Bacterial Production of Polyesters from Free Fatty Acids
Obtained from Natural Oils by Pseudomonas oleovorans
Baki Hazer,
1,2Oktay Torul,
3Mehlika Borcakli,
2Robert W. Lenz,
4R. Clinton Fuller,
4and
Steven D. Goodwin
4The carboxylic acids derived from olive oil, hazelnut oil, sesame oil and hamci(anchovy) oil were evaluated as substrates for cell growth and the production of reserve polyesters by Pseudomonas
oleovorans. Poly-3-hydroxy alkanoates containing both saturated(mainly 3-hydroxy-octanoate and
3-hydroxy-decanoate) and unsaturated repeating units with 8 to 20 carbon atoms, or more, were produced in 26 to 61 % yields based on cell dry weights. The number average molecular weights of these polymers varied from 45,000 to 68,000 Daltons.
In our investigations, the bacterium Pseudomonas
Oleo-vorans has proven to be very versatile for PHA
produc-tion because it can produce polyesters from a wide va-riety of carbon substrates, including alkanes, alcohols,
'Department of Chemistry, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey.
2TUBiTAK-Marmara Research Center, 41470 Kocaeli, Turkey. 'Department of Chemistry, Karadeniz Technical University, 61080
Trabzon, Turkey.
4 Polymer Science and Engineering Department, Biochemistry De-partment, Microbiology DeDe-partment, University of Massachusetts, Amherst, Massachusetts 01003.
and alcanoic acids which have 6 to 14 carbon atoms in their backbone [4-10]. The objective of the present study was to evaluate the types of PHAs produced by this bac-terium from carboxylic acids which were obtained either from agricultural products grown in Turkey or from a type of fish taken from the Black Sea.
EXPERIMENTAL Substrates
Olive oil used was a commercial product obtained from olives grown in western part of Turkey. Hazelnut oil was obtained from hazelnut fruits grown in northern part of Turkey. Sesame oil was a commercial product obtained from sesame seeds grown in southern Turkey. Hamci(anchovy) oil was obtained from hamci, which is a fish taken from the Black Sea. Each of these oils were hydrolyzed in a 10% solution of KOH in ethanol, after which the solution was neutralized with a 10% solution of sulfuric acid in water to obtain the carboxylic acid substrates. The acids were analysed by gas chromatog-raphy, GC, with a Hewlett Packard 439 model instru-ment equipped with a flame ionization detector, FID, at 6°C/min heating rate, programmed from 70 to 190°C.
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KEY WORDS: Carboxylic acid; biopolyesters; daltons.
INTRODUCTION
Poly-3-hydroxy alkanoates, PHAs, are a class of reserve polyesters produced by a large number of bac-teria when subjected metabolic stress [1-3]. PHAs have the general repeating units shown below, in which the structure of the R group is dependent upon the carbon substrate and the type of bacteria:
110 Hazer, Torul, Borcakli, Lenz, Fuller and Goodvin
Biosynthesis
Stock cultures of Pseudomonas oleovorans (ATCC 29347) were used in all growth and polymer production experiments as previously developed in this laboratory [7-11]. The strains were maintained at 4°C on nutrient agar plates using the modified mineral E* medium de-scribed below with a 20 mM n-nonanoic acid as the car-bon source. The culture was grown in a 3-L solution of mineral medium containing (NH4)2HPO4(l.lg),
K2HPO4(5.8g), K2HPO4(3.7g), 10 mL of 0.1M MgSO4
and 1.0 mL of a microelement solution (this micro-element solution contained FeSO4-7H2O(2.78g),
MnCl2-4H2O(1.98g), CoSO4-7H2O(2.81g), CaCl2
-2H2O(1.67g), CuCl2-2H2O(0.17g), and ZnSO4
-7H2O(0.29g) in 1-L of IN HC1). The natural oily acids
were used as a sole carbon source at a concentration of 20 mM and the pH was adjusted to 7.00. The cells were grown under aerobic conditions in 3 L cultures, which were agitated at 250 rpm at 30°C in a rotary shaker. Usually, these batch cultures were harvested after 24 h. The cells were harvested by centrifugation (Sorwall RC2-B; 4°C, 12,000 rpm). The cells were washed with methanol to discard unreacted natural oil and lyophi-lized on a Freeze Dry System (Labconco). The dry cell weights were determined gravimetrically. The polymer was extracted from lyophilized cells in a Soxhlet ex-tractor with 200 mL of chloroform. After the solvent was evaporated to 4-5 mL, the solution was filtered through glass wool and the polymer was precipitated into 300 mL of vigorously stirred methanol. After two pre-cipitation cycles, the polymer was dried under vacuum for 2 days. Total biomass and polymer yield are listed in Table I.
NMR Spectroscopy
'H NMR spectra were obtained on chloroform-d solutions at 17°C with a Bruker AC 200L instrument at 200 MHz. NMR spectra of all of the PHAs contained
the characteristic bands at d (ppm): 0.84 (C//3—),
1.2-1.6 (—CH2—CH2—), 2.0 (—CH2— next to the
double bond), 2.3-2.5 (C//2-C(O)-), 5.1
(—O—C//—), 5.3 (—CH=CH-). Figure 1 shows the comparison between 'H NMR spectra of hazelnut oily acid substrate and polyester obtained from this sub-strate.
Methanolysis and GC-MS Analysis
The methanolysis sulfuric reaction was carried out in chloroform/methanol/sulfuric acid (1 mL/0.85 mL/ 0.15 mL) at 100°C for 140 min following a procedure identical with that described previously [11]. The methyl esters obtained were assayed by gas chromatography and mass spectroscopy (GC-MS analysis) using a Hewlett Packard HP 5890 gas chromatograph with He carrier gas. After injection the column was maintained at 60°C for 4 minutes and then heated at 10°C/min to 270°C. A temperature program was used which efficiently sepa-rated the different methyl 3-hydroxy alka(e)noates. Each peak in the chromatogram was analyzed with a mass spectrometer. The data were processed with a Hewlett-Packard laboratory data system (including more than 250,000 GC-MS spectra of chemicals). GC-MS spectra of 3-hydroxy acids by methanolysis of biopolyesters ob-tained from fatty acids (hazelnut, olive, sesame and an-chovy) have been shown in Figure 2.
Molecular Weight Measurements
Molecular weights were determined by gel per-meation chromatography, GPC, with a Waters model solvent delivery system with a model 410 refractive in-dex detector, and with 2 ultrstyragel linear columns (HRI and HT6E) in series. Tetrahydrofuran was used as the eluent at a flow rate of 1.0 mL/min. Sample concentra-tion of 2-3 mg/mL and injecconcentra-tion volumes of 150 mL were used. A calibration curve was generated with six polystyrene standards having molecular weights of 3 x 106, 233 x 103, 22 x 103, 2.15 x 102, 580 and 92
Daltons.
RESULTS AND DISCUSSION
The acids obtained from the hydrolysis of olive, hazelnut, sesame and hamci oils included both saturated and unsaturated acids in different weight percentages. Table II contains the GC analysis results of the acid sub-strates which were in good agreement with the results reported in the reference [12]. Unsaturated acids,
in-Table I. Cell and Polymer Yields After 24 Hours
Natural oil acid Hazelnui Sesame Olive Hamci Dry cell" yield, g I 2.0 2.9 1.2 3.2 PHA yield g 1.2 1.2 0.31 0.90 % DW" 60 41 26 28 Molecular weight Mw x 10" 9.1 11 6.7 8.7 Mn x 10" 5.9 6.8 4.5 4.8 M J Ma 1.5 1.6 1.5 1.8 "DW is cell dry weight.
Fig. 1. 'H NMR spectra of Hazelnut oily acid substrate and its bipolyester.
eluding both oleic and linoleic acids, comprised approx-imately 90% by weight of the acids from the plant oils, with lesser amounts in the hamci oil. In the latter satu-rated acids, including both palmitic and stearic acids, were approximately 20% by weight of the samples used.
Hamci oily acid unlikely the others was included unsat-urated moieties containing di-, tri-, tetra-, penta- and hexaen's in 30%.
P. oleovorans was grown on the acids from the four
112 Hazer, Torul, Borcakli, Lenz, Fuller and Goodvin
isolated at a constant harvesting time of 24 h. Table I contains the results obtained on the amounts of PHAs produced in 3 L fermentation solution and their molec-ular weights. The highest PHA yield, based on cell dry weights, was 61 %, with the acids from hazelnut oil. The number average molecular weights of all of the PHAs were between 45,000 and 68,000 Daltons with polydis-persities ranging from 1.50 to 1.80.
Proton NMR spectra of the biopolyesters have in-dicated characteristic peaks such as olefin peak at 5.3, —CH—O- peak at 5.2 ppm. The typical 'H NMR spectrum of the biopolyester from hazelnut oily acid compared with hazelnut oily acid substrate can be seen in Figure 1.
The results from the analysis of the PHAs by meth-anolysis and mass spectrometry are listed in Table III. Each hydrolized and esterified sample of biopolyester was syringed to the instrument. The main GC-spectra of
each sample was drawn. Then every peak in the main spectrum was analyzed by means of mass spectrum. Then the computer program matches this mass spectrum to the reference compound into the library. Figure 2 shows the GC-spectra of the biopolyester repeating units. All of the PHAs were containing both unsaturated and saturated units. Each of these consisted of mainly C8, C10 and C16 saturated carboxilic acid units (retention
times: 11.4, 14.3 and 19.6 min, respectively). Addi-tionally, they have unsaturated units containing more than 12 carbon atoms appearing at the retention times, mostly, 18, 21, 25 min. Biopolyesters obtained were all assumed to be copolymers because P. oleovorans never produces homopolymers (11, 13). Biopolyester samples were all sticky, waxy and soft materials. We can say this property comes from biopolyesters have high car-bon chain repeating units. Interestingly, biopolyester from hazelnut has indicated higher unsaturation than the
others (43-55%). As an observation, the PHA from the hamci oil acid became crosslinked after several days on exposure to air.
ACKNOWLEDGMENT
Support for this work was provided by NATO Col-loborative Research Grant. The authors gratefully ac-knowledge Mine (Tir)Bilsel and Cemil Nas for GC-MS, GC and GPC measurements.
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Macromol. 12, 92. Table II. Fatty Acid Distribution for the Natural Oil Acid
Substrates Natural oil Fatty acid" Olive 14:0 -16:0 11 16:1 -18:0 0.5 18:1 74 18:2 10 18:3 1.5 20:1 20:2 20:4 20:5 22:1 22:4 22:5 22:6 -Hazelnut 6.0 — — 83 10 0.5 — — — — — — — — Sesame 9.3 — — 48 41 1.0 — — — — — — — — Hamci (Anchovy) 12 22 11 5.0 18 3.0 1.0 2.0 2.5 0.5 11 3.0 2.0 2.0 4.0 " First number indicates Carbon amount and the latter double bond in
the fatty acid.
Table HI. Composition of Biopolyesters Obtained from P. oleovorans Grown on the Natural Oily Acids
Types of repeating units" Saturated* C8 C,0 C,2 C,4 CI 6 C , g Hazelnut 14 11 5 1 16 10
Natural oily acid
Sesame Olive 28 26 — — 9 10 35 27 7 6 6 7 Hamci (anchovy) 24 27 10 — 6 13 Unsatu rated'' C|2-ciien
c,
6.
m C|6-dien C,8.cn C|8-dicn Cl8-crien C20-le(raen Total olefin* Total olefin' — — 2 28 13 — — 43 55 5 — 6 I I — — 5 27 13 -— — 12 — — — 12 15 — 8 — 5 — 7 — 20 10 "Subscript is number of carbon atoms in repeat unit.'Analysis by GC-Mass spectroscopy . 'Analysis by NMR.
