J. Bacteriol.-1950-Jezeski-645-58

THE ACTION OF MICROORGANISMS ON FATSI. OXYGEN UPTAKE BY BACTERIA IN THE PRESENCE OF LIPID SUBSTRATES1

J. J. JEZESKI,' H. 0. HALVORSON,S AND H. MACYDivisions of Bacteriology and Dairy Husbandry, University of Minnesota, Minneapolis and

St. Paul, Minnesota

Received for publication January 30, 1950

The ability of many bacteria to hydrolyze fats has been well established.On the other hand, the oxidative action of bacteria on this type of substratehas not been so well investigated. Both chemical and cell respiration studieshave demonstrated that bacteria can oxidize fats, but relatively little infor-mation has been obtained on the characteristics of this action on lipid sub-strates.

Chemical evidence of bacterial oxidation has been obtained on olive oil (Pigu-lewski and Chaxik, 1929), soybean oil (Horowitz-Wlassova and Livschitz, 1935),hardened cottonseed oil and leaf lard (Jensen and Grettie, 1933, 1937), andtriolein (Castell and Garrard, 1941). Corn oil was used as the substrate in theWarburg and Thunberg techniques by Mundt and Fabian (1944). A comparisonof the results revealed no agreement between the two methods. The Thunbergtechnique was used by Quastel and Whetham (1925) to show that B. coli-com-munis could dehydrogenate the lower fatty acids. The same species was able tooxidize the sodium salts of stearic, oleic, and palmitic acids in the Warburgapparatus (Mazza and Cimmino, 1933). At pH 7.5 stearate was oxidized at thegreatest rate, followed by oleate and palmitate. These results were confirmedby Singer and Barron (1945), who also showed that the oxidative enzyme con-tained active SH groups. Barron and Friedemann (1941) demonstrated thatseveral cultures of bacteria not capable of fermenting glucose were able tooxidize acetate, jpropionate, and butyrate, and that more than one enzyme mightbe responsible for the oxidation of saturated fatty acids. Streptococcus mitisoxidized butyrate aerobically with the accumulation of H202 (Niven et al.,1945).The purpose of this study was to gather additional information concerning

the metabolic response of various bacterial cultures on lipid substrates by use ofthe Warburg technique.

1 Taken from data presented in a thesis submitted to the Graduate Faculty of the Uni-versity of Minnesota by J. J. Jezeski in partial fulfillment of the requirements for the degreeof Doctor of Philosophy.

This work was supported in part by a grant from the National Institute of Health,Division of Research Grants and Fellowships.

Scientific Journal Series, Paper No. 2506, Minn. Agr. Expt. Sta.2 National Institute of Health Predoctorate Research Fellow.'Present address: Department of Bacteriology, University of Illinois, Urbana, Illinois.

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J. J. JEZESKI, H. 0. HALVORSON, AND H. MACY

METHODS

Oxygen utilization was measured by means of Warburg constant volumerespirometers of the single side arm type. Conventional techniques (Umbreit etal., 1945) were followed in all experiments. The fluid volume was 3.2 ml consistingof 1.0 ml buffer, 1.0 ml cell suspension, 0.5 ml distilled water, and 0.5 ml substratein solution or suspension in distilled water in the main body of the flask; whilethe center well contained a strip of filter paper moistened with 0.2 ml of 20 percent KOH to absorb the C02 developed during the oxidation. The flasks wereshaken at the rate of 120 oscillations per minute and the temperature of thewater bath was 30 1 0.05 C.The bacteria were grown on agar in Roux type bottles. The medium consisted

of 0.3 per cent beef extract, 0.3 per cent yeast extract, 0.5 per cent peptone, and1.5 per cent agar. The reaction was adjusted between pH 6.8 and 7.0. After 24hours of incubation at temperatures appropriate for each organism, the cultureswere harvested by washing the agar surface of each bottle twice with 5 ml ofchilled salt solution at pH 7.8, prepared according to Landy and Dicken (1942).These cells were then centrifuged and washed four times, after which the finalsuspensions were made up to 30 times the volume of the packed cells with thechilled salt solution. After such treatment it was found that pre-experimentalaeration did not significantly reduce cellular respiration. The cell preparationswere stored in the refrigerator at 3 to 5 C for periods not longer than 3 days,except for certain experiments.

Natural substrates used in these experiments were butter oil, cottonseed oil,and corn oil that had been caustic-refined to remove any traces of free fattyacids. The saturated fatty acid substrates, which included the free fatty acids,methyl esters, and triglycerides, were obtained from the Eastman Kodak Com-pany, Chemical Sales Division. These compounds were used without being sub-jected to any further purification. The methyl esters of oleic and linoleic acidswere obtained from the Hormel Institute, University of Minnesota, at Austin,Minnesota. The original peroxide values were less than 1.0 for methyl oleate andbetween 4.0 and 5.0 for methyl linoleate.

Soaps of the saturated fatty acids were prepared by neutralizing small amountsof the fatty acids with M/2 NaOH. The reaction was then adjusted to pH 7.5 to7.8 before the mixture was diluted to get the concentration of soap desired.

In the case of substrates that were insoluble in water, emulsification wasnecessary in order to expose an ample substrate surface for enzyme action.These insoluble substrates were suspended in distilled water by passing the mix-ture through a hand homogenizer four times. The resulting emulsion was rela-tively stable for the period of the experiments and usually for a much longerperiod. Each substrate was used at a concentration in excess of that required toproduce maximum oxygen uptake with a given suspension of cells.

RESULTS

A number of cultures isolated from various sources were tested for theirability to take up increased amounts of oxygen in the presence of fatty substrates.

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ACTION OF MICROORGANISMS ON FATS

Of the 44 cultures tested, 30 were able to increase their respiration in the presenceof fat and phosphate buffer (pH 7.0, M/90 final concentration).From these cultures, four, of differing morphological or physiological charac-

teristics, were selected for detailed study. These included Mycobacterium phlei(MlOa), an orange-pigmented micrococcus (B4) isolated from spoiled cannedbacon, and two cultures belonging to the genus Pseudomonas (P70 and P78),which had been isolated from defective butter. The latter two cultures possessedthe reactions of atypical Pseudomonas fluorescens strains and differed betweenthemselves only in the ability to hydrolyze butterfat. The presence of a lipasewas detected by the use of Nile blue sulfate and butterfat prepared according toKnaysi's method as cited by Stark and Scheib (1936). Cultures P78 and B4exhibited very strong lipolytic action, P70 possessed weak lipolytic ability, andMl0a was not able to hydrolyze butterfat according to the method given above.

The effect of the chemical composition of the buffer. Experiments with enzymesof animal origin capable of oxidizing fatty substrates have indicated that thetype of buffer and the presence of inorganic phosphate may influence the rateof oxidation (Lehninger, 1945a,b). In respiration experiments in which intactcells are used, the choice of buffers is limited to those of inorganic nature thatcould not serve as oxidizable substrates. Thus, acetate, citrate, and glycinebuffers were eliminated immediately. Borate, phosphate, and bicarbonate buffersat M/60 final concentration were used in these experiments with coconut oil andcorn oil serving as substrates. Potassium bicarbonate was substituted for thepotassium phosphate in the chilled salt solution used in the preparation of thecell suspensions.

Table 1 presents the results of these comparisons. It may be observed thatphosphate and bicarbonate buffers produced quite similar rates of oxygen uptakewith each substrate; however, in several instances the results with bicarbonatewere slightly lower. The significance of the differences is somewhat doubtful.The use of borate buffer resulted in considerable inhibition of the oxidationof these natural triglycerides by all cultures; however, B4 was inhibited to alesser extent. The reason for the borate inhibition is unknown, although it hasbeen suggested that it may be due to interference with the phosphorylationprocess.

Effect of phosphate buffer concentration on the rate of oxidation of coconut oil.In early experiments it was observed that there was considerable change in pHduring the course of the experiments when diluted buffers (M/60 final concen-tration) were used. In order to reduce the pH shift to a minimum, increasedconcentrations of buffer were used. Table 2 summarizes the effect of varying thephosphate buffer concentration on the rate of oxygen uptake in the presence ofcoconut oil.

Cell suspensions of P78 and B4 showed similar responses in that they ap-peared to be inhibited by increased buffer concentrations. Culture P78 appearedto be much more sensitive since strong inhibition was demonstrated againstfreshly prepared suspension. On aging in the cold this inhibitory effect was in-creased up to the point where no oxygen was taken up by the substrate at the

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M/6 buffer concentration. It should be noted that cultures B4 and P78 possessstrong lipolytic activities compared with the other two cultures tested.

Cultures P70 and MlOa were grouped together by reason of their similarbehavior. With freshly prepared suspensions, the rate of oxidation increased asthe buffer concentration became greater. It would appear that the rate of oxida-tion was somewhat dependent upon the phosphate concentration, for the fresh

TABLE 1The effect of the chemical composition of the buffer on the oxygen uptake of

several cultures in the presence of natural fat substratesMICROLITERS 01 OXYGEN CONSUMED IN PRESENCE OF

CULTURE Borate buffer Bicarbonate buffer Phosphate buffer

Corn oil Coconut oil Corn oil Coconut oil Corn oil Coconut oil

MlOa 138 166 195 257 178 297P70 _ 111 - 236 - 221P78 13 39 - 217 178 272B4 64 60 72 77 90 101

Data are corrected for endogenous respiration.Time, 90 min; buffer, pH 8.0, M/60 flask conc.; substrate, 10 mg per flask.

TABLE 2The effect of concentration of buffer on the oxygen consumption in the presence

of coconut oilMICROLITERS OF OXYGEN CONSUMD IN THE PRESENCE Ol

CULTURE SUBSTRATE./6 buffer x/12 buffer x/24 buffer K/48 buffer(final conc.) (final conc.) (final conc.) (final conc.)

MlOa Coconut oil 155 135 80 92None* 27 16

P70 Coconut oil 122 117 108 103None* 48 62

P78 Coconut oil 78 98 127 177None* 27 41

B4 Coconut oil 108 106 103 142None* 13 9

Time, 90 min; buffer (Clark's phosphate), pH 7.8; substrate,diluted to 1/60 in this experiment).

* Endogenous respiration.

10 mg per flask (cells

culture at least. As the suspensions were aged in the cold, this relationshipgradually disappeared.

The effect of the composition of natural and pure triglycerids on oxygen consump-tion. There are indications in the data previously presented that the type offat influences the oxygen uptake in a given bacterial culture. Thus, butter, corn,and coconut oils were compared as substrates in the presence of phosphate buffer.Butter oil contains large amounts of oleic and the saturated, long-chain (C14 to

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C18) fatty acids and is also relatively rich in the short-chain saturated acids,whereas coconut oil contains mostly saturated, long-chain (Cn to C16) fatty acids.Corn oil is composed of a very large percentage of the C18 unsaturated fattyacids, oleic and linoleic.The data are summarized in table 3. It is evident in all cases that the coconut

oil was oxidized at a greater rate than either corn or butter oil. The oxygenuptakes with corn and butter oil were almost identical in the case of three cul-

TABLE 3The influence of the type of natural fat on the oxygen consumption of four

cultures of bacteriaMICROITERS O OXYGEN CONSUMED IN PRESENCE OCULTURE ______________________

Butter fat Corn oil Coconut oil

MlOa 274 352P70 44 68 75P78 293 270 389B4 91 86 157

Data are corrected for endogenous respiration.Time, 90 min; buffer (Clark's phosphate), pH 7.8, M/60, flask conc.; substrates, 10 mg

per flask.

TABLE 4A comparison of oxygen uptake rates on various triglycerides

MICRtOLTRS 01 OXYGEN CONSUMED

Culture ................. MlOa P70 P78 B4Final buffer conc......... x/48 m/6 x/48 X/6 M/48 X/6 x/48 m/6

SubstrateTriacetin ........ 196 216 194 51 95 -33 22 -35Tributyrin ....... 108 267 124 -45 -24 -44 -3 -34Trilaurin........ 129 163 111 118 241 -11 174 -9Tristearin ....... 29 37 11 118 1 -12 7 4Coconut oil...... 138 277 112 128 233 -12 148 -4

Data are corrected for endogenous respiration.Time, 90 min; buffer (Clark's phosphate), pH 7.8; substrates, 25 mg per flask.

tures; the exception was culture P70, with which corn oil caused a significantlygreater uptake than butter oil. Even though corn oil and butter oil both containlarge amounts of unsaturated acids, other explanations for their similar behavioras substrates are not excluded by the data obtained.Four pure triglycerides of fatty-acid carbon-chain lengths varying from 2

to 18 carbon atoms were also used as substrates. Two buffer concentrations wereemployed to determine whether the changes observed on coconut oil also occurredwith the various pure triglycerides. The data are presented in table 4. In thepresence of M/48 buffer the strongly lipolytic cultures B4 and P78 again behaved

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similarly. Triacetin was weakly or moderately attacked, but there was no oxida-tion and even some inhibition of endogenous respiration in the presence of tri-butyrin. Trilaurin was oxidized at a vigorous rate, but no significant amount ofoxygen was taken up in the presence of tristearin. P70 and Ml0a followed anidentical pattern in oxidizing these substrates. Triacetin was oxidized at thegreatest rate. The rate decreased as the length of the carbon chain in the com-ponent fatty acid increased until tristearin was only weakly attacked. The useof M/6 buffer elicited the same response from these organisms on pure triglycer-ides as on coconut oil. The concentrated buffer stopped oxygen consumption inthe presence of the triglyceride with cultures B4 and P78; and in several instancesinhibition of endogenous respiration was evident. The results with P70 are notso clean-cut since there was some inhibition with triacetin and tributyrin as sub-strates, whereas none was shown with trilaurin, tristearin, and coconut oil. Anincrease in oxygen uptake due to increased buffer concentration was observed forculture MlOa in the presence of all substrates.There is the possibility that glycerol arising from lipase action may be the

oxidizable substrate in these experiments. Glycerol and coconut oil. were com-pared as substrates for the four cultures. Glycerol was oxidized very vigorouslyby culture B4, to a lesser extent by P70 and Ml0a, and relatively weakly byP78. A comparison showed that glycerol was oxidized by P78 at about one-fourth and by P70 and Ml0a at approximately one-half the rates observed withcoconut oil. On the other hand, B4 oxidized glycerol at twice the rate of coconutoil. Significant oxygen consumption due to glycerol produced when triglycerideswere used as substrates could be eliminated in the case of MlOa due to theabsence of active lipase and with P78 because of a low rate of oxidation of glyc-erol.The results obtained with natural fats and pure triglycerides indicate that

the composition of these substrates significantly influences oxygen consumptionby the cultures studied. However, no regular order of substrate specificity couldbe determined.

Oxidation of saturated fatty acid esters. Methyl and ethyl esters of the saturatedfatty acids were used in further substrate specificity studies. Results obtainedwith methyl alcohol and ethyl alcohol controls demonstrated that the action ofan esterase was unimportant in influencing the results obtained, since none ofthese cultures was able to cause a significant uptake of oxygen with either ofthese alcohol substrates. The data are presented in table 5. The four culturesresponded in a similar manner toward most of the substrates used. In general,it appears that there may be two enzyme systems responsible for the oxidationof these fatty acid esters. As the carbon chain of the fatty acid increased inlength, some oxygen consumption was observed with methyl acetate but littleor no oxidation was observed with the butyric and caprylic esters. The oxygenconsumption rates then increased to a maximum with esters from lauric topalmitic and fell again with the use of the stearic ester. Thus, in the presence ofM/48 phosphate buffer, two maxima were observed-one with the short-chainesters, usually acetic, and one with the longer-chain esters, lauric, myristic, orpalmitic.

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The use of M/6 phosphate buffer produced marked inhibition of oxygen con-sumption with most of the substrates in the case of culture P78, but culture P70was affected only when propionate and caprylate esters were used as substrates.

TABLE 5A comparison of oxygen uptake rates on various saturated fatty acid esters

MICROIITERS OF OXYGEN CONSUMED

Culture..................................... MlOa P70 P78 B4

Final buffer conc................................ x/48 m/48 m/6 ii/48 x/6 m/48

SubstrateMethyl acetate ................... 196 120 108 229 26 66Methyl propionate................ 154 108 30 203 10 4Methyl butyrate.................. 34 20 21 120 15 31Methyl caprylate................. 6 19 -62 -35 -37 -5Ethyl laurate..................... 115 182 149 249 -19 23Ethyl myristate.................. 202 98 144 273 -12 32Methyl palmitate................. 115 156 76 183 -24 13Methyl stearate ......... ......... 79 38 81 151 -21 -6Coconut oil....................... 144 176 144 283 -14 202

Data are corrected for endogenous respiration.Time, 90 min; buffer (phosphate), pH 7.8; substrates, 20 mg per flask.

TABLE 6A comparison of rates of oxygen consumption in the presence of sodium soaps

of saturated fatty acidsMICIRLITElLS OF OXYGEN CONSUXED

Culture.................................... MlOa P70 P78 B4

Final buffer conc.............................. x/48 m/48 m/48 x/6x /48SubstrateSodium acetate .......... ....... 108 546 -23 -17 18Sodium propionate.............. 69 327 64 -16 10Sodium butyrate................. 124 104 32 -21 4Sodium caproate................ 133 184 79 -19 14Sodium caprylate............... 276 302 120 -21 19Sodium caprate................. 219 292 117 -18 -16Sodium laurate .................. -41 286 102 -12 -18Sodium myristate............... 255 403 93 -2 12Sodium palmitate............... 260 320 61 -27 9Sodium stearate................. 153 369 52 -19 -10

Data are corrected for endogenous respiration.Time, 90 min; buffer (Clark's phosphate), pH 7.8; substrates, 0.033 M, flask conc.

The results obtained with culture B4 indicated a very low activity towardthese esters as compared with glycerol, natural fat, or pure triglyceride (trilaurin).In addition, it was the only culture tested that did not oxidize ethyl laurate atthe same rate as trilaurin or coconut oil. These observations plus the fact that

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glycerol was utilized much faster than coconut oil by this culture is evidence thatthe oxygen consumption of this organism in the presence of fat was due princi-pally to the oxidation of glycerol produced by the action of lipase.

Oxidation of the sodium salts (soaps) of the saturated fatty acids. The form of thesubstrate may be important in affecting the rate of oxygen consumption in theseexperiments. This is apt to be true particularly if the form of fatty acid substrateis changed from an ester to a sodium salt, since some of the physical and chemicalproperties of these compounds are quite different. Homogenizing an ester in

TABLE 7The influence of pH of buffer on oxygen consumption of several cultures in the

presence of saturated fatty acid estersMICROLITERS OF OXYGEN CONSUMED AT

CULTURE

pH 5.8 pH 6.1 p16.5 pH 7.0 pH 7.5 pH 7.8 pH 8.0

Methyl acetate

MlOa 126 111 104 102 99 107 91118* 86 94

P70 84 88 80 91 102 112 10638* 26 47

P78 98 81 78 95 101 89 9661* 34 64

B4 73 77 70 82 102 102 9146* 39 64

Ethyl laurate

MlOa 125 112 117 135 130 168 12286* 72 143

P70 218 233 238 249 245 251 226173* 175 169

P78 232 252 262 273 276 279 274178* 207 246

B4 76 81 71 74 89 93 8555* 38 84

Time, 90 min; buffer (Clark's phosphate), M/48, flask conc.; substrates, 0.033 M, flaskconc.

* All similar values corrected for endogenous,respiration.

distilled water produces an emulsion, whereas the sodium salts produce eithertrue solutions or colloidal solutions depending on the fatty acid involved.

Table 6 presents the data obtained on the activity of these cultures towardthe sodium salts of the saturated fatty acid series. There is little question thatthe sodium salts were utilized to much the same extent as the methyl esters,with, of course, a few individual exceptions. There is not, however, the consis-tency of results that is obtained with the methyl esters nor are the two maximain rate of oxygen uptake so apparent. Whereas with the esters the two maximawere fairly well defined with all cultures, the results on the sodium salts showed

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two definite maxima with only two of the cultures. As in the case of the methylesters, culture B4 was able to utilize these substrates only to a very limited extent.The inhibition by M/6 phosphate buffer is again demonstrated on this type ofsubstrate with culture P78.

Influence of pH on the oxidation of fatty acid esters. The effect of pH on thecourse of various enzyme reactions is well recognized. Esters of saturated fattyacids were used as substrates. Sodium salts are unsatisfactory because some areconverted to the free acid form at the pH levels used in these experiments.The results of these pH studies, using acetic and lauric esters, appear in table

7. With methyl acetate as a substrate and in the presence of a phosphate buffer,cultures B4 and P70 showed optima in the range of pH 7.4 to 7.8, whereas Ml0aexhibited an optimum at pH 5.8 or below. The data on P78 demonstrate oneoptimum at or belowppH 5.8 and another from pH 7.0 to pH 8.0. On ethyl laurate,

TABLE 8The influence of pH of buffer on oxygen constumption in the presence of pure

triglycerides and glycerolMICROLITERS OF OXYGEN CONSUMED AT

CULTURE SUBSTRATEpH 5.8 pH 6.2 pH 6.6 pH 7.0 pH 7.4 pH 7.6 pH 7.9

MlOa Triacetin 38 55 80 114 120 130 12924* 70 113 111

P70 Triacetin 155 167 170 201 220 269 296116* 122 212 216

P70 Trilaurin 192 244 261 271 286 317 362159* 216 251 275

P70 Glycerol 98 96 110 131 174 219 25367* 57 132 145

Time, 90 min; buffer (Clark's phosphate), m/48, flask conc.; substrates, 0.033 M, flaskconc.

* All similar values corrected for endogenous respiration.

the maximum rate of oxygen consumption took place in the vicinity of pH 7.4to 7.8 with culture B4, and pH 7.0 to 8.0 with P78. Relatively uniform activitywas recorded for P70 over the pH range studied. A rather sharp maximum at pH7.8 was shown by MlOa. Thus for this latter organism, at least, this is evidencethat the same enzyme does not attack acetate and laurate and that several en-zymes may act on substrates of the saturated fatty acid series.Table 8 presents a comparison of several pure triglycerides and glycerol as

substrates at pH levels similar to those previously used. These results includedata obtained on only two cultures, MlOa and P70. The data indicate that thetriglycerides tested and glycerol respond to changes in pH in a similar manner.Culture MlOa shows different responses when methyl acetate and triacetin areused as substrates and this, therefore, is evidence that the same enzyme systemdoes not attack triacetin and methyl acetate. The results obtained with cultureP70 are not so clear-cut in this respect.

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Experiments with unsaturated substrates. The oxygen consumption rates in thepresence of methyl esters of stearic, oleic, and linoleic acids were compared todetermine whether the degree of unsaturation would affect the rate of oxidation.Table 9 presents these results and some of the information is in harmony with

TABLE 9The influence of the degree of saturation of fatty acid esters on oxygen

consumption by several bacteriaICRlOITElRS OF OXYGEN CONSUMED IN MME PRESENCE OF

CUAGE(DAYS) Methyl stearate Methyl oleate Methyl linoleateFlask buffer conc.

x/6 x/48 M/6 x/48 _x/6 x/48

MlOa 3 40 48 100 128 131* 156*5 61 65 130 118

-23t -14tP70 3 78 89 45 249 154 315

6 48 168 66 254 94 306P78 1 62 180 46 122 60 154

3 -7 99 -5 77 83 170B4 1 24 18 15 10 1 2

These data are corrected for endogenous respiration and autoxidation of the unsat-urated esters.

Time, 90 min; buffer (Clark's phosphate), pH 7.8; substrates, 0.017 M, flask conc.* Peroxide value, methyl linoleate = 4.0 to 5.0 milliequivalents per gram.t Peroxide value, methyl linoleate = 19.0 to 20.0 milliequivalents per gram.

TABLE 10The influence of the pH of the buffer on oxygen consumption of culture P78 in

the presence of several fatty acid estersMICROLITERS OF OXYGEN CONSUMD AT

pH 6.1 pH 6.5 pH 7.0 pH 7.4 pH 8.0

Methyl stearate 119 133 144 142 14389* 109 101

Methyl linoleate 141 131 131 141 19595* 89 143

Time, 90 min; buffer (Clark's phosphate), M/48, flask cone.; substrates, 0.033 m, flaskcone.

* All similar values corrected for endogenous respiration.

data described earlier. Culture B4 produced only a small amount of oxidation onstearate and oleate, and linoleate was untouched. According to these data theoxidative ability of this organism toward the Cis esters decreased as the amountof unsaturation increased. The rate of oxidation of these esters by culture P70increased directly with the amount of unsaturation in the presence of M/48

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buffer. A similar response was shown by, culture Ml0a. P78 does not show thesame type of results since unsaturation does not cause increased oxygen consump-tion with young cells.A comparison of the results obtained with M/6 and M/48 buffer indicates that

the inhibition of oxidation of unsaturated substrates also takes place just as withother substrates tested in the case of two cultures, P70 and P78. It is of interestto note that with P78, though there is complete inhibition of oxygen uptake in thepresence of stearate and oleate with M/6 buffer, the linoleate undergoes consider-able oxidation. It should be noted that the actual decrease in oxygen utilized inthe presence of linoleate due to the use of the M/6 buffer is about equal to that de-crease observed in the case of complete inhibition with stearate and oleate as asubstrate. These results are, therefore, indicative of the presence of an enzymecapable of oxidizing linoleate (probably at the double bonds) that was rela-tively uninbibited by the higher concentration of phosphate.The peculiar results obtained with Ml0a on the linoleate substrate cannot be

explained readily except on the basis of inhibition due to autoxidation productsin the substrate. It should be observed that the peroxide values on the substrateincreased from 4.0 to 5.0 up to 19.0 to 20.0 milliequivalents per gram in the2-day interval. Several later experiments have shown that the inhibition takesplace only in the presence of substrate that showed evidence of autoxidation.

Further evidence that P78 may contain an enzyme specific for linoleate wasgathered in studies on pH optima. Table 10 shows the response of this cultureto various pH levels in the presence of stearate and linoleate esters. Methylstearate appears to be oxidized at maximum rates over a broad range of pH 7.0to 8.0, and the same is true for ethyl laurate. On the other hand, linoleate showsa sharp increase in oxygen consumption above pH 7.4, and the optimum appearsto be at or above pH 8.0 in the presence of phosphate buffer.

DISCUSSION

The results obtained in the experiments on buffer type comparisons may becompatible with those obtained on fat-oxidizing enzymes of animal origin,namely, that inorganic phosphate was required if adenylic acid, and not adenosinetriphosphate (ATP), was present. The results observed in the experiments couldbe explained on the basis that sufficient ATP was present in the cells so thatlittle difference was observed between the bicarbonate and the phosphate buf-fers, especially since the strength of the phosphate buffer was relatively weak.However, stimulation of oxidation by increased phosphate buffer concentrationsin the case of two cultures does indicate that inorganic phosphate may be in-volved in the process. The inhibition by borate buffer observed with the fourcultures studied is likewise observed in the case of an enzyme from rat livercapable of oxidizing long-chain fatty acids.The inhibition of oxygen consumption by M/6 phosphate buffer in the presence

of natural and pure triglycerides and saturated fatty acid esters and salts, aswell as unsaturated esters, occurred with the two strongly lipolytic cultures, B4

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and P78. Just what role a lipase plays in this phenomenon is questionable;nevertheless, the well-known inhibitory effect of phosphate buffer on the actionof lipases is not to be overlooked in this regard. A better explanation may liein the effect of the phosphate on some component of the respiratory system ofthe susceptible organism, for example, the effect of the buffer salts on certaintrace elements (Mn++ and Mg++) generally supposed to be required for fattyacid oxidation. This seems quite logical since the inhibition may take place in thepresence of a variety of fatty substrates and glycerol.However, the use of the sodium salts or soaps of the fatty acids presented

some problems since the method of preparation might influence their propertiesas substrates. The pH adjustments were particularly troublesome with thoselonger-chain soaps which formed colloidal systems at the pH values used inthese experiments. It is well known that the physical state of the micellesis influenced by the method of producing and neutralizing the soaps. The chargeon the micelle is likewise influenced by the pH of the system and the directionin which the pH is shifted. The inconsistencies, when vigorous oxidation oc-curred with one member of the series and inhibition of respiration took placewith the next homologue, are probably due to these inherent complexities of thesubstrate system.

In spite of these difficulties with the sodium salts, the data do show thatthe cultures studied differ in their behavior toward the various substrates used.Although the four cultures did not oxidize the substrates at equal rates, they ex-hibited similar relative behavior toward the various homologues of the series.The data obtained on the esters of the saturated fatty acids indicate that atleast two different enzymes are responsible for the oxidation of the members ofthe series. This statement is supported by data obtained in studies of substratespecificity and pH optima. Even though the pH optima recorded for the variouscultures were in general not sharply defined, it seems logical to assume that theyrepresent true optima, especially since, in all instances, the values corrected forendogenous respiration present the same picture.When unsaturated substrates were used, several cultures exhibited greater

oxidative activity as the amount of unsaturation increased. The data obtained onculture P78 indicate the possibility of the existence of an enzyme acting on thelinoleate that is different than the one attacking stearate, both from the resultsobtained on pH optima and the inhibition produced by M/6 phosphate buffer.The degree of inhibition produced by phosphate buffer on stearate and oleate ascompared with linoleate are indicative of the presence not only of an enzymecapable of ,8-oxidation according to the classical scheme, but also an enzymespecific for the two double bonds, since it is only in this respect that the sub-strates differ. Such an enzyme has been demonstrated in several natural materials.The peculiar inhibition of culture MlOa due to autoxidized linoleate wouldalso indicate that an enzyme specific for linoleate is present, especially sincemixing oxidized linoleate with stearate does not produce competitive inhibi-tion.

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SUMMARY

Four bacterial cultures of differing morphological and physiological charac-teristics were selected for detailed study from 30 cultures capable of showingincreased rates of oxygen consumption in the presence of fat.These organisms demonstrated similar rates of oxygen uptake in the presence

of bicarbonate and phosphate buffers (M/60, final concentration) but were defi-nitely inhibited in the presence of borate buffer. Increased concentrations of phos-phate buffer produced greater rates of oxygen consumption with coconut oil asa substrate in the case of two cultures. On the other hand, the two stronglylipolytic cultures were inhibited in their oxygen consumption by the increasedconcentrations of phosphate buffer (up to M/6) in the presence of all types oflipid substrates tested.

Coconut oil was oxidized by the four cultures at a greater rate than eithercorn oil or butterfat, whereas four pure triglycerides were attacked at varyingrates. Good evidence has been obtained that oxygen consumption in the presenceof fats may be due to the utilization of glycerol resulting from the action oflipase. This applies particularly to the Micrococcus culture (B4).The data indicate that at least two enzymes are involved in the oxidation of

compounds of the saturated fatty acid series by the individual culture; one isspecific for the short-chain and one for the long-chain fatty acids.The pH optima for the oxidation of the saturated fatty acid substrates by

Mycobacterium phli were in the vicinity of pH 7.8 for ethyl laurate and aboutpH 5.8 for methyl acetate. When triacetin was used as a substrate, the optimumwas in the range of pH 7.6 to 7.9. The other cultures showed optima in the rangeof pH 7.4 to 7.9 with most of the substrates used in these pH studies.The presence of an enzyme specific for methyl linoleate is suggested by the

data obtained on a Pseudomonas culture (P78). There is some evidence thatMycobacterium phlei (MlOa) may posses a similar type of enzyme.

REFERENCESBARRON, E. S. G., AND FRIEDEMANN, T. E. 1941 Studies on biological oxidations. XIV.

Oxidations by microorganisms which do not ferment glucose. J. Biol. Chem., 137,593-610.

CAST1ELL, C. H., AND GARRARD, E. H. 1941 The action of microorganisms on fat. III.Oxidation and hydrolysis of triolein by pure cultures of bacteria. Can. J. Research,19C, 106-110.

HOROWITZ-WLASSOVA, L. M., AND LIVSCHITZ, M. J. 1935 Zur Frage der Wirkung derMikroben auf Fette. Zentr. Bakt. Parasitenk., II, 92, 424-435.

JENSEN, L. B., AND GRETTIE, D. P. 1933 Action of microorganisms on fats. Oil andSoap, 10, 23-32.

JENSEN, L. B., AND GRETTIE, D. P. 1937 Action of microorganisms on fats. Food Re-search, 2, 97-120.

LANDY, M., AND DICKEN, D. M. 1942 A microbiological assay method for six B vitaminsusing Lactobacillus casei and a medium of essentially known composition. J. Lab.Clin. Med., 27, 1086-1092.

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LEHNINGER, A. L. 1945a The relationship of the adenosine polyphosphates to fatty acidoxidation in homogenized liver preparations. J. Biol. Chem., 157, 363-381.

LEHNINGER, A. L. 1945b On the activation of fatty acid oxidation. J. Biol. Chem.,161, 437-451.

MAZZA, F. P., AND CIMMINO, A. 1933 Sull'attivita deidrogenasica del B. coli communissugli acidi grassi superiori. Boll. soc. ital. biol. sper., 8, 531-534.

MUNDT, J. O., AND FABIAN, F. W. 1944 The bacterial oxidation of corn oil. J. Bact.,48, 1-11.

NIVEN, C. F., JR., EVANS, J. B., AND WHITE, J. C. 1945 Oxidation of butyric acid bystreptococci. J. Bact., 49, 105.

PIGUIWSKI, G., AND CHARIK, N. 1929 Zersetzung des Olivenols, unter dem Einflussder vitalen TAtigkeit, einiger Mikroorganismen: Umwandlung von Oleinsiure in Ke-tostearinsiure. Biochem.. Z., 200, 201-210.

QUASTEL, J. B., AND WHETHAM, M. D. 1925 Dehydrogenations produced by resting bac-teria. I. Biochem. J., 19, 520-531.

SINGER, T. P., AND BARRON, E. S. G. 1945 Studies on biological oxidations. XX. Sulf-hydryl enzymes in fat and protein metabolism. J. Biol. Chem., 157, 241-253.

STARK, C. N., AND SCHEIB, B. J. 1936 A study of fat-splitting and casein-digesting bac-teria isolated from butter. J. Dairy Sci., 19, 191-213.

UM[BREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F. 1945 Manometric techniques andrelated methods for the study of tissue metabolism. Burgess Publishing Co., Minne-apolis.

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