Research proposal

BIOETHANOL PRODUCTION BY AN
ETHANOL TOLERANT Bacillus cereus
STRAIN GBPS9 USING SUGARCANE
BAGASSE AND BANANA PEELS AS
FEEDSTOCKS
NAME: MALEEHA FATIMA
SEAT NUMBER: H1531046
ENROLLMENT NUMBER: SCI/BTC/KU-45635/2015
COURSE NUMBER: 516
COURSE TITLE: RESEARCH METHODOLOGY AND
TECHNIQUIES
SUBMITTED TO: DR. RAHEELA RAHMAT ZOHRA
SUBMISSION DATE: NOVEMBER 1st
, 2018

PROFORMA FOR THE SUBMISSION OF
RESEARCH PROPOSAL
NAME AND ADDRESS OF THE INSTITUTION: DEPARTMENT OF
BIOTECHNOLOGY, UNIVERSITY OF KARACHI
TITLE OF PROPOSED PROJECT: BIOETHANOL PRODUCTION BY AN
ETHANOL TOLERANT Bacillus cereus STRAIN GBPS9 USING SUGARCANE
BAGASSE AND BANANA PEELS AS FEEDSTOCKS
FIELD OF STUDY: ENVIRONMENTAL BIOTECHNOLOGY
NATURE OF PROJECT: APPLIED RESEARCH
PROJECT INVESTIGATOR (PI): DR. RAHEELA RAHMAT ZOHRA
PROPOSED DURATION OF THE PROJECT: TWO YEARS
TOTAL FUNDS REQUESTED: 1,994,130

i
Contents
1 Abstract ............................................................................................................................. 1
2 Introduction....................................................................................................................... 2
2.1 Biofuel ........................................................................................................................ 3
2.1.1 Biodiesel.............................................................................................................. 3
2.1.2 Bioethanol........................................................................................................... 3
2.2 Generations of biofuel............................................................................................... 3
2.2.1 First generation biofuels .................................................................................... 3
2.2.2 Second generation biofuels............................................................................... 4
2.2.3 Third generation biofuels................................................................................... 4
2.2.4 Fourth generation biofuels................................................................................. 4
2.3 Bacillus cereus strain GBPS9................................................................................... 5
2.4 Bioethanol .................................................................................................................. 5
2.4.1 Uses of bioethanol.............................................................................................. 5
2.4.2 Potential feedstock for bioethanol production ................................................. 6
2.4.3 Importance of using agricultural waste for bioethanol production................. 6
2.4.4 Use of banana peels and sugarcane bagasse for bioethanol production ...... 7
2.4.5 Bioethanol market overview .............................................................................. 9
2.4.6 Possible benefits from the use of bioethanol in Pakistan ..............................11
2.5 Rationale of the project ............................................................................................12
3 Objectives.........................................................................................................................13
4 Methodology ....................................................................................................................13
5 Budget ..............................................................................................................................16
6 References .......................................................................................................................25
7 Assignment…………………………………………….………………………………………….31

ii
Tables
Table 1 Current status of banana sub-sector......................................................................... 7
Table 2 Trends in area under sugarcane, national average cane yield, sugar production
and sugar recovery in Pakistan .............................................................................................. 8
Table 3 Annual ethanol fuel production by country (2007 to 2010) in top 10
countries/regional blocks (millions of U.S liquid gallons per year) ....................................10
Table 4 Bioethanol exports of Pakistan (million gallons).....................................................10
Table 5 List of equipments available ....................................................................................16
Table 6 Estimated cost of the project....................................................................................16
Table 7 Expenditure of salaries and allowances ..................................................................17
Table 8 Expenditure of equipments and supplies ................................................................17
Table 9 List of equipments.....................................................................................................18
Table 10 List of chemicals for first year ................................................................................19
Table 11 List of chemicals for second year ..........................................................................20
Table 12 List of glassware for first year ................................................................................21
Table 13 List of glassware for second year ..........................................................................22
Table 14 List of plasticwares..................................................................................................23
Table 15 List of anyother........................................................................................................24
Figure
Figure 1 Bioethanol market segmentation.................................................................. 9

1
1 ABSTRACT
Energy plays a vital role in the development of country. However fossil fuels, source
of non-renewable energy, creates negative impacts on the environment due to release
of greenhouse gases which are very harmful and due to inevitable depletion of World’s
energy supply, there has been an increasing interest worldwide in alternative sources of
energy. Bioethanol is a sustainable energy source which serves as an alternative fossil
fuel and contributes to a clean environment. In this study, bioethanol will be produced
from sugarcane bagasse and banana peels by an ethanol tolerant Bacillus cereus strain
GBPS9. The Bacillus cereus GBPS9 strain will be isolated from agro-wastes impacted
soil. The isolate will be selected on the basis of its cellulolytic ability, tolerance to
ethanol concentration and ability to ferment sugar into ethanol. Sugarcane bagasse and
banana peels will be subjected to acid, alkali and steam explosion pre-treatments to
increase cellulose content and therefore reduce lignin content. Cultural conditions of the
bacterium will be optimized to enhance cellulase production. Bioethanol will be
produced through simultaneous saccharification and fermentation (SSF) of pre-treated
feedstocks. Bioethanol concentration will be determined using Gas Chromatography-
Mass Spectrometry (GC-MS) system. Rapid increase in the generation of agricultural
wastes is one of the environmental crises. Large quantities of agricultural waste
materials are generated annually. Due to their abundant availability and renewable
nature, bioethanol production from such substrates (banana peels and sugarcane
bagasse) appears to have immense commercial potential. Bioethanol production from
sugarcane bagasse and banana peels will be profitable and have number of benefits
including economical, environmental, geopolitical.

2
2 INTRODUCTION
The universal energy demand is expanding rapidly. This expansion is one of the
results of many critical factors such as the rapid rise in world population and
industrialization. The standard sources of this energy are nonrenewable resources such
as fossil fuels, natural gas, coal, petroleum etc. According to the International energy
agency, 80% of the World’s energy utilization is based on oil, coal and natural gas. The
World oil demand is proposed to increase by 1.6% each year. One of the great
challenges for society in the 21st
century is that fossil fuels are depleting day by day and
considered as limited and non-renewable energy. In addition, the availability of these
nonrenewable energy resources will certainly decline as a result of the increase in
energy demands and the limitations of energy resources (Sheikh, R.A. et al., 2016). The
production of oil is expected to decline in the next 10-100 years (Croockes, R.J. 2006).
Due to the use of fossil fuel many environmental problems are created because gases
from the green house are released daily. Global warming significantly increases due to
the use of fossil fuels in the form of oil, coal and natural gas (Khan, R.A. et al., 2011).
Global warming is yet a big environmental issue. The burning of fossil fuels is the
largest source of heat trapping pollution producing about two billion tons of carbon
dioxide, every year. Coal burning power plants are the biggest pollutants.
Transportation contributes in pollution due to burning of petroleum, generates about 1.7
billion of carbon dioxide emissions a year causing global warming and many other
health issues (www.nrdc.org).Global warming distress forced many nations to reach
agreement called Kyoto protocol. Pakistan endorsed the Kyoto protocol on climate
change in 2004 and weighted the scientific innovation’s potential as a way to tackle
greenhouse gases emissions. About 27% of essential energy worldwide is utilized for
transportation. The shore of petroleum products is about 40% in the energy mix in
Pakistan. Its consumption has grown sharply, dominated by gasoline and fuel oil.
Gasoline is mainly consumed by transport sector by public and private sectors.
Therefore, transportation fuels are thus promising targets for reduction in greenhouse
gas emissions. Existing requirement of oil is around 12 million tons per day with a
projection to increase to 16 million tons per day by 2030. While 30% of the worldwide oil
utilization accounts for transport; a striking 60% of the rising demand is expected till
2030 (Arshad, M. 2009).Therefore, researchers are endeavoring to protect the
environment and reducing the dependence on petroleum and nonrenewable energy
sources and find alternative means of fossil fuels through biological ways and hence
much research has been put into producing biofuels (bioethanol) (Khan, R.A. et al.,
2011).

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2.1 BIOFUEL
Biofuel is energy made from plants or from agricultural wastes. It is obtained from
renewable sources, emits less gases when burnt and causes less pollution than
fossil fuels, and have received increasing attention in the transition to a low
carbon economy.
The two main biofuels currently in universal usage are:
2.1.1 Biodiesel
Biodiesel is made from vegetable oil and rapeseed oil, or it can be formed from
previously used cooking oil and tallow (animal fat), which would otherwise be
incinerated, put in a landfill or exported.
2.1.2 Bioethanol
Bioethanol is made from carbohydrate-rich crops such as corn, sugar beet,
wheat, potatoes and a variety of other starch crops. Bioethanol can also be
derived from cellulose found in common vegetation (cellulosic ethanol) or from
agricultural wastes like fruit peels (https://helpsavenature.com).
2.2 GENERATIONS OF BIOFUEL
2.2.1 First generation biofuels
First generation biofuels are produced directly from food crops by abstracting the
oils for used in biodiesel or producing bioethanol through fermentation. Crops
such as wheat and sugar are the most widely used feedstock for bioethanol while
oil seed rape has proved a very effective crop for use in biodiesel. However, first
generation biofuels have a number of associated problems. There is much
debate over their actually benefit in reducing greenhouse gas and carbon dioxide
emissions due to the fact that some biofuels can produce negative net energy
gains, releasing more carbon in their production than their feedstock’s capture in
their growth. However, the most contentious issue with first generation biofuels is
‘fuel vs food’. As the majority of biofuels are produced directly from food crops
the rise in demand for biofuels has leads to an increase in the volumes of crops
being diverted away from the global food market. This has been blamed for the
global increase in food prices over the last couple of years
(https://energyfromwasteandwood.weebly.com).

4
2.2.2 Second generation biofuels
Second generation biofuels have been developed to overcome the limitations of
first generation biofuels. They are produced from lignocellulosic raw materials
(sustainable feedstock that cannot be used directly for food production). The
production of second-generation bioethanol uses cellulose-released sugars. To
develop this generation of bioethanol, a number of cellulose-containing
agricultural by-products, such as wood trimmings, husks, straw, bamboo,
rapeseed oil, and sawdust, are used. One of the most important sources for
bioethanol production is residues that remain after production processes, such as
rice husks, bagasse, sesame hulls, and straw. Other important agroindustrial
biomass residues are the by-products of agriculture or its related industries,
including wheat, rice straw, cotton stalk, maize cobs, coconut shells, ricehusks
and different fruit peels for example : banana peels, orange peels, mango peels
etc (Demirbas, M.F. et at., 2009).
2.2.3 Third generation biofuels
The third generation of biofuels is based on improvements in the production of
biomass. It takes advantage of specially engineered energy crops such as algae
as its energy source. The algae are cultured to act as a low-cost, high-energy
and entirely renewable feedstock. It is predicted that algae will have the potential
to produce more energy per acre than conventional crops. Algae can also be
grown using land and water unsuitable for food production, therefore reducing
the strain on already depleted water sources. A further benefit of algae based
biofuels is that the fuel can be manufactured into a wide range of fuels such as
diesel, petrol and jet fuel.
2.2.4 Fourth generation biofuels
Fourth generation biofuels are aimed at not only producing sustainable energy
but also a way of capturing and storing carbon dioxide. Biomass materials, which
have absorbed carbon dioxide while growing, are converted into fuel using the
same processes as second generation biofuels. This process differs from second
and third generation production as at all stages of production the carbon dioxide
is captured using processes such as oxy-fuel combustion. The carbon dioxide
can then be geo sequestered by storing it in old oil and gas fields or saline
aquifers. This carbon capture makes fourth generation biofuel production carbon
negative rather than simply carbon neutral, as it is ‘locks’ away more carbon than
it produces. This system not only captures and stores carbon dioxide from the
atmosphere but it also reduces carbon dioxide emissions by replacing fossil fuels
(https://energyfromwasteandwood.weebly.com).

5
2.3 Bacillus cereus STRAIN GBPS9
Bacillus cereus is a gram positive, rod-shaped, aerobic, facultatively anaerobic
and motile. It is commonly found in soil and food (https://en.m.wikipedia.org). The
Bacillus cereus strain GBPS9 is a cellulolytic bacteria and is a unique candidate for
bioethanol production. It has ability to tolerant different ethanol concentrations and to
ferment sugar into bioethanol (Yan, H. et al., 2011).
2.4 BIOETHANOL
Fermentation-derived ethanol (CH3CH2OH) or ethyl alcohol is commonly known as
bioethanol. This organic chemical is a flammable, clear and colourless liquid. It is
biodegradable and less toxic. It is a principle fuel used as a petrol substitute for street
transport vehicles. It is expected it to be one of the potential renewable biofuels in the
transport sector within next 20 years (www.esru.strath.ac.uk). Bioethanol is a clean
burning renewable resource that can be produced from fermentation of glucose rich
substrates, such as agricultural waste materials and biomasses (Yu, Z. & Zhang, H.
2004). In the many parts of World, demand for bioethanol as an alternative fuel source
has steadily increased. Biomass based fuel development technology should rapidly gain
momentum and barrier imposed earlier have to be removed for successfully attempting
the production of bioethanol at commercial level. Fermentation of starchy materials
leads to the production of bioethanol which is economical (Rai, S. & Rajput, S. 2013).
2.4.1 USES OF BIOETHANOL
Bioethanol can be used as:
 Fuel for power generation by thermal combustion.
 Fuel for fuel cells by thermochemical reaction.
 Fuel in cogeneration systems.
 Feedstock in chemical industry (www.slideshare.net)
 Solvent.
 Antifreeze.
 Germicide.
 Automotive fuel (Promon, S.A. et al., 2018).

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2.4.2 POTENTIAL FEEDSTOCK FOR BIOETHANOL PRODUCTION
Food waste is a growing problem with around 1.3 billion tons produced globally.
These food wastes are land filled which have associated environmental and societal
impacts. Agricultural waste materials are inexpensively found outside the food chain of
human in large amount and can be obtained throughout the season. These agricultural
biomasses are the potential feedstock for bioethanol production, including the cellulosic
biomass, as well as starchy agricultural waste materials (Khan, R.A. et al., 2011).
Fermentation of cellulosic biomass, molasses, vegetables peels, fruits peels etc can be
considered as an economical process for bioethanol production due to easy supply of
inexpensive raw materials (www.esru.strath.ac.uk). Organic food waste is one of the
topmost suitable material. Solid food waste from household, restaurants or food
processing industries can be used as fermentation medium for bioethanol production
(Promon, S.K. et al., 2018).
2.4.3 IMPORTANCE OF USING AGRICULTURAL WASTE FOR BIOETHANOL
PRODUCTION
It is very important for human to generate bio-energy and other bio-based
products from agricultural waste because:
 It is renewable
 Huge volumes of starchy and cellulosic waste generated from agricultural and
industries
 Low cost
 Sustainable resources
 Improving the security of energy
 Developing the economy
 Cleaning the environmental and atmosphere by the disposing of problematic
solid waste and getting wealth out of wastes
 Inexpensive
 Available throughout the season (Khan, R.A. et al., 2011).

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2.4.4 USE OF BANANA PEELS AND SUGARCANE BAGASSE FOR BIOETHANOL
PRODUCTION
The cellulosic materials are less expensive and accessible in bounty yet their
change to ethanol includes numerous steps and is therefore expensive. Under such
conditions a novel methodology is fundamental to utilize inexhaustible substrates. For
example natural product i.e. fruit peels (Singh, A.K. et al., 2014).
2.4.4.1 Banana
It is a major fruit crop of Pakistan. It is grown on 34800 hectares with production
of 154800 tons (Memon, I.N. et al., 2016). It is the second largest produced fruit
after citrus contributing about 16% of the world total fruit production (Bhatia, L. &
Paliwal, S. 2010). The advantage of this fruit is its availability round the year and
can be easily maintained and grown on less fertile land that has been degraded
by farming (Barve, A. & Tarfe, K. 2017). As the banana peel is good source of
lignin, cellulose, starch, hemicelluloses, crude protein, carbohydrates etc so it
can be used as the substrate for bioethanol production (Bhatia, L. & Paliwal, S.
2010).
Table 1 Current status of banana sub-sector
A detailed description of the Current Status of Banana Sub-Sector is given in Table 1.
Year Area(000, ha) Production(000, tons)
Pakistan Sindh % Share
of Sindh
Pakistan Sindh % Share
of Sindh
2003-04 31.6 27.5 87.02 154.0 125.7 81.62
2004-05 33.1 29.0 87.61 158.0 129.6 82.02
2005-06 32.5 29.7 91.38 163.5 134.8 82.69
2006-07 34.9 32.2 92.26 150.5 126.3 84.2
2007-08 35.5 32.9 92.67 158.0 127.0 80.37
2008-09 36.0 33.4 92.77 157.3 128.9 81.94
2009-10 34.8 32.2 92.52 154.8 127.4 82.29
2010-11 29.6 26.8 90.54 141.2 113.4 80.31

8
2011-12 32.1 28.5 88.78 160.2 133.1 83.08
2012-13 33.2 29.8 89.75 159.4 134.0 84.06
Source: Agricultural Statistics of Pakistan, Islamabad (Memon, I.N. et al., 2016).
2.4.4.2 Sugarcane
It is an important cash crop of Pakistan. About two million tonnes of sugarcane
produce per annum. Therefore large amount of waste containing starch called
sugarcane bagasse is produce. Hence bioethanol production from sugarcane
bagasse using fermentation process is profitable.
Table 2 Trends in area under sugarcane, national average cane yield, sugar
production and sugar recovery in Pakistan
Year Area
(million
ha-1
)
Cane
yield
(t ha-1
)
Sugar
production
(million
tonnes)
Sugar
recovery
(%)
1986-87 0.762 39.27 1.256 8.67
1987-88 0.841 39.25 1.743 8.59
1988-89 0.8769 42.17 1.817 8.37
1989-90 0.8543 41.55 1.828 8.92
1990-91 0.8838 40.72 1.908 8.44
1991-92 0.8798 43.4 2.296 9.25
1992-93 0.8846 43.02 2.375 8.71
1993-94 0.9628 46.14 2.90 8.49
1994-95 1.009 46.75 2.983 8.72
1995-96 0.9631 47.0 2.449 8.70

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1996-97 0.9645 43.54 2.378 8.69
1997-98 1.0562 50.3 3.555 8.64
1998-99 1.155 47.77 3.53 8.21
1999-
2000
1.0098 45.80 2.42 8.32
Pakistan Sugar Mills Association, Annual Report 2000(www.pakissan.com).
2.4.5 BIOETHANOL MARKET OVERVIEW
The global bioethanol market was valued at $5,652 million in 2015, and is
expected to reach $9,544 million by 2022, growing at a CAGR of 7.6% from 2016 to
2022.
Figure 1 Bioethanol market segmentation
(https://www.alliedmarketresearch.com/bioethanol-market )

10
Table 3 Annual ethanol fuel production by country (2007 to 2010) in top 10
countries/regional blocks (millions of U.S liquid gallons per year)
(Khan, R.A. et al., 2011).
Table 4 Bioethanol exports of Pakistan (million gallons)
Year Bioethanol
production
2003 20.55
2004 33.20
2005 40.66
2006 56.35
2007 90.94
2008 105.18
Source:USDA, 2008. (Ali, T. et al., 2012).
Country 2010 2009 2008 2007
United States 13,230.00 10,600.00 9,000.00 6,498.60
Brazil 6921.54 6,577.89 6,472.2 5,019.2
European
Union
1,176.88 1,039.52 733.60 570.30
China 541.55 541.55 501.90 486.00
Thailand - 435.20 89.80 79.20
Canada 356.63 290.59 237.70 211.30
India - 91.67 66.00 52.80
Colombia - 83.21 79.30 74.90
Australia 66.04 56.80 26.40 26.40
Other - 247.27 - -
World Total 22,946.87 19,534.993 17,335.20 13,101.7

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2.4.6 POSSIBLE BENEFITS FROM THE USE OF BIOETHANOL IN PAKISTAN
2.4.6.1 Reduction in greenhouse gas emissions
Since biofuels are not composed of hydrocarbons, they produce much
lower levels of greenhouse gases upon combustion, and thus are much less
harmful to the atmosphere. Ethanol has the lowest carbon dioxide emission
among the major transportation fuels. Thus bioethanol contribute significantly to
climate change mitigation by reducing carbon dioxide emissions.
2.4.6.2 Increased employment
Pakistan is an agricultural country therefore large amount of waste is
generated which is used to produce bioethanol as a fuel. The existing production
capacity of fuel grade ethanol in the country is 270,000 tonnes per annum which
can be easily increased to 400,000 tonnes per annum with the increase in jobs.
This results in the loss of foreign exchange and increase in employment
opportunities.
2.4.6.3 Energy security and diminished reliance on oil imports
Pakistan energy demand is expected to grow exponentially each year.
Dependence on imported fuel leaves Pakistan economy vulnerable to possible
disruption in provisions, which may result in physical hardship and financial
burdens. Therefore renewable energy biofuels can help diversify energy supply
and increase energy security, offering a favorable trade balance with saving
foreign exchange.
2.4.6.4 Good fuel properties
Due to high heat of vaporization, high octane number and low flame
temperatures, bioethanol becomes an excellent fuel for transportation.
2.4.6.5 Safer to use
The flammability limit of ethanol is higher than that of petrol and similarly
the auto-ignition temperatures of bioethanol is higher than that of petrol. Thus,
bioethanol is safer than petrol due to lower likelihood of catching fire (Arshad, M.
2009).

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2.4.6.6 No mechanical changes required
Bioethanol and biodiesel can be used in existing automobile designs with
no or minimal changes to the engine. Bioethanol is already being used in many
countries - particularly Brazil - as an additive or even a substitute to conventional
fuel.
2.4.6.7 Made from renewable resources
Biofuels are made from crops that can potentially be grown in vast
quantities and from agricultural wastes thus carry a much lesser threat of running
out than conventional fossil fuels. This also means that biofuels can be produced
and utilized on an infinitesimally shorter time scale than fossil fuels, which need
millions of years to naturally decompose and form (https://helpsavenature.com).
2.4.6.8 Non toxic
Bioethanol is biodegradable and less toxic than fossil fuels. If spill occur, it
is easily biodegraded or diluted to non toxic concentrations (https://bioethanol-
np.blogspot.com).
2.5 RATIONALE OF THE PROJECT
Use of non-renewable resources as a source of energy such as fossil fuels creates
environmental pollution. Pakistan is facing acute shortage of energy which in turn, is
adversely affecting the economic growth besides enhancing discomforts for its vast
population. The rationale of this project will to produce Bioethanol, (alternative fuel
source) from agricultural waste such as: banana peels and sugarcane bagasse will be
profitable, resulting in the reduction of agricultural wastes, environmental pollution,
foreign exchange and increase the economy of the country.

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3 OBJECTIVES
 To collect banana peels and sugarcane bagasse
 To isolate cellulolytic bacteria from agro-wastes impacted soil
 To estimate the ethanol tolerance of the selected cellulolytic bacteria (Bacillus
cereus GBPS9)
 To produce bioethanol through simultaneous saccharification and fermentation of
feedstocks
 To save fossil fuels and minimize agro-based environmental pollution through
bioethanol production using agricultural wastes.
4 METHODOLOGY
Collection of banana peels and sugarcane bagasse
Wash, dry, grind,filter,store
biomass
Chemical analysis of feedstocks
Dry matter, acid and
neutral detergent fibre
content
(Milne, T.A. et al., 1992)
Crude fibre, total ash
(Sluiter, A. et al., 2008)
Crude protein, total
carbohydrate
(Sluiter, A. et al., 2011)

14
Isolation and screening of cellulolytic bacteria
(Apun, K. et al., 2000) (Behera, B.C. et al., 2014)
Ethanol tolerance test for cellulolytic bacteria
Acid method for banana
peels and sugarcane
bagasse
(Olanbiwoninu, A.A. and
Odunfa, S.A. 2012)
Pre-treatment of feedstocks
Steam explosion
method
(Sharma, N. et al., 2007)
(Fan, L.T. 1980)
Alkali method for
sugarcane bagasse
(Olanbiwoninu, A.A.
and Odunfa, S.A.
2012)
Selection of the fermentation bacterial candidates
Inoculum development
pH 7 ,24 hour incubation
Optimization of cultural conditions for cellulase and reducing sugar production
Effect of different
temperatures
Effect of different
nitrogen sources
Effect of different pH

15
Phenotypic and biochemical characterization of selected bacteria
(Holt, J.G. 1994) (MacFaddin, J.F. 2000) (Madigan, M.T. et al., 2012)
Estimation of enzyme activity
(Bailey, M.J. et al., 1992)
Molecular identification of isolates
DNA
Extraction
PCR amplification of bacterial 16S
rRNA gene
(Yamada, R. et al., 2000)
(Katsura, K. et al., 2011)
Agarose gel
electrophoresis
Sequence analysis
(Tamura, K. et al., 2013)
SSF of pre-treated feedstocks
(Kamble, R.D. & Jadhav, A.R. 2012)
Estimation of fermentation products using GC-MS
(Lin, Y.H. et al., 2013)
Statistical analysis
ANOVA

16
5 BUDGET
Table 5 List of equipments available
S.no Equipments Available at
1 Autoclave Department of Biotechnology, KU
2 Centrifuge Department of Biotechnology, KU
3 pH meter Department of Biotechnology, KU
4 Water bath Department of Biotechnology, KU
5 Water distillation
assembly
Department of Biotechnology, KU
6 Thermocycler KIBGE
7 Gel electrophoresis
assembly
Department of Biotechnology, KU
8 Gas chromatography
Mass spectrometry
GC/MS system
ICCBS
9 Spectrophotometer Centralized Science Lab
Table 6 Estimated cost of the project
Year Recurring Non-recurring Total in Rs.
1st
1,019,128 423,396 1,442,524
2nd
551,606 - 551,606
Total 1,570,734 423,396 1,994,130

17
Table 7 Expenditure of salaries and allowances
Table 8 Expenditure of equipments and supplies
S.no Post and pay Number
of posts
1st
year 2nd
year Total in Rs.
1 Project
investigator (PI)
1 80,000 80,000 160,000
2 Research Officer
20,000 per month
1 240,000 240,000 480,000
3 Lab attendant
8000 per month
1 96,000 96,000 192,000
4 Contigency - 50,000 50,000 100,000
Total 466,000 466,000 932,000
S.no Items 1st
year 2nd
year Total in Rs.
1 Equipments 298,333 - 298,333
2 Chemicals 301,225 59,527 360,752
3 Glassware 118,123 26,079 144,202
4 Plastic ware 133,780 - 133,780
5 Others 125,063 - 125,063
TOTAL 976,524 85,601 1,062,13

18
Table 9 List of equipments
S.no Items Quantity Brand
name
Catalogue
number
Total
in Rs.
1 Balance
digital
1 China IN 11 1995
2 Juster
adjustable
(1-1001)
1 Dragon IN 24 3059
3 Juster
adjustable
(10-10001)
1 Dragon IN 25 3059
4 Juster
adjustable
(100-
100001)
1 Dragon IN 26 3059
5 Hot plate 1 Labcore IN 36 2660
6 Vortex
mixture
1 Mini lab
dancer
IN 42 15,960
7 Incubator 1 Labcore IN 57 31,920
8 Refrigerator 1 Haier HRF-
618GG
87,000
9 Rotary
shaker
incubator
1 Nanbei HNY-200B 52,500
10 Blender 1 Philips HR 2001 4399
11 Drying
oven
1 FBL Bhg-9030
A
12,500
12 Kjeldahl
analyzer
1 Biobase BKN 18,750
13 Analytical
balance
1 HOCHOICE UTP 313 5625
14 Microscope 1 Jieruier XSZ-
107BN
12,500
Total 254,986
17% GST 43,347
Grand total 298,333

19
Table 10 List of chemicals for first year
S.no Chemiclas Quantity Brand Catalogue
number
Total in Rs.
1 H2SO4 100 ml Sigma Aldrich 258105 4860
2 KMnO4 100g Sigma Aldrich 31404 3510
3 Acetone 500 ml Sigma Aldrich 179124 3138
4 HgSO4 5g Sigma Aldrich 10029 1000
5 K2SO4 250g Sigma Aldrich P0772 5251
6 Zinc 100g Sigma Aldrich 209988 2187
7 Boric Acid 500g Sigma Aldrich 31146 4428
8 NaOH 25 g Sigma Aldrich 221465 2733
9 Methyl red 25 g Sigma Aldrich 250198 6291
10 Methylene blue 100 ml Sigma Aldrich 319112 3138
11 (NH2)2 SO4 5 g Sigma Aldrich 216046 3172
12 NaNO3 250 g Sigma Aldrich 58170 6142
13 K2HPO4 100 g Sigma Aldrich P8281 3172
14 KCl 500 g Sigma Aldrich P3911 5049
15 MgSO4 25 g Sigma Aldrich 230391 6885
16 Yeast extract 50g Sigma Aldrich 70161 1971
17 Glucose 100g Sigma Aldrich G8270 3831
18 Agar 50g Sigma Aldrich 05039 1944
19 Congo red 25g Sigma Aldrich C6767 3780
20 NaCl 25g Sigma Aldrich 59888 3273
21 Ethanol 1L Sigma Aldrich 32205 21,000
22 NaH2PO4 250g Sigma Aldrich 71496 2970
23 Na2HPO4 250g Sigma Aldrich 71640 3186
24 H3PO4 100g Sigma Aldrich 79622 3273
25 Dinitrosalicyclicacid 5g Sigma Aldrich 128848 3996
26 Sodium potassium
tartarte
500g Sigma Aldrich 52377 5130
27 Sodium citrate 25g Sigma Aldrich 54641 4792
28 HCl 25ml Sigma aldrich 258148 4050
29 Casein 5g Sigma Aldrich C7906 4826
30 NH4NO3 500g Sigma Aldrich A9642 6115
31 Peptone 100g Sigma Aldrich 70176 3678
32 Urea 250g Sigma Aldrich 15604 1998
33 Crystal violet 25g Sigma Aldrich C3886 4077
34 Iodine 5g Sigma Aldrich 207772 5231
35 Potassium iodide 100g Sigma Aldrich 7935832 5724
36 Safranin 250ml Sigma Aldrich 94635 2362
37 Malachite green 100ml Sigma Aldrich 38978 4306
38 NH4H2PO4 100g Sigma Aldrich 216003 4826
39 Bromothymol blue 5g Sigma Aldrich 114413 5703

20
40 Beef extract 20g Sigma Aldrich 8320 3381
41 Hydrogen peroxide 1L mp bio 18304 4455
42 p-dimethylamino
benzaldehyde
43 Isoamylalcohol 500g Sigma Aldrich W205702 4995
44 KH2PO4 5g Sigma Aldrich PHR1330 4387
45 Phenol red 5g Sigma Aldrich P3532 4083
46 α- naphthol 10g Sigma Aldrich N1000 2673
47 Potassium
hydroxide
48 Lactose 1g Sigma Aldrich PHR1025 6885
49 Sucrose 250g Sigma Aldrich 84100 22214
50 Iron 100g Sigma Aldrich 44890 4063
51 Starch 100g Sigma Aldrich 59765 3847
52 Gelatin 100g Sigma Aldrich 48724 2936
53 Maltose 100mg Sigma Aldrich M9171 3847
54 Mannitol 1g Sigma Aldrich PHR1007 6885
55 n- propanol 500g Sigma Aldrich 34871 7020
56 Isobutanol 500g Sigma Aldrich 34867 5163
57 Acetic acid 500g Sigma Aldrich 45754 6372
58 Ethyl acetate 500g Sigma Aldrich 34858 3881
Total 257,458
17% GST 43,767
Grand total 301,225
Table 11 List of chemicals for second year
S.no Chemicals Quantity Brand name Catalogue
number
Total
in Rs.
1 Ethanol 1L Sigma Aldrich 32205 21,000
2 Agar 50g Sigma Aldrich 05039 1944
3 Pepton 100g Sigma Aldrich 70179 3678
4 Beef extract 20g mp bio 8320 3381
5 Glucose 100g Sigma Aldrich G8270 3813
6 Sucrose 250g Sigma Aldrich 84100 2214
7 Phenol red 5g Sigma Aldrich P3532 4083
8 K2HPO4 100g Sigma Aldrich P8281 3172
9 NaOH 25g Sigma Aldrich 221465 2733
10 H2SO4 100ml Sigma Aldrich 258105 4860

21
Total 50,878
17% GST 8,649
Grand total 59,527
Table 12 List of glassware for first year
S.no Glasswares Quantity Brand name Catalogue
number
Total in
Rs.
1 Burette (25ml) 3 Normax GL22 4143
2 Burette (50ml) 5 Labcare GL28 9615
3 Burette (100ml) 5 Labcare GL29 11,160
4 Beaker (50ml) 10 Normax GL3 620
8 Conical flask
(100ml)
10 Normax GL32 2330
9 Conical flask
(250ml)
10 Normax GL33 2860
10 Conical flask
(500ml)
10 Normax GL34 4210
11 Cover slip 500pcs TED PELLA 260375-1 24,090
12 Glass rod 10 Normax GL81 400
13 Measuring cylinder
(100ml)
5 Normax GL90 1225
(500ml)
5 Normax GL92 3840
(250 ml)
5 Normax GL91 2380
16 Pipette graduated
(10ml)
20 Normax GL113 2920
17 Pipette graduated
(50ml)
10 Normax GL116 6650
18 Glass petri dish 300pcs HEQIGLASS - 3750

22
19 Test tube 300pcs Normax GL156 12,000
20 Slide 500 MIcroscope
slides
142595546982 4770
21 Clear glass bottles 4 Duran 21881285 1552
22 Weighing boats 5 Labcare GL198 200
Total 100,960
17% GST 17,163
Grand total 118,123
Table 13 List of glassware for second year
S.no Glasswares Quantity Brand name Catalogue
number
Total in Rs.
2 Beaker (100 ml) 5 Normax GL4 345
3 Beaker (250 ml) 5 Normax GL5 525
4 Burette (50ml) 3 Labcare GL28 5769
5 Conical flask
(250ml)
5 Normax GL33 1430
6 Conical flask
(500ml)
3 Normax GL34 759
7 Measuring
cylinder (100ml)
2 Normax GL90 490
8 Measuring
cylinder (250ml)
3 Normax GL91 1428
9 Pipette graduated
(10ml)
5 Normax GL113 730
10 Glass petridish 200 HEQIGLASS - 2500
11 Test tubes 200 Normax GL156 8000
Total 22,286
17% GST 3,788
Grand total 26,079

23
Table 14 List of plasticwares
S.no Items Quantity Brand
name
Catalogue
number
Total
in Rs.
1 Slide box 10 Labcare PW2 2530
2 Dropper (3ml) 1000 Labcare PW13 1862
3 Dropper (5ml) 1000 Labcare PW14 2394
4 Eppendorf
tube (0.5ml)
2000 Labcare PW19 3326
5 Eppendorf
tube (1.5ml)
6 Eppendorf
tube (2ml)
7 Petriplate 500 Labcare PW32 6500
8 Test tube rack 10 Labcare PW35 3720
9 Drying rack for
test tube
10 Tip yellow (1-
200µl)
11 Tip blue (200-
1000µl)
12 Tip white (5-
10µml)
13 Tip box (10-
20µl)
5 Labcare PW51 2330
14 Tip box (100-
1000µl)
5 Labcare PW52 2330
15 Beaker (50ml) 10 Labcare PW56 1000
16 Beaker
(100ml)
17 Beaker
(500ml)
18 Beaker
(1000ml)
19 Measuring
cylinder (50ml)
20 Measuring
cylinder
(100ml)
21 Measuring
cylinder
(250ml)
30 Labcare PW77 11,970
22 Measuring 30 Labcare PW78 13,980

24
cylinder
(500ml)
23 Wash bottle 10 Labcare PW98 2330
24 Funnel 10 Labcare PW108 1660
25 Micropipette 2000 Globalroll NLD453 2500
26 Mesh sieve 10 Navector NS-300 18,750
27 Polypropylene
bags
500 Congsony - 2500
Total 114,342
17% GST 19,438
Grand total 133,780
Table 15 List of anyother
S.no Others Quantity Brand name Catalogue
number
Total in
Rs.
1 DNA
extraction kit
1 Thermofisher
scientific
4463351 90,625
2 Whatman
Qualitative
filter paper
1 box
100pcs
Sigma Aldrich WHA1004042 1,147
3 Whatman
nylon filter
1 box
100 pcs
Sigma Aldrich WHA7404001 15,120
Total 106,892
17% GST 18,171
Grand total 125,063

25
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31
7 ASSIGNMENT
 Subject of experiment: Bacillus cereus strain GBPS9
 Variable of experiment: Bioethanol production
 Experimental treatment: Simultaneous saccharification and fermentation
(SSF) of banana peels and sugarcane bagasse using B.cereus
 Investigation of the project: Investigation of my project is series. As all
the steps are inter-connected with each other and all the results depends on the
previously conducted step. For example: after the feedstock will be collected and
comminuted, chemical analysis of feedstock will occur, after that the feedstock
will pass through different pre-treatment methods.

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