1. Production of biodegradable polymers: Polyhydroxyalkanoates
Dr. Ipsita Roy
School of Life Sciences
University of Westminster, London, UK

2. 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.

3. The general structure of Polyhydroxyalkanoates
R1/ R2 = alkyl groups (C1-C13)
x = 1,2,3,4

4. SCL and MCL Polyhydroxyalkanoates
Total Carbon chain length in monomer = 4-5;SCL PHAs
Total Carbon chain length in monomer =6-14;MCL PHAs
SCL-PHAs- Thermoplastics
MCL-PHAs-Elastomerics 4

5. Properties of SCL and MCL Polyhydroxyalkanoates
Type of
PHA
Melting
Temp
(oC)
Glass
Transition
Young’s
Modulus
(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 5

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

7. Production of SCL-Polyhydroxyalkanoates using Bacillus cereus SPV, a Gram positive bacteria

8. 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

9. PHAs produced by Bacillus cereus SPV using carbohydrates
Carbon source
Dry cell
weight
(g/litre)
PHA
concentration
3HB
fraction (mol%)
3HV
4HB
PHA yield
(% dry cell weight)
Glucose
Fructose
Sucrose
Gluconate
2.143
1.242
1.666
1.943
0.814
0.500
0.640
100 82 97 57
6.5 18 3
36.5
38.00
40.25
38.40
41.90
Valappil et al., 2007, Journal of Biotechnology, Volume 127(3),

10. P(3HB) production by Bacillus cereus SPV using glucose (Yield:38% dcw)MB
3HB
The GC chromatogram
of the methanolysed polymer
Mass spectrum of the methyl ester
of 3-hydroxybutyrate

11. PHAs produced by Bacillus cereus SPV using alkanoic acids
Carbon source
Dry cell
weight
(g/litre)
PHA
concentration
3HB fraction
(mol%)
3HV fraction (mol%)
PHA yield
(% dry cell weight)
Acetate
0.368
0.009
100
2.44
Propionate
0.120
0.004 90 10
3.33
Butanoate
0.487
0.012
2.57
Hexanoate
0.379
0.034
8.96
Heptanoate
0.278
0.005 83 17
1.90
Octanoate
0.289
0.029
10.5
Nonanoate
0.496
0.234 59 41
47.36
Decanoate
0.559
0.448
80.14
Do-decanoate
0.510
0.315
61.81
Valappil et al., 2007, Journal of Biotechnology, Volume 127(3),

12. P(3HB) and P(3HB-3HV) production by Bacillus cereus SPV using alkanoic acids Yield: up to 80% dcwMB MB
3HV
3HB
3HB
The GC chromatogram of the
methanolysed polymer produced
using even-chain alkanoic acids
The GC chromatogram of the
methanolysed polymer produced
using odd-chain alkanoic acids

13. Summary of the P(3HB) yield obtained by Bacillus cereus SPV using plant oils
Carbon source
Dry cell
weight
(g/litre)
P(3HB)
concentration
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

14. Large scale production of P(3HB) using batch fermentation in Kannan and Rehacek medium(Yield 30% dcw)
GLUCOSE
as the main
Carbon
Source
Valappil et al., 2007, Journal of Biotechnology, 132;

15. Large scale production of P(3HB) using fed batch fermentation in Kannan and Rehacek medium(Yield 38% dcw)
GLUCOSE
as the main
Carbon
Source
Valappil et al., 2007, Journal of Biotechnology, 132;

16. Large scale production of P(3HB) using batch fermentation in Kannan and Rehacek medium (Yield 49% dcw)
SUCROSE
as the main
Carbon
Source 16

17. Large scale production of P(3HB) using batch fermentation in modified G medium, MGM (Yield 60% dcw)
SUCROSE
as the main
Carbon
Source 17

18. Large scale production of P(3HB) using batch fermentation in modified G medium, MGM (Yield 67% dcw)
MOLASSES
as the main
Carbon
Source 18

19. Material and Thermal Properties of the P(3HB) produced
Type of
PHA
Melting
Temp
(oC)
Glass
Transition
Young’s
Modulus
(GPa)
Elongation at break
(%)
Tensile strength
(MPa)
P(3HB)
169
1.9
1.1 1 40 19

20. Production of MCL-Polyhydroxyalkanoates using
Pseudomonas mendocina, a Gram negative bacteria 20

21. Large scale production of P(3HO) using batch fermentation in MSM media
(Yield 31% dcw)
SODIUM
OCTANOATE
as the main
Carbon
Source

22. Material and Thermal Properties of the P(3HO) produced
Type of
PHA
Melting
Temp
(oC)
Glass
Transition
Young’s
Modulus
(GPa)
Elongation at break
(%)
Tensile strength
(MPa)
P(3HO) 49
-36
1.4
276 9

23. Large scale production of P(3HN-3HHP) using batch fermentation in MSM media
(Yield 20% dcw)
SODIUM
NONANOATE
as the main
Carbon
Source

24. Large scale production of P(3HO-3HX-3HD) using batch fermentation in MSM media
(Yield 15% dcw)
GLUCOSE
as the main
Carbon
Source

25. Large scale production of P(3HO-3HB) using batch fermentation in MSM media
(Yield 23% dcw)
SUCROSE
as the main
Carbon
Source
An SCL-MCL COPOLYMER!

26. Medical applications of PHAs
Valappil et al., 2006; Expert Review in Medical Devices 3(6):

27. Hard tissue engineering

28. 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):

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

30. Properties of the P(3HB)/Bioglass® composites
P(3HB) film
P(3HB)+
5 wt%
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

31. The acellular bioactivity of the P(3HB)/Bioglass® composites
HA peaks
SEM micrograph of the composite
showing the formation of hydroxyapatite
on 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
S.K.Misra et al., 2007 Biomacromolecules Jul;8(7):2112-9