7545 CURRENT MICROBIOLOGY Vol. 32 (1996). pp. 336-342 Current Microbiology An International Journal © Springer-Verlag New York Inc. 1996

Oleyl Oleate and Homologous Wax Esters Synthesized Coordinately from Oleic Acid by Acinetobacter and Coryneform Strains

T. Kaneshiro,1 L.K. Nakamura,Z ].1. Nicholson,3 M.O. Bagbyl

JOil Chemical Research. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street. Peoria. IL 61604. USA "Microbial Properties Research. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street. Peoria. IL 61604. USA 3Biopolymer Research. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street. Peoria. Illinois 61604. USA

Abstract. Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax esters (C30rC36:z) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered from 100-ml cultures ofAcinetobacter B-14920 supplemented with 810 mg oleic acid-oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty (CH-C lH ) alcohols and acids suggests a coordinated synthesis whereby the exog­ enous alcohol remains unaltered, and the fatty acid is partially oxidized with removal of Cz units before esterification. Consequently, CH-C lH primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation pool from which is produced a homologous series of liquid wax esters.

]ojoba (Simmondsia califomica) seeds are a source of A previous survey of cultures selected from liquid waxes, which are monoesterified mixtures of composted manure [14] revealed that 27 of 165 unsaturated fatty acids and unsaturated alcohols [18]. bacterial isolates converted exogenous oleic acid to Wax esters from jojoba contain an even number of solvent-extractable neutral products resembling tri­ carbon atoms from C34 to C4H [12, 18, 19]. This acylglycerols on thin-layer chromatograms (TLC). substitute for sperm whale oil is used in such diverse The present study examines chemical structures and commercial products as lubricants, cosmetics, solid probable biosynthetic paths of the oleate conversion wax coatings, and biofuel additives [1, 18, 20]. Wax products of four bacterial (identified as Acinetobacter esters containing CrCzo alcohols [10] or C l4-ClH spp. and a Gram-positive coryneform) and two yeast alcohols [8, 26] produced by Acinetobacter strains may isolates. afford alternative sources of waxes [3, 11, 22]. How­ Triacylglycerol is well established as a major ever, these C3o-C36 wax esters [4, 26] are mainly storage product of yeast cells and, consequently, may saturated and stored in cells grown on nitrogen have a similar function in bacteria [22]. Wax esters (N)-limited media containing succinate, hexadecane, may have a storage function also. Our oleic acid­ or hexadecanol as carbon sources [8]. containing medium [14] is confirmed to facilitate selection of acinetobacters and other bacterial types that accumulate liquid wax esters containing unsatu­ Names are necessary to report factually on available data; however. rated, even-numbered C3o-C36 chains, which re­ the USDA neither guarantees nor warrants the standard of the semble those in jojoba seeds [18]. The coordinated product. and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. conversion of oleic acid to homologous liquid waxes is deduced from the composition of solvent-extractable Correspondence 10: T. Kaneshiro products formed when cultures are supplemented T. Kaneshiro et al.: Ester Synthesis by Acinetobacter 337 with exogenous long-chained fatty alcohols and acids GLC and MS analyses. Wax esters extracted and concentrated in as well as with soybean oil. methanol-ethyl acetate were detected by capillary GLC isother­ mally at 260°e. All samples described above were chromato­ graphed on a SPB-l column (5 m x 0.32 mm id), and emergent Materials and Methods peaks were identified according to retention times relative to Microbial cultures. Microscopic examinations of isolates from standard compounds. The wax esters were analyzed further by composted manure [14] revealed that strains NRRL Y-17806 and GLC-MS (70 eV) after temperature-programmed separations at o Y-17807 were yeasts; and NRRL B-14920, B-14921, B-14922, and 160 -250°e. Wax esters gave characteristic molecular mass ion and B-14923 were bacteria. The four bacterial isolates were identified mass ion fragments (m/z) stemming from their fatty acid and further on the bases of utilization of 96 substrates (Biolog System) alcohol moieties [8.19,26]. and fatty acid profiles (MIDI System). Estimation ofwax esters (mg per 100 ml culture) was based on Cultures were maintained on tryptone-glucose-yeast extract extractable lipids recovered from duplicate 35-ml cultures. Each agar slants and stored at 4°e. For determination of oleic acid or extract was dissolved in 4.0 ml solvent before injection (1 fLl lipid conversions, cultures were transferred from the slants to a aliquot) into a capillary GLC column. In the absence of an nutrient-limited growth medium [27] which yielded less than adequate internal standard, a mean value of54 x 103 area units per optimal turbidimetric (suboptimal) growth and which contained fLg was used to estimate wax esters in normalized (100 ml) samples. The mean value was calculated from oleyl oleate and palmityl (per L): 5.0 g yeast extract, 4.0 g glucose, 4.0 g K2HP04, 250 mg oleate standards, which gave recorded GLC responses of 49 x 103 MgS04 ' 7H20, and 10 mg FeS04 . 7H20. The medium was ad­ justed to pH 7.3 before autoclaving. These inoculum cultures, and 59 x 103 area units/fLg, respectively. which were fully turbid by 24 h at 25°C (shaken at 200 rpm), were in turn transferred (I%, vol/vol) to 35 ml fresh medium in 125-ml Erlenmeyer flasks and incubated overnight for 16-17 h. To these Results early stationary-phase cultures were added oleic acid or other lipid substrates (0.30 ml or 0.27 g altogether). Although optimal condi­ Classification ofisolates. Strains capable oftransform­ tions were not determined. preliminary studies indicated that the ing oleic acid to triacylglycerol-like products were yields of wax esters were not enhanced significantly beyond 24 h. However, wax esters were increased appreciably by use of this isolated from composted manure [14]; these were turbidity-limiting growth medium [14]. designated NRRL accessions B-14920, B-14921, B-14922, B-14923, Y-17806, and Y-17807. Bacterial Biochemicals. Oleic acid, oleyl alcohol, palmitic acid, and palmi­ toleic acid were purchased from Sigma Chemical Co. (S1. Louis, strains B-14920, B-14921, and B-14923 were strictly Missouri). These and all other biochemicals were used without aerobic, oxidase negative, nonmotile, Gram-negative, further purification. Soybean oil was obtained from Riceland very short rods or cocci. Polyhydroxybutyrate storage Foods (Stuttgart, Arkansas). bodies were not observed in these strains. Strains Extraction, separation, and saponification. After 24 h, the cultures B-14920 and B-14921 were identified as Acinetobacter containing lipid supplements were acidified to pH < 3 and johnsonii, and strain B-14923 asAcinetobacter calcoace­ extracted twice with one volume of 1:9 (vol/vol) methanol-ethyl ticus. All three acinetobacters did not oxidize such acetate [14]. Subsequently, the solvents were removed in a rotary common sugars as D-fructose and D-glucose [2] and evaporator from the combined extracts. A standard wax ester, palmityl oleate, was fully recoverable by such an extraction regi­ were differentiated by differences in noncarbohy­ men. Before saponification, a neutral fraction was separated from drate utilization of either amino acids, organic acids, the lipid extract by column chromatography on Silica Gel 60 or fatty derivatives (Biolog). Profiles of two acineto­ (230-400 mesh; EM Science, Gibbstown, New Jersey). The neutral bacters gave 23-33% CI6:10 17-20% CI6:0, and 17-24% fraction was eluted by sequential treatment with 8 column-volumes C : fatty acids (MIDI). Strain B-14922, a strictly each of petroleum ether. 10:90 (vol/vol) ethyl acetate-petroleum IS I ether, 50:50 ethyl acetate-petroleum ether, and ethyl acetate. Each aerobic Gram-positive coryneform, was not identified fraction was monitored for neutral lipids by TLC Silica Gel 60 plate further. Yeast strains NRRL Y-17806 and Y-17807 (EC Science, Cherry Hill. New Jersey) chromatography as devel­ gave luxuriant growth on yeast extract-malt extract­ oped with a solvent mixture of hexane-ethyl acetate-acetic acid peptone-glucose agar medium at pH 6.5 and were (50:50:1, vol/vol), and the components were made visible by means identified by cursory, microscopic examinations only. of iodine vapor and vanillin spray. The respective Rfs of standard monoolein. oleic acid. and neutral triolein were 0.20. 0.55. and 0.80. Triacylglycerol of yeasts. The two yeast strains pro­ For mild saponification of triacylglycerols. the samples (10 mg duced a mean of 95 mg recovered lipid product when of neutral lipid fraction) were mixed with 1 ml of 2 N NaOH and heated at 65°C for 1 h. Saponified samples were acidified before approximately 810 mg oleic acid was added exog­ extraction with hexane (2 x 5 ml). and the fatty acids were then enously to 100-ml cultures. Separation of the lipid esterified with diazomethane for analysis by capillary GLC isother­ product over a silica gel 60 column yielded 30-50% mally at 185°C [14]. neutral lipid material that resembled triacylglycerols To saponify wax esters, samples (10 mg) dissolved in 0.5 ml by TLC analysis (R 0.80). The four bacterial strains methanol were mixed with 1.5 mIlO N NaOH and heated for 2 h at f 100°e. The resultant free fatty alcohols were extracted in diethyl NRRL B-14920, B-14921, B-14922, and B-14923 gave ether after sample adjustment to pH 8.0: free fatty acids were similar column chromatographic fractions that repre­ extractable with hexane at pH < 3. sented 26%, 37%, 63%, and 16% of the respective 338 CURRENT MICROBIOLOGY Vol. 32 (1996) crude lipid extracts. However, these bacterial neutral C:24 C:2S:0 A. C12:0 Alcohol lipids were eluted from the column (10-50% ethyl I I acetate in petroleum ether) slightly before the triacyl­ I glycerols (50-100% ethyl acetate). In addition, the I crude extract of B-14923 contained a highly polar, I unknown compound (Rr0.41, tail) that amounted to l J~:O 50% of the total recovered lipid. The IR spectrum of a bacterial neutral (wax ester) fraction was indistin­ guishable from that of the yeast triacylglycerol and B. C12:0 Alcohol + Oleic Acid from that of a triolein standard, and gave prominent ester (1740 em-I) and cis (3006 cm-I without 965 em-I) absorption bands. Mild saponification treatment, however, revealed significant differences between the neutral lipid frac­ tions of yeasts and bacteria. That the derivatized yeast samples contained > 97% methyl oleate sug­ I C. Oleic Acid gested a trioleylglycerol compound. When lyophilized I I to dryness, extracted with methanol, and then acety­ I lated with acetic anhydride, a water-soluble compo­ I I nent from saponified yeast Y-17806 yielded an acety­ I lated derivative having the same GLC retention time I and MS as triacetylglycerol. Neutral lipid fractions of I AI the four bacterial strains were not hydrolyzable under mild conditions. D. C18:1 Alcohol + Palmitoleic Acid Oleyl oleate of the acinetobacters. In the presence of 810 mg oleic acid, Acinetobacter strain NRRL B-14920 accumulated 210 mg lipid product, of which approxi­ mately 20 mg was oleyl oleate (C36:2; Fig. lC and Table 1). The neutral fractions recovered by column chromatography from the lipid products of all four bacterial strains gave, after saponification with metha­ 242628 30 32:2 34:2 36:2 Carbon Aloms:Unsaturation nolie 7.5 N NaOH, homologous series of alcohols having GLC retention times consistent with (mean o 10 20 30 40 Time (min) area %): C 1S:1 (73%), C I6:1 (16%), and C 14:1 (3%) alcohols. The identities of alcohols were confirmed by Fig.!. Gas chromatograms of accumulated lipid from Acinetobacter NRRL 8·14920 24·h cultures supplemented with: lauryl (C : ) MS. The fatty acid components of saponified wax I2 0 alcohol (A). combined C 12:0 alcohol and oleic acid (8). oleic acid esters gave GLC peaks (mean area %) for C 1S:1 alone (C). and combined oleyl (C IS:I) alcohol and palmitoleic (55%), C I6:I (30%), and other minor fatty acids. (CI6:Jl acid (D). Retention times are compared with expected wax Supplementation of 100-ml conversion cultures esters containing the specified C atoms and unsaturations. with 0.45 ml oleyl alcohol and 0.45 ml oleic acid (Table 1) significantly increased liquid wax ester production; oleyl oleate accounted for 56 mg or 20% other wax esters was deduced from an orderly produc­ of the extracted lipid (280 mg). The bacterial waxes tion of homologous wax esters and from the synchro­ generated from oleic acid conversions contained two nous production related to exogenous substrates. unsaturated hydrocarbon chains per ester molecule. Accordingly, cultures supplemented with oleic acid Consequently, MS ofoleyl oleate (molecular mass ion accumulated significant amounts (10-16 mg/IOO ml) m/z 532) gave distinct but weak mass ion fragments of of the C : homolog, which could be either palmitoyl m/z 283 for C : acid and m/z 250 for C : alcohol, 34 2 1S I 1S 1 oleate or oleyl palmitoleate (Fig. lC; Table 1). Mass analogous to fragmentation patterns of palmityl stea­ spectra of the C waxes from both oleic acid and oleic rate and stearyl palmitate [8, 26]. 34 acid-oleyl alcohol-supplemented cultures gave mass Coordinated synthesis of wax esters. In addition to fragment ions (m/z) from palmitoyl (222) and oleyl oleyl oleate, a coordinated synthesis/accumulation of (250) alcohols and oleate (283) and palmitoleate T. Kaneshiro et al.: Ester Synthesis by Acinetobacter 339

Table I. Accumulation of oleyl oleate (C36:2) and other liquid wax esters by Acinetobacter NRRL 8-14920 when oleic (CIS:I) acid and oleyl (CIs:1l alcohol" are exogenous substrates

Liquid wax esterC (mg) Extractable Substrate(s) Replicates lipid/> (mg) C3O:2 C32:2. C32:1 C34:2. C34:1

Oleic acid 6 210 tr 3. tr 10.

" Exogenous substrates were added in 0.9-ml (810 mg) amounts or 0.45 ml oleic acid-0.45 ml oleyl alcohol per 100-ml cultures at 25°C. Extractable lipid (gravimetric) and wax ester (GLC) determinations represent means from several cultures. /> Lipid extracted (normalized to 100-ml culture) with 1:9 (vol/vol) methanol-ethyl acetate.

C Wax esters were separated by carbon chain length and degree of unsaturation: oleyl oleate designated C36:2. Quantitative estimates based on 54 x 103 area units per IJ.g wax ester. Not detectable: tr. trace amounts.

Table 2. Alkyl alcohol substrates supplemented with oleic acid" for production of wax esters by Acinetobacter NRRL 8-14920

Alcohol added Extractable to C IS:I acid (Carbon atoms)" lipid/> (mg) Major wax ester productsC (mg)

Octyl (Cs:o) 720 C26 (6)' C2d<1) Decyl (Cmo ) no C2S (33)' C21> (8) C24 (2) Lauryl (CI2:0 ) 470 Co (lOO)' C2S (24) C26 (3) •..•...• lI

Myristyl (CI4:0 ) 360 C30 (8) C2S (4)

Oleyl (CIS:iJ 200 C36 (47)' C32 (2)

" Alkyl alcohols (405 mg of each supplemented with 405 mg oleic acid per ]()O-ml culture). Saturated myristyl alcohol is a solid at room temperature. The dotted (- ...) line denotes phase difference. h Lipid (normalized to lOO-ml culture) extractable with 1:9 (vol/vol) methanol-ethyl acetate.

C Wax esters designated by carbon length and determined quantitatively by GLC (see footnote c. Table I). Major wax ester homolog expected from alcohol substrate.

(255) carboxylates, which indicated uncoordinated other "intracellular" fatty acids. Intracellular fatty synthesis [8]. acids were expected [8] to be mainly saturated C1S­ By contrast in the culture supplemented with C l4 compounds. For example, addition of lauryl oleyl alcohol alone, wax synthesis appeared to be alcohol alone (Fig. lA) produced 47% CZ4:1h 25% coordinated with C34 :Z production mainly by oleyl CZS :1h and 13% C30:0 wax esters (approximately 18 mg palmitoleate (8 mg; fragment ions m/z 250 and 255). total wax esters per 100 ml culture), whereas a This nonrandom or orderly accumulation was more combination of lauryl alcohol and oleic acid produced readily apparent (Fig. lA IB) when shorter-chain 0.6% CZ4:lh 2% CZ6 :b 16% C ZS :b and 76% C30:1 wax

CS-C14 alcohols were added as substrates (Table 2). esters (150 mg total). Thus, the exogenous primary Also, coordination should be a time-related or syn­ alcohols remained unaltered, and both intracellular chronous event leading to such wax esters as decyl and exogenous carboxylates were incorporated into a oleate (approximately 33 mg C ZS :1; Table 2). When series of wax esters. addition of oleic acid was delayed 8 h, accumulation When various fatty acids were added in combina­ of CZS :1 wax ester decreased to 3 mg. tion with oleyl alcohol (Table 3), there were anoma­ The chain lengths of wax esters were controlled lies in wax ester accumulations. With oleyl alcohol­ more readily in cultures by chain lengths of exog­ decanoic, -palmitic, or -palmitoleic acid combinations, enous alcohols (Table 2) than by those of fatty acids the cultures produced a wide range of wax esters and, (Table 3). However, addition of such alcohols as oleyl consequently, appeared to generate significant (CIS:I) and lauryl (C1Z:0) without exogenous acids led amounts of the waxes from partially oxidized oleyl to a terminal -CHzOH oxidation, producing oleic alcohol (C36-C34 esters) instead of the exogenous and lauric acids, respectively, and esterified mixtures fatty acid. Thus, the oleyl alcohol-palmitic acid combi­ containing both the -COOH compounds as well as nation enhanced production of diunsaturated (35 mg 340 CURRENT MICROBIOLOGY Vol. 32 (1996)

Table 3. Production of wax esters by AcinClObacter sp. NRRL B-14920 cultures from fatty acids or soybean oil supplemented with oleyl alcohol"

Fatty acid added Extractable C to C IS:I alcohol (Carbon atoms)" lipid" (mg) Major wax ester products (mg)

Caprylic (Cso ) 500 C20 (8)' Decanoic (Cmu ) 700 C2S (7)' C-'4 «6) C-,d <3)

Palmitic (Clo:U) 590 C-'6 (29) C-'4(19)' C-,:(I)

Palmitoleic (CI6:Jl 540 C-'o (35) C-'4 (17)C C-'2 « I) Oleic (CIS:I ) 430 C-'6 (70)' C-'4 (24) C-,:(6) Linoleic (CIS:2) 340 C-'o (112)C C-'4 (23) C-'2 (4) Soybean oil (Triacylglycerols) 520 C-'6 (217)' C-'4 (42) C-'2 (2)

" Fatty acids designated by C-chain length and degree of unsaturation. Approximately 405 mg each were added along with 405 mg oleyl alcohol per 100-ml culture. Dotted line (...) denotes phase difference. "Lipid (IOO-ml culture) extractable with 1:9 (vol/vol) methanol-ethyl acetate.

C Wax esters designated by carbon chain length only and estimated by GLC (see footnote c. Table I). Major wax ester homolog expected from fatty acid substrate.

C36:2 and 8 mg C34:2) esters rather than monounsat­ X-Triacylglycerol urated (12 mg C34:1) ester attributed to exogenous : Lipase palmitic acid. With the oleyl alcohol-palmitoleic acid , X-"Oleic acid" X-Alcohol combination (Fig. ID) also, the C34:2 products gave multiple, unresolved peaks by GLC. In the latter instance, MS suggested that the principal C34:2 prod­ X-Oleic; , Esterase uct was oleyl palmitoleate (mass fragments m/z 250 Oxidized + X-Alcohol ... (Oleyloleate); Fatty Acids ~ and 255). Solid C1rC I6 and liquid palmitoleic (CI6:1) C3S -C 30 wax esters acids by themselves did not lead to significant amounts Pool O/R Enzymes of extractable wax esters. Fig. 2. Schematic summary ofwax ester syntheses by acinetobacters Esterification activity of cultures was not con­ that convert exogenous (X) oleic acid and other alcohol substrates fined to monounsaturated fatty acids; both polyunsatu­ into a homologous series of liquid waxes. rated linoleic- and soybean oil-oleyl alcohol combina­ tions were excellent substrates. The C36 products from both types of substrates gave skewed, unre­ The scheme of Fig. 2 attempts to reconcile the solved peaks by GLC, indicative of polyunsaturated extensive findings by others [8, 9, 25, 26] in relation to wax esters. Here, a polar capillary column would have been very useful for delineating the polyunsaturated the extractable wax esters derived from oleic acid wax esters produced. Thus, the major C product conversions. Others have shown that extracellular 36 lipases [3, and cell-bound esterase [15] may from cultures supplemented with linoleic acid alone 13] facilitate acinetobacter utilization of tributyrin [4, gave an MS consistent with a C36:4 wax ester (molecu­ lar ion m/z 528 and weak mass ion fragments m/z 281 15], olive oil [2, 4] and triacetylglycerol [11, 23] as C and 248). sources. However, wax esters of microorganisms are biosynthesized/accumulated by two pathways depen­ dent on either membranous (exogenous) or cytoplas­ Discussion mic fatty acids [3, 16] and by lipase-catalyzed esterifi­ cation of long-chain alcohols [22]. Six other enzymes Such compounds as triacylglycerols [Fig. 2: 22], al­ (three alcohol dehydrogenases and three aldehyde kanes [2, 11, 26], alcohols [9], and aromatics [6] may dehydrogenases in acinetobacters [9, 24, 25]) function be oxidized and reduced (O/R) effectively by acineto­ by distinctly different O/R mechanisms. These O/R bacters to supply growth and energy. Instead of dynamics [9, 21, 24] may explain the coordinated storing either triacylglycerol [22] or polyhydroxybutyr­ synthesislaccumulation we observe. Accordingly, ate [2], acinetobacters store wax esters when alkyl C 13-oxidations of exogenous fatty acids (Fig. 1 and sources are supplied in excess and the N source is Table 3) prior to wax ester synthesis are consistent limited [4, 8]. with a cellular pooling of long-chain acids. Subse- T. Kaneshiro et al.: Ester Synthesis by Acinetobacter 341 quently, the pooled fatty acids appear to react with 5. Bryn K Jantzen E. Bovre K (1977) Occurrence and patterns of waxes in Neisseriaceae. J Gen Microbiol102:33-43 intracellular a/R enzymes, which remove C2 units 6. Chalmers RM. Scott AJ, Fewson CA (1990) Purification of the stepwise to give a series of homologous, even­ benzyl alcohol dehydrogenase and benzaldehyde dehydroge­ numbered acids and to be activated as acyl-CoA nase encoded by the TOL plasmid PWW53 of Pseudomonas thioesters [3, 16, 21]. In addition, part of the exog­ plllida MT53 and their preliminary comparison with benzyl enous fatty acid is reduced to a corresponding alcohol alcohol dehydrogenase and benzaldehyde dehydrogenases I [26]. and II from Acinetobacter calcoaceticus. J Gen Microbiol Exogenous alkyl alcohol (Table 2), rather than 136:637-643 7. DeWitt S. Ervin JL. Howes-Orchison D. Dalietos D. Neide­ alkanoic acid (Table 3), dictates the retention of an Iman SL. Geigert J (1982) Saturated and unsaturated wax unaltered alcohol C chain in homologous wax esters. esters produced by Acinetobacter sp. HOI-N grown on C 16-C20 For example, lauryl and oleyl alcohols combined with n-alkanes. J Am Oil Chern Soc 59:69-74 oleic acid are efficient co-substrates that influence 8. Fixter LM, Nagi MN, McCormack JG. Fewson CA (1986) chain length. Coordinated synthesis/accumulation of Structure. distribution and function ofwax esters inAcinetobac­ tercalcoaceticus. J Gen MicrobioI132:3147-3157 the homologous (C30rC36:2) wax esters is indicated 9. Fox MGA. Dickinson FM. Ratledge C (1992) Long-chain further by nonrandom formation ofoleyl palmitoleate alcohol and aldehyde dehydrogenase activities in Acinetobacter homolog in cultures supplemented with oleyl alcohol calcoaceticlls strain HOI-N. J Gen Microbiol 138:1963-1972 and by its time-related accumulation upon supplemen­ 10. Gallagher IHC (1971) Occurrence of waxes in Acinetobacter. J tation with both oleyl alcohol and oleic acid. Gen Microbiol 68:245-247 11. Gutnick DL. Allon R. Levy C Petter R. Minas W (1991) Levels of diunsaturated, liquid wax esters in cells Applications of Acinetobacter as an industrial microorganism. are usually enhanced by either alkane [7, 17] or In: Towner KJ. Bergogne-Berezin E, Fewson CA (eds) The alkene [3, 16] growth media at low temperatures biology ofAcinetobacter. New York: Plenum Press. pp 411-441 (l5°-25°C) and vary over a wide range dependent on 12. Hamilton RJ. Raie MY. Miwa TK (1975) Structure of the bacterial species [5]. In the presence of exogenous alcohols derived from wax esters in jojoba oil. Chern Phys oleic acid and oleyl alcohol. acinetobacter cultures Lipids 14:92-96 13. Hou CT (1994) pH dependence and thermostability of lipases accumulate predominantly homologous liquid wax from cultures from the ARS Culture Collection. J Ind Micro­ esters resembling plant waxes, which have monounsat­ bioi 13:242-248 urated alcohol and monounsaturated acid moieties 14. Kaneshiro T. Nakamura LK Bagby MO (1995) Oleic acid [18]. The acinetobacter waxes, C3o-C36 in length, are transformations by selected strains ofSphingobactenllm thalpo­ philllm and Bacilllls cerellS from composted manure. Curr expected to melt at a lower temperature than C34-C4S Microbiol 31 :62-67 liquid waxes of jojoba seeds and may more closely 15. Kok RG. Christoffels VM. Vosman B. HellingwerfKJ (1993) resemble the C2S-C34 waxes of whale oil [1, 20]. Growth-phase-dependent expression of lipolytic system of Acinetobacter calcoacetiws BD 413: cloning of a gene encoding one of the esterases. J Gen Microbiol 139:2329-2342 ACKNOWLEDGMENTS 16. Lloyd GM. Russell NJ (1983) Biosynthesis of wax esters in psychrophilic bacterium Alicrococcus cryophillls. J Gen Micro­ We thank Dr. Morey E. Siodki for helpful discussion and suggested bioi 129:2641-2647 revisions. We gratefully acknowledge the technical assistance of 17. Makula RA. Lockwood PJ. Finnerty WR (1975) Comparative Katherine R. Berry. analysis of the lipids ofAcinetobacterspecies grown on hexadec­ ane. J BacterioI121:25D-258 18. Miwa TK (1973) Chemical aspects ofjojoba oil. a unique liquid Literature Cited wax from desert shrub Simillondsia califomica. Cosmet Perfum I. Bagby MO (1988) Comparison of properties and function of Jan 1973. pp 3255-3257 jojoba oil and its substitutes. In: Baldwin AR (ed) Proceedings 19. Miwa TK Spencer GF. Plattner RD (1976) Separation and of 7th International Conference on Jojoba and Its Uses. structure determination of jojoba oil components by high­ Champaign. Illinois: American Oil Chemists' Society. pp pressure liquid chromatography and gas chromatography! 190-200 mass spectrometry. In: Proceedings of the Second Interna­ ~ Baumann P, Doudoroff M. Stanier, RY (1968) A study of the tional Conference on Jojoba and Its Uses. Ensenada. Mexico. Moraxella group. II. Oxidative-negative species (Genus Acineto­ pp 187-197 bacter). J BacterioI95:152D-1541 20. Miwa TK Rothfus JA. Dimitroff E (1979) Extreme-pressure 3. Boulton CA (1989) Extracellular microbial lipids. In: Ratledge lubricant tests on jojoba and sperm whale oils. J Am Oil Chern C Wilkinson SG (eds) Microbial lipids. Vol 2. London: Soc 56:765-770 Academic Press. pp 669-694 21. Pieringer RA (1989) Biosynthesis of non-terpenoid lipids. In: 4. Breuil C Kushner DJ (1975) Lipase and esterase formation by Ratledge C Wilkinson SG (eds) Microbial lipids. Vol 2. psychrophilic and mesophilic Acinetobacter species. Can J London: Academic Press. pp 51-114 MicrobioI21:423-433 22. Ratledge C (1989) Biotechnology of oils and fats. In: Ratledge 342 CURRENT MICROBIOLOGY Vol. 32 (1996)

C. Wilkinson SG (eds) Microbial lipids, Vol 2. London: 25. Singer ME, Finnerty WR (1985b) Alcohol dehydrogenases in Academic Press, pp 567-668 Acinctobactcr sp. Strain H01-N: Hexadecane and hexadecanol 23. Shabtai Y. Gutnick DL (1985) Exocellular esterase and emul­ metabolism. J Bacterioll64: 1017-1024 san release from the cell surface ofAcinclObactcr calcoaccticlls. 26. Stewart JE, Kallio RE (1959) Bacterial hydrocarbon oxidation J BacterioI161:1176-1181 II. Ester formation from alkanes. J Bacteriol 78:726-730 24. Singer ME, Finnerty WR (1985a) Fatty aldehyde dehydroge­ 27. Wallen LL, Benedict RG. Jackson RW (1962) The microbio­ nases in Acinctobactcr sp. strain H01-N: role in hexadecane logical production of 10-hydroxystearic acid from oleic acid. and hexadecanol metabolism. J BacterioI164:1011-1016 Arch Biochem Biophys 99:249-253

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