Effects of lipids, fatty acids, and other detergents on ...

Effects of lipids, fatty acids, and other detergents on bacterial utilization of hexadecane COLETTE BREUIL'A N D D O N NJ. KUSHNER? Deptrrlt~letliof'Biolug.~, Utlicrrsiiy ( ~ ~ ' O ~ ~ NOIi Ii n. (~It,' ( rO, I I I . . C I I ~ I I I I I N K I N hN5

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Accepted October l I . 1979 B R E U I LC.. . and D. J. K U S H N E R1980. . Effects of lipids. fatty acids, and other detergents on bacterial utilization of hexadecane. Can. J. Microbiol. 26: 223-231. Fatty acids. lipids. and detergents affected hexadecane utilization by Ac~itrciohrrcrc,rIit*oJfi and Psc~rrt101~1ot1o.s rrc,rrrginosrr. Long-chain fatty acids (palmitic, oleic, and stearic) stimulated hexadecane utilization, as did triglycerides (tripalmitin, trioctanoin. trilaurin. and olive oil), lecithin. lysolecithin, hexadecanol, and wax esters (palmityl palmitate and palmityl stearate). i. acid Hexadecane in turn stimulated the utilization oftripalmitin forgrowth by A . 1 1 1 ~ d fPropionic caused diauxic growth. apparently being utilized before hexadecane. Isobutyric acid was not used for growth and inhibited hexadecane utilization. Caprylic and lauric acids, octan-2-01, and phenethyl alcohol inhibited hexadecane utilization. The detergents Triton X-100, Brij 35. and FL-70 were not themselves used for growth, but stimulated growth on hexadecane. In A . 111,oJfi.palmitic acid suppressed diauxic growth on a mixture of ethanol and hexadecane if added while ethanol was being oxidized, but not if added subsequently. Lecithin also suppressed diauxic growth. but Triton X-100 did not, though i t shortened the lag period before hexadecane utilization. Washed suspensions of cells grown on ethanol or hexadecane could not oxidize hexadecane unless Triton X-100 was also added. Then hexadecane-grown cells oxidized hexadecane about 3 times as fast as ethanol-grown cells. The results suggest that fatty acids and otherlipids stimulate growth on hexadecane partly. but not solely. by acting as emulsifying agents. They may also act as inducers of enzymes necessary for hexadecane breakdown. or they may be incorporated into cell membranes and change their properties so that hexadecane can more readily enter the cells. BREUIL. C.. et D. J . KUSHNER. 1980. Effects of lipids. fatty acids, and other detergents on bacterial utilization of hexadecane. Can. J. Microbiol. 26: 223-23 1. L'utilisation de I'hexadecane par Ac~it~c~iohrrcic~r 11t~oJfiet Psercdorrlotlcr.s (rer~rgitlosrretait effectuee par les acides gras, les lipides et les detergents. Les acides gras a chaines longues (palmitique, oleique et stearique) stimulaient I'utilisation de l'hexadecane au meme titre que les triglycerides (tripalmitine, trioctanoine. trilaurine et huile d'olive), la Iecithine, la lysolecithine, I'hexadecanol et les cires (palmitate de palmityle et stearate de palmityle). De son chte, I'hexadecane stimulait I'utilisation de la tripalmitine par une culture de A . In~offi.L'acide propionique provoquait une diauxie: il etait apparemment utilise avant I'hexadecane. L'acide isobutyrique n'etait pas utilise pour la croissance et inhibait l'utilisation de I'hexadecane. Les acides caprylique et laurique de mime que I'octan-2-01 et I'alcool phenethylique inhibaient I'utilisation de I'hexadecane. Les detergents Triton X-100, Brij 35 et FL-70 n'etaient pas euxmemes utilises pour la croissance mais ils stimulaient la croissance sur hexadecane. Chez A . Itvoffi. I'acide palmitique supprimait la croissance en diauxie sur un melange ethanol-hexadecane lorsqu'il etait ajoute au moment ou I'ethanol etait en voie d'oxydation; une addition ulterieure n'entrainait pas cet effet. La Iecithine supprimait aussi la diauxie. contrairement au Triton X-100. ce dernier ecourtait cependant la periode de latence precedant I'utilisation de I'hexadecane. Apres avoir ete produites sur ethanol ou hexadecane et avoir subi un lavage. les suspensions cellulaires ne pouvaient oxyder I'hexadecane a moins que du Triton X-100 ne soit ajoute. A ce moment. les cellules ayant ete produites sur hexadecane oxydaient ce substrat environ 3 fois plus rapidement que les cellules produites sur ethanol. Les resultats suggerent que les acides gras et d'autres lipides stimulent la croissance sur hexadecane en agissant entre autres comme emulsifiants. Ils peuvent aussi agir en induisant la production d'enzymes necessaires a la degradation de I'hexadecane ou en itant incorpores aux membranes cellulaires et en modifiant leurs proprietes afin de faciliter I'entrCe de I'hexadecane dans les cellules. [Traduit par le journal]

Introduction One of the major problems facing microorganisms that degrade hydrocarbons is how to 'Present address: National Research Council of Canada, Ottawa, Ont., Canada KIA OC6. 'Author to whom requests for reprints should be sent.

make proper contact with such insoluble substrates. Many of the microorganisms that can grow on petroleum or individual hydrocarbons as sole carbon source are able to produce substances that lower the surface tension ofthe growth medium and may serve to emulsify the growth substrate (Goma

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ct (11. 1973; Guire pt ( i / . 1973; Hisatsuka et a / . 1977; added separately before the experiment. The center well con0 . 2 m L 10% KOH. The substrate was introduced from suzuki et 1969; zajic et 1974; zajic c.t n l . tained the side arm of the Warburg flask. 1977). In experiments with radioactive compounds, flasks were We previously found that growth of bacteria on closed with a two-holed stopper. a slow stream of sterile air was alkanes was correlated with lipase production. O f a passed into the shaken flasks. and the air leaving the flasks number of freshwater bacteria, only those that passed through a Vigreux (Burnisson and Perez 1974) column a mixture of methanol and phenethylamine to trap could produce lipase could grow on hexadecane as containing CO,. The I4CO2trapped in the mixture was measured by liquid carbon source (Breuil et 1978). We ob- scintillation (Peterson 1969). Growth was measuredas turbidity served that Acinetobacterl~i~ojfi and Pse~rrfornor~as(610nm. 16-mm cuvette) as previously described (Breuil and (ierlrgirlo.y(i exhibited diauxic growth i n the pres- Kushner 1973, o r by determining the protein content of the ence of a mixtuse of ethanol and hexadecane. ~h~ culture. Protein was determined by the method of Lowry pr (11. ( 195 I), with bovine serum albumin (Sigma Chemicals, S t . Louis, and during the period Of MO) as standard. As noted before (Breuil ~t trl. 1978)there was a was used ethanol utilization no hexadecane was used. During strong correlation between increase in protein and optical denthe period of ethanol utilization, little lipase was sity, after an O D of 0.4 had been reached. In those experiments formed, but substantial ]ipase formation preceded in which growth was measured mainly by optical density. results were checked by protein concentration measurements. All exhexadecane utilization (Breuil et 1978). Such periments were repeated a t least twice; representative results results suggested, though they did not prove, that ,,,given. lipase was needed in some way for the utilization of Hexadecane (Humphrey Corp., North Haven, C T ) and hexadecane and other alkanes. Clearly, lipase can- analytical grade fatty acids and lipids were included in cultures not be acting directly upon the hydrocarbon. one at concentrations specified. Radioactive hexadecane was added to the mineral medium by mixing equal volumes of [I-'4C]hexapossibility considered was that lipase acted to make decane (0.1 14 mcilmol ( I Ci = 37 G B ~ )~, e England w Nuclear fatty acids which then acted as detergents in emul- carp., Boston, MA) and unlabelled hexadecane (final specific sifying the hydrocarbon substrate. As a continua- activity 0.057 mCilmoI), sterilizing by autoclaving at 15 Iblin." tion of this work, we have the effect of ( I Iblin.' = 6.89 kPa) for 15 min, then adding 0.5 mL t o 50 m L Radioactive tripalmitin, made by adding 1.6 mg certain fatty acids, lipids, and detergents on hydro- medium. [cclrbox~~l-14C]tripalmitin (12.7 Cilmol, New England Nuclear utilization two A . Corp.) to 23.4 mg unlabelled tripalmitin (final specific activity h110jfiand P . clerugirzosn. 813 mCilmol), was used at a concentration of 0.05% (wlv) in Materials and methods 0r~gcrt1i.stn.tcrrltl gron,th c o r ~ t l i t i o r ~ ~ Acir~rtoboctc~r In7offi O I 6 ,a facultative psychrophile described previously (Breuil ~t (11. 1975). and P. crerrcgirlosn, NRCC 5005, supplied by the National Research Council of Canada, were used in these studies. They were conserved on a semisolid medium (Breuil and Gounot 1972). Before being cultured on hydrocarbons, cells were grown for 48 h in complex liquid medium (2% peptone and 0.15% yeast extract, pH 7.0). One millilitre of this culture was transferred to 100 m L of mineral medium with hydrocarbon (containing (in glL) N H 4 N 0 3 , 2.0; K H 2 P 0 4 ,4.0; N a 2 H P 0 4 ,6.0; MgS04. 7 H 2 0 ,0.2; F e S 0 4 . 7 H 2 0 , 0.01; CaCI,. 2 H 2 0 , 0.05; hydrocarbon, 8; and NaOk; to achieve pH 7.5 f 0.1). which served as a preculture. The mineral medium used was that of Makula and Finnerty (1968) except that ( N H 4 ) 2 S 0 4was replaced by N H 4 N 0 3 . When the optical density of the preculture reached 0.8 (at 610 nm), 1.0 m L was transferred into 100 mL of the mineral medium containing the appropriate additions for studying hydrocarbon utilization. Growth in liquid cultures was carried out in Erlenmeyer flasks containing 20% volume liquid. The flasks were agitated on a reciprocal shaker at one hundred 2-cm strokes per minute at 20 f 2°C for A. I~rloffiand at 34 f 0.5"C for P. uerugirlosn. In experiments on the oxidation of hexadecane, at the end of the exponential phase the cells were centrifuged under aseptic conditions, washed, and resuspended in mineral medium without carbon source ( p H 7.0) to reduce the endogenous respiration. The cells were starved thus for 16 h after growth on hexadecane and for4 h after growth on ethanol. Theoxidation of hexadecane was investigated using a Gilson Warburg respirometer maintained at 20 k 0.5"C. The main compartment of each vessel contained 3 mL of bacterial cells; the side arm contained I mL of mineral medium to which the substrate was

mineral medium. Radioactive palmitic acid, made by adding 0.192 mg palmitic [I4C]acid (177 mCilmol, New England Nuclear c&.) to 49.8 mg unlabelled palmitic acid (final specific activity 177 mCilmol). was used at a concentration of 0.05% wlv. In experiments with [I-14C]ethanol,1 0 p L of this compound (5.85 mCilmol, New England Nuclear Corp.) was added to 0.05 m L of ethanol in 50 mL of mineral medium. These tracers were added to show the rir~lc,that oxidation of each substance began. rather than to account for the fate of each substrate. Calculations showed that only a fraction of each labelled substrate was released as CO,, the rest presumably being incorporated into cells o r cell products. In order to determine the amount of hexadecane remaining after growth, cultures were shaken with 4 volumes of 11-hexane, the hexane phase was separated by centrifuging, and extraction was carried out 2 more times. Under these conditions, hexane removes extracellular hydrocarbons (Walker and Cooney 1973). T h e extract also contained fatty acids and lipids, which were removed by passage through a silica gel column (Kates 1972). After concentrating t h e hexane eluate to dryness. the remaining hexadecane was determined by weighing. Thedetergents Brij 35 and FL-70 were obtained, respectively, from Atlas Chemical Industries Canada and from Fisher Scientific Co.

Results We showed previously that A . flzwfj could use a number of different alkanes as carbon sources for growth, and that among the different hydrocarbons hexadecane gave the highest growth yield in the shortest time at 20°C (Breuil et 01. 1978). If the inoculum used came from a single preculture on hexadecane, as it did in the experiments to be de-

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scribed, a lag phase of about 100 h preceded growth, although this could be shortened by several transfers on hexadecane (Breuil et a / . 1978). The effect of a number offatty acids and triglycerides on growth of A . lwof$ was tested (Table 1). For those substances that stimulated growth, the most striking effect was a reduction in the long lag phase. Such an effect is clearly seen with the long-chain fatty acids, palmitic and oleic (Fig. 1). The stationary phase was reached after 150 h when only hexadecane was present, but after only 50 h if 0.05% palmitic or oleic acids were also present. Similar results were obtained with stearic acid (not shown). Triglycerides (tripalmitin and olive oil) also stimulated growth on hexadecane (Fig. 2). At 20°C Time (h) tripalmitin, which is a solid, was not used for growth. However, it was used if hexadecane was FIG. 1. Growth of Acirzetohrrcfrr I\coJfi on 0.05% oleic acid present. In experiments with mixtures of I4C- (OA); 0.05% oleic acid plus 0.8% hexadecane (OA + HD); 0.870 labelled tripalmitin and unlabelled hexadecane, or hexadecane (HD); 0.0570 palmitic acid (PA);and 0.05% palmitic with labelled hexadecane and unlabelled tripal- acid plus 0.8% hexadecane (PA + HD). mitin, tripalmitin was used slightly before hexadecane, as judged by I4CO, release. Then the lipid and hydrocarbon were used simultaneously and finally, towards the end ofgrowth, lipid utilization slowed (Fig. 3). Analysis of the hexadecane remaining at the end of these experiments (Table 2) showed that half or more had been used. The presence of small amounts of tripalmitin or of palmitic or oleic acids increased the amount of cellular protein per gram of hexadecane used, probably because these lipid substances also served as carbon TABLE 1. Effect of fatty acids, esters, and lipids on total growth of A. lwoffi on hexadecane Growth (mg protein/mL) Lipid" added None Propionic acid Isobutyric acid Lauric acid Caprylic acid Palmitic acid Oleic acid Tripalmitin Olive oil Lecithin Lysolecithin Palmityl stearate Palmityl palmitate

Without hexadecane 0 0.100 0 0.152 0.210 0.270 0.250 0 0.280 0.087 0 0 0

(155)b (72) (187) (63) (90) (46) (63) (62) (50) (50) (72) (103) (1 13)

With hexadecane

OD,'

OD,' OD,' OD,'

1 .300 (155)b 1 ,380 (272) 1 .330 (187) 0.380 (72) 0.250 (90) 1.520 (67) 1.450 (73) 1.850 (62) 1.540 (72) 1.500 (63) 1 .400 (72) 1 ,525 (103) 1.575 (113)

OLipid added at a concentration of 0 . 0 5 z . bNumbers in parentheses show time in hours at which growth was measured. In all cases cells had reached stationary phase when measurements were made. =Forisobutyric acid, lysolecithin, palmityl stearate, and palmityl palmitate, only optical density was measured. An OD of 1.5 corresponds to about 1.2 mg protein/mL (Breuil el of. 1978).

Time (h)

F I G .2. Growth of AcinrfobacfrrIrvoffi on 0.05% tripalmitin HD); 0.8% hexadecane (HD); plus 0.8% hexadecane (TP 0.05% olive oil ( 0 0 ) ; and 0.05% olive oil plus 0.8% hexadecane (00+ HD).

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or energy sources for protein synthesis. It is known that hexadecane is converted to palmitic acid (Dunlap and Perry 1967; Makula and Finnerty 1968), and it is presumably used in this form for growth. Although detailed studies were not made of different concentrations of each lipid, we observed that 0.02-0.05% of tripalmitin had the same stimulatory effect on growth of A . lwoffi on hexa-

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TABLE2. Utilization of hexadecane under various culture conditions


Additions to hexadecane medium"

% Aerationb

Growth hexadecane duration (h) utilized

Amount of cell protein, mg/mL

None First transferc First transfer Second transferd Second transfer Tripalmitin

Palmitic acid

Oleic acid "Mineral medium with 0.8% (w/v) hexadecane. Lipids a n d fatty acids were 0.05x (w/v). addition l o shaking the flasks, air was allowed t o flow through the space above the medium. CInoculum from cells precultured in hexadecane mineral medium. dInoculum from the first transfer.

growth. If added to hexadecane medium, this carbon source was apparently used instead of hexadecane during the first phase of growth, then after a long lag phase hexadecane was metabolized. Isobutyric acid was not used; it extended the lag phase for growth on hexadecane. Caprylic and lauric acids were themselves utilized and seemed to shorten the lag phase for growth on hexadecane, but measurements of final turbidity and of protein showed that these acids greatly reduced or inhibited the amounts of growth on hexadecane (Table 1). Some alcohols (octan-2-01 and phenethyl alcohol) which could not be used as carbon sources, in a 0.1% concentration also prevented growth on hexadecane (not shown). E f f ~ ~oft sother lipids Small amounts of triglycerides (0.05% trioctanoin or trilaurin, not shown), phospholipids (0.02-0.05% lecithin or lysolecithin) (Table l), or wax esters (0.05% palmityl palmitate or palmityl stearate) (Table 1) stimulated the growth of cells on Time ( h ) hexadecane. The lag was reduced by 70 h on addiF I G . 3. Acit~c,tohllcter I ~ o j j utilization ? of hexadecane (HD) tion of triglycerides or phospholipids, and only by plus tripalmitin (TP). Growth shown as OD at 610nm (a). 50 h if wax esters were added to hexadecane. Utilization ofeach substrate in presenceoftheother is shown by Hexadecanol (0.05%) stimulated growth on hexatwo experiments: one in which the I4CO, release was measured decane, though to a lesser extent than palrnitic acid, from [l-14C]hexadecaneplus unlabelled tripalmitin (W), and the other in which the I4COZrelease was measured from unlabelled olive oil, or other triglycerides (results not shown). (0). hexadecane plus [carbo~yI-~~C]tripalmitin We also observed similar results with P. neruginosn. At 34"C, 0.05% palmitic acid, tripaldecane, but less than 0.02% tripalmitin had no ef- mitin, or lecithin, when added to hexadecane, refect. Similar observations were made for oleic acid. duced the lag phase by 20 h (Fig. 5). In contrast, short-chain fatty acids did not stimulate growth on hexadecane (Fig. 4). As stated Influence of detergents on hydrocarbon utilizcltion previously (Breuil et nl. 1978), propionic acid, With either organism the lag phase was greatly which could be utilized alone, caused diauxic reduced by addition of 0.01% Triton X-100 to hexa-


BREUIL A N D K U S H N E R

Time (h)

FIG.4. Growth of A(~ineroht1crerht.offi on 0.8% hexadecane (HD); 0.05% isobutyric acid plus 0.8% hexadecane (BA HD); 0.05% propionic acid (PrA); and 0.05% propionic acid plus hexadecane (PrA + HD).

+

branes, the concentration used did not seem to affect cell structure; treated cells still appeared phase dense and unchanged in form. The growth of P. aorugitzosa on hexadecane was similarly stimulated by two other detergents, Brij 35 and FL-70 (a biodegradable detergent) (Fig. 6B). These surfactants were effective a t concentrations of 0.0 1-0.001%.

Time (h)

FIG. 5. Growth of

Pseudo~nonns crerrrginosrr on 0.8%

hexadecane (HD); 0.05% palmitic acid plus 0.8% hexadecane (PA HD); 0.05% lecithin plus 0.8% hexadecane ( L e HD); and 0.05% tripalmitin plus 0.8% hexadecane ( T P HD). No growth occurred on tripalmitin alone. On palmitic acid alone growth in 70 h was 0.1 mg proteinlrnl; on lecithin alone growth in 70 h was 0.28 mg proteinlml.

+

+

+

decane medium (Fig. 6), and the total amount of growth was increased. This concentration of Triton X-100 did not support growth. Although this detergent is commonly used for disrupting cell mem-

Ejyect ofdijfkret~tsuhstat~ceson diauxic growth We showed previously that in a mixture of ethanol and hexadecane, diauxic growth of both A . l ~ ~ ~ oand j j i P. aerugitlosa occurred. In the first phase of growth ethanol was used, and hexadecane was used in the second phase. Almost no lipase activity was noted until after the end of the ethanol growth phase (Breuil et al. 1978). It seemed possible that lipase was necessary for growth on hexadecane and that it acted by supplying fatty acids which served as emulsifying agents. To test this possibility we examined the effect of fatty acids and other detergents on growth in the presence of a mixture of ethanol and hexadecane. Palmitic acid prevented diauxic growth; that is, growth occurred, but no longer in a diauxic manner if palmitic acid was added to a mixture of hexadecane and ethanol (compare Fig. 7 with Fig. 8). When ethanol, palmitic acid, or hexadecane was supplied in the I4C-labelled form, estimates of I4CO2evolution showed that ethanol was used first, followed shortly by palmitic acid. The release of I4CO, from hexadecane did not begin until that from palmitic acid was almost complete, but this does not necessarily mean that hexadecane was not being oxidized: hexadecane is probably oxidized through a palmitic intermediate, s o that, as long as any substantial amount of unlabelled palrnitic acid


C A N . J . MICROBIOL. VOL. 26. 1980

Time (h)

Time (h)

+ +

FIG.6 . (A) Growth ofAcitlrtohrrcter11i~offi on 0.8% hexadecane (HD); 0.01% Triton X- 100 plus 0.8% hexadecane(TX 100 HD); and 0.005% Triton X-I00 plus 0.8% hexadecane (TX100 HD). (B) Growth of Pserrdotnotias aerugitiosa on 0.8% hexadecane (HD); 0.01% Triton X-100 plus 0.8% hexadecane (TX100 HD); 0.01% Brij 35 plus 0.08% hexadecane (B HD); and 0.01% FL-70 plus 0.08% hexadecane ( F HD).

+

+

is present, this might mask the breakdown of labelled hexadecane. In another experiment (not shown), a similar pattern of CO, evolution from palmitic acid and hexadecane was observed when only these two substances were present. Palmitic acid prevented diauxic growth only if added while ethanol was still being oxidized. If palmitic acid was added during the lag phase following ethanol utilization, then an unusual growth curve resulted (not shown), expressed as a period of slow growth followed by a period of more rapid growth. We are unable to suggest any interpretation of these results. Lecithin also suppressed diauxic growth on hexadecane and ethanol (results not shown). When the detergent Triton X- 100 was added to an ethanol plus hexadecane system, the lag phase after ethanol utilization was shortened (by 67%) but the growth was still diauxic (Fig. 8).

Hexadecane oxidation by ethanol- and Izexadecane-grown A. lwoffi The fact that ethanol-grown cells could not utilize hexadecane until a long lag period had

+

elapsed suggested that some necessary enzyme(s) first had to be formed. Such enzymes could be those involved in making hexadecane accessible to degradative enzymes already present in the cell, o r they could be the degradative enzymes themselves. In order to examine the latter possibility we studied the oxidation of hexadecane by nongrowing ethanol- and hexadecane-grown cells in a Warburg respirometer (Fig. 9). If hexadecane alone was added, neither kind of cells showed an increase of oxygen uptake over the endogenous rate. Triton X-100 was not oxidized by ethanol-grown cells, though it slightly increased the rate of oxygen uptake by hexadecane-grown cells. Both kinds of cells showed an increased oxygen uptake if hexadecane emulsified with Triton X-100 was added. The increased rate of uptake for hexadecane-grown cells was about 3 times that of ethanol-grown cells, suggesting that growth on hexadecane increased the amounts of hexadecane-oxidizing enzymes. Discussion It is well established that some organisms, including Aci~zetobacterand Cnndida species, pro-

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BREUlL AND KUSHNER

Time ( h )

FIG.7. A c i t ~ e ~ o l ~ o c t c ~utilization r l ~ ~ ~ ( ~of f iethanol (EtOH) plus palmitic acid (PA) plus hexadecane (HD). Growth shown as O D at 610nm (a).Utilization of each substrate in presence of the others is shown by three experiments: one in which the IJCO2 release was measured from [I-'.'C]hexadecane plus unlabelled palmitic acid and ethanol (a),one in which the I4COZrelease was measured from [I-14C]ethanolplus unlabelled palmitic acid and hexadecane (0).and one in which IJCO2 release from palmitic [I4C]acid was measured in the presence of unlabelled ethanol and hexadecane (0).

Time ( h )

FIG.8. Growth of Acir~ctohacterIii~c~ffi o n 0.01% ethanol plus

+

hexadecane (HD EtOH); and 0.1% ethanol plus 0.8% hexadecane plus 0.01% Triton X-100(HD EtOH TX100).

+

+

not all alike. The effects of fatty acids depend on their chain lengths. Of those tested, C1, and C , , fatty acids stimulated growth on hexadecane whereas those of moderate length, C, and C I 2 ,inhibited it. The toxic effects of lower fatty acids duce extracellular products of lipid degradation have been described by a number of authors such as mono- and di-glycerides and fatty acids (Barker 1964; Hunkova and Fencl 1978; Khan and when grown in the presence of hydrocarbons (Klug Katamay 1969). Atlas and Bartha (1973) also reand Markovetz 1967; Makula et a / . 1975; Volfova ported that the presence of caprylic or lauric acids and Pecka 1973). Prokop et 01. (1972) suggested that completely inhibited crude oil biodegradation by long-chain free fatty acids. which are considered to Breuibncteriuln and by FInuobncte~.iumspp. Lipids (tripalmitin, olive oil, and lecithin) also be metabolites in hydrocarbon fermentation, play a role in decreasing the intelfacial tension. Goma et stimulated growth on hexadecane. It is possible, 01. (1973) showed that the solubility of hydro- but not certain, that they do so by first being hycarbons increases when growth occurs, since drolyzed to long-chain fatty acids. The stimulatory microorganisms produce surfactants that are re- action of tripalmitin is not one-sided, since at 200C sponsible for the decrease of oil drop size and the these cells do not use tripalmitin unless hexadecane subsequent formation of microemulsions (Goma et is also present. nl. 1972; Zajic et nl. 1977). However, Nakahara et Some nonionic detergents (surfactants), Triton nl. (1977) suggested that free fatty acids are not the X-100, FL-70, and Brij 35, show the same stimulaonly metabolites which can change the interfacial tory effect as do free fatty acids on the utilization tension, but that some sugars, peptides, lipids, and of hexadecane. Surfactants aid the formation and proteins will act in the same way (Erickson and the stabilization of oil emulsions. Whitworth et nl. Nakahara 1975; Hisatsuka et 01. 1977; Suzuki et 01. (1973) found that Triton X-100, which failed to sup1969). port growth of Ccrndidcl lipolyticn, had an inhibitory The present work shows that although fatty effect on the growth rate with alkane as substrate. acids, lipids, and nonbiological detergents all affect However, they found that some surfactants (inthe growth of A . hvoffi and P. nerugirzosa on hexa- cluding Triton X-100) used as emulsifying agents decane, their effects and mechanisms of action are with dodecane, enhanced the rate of oxidation of


C A N . J . MICROBIOL. VOL. 26. 1980

Time (min)

Time (min)

F I G .9. Oxidation of hexadecane by cells o f A c i t ~ e r o b a c ~ r e r I ~(A) ~ ~ oResponse jfi~ ofethanol-grown cells (2.6 rng protein/rnL); (@). endogenous respiration (cells): ( x 1. cells plus sonified hexadecane; (A), cells plus Triton X-100 (0.1%): and (0).cells plus sonified hexadecane ( 12.5 pL/mL) + Triton X-100(0.1%). (B) Responseof hexadecanegrown cells (2.5 mgprotein/mL); (@).endogenous respiration (cells); ( x ). cells plus sonified heuadecane(l2.5 pL/mL; (A). cellsplus Triton X- 100(0.l%): (0). cells plus Triton X-100(O. 1%) plus sonified hexadecane (12.5 pL/mL). Final concentrations are shown.

alkanes. In our experiments the rate of hexadecane oxidation by washed cells of A . 1u~)jfigrown either on hydrocarbon or on ethanol was negligible. It was only when hexadecane was emulsified with Triton X-100 that a measurable rate of oxidation could be shown. This experiment shows the importance of su~face-activeagents in permitting effective contact between cells and hexadecane. Although some fatty acids stimulate growth, they do not do so only by acting as emulsifying agents. The possibility of distinguishing between an emulsifying function and other functions of fatty acids is suggested by experiments on diauxic growth in the presence of ethanol and hexadecane. Adding a nonmetabolizable detergent, Triton X-100, to this system stimulates growth but does not abolish diauxie although it reduces the lag period between the utilization of the two substrates (Fig. 8). Palmitic acid both stimulates growth and abolishes diauxie. The fact that lipase formation is repressed during growth on ethanol (Breuil et (11. 1978) is consistent with this result: lipase activity might be necessary for the production of sufficient quantities of fatty acids. However, we cannot ascribe the lag after ethanol utilization solely to the time needed for lipase synthesis. We have now shown that there is a higher oxidative activity towards hexadecane in hexadecane-grown than in ethanol-grown cells. Several enzymes needed for hexadecane utilization may well be repressed during growth on ethanol. Our experiments raise several unanswered, but approachable, questions on the role of palmitic acid and other long-chain fatty acids in alkane utiliza-

tion. It is possible that palmitic acid induces the enzymes necessary for hexadecane utilization. by a type of product induction, that is, the induction of an enzyme degrading a substance by one of the first products in a degradative pathway. This seems to occur in different bacteria for the enzymes involved in glycerol and histidine breakdown (Mandelstam and McQuillen 1973). It should be recalled that when Aci~letobacterspp. are grown on hexadecane most of the fatty acids are C,,. whereas those grown on other substrates have much higher proportions of C18fatty acids (Makula et al., 1975; V. Vachon, J . McGarrity, and C . Breuil, unpublished results). Possibly, incorporation of palmitic acid in the cell membrane could permit a better entry of the hydrocarbon into the cell. Acknowledgments We are greatly indebted to Mr. Peter Lomax for technical assistance and to Dr. David Shindler for valuable discussions. This work was supported by grants from Environment Canada and from the National Research Council of Canada. A.1-LAS.R. M.. and R. BARTHA.1973. Inhibition by fatty acids of the biodegradation of petroleum. Antonie van Leeuwenhoek; J. Microbiol. Serol. 39: 257-27 1. BARKER. R. M. 1964. The bactericidal action of low molecular weight compounds. J. Appl. Bacterial. 27: 213-220. B R E U I L , C., and A. M. G o u ~ o - r . 1972. Recherches preliminaires sur les bacteries lipolytiques psychrophiles des sols et des eaux. Can. J. Microbiol. 18: 1445-1451. B R E U I L C., . and D. J . KUSHNER.1975. Lipase and esterase formation by psychrophilic and rnesophilic Acitlelobacler species. Can. J . Microbiol. 21: 423-433. BREUIL,C.. T . J . NOVITSKY, and D. J. K U S H N E R 1975. .


BREUIL AND KUSHNER

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