Vol. 37, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1979, p. 4-100099-2240/79/01-0004/07$02.00/0

Production of Surface-Active Lipids byCorynebacterium lepus

DAVID G. COOPER,* JAMES E. ZAJIC, AND DONALD F. GERSON

Biochemical Engineering, Faculty ofEngineering, The University of Western Ontario, London, Ontario,Canada

Received for publication 19 October 1978

Corynebacterium lepus was grown in 20-liter batch fermentations with keroseneas the sole carbon source. Critical micelle concentration measurements indicatedthe production of appreciable quantities of biosurfactants. This surface activity ofthe culture medium was due to lipids, which were extracted and identified.Samples of C. lepus whole broth were taken during a fermentation and monitoredfor surface tension, amount of surfactant present, and lipid content. The changesin the surfactant measured correlated with concentration changes of severalsurface-active lipids. An early dramatic increase in surfactant concentration wasattributed to the production of a mixture of corynomycolic acids (,8-hydroxy a-branched fatty acids). Surface activity at the end of the fermentation was due toa lipopeptide containing corynomycolic acids plus small amounts of severalphospholipids and neutral lipids which were identified by thin-layer chromatog-raphy.

Several species of microorganisms are re-ported to produce surface-active agents (1, 3-6,9-12, 17, 20, 21). These are usually organismswhich produce surface-active lipids when grow-ing on hydrocarbon substrates. The surface ac-tivity is observed as a substantial lowering of theculture medium surface tension, an emulsifica-tion of the medium and hydrocarbons, or relatedphenomena. Several different types ofbiosurfac-tants have been isolated and characterized.These include glycolipids (6, 9, 11, 21), lipopep-tides (1, 10), phospholipids (3, 12), and neutrallipids (17, 20). The complex lipids all containfatty acids (1, 3, 6, 9-12, 21), and often thesefatty acids have a hydroxyl function on thecarbon /8 to the carboxyl group (1, 10) or fartheralong the chain (6, 9, 11).Gerson and Zajic (5) reported the isolation of

Corynebacterium lepus which will grow on ker-osene. During growth, the surface tension of themedium quickly fell from 72 dyn/cm to under30 dyn/cm, and the active agent was producedin quantities greatly in excess of the criticalmicelle concentration (CMC; concentration ofsurfactant which causes micelle formation).With the best samples it was necessary to diluteportions of C. lepus culture medium 200 to 500times before the surface tension rose above 30dyn/cm. Gerson and Zajic (5) also reported thatthe CMC and, by implication the concentrationof biosurfactant present, varied dramaticallyduring batch fermentations of C. lepus on kero-sene.

This paper reports the nature of the surface-active lipids produced by C. lepus and theirchanges in concentration throughout a fermen-tation.

MATERIALS AND METHODS

Fermentation studies. Gerson and Zajkc (5) de-scribed the isolation, identification, and fermentationof C. lepus. For this work a 28-liter New Brunswickfermentor was used as described previously (5). Thetemperature was controlled at 250C. Air was intro-duced with a 50-mm stainless steel disk at 20 liters/minand a pressure of 4 lb/in2. A 25-ml inoculum in theearly stationary phase was used. The media used forboth the fermentations and the inocula were madewith standard mineral salts media (5). They containedthe following ingredients: NaNO3, 0.2%; K2HPO4,0.1%; KH2PO4, 0.05%; KCl, 0.01%; MgSO4, 0.05%;CaCl2, 0.001%; FeSO4, 0.001%; ethylenediaminetetra-acetate, 0.00015%; and trace amounts of B, Cu, Mn,Mo, and Zn. The medium usually contained 4% Im-perial Oil no. 9 kerosene and 0.01% nutrient broth.

Extraction of lipids from lyophilized C. lepusproduct. Samples of culture medium (ca. 60 g) of C.kepus were centrifuged to give an emulsion at theinterface between the aqueous medium and kerosene.This emulsion was collected by suction and lyophi-lized. Samples taken at the end of a fermentationyielded 2 to 3 g of a friable off-white solid per liter.

Lipids were extracted from this solid by using astandard method (reference 13, p. 351). The productwas stirred in chloroform-methanol-water (25:25:4; 10ml per g of product) for 6 h. An equal volume of waterwas added, and the mixture was stirred vigorously for1 h. The organic phase was isolated, washed with

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water, and evaporated to dryness. The resulting yellowoil was extracted with acetone at room temperature.The acetone-soluble fraction was obtained as a yel-

low oil after evaporation of the solvent. A typical yieldwas 2 to 5% of the original dry weight of productcollected from the culture medium.The acetone-insoluble fraction was a pale waxy solid

which contained polar lipids. This was dissolved inchloroform and filtered with Whatman 541 filter pa-per, and the solvent was removed. This was the majorlipid fraction, and typically the yield was 20 to 25% ofthe original dry weight.

Extraction of lipids from samples of culturemedium. This method was used for monitoring lipidsthroughout a fermentation. Samples of medium (ca.60 g) were weighed, shaken with 20 ml of pentane, andcentrifuged. The pentane above the product emulsionwas removed, and the extraction was repeated. Thepentane was evaporated from the combined extracts,and the residue was lyophilized. The resulting oil wastransferred to a 25-ml volumetric flask and made upto volume with pentane.The floating solid remaining in the pentane-washed

samples was removed and lyophilized. After the driedproduct was weighed, it was extracted with chloro-form-methanol-water as described above.

Extraction of lipopeptides from culture me-dium. It was possible to obtain a lipopeptide fractionby first adding sufficient HCI to a culture mediumsample to lower the pH to 2 and then extracting withchloroform. After the chloroform was removed, theresidue was washed with acetone and then lyophlized.The lipopeptide was obtained as a white powder inyields of up to 0.35 g/liter.

Calculation of CMC. The CMC is defined as theconcentration of a surfactant necessary to initiate mi-celle formation. If more of the surfactant is present,there will be no further decrease in surface tension (5).To obtain a measure of the CMC of a whole brothsample, portions were diluted by various amounts, andthe surface tension was obtained for each dilution.The CMC could then be estimated, from a plot ofsurface tension versus the log of the percent dilution,as the dilution at which the surface tension starts toincrease. The reciprocal of the CMC (CMC-') is pro-portional to the amount of surfactant present.TLC. Thin-layer chromatography (TLC) plates,

both analytical and preparative, were prepared withMerck G silica gel. Plates (20 by 20 cm) were spottedwith lipid extracts and known compounds and devel-oped in one of the following: solvent 1, chloro-form-methanol-water (65:25:4); solvent 2, chloro-form-methanol-acetic acid-water (25:15:4:2); sol-vent 3, chloroform-acetone-methanol-acetic acid-water (7:8:2:2:1); or solvent 4, hexane-isopropylether-acetic acid (15:10:1). After developing, the spotswere visualized with standard spray reagents (refer-ence 13, p. 435 -441). Ninhydrin generated a red orpurple color when a compound had an amine function.The Zinzadze reagent reacted with phosphate-contain-ing lipids, resulting in a blue color. Spraying the platewith an a-napthol solution, followed by concentratedsulfuric acid and then heating for 10 min, indicatedcarbohydrate by a red color. Certain compounds ap-peared as fluorescent spots under UV irradiation. Neu-

SURFACE-ACTIVE LIPIDS IN C. LEPUS 5

tral lipids which were not indicated by the abovemethods were visualized by spraying with rhodamine6G and observed under UV irradiation as yellow spots-All of the lipids could be visualized by charring theplates after spraying with potassium dichromate insulfiuric acid. Identifications were made on the basis ofcomparisons with published data (4, 7, 13, 15) andcorroborated with available phospholipids, includingphosphatidyl inositol, phosphatidyl seine, phospha-tidyl glycerol, phosphatidic acid, phosphatidyl choline,and lysophosphatidyl choline.

Instrumentation and chemicals. Infrared spec-tra were obtained on a Beckman IR-20 spectrometerby using chloroform solutions. UV and visible spectrawere obtained on a Perkin-Elmer spectrometer byusing pentane solutions. Protein was determined byusing the biuret method (reference 8, p. 244-249).Amino acids were determined with a Beckman aminoacid autoanalyzer. Solvents used were Fisher ScientificCo. Spectranalyzed grade. Standard lipids were ob-tained from Serdary Research Laboratories Inc., Lon-don, Ontario, Canada.

RESULTSVariations in surface tension and CMC-

during a fermentation of C. lepus. Figure 1contains some of the data from a typical fermen-tation of C. lepus, plotted as a function of sam-pling time. The surface tension of the culturemedium quickly dropped to below 30 dyn/cmand remained constant to the end of the fermen-tation. The plot of the reciprocal of the CMC,which is a measure of surfactant concentration,shows more variation. Initially there was insuf-ficient surfactant present to form micelles. At 25h the CMC-1 started to rise sharply, reaching amaximsum at about 44 h. It then decreased bymore than an order of magnitude and remainedconstant after 60 h. Although not observed inthis example, during many of the fermentationsthe CMC-1 had a second, small increase afterabout 70 h. The biosurfactant never completelydisappeared in any of the fermentations moni-tored.Biosurfactants present at the end of the

fermentation. Initially, a study was made ofthe compounds present in the culture mediumat the end of the fermentation. This correspondsto the region in Fig. 1 after 60 h when the plotof CMC-lhad leveled off. The surface activityof the culture medium was found to be due tothe fractions soluble in lipid solvents. Thesewere concentrated in a slimy white solid pro-duced in copious quantities by the end of thefermentation (ca. 3 g/liter). After removal of theproduct emulsion, the residual medium had asurface tension greater than 60 dyn/cm, and theamount of remaining surfactant was less thanthe CMC. If the dried product was redissolvedin distilled water, the minimum surface tension

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6 COOPER, ZAJIC, AND GERSON

was 49 dyn/cm. This was not as low as thatobtained for the whole broth; however, thewhole broth contained sufficient kerosene toinfluence the surface tension measurements.Samples of sterile medium did not yield detect-able amounts of lipids.The surface-active lipids were extracted from

the dried product (0.5 g/liter of original whole

C-

a,-)

cm

9.

87

7-

2.5

2.0

1.5

E

1.0

:0

0.51

OJ

IN40C:

C:0

.vi50.C:

0)

a)

:30LA0

Timne hoursFIG. 1. Data from a fermentation study of C. lepus

plotted against sampling time. Symbols: 0, CMC-';0, surface tension; O, yield of floating product; O,yield ofpolar lipids; and U, log of the cell count.

APPL. ENVIRON. MICROBIOL.

broth; minimum surface tension, 49 dyn/cm)and partitioned into acetone-insoluble polar lip-ids (88%) and acetone-soluble neutral lipids(12%).The polar lipids were resolved into several

compounds by using TLC (Table 1). Six phos-pholipids were observed. Phosphatidyl glycerol,phosphatidyl inositol, and a carbohydrate-posi-tive phospholipid, which is probably one of thephosphatidyl inositol mannosides often found incoryneform bacteria (7, 15, 16), were present inroughly equal amounts, and all were easily dis-tinguished. Phosphatidyl glycerol phosphateand cardiolipin could not be resolved at the endof the fermentation, but the study of lipids pres-ent throughout a fermentation indicated thatboth were present (vide infra). Only a very smallamount of phosphatidyl serine was observed. Asingle unidentified glycolipid was observed.The above lipids accounted for only a small

amount of the polar lipid fraction. At least 90%of the acetone-insoluble residue was a mixtureof ninhydrin-positive lipids. These lipopeptideswere a relatively long smear on the TLC plates,and there were probably more than one present.The lipopeptides could be obtained free of theother polar lipids by acidifying a sample of cul-ture medium and extracting it with chloroform.The minimum surface tension for the lipopep-tide added to distilled water was 52 dyn/cm.A biuret determination indicated a protein

concentration of about 35% by weight. Table 2shows the amino acid composition. About 20%of the residues present were acidic (i.e., glutamicor aspartic acids), and most of the rest wereneutral amino acids.The remainder of the lipoprotein was found to

be 25% saturated fatty acids and 75% coryno-mycolic acids. The isolation and characterizationofthese acids will be reported in full in a separate

TABLE 1. TLC data for lipids isolated from C. lepusRf, in solvent: Phos- Carbo- Time (h) of

Lipid phate teste hy- aper1 2 3 teat test drate anceatest

Lipopeptides 0 0.04-0.15 0.07-0.22 + 21Phosphatidyl glycerol 0.52 0.8 0.75 + 19Phosphatidyl inositol 0.22 0.5 0.3 + 19Phosphatidyl inositol 0.55 0.58 0.22 + + 19mannoside

Phosphatidyl serine 0.17 0.63 0.51 + + 20Cardiolipin 0.7 0.94 0.90 + 27Phosphatidyl glycerol 0.90 + 20phosphateb

Glycolipid 0.57 0.35 0.5 + 27Neutral lipids 0.5-0.75 1.0 1.0 30-45

a From a monitored fermentation; inoculation was zero time.b These two phospholipids were not resolved except in samples taken at from 20 to 50 h.

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publication. Corynomycolic acids are,/-hydroxya-branched fatty acids which have been isolatedpreviously from coryneform bacteria (2, 16). Themixture of fatty acids obtained by saponification(alcoholic potassium hydroxide; reference 13, p.363) had a very low minimum surface tension indistilled water (32 dyn/cm).The acetone-soluble neutral lipids were only

about one-tenth ofthe total lipid extract but hada relatively low surface tension in water (40dyn/cm). Table 3 lists the components whichwere identified in this fraction. The various glyc-erides were present in very small amounts andwere only identified by comparison on the TLCplates with known compounds. The other com-ponents were present in sufficient amounts toobtain samples by preparative TLC and to con-firm identification by infrared spectroscopy ofchloroform solutions. The most prominent spoton the plates was usually due to corynomycolic

TABLE 2. Amino acids found in lipopeptide from C.epus

Amino acid Mole compositionaAmmoacld ~~~~~~~(%)Alanine........................ 17.4Glutamic acid .................. 15.6Glycine ........................ 13.0Leucine........................ 10.6Serine ....................... 8.4Threonine ..................... 7.7Phenylalanine ............... 6.4Aspartic acid ................... 5.9Proline ........................ 5.5Methionine .................... 3.0Isoleucine ............ 2.6Valine......................... 2.1Lysine ......................... 1.8

a Expressed as a percentage of the total moles ofamino acids in the sample.

TABLE 3. TLC data for neutral lipids isolated fromC. lepus with solvent 4

Identification'Lipid Rf Stan- In-

dard fraredMonoglyceride 0.03 +1,2-Diglyceride 0.2 +1,3-Diglyceride 0.25 +Corynomycolic acids 0.3-0.4 +Fatty acids 0.6 + +Triglyceride 0.7 +Ketone 0.85 +Kerosene residue 0.9-1.0 + +

a Lipids were identified by comparison with stan-dard lipids and by infrared spectroscopy when possi-ble. The four glyceride references were obtained fromolive oil, and palmitic acid was used as a fatty acid.

SURFACE-ACTIVE LIPIDS IN C. LEPUS 7

acids (POH, alcohol 3,530 cm-'; vOH, carboxyl3,000 and 2,700 cm-'; vC==O 1,710 cm-'; vC-O1,100, 1,290 cm-'). There were also smalleramounts of simple fatty acids and a compoundwith vC=O at 1,740 cm-', which was tentativelyidentified as a ketone (e.g., neither vC-O, ex-pected for an ester, nor 'C(O)-H, expected foran aldehyde, were observed [13]). Finally, therewas a significant component moving with thesolvent front, which was identified as a nonvol-atile kerosene residue.Variation in surfactant concentration

during a fermentation of C. lepus. In anattempt to explain the behavior of the CMC-1data during a fermentation, samples of the cul-ture medium were taken at various times duringthe fermentation shown in Fig. 1. These wereanalyzed for changes in surface-active lipids.The extraction procedure for samples of wholebroth outlined above resulted in three types ofsamples: (i) the aqueous supernatant, (ii) thepentane extract, and (iii) the lipid extract of thefloating product.

(i) Aqueous supernatant. The aqueousphase which had been exhaustively extracted forlipids showed little surface activity throughoutthe fermentation. The surface tension did not gobelow 60 dyn/cm. The optical density at 270 nmof the aqueous phase increased throughout thefermentation due to the presence of a small butsteadily increasing amount of protein. As thisdid not contribute significantly to the surfacetension of the whole broth, it was not studiedfurther.

(ii) Pentane extracts. Pentane solutions ofthe neutral lipids were used for the followingmeasurements. As it was impossible to take ex-actly the same weight of whole broth with eachsample, when feasible the data were normalizedrelative to the measured sample weights. If thiswas not possible due to the nature of the mea-surement, only data from samples whichweighed 60 + 5 g were considered. The surfacetension data were obtained from a distilled watersolution prepared by layering 1 ml ofthe pentanesolution on 5 ml of water and evaporating thepentane in a stream of air.A plot of CMC-1 from the pentane-soluble

lipids versus sampling time (Fig. 2) is similar tothe plot of CMC-1 of culture medium (Fig. 1).At about 25 h it began to increase sharply,reaching a maximum between 40 and 45 h. Itthen decreased by an order of magnitude by 60h. The pentane samples at this peak in surfac-tant concentration had a noticeable yellow colorwhich was not seen earlier or later in the fer-mentation. The optical densities of the pentanesolutions at 258 nm are also plotted in Fig. 2.

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E50-

c

b30

0

L-

301

0.8j

00.2~C0.4

u0.20

0

0 40Time hours

FIG. 2. Properties ofpentane extracts cfermentation samples versus sampling time0, CMC-'; 0, surface tension; and 0, optic

The peak in optical density closely mpeak in CMC-G. However, the absorbindisappeared completely by 60 h. Th4plot indicated that some surfactant Epeared, as it displayed a significant incr460 h. The final plot in Fig. 2 is the inititension before diluting the solutionsCMC data. This curve is not as dramafirst two. However, there was a miinsurface tension (36 dyn/cm) at the samthe maxima in CMC-1 and optical densurface tension then increased to ldyn/cm at the end of the fermentation

Micropipettes were used to sp(amounts of several pentane solutions aedge of a single TLC plate which ideveloped in solvent 4. Relatively smaiwere applied to the plates, and, even aof the fermentation, most of the neutrecorded in Table 3 were not observEnomycolic acids were observed afterthe plates with rhodamine 6 G. Theypeared in the sample taken at 29 h anda peak in concentration between 40 a

They then decreased in concentration, althoughthey were still observed as a faint spot by theend of the feripentation. Two other compoundswere observed as fluorescent blue spots underUV irradiation before spraying with rhodamine6 G (RJs in solvent 4,0.75 and 0.85, respectively).Both first appeared before 30 h, reached a max-

0O imum concentration between 40 and 45 h, andwere not detected by 60 h. As neither was pres-ent at the end of the fermentation, they had notbeen observed in the preliminary study of theneutral lipids (Table 3). Even with preparativeTLC of the samples taken at 40 to 45 h, it wasimpossible to obtain sufficient quantities ofthesetwo compounds to identify them.The final analysis of the pentane samples was

to concentrate portions and use these to spotTLC plates in an attempt to observe the otherneutral lipids. Although this made it more diffi-cult to observe the behavior of individual lipids,all of the components listed in Table 3 werepresent by 40 h. For each lipid there was ageneral increase in concentration to a plateauafter 60 h. This was synchronous with the secondincrease in CMC-1 and the leveling-off of thesurface tension shown in Fig. 2.

(iii) Lipid extract of floating product. Theyield of floating product increased steadily

80 throughout the fermentation of C. lepus (Fig. 1).The yield of polar lipid extracted from the float-

)f C. lepus ing product also increased throughout the fer-m.SymbOl: mentation (Fig. 1). There was no evidence foral density. maxima in these concentration curves corre-

sponding to the maximum in the CMC-' plot ofimics the the whole broth. Instead, the maximum concen-ig species trations were observed at the end of the fermen-e CMC-1 tation.iad reap- The polar lipid samples were subjected to aease after comparative TLC study similar to that describedal surface for the pentane extracts except that solvent 2to obtain was used. No lipids were observed which hadLtic as the not been identified previously in the productsiimum in collected from C. lepus fermentations in lateie time as growth phase (Table 1). Table 1 also lists theLsity. The time of first appearance of each polar lipid. Theabout 46 first were observed at 19 h and by 30 h all of thel. phospholipids, the lipopeptides, and the glyco-Dt equal lipid had appeared. There were no apparentilong one concentration maxima for the polar lipids be-was then tween 40 and 45 h, as was observed for some of1 samples the neutral lipids. Instead, the polar lipid con-Lt the end centrations increased steadily until about 70 h,ral lipids after which they appeared to remain constant.ed. Cory- Monitoring the phospholipid concentrationsspraying as a function offermentation time confirned thefirst ap- occurrence of both phosphatidyl glycerol phos-

1 reached phate and cardiolipin, which could not be re-md 45 h. solved in the initial TLC studies. Phosphatidyl

AkPPL. ENVIRON. MICROBIOL.

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SURFACE-ACTIVE LIPIDS IN C. LEPUS

glycerol phosphate was observed first at 20 h as

a small phosphate-positive spot at Rf 0.90. At 27h a second small spot appeared at Rf 0.94 touch-ing the original slightly lower spot, and this wasattributed to cardiolipin. As these two spotsincreased in size in later samples, they coalescedinto a single spot.

DISCUSSIONAll of the surface-active agents isolated from

C. lepus were lipids. The CMC data of theculture medium samples taken during severalfennentations indicated early, large increases insurfactant concentration followed by rapid dis-sipation. In the fermentation represented by Fig.1 and 2, there was a close correlation betweenthe behavior of the CMC-' of the whole brothand the amount of corynomycolic acids[R1-CH(OH)-CHR2-COOH] present in thewhole broth which could be extracted into pen-

tane. Two unidentified neutral lipids, which flu-oresced blue under UV irradiation of the TLCplates, were the only other lipids to show con-

centration maxima at the same time. However,neither was present in more than trace amounts.Thus the production of corynomycolic acidsseems to be the cause of the early sharp rise inCMC-1.Because the corynomycolic acids did not ab-

sorb at 258 nm, their production cannot be thecause of the peak in UV absorption observed forthe pentane extracts (Fig. 2) at 40 to 45 h. Thisphenomenon may be due to one or both of thetwo fluorescent neutral lipids produced at thistime. It is interesting that the biosynthesis ofcorynomycolic acids is thought to involve thecondensation of two simple fatty acids to give,8-keto acids [R1-C(O)-CHR2-COOH],which are reduced to corynomycolic acids (2,22). These fi-keto intermediates are unstableand decarboxylate to ketones if not reduced toproduct (18). However, a UV spectrum has beenreported for a methyl ester derivative, and thereis a maximum at about 260 rm (18). The pentanesolutions demonstrate the increased concentra-tion of a compound with a UV maximum at 258rim at the same time that the concentration ofthe corynomycolic acids is increasing rapidly.Conceivably, one of the unknown neutral lipidswas a mixture of the 18-keto acids produced as

intermediates in corynomycolic acid synthesis.Although the surfactant concentration

(CMC-1) decreases after the initial maximum,

there is still an appreciable amount of sirfactantpresent at the end of the fermerxtation.-On sev-

eral monitored fermentations of C. lepus, thevalues of CMC-1 started to increase again latein the fermentation (usually after 70 h). This

continuing surface activity is probably due to acombination of lipids, including a very smallresidue of free corynomycolic acids, the otherneutral lipids, and the polar lipids.The correlation between the CMC-1 and TLC

data of the pentane-soluble lipids illustrates theimportance of changing concentrations of differ-entlipids to the overall surface tension. The firstlarge maximum in CMC-' corresponds to themaximum in corynomycolic acid concentration.The second, small increase in CMC-1 corre-sponds to maxima in the concentrations of theother neutral lipids.The concentration of polar lipids reached a

plateau of maximum concentration between 50and 60 h, as the corynomycolic acid concentra-tion was becoming negligible. After this time theamount of polar lipids present was about 10times the amount of neutral lipids. In particular,the lipopeptides accounted for 80 to 90% of thetotal lipid late in the fermentation. This lipidhas been isolated and exhibited surface activity(52 dyn/cm). Thus, the lipopeptide probablyaccounts for most of the surface activity of theculture medium at the end of the fermentation.The relative concentrations of the two major

biosurfactants produced by C. lepus are inter-esting because the one type, the lipopeptides, isabout 50% by weight corynomycolic acids, theother type. The concentration of corynomycolicacids imizned early in the fermentation, andthe lipopeptides can be observed in the culturemedium at this time. As the concentration of thelipopeptides starts to increase, the free coryno-mycolic acids disappear from solution. This isconsistent with the free corynomycolic acidsbeing removed from the culture medium andincorporated into the lipopeptides.Some of the lipids isolated have taxonomic

significance. The corynomycolic acids producedby C. lepus are typical of the f8-hydroxy a-branched fatty acids found in Corynebacteriumand related bacteria (2, 14). The phospholipidcomposition, in particular the complete absenceof phosphatidyl ethanolamine, is characteristicof the low-guanine-cytosine-content (DNA)group of Corynebacterium (4, 16, 19).

ACKNOWVLEDGMEENTSThis work was supported by the department of Energy,

Mines and Resources of the Government of Canada.We express our appreciation to D. S. Montgomery for

useful and interesting discussions.

LITERATURE CITED

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