done to deter-
mine the distribution of naphthalene when hepta-methylnonane containing 0.015 g ofnaphthalene perml was added to flasks containing 500 ml of BMSsolution. The solubility of naphthalene in water is0.030 g/liter (4). The absorbance of naphthalene inthe aqueous phase was determined by the sameprocedure described for the previous experiments,and the approximate absorbance of naphthalene inthe heptamethylnonane phase was calculated.
Detection of undissolved hydrocarbon in BMSsolution saturated with hydrocarbon. When mediumwas made with dissolved diphenylmethane as thesole carbon source, it was essential to demonstratethe absence of undissolved diphenylmethane. Thiswas done by determining whether or not dilutions ofthe diphenylmethane-BMS medium followed Beer'slaw at 258 nm (Xmax). The absorbance of the BMSsolution saturated with diphenylmethane and di-luted to one-half strength with BMS solution was49.7% that of the BMS solution saturated with di-phenylmethane. The results obtained are the resultsexpected if no solid, undissolved diphenylmethanewas present in the saturated solutions. It was deter-mined previously that the naphthalene-BMS solu-tions were free of solid, undissolved naphthalene (6).
RESULTS
Rate of growth of the Pseudomonas cultureon diphenylmethane at 30°C. In fermentationsin the presence of 0.5 g and 0.05 g of liquiddiphenylmethane per liter, a lack of dissolvedoxygen stopped growth at the end of the experi-ments, and at least 70% of the observed oxygenutilization occurred when the cells were grow-ing exponentially. In fermentations with onlydissolved diphenylmethane as the carbonsource, approximately 30% of the available dis-solved oxygen was used when exponentialgrowth ended. After exponential growth ended,oxygen was used at a nonexponential rate untilit was depleted. There are no data to supportany explanation of why exponential growthended.An oxygen probe in a fermenter filled with
air-saturated BMS solution or with air-satu-rated BMS solution and diphenylmethaneshowed a decrease in dissolved oxygen contentof 2% in 30 h at 300C. Thus, the oxygen probesused were stable. When fermentations were
done without added substrate, the decrease indissolved oxygen after 24 h was approximately6%.The generation times obtained for the culture
on diphenylmethane at 30°C are shown in Table1. The slowest growth was obtained when themedium contained only dissolved diphenylme-thane. The faster growth was obtained on themedium with the greatest amount of undis-solved, liquid diphenylmethane.Growth in the presence of various amounts
of solid diphenylmethane at 20°C. The genera-tion times obtained in the presence of variousamounts of solid diphenylmethane at 20°C areshown in Table 2. The generation times wereindependent of the amount of solid diphenylme-thane present. In fermentations in the presenceof 0.5 and 0.05 g ofdiphenylmethane per liter, alack of dissolved oxygen stopped growth at theend of the experiment, and at least 70% of theobserved oxygen utilization occurred when thecells were growing exponentially. In fermenta-tions containing 0.015 g of undissolved diphen-ylmethane per liter, approximately 40% of theavailable dissolved oxygen was used before ex-ponential growth ended. After exponentialgrowth ended, oxygen was used at a nonexpo-nential rate until it was depleted.Growth on dissolved diphenylmethane at
20°C. The generation time of the Pseudononasculture on dissolved diphenylmethane alone inan air-saturated medium at 20°C could not beobtained. There was uptake of dissolved oxy-gen, but it was not sufficient to measure accu-rately the generation time.The generation time ofthe Pseudomonas cul-
ture on dissolved diphenylmethane at 20°C (Ta-
TABLE 1. Generation times of Pseudomonas sp. ondiphenylmethane at 30°C
Initial amt of liquid No. of Generation(g/liter) trials time + SDa
thane were done in medium in which the oxygenconcentration was one-half air saturation.
bSD, standard deviation.
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ble 2) was determined in a medium in which theinitial oxygen concentration was one-half airsaturation. Approximately 30% ofthe availabledissolved oxygen was used when exponentialgrowth ended. After exponential growth ended,oxygen was used at a nonexponential rate untilit was depleted. In this experiment, the ob-served exponential phase occurred between 20and 40 h of cultivation. An inoculated controlcontaining no added substrate showed a 6%decrease in dissolved oxygen during the 20- to40-h period, and only a 1% decrease from 0 to 20h.The average generation time for the culture
growing in the presence of dissolved diphenyl-methane alone was the same as that for theculture growing in the presence of both dis-solved and various amounts of solid diphenyl-methane.
Distribution of naphthalene in solutions ofheptamethylnonane and BMS solution. It wasimportant to know the distribution of naphtha-lene between the aqueous and heptamethyl-nonane phases because this distribution givesan indication of the concentrations of naphtha-lene available to the bacteria in the aqueous
and heptamethylnonane phase. Table 3 showsthe distribution of naphthalene between theaqueous and the heptamethylnonane phaseswhen increasing amounts of heptamethylnon-ane were added to naphthalene-BMS solutions.It is apparent that as more heptamethylnonaneis added to the system, the concentrations ofnaphthalene in the aqueous and in the hepta-methylnonane phases decrease.Table 4 shows the distribution of naphtha-
lene between the two phases when increasingamounts of a naphthalene-heptamethylnonanesolution were added to BMS solution. Each mil-liliter of heptamethylnonane added contained
TABLE 3. Generation times ofa Pseudomonas sp. on
naphthalene, and the distribution of naphthalenebetween aqueous and hydrocarbon phases when
heptamethylnonane is added to 500 ml ofnaphthalene-BMS solution
Heptame-Calculated
thylnonane A275 of A275 of hep- Generationadded aqueous tamethyl- time + SDa
a Generation times are the average of a minimumof six determinations. SD, standard deviation.
b Data reported previously by Wodzinski and Ber-tolini (6).
TABLE 4. Generation times ofa Pseudomonas sp. onnaphthalene, and the distribution of naphthalenebetween aqueous and hydrocarbon phases when
various amounts of heptamethylnonane containing0.015 g ofnaphthalene per ml are added to 500 ml of
BMS solution
Heptame- Calculatedthylnonane A275 Of A275 of hep- Generation
added aqueous tamethyl- time t SDa(ml) phase nonane (h)
phase
1 0.203 545 1.7 ± 0.13 0.243 608 1.2 0.1
12 0.232 637 1.1 0.1
a Generation times are the average of a minimumof six determinations. SD, standard deviation.
0.015 g of naphthalene. The absorbances shownin Table 4 indicate that the concentration ofnaphthalene remains relatively constant inboth the aqueous and heptamethylnonanephases as more naphthalene-heptamethylnon-ane mixture is added to BMS solution.Growth on naphthalene in heptamethyl-
nonane-BMS solution. In all growth experi-ments on naphthalene, a lack of dissolved oxy-gen stopped growth at the end of the experi-ment, and at least 70% of the dissolved oxygenwas consumed during the exponential phase ofgrowth. When fermentations were done with-out naphthalene in the presence of 12 ml ofheptamethylnonane, there was an 8 to 9% up-take of dissolved oxygen. It was apparent thatgrowth observed in the presence of naphthalenecould not have been due to utilization of impuri-ties in the heptamethylnonane.
In growth experiments, cells were grown in anaphthalene-BMS medium to which increasingamounts of heptamethylnonane were added,giving the distribution of naphthalene shown inTable 3. The resulting generation times shownin Table 3 indicate that the cells grew moreslowly when heptamethylnonane was presentthan when no heptamethylnonane was added,and that growth rates decreased when moreheptamethylnonane was added.A second group of growth experiments was
done in which cells were grown on BMS solu-tion to which was added a mixture of 0.015 g ofnaphthalene per ml of heptamethylnonane.The generation times from these experimentsindicate that the bacteria grew faster when 3and 12 ml of naphthalene-heptamethylnonanemixture were added than when 1 ml was added(Table 4).Microscopic observations. When the Pseu-
domonas sp. that utilizes naphthalene wasadded to BMS solution that contained solid par-ticles of naphthalene, microscopic observation
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did not reveal any affinity for the surface of thesolid particles. However, the bacteria did showa definite affinity for the surface of heptame-thylnonane or naphthalene-heptamethylnon-ane droplets suspended in the BMS solution.
DISCUSSIONBecause of the small amounts of substrate
metabolized compared to amounts used to satu-rate solutions or added to some fermenters, itwas necessary to show that the growth ob-served could not be on impurities in the sub-strates. In fermentations on diphenylmethane-saturated medium, 30% of the available dis-solved oxygen was utilized when exponentialgrowth ended. If one assumes that this expo-nential growth was on water-soluble impuritiesthat were present in the 0.5 g of diphenylme-thane per liter used to saturate the medium, itis difficult to explain why, in fermentations inthe presence ofdiphenylmethane-saturated me-dium with an additional 0.05 g of liquid diphen-ylmethane per liter, 70% of the available dis-solved oxygen was utilized when exponentialgrowth ended. It was shown previously that thegrowth observed during fermentations onnaphthalene were not due to impurities in thesystem (6).The growth rates of the culture growing in
the presence ofvarious amounts of solid diphen-ylmethane are the same, and these rates ofgrowth are the same as the rate of growth ondissolved diphenylmethane alone. Similar datawere obtained for the solid aromatic hydrocar-bons, naphthalene (6), phenathrene (7), andbibenzyl (6). The data support the hypothesisthat the bacterium utilizes dissolved diphenyl-methane and does not obtain hydrocarbon di-rectly at the surface of solid particles suspendedin the medium. The results obtained are con-sistent with the kinetic model of Dunn (2),which predicts that for cells growing on dis-solved hydrocarbon as opposed to growth at anaqueous-hydrocarbon interface, the exponen-tial growth rate is independent ofthe amount ofsolid, undissolved hydrocarbon present.The growth rates of bacteria in the presence
of various amounts of liquid diphenylmethanereveal a different pattern. The rates of growthof bacteria growing in the presence of variousamounts of liquid diphenylmethane increasewith increasing amounts of liquid diphenylme-thane, and these rates are higher than the rateof growth obtained on medium containing onlydissolved diphenylmethane. The data supportthe hypothesis that the bacteria can obtain hy-drocarbon directly from the droplets of diphen-ylmethane suspended in the medium. The re-
sults are consistent with the kinetic model of
Dunn (2), which predicts that for cells growingat an aqueous-hydrocarbon interface, the expo-nential growth rate will increase as the interfa-cial area of the hydrocarbon increases. Thus,for the same hydrocarbon, the pattern ofgrowth kinetics depends on the physical state ofthe undissolved hydrocarbon.
Cells grow slower when heptamethylnonaneis present than when no heptamethylnonane ispresent. Growth rates also decrease as moreheptamethylnonane is added to the system.One possible explanation for the observed gen-eration times is that the cells can obtain naph-thalene at the water-hydrocarbon interface,and the concentration of naphthalene in theheptamethylnonane phase decreases with in-creasing amounts of added heptamethylnon-ane, which results in slower growth. This ex-periment is supported by the data of the secondgroup of experiments shown in Table 4. Whencells were grown in BMS solution to which amixture of 0.015 g naphthalene per ml of hepta-methylnonane was added, the concentrations ofnaphthalene in the heptamethylnonane andaqueous phases did not vary significantly asmore naphthalene-heptamethylnonane mix-ture was added to the BMS solution. The onlysignificant change in this system as morenaphthalene-heptamethylnonane mixture wasadded was that the total surface area of hepta-methylnonane increased. The increase in growthrate during the addition of more naphthalene-heptamethylnonane mixture indicates that thecells can utilize the naphthalene dissolved inthe heptamethylnonane phase at the interfacebetween the heptamethylnonane and BMS so-lution. The faster growth rate with increasinginterfacial-surfacial area agrees with Dunn'smodel (2) for cells growing on an interface.Visual observation of cells concentrated at theinterface of naphthalene-heptamethylnonanedroplets supports (but does not prove) the hy-pothesis that cells obtain naphthalene at theinterface of the heptamethylnonane and waterphases.Although the conclusions made are the sim-
plest explanations of the data obtained, alter-nate explanations are possible. For example,the results obtained in the presence of liquidhydrocarbons could be due to the undissolvedhydrocarbon altering the cell's affinity for thedissolved hydrocarbon in the aqueous phase.
Previous work indicating that aromatic hy-drocarbons and arenes are utilized in the dis-solved state (6, 7), if generally applicable, mayonly be true for the solids.
It was suggested (6, 7) that the solubility ofan aromatic hydrocarbon could influence howrapidly the hydrocarbon is degraded by bacte-
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ria. This hypothesis may not be applicable toconditions found in the environment. For ex-ample, a solid aromatic hydrocarbon that isvery insoluble in water could possibly be con-centrated in a hydrophobic liquid and be read-ily available to cells at the aqueous-hydropho-bic liquid-phase interface.
ACKNOWLEDGMENTDavid Larocca was supported by Undergraduate Re-
search Participation Grant number GY-11175 from the Na-tional Science Foundation.
LITERATURE CITED
1. Borokowski, J. D., and M. J. Johnson. 1967. Long-livedsteam-sterilizable membrane probes for dissolved ox-ygen measurement. Biotechnol. Bioeng. 9:635-639.
2. Dunn, I. J. 1968. An interfacial kinetics model for hy-
BACTERIAL GROWTH KINETICS 665
drocarbon oxidation. Biotechnol. Bioeng. 10:891-894.3. Focht, D. D., and M. Alexander. 1971. Aerobic co-
metabolism of DDT analogues by Hydrogenomonassp. J. Agric. Food Chem. 19:20-22.
4. Hodgman, C. D., R. C. Weast, R. S. Shankland, and S.M. Selby (ed.). Handbook of chemistry and physics,43rd ed. Chemical Rubber Publishing Co., Cleveland,Ohio.
5. Rogoff, M. H. 1962. Chemistry of oxidation ofpolycyclicaromatic hydrocarbons by soil pseudomonads. J. Bac-teriol. 83:998-1004.
6. Wodzinski, R. S., and D. Bertolini. 1972. Physical statein which naphthalene and bibenzyl are utilized bybacteria. Appl. Microbiol. 23:1077-1081.
7. Wodzinski, R. S., and J. E. Coyle. 1974. Physical stateof phenanthrene for utilization by bacteria. Appl.Microbiol. 27:1081-1084.
8. Wodzinski, R. S., and M. J. Johnson. 1968. Yields ofbacterial cells from hydracarbons. Appl. Microbiol.16:1886-1891.
