1) The document presents a feasibility study for the production of bioethanol from glycerol using Enterobacter aerogenes TISTR1468. It discusses the process selection, raw materials, design constraints, and site location analysis. 2) A continuous process is proposed using two main fermenters in series. Mass and energy balance calculations show the process can produce 15,000 tonnes of ethanol per year. 3) Downstream separation will involve disc stack centrifugation, distillation, and molecular sieves to obtain concentrated ethanol and separate water and glycerol byproducts.

1. KKKB 4774
BIOPROCESS PLANT DESIGN
PROJECT I
Lim Kah Huay A132816
Low Bee Chan A132764
Sonia Dilip Patel A/P Dilip Kumar A133115
Fatin Atikah Binti Kassim A132739
Jamilah Binti Ahmad A133159
Muhammad Khairil Azim bin Abdullah A133275
PRODUCTION OF BIOETHANOL FROM GLYCEROL
USING Enterobacter aerogenes TISTR1468
FEASIBILITY STUDY
Group KB4

2. Level 1
Level 2
Level 3
Level 4
Feasibility Study Outline
• Process Selection
• Chemical reaction
• Details for raw material and product
• Design constraint
• Site location
• Physical / Chemical properties
• Synthesis of Input-Output Structure
• Design variable
• Design capacity
• Mass balance
• Curve of FEP 2
• Number of bioreactor
• Limiting reactant balance
•Bioreactor selection
• Bioreactor sizing
• Cost of bioreactor
• Curve of FEP 3
• Disc Stack centrifuge
• Distillation column
• Molecular sieve
• Storage tank
• Curve of FEP 4

3. • Higher octane number
• Reduced particulate and NOx emission
• Higher flame speed
• Higher heat of vaporization
• SO2 & PM emission decreased
• Reduce emission of hydrocarbon – depletion of ozone
• Lower volatility and photochemical reactivity – smog
• Octane enhancer (cancer-causing) – emission reduced by half
• Broader flammability limits
Source: Renewable Fuels Association (2008)
Level 1
Why Ethanol?

4. • The United States
• Brazil
• Canada
• Sweden
• India
• China
Ethanol as Fuel Worldwide
Other Usage
• Industries
 Pharmaceutical
 Personal care
 Cleaning products
 Paint
 Food
 Beverage
Product Usage

5. Property Gasoline Ethanol
Specific gravity 0.73 0.79
30 - 225 78.3
Specific heat (MJ/kg) 43.5 27.0
Heat of vaporization (kJ/kg) 400 900
Octane number 91-100 108
-40 13
300 366
Heat of formation (kcal/mole) -52.78 -51.95
Latent Heat (kcal/kg) 90.82 210.7
Molecular weight (g/mol) 113.228 46.070
Toxicity Toxic Less toxic than gasoline
Solubility in water No Yes
Smoke Produce visible
smoke
Does not produce
visible smoke
Source: Shah (2010)
GASOLINE & ETHANOL
Advantages of Ethanol Over Gasoline
 Exhibits a higher octane number which enables engine to have higher
compression
 Oxygenated fuel that contains 35% oxygen
 Reduced particulate and NOx emission from combustion
 Ethanol based on fermentation produces no net increase in carbon dioxide in
atmosphere
 Octane enhancing additive
 Removes free water which can plug fuel lines in cold climates
 Broader flammability limits
 Higher flame speeds
 Higher heat of vaporization
 Lower volatility and photochemical reactivity (Reduced smog formation)
 Lower toxicity compared to gasoline
Source: Srivastava (2008)

6. Crude glycerol
Components Concentration
Glycerin >60%
Water < 20 %
Sodium Chloride < 5 %
Methanol < 1%
Ash < 5 %
Fatty Acid Ester < 5 %
Table 1.2 Physical and chemical properties of glycerol
Physical Properties Chemical Properties
Amber coloured Density of 1.22-1.24 g/m3
Grain-like odour Melting point of 18oC
Liquid state Boiling point : >130 oC
Molecular weight of 92.09 Vapour density : 3.17
Specific gravity : 1.26 Flash point : >120 oC
Raw Material
Source: Eastridge (2009)
Source: Pangliaro & Rossi (2008)
Table 1.1 Chemical composition of crude glycerol
Advantage over sugar
because of highly
reduced nature of
carbon atoms

7. Bacterial strain Condition Yield of
ethanol
Ethanol
concentration
Reference
Aerobacter
aerogenes 1033
pH 6.5, 35°C, batch
culture containing 10%
glycerol for 18h.
0.86 mol/mol
glycerol
0.54 g/l Megasanik
(1953)
Enterobacter
aeogenes HU
101
pH of 6.8, 37°C 0.8 mol /mol
purified
glycerol
0.51 g/l Ito et al
(2005)
Enterobacter
agglomerans
CNCM 1210
pH 7.0, 30°C,
20 g/L of glycerol
0.23
mol/0.05
mol glycerol
2.91
g/l
Barbirato
et al
(1997)
Klebsiella
planticola DR3
Initial pH 7.2 - 7.4, 37°C
10g/L of glycerol for 48h
30 mmol/L 2.76 g/l Jarvis et al
(1996)
Clostridium
butylicum B593
Initial pH 6.5, 35°C 0.54 mM 0.002 g/l Forsberg
(1986)
Klebsiella
pneumoniae
M5a1
pH 6.8, 37°C 34.0 + 0.4
mmol/L
1.57 g/l Lin et al
(2005)
Enterobacter
aerogenes
TISTR 1468
Crude glycerol, 30°C 0.94 mol/mol 24.5 g/l Ciptanto
(2009)

8. Kingdom Bacteria
Phylum Proteobacteria
Class Gamma Proteobacteria
Order Enterobacteriales
Family Enterobacteriaceae
Genus Enterobacter
Facultative anaerobe
Gram negative
Rod shaped
Enterobacter aerogenes TSISR 1468
Source: Microbial Library (2010)
Biochemical Pathway
Mutated pathway to maximize ethanol produced.
Insignificant amount of butanol produced.
Table 1.3 Hierarchy for Enterobacter aerogenes

9. p/s: The ratings are to the scale of 5.
Factors of Consideration Port Klang Pasir Gudang Pengerang
Availability of raw material 5 5 5
Proximity to market 5 5 4
Labor Availability 5 5 5
Transport and facilities 5 5 5
Effluent Disposal facilities 4 4 4
Product storage availability 3 3 5
Utilities (services) 5 5 4
Political and strategic considerations 4 4 5
Total 36 36 37
Table 1.3 Factors of considerations for site location
Why Pengerang?
Malaysian Federal Government and Johor State Government
give full support to develop this area.
Located in the middle of oil and gas trading route –Iskandar
Malaysia Integrated Development.
Potential in becoming major regional manufacturer of oil
refinery – attraction of investor for bioethanol plant.
Close to major trading hubs – attract investor/customer.
Availability of sufficient land 20 000 acres

10. 0
50
100
150
200
250
300
350
400
2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Bioethanol
(billion
litres)
Years
World Bioethanol Supply and Demand Capacity from Year 2013
- 2022 (Projections)
demand
supply
Source: OECD-FAO Agricultural Outlook 2013 – 2022 (2013)
Market Analysis
93
93.5
94
94.5
95
95.5
96
96.5
97
2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Bioethanol
(millions
of
litres
)
Years
Malaysia Bioethanol Supply and Demand Capacity from Year
2013 - 2022 (Projections)
supply
demand
Demand – 95.7 million litres
Supply – 95 million litres
Shortage – 0.7 million litres

11. Generation of Productin Capacity Basis
According to OECD-FAO Agricultural Outlook 2013,
Shortage = 0.7 million litres / year
= 552.44 tonnes / year
However from Biofuels Digest 2012,
Japan’s Toyo Engineering Co. in a joint venture with Glycos
Biotechnologies and Malaysian developer Bio-XCell will
build a 10,000 ton per year ethanol plant in Johor Bahru by Q2
2013. The facility that will use from crude glycerin from the
production of palm methyl ether as feedstock will expand to
30,000 tons per year by 2014.
Therefore, an average of 15,000 tonnes / year of ethanol
production will be taken as the plant production capacity basis.

12. Comparison between Batch & Continuous process
Process
Criteria
Batch Continuous
Capital cost Low High
Rate of production Low High
Raw material product Processed differently
in various pieces
equipment
Processed in
identical
fashion/equipment
Workforce More Less
Ease on automation Relatively difficult Relatively easy
Energy efficiency Large peak demand Small but continuous
loads
Down time Long Short

13. INPUT-OUTPUT STRUCTURE
CONTINUOUS PROCESS
Crude glycerol
Ammonia
Carbon dioxide
Biomass
Ethanol
Nitrogen, Oxygen
Water
Process Output Destination
Product Boiling Point at 1
atm (°C)
Class Destination
Ethanol 78.3 Main Product Main product
Biomass N/A Waste Waste treatment
Carbon Dioxide -78.5 Gas byproduct Vent
Water 100 Waste Waste water treatment
plant

14. Price of ethanol – RM 3.06 / kg (Source: OECD-FAO Agricultural Outlook 2013)
Price of glycerol – RM 0.64 / kg (Source: Petrosil Glycerin Report 2013)
-300.00
-275.00
-250.00
-225.00
-200.00
-175.00
-150.00
-125.00
-100.00
-75.00
-50.00
-25.00
0.00
25.00
50.00
75.00
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Economic
Potential
2
(RM
million/yr)
Glycerol Conversion
Economic Potential Graph Level 2 vs Glycerol Conversion
552.44 MT/yr
10, 000 MT/yr
30, 000 MT/yr
15, 000 MT/yr

15. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Yield(mol/mol
)
Conversion
Yield versus Conversion
ethanol succinic lactic acetic
1.05
0.75
Yield
(mol/mol)
1.05
Yield (g/g) 0.52
Conversion 75%
Conversion and Stoichiometry
𝐶3 𝐻5 (𝑂𝐻)3 + 0.06594 𝑁𝐻3 + 0.047775 𝑂2 →0.27475 𝐶𝐻1.78𝑂0.33𝑁0.24 + 1.05 𝐶2𝐻5𝑂𝐻 +
0.62525𝐶𝑂2 + 0.704382 𝐻2𝑂

16. Comparison Between 1 Fermenter and 2 Fermenters In Series
0
5000
10000
15000
20000
25000
30000
35000
0.27 0.49 0.63 0.74
Volume,
L
Conversion
Fermenter Volume versus Conversion 2 fermenters 1 fermenter
-40.00
-30.00
-20.00
-10.00
0.00
10.00
20.00
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
FEP
3
(RM
million/YR)
Conversion
Economic Potential Graph Level 3 Versus Conversion
1 fermenter 2 fermenters

17. Bioreactor
1 seed fermenter 2 main fermenters in series
8.9
6.7
Diameter of Fermenter, m 1.56
Height of Fermenter, m 4.67
Diameter of impeller, m 0.52
Aeration rate, vvm 0.5
Agitation, rps 0.169
Material SS304
64.77 2.47
48.58 1.85
Diameter of Fermenter,
m
3.02 1.02
Height of Fermenter, m 9.05 3.05
Diameter of impeller,
m
1.00 0.34
Aeration rate, vvm 0.5 0.5
Agitation, rps 0.083 0.225
Material SS304 SS304

18. Component Mr Ni
(kmol/h)
Fi (kg/h) No
(kmol/h)
Fo (kg/h) Ni
(kmol/h)
Fi (kg/h) No
(kmol/h)
Fo (kg/h)
Glycerol 92.09 5.05 465 1.24 113.77 51.73 4763.77 12.66 1165.56
Ammonia 17 0.25 4.27 0 0 2.51 42.75 0 0
Oxygen 32 0.09 2.92 0 0 0.91 29.23 0 0
Biomass 22.5 0 0 1.05 23.58 1.05 23.58 11.78 265.11
Ethanol 46.07 0 0 4.00 184.48 4.00 184.48 45.03 2074.42
Carbon
Dioxide
44 0 0 2.32 102.01
2.32 102.01 26.07 1147.12
Water 18 232.26 4180.73 234.95 4229.08 2614.40 47059.24 2641.92 47554.61
∑ 4652.92 4652.92 52205.07 52206.83
Mass Flow Rate(kg/h) Mass Balance Superpro simulation Error %
Total output from fermenters 52205.07 52761.08 1.05
Comparison of mass balance manual calculation with superpro simulation
Mass balance of bioreactor (fermentors)
Seed
Main
rlimiting = Nik /- αk
Glycerol flow rate,
Ng(mole/hr)
Glycerol limiting,
rg, limiting
Ammonia flow
rate,
Na(mole/hr)
Ammonia
limiting, ra,
limiting
Seed Fermenter 5049.2 5049.2 251.5 3813.8
Main Fermenter 51727.4 51727.4 2514.8 38138.0
Excess or Limiting Reactant
Mass Balance

19. IMSK
(recent)
IMSD (old)
Fm (material
construction
factor)
Fp (pressure
factor)
Fi
(installation
factor)
1512.5 280 3.75 1 1.5

20. Downstream Separation General Structure
Distillation
Column
Disk Stack
Centrifuge
Molecular
Sieve
Ethanol
Biomass
Water
Biomass
Concentrated
Ethanol(99.5%)
Water
Water
Glycerol

21. Downstream Separation
• Disc-stack
Separation of finely
dispersed particles.
Easy to operate and control
through continuous and
automatic operation.
Disc split the stream into a
large number of very thin
layers thereby improving
separation.
No filter cloth, additives or
flocculants necessary.
Source: Alfa Laval (2008)

22. Molecular sieves
Minimal labor requirement
The process is inert
The molecular sieve desiccant
material used has a very
long potential service life
Regenerable process
Source: Ethanol India (2011)

23. • 𝐾𝑐𝑒𝑛𝑡𝑟𝑖𝑓𝑢𝑔𝑒 = 1.6 65
𝑀𝑆𝐼
800
𝑄0.73𝑥
𝑅𝑀 3.18
$ 1.00
• 𝐾𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑎𝑡𝑖𝑜𝑛 𝐶𝑜𝑙𝑢𝑚𝑛 =
𝑀𝑆𝐼
280
× 4.7 × 𝐷1.55 × 𝐻 × 𝐹𝑐 ×
𝑅𝑀 3.18
$1.00
• 𝐾𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑠𝑖𝑒𝑣𝑒 =
•
𝑀𝑆𝐼
280
𝑥
7775.3
3
𝑥 𝐷𝑅
1.066
𝑥 𝐿𝑅
0.82
𝑥 0.82 + 𝐹𝑚 𝐹𝑝 𝑥 𝐹1 𝑥
𝑅𝑀 3.18
$ 1.00
• 𝐾𝑆𝑡𝑜𝑟𝑎𝑔𝑒 𝑡𝑎𝑛𝑘 = 1.97
𝑀𝑆𝐼
800
× 8800 × 0.80.55
×
𝑅𝑀 3.18
$1.00
Source: Doughlas (1988)

24. Glycerol
conversion
K centrifuge K distillation K Molecular
sieve
K storage tank K total (in
million)
0.0 164 760.42 167 833.8 755 252.2 3 386.23 1.0912
0.1 165 624.54 168 098.5 756 443.1 151 133.90 1.2413
0.2 166 491.86 168 363.2 757 634.6 221 273.00 1.3138
0.3 167 362.37 168 628.2 758 826.7 276 553.20 1.3714
0.4 168 228.79 169 025.8 760 616.2 323 962.70 1.4218
0.5 169 095.98 169 291.1 761 810.1 366 265.10 1.4665
0.6 169 959.11 169 556.6 763 004.6 404 897.60 1.5074
0.7 170 818.21 169 822.2 764 199.7 440 723.20 1.5456
0.8 171 680.53 170 087.9 765 395.6 474 309.20 1.5815
0.9 172 538.85 170 486.8 767 190.6 506 052.30 1.6163
1.0 173 311.67 170 048.8 767 190.6 536 240.30 1.6472
K Values of different unit separation

25. K Values for Different Types of Separation
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
0 0.2 0.4 0.6 0.8 1 1.2
Economy
Potential
(RM
Million/year)
Glycerol conversion
Graph of Economy Potential against Glycerol Conversion
Absorber
Storage tank
Centrifuge
Distillation

26. Comparison of Economic Potential Curve Level 3 (FEP 3)
and Level 4 (FEP 4)
-180
-160
-140
-120
-100
-80
-60
-40
-20
0
20
40
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Economy
Potential
(RM
Million/year)
Glycerol Conversion
FEP 3
FEP 4

27. • James M.Douglas. 1988. Conceptual Design of Chemical Process. McGraw-
Hill Book.
• Shalabh Srivastava. 2008. Numerical Simulation of a Direct Injection Spark
Ignition Engine Using Ethanol As Fuel (2008): 2-5.
• Renewable Fuels Association. 1981.
http://www.ethanolrfa.org/pages/philosophy.
• Saon Ray, Smita Miglani & Amrita Godlar. 2011. Ethanol Blending Policy
in India: Demand and Supply Issues. ICRIER Policy Series.
• Vishal Shah 2010. Emerging Environmental Technologies: Volume II (2010):
2-5.
• Renewable Fuels Association. 1981.
http://www.ethanolrfa.org/pages/philosophy.
• M. Wang, C.Saricks & D.Santini. 1999. Effects of Fuel Ethanol use on Fuel-
Cycle Energy and Greenhouse Gas Emission. United States Department of
Energy.
REFERENCE

28. Thank
You!!