Production of novel polyhydroxyalkanoates by Pseudomonas stutzeri 1317 from glucose and soybean oil

FEMS Microbiology Letters 169 (1998) 45^49

Production of novel polyhydroxyalkanoates by Pseudomonas stutzeri 1317 from glucose and soybean oil Wennan He a , Weidong Tian b , Guang Zhang b , Guo-Qiang Chen b; *, Zengming Zhang a b

a Department of Chemical Engineering, Tsinghua University, Beijing 100084, China Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

Received 6 August 1998; received in revised form 27 September 1998; accepted 1 October 1998

Abstract A strain of Pseudomonas stutzeri coded 1317 isolated from oil-contaminated soil was found to grow well in glucose and soybean oil as a sole carbon source, respectively. Polyhydroxyalkanoates containing medium chain length monomers of hydroxyalkanoates ranging from C6 to C14 were synthesized up to 52% of cell dry weight of P. stutzri 1317 grown on glucose mineral media. In mineral media containing 10 g l31 soybean oil, P. stutzeri 1317 produced up to 63% of polyhydroxyalkanoates containing mainly a novel monomer of 3,6-epoxy-7-nonene-1,9 dioic acids making up to 63% of the polyhydroxyalkanoates, together with minor monomers of C8 and C10. 1 H- and 13 C-nuclear magnetic resonance spectroscopy and electron impact gas chromatography-mass spectroscopy and chemical ionization gas chromatography-mass spectroscopy were used to study the structures of polyhydroxyalkanoates synthesized by P. stutzeri 1317. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Pseudomonas stutzeri; Polyhydroxyalkanoates ; Polyhydroxyalkanoate ; Glucose ; Soybean oil

1. Introduction Fluorescent pseudomonads, such as Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas aeruginosa and many other microorganisms, can produce polyhydrohyalkanoates (PHAs) with various structures as energy and carbon storage materials when the cells are cultivated in the presence of various carbon sources [1,2]. The properties of PHA can be varied to a great extent according to di¡erent alkyl * Corresponding author. Tel.: +86 (10) 6278-3844; Fax: +86 (10) 6278-8784; E-mail: [email protected]

side chain lengths, di¡erent chemical structures of the alkyl side chain [3]. P. putida and P. oleovorans are two organisms that have commonly been found to be able to incorporate functional groups including cyano, pheny, vinyl, halogen, and phenoxy into monomers of PHAs [4^6]. Normally, most of the building blocks of PHA are incorporated into the polymer only if the cells are cultivated on a carbon source whose carbon skeleton is related to the structure of the constituents monomer, such as the production of PHAs containing the above functional groups [4^6]. Exception to this rule, it has been referred to this capability as PHA biosynthesis from unrelated substrates [3,7]. This is very

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 6 2 - 5

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important as they allow synthesis of PHAs from simple substrates rather than from expensive or relatively toxic substrates [3]. Pseudomonas aeruginosa and other £uorescent pseudomonads, including P. putida, P. mendocina etc. were reported to produce medium chain length (mcl) PHAs from unrelated carbon source gluconate [8]. Several strains of P. stutzeri were reported to produce no PHAs from gluconate and octanoate, respectively [8]. In this paper, we found that a strain of P. stutzeri coded 1317 isolated from oil-contaminated soil in an oil ¢eld in northern China was able to synthesize mcl PHAs from glucose, an unrelated substrate to mcl PHAs. Also, a novel monomer structure-3,6-epoxy-7-nonenoic acid was the major constituent in PHAs synthesized by the strain grown on soybean oil.

hypochloride as described by Hahn et al [9]. In brief, 1 g dry biomass was added to a 500 ml conical £ask containing 15 ml of chloroform and 15 ml of a diluted sodium hypochloride solution (30 vol%), the suspension was incubated on the NBS shaker at 30³C for 90 min. The mixture was then centrifuged at 5000Ug for 18 min. The lower organic phase was washed with distilled water twice and the PHAs were precipitated with 95% ethanol (10 times volume of chloroform). The precipitated PHAs were separated from the liquid by centrifugation (2000Ug), washed with ethanol and air dried.

2. Materials and methods

2.4. GC-MS analysis

2.1. Bacterial strains and growth conditions

Gas chromatography (GC) and mass spectroscopy (MS) of the methanolyzed polyester was used to determine the compositions of the polymers produced. The procedure of methanolysis was performed as described by Abe and Doi [10]. The chloroform phase was decolored using activated charcoal and then subjected to GC-MS analysis. A Shimadzu GC-17A gas chromatograph equipped with a capillary column (30 mU0.25 mm) was used for the PHA esteri¢ed monomer separation. The column temperature was programmed to remain at 120³C for 2 min and then increase at a rate of 10³C/min to 250³C. It remained for 10 min at 250³C. The injection temperature was 280³C. Mass spectra and gas chromatograms were acquired and processed with a Shimadzu QP5050 mass spectrometer. Spectra were obtained as electron impact (EI) spectra or as chemical ionization (CI) spectra.

A strain coded 1317 was isolated from an oil-contaminated soil sample collected from Dagang oil ¢eld in northern China. It was identi¢ed as a strain of P. stutzeri by the Institute of Microbiology, Academia Sinica at Beijing, China. The strain was grown at 30³C in mineral salt media containing 10 g l31 glucose and 10 g l31 soybean oil, respectively, as the sole carbon source in 1000 ml conical £asks containing 400 ml culture on a reciprocal shaker (NBS, Series 25D, New Brunswick, USA). The mineral salt media consisted of (g l31 ) 0.5 (NH4 )2 SO4 , 0.4 MgSO4 W7H2 O, 9.65 Na2 HPO4 W12H2 O, 2.65 KH2 PO4 in distilled water. In addition, 1 ml of a microelement solution was added to the medium. The microelement solution contained (g): 20 FeCl3 W6H2 O, 10 CaCl2 WH2 O, 0.03 CuSO4 W5H2 O, 0.05 MnCl2 W4H2 O, and 0.1 ZnSO4 W7H2 O in 1 l 0.5 N HCl. The cells were cultivated in the above media for 48 h under aerobic condition (200 rpm), harvested by centrifugation (5000Ug), and then lyophilized. 2.2. Isolation of intracellular PHAs Polyesters were extracted from the lyophilized cells using dispersions of chloroform and aqueous sodium

2.3. Determination of PHA contents in cells The content of polyester in the cells was calculated as the ratio of weight of extracted PHA to the cell dry weight, from which the PHAs were extracted.

2.5. NMR analysis The 1 H- and 13 C-nuclear magnetic resonance (NMR) were recorded on a Bruker AM-500 NMR Spectrometer. The 500 MHz 1 H-NMR spectrum was taken from a CDCl3 solution of the polymer at 23³C, with a 4 Ws pulse width, 7000 Hz spectral

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width, 16 000 data points, and 32 accumulations. The 125 MHz 13 C-NMR spectrum was taken from a CDCl3 solution of the polymer at 23³C, with a 4 Ws pulse width, 27777.8 Hz spectral width, 32 000 data points, and 2048 accumulations.

3. Results and discussion 3.1. PHAs production by P. stutzeri 1317 from glucose mineral media P. stutzeri 1317 grew well in glucose mineral media containing up to 40 g l31 glucose at 30³C. Up to 52% of mcl PHAs were accumulated by P. stutzeri 1317 grown in 10 g l31 glucose mineral medium. The cell dry weight reached 2.3 g l31 at the end of the growth. The compositions in the PHAs were studied using GC-MS and NMR spectroscopy. It was found that 3-hydroxydecanoate (HD or C10) was the most important constituent among PHA compositions, making up 63% of the total monomers in the polyester (Fig. 1), with 21% of 3-hydroxyoctanoate (HO or C8) being the second most common monomers in the polymer (Table 1). Other mcl HA monomers were mainly 3-hydroxyhexanoate (HHx or C6), 3-hydroxy-5-dodecenoate (3H5DD), 3-hydroxydodecanoate (HDD) and 3-hydroxytetradecanoate (3HTD) (Table 1). The PHAs has a glass transition temperature (Tg ) and melting temperature (Tm ) of 352³C and 50³C, respectively, typical for the mcl PHA copolyesters. It is interesting to note that glu-

47

cose is an unrelated substrate to the above mcl PHAs and PHAs made up 52% of the cell dry weight, by optimizing the growth and PHA formation conditions, PHA contents should be able to increase further. Compared with strain Pseudomonas putida KT2442, the most productive strain able to synthesize mcl PHAs from the glucose mineral medium [5], P. stutzeri 1317 seems to be a more interesting strain for the mcl PHA production study both in terms of mcl PHA accumulation (52 vs 20.5%) and cell dry weight (2.7 vs. 1 g L31 ). 3.2. PHAs production by P. stutzeri 1317 from soybean oil mineral media The strain showed a better growth in mineral media containing soybean oil as a sole carbon source under the conditions similar to the above one. PHAs were accumulated up to 63% of the cell dry weight after 48 h of incubation at 30³C in a 10 g l31 soybean oil mineral medium. The cell dry weight was 2.7 g l31 after 48 h of cultivation. The polymer demonstrated elasticity similar to the copolyester obtained from the glucose growth medium. GC-MS was applied to investigate the polymer structures and compositions. The EI GC-MS spectrum revealed that 3,6-epoxy-7-nonene-1,9-dioic acid (ENN) formed the major constituents of the polyester. Above 63% of the monomers in the PHAs were ENN (Table 2, Fig. 2). This monomer structure was not reported in the literature so far. Similar to PHAs synthesized from glucose media, other monomers including

Fig. 1. EI GC-MS spectrum of methyl ester of 3-hydroxydecanoic acid (HD), a major monomer in PHAs synthesized by Pseudomonas stutzeri 1317 cultivated in glucose mineral media.

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Table 1 GC-MS analysis of methanolyzed samples of PHAs produced by P. stutzeri 1317 grown in a glucose mineral medium under aerobic condition at 30³C for 48 h m/e (EI)a

MWb (CI)

Methyl esteri¢ed PHA monomer structure

Monomer (mol%)

2.294

43, 55, 71, 74, 97, 103, 104, 117, 131, 145

146

Methyl ester of 3-hydroxy-hexanoic acid

3HHx (2.4)

4.223

43, 55, 71, 74, 83, 96, 103, 104, 156, 173

174

Methyl ester of 3-hydroxy-octanoic acid

3HO (21.3)

7.036

41, 43, 55, 71, 74, 101, 103, 201

202

Methyl ester of 3-hydroxy decanoic acid

3HD (63.2)

9.497

41, 43, 55, 71, 84, 97, 103, 126, 136, 150, 210

228

Methyl ester of 3-hydroxy 5-dodecenoic acid

3H5DD (6.1)

9.761

41, 43, 55, 71, 74, 83, 96, 103, 138, 229

230

Methyl ester of 3-hydroxy dodecanoic acid

3HDD (4.2)

41, 43, 71, 74, 83, 96, 103, 124, 141, 183, 208

258

Methyl ester of 3-hydroxy tetradecanoic acid

3HTD (1.0)

Retention time (min)

12.280

The above table lists major PHA monomers detected by GC-MS. a The monomer structures of PHAs were obtained by GC-MS based on electron impact (EI) semi-quantitatively. b Determined by chemical ionization (CI). MW stands for molecular weight.

L-oxidation and synthesis pathway. One carboxyl group was derived from degradation of polyester, another carboxyl group should be coming from the soybean oil. The above results showed that P. stutzeri 1317 had the ability to grow both in hydrophilic substrate, such as popular glucose, and hydrophobic substrate, such as soybean oil. Medium chain length PHAs

HO, HD and HDD were observed as important compositions in the PHAs. The formation of 3,6-epoxy group suggested that the hydroxyl group on C3 of HA monomers was dehydrated with hydroxyl group on C6. EI GC-MS indicated the presence of carboxyl groups at each end of the monomer. (Fig. 2), this unusual structure must be derived from the soybean oil through some

Table 2 GC-MS analysis of methanolyzed samples of PHAs produced by P. stutzeri 1317 grown in a soybean oil mineral medium under aerobic condition at 30³C for 48 h Retention time (minute)

m/e (EI)

MWa (CI)

Methyl esteri¢ed PHA monomer structure

Monomer (mol%)

4.161

41, 55, 71, 75, 83, 115, 117, 143, 158, 173

188

Methyl ester of 3-hydroxy-octanoic acid

3HO (8.2)

6.791

41, 43, 55, 69, 75, 101, 111,117, 143, 186, 201

216

Methyl ester of 3-hydroxy-decanoic acid

3HD (7.1)

8.794

41, 43, 55, 69, 83, 101, 111,116, 143, 155, 228

228

Dimethyl esters of 3,6-epoxy-7trans-nonenoic acid

+ENN (25.2)

8.913

41, 43, 69, 83, 101, 111, 116, 143, 155, 228

228

Dimethyl esters of 3,6-epoxy-7cis-nonenoic acid

3ENN (38.2)

9.483

41, 43, 55, 69, 71, 75, 83, 97, 117, 155, 171, 214, 229

244

Methyl ester of 3-hydroxy-dodecanoic acid

3HDD (2.6)

The above table lists major PHA monomers detected by GC-MS. a The monomer structures of PHAs were obtained by GC-MS based on electron impact (EI) semi-quantitatively. b Determined by chemical ionization (CI). MW stands for molecular weights.

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Fig. 2. EI GC-MS spectrum of dimethyl esters of 3,6-epoxy-7-nonenoic acid (ENN), a major monomer in PHAs synthesized by Pseudomonas stutzeri 1317 cultivated in soybean oil mineral media.

were synthesized from unrelated carbon source, glucose. This promises some possibility to produce the mcl PHAs in an economic way. Furthermore, strain 1317 was able to produce PHAs with a novel structure ENN not reported in the literature so far when it was grown in soybean oil. The strain seems to have a unique PHAs synthase system that could work on a wide substrate spectrum and produce some more novel structures. Studies are in progress to investigate the synthesis of more novel PHAs from some unusual carbon sources.

[2]

[3]

[4]

[5]

Acknowledgments [6]

The authors wish to thank Professor Zhang Riqing of the State Key Laboratory of Biomembrane and Membrane Biotechnology at Tsinghua University for the NMR analysis of the PHA samples and Professor Wang Guanhui, Institute of Chemistry, Academia Sinica, Beijing, China for assistance in GC-MS analysis and many useful discussions. The State 95 Key Project and the State Cross Century Foundation provided partial funding for this research.

References

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