production and characterization of medium-chain-length polyhydroxyalkanoates by pseudomonas mosselii...

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Journal of Bioscience and BioengineeringVOL. xx No. xx, 1e8, 2014

Production and characterization of medium-chain-length polyhydroxyalkanoatesby Pseudomonas mosselii TO7

Yi-Jr Chen,1,2 Yan-Chia Huang,1 and Chia-Yin Lee1,*

Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan1 and Department of Nursing, Chang Gung University of Science and Technology,Tao-Yuan 33333, Taiwan2

Received 5 August 2013; accepted 27 January 2014Available online xxx

* CorrespondTaiwan Univer33664812; fax

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The polyhydroxyalkanoate (PHA) production and growth of Pseudomonas mosselii TO7, a newly isolated Pseudomonasspecies from the wastewater of a vegetable oil manufacturing facility, was analyzed. Phenotypic analysis and phyloge-netic analysis of the 16S rRNA gene revealed that it is closely related to Pseudomonas mosselii. In the presence of palmkernel and soybean oils, P. mosselii TO7 produced up to 50% cell dry weight (CDW) medium-chain-length (MCL) PHAscomprising high poly(3-hydroxyoctanoate) (P(3HO)) content; P(3HO) content increased to 45% CDW when grown inoctanoate using a single-step culture process. The PHA monomer was identified by 13C nuclear magnetic resonancespectroscopy. The average molecular weight and polydispersity index of PHA were 218.30 ± 31.73 and 2.21 ± 0.18,respectively. The PHA produced by P. mosselii TO7 in the presence of palm kernel oil had two melting temperature (Tm)values of 37.2�C and 55.7�C with melting enthalpy (DHm) values of 51.09 J gL1 and 26.57 J gL1, respectively. Inhibitionanalyses using acrylic and 2-bromooctanoic acids revealed b-oxidation as the primary pathway for MCL-PHA biosyn-thesis using octanoic acid. Moreover, Pseudomonas putida GPp104 PHAL, harboring the PHA synthase genes ofP. mosselii (phaC1pm and phaC2pm) was used for heterologous expression, which demonstrated that phaC1pm is themain PHA synthesis enzyme, and 3-hydroxyoctanoyl-CoA is its major substrate. This was the first report of a P. mosseliiTO7 isolate producing high-yield P(3HO) through utilization of plant oils.

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[Key words: Carbon/metabolism; Fatty acids; Plant oils; Polyhydroxyalkanoates; Pseudomonas mosselii]

Polyhydroxyalkanoates (PHAs), one of the largest groups ofthermoplastic polyesters, consist of biopolymers composed of (R)-3-hydroxy fatty acid monomers that are synthesized by variousbacteria for carbon and energy storage by PHA synthases (1,2).PHAs are classified as short-chain-length and medium-chain-length PHAs (SCL-PHAs and MCL-PHAs, respectively), depending ifthe monomers contain 3e5 or 6e14 carbon atoms, respectively (3).While SCL-PHAs are brittle and stiff, MCL-PHAs possess greaterelasticity and are more biocompatible (4,5). Given that PHAs arebiodegradable, they have the potential to be used as a substitute fornon-degradable plastics in packaging materials, agriculture, andbiomedicine (6e8). Specifically, the flexible nature of poly(3-hydroxyoctanoate) (P(3HO)) makes it a potentially suitablecandidate as a biomaterial for soft tissue engineering and drugdelivery (9e11). In addition, PHAs and their composites have beenextensively used in medical devices, including but not limited tostents, sutures, repair/regeneration devices, and wound dressings(12). However, studies on MCL-PHAs are rare because large quan-tities of these polymers are often unavailable due to their high costof production.

Cupriavidus necator is the most commonly used PHA producer;the most frequently studied MCL-PHA producers include

ing author at: Department of Agricultural Chemistry, Nationalsity, 1 Sec 4, Roosevelt Road, Taipei 10617, Taiwan. Tel.: þ886 2: þ886 2 23660581.ress: [email protected] (C.-Y. Lee).

e see front matter � 2014, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2014.01.012

this article in press as: Chen, Y.-J., et al., Production and cas mosselii TO7, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1

Pseudomonas putida, P. oleovorans, and P. mendocina. However, themonomers produced differ between Pseudomonas strains. Forexample, culture of P. putida IPT046 on rice oil produced 61.8% celldry weight (%CDW) of PHA, which was primarily 3-hydox-ydecanoate (3-HD); however, only 19.6%CDWof PHAwas observedin Pseudomonas aeruginosa IPT171 under the same culture condi-tions, which was primarily P(3HO) (3,13).

Because the high price of PHA production (i.e., costly substrate)is the major drawback for their use in various new applications andreplacement of petroleum-derived plastics, the potential of inex-pensive carbon sources are being explored (3). For example, agenetically engineered P. putida KT2440 that expressed xyloseisomerase (XylA) and xylulokinase (XylB) from Escherichia coliW3110 was able to synthesize MCL-PHAs from xylose and octanoicacid (14). In addition, production of PHA from pyrolysis of wastepolystyrene by P. putida CA-3 has been reported (15). Furthermore,plant oils (e.g., palm and soybean oils) are considered desirable,renewable feedstocks for PHA production given their high carboncontent and low cost (16e18).

The objective of this study was to characterize the PHA pro-duction and growth of Pseudomonas mosselii TO7, a newly isolatedPseudomonas species from the effluent of a vegetable oilmanufacturing facility. After culturing in the presence of differentcarbon sources (i.e., fatty acids, carbohydrates and plant oils), thepolymer composition and yield of PHA were determined. In addi-tion, the physical properties of the PHA produced by P. mosselii TO7were examined. Furthermore, inhibition analyses were undertaken

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2 CHEN ET AL. J. BIOSCI. BIOENG.,

using octanoic acid and gluconate to identify the PHA synthesispathway, and the PHA polymerase gene of P. mosselii was isolatedfor subsequent genetic engineering in P. putida GPp104 PHA�.Identification and characterization of the new P. mosselii TO7 maylead to more cost-efficient production of PHA and therefore widenits industrial use.

FIG. 1. Isolation and identification of P. mosselii TO7. A phylogenetic tree based on the16S rRNA nucleotide sequences of P. mosselii TO7 and the sequences of neighbors wasgenerated using the neighbor-joining method to illustrate evolutionary relationships.The distance was calculated using the Kimura 2-Parameter algorithm. The sequence ofP. mosselii TO7 was deposited in GenBank under accession number JQ779914. Thenumber on the branch indicated the bootstrap value over 70% (1000 replicates). Thescale bar indicates the number of substitutions per nucleotide position.

MATERIALS AND METHODS

Isolation and identification of PHA-producing bacterium Samples werecollected from wastewater in a vegetable oil manufacturing factory in Taiwan. Thebacterial strains were isolated as described previously (19) with minormodification. Samples were incubated on mineral salt (MS) agar plates (20),containing 0.5% (v/v) octanoic acid as the sole carbon source and cycloheximide(0.06 g/L). The growing colonies were transferred to a new MS agar plate andincubated at 30�C for 72 h to detect accumulation of PHAs by staining colonieswith Nile red (0.5 mg/L) (21). The isolated strains that produced PHAs weresub-cultured in flasks containing MS liquid medium with 0.5% (v/v) octanoicacid. The culture was shaken at 200 rpm for 72 h at 30�C to accumulate PHAfor its subsequent analysis.

The isolated strain, TO7, was grown at 30�C in 2�YT medium (16 g of Tryptone,10 g of yeast extract, and 5 g of NaCl per liter) with shaking at 200 rpm in an orbitalshaker (model 703R, Hotech, Taiwan).

The 16S rRNA gene of the TO7 cells was amplified by PCR (ABI 9700,PerkineElmer Applied Biosystems, Carlsbad, CA, USA) using the prokaryotic 16SrRNA universal primers: pA (50-AGAGTTTGATCCTGGCTCAG-30) and pH (50-AAG-GAGGTGATCCAGCCGCA-30) (22) under the following conditions: one cycle at 95�Cfor 5min; 30 cycles at 94�C for 50 s, 60�C for 50 s, 72�C for 90 s; and one cycle at 72�Cfor 7 min.

PHA production, extraction, and analysis A one-step culture process wasused for production of PHA by TO7 using different carbon sources, including glu-conate, fructose, sucrose, hexanoic acid, octanoic acid, decanoate, dodecanoate(Sigma, St. Louis, MO, USA), soybean oil (Uni-President, Tainan, Taiwan) and palmkernel oil (Chant Oil Co., Ltd., New Taipei City, Taiwan). Palm kernel oil was derivedfrom the nut of Elaeis guineensis fruit. It includes the triglycerides of the followingfatty acids: 48.5% lauric acid (C12:0), 16.2% myristic acid (C14:0), 15.7% oleic acid(C18:1), 3.9% caprylic acid (C8:0), 3.5% capric acid (C10:0), 7.5% palmitic acid (C16:0),2.6% stearic acid (C18:0) and 2.1% linoleic acid (C18:2) (23). The fatty acid profile ofsoybean oil contained the following: 54.8% linoleic acid (C18:2), 22.7% oleic acid(C18:1), 10.2% palmitic acid (C16:0), 7.8% a-linolenic acid (C18:3), 4.5% stearic acid(C18:0) and 0.3% arachidic acid (C20:1) (24).

Briefly, bacteria were cultivated in 30 mL MS medium supplemented with acarbon source and incubated at 28�C and 200 rpm for 68 h to accumulate PHA (20).Cells were harvested by centrifugation, washed with a mixture of cold distilledwater and cold hexane for removal of residual palm kernel oil (25), and thenlyophilized overnight after which %CDW was determined. Approximately 10 mg ofthe lyophilized cells underwent methanolysis in a solution of 0.85 mL methanol,0.15 mL 98% sulfuric acid, 2 mg benzoic acid, and 1 mL chloroform at 100�C for140 min for measurement of PHA content and polymer composition (26). Themethylated monomer was determined by gas chromatography as previouslydescribed (20).

PHA chemical structure and molecular weight analysis PHA was extrac-ted from 40 mg of lyophilized cells as described elsewhere (27,28). Briefly, PHA wasextracted from 40 mg of lyophilized cells in 20 mL chloroform for 12 h withcontinuous stirring. The mixture was then filtered, and the polymer wasprecipitated in 10 volumes of chilled methanol for 24 h. The polymer was driedusing a rotary vacuum evaporator SPD111V (Thermo, Waltham, MA, USA). PHAchemical structure and molecular weight was determined using 13C nuclearmagnetic resonance spectroscopy (NMR) and gel permeation chromatography(GPC), respectively. PHA samples were dissolved in deuterated chloroform (CDCl335 mg/L), and the 125-MHz 13C NMR spectrum was determined using a BrukerAvance 500 NMR spectrometer (Bruker, Coventry, UK) at a probe temperature of25�C. The chemical shifts were reported in ppm.

A PHA sample of 2.5 mg was dissolved in 1 mL tetrahydrofuran. The injectionvolume was 0.1 mL, and it was eluted at a flow rate of 1.0 mL/min at 40�C. Sixpolystyrene molecular weight standards (American Polymer Standards Co.,Mentor, OH, USA), with molecular weights ranging from 104 to 382,100 g/mol,were used for calibration. The GPC system was equipped with two Phenomenexcolumns (Phenogel 5U M2 New Column 300 � 7.8 mm and Phenogel 5U 104 �ANew Column 300 � 7.8 mm, Phenomenex, Torrance, CA, USA), and a differentialrefractometer (model T60A, Viscotek Co. Houston, TX, USA) was used to detectthe eluted polymer.

PHA purity was determined as previously described (29). Briefly, aliquots oflyophilized bacteria were used to analyze PHA content using gas chromatography toestimate the weight of PHA (A) as well as PHA extraction and weighing (B). Thepurity of the PHA was 92.30 � 2.45%, which was determined using the followingequation: (A/B) � 100%.

Please cite this article in press as: Chen, Y.-J., et al., Production and cPseudomonas mosselii TO7, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1

Thermal property analysis of PHA After PHA was extracted from 40 mg oflyophilized cells as previously described, its thermal properties were evaluatedusing differential scanning calorimetry (DSC) analysis using a Perkin Elmer Pyris 6(PerkineElmer, Norwalk, CT, USA) under a nitrogen atmosphere. After a 5mg samplewas encapsulated in an aluminum pan, the temperature range was scannedfrom �100�C to 200�C with a heating rate of 10�C/min. The DSC endothermal peakvalues and areas of the second scan were used to evaluate the thermal properties ofPHA, including glass transition temperature (Tg), melting temperature (Tm), andmelting enthalpy (DHm).

Inhibition experiments A two-step culture process was used for the inhi-bition experiments to observe the accumulation of PHA by TO7 cells. Bacteria werefirst cultured in 30 mL 2�YT broth at 30�C for 14 h and thenwashed with 20 mL MSmedium. Various concentrations of acrylic acid and 2-bromooctanoic acid (Sigma, St.Louis, MO, USA) were added after the cells were transferred to 30 mL of MS medium(pH 9) containing 2% (w/v) gluconate and 0.5% (v/v) octanoic acid. The bacteria werecultured for an additional 30 h at 28�C and 200 rpm.

PHA polymerase gene cloning The PHA polymerase genes, phaC1pm andphaC2pm, were amplified by PCR using the following primer pairs: 50-


TABLE 1. Production of polyhydroxyalkanoates by strain TO7 when supplied various carbon sources.

Carbon sourcea CDWb (g/L) Yield of PHA (% CDW) Polymer composition (mol%)c

3-HB 3-HHX 3-HO 3-HD 3-HDD

Gluconate 1.35 � 0.13 1.85 � 0.20 ND ND 37.43 � 5.54 62.57 � 5.54 NDFructose 1.78 � 0.12 5.45 � 1.39 ND ND 21.44 � 0.51 78.56 � 0.51 NDSucrose No growth NDd ND ND ND ND NDHexanoic acid 1.12 � 0.26 4.64 � 0.94 ND 46.79 � 2.39 49.51 � 0.43 3.70 � 2.82 NDOctanoic acid 1.70 � 0.71 45.20 � 1.10 ND 3.57 � 0.25 93.79 � 1.08 2.64 � 0.82 NDDecanoate 2.54 � 0.01 46.54 � 2.44 ND 2.01 � 2.32 61.22 � 0.01 36.76 � 2.31 NDDodecanoate 2.45 � 0.39 45.42 � 1.93 ND 3.30 � 0.40 49.85 � 4.93 34.12 � 2.09 12.74 � 3.24Soybean oil 3.76 � 0.06 49.82 � 5.17 ND 5.59 � 0.87 47.72 � 0.34 35.63 � 0.09 11.06 � 0.43Palm kernel oil 4.31 � 0.52 47.10 � 0.84 ND 3.90 � 0.65 43.79 � 2.32 36.66 � 1.28 15.65 � 1.69

a 2% (w/v) gluconate, 1.5% (w/v) fructose, 1.5% (w/v) sucrose, 0.5% (v/v) hexanoic acid, 0.5% (v/v) octanoic acid, 0.5% (w/v) decanoate and 0.5% (w/v) dodecanoate, 0.5% (v/v)soybean oil and 0.5% (w/v) palm kernel oil.

b CDW, cell dry weight.c 3-HB, 3-hydroxybutyrate; 3-HHx, 3-hydroxyhexanoate; 3-HO, 3-hydroxyoctanoate; 3-HD, 3-hydroxydecanoate; 3-HDD, 3-hydroxydodecanoate.d ND, not detected.

TABLE 2. Time course of cell dry weight and PHA production from P. mosselii TO7.

Culture time (h)a CDWb (g/L) Yield of PHA (% CDW) PHA productivity (g PHA/L/h) Polymer composition (mol%)c

3-HB 3-HHX 3-HO 3-HD 3-HDD

1 0.04 � 0.01 0.00 � 0.00 0.00 � 0.00 NDd ND ND ND ND2 0.05 � 0.00 0.00 � 0.00 0.00 � 0.00 ND ND ND ND ND4 0.07 � 0.02 0.00 � 0.00 0.00 � 0.00 ND ND ND ND ND6 0.23 � 0.06 3.54 � 0.96 0.13 � 0.00 ND ND 100.00 � 0.00* ND ND8 0.37 � 0.04 4.45 � 0.81 0.21 � 0.06 ND ND 99.00 � 0.23* 1.00 � 0.23* ND16 1.28 � 0.23* 18.72 � 7.84* 1.56 � 0.90* ND 3.86 � 0.34* 93.81 � 0.23* 2.33 � 0.11* ND24 1.45 � 0.09* 29.54 � 3.21* 1.79 � 0.31* ND 3.63 � 0.05* 94.52 � 0.39* 1.85 � 0.34* ND32 1.59 � 0.00* 38.73 � 3.09* 1.92 � 0.15* ND 3.78 � 0.14* 94.10 � 0.76* 2.12 � 0.91* ND40 1.83 � 0.07* 44.73 � 2.84* 2.05 � 0.21* ND 4.03 � 0.09* 94.58 � 0.07* 1.39 � 0.02* ND48 1.92 � 0.25* 45.44 � 2.00* 1.82 � 0.32* ND 4.10 � 0.29* 94.79 � 0.13* 1.11 � 0.16* ND68 1.51 � 0.01* 48.88 � 5.02* 1.09 � 0.12* ND 3.99 � 0.35* 94.70 � 0.33* 1.31 � 0.02* ND

*P < 0.05 compared to a culture time of 1 h.a P. mosselii TO7 was cultured in mineral salt medium containing 0.5% (v/v) octanoic acid using a one-step culture process.b CDW, cell dry weight.c 3-HB, 3-hydroxybutyrate; 3-HHX, 3-hydroxyhexanoate; 3-HO, 3-hydroxyoctanoate; 3-HD, 3-hydroxydecanoate; 3-HDD, 3-hydroxydodecanoate.d ND, not detected.

VOL. xx, 2014 MCL-PHA PRODUCTION BY P. MOSSELII TO7 3

ACTGAATTCAGGACAAAGGAGCGTCGTAGATGAG-30 (EcoRI site is underlined) and 50-AGGATCCCCACGGCGGTTCGGCTCAAC-30 (BamHI site is underlined) for phaC1pm; 50-ATGGAATTCACGAAGGAGTGTTGGCATGAAGG-30 (EcoRI site is underlined) and 50-TCGGATCCGGGTCTTCATCCGGTCAG-30 (BamHI site is underlined) for phaC2pm. Theprimer synthesis and resulting PCR products were sequenced by Tri-I Biotech, Inc.(Taipei, Taiwan).

Heterologous expression To construct the pBBR-phaC1pm and pBBR-phaC2pm heterologous expression plasmids, the DNA fragments of phaC1pm andphaC2pmwere digested with EcoRI and BamHI (NEB Biolabs, Schwalbach, Germany)and introduced into the respective sites of pBBR1MCS2 (30). The pBBR-phaC1pmand pBBR-phaC2pm were introduced into P. putida GPp104 PHA�, a PHA negativemutant of P. putida KT2442 (31) by electroporation (Gene Pulser Xcellelectroporation system, Bio-Rad, Hercules, CA, USA). Recombinant P. putidaGPp104 PHA� were first cultivated in 30 mL 2�YT broth at 30�C for 14 h, washedwith 20 mL MS medium and transferred to 30 mL of MS medium (pH 8.5)supplemented with 2% (w/v) gluconate, 1.5% (w/v) fructose, 0.5% (v/v) hexanoicacid, 0.5% (v/v) octanoic acid, 0.5% (w/v) decanoate and 0.5% (w/v) dodecanoatefor an additional 30 h at 30�C and 130 rpm.

Data analysis Continuous data are presented as means � standard de-viations (SDs). The differences among three or more groups were detected byanalysis of variance (ANOVA). Pair-wise post-hoc tests using Bonferroni correctionwere applied for those significant differences in ANOVA. The statistical analyseswere performed with SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA). Atwo-tailed P-value <0.05 indicated statistical significance.

RESULTS AND DISCUSSION

Isolation and identification of P. mosselii TO7 Wastewatersamples were initially grown onMS agar plates containing octanoicacid as the sole carbon. Those that produced PHA were sub-cultured in MS medium supplemented with a carbon source andincubated at 28�C and 200 rpm for 68 h to accumulate PHA. Finally,a strain that produced high yields of MCL-PHAwith unusually high


P(3HO) monomer content, strain TO7, was identified. Subsequentphenotypic analysis revealed that TO7 was a Gram-negative, rod-shaped, motile, asporogenous, catalase-negative and oxidase-pos-itive bacterium. It was an aerobic bacterium that could not growunder anaerobic conditions.

After amplification and sequence analysis of the near-complete16S rRNA gene, comparison of the obtained sequence with those inthe NCBI database indicated that strain TO7 was closely related tothe genus, Pseudomonas. As shown in Fig. 1, a phylogenetic tree wasgenerated using Molecular Evolutionary Genetics analysis softwareversion 5.0 (32), which confirmed that strain TO7 is more closelyrelated to a type strain of P. mosselii than other known Pseudomonasspecies.

Production of PHA on different carbon sources P. mosseliiTO7 was cultured in the presence of various carbon sources toidentify the pathway used for PHA production using a one-stepculture process since the yield of PHA and polymer compositionwere similar to that observed using a two-step culture procedure(Table S1). As shown in Table 1, the 3-hydroxybutyrate (3-HB)monomer of PHA never appeared when the 3-hydroxyoctanoate(3-HO) and 3-hydroxydecanoate (3-HD) were present regardlessof the carbon source. Therefore, we concluded that P. mosselii TO7was an MCL-PHA producer. When supplied with C8eC12 fattyacids as the sole carbon source, larger quantities of PHA weresynthesized than when cultured in the presence of a C6 fattyacid, gluconate, or fructose. However, P. mosselii TO7 did not growwhen sucrose was the sole carbon source (Table 1).

3-HO was the primary PHA composition of cells cultured usingC8eC12 fatty acids as the sole carbon source, whereas 3-HD,


FIG. 2. 13C NMR spectrum of the near P(3HO) homopolymer produced by P. mosselii TO7.


followed by 3-HO, were the primary PHA composition of cellscultured using gluconate and fructose as the sole carbon sources(Table 1). These results indicated that P. mosselii TO7 preferredusing C8eC12 fatty acids to produce large quantities of MCL-PHA,especially using octanoic acid as a carbon source to produce a high-yield P(3HO).

In the presence of palm kernel oil as the sole carbon source,production of high-yield MCL-PHA by P. mosselii TO7 wasobserved; approximately 47% (w/w) of the cell dry weight wasMCL-PHA. In addition, cell growth was demonstrated using 0.5%(w/v) palm kernel oil in MS medium. Using palm kernel oil as thesole carbon source, 3-HO was the most prevalent PHA producedfollowed by 3-HD. Other monomers might have been also present,including saturated 3-hydroxytetradecanoate (C14:0), unsatu-rated dodecenoate (C12:1) and tetradecenoate (C14:1) (33). Cul-ture of P. mosselii TO7 with soybean oil as the sole carbon sourcealso supported growth and produced high yields of MCL-PHA; theyield was 49.82 � 5.17 %CDW and the CDW was 3.76 � 0.06 g/L(Table 1).

Under nitrogen limitation and carbon excess, intracellularpolymers were usually formed to store energy and carbon for cellgrowth. Because PHA production was enhanced under nitrogenlimitation conditions in P. putida (34,35), the effects of nitrogenlimitation on PHA production by P. mosselii TO7 were determined.As shown in Table S2, PHA production was decreased in MS me-dium containing 1.5 g/L (NH4)2SO4 and having a C/N ratio of 16.67.In these conditions, the dominant composition of PHAwas 3-HO. Inaddition, when NH2SO4 was absent, there was a significantdecrease in CDW.


Effects of cell growth on PHA production Previous studiesusing Cupriavidus necator or Aeromonas hydrophila indicated thatmaximal PHA production occurred in the stationary phase or underconditions of limited cell growth (36,37). Therefore, PHAproduction by P. mosselii TO7 was analyzed at different timepoints that included various phases of cell growth. As shown inTable 2, PHA production by P. mosselii TO7 began after 6 h in MSmedium containing an 8C fatty acid as the sole carbon source.CDW and PHA productivity peaked at 48 h and 40 h, respectively.The maximum PHA productivity of the P. mosselii TO7 wascalculated as 2.05 � 0.21 g PHA/L/h, which occurred after 40 h inculture. Thus, production of PHA began in the early log phase andpeaked during the stationary phase of cell growth, whichsuggested that the optimum time to harvest the cells to acquirethe maximum yield of mcl-PHA was during the stationary phase.In addition, the polymer composition of the PHA changed overtime with 3-HO appearing first at 6 h followed by a little 3-HD at8 h, and 3-HHX at 16 h.

Characterization of PHA chemical structure and molecularweight produced by P. mosselii TO7 The chemical structureand molecular weight of the PHA were determined using 13C NMRspectrum and GPC, respectively. As shown in Fig. 2, 13C NMRanalysis revealed eight peaks, corresponding to P(3HO)monomersobserved by Huijberts et al. (38). The C1 (C]O group) wasobserved by the peak at 169.4 ppm, C2 (eCH2 group) at39.14 ppm, C3 (eCH group) at 70.88 ppm, C8 (eCH3 group) at13.96 ppm, and C4, C5, C6 and C7 (eCH2 group) at 33.78, 24.74,31.55, and 22.52 ppm, respectively. The molecular weight of the


FIG. 3. Effects of acrylic acid and 2-bromooctanoic acid inhibitors on the PHA synthesis by P. mosselii TO7. (A, B) PHA content (wt%) and (C, D) cell dry weight (CDW g L�1) by weredetermined with increasing concentration of (A, C) acrylic acid and (B, D) 2-bromooctanoic acid. In each of experiments, 0.5% (v/v) octanoic acid and 2% (w/v) gluconate was used asthe sole carbon source, respectively. Data are presented as means � SDs of two independent experiments. Calculation of inhibition of PHA was as follows: {[(PHA content wt%) noinhibitor � (PHA content wt%)inhibitor]/(PHA content wt%) no inhibitor}. Calculation for CDW inhibition was as follows: {[(CDW no inhibitor) � (CDW inhibitor)]/(CDW no in-hibitor)}. *P < 0.05 compared to concentration 0 mM.


PHA produced by P. mosselii TO7 cultured inMSmedium containing0.5% (v/v) octanoic acid as the sole carbon source for 68 h was218.30 � 31.73 kDa; the polydispersity index was 2.21 � 0.18.These values coincided with those of MCL-PHAs produced byother Pseudomonas spp. (28).

Thermal properties of MCL-PHA from palm kernel oilproduced by P. mosselii TO7 The thermal properties of theMCL-PHA produced by P. mosselii TO7 from palm kernel oil werenext analyzed. As shown in Fig. S1, two Tm values of 37.2�C and55.7�C were detected, and the DHm values were 51.09 J g�1 and26.57 J g�1, respectively. The Tg value was �42.8�C (Fig. S1).Moreover, the MCL-PHA produced in the presence of soybean oilhad Tm and heat of melting fusion (DHm) values of 46.0�C and1.480 J g�1, respectively (data not shown).

Effect of inhibitors of fatty acid metabolism on theproduction of PHA To determine the relationship betweenfatty acid metabolism and the generation of precursors for PHAsynthesis, two inhibitors, acrylic acid and 2-bromooctanoic acid,were employed as previously described (38e40). Acrylic acid is aknown inhibitor of acyl-CoA synthase, which catalyzed the acyl-CoA formation from fatty acids (38). 2-Bromooctanoic acidinhibited (R)-3-hydroxyacyl-ACP:CoA transferase (PhaG) andconsequently inhibited PHA synthesis from saccharides inbacterium (39); it also partially inhibited specific b-oxidationpathway enzymes (39). To avoid suppression of bacterial growthby the inhibitors and the subsequent impact on PHA yield,P. mosselii TO7 was cultured using a two-step culture process. As


shown in Fig. 3A, the production of MCL-PHA was almostcompletely inhibited by 2.5 mM acrylic acid; the CDW was alsoreduced when 0.5% (v/v) octanoic acid was the sole carbon source(Fig. 3C). However, the production of MCL-PHA and CDW was notinhibited by 2.5e5 mM acrylic acid when 2% (w/v) gluconate wasused as the sole carbon source (Fig. 3A and C). These resultssuggested that PHA synthesis by P. mosselii TO7 occurs via the b-oxidation pathway when octanoic acid is the carbon source.

The production of MCL-PHAwas also gradually inhibited to 90%with 2.5e5 mM 2-bromooctanoic acid (Fig. 3B). However, the CDWwas not significantly affected by 2.5e5 mM 2-bromooctanoic acidwhen 2% (w/v) gluconate was the sole carbon source (Fig. 3D). Theproduction of MCL-PHA and CDWwas rescued by 21 and 44% by 2.5and 5 mM 2-bromooctanoic acid, respectively when 0.5% (v/v)octanoic acid was the sole carbon source (Fig. 3D).

PHA polymerase gene cloning and its heterologousexpression in P. putida GPp104 PHAL Because P. mosselii wasa recently recognized species (22), the PHA polymerase gene hadnot been previously analyzed. In this study, the phaC genesencoding PHA polymerases of this strain were cloned, sequenced,and compared with other genes. The phaC1pm and phaC2pmsequences were subsequently deposited in the EMBL andGenBank databases under accession numbers JN123471 andJN123472, respectively. A considerable percentage of identity, 91%for the gene and 97% for the amino acid sequence, between thePHA polymerase phaC1pm gene isolated from P. mosselii TO7 andthat of the Pseudomonas entomophila strain L48 was observed. In



addition, the phaC2pm gene product of TO7 had the greatest aminoacid identity (91%) to that of P. entomophila strain L48.

Heterologous expression of phaC1pm and phaC2pm in P. putidaGPp104 PHA� was undertaken to identify the PHA polymeraseactivities of phaC1pm and phaC2pm gene products. The recombi-nant P. putida GPp104 PHA� (phaC1pm) produced high yields ofPHA when cultured in an MS medium containing C8eC12 fattyacids (Fig. 4). 3-HO was the primary PHA produced using P. putidaGPp104 PHA�. Regardless of the type fatty acid or sugar used as thecarbon source, the PHA production of the recombinant strains,P. putida GPp104 PHA� (phaC2pm) and P. putida GPp104 PHA�,harboring the pBBR1MCS2 vector was low (Fig. 4).

This study characterized an environmentally isolated bacterium,P. mosselii TO7 that produced high-yield P(3HO) when grown inoctanoate using a single-step culture process. This strain continuedto produce high levels of MCL-PHAs in the presence of plant oilsprimarily through the b-oxidation synthesis pathway.

P. mosselii is an environmental species observed in soil in therhizosphere. This species exhibits suitable phosphate solubilizationpotential and antifungal activity; thus, it has been used for plantdisease control and growth promotion (41e43). In the presentstudy, the PHA composition produced by P. mosselii TO7 wasanalyzed after culturing with hexanoic acid (C6), octanoic acid (C8),decanoate (C10) and dodecanoate (C12) as the sole carbon sources.The results of this analysis indicate that the order of the mostsuitable PHA polymerase substrates were (R)-3-hydroxyoctanyl-CoA, (R)-3-hydroxydecanyl-CoA, and (R)-3-hydroxydodecanyl-CoA,and (R)-3-hydroxyhexanyl-CoA (C6); the smaller (R)-3-hydrox-yacyl-CoA were unsuitable substrates.

Given the expense of pure fatty acids for PHA production, plantoils have been suggested as a possible alternative carbon source(44). However, few studies have analyzed the potential of bacterialspecies to produce MCL-PHAs in the presence of plant oils(3,13,16,45). Previous analysis of PHA production by P. putidaIPT046, P. aeruginosa IPT169, P. aeruginosa IPT171 and P. putidaBet001 cultured with rice, canola, sunflower, corn, soybean andpalm oils revealed a maximal PHA % CDW of 61.8 with P. putidaIPT046 cultured in rice oil using a one-step culture process (13). Inthe present study, high yields of MCL-PHA by P. mosselii TO7 werealso observed in the presence of soybean and palm kernel oils,reaching up to 50% CDWwith most consisting of P(3HO) and 3-HD.This is similar to that observed for Comamonas testosterone culturedwith a wide variety of plant oils, including castor seed, coconut,mustard, cotton seed, groundnut, olive, and sesame oils (46).

In addition to plant oils, Ward et al. (15) reported successful PHAproduction from P. putida CA-3 using styrene oils that had under-gone pyrolysis. In this way, plastic waste could be converted into

FIG. 4. PHA content synthesized by recombinant P. putida GPp104 PHA� expressingphaC1pm and phaC2pm. Data are means of two independent experiments, and errorbars indicate standard deviations. *P < 0.05 as compared to phaC2pm-expressing cells;yP < 0.05 as compared to plasmid control (pBBR1MCS2).


biodegradable plastics. Further studies will assess the ability ofP. mosselii TO7 to also convert petroleum-based plastics to PHA.

In the presence of palm kernel oil, the PHA produced had two Tmvalues of 37.2�C and 55.7�C. This is different from that reported forthe PHA produced by P. putida PGA1 cultured in palm kernel oil,which had no Tm value and low 3-HD and 3-HDD (3-hydrox-ydodecanoate) monomer composition (4.5% and 1.4%, respectively)(29). However, two Tm values were also observed with high 3-HDDmonomer content synthesized by P. entomophila (47), which isconsistent with the present study.

Previous studies indicate that greater 3-HD and 3-HDD mono-mer fractions of PHA had high crystallinity and improved proper-ties, including greater tensile strength (47e49). The 3-HD and 3-HDD monomer content of PHA in P. mosselii TO7 was higher thanobserved for P. putida PGA1 (33), suggesting that b-oxidation wasslower than P. putida PGA1 at the step in which two carbons areremoved given that the predominant fatty acid in palm kernel oilwas lauric acid (C12:0).

PHA polymer synthesis by bacteria cells requires the PHA poly-merase, which polymerizes 3-hydroxyacyl-moiety in (R)-3-hydroxyacyl-CoA (50). Previous in vitro studies have confirmed thatPHA polymerase substrates are (R)-3-hydroxyacyl-CoA metabolites(51); therefore, (R)-3-hydroxyacyl-CoA is a precursor for PHA syn-thesis. The MCL-PHA precursor is supplied through the followingthree metabolic pathways, which diverge depending on the carbonsource. b-Oxidation is the primary pathwaywhen the carbon sourceis a fatty acid. Fatty acid de novo synthesis is another pathwaywhenacetyl-CoA from carbohydrate oxidation is the carbon source (52).Finally, acyl chain elongation occurs when acetyl-CoA moieties arecondensed to 3-ketoacyl-CoA (40). Inhibition of enzymeswithin thePHA synthesis pathways candramaticallyalter the PHAcompositionas was observed with gene deletion of fadA and fadB encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase inP. putidaKT2442 (49). In thepresent study, PHA synthesis precursorsin P.mosseliiTO7were suppliedprimarily through b-oxidationwhengrown using octanoic acid. In the presence of acrylic acid, an in-hibitor of acyl-CoA synthase, the 3-hydroxyhexanoate (3-HHx)monomer disappeared (data not shown), indicating inhibition of 3-ketoacyl-CoA thiolase that catalyzes the acetyl-CoA release from 3-ketoacyl-CoA (38). However, 2-bromooctanoic acid had no inhibi-tory influence on cell growth, suggesting that PHA synthesis byP. mosselii TO7 is via the de novo fatty acid synthesis pathway whengluconate is present in the culture medium.

Recently, the flexible semi-crystalline P(3HO) is a promisingnovel biomaterial for the industrial production of bioplastics (53).In this study, the 3-HO monomer ratio was 85 mol% when octanoicacids were used as the sole carbon source for P. putida GPp104PHA� (phaC1pm). P. mosselii TO7 produced a higher ratio of the 3-HOmonomer (94 mol%), which is not only relevant to the substratespecificity of phaC1pm, but also the result of other enzymesinvolved in PHA synthesis, such as (R)-specific enoyl-CoA hydratase(PhaJ), NADPH-dependent 3-ketoacyl-CoA reductase (FadG), and 3-hydroxyacyl-CoA epimerase (FadB) from P. mosselii TO7.

In the present study, the recombinant strain, P. putida GPp104PHA� (phaC1pm) produced significantly greater PHA as comparedto the control strain; however, no such induction of PHA productionwas observed in the P. putida GPp104 PHA� (phaC2pm) recombi-nant strain.

A novel wild-type Pseudomonas, P. mosselii TO7, that producesup to 48 wt% of P(3HO) with more than 94 mol% monomer contentwithin 68 h when grown in octanoate, was isolated and identified.This is the first study to report high-yield P(3HO) production fromP. mosselii, as well as direct utilization of plant oils by the P. mosseliiTO7 for production of MCL-PHA. The novel MCL-PHA produced byP. mosselii TO7 using plant oil consisted of long-chain monomerswith improved thermal properties. In addition, the MCL-PHA



possessed elastomeric and flexible properties that are required forapplication as biomedical materials, and further studies will assessthe molecular weights and polydispersity indices of the PHAs.However, studies regarding the application of MCL-PHA remainsparse due to the limited quantities of the polymer. Given theunique properties of the MCL-PHA produced by P. mosselii TO7utilizing plant oil substrates, further studies are necessary toexplore the full potential of these biomaterials.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jbiosc.2014.01.012.

ACKNOWLEDGMENTS

The authors thank Professor Wen-Yen Chiu (NTU) for help withthe GPC analysis. This work was supported in part by grants fromthe National Science Council of Taipei, Taiwan (NSC93-2313-B-002-085 and 94-2313-B-002-027).

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