INTEGRATED PROJECT PRESENTATION

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

INTRODUCTION

PHB

INTRODUCTION:

~First discovered by Lemoigne (1925)

~Accumulated in intracellular granules by Gram+ & - microorganusm.

~ Required the limitation of an essential nutrient element in the presence carbon source for efficient synthesis of PHB.

CHARACTERISTICS:

~Partially crystalline thermoplastic

~Good material for producing biodegradable and/or biocompatible plastic

~ Stiff and brittle

~Water insoluble & relatively resistant to hydration degradation

~Good in ultra-violet resistance but poor in resistance to acid and bases.

Alcaligenes eutrophus:

~ Gram - & non-spore forming bacillus

~Optimal growth at 30°C

~Obligate aerobe, facultative chemolithoautotroph

~ Up to 80% of the dry weight of A.eutrophus can be composed of PHB inclusions

PRODUCT USAGEMEDICAL INDUSTRY PHAMACEUTICAL INDUSTRY PACKING INDUSTRY

Biodegradable sutures, surgical mesh, screws and plates for bone fixation, periodontal membranes and wound dressing.

Bioabsorbable surgical sutures= Possess necessary strength for healing of myofascial wounds.= High tensile strength and longer strength retention characteristics.= Lighter inflammation when compare with other type material.= Changes in surface morphology can be determine by SEM & AFM

Biodegradable screw & plates= Avoidance secondary removal of hardware = Do not cause imaging or radiotherapy interface or discomfort.

Drug delivery on the base of PHB films= used as drug delivery matrix for sustaining the release of various drugs such as DP.= Via diffusion & degradation= release diffusion depends on its nature, thickness, weight ration & molecular weight of PHB.= Regulate the rate by changing the MW.

Drug delivery on the base microsphere & microcapsule= release coefficient depend on diameter of microspheres.= possible produce a system with prolonged uniform drug release.

normally used in food industry based on its biodegradable characteristics / bioplastics.

used in food related applications such as films for food wrapping and thermoplastics for food packing and food container such as bowls, plates & cups.

Also used to produce container such as shampoo bottles, laminated foils, one way cup & agriculture foils.

ECONOMIC ASPECT

WORLD BIOPLASTICS DEMAND (thousand metric tons)

% Annual Growth

Item 2005 2010 2015 2005-2010

2010-2015

Bioplastic DemandNorth America

Western EuropeAsia/Pacific

Other Regions

1303460333

300801258312

1025242347320116

18.218.715.820.332.0

27.924.822.731.057.4

• 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.

Table 2.1 World demand for bioplastics to exceed 1 million tons in 2015 Greener Package

Source: Mohan 2011

2003 2007 2009 (Projection)

2013 (Projection)

2020 (Projection)

0

0.5

1

1.5

2

2.5

3

3.5

Other

Bio-based Monomers

PHA

Bio-based Ethylene

PLA

Starch Plastics

Cap

acit

y (

million

ton

nes p

er

year)

Figure 2.1 Estimated Worldwide capacities of bio-based plastics until 2020 based on company announcements.

Source: (Shen et.al 2009)

• Production of bioplastics based on PHA in 2013 has been projected to reach 0.5 billion kg.

• Therefore, Demand – Supply = (0.9 – 0.5) billion kg = 0.4 billion kg• 0.4 billion kg x 0.1% = 400 000 kg per annum

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.

PROCESS DESCRIPTION

Lower surface tension

T = 30°C, P = 1 bar, pH =7

Homogenizer blended with chloroform

together with enzymatic

method

Fed-batch mode

To obtain more

concentrated product

Homogenizer

Synthesis of PHB

Spray dryer

Disc-stack

centrifuge Blending

tank

Mixing chamberCentrifuge

Extractor

EvaporatorCultivation

STOCHIOMETRY EQUATION

C6H12O6 + 2.5027O2 + 0.6689NH3 2.2676CH1.75O0.41 N0.25 + 0.2676C4H6O2 + 2.6620CO2 + 4.2164H2O

MASS BALANCE 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.

In stream

Out stream

  Glucos

e

NH

3

O2 Dry

Biomass

PHB CO2 H20 Total

Feed 380 24 0 0 0 0 2596 3000

O2 gas 0 0 168.7

4

0 0 0 0 168.

74

Produ

ct

- - - - - - - -

Off-

gas

- - - - - - - -

Total 380 24 168.7

4

0 0 0 2596

  Gluco

se

NH3 O2 Dry

Biomass

PHB CO2 H20 Total

Feed - - - - - - - -

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

ENERGY BALANCE

For inlet of fermenter,

For outlet of fermenter,

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 345 380 2111.11 728.33Ammonia 4240.9 24 1411.76 5987.13Oxygen 88.45 168.74 5273.13 466.41Water 225.83 2596 144222.22 32569.70Σ 3169   39 751.57

Outlet Components

Enthalpy change,

ΔHo (J/mol)

Mass flow rate, ṁo

(kg/h)

Molar flow rate, N0

(mol/h)

Total enthalpy change, ΔH0N0

(kJ/h)Glucose 345 3.6 20 6.9PHB 33.65 48.5 563.95 18.98Carbon dioxide

113.35 247 5613.64 636.31

Water 225.83 2756 153 111.11 34577.08Biomass 34.62 113.8 4779.50 165.47Σ 3169   35404.74

 

•Sterilization refers to physical, chemical or mechanical process that 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 : Lee 2001)

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

BIOREACTOR II

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

Sterilization

Heating section

Holding section

Cooling section

Plate-and-frame heat exchanger

Shell-and-tube exchanger

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.

DESIGN OF BIOREACTORFour

Baffles

Sigma 298 silicon

antifoam

CRITERIA FOR SCALE-UP

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 10,000

Diameter of the vessel (m) 0.36 1.85

Diameter of impeller (m) 0.12 0.61

Height of liquid media (m) 0.72 3.70

Table 7.3 Values of scale-up operations for 75l and 10,000l bioreactor

The impeller rotation number after scale-up is 0.34. The energy input can be calculated as 133.33; the impeller diameter can be assumed as 5.33; pump rate of impeller 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 complete reference 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 constant power 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

Impeller diameter 1.0 5.0

Pump rate of impeller 1.0 42.5

Pump rate of

impeller/volume

1.0 0.34

Maximum impeller

speed (max.shearing

rate)

1.0 1.7

Reynolds number 1.0 8.5

Table 7.2 Interdependence of scale-up parameters

Source: Shuler & Kargi 2002

Cell disruption

Non-mechanical

Lysis

Physical

(osmotic shock, thermolysis)

Enzymatic

Chemical

(osmotic, solubilisation, lipid dissolution, alkali

treatment)

Dessication

Mechanical

Solid shear

(bead mill, grinding,

Hughes press)

Liquid shear

(homogenization, untrasonic, French

press)

Figure 8.1 Hierarchy chart for cell disruption methods

BIOSEPARATION

The release of intracellular bioplastic PHB granules from fed-batch cultured gram-negative bacterium Alcaligenes eutrophus using combinations of non-mechanical and mechanical methods to disrupt the first and second layers of the cells. Non-mechanical : enzymatic pretreatment of bacterium with lytic enzyme from Cytophaga;

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.

Wash & suspend in phosphate-

buffered saline, pH 7.4

5 fold dilution of cell

suspension in 50mM Tris-HCl buffer, pH 7.3.

5mM of EDTA is used to

destabilize the outer

membrane

ln (1 − R)=−kN Pa

BIOMATERIAL ENGINEERINGNonviable material used in medical device which is

intended to interact with biological systems( Williams 1987).

Unit operation

• Cyclone column bioreactor

Product usage

• Medical • Phamaceutic

al• packing

Biological response

• Bioabsorbable suture

• Drug delivery system

Cyclone Column Bioreact

or

i. Body Construction~ Stainless Steel Grade 316L~Excellent in a range of atmospheric environment & many corrosive media~intermittent service to 870°CIn continuous service to 925°C~ Solution treatment: heat to 1038-1149°C then rapid quenchExample: Heat exchanger

ii. Impeller~ Downward pumping hydrofoil & Rushton turbine~ Stainless Steel Grade 316L

iii. Baffles~ Prevent formation of vortex around walls of vessel~ made from metal strip & Stainless Steel Grade 316L

iv. Temperature Probe~ Temperature deviation by a couple of degree can dimishish dramatically the growth and biosynthesis productivity~Stainless Steel Pt 100

v. DO Probe~ Polarographic DO Probe~ anodemade from silver; cathode made from gold

vi. pH Probe~ Speed of a reaction & solubility of compound ~ made from glass tube & silver chloride covered silver wire located inside solution in glass tube

vii. Sealing~ between top plate and vessel~glass&glass;glass & metal; metal & metal~ fabric-nitryl or butyl rubber~ gasket, lipseal and ‘O’ ring

CYCLONE COLUMN BIOREACTOR

BIOLOGICAL RESPONSEBioabsorbable 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.

MATLAB

• Stoichiometry Calculation• Material Balance Of Fermenter• Energy Balance Of Fermenter • Economic Aspect

* Script files attached in the submitted CD

ENVIRONMENT AND SAFETY• Waste Generation – waste water, carbon

dioxide, biomass• Discharge limit for waste, carbon dioxide• Relevant Environmental Act• Safety Precautions – Production plant,

personal

CONCLUSION

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.

Thank you for your attention