PRODUCTION OF POLYHYDOXYBUTYRATE USING Alcaligenes eutrophus

Integrated Project Presentation Group KB5

NAME MATRIC NO.

CHUNG KEN VUI A 98753TEE ZHAO KANG A 132597RAJESWARI A/P JAYARAMAN A 133999

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

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

Source: Hall 2012

TEM of R. eutropha showing the PHB inclusion bodies

Source: Porter & Yu 2011

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

PHB: Properties

Properties PHB PP PHB-HV

P(3HB-4HB)

PLA PGA PCL

Tm (°C) 171 to 182

171 to 186

137 to 179

150 to 177

150 to 162

220 to 233

58 to 65

Tg (°C) 5 to 15 -10 -6 to 10 4 to 23 45 to 60 35 to 45 -65 to -60

ρ (g/cm3) 1.23 to 1.25

0.905 1.20 1.21 to 1.25

1.5 to 1.71

1.11 to 1.15

σ (Mpa) 40 38 30 to 40 10 to 43 21 to 60 60 to 99.7

20.7 to 42

E (Gpa) 3.5 to 4 1.7 0.7 to 3.5

0.35 to 3.5

6 to 7 0.21 to 0.44

ε (%) 5 to 8 400 8 to 10 5 to 511 2.5 to 6 1.5 to 20

300 to 1000

Comparing physical properties of PHB with PP, other PHAs and biopolymers

Sources: Mark 2003; Van de Velde & Kiekens 2002

PHB: Applications and Usages

Uses of bioplastics in Europe in 2008 (reproduced on MATLAB®)

Source: Barker & Safford 2009

Thermoplastic polymer

Medical devices

Plastic Mulch

2009 2010 2011 20160

1000

2000

3000

4000

5000

6000Global Production Capacity for Bioplastic

Year

Capacity P

roduction (

in 1

06 k

g)

Biodegradable

Bio-based, Non-biodegradable

PHB: Production

Global production capacity of bioplastics from 2009 to 2016 (reproduced on MATLAB®)

Source: European Bioplastics & Institute for Bioplastics and Biocomposites 2012

776

5003

486

675

PHB: Demand

2005 2010 20150

50

100

150

200

250

300

350Global Demand for Bioplastic

Year

Dem

and (

in 1

06 k

g)

North America

Western EuropeAsia Pacific

Other Regions

World bioplastics demand from 2005 to 2015 (reproduced on MATLAB®)

Source: Mohan 2011

Total demand: 1.025 billion kg in 2015

2009 2010 2011 20160

1000

2000

3000

4000

5000

6000

226 342 486 77623 674 675

5003

Global production capacity of bio-plastics from 2009 to 2016

Biodegradable Biobased/Non-biodegradableYear

Production(million kg)

Types % Production capacity (million kg)

Biobased/non-biodegradableBio-PET 30 38.9 451.71Bio-PE 17.2 199.73Bio-PA 1.6 18.58Other non-biodegradable 0.4 4.64BiodegradablePLA 16.1 186.95Biodegradable starch blend 11.3 131.22Bio-polyesters 10 116.12Regenerated cellulose 2.4 27.87PHA 1.6 18.58Other biodegradable 0.5 5.81TOTAL 100.

01161.20

Sources: modified from European Bioplastics & Institute for Bioplastics and Biocomposites 2012

Mode of operation: Fed-batch fermentation

Total annual Production:PHB = (1.6/41.9)*(776 million kg)

≈ 30 million kg

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)

PROPOSED PRODUCTION

PROCESS FLOW DIAGRAM

MATERIAL BALANCE

Mass balance for components of fermentor in kg/hrStream In Out

Feed Gas Total Product Gas-off TotalGlucose 500 0 500 16.46 0 16.46O2 0 253.93 253.93 0 0 0

NH3 2.97 0 2.97 0 0 0

Biomass 0.5 0 0.5 21.18 0 21.18PHB 0 0 0 140.00 0 140.00CO2 0 0 0 0 386.27 386.27

H2O 7496.53 0 7496.53 7689.95 0 7689.95

Total 8000 253.93 8253.93 7867.58 386.27 8253.86

Comparison between manual calculations with SUPERPRO®

Element Error Percentage (%)

Glucose 68

Oxygen -

Biomass -6.88

PHB -7.19

Carbon dioxide -8.14

Water -0.182

Balanced!!!

Sources: Nielsen et al. 2003, Shuler & Kargi 2002

ENERGY BALANCE Energy balance for components of fermentor in kJ/hr

Inlet Components

Enthalpy change, ΔHi (J/mol)

Mass flow rate, ṁi

(kg/h)

Molar flow rate, Ni (mol/h)

Total enthalpy change, ΔHiNi

(kJ/h)

Glucose 460.000 500.00 2777.78 1277.78

Ammonia 143.989 2.97 174.71 25.16

Oxygen 105.301 253.93 7935.31 835.60

Water 300.960 7497.03 416501.67 125350.34

Σ 8253.93 127488.87

Outlet Components

Enthalpy change, ΔHi (J/mol)

Mass flow rate, Ni (kg/h)

Molar flow rate, Ni (mol/h)

Total enthalpy change, ΔHiNi

(kJ/h)

Glucose 460.000 16.46 91.44 42.06

Biomass 46.800 20.68 824.890 38.60

PHB 581.016 140.00 1627.907 945.84

Carbon dioxide 151.065 386.27 8778.86 1326.18

Water 300.960 7690.45 427247.22 128584.32

Σ 8253.86 130937.01

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)

Qgen = 130937.01 - 127488.87 + (-3650.24) = -202.10 kJ/hExothermic

BIOREACTOR: Sterilization Batch versus Continuous Sterilization

Steam

Glucose

Ammonium chloride

Air

Carbondioxide gas

Fermentation broth

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

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/VFermentation 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

Power requirement without aeration, Pmo1105.8 W 1105.1 kW

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

BIOREACTOR: Scale Up and Design (2)

Impeller type: Flat-blade turbine Baffle width = 0.62 m Impeller diameter, Di = 2.05 m Impeller spacing, Hi = 4.10 m Impeller blade length, Li = 0.51 m Impeller blade height, Wi = 0.41 m

Height of liquid media = 12.40 m Location of sparger from bottom of

the tank = 1.03 m Number of impeller blade: 6, 3 levels

Source: Komline-Sanderson

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

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

BIOSEPARATION: Rotary Filtration (2)

COMPUTATION USING MATLAB®

Global Production >> Here Global Demand >> Here Mass and energy balance:

Displaying the result on the Command Window:

BIOMATERIAL: Material to Build Fermentor

Property Values

Density 8000kg/m3

Young’s Modulus 193GPa

Maximum Withstand Temperature 925oC

Thermal Conductivity 21.5W/m.K

Stainless steel

Source: Modified from Atlas Steels Australia 2001

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

BIOMATERIAL: Improving PHB Properties

Modification

Compatible plasticizers

Citrate ester, Low molecular weight

PEG, salicylic ester, etc

Copolymer with HA units PHB-HV, P(3HB-4HB)

Nano-technology ??

Source: Wang et al. 2007

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-could-build-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.

THANK YOU