Biosynthesis and Characterization of Polyhydroxyalkanoates with

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Biosynthesis and Characterisation of Polyhydroxyalkanoates with Controlled Composition and Microstructure Anna Ferre-Guell, and James Winterburn Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01788 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Biomacromolecules

1

Biosynthesis and Characterisation of

2

Polyhydroxyalkanoates with Controlled

3

Composition and Microstructure

4

Anna Ferre-Guell, James Winterburn*

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School of Chemical Engineering and Analytical Science, The Mill, The University of

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Manchester, Manchester M13 9PL, UK

7

ABSTRACT

8

Volatile fatty acids (VFA) C2:0 to C6:0 were used as a sole carbon source the production of

9

poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

(PHBV)

production

with

controllable

10

composition and microstructure in Haloferax mediterranei. Feeding carbon-even VFA gave

11

>90 mol% poly(3-hydroxybutyrate) (3HB) PHBV while carbon-odd VFA generated >87 mol%

12

poly(3-hydroxyvalerate) (3HV) PHBV. Bespoke random, block and blend copolymers with

13

0-100 mol% 3HV were synthesized using C4:0/C5:0 mixtures. The copolymer 3HV fraction is

14

proportional to the %C5:0 in the feed mixture, allowing control over composition.

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Microstructure depends on the substrate addition order: co-feeding generated random

16

copolymers whilst sequential feeding created block and blend copolymers. On average the

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PHBV had an ultra-high molecular weight of 3×106 g/mol. 3HV rich PHBV showed lower

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melting temperature, enhanced elasticity and ductility. H. mediterranei is ideal for large-scale

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production of PHBV due to its inherent bioprocessing advantages. Control over the composition

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and microstructure of PHBV will facilitate the production of biopolymers capable of meeting

21

industrial criteria for specific applications.

22

KEYWORDS

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Halophile, Haloferax mediterranei, PHBV, block copolymer, volatile fatty acids (VFA),

24

material properties

25 26

INTRODUCTION

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Interest in the development of environmentally friendly alternatives to oil-based plastics is

28

growing due to the problems associated with the processing and disposal of traditional plastics.

29

Biopolymers such as polyhydroxyalkanoates (PHA) have attracted considerable attention as

30

potential candidates to replace some oil-based plastics because of their biodegradability,

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biocompatibility and similarity in terms of mechanical properties. PHA have the added benefit of

32

being produced from renewable resources and thus being independent of crude oil price and

33

availability1,2.

34

PHAs are a family of natural polyesters that accumulates inside different bacteria and archaea

35

as intracellular granules acting as a carbon and energy reservoir. More than 150 different types of

36

PHA monomers have been identified with the type and distribution of these monomers dictating

37

the final polymer properties, extending from rigid plastics to elastic rubbers. The two most

38

investigated

39

poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)3. PHB is hard to process into

40

commodity goods because is stiff and brittle, has a high degree of crystallinity and the melting

41

temperature is close to its decomposition temperature, which restricts its use. The introduction of

types

of

PHA

are

poly(3-hydroxybutyrate)

(PHB)

and


2

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Biomacromolecules

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3-hydroxyvalerate (3HV) units to form PHBV disrupts the highly crystalline PHB structure,

43

resulting in a polymer with enhanced mechanical properties, quicker degradation rates, improved

44

processing and greater potential for commodity applications4–6.

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Commercial PHA production is typically done with pure cultures of wild-type or genetically

46

modified microorganisms7. The main factors restricting wider PHA utilization are

47

inconsistencies in PHA structure and hence properties; high production costs associated with the

48

use of refined carbon sources; the discontinuous production in batch or fed-batch; the prevention

49

of contamination and the use of large volumes of solvent in downstream processing8–10.

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Halophiles, salt loving microorganisms, are promising candidates with increasing interest for the

51

economical large scale production of PHA11 because they have simple growth requirements and

52

can utilize a wide variety of carbon sources as substrates12. More importantly, the extremely high

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salinity of the media drastically reduces the risk of contamination and the requirement for media

54

and equipment sterilisation13, which in turn opens the possibility for the development of longer

55

and continuous production campaigns or production in open ponds14,15. Amongst the halophiles,

56

the extreme archaea H. mediterranei is of interest for the production of PHBV since it naturally

57

produces the copolymer without the addition of precursors or structurally related carbon sources3

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and the polymer accumulation is associated with cell growth, hence no specific nutrient

59

limitation is required leading to straight-forward fermentations. Furthermore, recovery of the

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biopolymer is solvent-free because in a hypotonic environment the high intracellular salinity

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leads to cell lysis, thus reducing the use of toxic solvents and contributing towards the

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development of greener, cheaper and potentially more sustainable processes16,17.

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The number of publications in which H. mediterranei is used as PHA producer has increased

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in recent years with the main focus on the utilisation of different wastes as cheap substrates18–21,


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the development of new fermentation techniques22,23 and the study of H. mediterranei

66

metabolism3,24–27, with only a few dedicated to the investigation of the PHA characteristics6,28,29.

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The thermal properties of PHA from H. mediterranei have been frequently reported6,19–21,28 but

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there is limited information regarding the biopolymer mechanical properties29. Koller30, Zhao5

69

and co-workers found significant differences between the PHBV from haloarchaea and bacteria,

70

so the biopolymer properties from these microorganisms might not be directly comparable. Thus,

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the characterisation of biopolymers from haloarchaea is required to identify potential

72

applications based on the unique PHA properties. Additionally, the production of PHBV with

73

high 3HV fraction is normally limited by the 3HV precursor toxicity. For example, Han and

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coworkers29 reported the production of PHBV with 60 mol% 3HV at expenses of biomass and

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PHA synthesis and when sufficient amount of cells were generated the final copolymer only had

76

an 3HV content of 46.8 mol%. Similarly, McChalicher and coworkers31 wanted to study the

77

properties of block copolymers with fractions containing 60 mol% 3HV but such composition

78

was not attainable using fructose and valerate co-feeding. Thus, examples in literature of such

79

copolymers are scarce and their potential is not fully explored.

80

The genome of H. mediterranei32 encodes enzymes for the β-oxidation pathway and it has

81

been suggested that the strain can utilise short-chain fatty acids as a carbon source29. Han and

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coworkers29 started to explore the possibility using short-chain fatty acids by co-feeding C5:0

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and glucose and synthesised PHBV with 3HV content ranging from 7 to 57 mol% that showed

84

good haemostatic properties. However, there are no reports regarding the utilization of fatty

85

acids as sole substrates for cell growth and PHA production. In this study VFA C2:0, C3:0, C4:0,

86

C5:0 and C6:0 have been used as a sole carbon source for H. mediterranei growth and PHA

87

production. PHBV with a wide range of composition was produced, from PHB rich copolymers,


4

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Biomacromolecules

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when feeding solely carbon-even VFA, to PHV rich copolymers, when only carbon-odd VFA

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were supplied. More importantly, it was found that when cells were grown with C4:0/C5:0

90

mixtures, the 3HV fraction in the PHBV copolymer was directly proportional to percentage of

91

C5:0 in the mixture. Co-feeding or sequential feeding led to the generation of random or block

92

and blend copolymers respectively. Thus, the composition and microstructure of the copolymers

93

can be controlled by regulating the C4:0:C5:0 ratios in the feed and the order of addition. The

94

different PHBV produced had ultra-high molecular weight and showed excellent thermal and

95

mechanical properties. This study demonstrates that it is possible to produce bespoke PHBV

96

polymers with specific characteristics by controlling the feed composition order of addition to

97

meet the criteria for commodity uses.

98 99

EXPERIMENTAL

100

Microorganism and culture conditions

101

The microorganism used in all the experiments was H. mediterranei DSM 1411. A 20 mL seed

102

culture was initiated from vials stored at −80°C33 and grown for 24 h at 37°C and 200 rpm in

103

minimal synthetic medium (MSM) supplemented with 10 g/L of glucose. The culture was then

104

transferred to a second 200 mL seed culture grown under the same conditions for 36 h in order to

105

generate sufficient biomass to inoculate the production flask. Prior to the inoculation of the

106

production flask, the second seed culture was concentrated by centrifugation at 7000 rpm for

107

5 min (Sigma 6-16S, SciQuip) and re-suspended with MSM. The production flasks were

108

inoculated to an initial OD600 of 1.4. The production flasks contained MSM supplemented with

109

different fatty.


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For the screening experiment (exp. A-E 1-3), cells were grown in 100 mL of MSM with

111

saturated fatty acids from C2:0 to C11:0, namely ethanoic acid (C2:0), propanoic acid (C3:0),

112

butanoic acid (C4:0), pentanoic acid (C5:0), hexanoic acid (C6:0), heptanoic acid (C7:0),

113

octanoic acid (C8:0), nonanoic acid (C9:0), decanoic acid (C10:0) and undecanoic acid (C11:0).

114

The fatty acids were added at carbon concentrations of 0.10, 0.25 and 0.50 M, see Table 1

115

(supporting information). The cells were cultivated for 94 h to 335 h until no further growth was

116

detected.

117

The remaining experiments (A4-E4, F1-F3, G1, and G2) were carried out in 200 mL of MSM

118

with different VFA added in a fed-batch feeding strategy, see Table 1 and Table 2 for details

119

(supporting information). For the fed-batch feeding strategy, the initial carbon concentration in

120

the production flasks was 0.1 M, with two subsequent 10 mL additions of MSM containing 4 M

121

carbon of the corresponding VFA to achieve a final carbon concentration of 0.50 M. For

122

experiments A4-E4 ethanoic acid (C2:0), propanoic acid (C3:0), butanoic acid (C4:0), pentanoic

123

acid (C5:0), hexanoic acid (C6:0) were used. The cells were cultivated for 144 h to 262 h until no

124

further growth was detected. Experiments F1-F3 were carried out for 213 h by co-feeding a

125

mixture of butanoic and pentanoic acid (C4:0:C5:0) at carbon ratios of 25:75, 50:50 and 75:25.

126

For experiments G1 and G2, sequential feeding of butanoic acid (C4:0) and pentanoic acid

127

(C5:0) was performed. Specifically, in experiment G1 cells were initially grown in C4:0 and

128

sequentially fed first at 92 h with one addition of C4:0 followed by a second addition of C5:0 at

129

160 h; and in experiment G2 cells initially grown in C5:0 and sequentially fed with first at 92 h

130

one addition of C5:0 followed by a second addition of C4:0 at 160 h. The total length of the

131

cultivation was 213 h.


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Biomacromolecules

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All the experiments were carried out at 37°C and 200 rpm and were performed in duplicate

133

(experiments A-E 1-3, F1-F3, G1, G2) or quadruplicate (A4-E4). Sterilisation of the glassware

134

and media was not done at any stage during the experiment and potential contamination was

135

monitored via regular microscopic observations and streaking on agar plates.

136

The minimal synthetic medium (MSM) contained (g/L): 156 NaCl, 13 MgCl2 • 6 H2O,

137

20 MgSO4 • 7 H2O, 1 CaCl2 • 6 H2O, 4 KCl, 0.2 NaHCO3, 0.5 NaBr, 2 NH4Cl, 0.5 KH2PO4,

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0.005 FeCl3, 15 1,4-Piperazinediethanesulfonic acid (PIPES) and 10 mL/L21 trace element

139

solution

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0.03 MnCl2 • 4 H2O, 0.3 H3BO3, 0.2 CoCl2 • 6 H2O, 0.01 CuCl2 • 2 H2O, 0.02 NiCl2 • 6 H2O,

141

0.03 Na2MoO4 • 2 H2O. The medium and different substrate additions were adjusted to pH

142

6.8±0.2 with NaOH prior to use.

SL6.

Trace

element

solution

SL6

contained

(g/L):

0.1 ZnSO4 • 7 H2O,

143 144

Determination of optical density and cell dry mass

145

Samples for optical density (OD600) determination were centrifuged at 13000 rpm for 5 min

146

(MiniSpin, Eppendorf), the cells re-suspended in 10% (w/v) NaCl solution and optical density

147

was measured at 600 nm in a UV-visible spectrophotometer (UVmini-1240, UV-VIS

148

spectrophotometer, Shimadzu). For the cell dry mass (CDM) measurement, 2 mL cell culture

149

were transferred in ceramic crucibles, dried at 105°C to constant weight and subsequently heated

150

in a P300 furnace (Nabertherm) at 400°C for 4h. CDM was calculated as the weight difference

151

between the samples before and after the furnace heating step.

152

Quantification of PHA and composition characterization with GC-FID

153

For PHA quantification, 2 mL of cell culture was centrifuged at 13000 rpm for 5 min

154

(MiniSpin, Eppendorf). The pellet was re-suspended with distilled water, transferred to pressure


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tubes and dried at 105°C to constant weight. The methanolysis sample preparation procedure and

156

gas chromatography method was used as previously described

157

(mPHA/CDM) was calculated by divining the PHA produced by the CDM obtained.

33

. The PHA accumulation

158

PHA recovery, purification and film preparation

159

To recover the PHA from the biomass, the samples were first centrifuged at 7000 rpm for

160

10 min (Sigma 6-16S, SciQuip), the pellet re-suspended with the same volume of distilled water

161

with 0.1% sodium dodecyl sulphate (SDS) and then continuously agitated for 30 min. This

162

procedure was repeated 2 to 5 times until a white PHA pellet was obtained. The PHA was

163

washed one final time with distilled water and the PHA pellet was dried.

164

For PHA purification the PHA pellet was dissolved in CHCl3 to an approximate final

165

concentration of 10 g/L, and the obtained solution was then precipitated using 10 volumes of

166

cold methanol under continuous stirring and then agitated for 20 min. Finally, the PHA was

167

recovered by decantation or centrifugation at 7000 rpm for 5 min and dried at 30°C.

168

PHA films were prepared using the solvent casting technique. Approximately 0.1 g of purified

169

PHA was dissolved in 10 mL CHCl3 and the solution poured into 60 cm PTFE dishes. The dishes

170

were covered with a glass lid and the CHCl3 left to evaporate. The films obtained were semi-

171

transparent with an average thickness of 32 ± 8 μm and were stored at room temperature for a

172

minimum of 2 weeks before testing.

173

Quantification of fatty acids with HPLC and yields calculation

174

Fatty acids in the cell-free supernatant were quantified using high-performance liquid

175

chromatography (UltiMate3000 Dionex HPLC system, Thermo Scientific) equipped with an

176

Aminex HPX-87H (Biorad) column coupled to a UV detector (HPLC-UV). The mobile phase


8

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Biomacromolecules

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was 5 mM H2SO4, the flowrate was set to 1.0 mL/min, the operating temperature was 50°C and

178

the detecting wavelength was 210 nm.

179

The biomass yield (YX/S) was calculated by dividing the C-biomass generated by the C-

180

substrate consumed. The biomass value was obtained by subtracting the amount of PHA from

181

the cell dry mass and taking the elemental composition of the cell biomass as

182

CO0.63H1.57N0.13P0.02S0.01 with molar mass 26.4 g/mol34. The PHA yield (YPHA/S) was calculated

183

by dividing the C-polymer synthesized by the C-substrate consumed.

184

Determination of molecular weight with GPC

185

The determination of PHA molecular weight was done as previously described 33.

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Determination of PHBV microstructure and composition with NMR

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Purified PHA was dissolved in CDCl3 to a concentration of approximately 20 mg/mL and a

188

B500 MHz Avance II+ (Bruker) was used to record 1H NMR and

189

temperature. PHBV composition was calculated from the 1H NMR spectra using the relative

190

intensities of the methyl peaks corresponding to 3HB and 3HV units. 13C NMR data was used to

191

calculate nearest neighbour statistics D value35, see equation 1, and determine the PHBV

192

microstructure. D value is indicative of the distribution of the 3HB and 3HV monomer units:

193

statistically random distribution are characterised by D values close to 1, block polymers show D

194

values greater than 1 and alternating copolymers have D values smaller than 135,36. D =

13

C NMR spectra at room

FHBHB × FHVHV FHBHV × FHVHB

195

Equation 1. FHBHB is fraction of 3HB adjacent to 3HB monomers, FHVHV is fraction of 3HV

196

adjacent to 3HV monomers, FHBHV is fraction of 3HB adjacent to 3HV monomers and FHVHB is

197

fraction of 3HV adjacent to 3HB monomers.

198

Determination of the thermal properties using DSC and TGA


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A differential scanning calorimeter DSC Q100 (TA Instruments) was used for the

200

determination of the thermal properties of the obtained PHA samples. Briefly, 2.5 ± 0.8 mg of

201

polymer were initially heated to 200°C at 10°C/min and maintained at this temperature for

202

5 min, the sample was then cooled to −20°C at 10°C/min, heated up to 200°C at 10°C/min and

203

cooled to −20°C. The glass transition (Tg) and crystallization (Tc) temperature were determined

204

from the onset point and the melting temperature (Tm) was determined at the minimum of the

205

peak.

206

The thermal stability of the PHA was investigated by thermogravimetric analysis with

207

TGA550 (TA instruments). The samples were heated at 5°C/min to 500°C and the gas flow rate

208

was set to 25 mL/min. The decomposition temperature (Td) was determined at the onset point.

209

Tensile testing of PHA films

210

Solvent cast film strips of 5 × 25 mm were tested with a 3344 Testing System (Instron)

211

equipped with a 10 N load cell at a tensile rate of 5 mm/min at room temperature. The Young’s

212

modulus, tensile strength and elongation at break were calculated from the stress-strain curve.

213 214

RESULTS AND DISCUSSION

215

PHA production using fatty acids from C2:0 to C11:0

216

Fatty acids were used as the sole carbon source for H. mediterranei growth and PHBV

217

production. Saturated fatty acids with chain lengths varying from C2:0 to C11:0, at three

218

different carbon concentrations of 0.10 M, 0.25 M and 0.50 M, were initially tested in a

219

screening experiment (exp. A-E 1-3). The results obtained show that high fatty acid

220

concentrations were toxic for the cells and inhibited cell growth, hence a new set of experiments

221

was performed following a fed-batch feeding strategy (exp. A-E 4): the initial fatty acid carbon


10

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Biomacromolecules

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concentration in the culture media was set to 0.1 M and two additions of 0.2 M were performed

223

to reach a final concentration of 0.5 M.

224

The results for the screening and fed-batch experiments are summarised in Table 1 (supporting

225

information). Cell growth was compared by determining the maximum specific growth rate

226

(µmax) and cell dry mass (CDM). In the screening experiment, cell growth was only observed

227

with C2:0 to C5:0 at all carbon concentrations tested and C6:0 at 0.10 and 0.25 M. No biomass

228

was detected for cultures containing C6:0 at 0.50 M or C7:0 to C11:0 at any concentration tested.

229

The variation in µmax for C2:0, C3:0 and C4:0 was relatively small for the concentrations tested

230

and the CDM obtained increased or remained constant with increasing amount of substrate, from

231

an average of 2.4 g/L for 0.10 M to a maximum of 6.70 g/L for condition B3. There was a

232

substantial decline in µmax as the carbon concentration of C5:0 or C6:0 increased: µmax decreased

233

from 0.017 h-1 to 0.006 h-1 for C5:0 and from 0.011 h-1 to no growth for C6:0. A similar trend

234

was observed in CDM with increasing C5:0 and C6:0 concentrations. The results clearly showed

235

that substrate toxicity increased with fatty acid length and carbon concentration. It is often

236

reported in literature that the PHV precursors propionate (C3:0) and valerate (C5:0) inhibit cell

237

growth. Han and coworkers29 observed a significant reduction in maximum CDM when

238

H mediterranei was grown on glucose and 17mM of valerate (equivalent to a carbon

239

concentration of 0.085 M). To overcome the toxicity problem, a fed-batch feeding strategy was

240

successfully employed (exp. A-E 4). The fed-batch feeding allowed the production of similar or

241

higher amounts of CDM compared to 0.5 M batch cultures (exp. A-E 3), thus allowing the

242

generation of sufficient biomass and PHA under otherwise toxic conditions. Interestingly, µmax

243

for fed-batch (exp. A-E 4) cultures and for the cultures fed with a carbon concentration of 0.1 M

244

(exp. A-E 1) are comparable, indicating that the subsequent addition of substrate in the fed-batch


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245

experiment did not have a negative effect on cell growth. Fed-batch cultivation was especially

246

beneficial when C5:0 and C6:0 were used as a carbon source as it permitted biomass growth and

247

polymer accumulation.

248

The intracellular PHA content ranged from 5% to 23% in batch cultures and was between 11%

249

and 27% for fed-batch cultures, see Table 1. The values are comparable to other flask

250

fermentations reported in literature26,37,38, although higher PHA accumulations can be achieved

251

in bioreactor fermentations or in complex media containing hydrolysates or wastes19,21,28,39,40.

252

The polymer composition was determined by GC and 1H NMR and similar results were obtained

253

with each technique. PHBV copolymers with varied composition were obtained: carbon-even

254

VFA produced a PHBV polymer rich in 3HB fraction (3HB > 90 mol%) while carbon-odd VFA

255

generated a PHBV polymer rich in 3HV fraction (3HV > 87 mol%). When C2:0 was used as

256

carbon source a copolymer with an 8.2 mol% 3HV synthesised, a composition similar to the

257

10 mol% 3HV obtained from glucose33. Since glucose is metabolised to acetyl-CoA to produce

258

PHBV, a likely explanation is that C2:0 is converted to the same intermediate and follows the

259

same pathway to generate PHBV. In that case, the end polymer would have a similar

260

composition irrespective of the original substrate used. The PHBV polymer with 86.9 mol%

261

3HV obtained from C3:0 indicates that the substrate is used to generate both 3HB and 3HV

262

units, a phenomenon well documented in literature4,41–43. Bhubalan4, Doi42 and co-workers

263

reported that twice the amount of C3:0 was required to generate PHBV polymer with a certain

264

3HV content when compared to C5:0 since some of the C3:0 supplied was directed towards 3HB

265

synthesis. The loss of the 3HV precursor could be due to oxidative decarboxylation, which

266

converts propionyl-CoA to acetyl-CoA, hence a 3HV precursors is converted to a

267

3HB precursor44. High purity homopolymer PHB was generated from C4:0 and C6:0 and PHV


12

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Biomacromolecules

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from C5:0. This indicates that C4:0 and C5:0 are directly incorporated into the polymer chain

269

without breaking the carbon skeleton41,42. However, C6:0 is first converted into a PHB precursor

270

and then incorporated into the polymer chain as 3HB.

271

The results presented show that VFA are promising substrates for the production of PHBV by

272

H. mediterranei. VFA can be economically obtained by anaerobic fermentation of different

273

organic wastes45, which could contribute towards a reduction of the production cost.

274

Additionally, scaling up the process into multistage batch46 or continuous reactors23 could

275

enhance polymer yields, thus making the biopolymer production more economically viable.

276

Production of PHBV with controlled composition and microstructure

277

The experiments described in the previous section revealed that when C4:0 and C5:0 were

278

used as the sole carbon source in H. mediterranei cultures, high purity PHB and PHV were

279

obtained. In light of these results, a new set of experiments was designed to control the PHBV

280

composition and microstructure by regulating the C4:0/C5:0 proportions in the substrate mixture

281

and the time-sequence of addition. For the generation of random PHBV polymers, C4:0/C5:0

282

mixtures were co-fed to cell cultures; the mixtures contained 25%, 50% or 75% carbon coming

283

from C4:0, resulting in C4:0:C5:0 molar ratios of 29:71, 56:44 and 79:21 (exp. F1-F3). For the

284

generation of block and blend polymers, sequential feeding of C4:0 followed by C5:0 and

285

vice versa was performed; the C4:0:C5:0 molar ratios tested were of 65:35 and 45:54 (exp. G1,

286

G2). The total carbon fed in all cases was 0.50 M, the initial carbon concentration was set to

287

0.1 M and two additions of 0.2 M were performed.

288

The results for experiments F1-F3, G1, G2 are summarised in Table 2 (supporting

289

information). Similar cell growth, PHBV production and accumulation were obtained with the

290

co-feeding or sequential feeding of C4:0/C5:0 mixtures and also with the cultures grown with


13


Page 14 of 39

291

pure C4:0 and C5:0 (see Table 1, exp. C4 and D4). The biomass yield (YX/S) and PHA yield

292

(YPHA/S) reported in Table 2 are comparable to the 0.30 and 0.31 Cmol/Cmol biomass yield and

293

the 0.12 and 0.15 Cmol/Cmol PHA yield obtained in experiments C4 and D4 respectively. Thus,

294

the cell cultures have similar performances in terms of cell growth and PHBV synthesis

295

regardless of the substrate used and the feeding sequence, indicating that the composition of the

296

feed only influences the proportion of 3HB and 3HV in the final copolymer, rather than the

297

efficiency of PHBV production.

298

Polymer composition was determined by GC and 1H NMR and comparable results were

299

obtained using both techniques. In all cases, the polymer obtained was a PHBV copolymer with

300

increasing 3HV fraction as the C5:0 percentage in the mixture increased, see Table 2. Strikingly

301

there is a strong linear correlation between the percentage of C5:0 in the feed and the 3HV

302

fraction in PHA polymer, with a correlation coefficient (R2) greater than 0.98, see Figure 1. This

303

correlation exists regardless of the feeding strategy used, co-feeding or sequential feeding, and

304

covers the complete spectrum of PHBV compositions, from pure PHB to pure PHV. Therefore, it

305

is possible to produce PHBV polymers with a pre-defined composition by simply feeding the cell

306

culture with a C4:0/C5:0 mixture with the same C5:0 molar ratio as the desired 3HV fraction.

307


14

Page 15 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

308 309

Figure 1. Relationship between percentage of C5:0 in the feed and 3HV fraction in PHBV

310

polymers obtained with co-feeding or sequential feeding of C4:0/C5:0 mixtures (random PHBV

311

square solid symbols, F1-F3; block PHBV circle, G1; blend PHBV triangle, G2). Error bars

312

show the standard deviation from the mean.

313

PHBV copolymers are desired because of their improved thermal and mechanical properties.

314

However, PHBV copolymers with more than 50 mol% 3HV cannot be easily produced because

315

3HV precursors are normally toxic for the cells

316

of 3HV rich PHBV copolymers with a pre-defined composition can be easily accomplished

317

when C4:0 and C5:0 mixtures are used as sole carbon source in H. mediterranei cultures. The

29,31

. This study demonstrates that the synthesis


15


Page 16 of 39

318

simple and direct control over the complete range of PHBV composition demonstrated in this

319

study is a significant step forward towards the study of such polymers and towards the

320

production of bespoke polymers for specific applications such as biomedical implants or food

321

packaging.

322

For experiments F1-F3 and G1-G2, PHBV content and composition was analysed at the start,

323

before each subsequent addition of fatty acid (addition 1 at 92 h and addition 2 at 160 h) and at

324

the end of the experiment at 213 h. Figure 2 shows the change in polymer composition over time.

325

The 3HV content of the PHA samples obtained from co-feeding of C4:0/C5:0 mixtures (exp. F1-

326

F3) showed minor variations throughout the cultivation period, indicating that both substrates are

327

metabolised simultaneously and thus it can be concluded that H. mediterranei does not have a

328

preference between C4:0 and C5:0. In the sequential feeding experiment G1, when C4:0 was

329

present in the initial medium as well as in addition 1 only PHB was synthesised (in Figure 2,

330

displayed as 0 3HV mol%) and after addition 2, when C5:0 was added, a 3HV fraction was

331

detected. Similarly, culture G2 grew with C5:0 until addition 2, thus 3HV fraction in the polymer

332

remained above 90% until addition 2, after which the 3HV fraction decreased to 60.5 mol% due

333

to the addition of C4:0 and production of 3HB.


16

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Biomacromolecules

334 335

Figure 2. Variation of the 3HV fraction of the PHBV over time from the copolymers obtained

336

with co-feeding and sequential feeding of C4:0/C5:0 (random PHBV square solid symbols,

337

F1 dash-dot, F2 dash, F3 dot; block PHBV open circle, G1; and blend PHBV open triangle, G2.

338

For G1 and G2, the substrate added at each stage is denoted in bold and the substrate from

339

previous additions is denoted in grey). Error bars show the standard deviation from the mean.

340

To discern the PHBV microstructure of the polymers obtained with co-feeding and sequential 13

C NMR data was used to determine the nearest neighbour statistics D value35. The D

341

feeding,

342

value is the ratio between the fraction of the same type of adjacent units (3HV*3HV and

343

3HB*3HB) over fraction of different type adjacent units (3HV*3HB and 3HB*3HV). Table 3


17


Page 18 of 39

344

(supporting information) shows that co-feeding of C4:0 and C5:0 mixtures (exp. F1-F3)

345

produced random copolymers with D values around 1 and that sequential feeding (G1, G2)

346

generated PHBV with D values much greater than 1, suggesting the formation of block

347

copolymers. Specifically, a PHBV characterised with a D value of 21 was obtained in the

348

sequential feeding experiment G1, showing that 3HV and 3HB units are found next to each other

349

in the polymer chain thus forming a block polymer, see Figure 3. However, the PHBV from

350

experiment G2 had a D value greater than 5000, an indication that the incidence of 3HV units

351

next to 3HB units is nearly non-existent as shown in figure 3, i.e. there is an absence of polymer

352

changing points between 3HB and 3HV regions. Thus, the PHBV from G2 is in fact a blend of

353

PHB and PHV polymer chains. The thermal analysis confirmed the block and blend nature of

354

such polymers. In experiment G2, cell growth decelerated just before addition 2 likely due to

355

substrate depletion (data not shown). When addition 2 was performed, cell growth and polymer

356

synthesis continued but the temporary substrate depletion might have influenced PHA formation.

357

GC results show that the amount of 3HV remained constant between addition 2 until the end of

358

the cultivation and during that period only PHB was produced. A possible explanation for the

359

synthesis of a blend polymer is that after addition 2, the new substrate fed was used to synthesise

360

new polymer chains rather than to elongate existing ones. The coexistence of the two types of

361

polymer chain could explain the high polydispersity index of 2.8 obtained for experiment G2.

362

The results highlight the importance of addition time when performing sequential feeding. The

363

control over the microstructure of the polymer allows for the design of materials with unique

364

properties. Block polymers can display the characteristics of each constituent unit and potentially

365

show new properties that couldn’t be achieved otherwise47, such as improved mechanical and


18

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Biomacromolecules

366

thermal properties48,49. Block polymers can also have improved cell adhesion, a desired

367

characteristic for scaffolding, wound healing and biomedical applications.

1 6 9 .7 1 6 9 .6 1 6 9 .5 1 6 9 .4 1 6 9 .3 1 6 9 .2

7 2 .2 7 2 .1

7 2 .0 7 1 .9 3 9 .0 3 8 .9

3 8 .8

3 8 .7

2 7 .0

2 6 .9 2 6 .8

9 .5

9 .4

368 369

Figure 3. a) PHBV chemical structure and

370

molecule. b) 13C-NMR resonance splittings for 3HV units from random, block and blend PHBV.

371

Asterisk (*) designates interaction between units; interaction between different units (3HB*3HV

372

or 3HV*3HB) is shown in italics.

13

C-NMR chemical shifts of carbons from PHBV

373

PHBV characterisation

374

PHBV with 3HV fractions above 50 mol% are rarely reported and with no characterisation of

375

their mechanical or thermal properties. Furthermore, there is limited information about the


19


Page 20 of 39

376

mechanical properties of PHA from halophiles. This section contributes to an expansion of

377

knowledge in the aforementioned areas. The molecular weight and thermal properties of random,

378

block and blend HBV copolymers obtained from cultures grown in C2:0 to C6:0 (exp. A-E 4)

379

and C4:0/C5:0 mixtures (F1-F3, G1-G2) were investigated. In addition, the mechanical

380

properties of cast films were also measured. The results obtained are summarised in Table 3.

381

Molecular weight

382

PHBV from C2:0 (exp. A4) had the lowest average molecular weight (Mw) of 0.46 × 106 g/mol

383

and a remarkably low polydispersity index (PDI) of 1.3. The rest of the PHBV copolymers

384

obtained showed Mw in the range of 1.7 to 3.4 × 106 g/mol with typical values for PDI between

385

1.8 and 2.8, see Table 3. PHBV obtained from C4:0 and C5:0 mixtures (F1-F3, G1-G2) exhibited

386

a Mw around 3 × 106 g/mol and could be considered ultra-high Mw PHA (Mw > 3 × 106 g/mol), a

387

preferable type of PHA because of its enhanced mechanical strength50,51. PHA from halophiles

388

often shows relatively low Mw of 0.1 to 0.6× 106 g/mol20,21,28,52,53 although PHA with relatively

389

high degree of polymerisation of 1 to 2 × 106 g/mol has been previously synthesised5,6,29,54–56.

390

However, this is the first time ultra-high Mw PHA has been reported. Han and coworkers29

391

obtained PHA with 1 to 2 × 106 g/mol when glucose and fatty acids were used in H. mediterranei

392

cultures. Results from this study and from Han and coworkers suggest that fatty acids are

393

promising substrates for the generation of ultra-high Mw biopolymer with H. mediterranei,

394

without the need for genetic manipulation.

395

Results from Table 3 show that the 3HV content in the polymer did not influence the Mw of the

396

molecule, thus the incorporation of 3HV units did not disrupt the polymer chain growth.

397

However, cells which grow slower synthesised PHBV with higher Mw (except for sample F2).

398

This result might reflect that a slower metabolism is beneficial to produce polymers with high


20

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Biomacromolecules

399

degree of polymerisation. As expected PDI values increased with Mw, demonstrating a more

400

diverse polymer chain population with longer chain length polymers.

401

Mechanical properties

402

In order for a material to be appropriate within a given application, it is important to

403

understand its mechanical properties. In this regard, PHBV films were prepared using the

404

cast-solvent method with chloroform and the obtained films were subjected to an uniaxial

405

extension test to determine the following mechanical properties: Young’s modulus, a measure of

406

polymer stiffness; ultimate tensile strength, a measure of polymer toughness used to determine

407

the brittleness or ductility of a material; and elongation at break, a measure of polymer strength

408

prior to permanent plastic deformation31,57.

409

PHB obtained from C4:0 and C6:0 (experiments C4 and E4) had the highest Young’s modulus

410

above 1000 MPa, see Table 3, which is in agreement with 1500 MPa for pure PHB58 and similar

411

to 1700 MPa for polypropylene (PP)59. For random polymers the introduction of 3HV units into

412

the polymer chain interrupted the highly crystalline PHB structure, which reduced the Young’s

413

modulus and thereby improved the polymer elasticity, see Figure 4. However, the decrease in

414

stiffness was only observed for PHBV with 0-44 mol% 3HV fractions and above this percentage

415

the biopolymers had similar elasticity, with Young’s modulus values close to 390 MPa58 for

416

PHV and comparable to 200 MPa from low-density polyethylene (LDPE)59. Block copolymer

417

G1 showed a comparable degree of elasticity to random PHBV with >40 mol% 3HV despite

418

having an 3HV content of 26 mol%. On the other hand, the 60 mol% 3HV copolymer blend G2

419

had a Young’s modulus of 782.3 MPa, showing an elastic behaviour more similar to PHB rich

420

polymers due to the nature of its microstructure. These results highlight the importance of 3HV

421

being incorporated together with 3HB into the chain to increase elasticity.


21


Page 22 of 39

422 423

Figure 4. Young’s modulus (solid symbols) and tensile strength (open symbols) in relation to the

424

3HV fraction found in PHBV random copolymers (squares), block copolymer (circle) and blend

425

(triangle) polymer. Error bars show the standard deviation from the mean.

426

PHB rich polymers were the strongest with tensile strength of approximately 20 MPa and

427

comparable to 18 MPa for PHB57. The introduction of 3HV units into the polymer chain

428

decreased the tensile strength, see Figure 4, thus making the copolymers easier to process in

429

comparison to PHB. Again, the copolymer blend from exp. G2 was the exception and despite

430

having 60.5 mol% 3HV showed a relatively high strength value of 14.5 MPa due to its

431

microstructure. The films from experiments C4, E4, F3 and G2 had similar strength to the

432

10 MPa from LDPE59 or to the 16 MPa from polycaprolactone (PLC)60.


22

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Biomacromolecules

433

PHB and PHBV copolymers are known for their brittleness31,57. Elongation at break between

434

6.1 and 13.2% were observed and ductility increased at higher 3HV fractions regardless of the

435

microstructure. PHB rich polymers showed the smallest elongation at break of approximately

436

2.5%, in agreement to 3-5% of PHB58,61. Although PHBV copolymers were less brittle than pure

437

PHB, the copolymers broke at low elongations compared to some reports in literature. On the

438

other hand, the films were tested two weeks after preparation and PHBV quickly ages and

439

becomes brittle in less than 3 days31; therefore even if the films from random PHBV might have

440

been more ductile initially, by the time the test was performed they would had lost their ductility

441

and therefore break at low elongations.

442

Similar values for Young’s modulus and tensile strength of random and block copolymers

443

were obtained, suggesting that the composition, rather than the microstructure, had a greater

444

influence on the elasticity and strength of the polymers. The previous observation agrees with the

445

investigations of structure-property relationship of PHBV copolymers performed by

446

McChalicher and coworkers31. However, blend copolymer from G2 was stiffer and stronger than

447

expected for its composition, indicating than in this case the microstructure is the main driving

448

force determining the polymer properties. PHBV copolymers show good strength and elasticity,

449

comparable with oil-derived plastics such as PP, LDPE or PLC, but the extreme brittleness still

450

hinders its wider use in commodity applications. Polymer toughness could be improved by the

451

synthesis of block polymers, which can be easily performed in H. mediterranei cultures with the

452

controlled sequential feeding of C4:0 and C5:0.

453

Thermal properties

454

The thermal characterisation of PHBV samples was performed with DSC and results are

455

summarised in Table 3. The glass transition temperature (Tg) was only detected for random


23


Page 24 of 39

456

polymers with 3HV fractions greater than 20 mol%. The Tg for PHV is between −11°C and

457

−16°C62, in agreement with −17.8°C recorded for PHV sample D4. A linear decrease in Tg from

458

−0.5°C to −17.8°C was observed with increasing 3HV fractions, indicating an increase in

459

polymer chain molecular mobility in the amorphous phase36,63. Crystallisation temperature (Tc)

460

varied between 34.7°C and 117.2°C. The absence of Tc peaks could be an indication of the

461

amorphous nature of random polymers48.

462

The melting temperature (Tm) of PHB polymer from samples C4 and E4 was around 165°C,

463

which is similar to 160-177°C for pure PHB64. PHV from sample D4 showed a melting peak at

464

108.0°C, also in agreement with 107-112°C reported for pure PHV62,65. Polymers with

465

8 - 72 mol% 3HV showed two melting peaks in the DSC curve, while 3HB or 3HV rich

466

copolymers only had one Tm similar to the pure polymer, see Figure 5. The block and blend

467

copolymer G1 and G2 showed a Tm around 110°C, corresponding to the 3HV fraction of the

468

polymer; and a Tm around 165°C, for the 3HB fraction of the polymer. Specifically, sample G2

469

showed two Tm at 167.8°C and 175.5°C probably due to a melt-recrystallisation process63. The

470

presence of two well separated melting peaks in the samples G1 and G2 with a similar Tm to pure

471

PHB and PHV indicate the coexistence of two main fractions immiscible in the melt, being a

472

clear indication of the block and blend nature of the samples, providing further confirmation of

473

the microstructure results obtained with

474

due to the interaction between 3HB and 3HV units. In random copolymers, Tm decreased with

475

3HV fraction and PHBV polymers with >70 mol% 3HV have Tm equal or below 110°C, see

476

Figure 5. A minimum of the melting point versus composition has been previously observed at

477

around 40-50 mol% 3HV; the change has been attributed to the changeover from PHB crystalline

478

lattice to PHV crystalline lattice observed between 47 and 52 mol% 3HV36,62.

13

C-NMR. Random copolymers have shifted Tm peaks


24

Page 25 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

479 480

Figure 5. Melting temperatures (Tm) in relation to the 3HV fraction for PHBV random

481

copolymers. Error bars show the standard deviation from the mean.

482

Polymer decomposition temperatures (Td) were analysed by TGA and the results are

483

summarised in Table 3. Td obtained for all samples was between 220.8 to 267.4°C. Random

484

PHBV copolymers A4, B4 and F1 showed two Td, the first one around 225°C and the second one

485

at 360°C approximately. The weight loss after the first Td was 85%, 96% and 98% respectively.

486

For the remaining samples, the weight loss after Td was greater than 99%. One of the

487

disadvantages of PHB is its thermal instability since the temperature processing window, the


25


Page 26 of 39

488

difference between melting and decomposition temperature, is small (35°C28), thus it is hard to

489

mould into commercial items. Figure 6 shows that the incorporation of 3HV units in the polymer

490

chain significantly increases the temperature processing window from 50-60°C for pure PHB to

491

above 120°C for polymers with 3HV content above 40 mol%. The blend copolymer G2 had a

492

similar Tm value to pure PHB despite having 60 mol% 3HV, which explains the similarity

493

between the temperature processing window of this copolymer and for rich PHB polymers.

494 495

Figure 6. Variation of temperature processing window with 3HV fraction for random PHBV

496

polymers. For samples with more than one Tm or Td, the highest Tm and lowest Td was used to


26

Page 27 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

497

calculate the temperature processing window. Error bars show the standard deviation from the

498

mean.

499

The results for the thermal analysis confirmed the synthesis of PHBV block and blend

500

polymers. The incorporation of 3HV units into PHB chain significantly decreased the melting

501

temperature and increased the temperature processing window. The blend PHB and PHV

502

copolymer showed similar characteristics to pure PHB, making random or block copolymers

503

more desirable in terms of thermal stability and ease of processing to finished goods.

504

CONCLUSIONS

505

Cell growth and PHBV production in H. mediterranei from fatty acids C2:0, C3:0, C4:0, C5:0

506

and C6:0 was demonstrated. Feeding solely C4:0 or C5:0 generated 3HB and 3HV rich polymers

507

respectively but more importantly copolymer composition and microstructure can be

508

manipulated by regulating the C4:0:C5:0 ratio in the feed and the order of addition. The

509

polymers obtained had an ultra-high Mw around 3 × 106 g/mol and showed similar elasticity and

510

strength as oil-derived plastics such as PP, LDPE or PLC. Copolymers with a 3HV fraction

511

greater than 50 mol% had lower melting temperatures in comparison to 3HB rich copolymers

512

and exhibited a wider range of temperature in which the copolymers can be processed. The PHB

513

and PHV blend had similar thermal and tensile characteristics to PHB, making the random and

514

block copolymers more desirable. Thermal and mechanical properties are key characteristics

515

when selecting materials for specific applications. Such properties can be enhanced by

516

manipulating copolymer composition as well as chemical microstructure. A suitable strategy to

517

control 3HV fraction in PHBV copolymers as well as its microstructure has been demonstrated,

518

with the ultimate goal to create a polymer with bespoke properties and greater industrial

519

potential.


27


520

ASSOCIATED CONTENT

521

Supporting information

522 523 524 525

Page 28 of 39

Table 1 Cell growth, PHBV production and composition for cultures grown in C2:0 to C6:0 at different carbon concentrations and feeding strategy. Table 2 Cell growth, PHBV production and composition for cultures co-fed or sequentially fed with C4:0/C5:0 mixtures.

526

Table 3 Molecular weight, mechanical and thermal properties of PHBV copolymers obtained

527

from pure fatty acids (A4-E4), co-feeding or sequentially feeding C4:0/C5:0 mixtures (F1-F3 or

528

G1, G2 respectively).

529

AUTHOR INFORMATION

530

Corresponding Author

531

E-mail: *[email protected]

532

ACKNOWLEDGMENTS

533

The authors thank Dr. Matthew Cliff for carrying out the NMR analysis and Kamil Oster for

534

performing the DSC measurements. This research was funded by the Biotechnology and

535

Biological Research Council (BBSRC) grant BB/J014478/1.

536 537

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