ARTICLE pubs.acs.org/Biomac
Biosynthesis and Characterization of Polyhydroxyalkanoate Block Copolymer P3HB-b-P4HB Die Hu,†,‡ Ah-Leum Chung,†,‡ Lin-Ping Wu,§ Xin Zhang,‡ Qiong Wu,‡ Jin-Chun Chen,*,‡ and Guo-Qiang Chen*,‡ ‡
MOE Key Lab of Protein Sciences, Department of Biological Sciences and Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China § Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark ABSTRACT:
Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into betaoxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (Tm) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.
’ INTRODUCTION The growing interest of biorenewable resources to supplement or replace the need of nonrenewable resources for a sustainable economy led to an exploration into biologically derived plastics over the past years.13 Polyhydroxyalkanoates (PHA) are a family of bacterial polyesters, one of the representatives of biopolymers that are currently under extensive investigation due to their biodegradable, biocompatible, and sustainable properties.4 Recently, PHA-based research has slowly evolved into an industrial value chain, including industrial fermentation, packaging materials, medical implants, biofuel, fine chemicals, and drugs.5,6 PHA are synthesized by many microorganisms as carbon source and energy storage substance during unbalanced growth.7 More than 100 different monomers can be combined within this family to generate materials with different properties.4 Based on the monomer structures, PHA are divided into short-chain-length (scl) PHA, commonly consisting of C3C5 hydroxyl fatty acids such as 3-hydroxypropionate (3HP), 3-hydroxybutyrate (3HB), r 2011 American Chemical Society
4-hydroxybutyrate (4HB), and 3-hydroxyvalerate (3HV); medium-chain-length (mcl) PHA, containing C6C14 hydroxyl fatty acids including typically 3-hydroxyhexanoate (3HHx), 3-hydroxyheptanoate (3HHp) to 3-hydroxytetradecanoate (3HTD); copolymers of scl and mcl PHA containing the above scl and mcl monomers.4,8 Poly(3-hydroxybutyrate) (P3HB), one of the well-studied scl PHA, is difficult to process and use due to its poor thermal and mechanical properties.8 Typical mcl PHA copolymers are generally sticky amorphous materials without useful thermal and mechanical advantages.8,9 PHA with improved thermal and mechanical properties can be obtained by adjusting the composition of monomers. For example, incorporation of 4HB as a monomer into P3HB has resulted in the production of commercially attractive copolymers,10 copolymers of scl and mcl monomers combine the Received: May 14, 2011 Revised: July 8, 2011 Published: August 24, 2011 3166
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Table 1. Bacterial Strains and Plasmids Used in This Study plasmids/strains
relevant characteristics
references
Plasmids pK18mobsacB pSPK02
Suicide plasmid for gene knockout, KanR
33 R
36
Suicide plasmid for phaC1-phaZ-phaC2 operon knockout, Kan R
37
pKSSE5.3
Amp , containing phbC of R. eutropha (Class I PHA synthase) and 4-hydroxybutyrate-
pBBR1MCS2
Broad-host-range plasmid, KanR
34
pBHH01
A fragment from pKSSE5.3 containing partial DNA sequence including phbC
this study
CoA transferase encoding gene orfZ from C. kluyveri
(Class I PHA synthase) and 4-hydroxybutyrate-CoA transferase encoding gene orfZ from C. kluyveri was inserted in pBBR1MCS2 Strains E. coli S17-1
recA; harboring the tra genes of plasmid RP4 in the chromosome, proA, thi-1
35
KTOY08
P. putida KT2442 mutant (ΔfadB2x, ΔfadAx, ΔfadB, ΔfadA)
41
KTOY08ΔG
P. putida KT2442 mutant (ΔfadB2x, ΔfadAx, ΔfadB, ΔfadA, ΔphaG)
32
KTOY08ΔGC
P. putida KT2442 mutant (ΔfadB2x, ΔfadAx, ΔfadB, ΔfadA, ΔphaC-phaZ-phaC2, ΔphaG)
this study
KTHH06
KTOY08ΔGC harboring PHA synthase phbC of R. eutropha (Class I PHA synthase) and 4-hydroxybutyrate-
this study
CoA transferase encoding gene orfZ genes from C. kluyveri
strength of P3HB and the flexibility of mcl PHA.11,12 So far, random copolymers including poly(3-hydroxybutyrate-co-3-polyhydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-polyhydroxyhexanoate) (PHBHHx) have been found to exhibit useful thermal and mechanical properties,8,13,14 although they still suffer from property detrimental aging effects.1519 P3HB, P4HB, PHBV, PHB4HB, and PHBHHx are commercially available.5 Block copolymerization is another approach to gain novel polymers with new properties.2022 Block polymer chains contain two or more different regions (or “blocks”) of polymer covalently bonded together.23 The structures of block copolymers include A-B diblock, A-B-A or A-B-C triblock, and (A-B)n repeating multiblocks. Block copolymerization can lead to additional physical properties that cannot be obtained by random copolymerization, polymer blending, or filler-adding techniques, because this block structure captures the properties of each block.24 Traditional chemical synthesis techniques have been successful in generating PHA block copolymers, such as PHB blocks balanced with blocks of poly(6-hydroxyhexanoate),25 poly(3-hydroxyoctanoate),26 monomethoxy-terminated poly(ethylene glycol) (mPEG),27 and poly(ethylene glycol).28 However, microbial synthesis of PHA block copolymers has been only limited by PHBb-PHBV22 and PHB-b-PHVHHp.29 Pseudomonas putida KT2442 is a typical mcl PHA producer. Its derivative, P. putida KTOY08, is fadB2x, fadAx, fadA, and fadB genes knocked out mutant with a weakened β-oxidation.30 By knocking out phaG and phaC genes from P. putida KTOY08, a mutant termed KTOY08ΔGC was constructed. P. putida KTOY08ΔGC harboring phbC of Ralstonia eutropha H16 and orfZ of Clostridium kluyveri that encodes a 4-hydroxybutyrate (4HB)-CoA transferase31 (this strain was named KTHH06) was found to be able to use fatty acids more efficiently in producing C4 monomers containing scl PHA due to the specificity of PHA synthase PhaC of R. eutropha32 that favors only scl monomers. In this study, for the first time, KTHH06 was used for biosynthesis of P3HB and P4HB containing block copolymers. The block copolymer showed better physical properties, especially in yield strength and tensile strength compared with random copolymer and
blend of homopolymers of P3HB and P4HB. The novel properties of the block polymer P3HB-b-P4HB would be interesting in PHA research field.
’ MATERIAL AND METHODS Plasmids Construction. Plasmid pK18mobsacB is a suicide vector donated by Professor Andreas Sch€afer, University of Bielefeld, Germany.33 Broad-host-range plasmid pBBR1MCS2 was kindly provided by Dr. Philip Green, Procter and Gamble, U.S.A.34 Escherichia coli S17-1 was used as the host for plasmid construction and a vector donor in conjugation.35 All plasmids and strains used in this study are listed in Table 1. Plasmid pSPK02 were pK18mobsacB derivatives.36 The method to generate a defined gene knockout mutant was described by Sch€afer et al.33 Briefly, in this study, to disable phaC activity in the P. putida mutant, knockout mutation was carried out. Plasmid pSPK02 was first transformed into E. coli S17-1 by electroporation, then transconjugation of P. putida strains and E. coli S17-1 harboring suicide plasmids were carried out. Because pSPK02 could not be replicated in P. putida, transconjugants were only obtained after integration of the whole suicide plasmid into the chromosome via single crossover event. LB agar plate containing 100 mg/L ampicillin and 50 mg/L kanamycin was used to select transconjugants. An insertion mutant with kanamycin resistance was constructed. To select knockout mutant, a single colony of the insertion mutant obtained in the previous step was cultured in a nonantibiotic containing LB medium at 30 °C for 24 h. Approximately 106 cells (about 10 μL of cultivation broth) were plated onto nonantibiotics LB medium supplemented with sucrose (LBS) agar plate (LB agar plate supplemented with 80 g/L sucrose) and incubated for 48 h. The resulting colonies sensitive to kanamycin indicated the occurrence of a second crossover event, these strains were selected. The colonies which were knockout mutants were confirmed by PCR verifications and DNA sequencing.36 Plasmids pKSSE5.337 and pBBR1MCS2 were both double digested by BamHI and EcoRI. A 5,419 bp fragment of pKSSE5.3 containing phbC and orfZ was retrieved and inserted into corresponding pBBR1MCS2 vector to form pBHH01. The plasmids were first transformed into E. coli S17-1. The transconjugation of P. putida and E. coli S17-1 harboring recombinant plasmids were carried out as described by Friedrich et al.35 Kanamycin of 50 mg/L and/or ampicillin of 100 mg/L were used for screening whenever necessary. 3167
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Table 2. Effect of Cell Growth and PHA Production by P. putida KTHH06 Grown in Various Concentrations of Butyrate (C4) or γ-butyrolactone (C4*)a (a) C4* (g/L)b
CDW (g/L)
PHA (g/L)
PHA (wt%)
3HB (wt%)
4HB (wt%)
1
6.07 ( 0.27
0.84 ( 0.07
13.82 ( 0.85
0
100
2.5
7.10 ( 0.23
1.56 ( 0.16
22.11 ( 2.94
0
100
5
6.93 ( 0.97
2.30 ( 0.45
32.80 ( 2.17
0
100
C4 (g/L)c
CDW (g/L)
PHA (g/L)
PHA (wt%)
3HB (wt%)
4HB (wt%)
(b)
1
5.65 ( 0.48
0.29 ( 0.04
5.19 ( 1.04
100
0
2.5
5.76 ( 0.19
0.63 ( 0.08
10.96 ( 1.36
100
0
5
2.94 ( 0.27
0.08 ( 0.07
2.81 ( 0.85
100
0
Bacteria were cultured in LB medium for 12 h at 30 °C in a 500 mL shake flask containing 100 mL LB as described in the Material and Methods section. The total cultivation time was 48 h. All data were express as average value ( SD and represented the average value of three parallel experiments. b 1, 2.5, and 5 g/L γ-butyrolactone (C4*) was added each at 12 and 24 h, respectively. c 1, 2.5, and 5 g/L sodium butyrate (C4) was added each at 12 and 24 h, respectively. a
Cultivation of Bacteria. All bacteria listed in Table 1 were stored
at 80 °C using glycerol as preservation agent and then transferred into LuriaBertani (LB) medium. A 1% v/v inoculation was conducted in the LB medium at 30 °C and 200 rpm for 12 h in order to activate the cold stored cultures. A 5% v/v seed culture of P. putida KTHH06 grown for 12 h was first inoculated into a 500 mL shake flask containing 100 mL LB (5 g/L yeast extract, 10 g/L tryptone, 10 g/L sodium chloride) for all cultivation. Different concentration of carbon sources were added into medium and cultivated 48108 h for better polymer production. Polymer Extraction. Bacterial cells grown in the fermentor were recovered by centrifugation at 8000 g for 5 min and washed twice using distilled water, and then lyophilized. PHA extraction was conducted using 10 times chloroform (v/w) added to the lyophilized cells. Extraction was carried out at 90 °C for 4 h. The chloroform PHA solution was cooled to room temperature, residual biomass was removed via filtration. The PHA was precipitated with 10-fold volume quantities of cold ethanol (v/v). Gas Chromatography. About 10 mg of PHA sample or at least 20 mg biomass was added into 2 mL chloroform and 2 mL acidic methanol (3% v/v H2SO4 in methanol), 1 g/L decanoic acid was used as an internal standard.30 Methanolysis was conducted at 100 °C for 4 h. PHA content and composition were determined by gas chromatography (GC) using Hewlett-Packard 6890 equipped with 30-m HP-5 capillary column. Physical Properties. The molecular weight and molecular weight distribution were measured by using gel permeation chromatography (GPC) equipped with a refractive index (RI) detector (Wyatt Optilab rEX). The measurements were carried out at 35 °C using a PLgel 5 μm mixed-D column, which was calibrated with polystyrene standards. THF was used as the eluent, and the flow rate was 1.0 mL/min. Differential scanning calorimetry (DSC) was performed on Shimadzu DSC-60 under a nitrogen flow rate of 50 mL/min. Sample of 25 mg in an aluminum-sealed pan was cooled to 90 °C, heated from 90 to 180 °C at a rate of 10 °C/min, and maintained for 2 min. Then the pan was quenched to 90 °C and reheated from 90 to 180 °C at a rate of 10 °C/min. Data were collected during the two heating runs. Melting temperature (Tm) and apparent heat of fusion (ΔHm) were determined from the DSC endothermal peak value and areas of the first scan. The midpoint of transition temperature range was determined as the glass transition temperature (Tg). Nuclear Magnetic Resonance. Proton, carbon, and 2D magnetic resonance spectra (1H and 13C NMR) were recorded at room temperature in deuterated chloroform (CDCl3) at a concentration of 20 mg/mL
on a JEOL JNM ECA-600 NMR spectrometer, 1H NMR spectra were used to determine the polymer composition of 3HB and 4HB units, and 13 C signals and 2D NMR 1H13C HMBC spectrum were used to determine the chemical microstructure and the monomer sequence. The long rang J constant was set as 4 Hz in the HMBC spectrum. Tetramethylsilane was used as the internal standard.38 Mechanical Properties. Dumbbell-shaped samples of polymer films with a thickness of 0.050.1 mm, base width 10 mm, and base length 5 cm, were used for mechanical property tests. The elongation at break, tensile strength, and Young’s modulus of the tested samples were determined using a CMT 4204 universal testing machine (Sans, China) at room temperature at an extension rate of 5 mm/min. The values were the average values of three parallel studies. PHBHHx materials were produced on a small scale by Lukang Pharma Co. Ltd. (Shandong, China) as a gift for this lab.
’ RESULTS AND DISCUSSION As P. putida KTHH06 is able to use fatty acids more efficiently for the production of 3HB and 4HB containing PHA due to the weakened β-oxidation ability,36 sodium butyrate (C4) and γbutyrolactone (C4*) were used for biosynthesis of P3HB and P4HB, respectively. P. putida KTHH06 was able to produce homopolymers of 3HB and 4HB and a block copolymer of P3HB and P4HB when related substrates were added (Tables 2 and 3). Production of Homopolymers. When grown on sodium butyrate (C4), P. putida KTHH06 produced only homopolymer P3HB. While only homopolymer P4HB was produced when grown on γ-butyrolactone (C4*; Table 2). When different concentrations of C4 or C4* were added to the medium as a carbon source, the strain showed different cell growth and PHA accumulation (Table 2). When 10 g/L of C4* was fed, the PHA content was higher than that under 5 or 2 g/L C4* (Table 2a), and when 5 g/L C4 was fed, the highest CDW and PHA production were obtained (Table 2b), which could be attributed to the cytotoxicity of C4, suggesting that 10 g/L C4* and 5 g/L C4 could be used to prepare block copolymers for enhanced CDW and PHA production. Microbial Synthesis of the Block Copolymer. Based on the above results that P. putida KTHH06 produced only P3HB on sodium butyrate and P4HB on γ-butyrolactone (Table 2), it was concluded that concentration of the two substrates can be used to 3168
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Table 3. Effect of Cell Growth and PHA Production by P. putida KTHH06 Grown under Various Glucose Addition Conditionsa total time (h)
time (h) of glucose added
CDW (g/L)
PHA (g/L)
PHA content (wt%)
3HB (mol %)
4HB (mol %)
60b
12, 24, 36, 48
3.54 ( 0.74
1.01 ( 0.64
25.75 ( 12.77
18.87 ( 10.69
81.13 ( 10.69
84c
none
3.14 ( 0.10
0.36 ( 0.06
11.40 ( 2.08
27.81 ( 2.85
72.19 ( 2.85
84c
0, 24, 48, 60
5.17 ( 0.13
2.58 ( 0.12
49.93 ( 1.15
13.33 ( 4.59
86.67 ( 2.85
84c
12, 24, 48, 60
5.06 ( 0.23
1.44 ( 0.29
28.15 ( 4.38
13.68 ( 5.72
86.32 ( 5.72
108d
0, 12, 36, 60, 84
5.47 ( 0.27
2.71 ( 0.07
49.58 ( 1.76
19.69 ( 5.27
80.31 ( 5.27
Bacteria were cultured in LB medium for 12 h at 30 °C in a 500 mL shake flask containing 100 mL of LB, as described in Material and Methods. All data were expressed as average value ( SD and represented the average value of three parallel experiments. b 5 g/L γ-butyrolactone (C4*) was added as the carbon source at 12 and 24 h, respectively. 2.5 g/L sodium butyrate (C4) was fed to the culture at 36 and 48 h, respectively. c 5 g/L γ-butyrolactone (C4*) was added as carbon source at 12 and 24 h, respectively. 2.5 g/L sodium butyrate (C4) was fed to the culture at 48 and 60 h, respectively. d 5 g/L γbutyrolactone (C4*) was added as carbon source at 12 and 36 h, respectively. 2.5 g/L sodium butyrate (C4) was fed to the culture at 60 and 84 h, respectively. a
adjust the composition of a possible block copolymer of P3HB and P4HB. A total of 10 g/L C4* and 5 g/L C4 were used as carbon sources for production of the block copolymer mentioned above by P. putida KTHH06. To investigate a suitable time for C4 addition, a HPLC was employed to follow the consumption of C4*. After approximately 24 h of cell growth, less than 0.2 g/L C4* was observed from an initial concentration of 5 g/L C4*, suggesting that the suitable time for C4 feeding to form the second P3HB block polymer could be 24 h after the cell growth on C4*. Glucose was added during the fermentation process as a nutrient for cell growth. When 20 g/L glucose was added at 0, 24, 48, and 60 h, the cell growth was significantly improved to 5.17 g/L, of which nearly 50 wt % of PHA was synthesized. Glucose could also be added at the beginning of cultivation with fed-batch addition of related carbon sources to avoid inhibition. An incubation lasting 84 h resulted in more CDW and PHA contents compared to results of a 60 h growth (Table 3). Longer hour of incubation seemed to be necessary for cell growth in toxic substances like butyrate or other short chain fatty acids at inhibitory concentrations to accumulate more CDW consisting of more PHA. The strain was first cultivated in LB medium for 12 h in a 500 mL shake flask containing 100 mL of LB, then, when 5 g/L γ-butyrolactone (C4*) was added as carbon source at 12 and 36 h, respectively, P4HB was formed during the C4* consuming process. As the C4* was consumed to a very low concentration level (barely detectable level), 2.5 g/L sodium butyrate (C4) was fed to the culture at 60 h. Incubation was continued for another 48 h to utilize the C4 for P3HB block formation. The block polymer P3HB-bP4HB was obtained after 108 h of a fermentation process. 5.47 g/L CDW was obtained from the recombinant strain P. putida KTHH06 on related carbon sources at the end of the cell growth. The PHA accumulation reached 2.71 g/L, which was the highest yield observed. The resulting P3HB-b-P4HB block copolymer consisted of 20 mol % P3HB and 80 mol % P4HB in their respective block (Table 3). Physical Characterization of the Novel Block Copolymer. The suspected block copolymer P3HB-b-P4HB produced as mentioned above was characterized and compared with a blend PHA containing homopolymers P3HB and P4HB of the same compositions as that of the block copolymer, and with a random copolymer of P3HB4HB. GPC showed that the block copolymer had a number average molecular weight of 50000 D and a polydispersity of 3.1 (data not shown).
NMR. The NMR analysis revealed very different spectra among the blend and the suspected block copolymer samples (Figure 1). In the blend of P3HB and P4HB, monomers of 4HB and 3HB have only one chemical environment, respectively (Figure 1a). While in the block copolymer P3HB-b-P4HB, the 4HB and 3HB monomers (Figure 1a, gray color) has different chemical environments, they had cross-correlation with each other. The different chemical environments showed clear influences with the adjacent carbonyl group {3HB (1), 4HB (1)} (Figure 1b), the diad-dependent carbonyl resonances in the 13C NMR spectrum of the block polymer P3HB-b-P4HB was split into four peaks (Figure 1c) with relative intensities, as described by a Bernoullian model in eq 1.39 If the polymer is a statistically random copolymer described by the Bernoullian statistics, F4HB*4HB, F4HB*3HB, F3HB*4HB and F3HB*3HB (FX*Y represents the molar fraction of XY sequence) can be expressed with the molar fraction of 4HB F4HB, as follows:
F4HB4HB ¼ F4HB 2 F4HB3HB ¼ F3HB4HB ¼ F4HB ð1 F4HB Þ F3HB3HB ¼ ð1 F4HB Þ2
ð1Þ
To determine whether a polymer is a random copolymer or not, a parameter D is defined as follows: D ¼ ðF4HB4HB F3HB3HB Þ=ðF4HB3HB F3HB4HB Þ
ð2Þ
The fractions were examined with 13C NMR to determine the monomer sequence distribution. Analysis of the diad peaks revealed the nearest monomer neighbor distribution in the fraction and the D statistic of eq 2 was determined where F4HB*4HB represents the fraction of 4HB neighboring a 4HB monomer, and so forth. Generally speaking, from eq 2, random copolymers would have a D value near 1; the D value for a block copolymer should be much larger than 1, while that of an alternating copolymer should be smaller than 1. Analysis of our fractions yielded block polymer P3HB-b-4HB according to 13C NMR spectrum (F4HB*4HB = 0.657, F3HB*3HB = 0.296, F4HB*3HB = 0.026, F3HB*4HB = 0.021), the D value was 356.2, which was much larger than 1, confirming that this sample was of a block polymer P3HB-b-4HB. To further confirm the microstructure of the block copolymer, 2D NMR 1H13C HMBC spectra of the block copolymer and the blend sample were recorded. It was clear that there were crosscorrelation signals between the OCH2 group {1H NMR δ4.12 ppm, 4HB (4)} of 4HB and the two adjacent carbonyl 3169
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Figure 1. NMR study of the PHA microstructure: (a) The chemical structures of the block polymer and blend of P3HB and P4HB; (b) The 13C NMR spectrum of the blend of PHB and P4HB; (c) The 13C NMR spectrum of the block copolymer of P3HB and P4HB.
groups of 4HB {13C NMR δ172.75 ppm, 4HB(1)} and 3HB {13C NMR δ169.2 ppm, 3HB (1)} (Figure 2a). The OCH2 group of P4HB not only correlated to P4HB itself, but also correlated to the carbonyl group of P3HB, indicating that P3HB was blocked with P4HB. On the other hand, there was no crosscorrelation between the OCH {1H NMR δ5.26 ppm, 3HB (3)} and carbonyl groups of 4HB {13C NMR δ172.75 ppm, 4HB (1)}, except the carbonyl groups of P3HB itself, suggesting that P3HB-b-P4HB was the predominate microstructure in this polymer. Compared with block P3HB-b-P4HB, there was only correlation with its own monomer respectively in the blend of P3HB and P4HB (Figure 2b). The 2D NMR spectrum provided solid evidence for the existence of the block copolymer P3HB-b-P4HB. Thermal Properties. The block, random copolymers and blend polymer showed different thermal behaviors due to their different microstructures (Figure 3, Table 4). The random copolymer had a single Tg with small Tm and without Tc, demonstrating its amorphous nature as a result of the random assembly of its components in the chain segment instead of repeated chain segments. While, the block copolymer revealed two Tm of 54 and 161 °C, but only one Tg of 47 °C was observed in DSC curve, the second glass transition of P3HB-bP4HB should be around 0 °C which belong to P3HB block, but
the trace of cooling crystallization peak (Tc) among 20 to 5 °C covered the second Tg, and the amount of P3HB was just 20% in block copolymer, with the interferer of Tc, the second Tg was too weak to observe in the DSC thermograms (Figure 3b). Two Tm of 56 and 171 °C were found in the blend polymer, corresponding to the typical Tm of homopolymers P4HB and P3HB. Also singular Tg of 48 °C was observed in blend of P3HB and 4HB, because the cooling crystallization peak (Tc) covered the second Tg belonging to P3HB, it cannot observe the second Tg around 0 °C. And the Tg of 48 °C belong to P4HB in blend was almost equal to Tg of P4HB homopolymer, demonstrating the immiscibility of P4HB with P3HB. Compared with the Tm of PHB segment in block and blend samples, only the Tm of PHB in block copolymer was shift to low temperature 161 °C, while the homopolymer P3HB was 171 °C, indicating that there was not simple mixture of P3HB segment and P4HB segment in block copolymer, but a strong interaction with each segment. Mechanical Properties. Mechanical properties of the block copolymer P3HB-b-P4HB illustrated higher yield strength and tensile strength compared with its random copolymer and blend despite the lowest elongation at break due to its microstructure (Table 5).40 Yet the block copolymer still had a much larger elongation at break of over 300% compared with that of 3170
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Figure 2. 2D NMR 1H13C HMBC spectra of block copolymer P3HB-b-P4HB (a) and blend sample of P3HB and P4HB (b).
PHB of only 5.9%. The novel properties of the resulting block copolymer point to possibility of property manipulation based on changing block compositions (Table 5), as reported by Pederson et al., elongation at break increased significantly from 250 to 575% with increasing 3HV components from 15 to 25 mol %.22 Although PHB has better tensile strength and Young’s modulus, it suffers from its brittleness and instability at its melting point. Therefore, application of PHB is still limited
although it has the lowest cost among all PHA known. Compared with other commercially available PHA, especially PHBHHx, the novel P3HB-b-P4HB block copolymer showed improved properties in tensile strength and elongation at break. Compared to other scl PHA, mechanical properties of the block copolymer appears to be significantly improved. The crystallization rate due to different size polymer domains may cause differences between random copolymer and block copolymer.22 New properties are waiting be developed based on the 3171
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Table 5. Mechanical Properties of P3HB4HB Random Copolymer, P3HB-b-P4HB Block Copolymer, and Blend Sample of P3HB and P4HB Compared to Commercially Available PHBHHx a sample
yield strength (MPa)
tensile strength (MPa)
elongation at break (%)
PHBHHx copolymerb
12.5
7.0
400
random copolymerc
7.3
9.9
729
blend sampled
10.9
16.5
357
block copolymerd
13.3
19.9
438
a
PHBHHx consists of 12 mol % 3HHx; Random copolymer: P(3HBco-70 mol % 4HB); Blend sample: P3HB (20%) + P4HB (80%); Block sample: P3HB (20%)-b-P4HB (80%). b Data cited from Li et al.29 c Data cited from Li et al.42 d Samples were prepared by P. putida KTHH06.
proportion of each blocks in the polymers and the monomer structures on each block.
’ CONCLUSIONS For the first time, diblock copolymer P3HB-b-P4HB consisting of approximately 80 mol % P4HB and 20 mol % P3HB were synthesized by Pseudomonas putida KTHH06 when γ-butyrolactone (C4*) and butyrate (C4) were supplied as related carbon source in a sequence order, NMR, DSC, and mechanical property studies confirmed the existence of the block copolymer and its improved thermal and mechanical properties over the relevant blend and random copolymer. Polymer property manipulations could be realized if ratios of the two blocks are changed. Microbial block copolymerization has opened a new area for PHA structure and property diversification. ’ AUTHOR INFORMATION Corresponding Author
*Tel.: +86-10-62783844 (G.-Q.C.). Fax: +86-10-62794217 (G.-Q.C.). E-mail:
[email protected] (G.-Q.C.); chenjc@mail. tsinghua.edu.cn (J.-C.C.). Figure 3. (a) DSC thermograms of the second heating process for block copolymer P3HB (20%)-b-P4HB (80%) (1), blend sample of P3HB (20%) and P4HB(80%) (2), random copolymer of P(3HB-co70 mol % 4HB) (3), and P3HB (4). (b) The amplification of Tg DSC trace.
Table 4. Thermal Properties of P3HB4HB Random Copolymer, P3HB-b-P4HB Block Copolymer and Blend Sample of P3HB and P4HB Homopolymers Compared with Their Individual Homopolymersa sample (PHA)
a
Tg (°C)
Tm (°C)
Tc (°C)
P3HB
0.7
171.6
41.0
P4HB random copolymer
45.67 22.4
50.1 50.1
0.69
blend sample
48.6,b
56.0, 171.2
12.7
block copolymer
47.3,b
54.2, 161.4
6.7
Random copolymer: P(3HB-co-70 mol % 4HB); Blend sample: P3HB (20%) +P4HB (80%); Block sample: P3HB (20%)-b-P4HB (80%). b Cannot detect from the DSC thermograms since the cooling crystallization peak covered the second Tg.
Author Contributions †
These authors contributed equally to this paper.
’ ACKNOWLEDGMENT We are very grateful for the kind donation of plasmid pK18mobsacB from Dr. Andreas Sch€afer of the University of Bielefeld (Bielefeld, Germany). Broad-host-range plasmid pBBR1MCS2 was a gift from Dr. Philip Green, Procter and Gamble, U.S.A. This research was financially supported by National High Tech 863 Grants (Project No. 2010AA101607) and 973 Basic Research Fund (Grant No. 2007CB707807), as well as the Tsinghua University Initiative Scientific Research Program 2009THZ01005. ’ REFERENCES (1) Luengo, J. M.; Garcia, B.; Sandoval, A.; Naharro, G.; Olivera, E. R. Curr. Opin. Microbiol. 2003, 6, 251–260. (2) Lawson, J. L. Plast. Eng. 1998, 54, 12–12. (3) Warwel, S.; Bruse, F.; Demes, C.; Kunz, M.; Ruschgen Klaas, M. Chemosphere 2001, 43, 39–48. (4) Steinb€uchel, A.; Valentin, H. E. FEMS Microbiol. Lett. 1995, 128, 219–228. 3172
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