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Production of PHB



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  • 1. Topic: Production of PHB using Alcaligenes eutrophus KB Group 2: 1. Sonia Dilip Patel A133115 2. Tan Yi Von A132788 3. Chin Lee Nee A132359 4. Judy Loh Ea Ea A132395
  • 2. CHARACTERISTICS: ~Partially crystalline thermoplastic ~Good material for producing biodegradable and/or biocompatible plastic ~ Stiff and brittle ~Water insoluble & relativelyINTRODUCTION: resistant to hydration~First discovered by Lemoigne degradation(1925) ~Good in ultra-violet resistance Alcaligenes eutrophus:~Accumulated in intracellular but poor in resistance to acid ~ Gram - & non-sporegranules by Gram+ & - and bases. forming bacillusmicroorganusm. ~Optimal growth at 30 C~ Required the limitation of an ~Obligateessential nutrient element in the aerobe, facultativepresence carbon source for chemolithoautotrophefficient synthesis of PHB. ~ Up to 80% of the dry weight of A.eutrophus can PHB be composed of PHB inclusions
  • 3. MEDICAL INDUSTRY PHAMACEUTICAL INDUSTRY PACKING INDUSTRY Biodegradable sutures, surgical Drug delivery on the base of PHB normally used in food industrymesh, screws and plates for bone films based on its biodegradablefixation, periodontal membranes and = used as drug delivery matrix for characteristics / bioplastics.wound dressing. sustaining the release of various used in food related applications drugs such as DP. such as films for food wrapping and Bioabsorbable surgical sutures = Via diffusion & degradation thermoplastics for food packing and= Possess necessary strength for = release diffusion depends on its food container such as bowls, plateshealing of myofascial wounds. nature, thickness, weight ration & & cups.= High tensile strength and molecular weight of PHB. Also used to produce container suchlonger strength retention = Regulate the rate by changing the as shampoo bottles, laminated foils,characteristics. MW. one way cup & agriculture foils.= Lighter inflammation whencompare with other type material. Drug delivery on the base= Changes in surface morphology can microsphere & microcapsulebe determine by SEM & AFM = release coefficient depend on diameter of microspheres. Biodegradable screw & plates = possible produce a system with= Avoidance secondary removal of prolonged uniform drug release.hardware= Do not cause imaging orradiotherapy interface or discomfort.
  • 4. Table 2.1 World demand for bioplastics to exceed 1 million tons in 2015 Greener Package WORLD BIOPLASTICS DEMAND (thousand metric tons) % Annual Growth Item 2005 2010 2015 2005-2010 2010-2015 Bioplastic Demand 130 300 1025 18.2 27.9 North America 34 80 242 18.7 24.8 Western Europe 60 125 347 15.8 22.7 Asia/Pacific 33 83 320 20.3 31.0 Other Regions 3 12 116 32.0 57.4 Source: Mohan 2011 • Global demand for bioplastics that derived from plant-based sources, has been estimated to 0.9 billion kg in 2013, valued at approximately RM7.6 billion. (Freedonia Group 2012). • Factors: customer demand for more environmentally-sustainable products, development of bio-based feed stocks for commodity plastic resins, increasing restrictions on the use of nondegradable plastic products and high rise of crude oil and natural gas prices.
  • 5. 3.5Capacity (million tonnes per Other • Production of 3 Bio-based Monomers bioplastics based on 2.5 PHA PHA in 2013 has been 2 projected to reach 0.5 year) Bio-based Ethylene PLA billion kg. 1.5 • Therefore, Starch Plastics 1 Demand – Supply 0.5 = (0.9 – 0.5) billion kg 0 = 0.4 billion kg 2003 2007 2009 2013 2020 • 0.4 billion kg x 0.1% (Projection) (Projection) (Projection) = 400 000 kg per Figure 2.1 Estimated Worldwide capacities of bio-based annum plastics until 2020 based on company announcements. Source: (Shen 2009)Malaysia Demand And Supply Of Bioplastic• Malaysia’s first fully automated PHA Bioplastics Pilot Plant was launched by Science, Technology and Innovation Minister Datuk Seri Dr. Maximus Johnity Ongkili at Jalan Beremban.• Scaled-up to 2,000 L, the bioreactor facilities and integrated manufacturing process of the plant are able to produce various options of PHA materials from crude palm kernel oil and palm oil mill effluent.
  • 6. T = 30°C, P = 1 Fed- bar, pH =7 batch mode Lower Homogenizersurface blended withtension chloroform together with enzymaticTo obtain more method concentrated product
  • 7. Synthesis Mixing Evaporator of PHB chamberCultivation Centrifuge Disc-stack centrifuge Blending Spray Homogenizer Extractor dryer tank
  • 8.  C6H12O6 + 2.5027O2 + 0.6689NH3 2.2676CH1.75O0.41 N0.25 + 0.2676C4H6O2 + 2.6620CO2 + 4.2164H2O 0.1 % of 0.4 billion kg = 400 000 kg per year. *1 batch = 62 hours 133 batches/year= 8246 kg of PHB/year = Production of 48.5 kg/hour *21 days off production for maintenance etc.
  • 9. Glucos NH O2 Dry PHB CO2 H20 Total e 3 Biomass Feed 380 24 0 0 0 0 2596 3000 O2 gas 0 0 168.74 0 0 0 0 168.7 4 In stream Produ - - - - - - - - ct Off-gas - - - - - - - - Total 380 24 168.74 0 0 0 2596 Gluco NH3 O2 Dry PHB CO2 H20 Total se Biomass Feed - - - - - - - - Out stream O2 gas - - - - - - - - Product 3.6 - - 113.8 48.5 0 2756 2921. 9 Off-gas 0 0 0 0 0 247 0 247 Total 3.6 0 0 113.8 48.5 247 2756
  • 10. For inlet of fermenter, Inlet Enthalpy Mass flow Molar flow Total Componen change, ΔHi rate, ṁi rate, Ni enthalpy ts (J/mol) (kg/h) (mol/h) change, ΔHiNi (kJ/h) Glucose 345 380 2111.11 728.33 Ammonia 4240.9 24 1411.76 5987.13 Oxygen 88.45 168.74 5273.13 466.41 Water 225.83 2596 144222.22 32569.70For outlet of fermenter, Σ 3169 39 751.57 Outlet Enthalpy Mass flow Molar flow Total Componen change, rate, ṁo rate, N0 enthalpy ts ΔHo (J/mol) (kg/h) (mol/h) change, ΔH0N0 (kJ/h) Glucose 345 3.6 20 6.9 PHB 33.65 48.5 563.95 18.98 Carbon 113.35 247 5613.64 636.31 dioxide Water 225.83 2756 153 111.11 34577.08 Biomass 34.62 113.8 4779.50 165.47 Σ 3169 35404.74
  • 11. •Sterilization refers to physical, chemical or mechanical processthat completely destroys or removes all form of viable microorganisms.•Mode of sterilization methods : a) continuous b) batch•Advantages of continuous sterilization are shown below: (Source : Lee2001)i. Running costs are lessii. Ease in scaling-up of the process.iii. Easier to automate the process and therefore less labor intensive.iv. Requires less steam by recovering heat from the sterilized medium and thus requires less cooling water. It can averagely save about 30% steam and 40% of cooling water
  • 12. Figure 7.1 Comparison of a batch (A) with a continuous sterilization strategy (B) for the temperature profile of the medium sterilized Source: Shuler & Kargi 2002
  • 13. Sterilization Heating section Cooling Holding section sectionPlate-and-frame heat exchanger Shell-and-tube exchanger
  • 14.  There are three sections in the sterilization: a)heating section b) holding section c) cooling section Indirect heating in plate-and-frame heat exchanger is chosen and it can be used for cooling purpose. The heated medium need to pass through holding section which is composed of long tubes as the temperature is assumed to be constant . Therefore, the time needed for heating, holding and cooling are 17.27s, 11.45s and 12.73s respectively, the sum of time required is 41.45s and equal to 0.012 h.
  • 15. Four BafflesSigma 298 silicon antifoam
  • 16.  Case that can be selected as the criteria of scale-up: scale up based on constant power input (P0/V) implies constant OTR.Volume of bioreactor (l) 75 10,000Diameter of the vessel (m) 0.36 1.85Diameter of impeller (m) 0.12 0.61Height of liquid media (m) 0.72 3.70Table 7.3 Values of scale-up operations for 75l and 10,000l bioreactor
  • 17. The impeller rotation number after scale-up is 0.34. The energy input can becalculated as 133.33; the impeller diameter can be assumed as 5.33; pump rate ofimpeller can be assumed as 45.33; pump rate of impeller over volume is 0.36;maximum impeller speed is 1.81 and Reynolds number is assumed as 9.0. There are some additional information that need to calculated as completereference in scale-up operations:(1)Aeration rate = 6.25 x 10-4 m3/s for 0.5vvm(2)Gas superficial velocity = 43.48m/h (3)Power calculation Pg1 = 15.71 hp; PI =579.42hp;(4) Rotational speed 350rpm for N1 while N2 = 118.98 rpm for constantpower input and N2 = 68.85 rpm for constant input velocity. Scale-up criterion Small fermenter,80l Constant Po/V Energy input 1.0 125 Energy input/volume 1.0 1.0 Impeller rotation number 1.0 0.34 Table 7.2 Interdependence of Impeller diameter 1.0 5.0 scale-up parameters Pump rate of impeller 1.0 42.5 Pump rate of 1.0 0.34 Source: Shuler & impeller/volume Kargi 2002 Maximum impeller speed 1.0 1.7 (max.shearing rate) Reynolds number 1.0 8.5
  • 18. Cell disruption Non-mechanical Mechanical Lysis Dessication Solid shear Liquid shear (bead mill, (homogenization, Physical grinding, untrasonic, French (osmotic Enzymatic Hughes press) press) shock, thermolysis) Chemical(osmotic, solubilisatio n, lipid dissolution, alkali treatment) Figure 8.1 Hierarchy chart for cell disruption methods
  • 19. The release of intracellular bioplastic PHB granules from fed-batchcultured gram-negative bacterium Alcaligenes eutrophus usingcombinations of non-mechanical and mechanical methods to disruptthe first and second layers of the cells.Non-mechanical : enzymatic pretreatment of bacterium with lytic enzyme from Cytophaga; 5 fold dilution of Wash & suspend 5mM of EDTA cell suspension in phosphate- is used to in 50mM Tris- buffered saline, destabilize the HCl buffer, pH pH 7.4 outer membrane 7.3.Mechnical : disruptive by using an APV-Gaulin 15M-8BA and 30CD high-pressure homogenizer with a ceramic valve seat. three passes at 60-70 Mpa for complete disruption two-stage process: primary point break of the cell envelope & further breakage of the cell wall and degradation of cellular debris.
  • 20. Nonviable material used in medical device which is intended to interact with biological systems ( Williams 1987). Biological Unit operation Product usage response• Cyclone • Medical • Bioabsorbable column • Phamaceutical suture bioreactor • packing • Drug delivery system
  • 21. iv. Temperature Probe v. DO Probe iii. Baffles ~ Temperature deviation by a ~ Polarographic DO ~ Prevent formation of vortex couple of degree Probe around walls of vessel can dimishish ~ anodemade from ~ made from metal strip & dramatically the silver; cathode made Stainless Steel Grade 316L growth and from gold ii. Impeller biosynthesis productivity vi. pH Probe ~ Downward pumping hydrofoil & Rushton ~Stainless Steel Pt ~ Speed of a reaction & solubility turbine 100 of compound ~ Stainless Steel Grade ~ made from glass tube & silver 316L chloride covered silver wire located inside solution in glassi. Body Construction tube~ Stainless Steel Grade 316L vii. Sealing~Excellent in a range of ~ between top plate andatmospheric environment & Cyclone vesselmany corrosive media~intermittent service to 870°C Column ~glass&glass;glass & metal; Bioreactor metal & metalIn continuous service to 925°C ~ fabric-nitryl or butyl rubber~ Solution treatment: heat to ~ gasket, lipseal and ‘O’ ring1038-1149°C then rapid quenchExample: Heat exchanger
  • 22. Bioabsorbable surgical suture• Biodegradable P3/4HB monofilament suture has better tissue compatibility than nature and chemosynthesis biodegradable suture.• The tissue response for P3/4HB is less serious than chromic catgut and Vicryl.• Inflammation process will reduced slowly by indicated by disappearing of leucocytes. Drug delivery system• Suitability depend on its biodegradation properties and also biocompatibility.• Slight inflammation in capsule zone during implantation period changed from the mostly neutrophils granulocytes to mostly lymphocytes.• Typical host reaction to foreign implant.• PHB did not inhibit growth of the cells.
  • 23. • Stoichiometry Calculation• Material Balance Of Fermenter• Energy Balance Of Fermenter• Economic Aspect* Script files attached in the submitted CD
  • 24. • Waste Generation – waste water, carbon dioxide, biomass• Discharge limit for waste, carbon dioxide• Relevant Environmental Act• Safety Precautions – Production plant, personal
  • 25.  The demand of PHB keeps increasing. Our production of 0.1% of 0.4 billion had a total mass in and out of 3169 kg/h. The total time of sterilization required is 0.012h. Scale up – from 75l to 10 000l. The criteria of bioreactor for production was calculated. Operating way of homogenizer was understood. Material of bioreactor was studied in deep together with biological response of PHB. MATLAB coding comparison and SuperPro usage was understood.
  • 26.  Thank you for your attention 