Production of biodegradable polymers:

Polyhydroxyalkanoates

Dr. Ipsita RoySchool of Life Sciences

University of Westminster, London, UK

Polyhydroxyalkanoates, the biodegradable and biocompatible

polymers Polyhydroxyalkanoates are water-insoluble storage

polymers which are polyesters of 3-, 4-, 5- and 6-hydroxyalkanoic acids produced by a variety of bacterial species under nutrient-limiting conditions. They are biodegradable and biocompatible, exhibit thermoplastic properties and can be produced from renewable carbon sources. Hence, there has been considerable interest in the commercial exploitation of these biodegradable polyesters.

The general structure of

Polyhydroxyalkanoates

O

CH

(CH2)x

C

O

R2 OOR1

O

CH

(CH2)x

C[ ]n

R1/ R2 = alkyl groups (C1-C13)x = 1,2,3,4

SCL and MCL Polyhydroxyalkanoates

O

CH

(CH2)x

C

O

R2 OOR1

O

CH

(CH2)x

C[ ]n

Total Carbon chain length in monomer = 4-5;SCL PHAsTotal Carbon chain length in monomer =6-14;MCL PHAs

SCL-PHAs- ThermoplasticsMCL-PHAs-Elastomerics

Properties of SCL and MCL Polyhydroxyalkanoates

Type ofPHA

MeltingTemp(oC)

GlassTransitionTemp(oC)

Young’sModulus

(GPa)

Elongation at break

(%)

Tensile strength(MPa)

P(3HB) 171 2.7 3.5 1 40

P(3HB-co-20%3HV)

145 -1 1.2 3.84 32

P(4HB) 60 50 0.149 1000 104

P(3HB-co-16%4HB)

152 8 ND 444 26

P(3HO-co-18%3HHx)

61 35 0.008 400 9

P(3HB-co-3HHx)

120 -2 0.5 850 21

Polyhydroxyalkanoates as inclusions in bacteria

A

B

Roy et al.,2008 in “Biotechnology: Research, Technology and Applications.”Nova Science Publishers, Inc., New York, USA pp 1-48.

Production of SCL-Polyhydroxyalkanoates using

Bacillus cereus SPV,a Gram positive bacteria

PHA biosynthesis in Bacillus cereus SPV

• Bacillus cereus SPV is a newly characterised PHA producing species• It produces up to 60% dcw of PHA• It is capable of using a range of different carbon sources for PHA

production including, glucose, fructose, sucrose, gluconate, a range of alkanoic acids and plant oils

• The polymer produced lacks lipopolysaccharides, the known immunogen and is hence more suited for medical applications

PHAs produced by Bacillus cereus SPV using carbohydrates

Carbon source Dry cellweight(g/litre)

PHAconcentration

(g/litre)

3HBfraction (mol

%)

3HVfraction (mol

%)

4HBfraction (mol

%)

PHA yield(% dry cell

weight)

GlucoseFructoseSucroseGluconate

2.1431.2421.6661.943

0.8140.5000.6400.814

100829757

000

6.5

018336.5

38.0040.2538.4041.90

Valappil et al., 2007, Journal of Biotechnology, Volume 127(3), 475-487

P(3HB) production by Bacillus cereus SPV using glucose

(Yield:38% dcw)

RT: 0.00 - 24.00

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

9.47

6.93

11.37

17.6010.73 15.0113.68

12.40 18.5215.95 16.955.48 12.488.42 23.106.36 19.239.81 19.924.56 20.774.17

NL:3.59E7

TIC MS 04GC128

04GC138 #343-346 RT: 6.86-6.88 AV: 4 SB: 37 7.52-7.76, 6.47-6.52 NL: 1.24E6T: {0,0} + c EI det=350.00 Full ms [ 20.00-540.00]

20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

43.2

74.145.2

71.1

103.187.142.2

59.1

41.2 85.129.2 58.1 75.1 100.169.127.2 46.1 104.157.1 88.1 117.162.1 76.1 99.125.2 125.0 181.7 188.5130.1 139.1 154.2 159.9 170.2 198.4145.2

The GC chromatogram of the methanolysed polymer

Mass spectrum of the methyl ester of 3-hydroxybutyrate

MB3HB

PHAs produced by Bacillus cereus SPV using alkanoic acids

Carbon source Dry cell weight (g/litre)

PHAconcentration(g/litre)

3HB fraction (mol%)

3HV fraction (mol%)

PHA yield(% dry cell weight)

Acetate 0.368 0.009 100 0 2.44

Propionate 0.120 0.004 90 10 3.33

Butanoate 0.487 0.012 100 0 2.57

Hexanoate 0.379 0.034 100 0 8.96

Heptanoate 0.278 0.005 83 17 1.90

Octanoate 0.289 0.029 100 0 10.5

Nonanoate 0.496 0.234 59 41 47.36

Decanoate 0.559 0.448 100 0 80.14

Do-decanoate 0.510 0.315 100 0 61.81

Valappil et al., 2007, Journal of Biotechnology, Volume 127(3), 475-487

P(3HB) and P(3HB-3HV) production by Bacillus cereus SPV using alkanoic acids

Yield: up to 80% dcw

RT: 0.00 - 24.00

0 2 4 6 8 10 12 14 16 18 20 22

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

9.53

6.94

8.09

10.088.4314.546.35 15.03

11.12 12.28 13.314.10 15.74 16.99 17.61 19.244.81 20.05 21.24 22.42

NL:5.20E7

TIC MS 04GC141

RT: 0.00 - 24.00

0 2 4 6 8 10 12 14 16 18 20 22

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

9.53

7.00

17.62

15.7514.7716.96

8.4314.26

9.8216.0711.136.37 13.69 18.728.84 12.517.57 18.954.554.10 20.315.07 21.60 22.40

NL:3.44E7

TIC MS 04GC138

The GC chromatogram of the methanolysed polymer producedusing even-chain alkanoic acids

The GC chromatogram of the methanolysed polymer producedusing odd-chain alkanoic acids

3HB

MB

3HB

3HVMB

Summary of the P(3HB) yield obtained by Bacillus cereus SPV using plant oils

Carbon source Dry cellweight(g/litre)

P(3HB)concentration

(g/litre)

P(3HB) yield(% dry cell weight)

Olive oil 1.013 0.150 14.8

Rapeseed oil 0.636 0.018 2.80

Corn oil 0.557 0.183 32.8

Ground nut oil 1.094 0.143 13.1

Mustard oil 0.868 0.080 9.20

Time (hours)

0 10 20 30 40 50 60

Dry

cel

l wei

ght (

g/l)

0

1

2

3

%D

OT

0

10

20

30

40

50

60

70

80

90

100

PH

B %

dry

cel

l wei

ght

,

0

5

10

15

20

25

30

pH

0

2

4

6

8

10

Valappil et al., 2007, Journal of Biotechnology, 132; 251-258

GLUCOSEas the main Carbon Source

Large scale production of P(3HB) using batch fermentation in Kannan and Rehacek

medium(Yield 30% dcw)

Time (hours)

0 5 10 15 20 25 30 35 40 45 50 55 60

Dry

cel

l wei

gh

t (g

/l)

0

1

2

3

pH

0

2

4

6

8

10

%D

OT

0

10

20

30

40

50

60

70

80

90

100

PH

B %

Dry

cel

l wei

gh

t0

10

20

30

40

Valappil et al., 2007, Journal of Biotechnology, 132; 251-258

Large scale production of P(3HB) using fed batch fermentation in Kannan and Rehacek

medium(Yield 38% dcw)

GLUCOSEas the main Carbon Source

Large scale production of P(3HB) using batch fermentation in Kannan and Rehacek

medium (Yield 49% dcw)

SUCROSEas the main Carbon Source Time (hours)

0 5 10 15 20 25 30

Dry

cel

l wei

ght (

g/L)

0

1

2

3

4

P(3

HB

) % D

ry c

ell w

eigh

t0

10

20

30

40

50

60

P(3

HB

) (g/

L)

0

1

2

3

Suc

rose

con

sum

ptio

n (g

/L)

0

5

10

15

20

25

Large scale production of P(3HB) using batch fermentation in modified G medium,

MGM (Yield 60% dcw)

SUCROSEas the main Carbon Source Time (hours)

0 10 20 30 40 50 60

Dry

cel

l wei

gh

t (g

/L)

0

2

4

6

8

P(3

HB

) %

dry

cel

l wei

gh

t0

10

20

30

40

50

60

P(3

HB

) (g

/L)

0

1

2

3

4

5

6

Large scale production of P(3HB) using batch fermentation in modified G medium,

MGM (Yield 67% dcw)

MOLASSESas the main Carbon Source

Time (hours)0 10 20 30 40 50 60

Dry

cel

l wei

ght (

g/L)

0

2

4

6

8

10

PH

B %

Dry

cel

l wei

ght

0

20

40

60

PH

B (g

/L)

0

2

4

6

8

0

2

4

6

8

pH

Material and Thermal Properties of the P(3HB) produced

Type ofPHA

MeltingTemp(oC)

GlassTransitionTemp(oC)

Young’sModulus

(GPa)

Elongation at break

(%)

Tensile strength(MPa)

P(3HB) 169 1.9 1.1 1 40

Production of MCL-Polyhydroxyalkanoates using

Pseudomonas mendocina,a Gram negative bacteria

Large scale production of P(3HO) using batch fermentation in MSM media

(Yield 31% dcw)

SODIUM OCTANOATEas the main Carbon Source

Material and Thermal Properties of the P(3HO) produced

Type ofPHA

MeltingTemp(oC)

GlassTransitionTemp(oC)

Young’sModulus

(GPa)

Elongation at break

(%)

Tensile strength(MPa)

P(3HO) 49 -36 1.4 276 9

Large scale production of P(3HN-3HHP) using batch fermentation in MSM media

(Yield 20% dcw)

SODIUM NONANOATEas the main Carbon Source

Large scale production of P(3HO-3HX-3HD) using batch fermentation in MSM media

(Yield 15% dcw)

GLUCOSEas the main Carbon Source

SUCROSEas the main Carbon Source

Large scale production of P(3HO-3HB) using batch fermentation in MSM media

(Yield 23% dcw)

An SCL-MCL COPOLYMER!

Medical applications of PHAs

Valappil et al., 2006; Expert Review in Medical Devices 3(6): 853-868

Hard tissue engineering

Production of P(3HB)/Bioglass® composites

Bioglass® (type 45S) is a Class A bioactive material and in the context of bone and cartilage tissue engineering, it is osteoproductive, osteoconductive and osteoinductive.

S.K.Misra et al., 2006 Biomacromolecules Aug;7(8):2249-58

Production of P(3HB)/Bioglass® composites

P(3HB)/20 wt% Bioglass® film Cross section of a P3HB/20 wt% Bioglass® film

Properties of the P(3HB)/Bioglass® composites

Properties P(3HB) film P(3HB)+5 wt%

P(3HB)+20 wt%

Tm (0C) 171.56±1.48 152/169 156/172

Tg (0C) -6.3±0.6 -5±3 -1.2±0.5

Xc (%) 73.45±1.01 60.45±1.95 60.11±3.64

ΔHf (J/g) 0.492±0.007 0.405±0.01 0.402±0.02

E( GPa) 1.1±0.3 0.8±0.1 0.84±0.05

Mw 285000 245000 261000

S.K.Misra et al., 2007 Biomacromolecules Jul;8(7):2112-9

The acellular bioactivity of the P(3HB)/Bioglass® composites

SEM micrograph of the composite showing the formation of hydroxyapatiteon the surface of the composite after one month of immersion in SBF XRD patterns of in vitro degraded P(3HB)/Bioglass®

composites (20wt%) , showing the emergence of the hydroxyapatite peaks after (a) 0 days, (b) 2 days, (c) 7 days and (d) 28 days of immersion in SBF

HA peaks

S.K.Misra et al., 2007 Biomacromolecules Jul;8(7):2112-9