Production of PHB

1. PRODUCTION OF
POLYHYDOXYBUTYRATE USING
Alcaligenes eutrophus
Integrated Project Presentation Group KB5
NAME
CHUNG KEN VUI
TEE ZHAO KANG
RAJESWARI A/P JAYARAMAN
MATRIC NO.
A 98753
A 132597
A 133999

2. CONTENTS

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




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Literature Review: R. eutropha & PHB
Usage of PHB
Economy Analysis of PHB: Production & Demand
Process Descirption with PFD
Calculation of material and energy balance in the
fermentor
Bioreactor: Sterilization, Scale up and Basic Design
Bioseparation: Rotary Filtration
Computation Engineering
Biomaterial Engineering: Fermentor & Biological
Responses

3. Ralstonia eutropha (ATCC 17699)




Formerly known as Alcaligenes eutrophus
Motile, rod-shaped, Gram-negative, non-sporing bacterium,
major strains: H16 and JMP 134 (Larsen & Pogliano 2007)
Optimal temperature is 30°C, optimal pH is 7 and non-halophilic
(Larsen & Pogliano 2011)
Produces PHB inside the inclusion bodies under limited nitrogen
but excessive carbon sources (Ojumu et al. 2004)
SEM of Ralstonia eutropha
TEM of R. eutropha showing the PHB inclusion bodies
Source: Hall 2012
Source: Porter & Yu 2011

4. POLYHYDROXYBUTYRATE (PHB)




Member of the polyhydroxyalkanoates, a polymer of
polyesters (Tan 2007)
Linear homopolymer of four carbon D-3-hydroxybutyrate
(Dawes 1988)
Chemical composition is [-COCH2CH(CH3)O-]n or
[C4H6O2]n
Water-insoluble, biocompatibility and non-toxic; but
brittle (Kok & Hasirci 2003)
Molecular structure for the linear chain of
PHB
Source: Modified from Dawes 1988

5. PHB: Properties
Comparing physical properties of PHB with PP, other PHAs and biopolymers
Properties
PHB
PP
PHB-HV
171 to
182
5 to 15
171 to
186
-10
137 to
179
-6 to 10
0.905
1.20
σ (Mpa)
1.23 to
1.25
40
38
30 to 40
E (Gpa)
3.5 to 4
1.7
0.7 to 3.5
5 to 8
400
8 to 10
Tm (°C)
Tg (°C)
ρ (g/cm3)
ε (%)
P(3HB4HB)
150 to
177
4 to 23
PLA
PGA
PCL
150 to
162
45 to 60
220 to
233
35 to 45
58 to 65
10 to 43
1.21 to
1.25
21 to 60
5 to 511
0.35 to
3.5
2.5 to 6
1.5 to
1.71
60 to
99.7
6 to 7
Sources: Mark 2003; Van de Velde & Kiekens 2002
1.5 to 20
-65 to 60
1.11 to
1.15
20.7 to
42
0.21 to
0.44
300 to
1000

6. PHB: Applications and Usages
Plastic Mulch
Medical devices
Thermoplastic
polymer
Uses of bioplastics in Europe in 2008 (reproduced on MATLAB®)
Source: Barker & Safford 2009

7. PHB: Production
Global Production Capacity for Bioplastic
6000
Biodegradable
Bio-based, Non-biodegradable
5003
Capacity Production (in 106 kg)
5000
4000
3000
2000
675
776
1000
486
0
2009
2010
2011
2016
Year
Global production capacity of bioplastics from 2009 to 2016 (reproduced on MATLAB®)
Source: European Bioplastics & Institute for Bioplastics and Biocomposites 2012

8. PHB: Demand
Global Demand for Bioplastic
350
300
North America
Western Europe
Asia Pacific
Other Regions
Demand (in 106 kg)
250
Total demand: 1.025
billion kg in 2015
200
150
100
50
0
2005
2010
Year
World bioplastics demand from 2005 to 2015 (reproduced on MATLAB®)
Source: Mohan 2011
2015

9. PROPOSED PRODUCTION
Global production capacity of bioplastics
from 2009 to 2016
Production
(million kg)
6000
Mode of operation: Fed-batch
fermentation
5000
4000
Total annual Production:
PHB
= (1.6/41.9)*(776 million kg)
≈ 30 million kg
5003
3000
2000
1000
23
226
674
342
675
486
776
2009
0
2010
2011
2016
Biodegradable
Types
Biobased/Non-biodegradable
%
Biobased/non-biodegradable
Bio-PET 30
Bio-PE
Bio-PA
Other non-biodegradable
Biodegradable
PLA
Biodegradable starch blend
Bio-polyesters
Regenerated cellulose
PHA
Other biodegradable
TOTAL
Production
capacity (million
kg)
38.9
17.2
1.6
0.4
451.71
199.73
18.58
4.64
16.1
11.3
10
2.4
1.6
0.5
100.0
186.95
131.22
116.12
27.87
18.58
5.81
1161.20
Year
Proposed annual Production:
PHB
=3.3% of total annual
production
= 1 million kg
Capacity = 1 million kg/150 cycles
= 6667 kg per cycle
≈ 140 kg/hr
Cost of production for PHB is estimated
to be around RM6 per kg (The Star
2011)
Sources: modified from European Bioplastics & Institute for Bioplastics and Biocomposites 2012

10. PROCESS FLOW DIAGRAM

11. MATERIAL BALANCE
Sources: Nielsen et al. 2003, Shuler & Kargi 2002

Mass balance for components of fermentor in kg/hr
Stream
Glucose
O2
NH3
Biomass
PHB
CO2
H2O
Total

In
Feed
500
0
2.97
0.5
0
0
7496.53
8000
Gas
0
253.93
0
0
0
0
0
253.93
Total
500
253.93
2.97
0.5
0
0
7496.53
8253.93
Out
Product
16.46
0
0
21.18
140.00
0
7689.95
7867.58
Gas-off
0
0
0
0
0
386.27
0
386.27
Total
16.46
0
0
21.18
140.00
386.27
7689.95
8253.86
Comparison between manual calculations with SUPERPRO®
Element
Error Percentage (%)
Glucose
68
Oxygen
-
Biomass
-6.88
PHB
-7.19
Carbon dioxide
-8.14
Balanced!!!

12. ENERGY BALANCE

Energy balance for components of fermentor in kJ/hr
Glucose
Ammonia
Oxygen
Water
Σ
Outlet
Components


Mass flow rate, ṁi
(kg/h)
Molar flow rate, Ni
(mol/h)
Total enthalpy
change, ΔHiNi (kJ/h)
500.00
2.97
253.93
7497.03
8253.93
Mass flow rate, Ni
(kg/h)
2777.78
174.71
7935.31
416501.67
1277.78
25.16
835.60
125350.34
127488.87
Total enthalpy
change, ΔHiNi (kJ/h)
Enthalpy change,
ΔHi (J/mol)
Glucose
Biomass
PHB
Carbon dioxide
Water
Σ
Qgen
Enthalpy change,
ΔHi (J/mol)
460.000
143.989
105.301
300.960
Inlet Components
460.000
46.800
581.016
151.065
300.960
Molar flow rate, Ni
(mol/h)
16.46
20.68
140.00
386.27
7690.45
8253.86
91.44
824.890
1627.907
8778.86
427247.22
42.06
38.60
945.84
1326.18
128584.32
130937.01
= 130937.01 - 127488.87 + (-3650.24) = -202.10 kJ/h
Exothermic
Energy or heat generated by the operation in fermentor is -202 kJ/h
Requires 9.67 kg/h of cooling water from reservoir to be pumped into the
cooling jacket (maintaining output T at 35°C)

13. BIOREACTOR: Sterilization

Batch versus Continuous Sterilization
Steam
Carbon
dioxide gas
Glucose
Ammonium
chloride
Fermentation
broth
Air
Batch
Continuous
43.86
Total Del Factor
43.86
8.74
Heating Del
-
17.73
Cooling Del
-
17.39
Holding Del
43.86
5.32 min
Holding time
2.43 min

Continuous sterilization is more economical as it requires less holding
time

Requires 24.37 m of pipe length and steam generator capable to flow
about 1589 kg/h of steam

14. BIOREACTOR: Scale Up and Design (1)

Scale up from pilot plant 0.37 m3 to production plant volume determined.

Stirred tank reactor is used.

Criterion of scale-up: Constant P/V
Fermentation System
Model
Prototype
Working volume (m3)
0.37
373
Volume of fermentor (m3)
0.5
500
Tank diameter, Dt (m)
0.62
6.20
Diameter of impeller, Di (m)
0.20
2.05
Height of liquid media (m)
1.24
12.40
Height of fermentor, Ht (m)
1.66
16.56
Agitator/Impeller speed , N (rpm)
500
107.7
1105.8 W
1105.1 kW
Power requirement without aeration, Pmo
Gas hold up, H
0.002
Sauter-mean diameter, D32 (mm)
1.2
Interfacial area, a (1/m)
10
Volumetric mass transfer coefficient, Kla (1/s)
0.0047

15. BIOREACTOR: Scale Up and Design (2)

Impeller type: Flat-blade turbine

Height of liquid media = 12.40 m

Baffle width = 0.62 m


Impeller diameter, Di = 2.05 m
Location of sparger from bottom of the
tank = 1.03 m

Impeller spacing, Hi = 4.10 m

Number of impeller blade: 6, 3 levels

Impeller blade length, Li = 0.51 m

Impeller blade height, Wi = 0.41 m

16. BIOSEPARATION: Rotary Filtration (1)

Continuous separation as large volume of fermentation broth is
flowed into this unit

PHB are intracellular components while the biomass has to be
filtered out together with the product inside

Rotation of the drum is 1.0 rpm
Source: Komline-Sanderson

17. BIOSEPARATION: Rotary Filtration (2)

Area of rotary drum, A’ = 2.63 m2

Rate of filtration = 9.972 L/m2s

Assume that 4% fermentation broth left in
the cake and washing efficiency is 65%

Cake formation time = 60s

Washing time = 124.5 s

18. COMPUTATION USING MATLAB®

Global Production >> Here

Global Demand >> Here
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Mass and energy balance:

Displaying the result on the Command Window:

19. BIOMATERIAL: Material to Build Fermentor
Stainless steel
Property
Values
Density
8000kg/m3
Young’s Modulus
193GPa
Maximum Withstand Temperature
925oC
Thermal Conductivity
21.5W/m.K
Source: Modified from Atlas Steels Australia 2001

20. BIOMATERIAL: Biological Responses to PHB

PHB microspheres are the best injection-prolonged-action drug delivery
system (Anderson & Shive 1997)

Does not causes necrosis, abscess and tumorigenesis – biocompatible
and non-toxic (Qu et al. 2006)

Exudation and proliferation phases: Neutrophil, macrophage and
fibroblast (Shishatskaya et al. 2008)
Microscopic picture of tissue at the site
of PHB microspheres implantation.
Source: Shishatskaya et al. 2008

21. BIOMATERIAL: Improving PHB
Properties
Compatible
plasticizers
Modification
Citrate ester, Low
molecular weight PEG,
salicylic ester, etc
Copolymer with
HA units
PHB-HV, P(3HB-4HB)
Nanotechnology
??
Source: Wang et al. 2007

22. REFERENCES

Anderson, J.M. & Shive, M.S. 1997. Biodegradation and Biocompatibility of PLA and PLGA Microspheres. Advance Drug Delivery Review 28:5-24.

Anon. 2011. Malaysia’s Pioneer Bioplastics Pilot Plant is Operational. The Star, 13 July.
http://biz.thestar.com.my/news/story.asp?file=/2011/7/13/business/20110713141942&sec=business [10 November 2012].

Barker, M. & Safford, R. 2009. Industrial Uses for Crops: Markets for Bioplastics. London: HGCA.

Cramm, R. 2008. Genomic View of Energy Metabolism in Ralstonia eutropha H16. Journal of Molecular Microbiology and Biotechnology 16: 38-52.

Dawes, E.A. 1988. Polyhydroxybutyrate: an Intriguing Biopolymer. Bioscience Reports 8(6): 537-547.

European Bioplastics & Institute for Bioplastics and Biocomposites. 2012. European Bioplastics: Fivefold growth of the bioplastics market by 2016.
http://en.european-bioplastics.org/wp-content/uploads/2012/10/PR_market_study_bioplastics_ENG.pdf [30 October 2012]

Hall, C. 2012. Energy Digital: Electrofuel System Could Build Alternative Fuels. http://www.energydigital.com/green_technology/electrofuel-system-couldbuild-alternative-fuels [24 October 2012].

Kok, F. & Hasirci, V. 2003. Polyhydroxybutyrate and Its Copolymers: Applications in the Medical Field. Tissue Engineering and Novel Delivery Systems.
Boca Raton: CRC Press.

Lakshimi, R.S., Hema, T.A., Raj, D.Y. & Starin, S.T. 2012. Production and Optimization of Polyhydroxybutyrate from Rhizobium sp. Present in Root
Nodules. Journal of Pharmacy and Biological Sciences 3(2): 21-25.

Larsen, R. & Pogliano, K. 2011. Ralstonia eutropha. Kenyon Microbewiki. http://microbewiki.kenyon.edu/index.php/Ralstonia_eutropha [22 October 2012].

Mark, H.F. 2003. Poly(3-hydroxyalkanoates). Encyclopedia of Polymer Science and Technology. Third Edition. New Jersey: John Wiley & Sons, Inc.

Mohan, A.M. 2011. World Demand for Bioplastics to Exceed 1 Million Tons in 2015.
http://www.greenerpackage.com/bioplastics/world_demand_bioplastics_exceed_1_million_tons_2015 [31 October 2012]

Ojumu, T.V., Yu, J. & Solomon, B.O. 2004. Production of Polyhydroxyalkanoates, a Bacterial Biodegradable Polymer. African Journal of Biotechnology
3(1): 18-24.

Porter, M. & Yu, J. 2011. Monitoring the In Situ Crystallization of Native Biopolyester Granules in Ralstonia eutropha via Infrared Spectroscopy. Journal of
Microbiological Methods 87(1): 49-55.

Qu, X., Wu, Q., Zhang, K. & Chen, G.Q. 2006. In-vivo Studies of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Based Polymers: Biodegradation and
Tissue Reactions. Biomaterials 27:3540-8.

Skrifvars, M., Rajan, R. & Joseph, K. 2009. Assessing Thermal Characteristics of Polyhydroxybutyrate Based Composites Reinforced with Different
Natural Fibres. Second International Conference on Innovative Natural Fibre Composites for Industrial Applications, Rom 2009 Slide Presentation.
http://www.namateco.com/attachments/093_Skrifvars%20presentation%20Rome%202009.pdf [24 October 2012].

Tan, K.P. 2007. Polyhydroxyalkanoates. Kirk-Othmer: Concise Encyclopedia of Chemical Technology. Fifth Edition. New Jersey: John Wiley & Sons, Inc.

Wang, L., Zhu, W., Wang, X., Chen, X., Chen, G. & Xu, K. 2007. Processability Modifications of Poly(3-hydroxybutyrate) by Plasticizing, Blending, and
Stabilizing. Journal of Applied Polymer Science 107(1): 166-73.

23. THANK YOU