Applied Microbiology and Biotechnology

, Volume 89, Issue 6, pp 1721–1727

Production of 7,10-dihydroxy-8(E)-octadecenoic acid from olive oil by Pseudomonas aeruginosa PR3

Authors

  • Min-Jung Suh
    • Department of Animal Science and BiotechnologyKyungpook National University
  • Ka-Yeon Baek
    • Department of Animal Science and BiotechnologyKyungpook National University
  • Beom-Soo Kim
    • Department of Chemical EngineeringChungbuk National University
  • Ching T. Hou
    • Renewable Product Technology Research UnitNational Center for Agricultural Utilization Research, ARS, USDA
    • Department of Animal Science and BiotechnologyKyungpook National University
Biotechnological Products and Process Engineering

DOI: 10.1007/s00253-010-3040-2

Cite this article as:
Suh, M., Baek, K., Kim, B. et al. Appl Microbiol Biotechnol (2011) 89: 1721. doi:10.1007/s00253-010-3040-2

Abstract

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.

Keywords

Hydroxy fatty acidOlive oilBioconversionPseudomonas aeruginosaDOD

Introduction

Microbial conversion of synthetic or naturally finding compounds often leads to the formation of novel products. Accordingly, microbial fermentation with a specific substrate has been well studied as an efficient way to transform natural compounds into new value-added products occasionally carrying novel biological activities. Among substrates used for bioconversion, naturally occurring unsaturated fatty acids have been well studied as good substrates to cause novel chemical changes on the substrate. Since Wallen et al. (1962) reported the first bioconversion of oleic acid to 10-hydroxystearic acid by a Pseudomonad, various trials using several bacterial strains had been focused on the microbial oxidation of fatty acids leading to the formation of hydroxy fatty acids (HFA).

HFAs, originally found mainly from plant systems (Zimmerman 1966), are known to have special properties such as higher viscosity and reactivity compared to other normal fatty acids (Bagby and Calson 1989). These special properties augmented HFAs to contain high industrial potentials in a wide range of applications including resins, waxes, nylons, plastics, lubricants, cosmetics, and additives in coatings and paintings. Some HFAs are also reported to contain antimicrobial activities against plant pathogenic fungi and some of food-born bacteria (Bajpai et al. 2004; Hou and Forman 2000; Kato et al. 1984; Shin et al. 2004).

Among microbial strains studied to produce HFAs from fatty acid substrates, Pseudomonas aeruginosa PR3 is well known to convert various unsaturated fatty acids into mono-, di-, and trihydroxy fatty acids. Of those fatty acid substrates, oleic acid was used as a most efficient substrate for strain PR3 to produce 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) with 60% yield (Hou et al. 1991), and its production yield was improved over 80% through the modification of culture conditions (Kuo et al. 1998). Recently, we have reported that triolein, the triacylglyceride form of oleic acid, was successfully utilized as a substrate to produce DOD by strain PR3 via triolein-induced lipase activity (Chang et al. 2007). However, triolein is still expensive material since it is a synthetic lipid, making it difficult to be used as economic industrial resource specifically for the production of DOD.

Therefore, in this study, we tried to use olive oil, one of the natural vegetable oils carrying high content of oleic acid as a substrate for DOD production and firstly report that olive oil could be efficiently utilized to produce DOD by P. aeruginosa PR3.

Materials and methods

Chemicals

Olive oil was purchased from the market, and heptadecanoic acid (C17:0) was purchased from Nu-Chek Prep, Inc. (Elysian, MN, USA). Mixture of trimethylsilylimidazole (TMSI) and pyridine (1:4, v/v) was purchased from Supelco Inc. (Bellefonte, PA, USA). All other chemicals were reagent grade and were used without further purification. Thin-layer pre-coated Kieselgel 60F254 plates were obtained from EM Science (Cherry Hill, NJ, USA). Other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA), unless mentioned otherwise.

Microorganism and bioconversion

P. aeruginosa NRRL strain B-18602 (PR3) was kindly provided by Dr. Hou of USDA/NCAUR. The strain was grown at 28°C aerobically in a 125-ml Erlenmeyer flask containing 50 ml of standard medium with shaking at 200 rpm. The standard medium used hereafter contained (per liter) 4 g dextrose, 4 g K2HPO4, 1 g (NH4)2HPO4, 1 g NH4NO3, 1 g yeast extract, 0.056 g FeSO4·7H2O, 0.1 g MgSO4, and 0.01 g MnSO4·7H2O. The medium was adjusted to pH 7.0 with diluted phosphoric acid. For optimization study, each nutritional component was replaced as needed from the standard medium, unless mentioned otherwise. For the production of hydroxy fatty acids, olive oil (0.5 g) containing 73% of oleic acid out of total fatty acid content was added to 24-h-old culture followed by additional incubation for 72 h. At the end of cultivation, the culture broth was acidified to pH 2 with 6 N HCl followed by immediate extraction twice with an equal volume of ethyl acetate and diethyl ether. The solvent was evaporated from the combined extracts with a rotary evaporator. Cell growth was determined spectrophotometrically by measuring absorbance of cell culture at 610 nm.

Analysis of products

The extracted reaction products were analyzed by thin-layer chromatography (TLC) and gas chromatography (GC). The TLC was developed with a solvent system consisting of toluene:dioxane:acetic acid (79:14:7, v/v/v). Spots were visualized first by iodine vapor and then spraying the plate with 50% sulfuric acid and heating in a 100°C oven for 10 min. For GC analysis, the samples were first methylated with diazomethane for 5 min at room temperature followed by derivatization with a mixture of TMSI and pyridine (1:4, v/v) for at least 20 min at room temperature. The TMS-derivatized sample was analyzed with Younglin AMCE 6100 GC (Younglin, Seoul, Korea) equipped with a flame-ionization detector and a capillary column [SPB-1™, 15 m × 0.32 mm i.d., 0.25 μm thickness (Supelco Inc., Bellefonte, PA, USA)]. GC was run with a temperature gradient of 20°C/min from 70°C to 200°C, holding 1 min at 200°C, and then 0.7°C/min to 240°C, followed by holding for 15 min at 240°C (nitrogen flow rate = 0.67 ml/min). Injector and detector temperatures were held at 250°C and 270°C, respectively. Heptadecanoic acid (C17:0) was added to the sample before derivatization as an internal standard for quantification.

Chemical structure of target compound was confirmed by GC/mass spectrometry (GC/MS) analysis. Electron-impact (EI) mass spectra were obtained with a Hewlett Packard (Avondale, PA, USA) 5890 GC coupled to a Hewlett Packard 5972 Series Mass Selective Detector. The column outlet was connected directly to the ion source. Separation was carried out in a methylsilicone column (30 m × 0.25 mm i.d., 0.25 μm film thickness) with a temperature gradient of 20°C/min from 70°C to 170°C, holding 1 min at 170°C and 5°C/min up to 250°C followed by holding for 15 min (helium flow rate = 0.67 ml/min).

Results

Production and identification of target compound

For the bioconversion of olive oil by P. aeruginosa PR3, olive oil (1% of culture volume) was added to the 24-h-old culture followed by additional 72 h incubation. Crude extract of the bioconverted olive oil was prepared and analyzed for identification of the target product. TLC analysis of the crude extract revealed that the plausible DOD spot became dominant time-dependently while the size of olive oil spot reduced, indicating that the olive oil was transformed into new spot, saying DOD (Fig. 1). The crude extract after 72 h incubation was used for GC analysis. GC analysis of the crude extract shown in Fig. 2 confirmed that the relative retention time of the major new spot (indicated by arrow) corresponded exactly to that of DOD (Chang et al. 2007). The area percent of the major peak was 67% of total peaks except internal standard (data not shown). This result was well supported by the result from TLC analysis in that the size of target spot was major.
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Fig. 1

Thin-layer chromatography analysis of the crude extract produced from olive oil by P. aeruginosa PR3. Lane 1 olive oil, lane 2 12 h incubation, lane 3 24 h incubation, lane 4 48 h incubation, lane 5 72 h incubation. DOD-like spots were indicated by arrow. Running and developing conditions are given in “Materials and methods” section

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Fig. 2

Gas chromatography analysis of the crude extract produced from olive oil after 72 h incubation by P. aeruginosa PR3. DOD-like peak was indicated by arrow. I.S represented internal standard. Reaction and GC running conditions are explained in “Materials and methods” section

Structure determination

The plausible major DOD product was further analyzed by GC/MS for structure determination. The electron impact (EI) GC/MS data of TMS derivative of the methylated product was given in Fig. 3. This was consistent with the TMS derivative of a methylated C18 dihydroxy monoenoic fatty acid with a molecular mass of 472. The locations of hydroxyl groups were apparent from the fragments observed in the EI spectrum of the TMS derivative of the methylated fatty acid. The intense fragment arising from alpha cleavage to the derivatized hydroxyl group toward methyl end gave fragments containing TMS at m/z 215 and both two TMS and a double bond at 343 m/z. Other two intense fragments arising from alpha cleavage to the derivatized hydroxyl group toward the methylated carboxyl end were observed at 231 m/z containing TMS and at 359 m/z containing both two TMS and a double bond. These fragments allocated the hydroxyl groups at C7 and C10 and a double bond at C8–9. These fragmentation data were exactly matched to those of DOD produced from triolein by the same strain (Chang et al. 2007). These results confirmed that the major product of the crude extract of the bioconverted olive oil was DOD, indicating that strain PR3 could produce DOD efficiently from olive oil.
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Fig. 3

Electron-impact mass spectrum of TMS derivatives of the methylated major product from olive oil by P. aeruginosa PR3. Sample preparation and running conditions are explained in “Materials and methods” section

Time-coursed DOD production from olive oil

Since it was confirmed that strain PR3 could produce DOD efficiently from olive oil, optimal incubation time for DOD production was studied. DOD production was monitored at every 24 h for 7 days after substrate was added to the 24-h-old culture. As shown in Fig. 4, DOD production increased time-dependently up to 72 h incubation followed by sharp decrease, indicating that DOD produced was possibly degraded or consumed by microorganism. Maximum amount of DOD produced at 72 h incubation was 198 mg per 50 ml culture, representing 40% and 55% production yields from olive oil and oleic acid contained in olive oil, respectively. However, cell growth increased until 24 h incubation and was maintained plateau during incubation. Based on this result, incubation time for DOD production from olive oil was standardized with 72 h wherever other variables were evaluated.
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Fig. 4

Time-coursed production of DOD from olive oil by P. aeruginosa PR3. Incubation time represented reaction time after olive oil was added to the 24-h-old culture. Closed circle and square represented DOD production and cell growth, respectively. Cell growth was determined at 24-h pre-incubation before substrate was added

Optimum conditions for the production of DOD from olive oil

Several critically important factors were studied for optimal production of DOD from olive oil by strain PR3. First, eight different carbon sources were tested independently for the production of DOD (Table 1). Among the carbon sources tested, galactose was most effective for DOD production followed by glucose (control), maltose, fructose, and lactose. Maximum amount of DOD produced with galactose was 210.8 mg per 50 ml culture. Xylose and sucrose represented 41.8% and 34.8% of DOD production compared to the maximum value with galactose, respectively. However, cell growth showed quite different results according to the carbon sources tested. Maximum cell growth was obtained with glucose followed by galactose, glycerol, and xylose. In the case of maltose, cell growth was 32.4% of the maximum value with glucose while DOD production was 97% of that with glucose. Glycerol was highly effective for cell growth, representing 81% of the maximum value with glucose while DOD production was not detected. These results indicated that requirement of carbon source for cell growth and DOD production from olive oil was different.
Table 1

Effect of carbon sources on DOD production from olive oil by PR3

Carbon source (0.4%)

Cell growth (OD at 610 nm)a

Total DOD production (mg/50 ml culture)

Glucose

3.06 ± 0.24

180.7 ± 8.5

Galactose

2.63 ± 0.13

210.8 ± 12.0

Fructose

0.96 ± 0.04

135.5 ± 10.5

Xylose

2.38 ± 0.21

88.1 ± 6.8

Sucrose

0.60 ± 0.02

73.3 ± 8.2

Maltose

0.99 ± 0.11

175.4 ± 9.9

Lactose

1.09 ± 0.14

117.1 ± 8.9

Glycerol

2.48 ± 0.27

nd

nd not detected

aFinal OD value representing cell growth was obtained by calculation from the original value of the diluted sample

For optimal nitrogen source, combined nitrogen sources of the control medium containing yeast extract (0.1%), ammonium phosphate dibasic (0.1%), and ammonium nitrate (0.1%) was replaced with various organic and inorganic nitrogen compounds individually (Table 2). As shown in the table, glutamine was most effective, even more effective than the control, for DOD production from olive oil, representing 122% of the control medium. Cell growth was also most efficient with glutamine. Other nitrogen sources showed relatively low DOD production, representing below 50% of the control medium. Peptone and malt extract were very weak for cell growth and DOD production as well.
Table 2

Effect of nitrogen sources on DOD production from olive oil by PR3

Nitrogen source

Cell growth (OD at 610 nm)b

Total DOD production (mg/50 ml culture)

Controla

3.21 ± 0.12

178.8 ± 5.6

Yeast extract (0.1%)

2.79 ± 0.19

40.5 ± 3.2

Malt extract (0.1%)

0.37 ± 0.02

7.1 ± 0.3

Peptone (0.1%)

0.24 ± 0.03

2.4 ± 0.3

Tryptone (0.1%)

1.88 ± 0.20

38.5 ± 2.7

Glutamine (25 mM)

3.32 ± 0.25

218.6 ± 12.2

NH4NO3 (25 mM)

1.98 ± 0.11

nd

(NH4)2HPO4 (25 mM)

2.85 ± 0.17

89.8 ± 4.2

(NH4)2SO4 (25 mM)

2.59 ± 0.24

nd

Urea (25 mM)

2.45 ± 0.18

21.4 ± 1.4

nd not detected

aControl represented the standard medium

bFinal OD value representing cell growth was obtained by calculation from the original value of the diluted sample

Generally, metal ions are known to play important role in enzyme action as a cofactor. Therefore, several metal ions in sulfate or chloride salts were tested individually for DOD production (Table 3). Each metal ion tested was replaced as a sole metal ion source with the standard medium. Among nine metal ions tested, magnesium was most effective for DOD production and cell growth. DOD production was 228.8 mg per 50 ml culture, representing 108% compared to the control standard medium. However, all other metal ions were very poor for DOD production and cell growth as well in that DOD production were not detected with other metal ions except potassium ion. These results indicated that magnesium ion as a singular metal ion was critically required and fully efficient for both cell growth and DOD production.
Table 3

Effect of metal ions on DOD production from olive oil by PR3

Metal ions(1 mM)

Cell growth (OD at 610 nm)b

Total DOD production (mg/50 ml culture)

FeSO4

0.85 ± 0.10

nd

ZnSO4

0.64 ± 0.07

nd

CuSO4

0.63 ± 0.05

nd

MgSO4

2.68 ± 0.17

228.8 ± 16.1

MnSO4

0.56 ± 0.04

nd

CoCl2

0.34 ± 0.02

nd

KCl

0.66 ± 0.07

1.5 ± 0.2

CaCl2

0.98 ± 0.10

nd

NaCl

0.91 ± 0.08

nd

Controla

2.93 ± 0.24

211.7 ± 9.8

nd not detected

aControl represented the standard medium

bFinal OD value representing cell growth was obtained by calculation from the original value of the diluted sample

Optimal incubation temperature and initial medium pH for DOD production were also studied. As shown in Fig. 5, DOD production increased significantly according to temperature increase up to 28°C, at which DOD production was maximized and reduced to 67% of the maximum value at 30°C. Over 35°C, DOD was not detected. However, cell growth was similarly efficient in the range of between 20°C and 30°C, indicating that DOD production but not cell growth was very sensitive to temperature. The effect of initial medium pH on DOD production was studied in the range of from 5.0 to 10.0 (Fig. 6). Overally, alkaline pH was good for DOD production in that DOD production was maximized between pH 8.0 and 9.0. Below pH 6.0, DOD was not detected. However, cell growth was optimal at pH 7.0 and declined thereafter. At pH 6.0, cell growth was 63.6% of the maximum value at pH 7.0, but DOD was not produced at all. These results indicated that enzyme system involved in DOD synthesis from olive oil by strain PR3 could use plausibly saponified free oleic acid under alkaline condition, although this should be further confirmed.
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Fig. 5

Effect of incubation temperature on DOD production from olive oil by P. aeruginosa PR3. Gray bar and closed circle line represented DOD production and cell growth, respectively. Cell growth was determined at 24-h pre-incubation before substrate was added

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Fig. 6

Effect of initial medium pH on DOD production from olive oil by P. aeruginosa PR3. Gray bar and closed circle line represented DOD production and cell growth, respectively. Cell growth was determined at 24-h pre-incubation before substrate was added

Discussion

Microbial fermentation is widely used as an efficient and economical way to cause structural modification of natural compounds leading to promotion of the value. Microbial oxidation of natural fatty acid leading to the formation of hydroxy fatty acid is a good example. In this study, we tried to use olive oil as a substrate to produce DOD by P. aeruginosa PR3. The major product from bioconversion of olive oil by strain PR3 was identified to be DOD which was the same product obtained from oleic acid by PR3. Optimal incubation time for DOD production was 72 h after substrate was added to the culture. This result was in good agreement with our previous report about DOD production from triolein (Chang et al. 2007). Amount of DOD produced from olive oil and triolein were similar representing 198 and 206 mg out of 500 mg substrate, respectively. However, DOD production yield out of oleic acid in olive oil was a little higher by 30% than that with triolein. This difference was caused from the different oleic acid contents of substrates in that oleic acid content of olive oil was 74.1%, while triolein was 100%. This optimal incubation time was quite different from that for DOD production from free oleic acid by the same strain (Hou and Bagby 1991) in that DOD production was peaked at 48 h after substrate addition. This discrepancy could be explained by that PR3 required additional time to induce lipase activity for the release of oleic acid from oil substrate before the released oleic acid was used as real substrate for DOD production (Chang et al. 2007). All the cases with olive oil, triolein, and oleic acid, DOD production decreased after certain optimal incubation time, indicating that DOD produced was possibly degraded or further metabolized by microorganism. If this result was caused from further metabolic consumption of DOD as an alternative carbon source since the carbon source in the medium exhausted according to cell growth, this problem might be solved by supplement of new carbon source in the middle of fermentation. Based on this assumption, following study should be focused on this point to increase the production yield.

Nutritional requirements for DOD production from olive oil and triolein were similar but quite different from those for the production of trihydroxy octadecenoic acid (THOD) from linoleic acid by the same strain. Optimal carbon sources for DOD production from olive oil and triolein included glucose, galactose, and fructose (Chang et al. 2007). However, THOD was not produced with fructose. Requirement of metal ions for DOD production and THOD production from their corresponding substrates by the same strain were quite different in that iron or copper ion was essential for THOD production from linoleic acid (Kim et al. 2000), while magnesium ion was essentially required for DOD production (in this study, Chang et al. 2008). From these differences, we could assume that enzyme system involved in DOD production was possibly different from that for THOD production. It has been reported that the catalytic amount of iron ion was required for induction of lipid peroxidation catalyzed by lipoxygenase (Gardner et al. 1976). Lipoxygenase (LOX) is a nonheme iron-containing dioxygenase that catalyzes the oxidation of unsaturated fatty acid with a 1Z,4Z-pentadiene moiety leading to the formation of conjugated hydroperoxy fatty acid (Yamamoto 1991; Zhang et al. 1993; Schilstra et al. 1994; Brash 1999). 1Z,4Z-pentadiene moiety is found in linoleic acid but not in oleic acid. Based on these information, it is highly possible that LOX could possibly be involved in the conversion of linoleic acid but not in conversion of oleic acid by P. aeruginosa PR3. The next study should be focused on identification of enzyme system involved in DOD production by PR3 strain.

In conclusion, from the results from this study and our previous report (Chang et al. 2007), we confirmed that strain PR3 was able to utilize efficiently olive oil as a substrate for DOD production. These results demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of value-added DOD by microbial bioconversion. Further work should be performed to scale-up production of DOD from olive oil in a large-scale fermenter.

Acknowledgement

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) [No. R01-2008-000-20067-0(2008)].

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