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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