PRODUCTION OF
POLYHYDOXYBUTYRATE USING
Alcaligenes eutrophus
Integrated Project Presentation Group KB5
NAME
CHUNG KEN VUI...
CONTENTS









Literature Review: R. eutropha & PHB
Usage of PHB
Economy Analysis of PHB: Production & Dema...
Ralstonia eutropha (ATCC 17699)




Formerly known as Alcaligenes eutrophus
Motile, rod-shaped, Gram-negative, non-...
POLYHYDROXYBUTYRATE (PHB)




Member of the polyhydroxyalkanoates, a polymer of
polyesters (Tan 2007)
Linear homop...
PHB: Properties
Comparing physical properties of PHB with PP, other PHAs and biopolymers
Properties
PHB
PP
PHB-HV
171 ...
PHB: Applications and Usages
Plastic Mulch
Medical devices
Thermoplastic
polymer
Uses of bioplastics in Europe in 2008 ...
PHB: Production
Global Production Capacity for Bioplastic
6000
Biodegradable
Bio-based, Non-biodegradable
5003
Capacity ...
PHB: Demand
Global Demand for Bioplastic
350
300
North America
Western Europe
Asia Pacific
Other Regions
Demand (in 106...
PROPOSED PRODUCTION
Global production capacity of bioplastics
from 2009 to 2016
Production
(million kg)
6000
Mode of ope...
PROCESS FLOW DIAGRAM
MATERIAL BALANCE
Sources: Nielsen et al. 2003, Shuler & Kargi 2002

Mass balance for components of fermentor in kg/hr
S...
ENERGY BALANCE

Energy balance for components of fermentor in kJ/hr
Glucose
Ammonia
Oxygen
Water
Σ
Outlet
Components
...
BIOREACTOR: Sterilization

Batch versus Continuous Sterilization
Steam
Carbon
dioxide gas
Glucose
Ammonium
chloride
Fer...
BIOREACTOR: Scale Up and Design (1)

Scale up from pilot plant 0.37 m3 to production plant volume determined.

Stirre...
BIOREACTOR: Scale Up and Design (2)

Impeller type: Flat-blade turbine
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Height of liquid media = 12.40 m
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Baffle ...
BIOSEPARATION: Rotary Filtration (1)

Continuous separation as large volume of fermentation broth is
flowed into this un...
BIOSEPARATION: Rotary Filtration (2)

Area of rotary drum, A’ = 2.63 m2

Rate of filtration = 9.972 L/m2s

Assume ...
COMPUTATION USING MATLAB®

Global Production >> Here

Global Demand >> Here

Mass and energy balance:

Displayin...
BIOMATERIAL: Material to Build Fermentor
Stainless steel
Property
Values
Density
8000kg/m3
Young’s Modulus
193GPa
M...
BIOMATERIAL: Biological Responses to PHB

PHB microspheres are the best injection-prolonged-action drug delivery
system ...
BIOMATERIAL: Improving PHB
Properties
Compatible
plasticizers
Modification
Citrate ester, Low
molecular weight PEG,
sal...
REFERENCES

Anderson, J.M. & Shive, M.S. 1997. Biodegradation and Biocompatibility of PLA and PLGA Microspheres. Advance...
THANK YOU
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Polyhydroxybutyrate IP

Production of PHB
Published on: Mar 4, 2016
Published in: Education      Technology      Business      
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Transcripts - Polyhydroxybutyrate IP

  • 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          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  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