1. UNIVERSITA’ DEGLI STUDI DI CATANIA
FACOLTA’ DI FARMACIA
PhD in Medicinal Chemistry
GIUSEPPE PUZZO
BACTERIAL FERMENTATION AND MICROWAVE-
ASSISTED SYNTHESIS FOR THE PRODUCTION OF
BIODEGRADABLE AND BIOCOMPATIBLE
POLYMERS USABLE IN THE PHARMACEUTICAL
FIELD.
Coordinator: Tutor:
Prof. Giuseppe Ronsisvalle. Prof. Alberto Ballistreri.
Ciclo XXIV

2. Introduction
Not biodegradable
plastics
Biodegradable
plastics

3. Biodegradable polymers on the market.
Biomax® Ecoflex® Mater-Bi®
® PHA
EcoPLA

4. Polyhydroxyalkanoates (PHA)
P
Poly(3-hydroxyalkanoates)
with R= alkyl or functional group
n
PHASCL: short-chain length PHA
3-5 carbon atoms
PHAMCL: medium-chain length PHA
6-14 carbon atoms

5. Physical and chemical properties.
•Average molecular weight ranging between 5·104 and1·106 Da
•Enantiomerically pure
•Biodegradable and biocompatible
Extension at
Polymer T
Tg(°C) T
Tm (°C) C
Cristallinity (%)
b
break (%)
P
P(3HB) 15 175 50-80 5
P(3HB-co-3HV) -1 145 56 50
P(3HB-co-4HB) -7 150 43 444
PP -15 176 50 400

6. Applications of PHAs in medicine and pharmaceuticals.
•Sutures. •Bone graft substitutes.
•Temporary heart valves. •Carrier for drug delivery.

7. Role of PHAs in tissue engineering.

8. The aim of the thesis
Explore new strategies for obtaining new polymers which, in the
pharmaceutical field, have feature of biodegradability and
biocompatibility with wider opportunity of utilization with respect to
poly(3-hydroxybutyrate) (PHB) by:
1. The study on the capabilty to P. aeruginosa to grow and
synthesize PHAs from Long Chain Fatty Acids (LCFA) or
vegetable oils, with better yields or with new structures and new
properties.
2. Chemical synthesis of new coplymers and terpolymers by
transesterification reaction microwave assisted.

9. PHA’s production by microorganisms.

10. Substrates for PHA’s production.
Corn, sugar cane,
Glucose Carbohydrate potato etc
Agriculture, waste materials
Alkanoates
(propionic acid, Vegetable oils
butyric acid, Fatty acid and fats
valeric acid etc.)

11. Table 1. PHA production from P. aeruginosa cultured
using odd carbon atoms fatty acids as carbon source.
Fatty acid Dry cell weight PHA content PHA yield
(mg/L) (% dry cell weight) (mg/L)
Eptadecanoic -N 1600 9,8 157
Nonadecanoic -N 2370 5,3 127
Eneicosanoic-N 2 737 0,25 7

12. GC trace of the products prepared by methanolysis of
PHA from nonadecanoic acid.
V= 3-hydoxyvalerate; H= 3-hydoxyheptanoate; O= 3-hydroxyoctanoate;
N= 3-hydroxynonanoate; D= 3-hydroxydecanoate; U= 3-hydroxyundecanoate;
Θ= 3-hydroxytridecanoate; P = 3-hydroxypentadecanoate

13. 200 MHz 1H-NMR spectra of the PHAs obtained from P.
aeruginosa grown on (a) heptadecanoic; (b) nonadecanoic and
(c) eneicosanoic acid.

14. 50 MHz 13C-NMR spectra of the PHAs obtained from P.
aeuruginosa grown on (a) nonanoic; (b) heptadecanoic and (c)
eneicosanoic acid.

15. Chemical structure of the PHAs.
V H N U Θ P
Δ
3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1
O CH CH CO O CH CH CO O CH CH CO O CH CH CO O CH CH CO O CH CH CO
2 l 2 m 2 n 2 o 2 p 2 q
4CH 4CH 4 CH 4 CH 4 CH 4 CH
2 2 2 2 2 2
5CH 5CH 5 CH 5 CH 5 CH 5 CH
3 2 2 2
6CH 6 CH 6 CH 6 CH 6 CH
2 2 2
7CH 7 CH 7 CH 7 CH 7 CH
3 2 2 2 2
8 CH 8 CH 8 CH 8 CH
2 2 2 2
9 CH 9 CH 9 CH 9 CH
3 2 2 2
10CH 10CH 10CH
2 2 2
11CH 11CH 11CH
3 2 2
12 CH 12 CH
2 2
13 CH 13 CH
3 2
14 CH
2
15CH
3

16. Table 2. Physical characteristics of the PHAs isolated from P.
aeruginosa grown on C-odd fatty acids.
Fatty acids Tg (°C) Tm (°C) ∆Hm (J/g) Mw x 10-3 Mw/Mn
Eptadecanoic -41 50 7,9 77 1,6
Nonadecanoic -43 58 12 97 2
Eneicosanoic -39 49 5,7 188 1,7

17. Thermal degradation of PHAs

18. Assuming a Bernoullian (random) distribution of repeating units
in these copolymers, the probability of finding a given Ax, By…
Nz can be calculated by the Leibnitz formula as follows:
A measure of the fit of the calculated oligomers intensities to
the experimental ones is given by the agreement factor (AF);
the lower AF, the closer fit.
∑(I + I
expi
. calci
. )2
AF= i
∑I 2
expi
.
i

19. R R
R CH CH CO [O CH CH2 CO ]
n
O CH CH2 COOH
Negative ion ESI mass spectrum of the partial pyrolisate of the PHA from
enicosanoic acid. R may be an un n-etyl, n-butyl, n-hexyl, n-octyl, n-
decyl and n-dodecyl group.

20. Table 3. Experimental and calculated relative amounts of
the partial pyrolisis products of P. aeruginosa from
eneicosanoic acid.
m/z ESI Calculated
Dimers
V-H 227 4 4
V-N; H2 255 15 15
V-U; H-N 383 18 18
V-Θ; H-U; N2 311 26 26
V-P; N-U; H-Θ 339 20 20
H-P; U2; N-Θ 367 12 13
U-Θ; N-P 395 5 5
Trimers
C23 411 8 9
C25 439 14 16
C27 467 23 22
C29 495 21 24
C31 523 15 18
C33 551 10 10
C35 579 3 0

21. Brassica carinata
production’s seeds
Remaining
Oil De-oiling flour
Modified As such As such Formulation
Lubrificants Fertilizer Soil products
Lubricants
Energy products
Biofuels
Agricoltural
oils

22. Table 4. PHA production from P. aeruginosa cultured on
differents substrates.
Substrate Dry cell weight PHA content PHA yield
(mg/L) (% dry cell weight) (mg/L)
B. Carinata oil 1000 5,0 50
Oleico acid 380 15,0 57
Erucic acid 2 866 9,3 81
Nervonic acid 416
416 10
10.0 42

23. Table 5. Comonomer composition (mol%) of PHA obtained
from varius carbon sources, determined by GC.
Substrate C O O:1 D D:1 Δ Δ:1 T:1 T:2 T:3
B.Carinata oil 3 34 3 32 3 10 1 9 2 3
Oleico acid 4 55 - 27 - 8 - 6 - -
Erucico acid 3 43 - 36 - 10 - 8 - -
Nervonico acid 4 28 - 43 - 14 - 11 - -
C= 3-hydroxyhexanoate; O= 3-hydroxyoctanoate; O:1= 3-hydroxy-5-
octenoate; D= 3-hydroxydecanoate; D:1= 3-hydroxy-7-decenoate; Δ= 3-
hydroxydodecanoate; Δ:1= 3-hydroxy-6-dodecenoate; T:1 = 3-
hydroxy-5-tetradecenoate; T:2= 3-hydroxy-5,8-tetradecadienoate; T:3= 3-
hydroxy-5,8,11-tetradecatrienoate.

24. C5, O5-7, D5-9, C6, O8, D10,
Δ5-11, T:1 8-13 Δ12, T:1 14
C/O/D/Δ
C/O/D/Δ/T:1 C/O/D/Δ/T:1
4
3 2
T:1 T:1
T:1 T:1
4 7
6 5
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
(ppm)
200 MHz 1H-NMR spectra of the PHA obtained from erucic acid.

25. D8 D6-7 D9
O/D/Δ Δ10 O6 Δ6-9 Δ11 D10
T:1
3 T:1 T:1 T:1 Δ12
4
12 8-11 13 T:1
C/O/D/Δ O7 O8
C/O/D/Δ O5 14
2
1 D5
O/D/Δ
D5
4
T:1
T:1 C3 T:1 2 T:1
1 T:1 T:1
3 7 C5 C6
6 C4
5
170 130 120 70 40 35 30 25 20 15
(ppm)
50 MHz 13C-NMR spectra of the PHA obtained from erucic acid.

26. Chemical structure of the PHAs from oleic, erucic and
nervonic acids.
C O D Δ T:1
Δ
3 2 1 3 2 1 3 2 1 3 2 1 3 2 1
O CH CH CO O CH CH CO O CH CH CO O CH CH CO O CH CH CO
2 m 2 n 2 o 2 p 2 q
4CH 4CH 4 CH 4 CH 4 CH
2 2 2 2 2
5 CH 5CH 5 CH 5 CH 5 CH
2 2 2 2
6CH 6CH 6 CH 6 CH 6 CH
3 2 2 2
7CH 7 CH 7 CH 7 CH
2 2 2 2
8 CH 8 CH 8 CH 8 CH
3 2 2 2
9 CH 9 CH 9 CH
2 2 2
10 CH 10 CH 10 CH
3 2 2
11CH 11CH
2 2
12 CH 12 CH
3 2
13 CH
2
14 CH
3

27. O:1 8, D:1 10, Δ:112, T:2/T:3 14
O:1 5
D:1 5, Δ:1 9-11, T:2 11-13
D:1 7-8
Δ:1 6-7 O:1 7 D:1
T:2 5, 8-9 D:1 6, 9 Δ:1
T:3 5, 8-9, Δ:1 5, 8 4
11-12O /D /Δ /T /T O:1/D:1/Δ:1/T:2/T:3 T:2 10
:1 :1 :1 :2 :3
3 2 T:3 13
O:1 6 O:1/T:2/T:3
T:2/T:3 6 4
T:2 7
T:3 7, 10
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
(ppm)
200 MHz 1H-NMR spectra of the PHA obtained from B. carinata oil.

28. O:1 7, D:1 6/9, Δ:1 5, 8 T:2 10, T:3 13 }
Δ:1
O:1 T:2 7
D:1 D:1 12
T:2 T:3 7, 10
Δ:1 Δ:1 T:2
T:3 O:1
D:1 3 2O Δ:1 14
:1 4
Δ:1 O:1 8
T:2 9
O:1 T:2 D:1
1
T:3
T:2 T:3 10
2 T:3
T:3 3 D:1
1 14
Δ:1
4
170 130 120 70 40 35 30 25 20 15
(ppm)
50 MHz 13C-NMR spectra of the PHA obtained from B. carinata oil.

29. T:3 T:3
12 T:3 8, 11
T:1 D:1 T:2 Δ:1 T:1
6 T:2
6 T:2 6
8 9 D:1 5
O:1 Δ:1 8
6 O:1
6 7 7
T:3 T:2 5
T:3
9 5
5
138 136 134 132 130 128 126 124 122 120
(ppm)
C-NMR spectra of the PHA obtained from B. carinata oil in the region
13
of the olefinc signals.

30. Chemical structure of the PHA from B. carinata oil. This PHA
is made up of all the repeating units constituting the PHA
from erucic acid, plus the unsatureted ones shown here.
O:1 D:1 Δ:1 T:2 T:3
3 2 1 3 2 1 3 2 1 3 2 1 3 2 1
O CH CH CO O CH CH CO O CH CH CO O CH CH CO O CH CH CO
2 n 2 o 2 p 2 r 2 s
4CH 4CH 4 CH 4 CH 4 CH
2 2 2 2 2
5CH 5CH 5 CH 5 CH 5 CH
2 2
6CH 6CH 6 CH 6 CH 6 CH
2
7CH 7CH 7 CH 7 CH 7 CH
2 2 2
8CH 8 CH 8 CH 8 CH 8 CH
3 2
9CH 9 CH 9 CH 9 CH
2 2
10 CH 10 CH 10CH 10CH
3 2 2 2
11CH 11 CH 11CH
2 2
12CH 12CH 12CH
3 2
13CH 13CH
2 2
14CH 14CH
3 3

31. Table 6. Physical characteristics of the PHAs isolated
from P. aeruginosa grown on B. carinata oil and on oleic,
erucic and nervonics acids.
Sustrate Tg (°C) Tm (°C) ΔHm (J/g) Mw x 10-3 Mw/Mn
B.carinata oil -47 - - 56 1,8
Oleico acid -52 - - 57 2,2
Erucic acid -46 50 16,1 122 1,9
Nervonic acid -43 50 15,5 114 2

32. Dimeri
R R
311 R CH CH CO [O CH CH2 CO ] n
O CH CH2 COOH
100 283
% Intensità 339
Trimeri
Tetrameri
60 453 481
255 367 425 509 595 623
393 535 567 651 679
20
300 400 500 600 700
x3 (m/z)
100
Pentameri
% Intensità
Esameri
765 Eptameri
60 737 793 907
879 935 1049
709 821 1077
851 963 1021 1105
991 1133
1161
20
700 800 900 1000 1100
(m/z)
Negative ion ESI mass spectrum of the partial pyrolisate of the PHA
from erucic acid. R may be a n-propyl, n-pentyl, n-heptyl, n-nonyl or n-
undecenyl group.

33. Table 7. Experimental and calculated relative amounts of
the partial pyrolisis products of the PHA produced by P.
aeruginosa from erucic acid.
m/z ESI Calculated
Dimers
C-O 255 10 9
C-D; O2 283 24 24
C-Δ; O-D 311 26 26
O-Δ; D2 339 18 19
O-T:1 365 6 7
D-Δ 367 8 7
D-T:1 393 6 4
Δ-T:1 421 2 2
Trimers
C22 397 4 4
C24 425 12 12
C26 453 20 19
C28 481 18 20
C30 :1 507 5 7
C30 509 13 13
C32:1 535 10 9
C32 537 6 5
C34:1 563 8 6

34. R R
Dimeri
R CH CH CO [O CH CH2 CO ]
n
O CH CH2 COOH
311
100
283
339 Trimeri
% Intensità
60 255 Tetrameri
453 481
367 425 509 623 651
393 535 567 595 679
20
300 400 (m/z) 500 600 700
100 x 4
Pentameri
737 765 793 Esameri
% Intensità
60 709 821 907 Eptameri
879 935 963
849
991 1021 1049 107711051133
20
700 800 900 (m/z) 1000 1100
Negative ion ESI mass spetrum of the partial pyrolisate of the PHA
from B. carinata. R may be a n-pentaenyl, n-heptaenyl, n-nonaenye, n-
undecadieyil or n-undecatrienyl group.

35. Design For Efficient Energy: Energy requirements should be recognized for their
environmental and economic impacts and should be minimized. Synthetic methods should be
conducted at ambient temperature and pressure.
Heating mechanisms heat exchange Heating with Microwave
Benefits:
 Energy saving
 Process Efficiency
 Restrictions on the use of halogenated
solvents

36. What are the microwave
The microwaves are not ionizing electromagnetic waves having a
wavelength between 1 mm (ν = 300 GHz) and 1 m (ν = 300 MHz),
they are located in the area of the spectrum between the
frequencies of the infrared and the radio waves.
The frequency of 2.45 (± 0.05) GHz, corresponding in vacuum at a
wavelength (λ) of 12.2 cm, is that used for applications in the
domestic field, scientific, medical, and for many industrial
processes.

37. Chemical synthesis of copolyesters.
CH 3 O O
O CH CH 2 C + O CH 2 CH 2 CH2 CH2 CH 2 C
PHB n PCL m
1. PTSA·H2O, Chloroform, Toluene (reflux)
2. Azeotropic (dehydration)
CH 3 O O
O CH CH 2 C O CH 2 CH 2 CH 2 CH 2 CH 2 C
n m
P(HB-co-CL)

38. Table 8. Transesterification Conditions, Yields, Molecular
Weights, and Degree of Transesterification of P(HB-co-CL)
Copolymers.
Sample HB/CLa Yield (%) Mw·103 b Mw/Mn c DT d DR e RT(h) f
Conventional
heating
A 54/46 15 7.8 1,41 0,16 0,3 1/2
B 45/55 23 n.d. n.d. 0,21 0,52 2/2
C 75/25 19 n.d. n.d. 0,42 0,92 3/2
D 55/45 10 7.9 1,3 0,37 0,74 5/2
Microwave
heating
E 55/45 52 5.2 1,3 0,1 0,21 1/2
F 48/52 49 6.4 1,27 0,12 0,25 2/2
G 55/45 30 9 1,2 0,17 0,36 3/2
H 46/54 26 12 1,24 0,31 0,63 5/2
a
Molar composition of the resulting copolymers. b Weight-average molecular weight.
c
Molecular weight distribution. d Degree of transesterification at the end of the second
stage of the reaction. e Degree of randomness at the end of the second stage of the
reaction. f Duration in hours of the two transesterification stages. n.d.: not detemined.

39. a
O CH3 O
O CH2 CH2 CH 2 CH 2 CH2 C O CH CH2 C
e f g h i l m b c d n
a
e g
f+h
i
c
b
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the copolymer P(HB–co-45%mol CL)
(sample D) obtained with conventional heating.

40. m’ n’ a
H HO O CH O
3
H3C S O CH CH2 CH 2 CH 2 CH2 C O CH CH2 C
n e 2 f g h i l m b c d n
H HO
m n
e
g
f+h
a
i
c
m+m’ n+n’ b
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the copolymer P(HB–co-54%mol CL)
(sample H) obtained with microwave heating.

41. a
O CH3 O
O CH2 CH2 CH 2 CH 2 CH2 C O CH CH2 C
e f g h i l m b c d n
f g
e h
i
c
a
b
l d
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR spectra of the copolymer P(HB–co-45%mol CL)
(sample D) obtained with conventional heating.

42. m’ n’ a
H HO O CH O
3
H3C S O CH CH2 CH 2 CH 2 CH2 C O CH CH2 C
e 2 f g h i l m b c d n
H HO
m n h
g
e i f
c
a
l b
d m+m’ n+n’
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR spectra of the copolymer P(HB–co-54%mol CL)
(sample H) obtained with microwave heating.

43. CCC
BCC
BBC
BBB
BCB CBB
CCB
CBC
174.5 174.0 173.5 173.0 172.5 172.0 171.5 171.0 170.5 170.0 169.5 169.0 168.5
(ppm)
13
C-NMR spectral expansion of the carbonyl region of the copolymer
(sample H).

44. 2X B 2XC
LB = LC =
( I BC + I CB )
( I BC + I CB )
where XB and XC are the dyad mole fractions of HB and CL calculable by the
equations:
X B = I BB + 1 2 ( I BC + I CB ) X C = I CC + 1 2 ( I BC + I CB )
DT = I BC + I CB DR = 1 LB + 1 LC
For a random copolymer of 1:1 composition, these parameters are expected
to assume the values LB = LC = 2, DT = 0.5 and DR = 1.

45. : Spettro MALDI-TOF della frazione eluita dopo il massimo del tracciato GPC del copolimero P(HB-co- 45 mol%CL) (campione D).
5894
5810
5838
5754
5866
5782
5922
5950
1000
800
5750 5850 5950
600 (m/z)
400
200
4500 5000 5500 6000 6500 7000 7500
(m/z)
MALDI-TOF mass spectrum of the fraction eluting after the maximum
of the GPC trace of the copolimer P(HB-co- 45 mol%CL) (sample D).

46. : Spettro MALDI-TOF della frazione eluita dopo il massimo del tracciato GPC del copolimero P(HB-co- 45 mol%CL) (campione D).
Chemical synthesis of terpolyesters.
CH3
O CH O CH2 O
3
O CH2 CH2 CH2 CH2 CH2 C + O CH CH2 C O CH CH2 C
m n o
PCL P(HB-co-HV)
1. PTSA·H2O, Chloroform, Toluene (reflux)
2. Azeotropic (dehydration)
CH
3
O CH O CH2 O
3
O CH2 CH2 CH2 CH2 CH2 C O CH CH2 C O CH CH2 C
m n o
P(HB-co-HV-co-CL)

47. Table 9:Transesterification Conditions, Yields, Molecular
Weights, and Degree of Transesterification of P(HB-co-HV-
co-CL) Terpolymers.
Sample HB/HV/CLa Resa (%) Mw·103 b Mw/Mn c DT d DR e RT(h) f
Conventional
heating
L 51/15/34 30 6.7 1,36 0,61 1,05 1/2
M 47/12/41 19 11.3 1,16 0,71 1,41 2/2
N 48/13/39 13 8.1 1,12 0,81 1,54 3/2
Microwave
heating
P 62/14/24 51 8.1 1,3 0,64 1,64 1/2
Q 58/15/27 37.5 9.1 1.9 0,7 1,27 2/2
R 68/13/19 35 6.7 1,2 0,75 1,47 3/2
a
Molar composition of the resulting terpolymers. b Weight-average molecular weight.
c
Molecular weight distribution. d Degree of transesterification at the end of the second
stage of the reaction. e Degree of randomness at the end of the second stage of the
reaction. f Duration in hours of the two transesterification stages.

48. Spettro 1H-NMR a 200 MHz del terpolimero P(3HB-co-12%mol 3HV-co-41%mol CL) (campione M).
m
g CH
3
O CH O n CH O
3 2
O CH CH CH CH CH C O CH CH C O CH CH C
2 2 2 2 2 2 p 2 q
a b c d e f m h i l n o o
g+n
a b+d
i+p e
m
h+o c
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the terpolymer P(HB-co-12%mol HV-
co-41%mol CL) (sample M).

49. m
x’ y’ CH
g 3
H HO O CH O n CH O
3 2
H3C S O CH CH CH CH CH C O CH CH C O CH CH C
a 2 b 2 c 2 d 2 e2 f m h i 2 l
n o p2 q o
H HO
x y
g+n
i+p b+d
a
e m
y+y’ x+x’ h+o
c
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the terpolymer P(HB-co-15%mol HV-
co-27%mol CL) (sample Q).

50. m
CH3
g
O CH3 O n CH O
2
O CH CH2 CH2 CH CH2 C O CH CH C O CH CH C
2 2 2 l o p2 q o
a b c d e f m h i n
g
i
h
ed
a
b c
l+q
n m
f o p
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR of the terpolymer P(HB-co-12%mol HV-co-41%mol CL)
(sample M).

51. m
x’ y’ CH3
g
H HO O CH3 O
nCH O
2
H3C S O CH2 CH CH2 CH CH C O CH CH2 C O CH CH 2 C
a b2 c d2 e 2 f m h i l n o p q o
H H O
x y g
i
h
e
l+q a
b cd
m
o n
y+y’ x+x’ p
f
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR spectra of the terpolymer P(HB-co-15%mol HV-
co-27%mol CL) (sample Q).

52. Espansione dello spettro 13C NMR della regione dei carbonili del terpolimero M.
BB
BV,VB
CC
BC
BC
CV
VC VV
173.6 173.2 172.8 172.4 172.0 171.6 171.2 170.8 170.4 170.0 169.6 169.2 168.8
(ppm)
13C-NMR spectral expansion of the carbonyl region of the terpolymer
(sample M).

53. 2 XB 2 XV
LB = LV =
( I CB + I BC + I CV + I BV + IVB ) ( I CB + I BC + I CV + I BV + I VB )
2 XC
LC =
( I CB + I BC + I CV + I BV + I VB )
where XB, XV, and XC are the dyad mole fractions of HB, HV and CL calculable by
the equations:
X B = I BB + 1 2 ( I CB + I BC + I CV + I BV + I VB ) X V = I VV + 1 2 ( I CB + I BC + I CV + I BV + I VB )
X C = I CC + 1 2 ( I CB + I BC + I CV + I BV + I VB )
DR = 1 LB + 1 LC + 1 LV DT= ICB+IBC+ICV+IVC+IBV+IVB/2XB XC+2XCXV+2XBXV

54. Conclusion 1
Through bacterial fermentation were obtained for the first
time PHA using very long chain fatty acids (VLCFA), more
than 20 C atoms and B. carinataI oil. The PHA produced by
fatty acid with odd number of carbon atoms are flexible
materials whose physical characteristics do not vary
significantly as a function of the side chain, although longer
pendant groups confer a greater speed of recrystallization.
The PHA produced by using erucic and nervonic acids, are
transparent as well, partially crystalline and therefore they
show rubber-like characteristics. Their proposed use is as
scaffold in tissue engineering and in the pharmaceutical
delivery system.
The PHA from B. carinata oil is a transparent material, totally
amorphous. The presence of double bonds allows the
derivatization and functionalization.

55. Conclusion 2
By chemical synthesis were obtained biodegradable and
biocompatible copolymers and terpoIymers. The structure of
these polymers is random or microblock depending on the
duration of the reaction or the amount of catalyst used and
the type of heating used. At equal number of hours of
reaction, and degree of transesterification catalyst used, the
use of microwaves has allowed to obtain higher yields for
both copolymers that for the terpolymers.
Copolymers and terpolymers obtained by this method are
capable of producing micro-and nanoparticles used in the
drug delivery system.