Report on

A Comparative Study on Biomass Potential and

Lipid Profile of Micro Algal Species

This Project Report is submitted as the partial fulfillment of the degree of B.

Tech in Chemical Engineering conferred by MAKAUT in the year of 2016-2017

Under the guidance of Mr. Rajesh Ghosh

Asst. Professor, Chemical Engineering Dept., Calcutta Institute of Technology.

Submitted By :

Subham Chakraborty
 ACKNOWLEDGEMENT

I would like to express my profound gratitude and grateful indebtedness to Mr.

Rajesh Ghosh, Dept. of Chemical Engineering, Calcutta Institute of Technology
for his valuable guidance.

Furthermore, I would like to acknowledge with much appreciation, the role of

Mr. Manas Chakraborty, Dept. of Microbiology, Calcutta Institute of
Pharmaceutical Technology, who helped us in numerous ways and gave us the
permission of using all the necessary equipment and material from his lab.

I would also like to extend my sincere gratitude to Mrs.(Dr.) Mahua Ghosh,

Professor, Dept. of Chemical Technology, Calcutta University and Ms. Swarnali
Chakraborty, Dept. of Chemical Technology for their help and suggestions.

I would also like to thank Dr. B. K. Behra, Principle Scientist, Central Inland
Fisheries Research Institute, Barrackpore for his contributions to this project
and also for letting us use his research facility.

Last but not the least, I would like to thank all my team mates and all of my
teachers of Chemical Engineering Department of C.I.T.
 To Whom It May Concern
This is to certify that, Subham Chakraborty, a final year B. Tech student of the
department of Chemical Engineering, Calcutta Institute of Technology, has
successfully carried out the project entitled “A Comparative Study on Biomass
Potential and Lipid Profile of Micro Algal Species” as a part of fulfillment of his
course of four years B. Tech in Chemical Engineering in my supervision. He is very
much sincere in his work and also extremely hard working. I wish him a very
prosperous and bright future.

Date: (Mr. Rajesh Ghosh)

Supervisor

Date: (Mr. Dipankar Bhattacharyay)

Head , Department of
Chemical Enginering
BIODIESEL: BIOMASS POTENTIAL AND LIPID PROFILLING:

1. Introduction

2. Aims & objectives

3. Literature Review

4. Materials & Methods

4.1 Collection of Species

4.2 Media Preparation

4.3 Sterilization of Media

4.4 Inoculation of Sample

4.5 Incubation

4.6 Centrifuge

4.7 Study of Growth Kinetics

4.8 Lipid Extraction

4.8.1 Soxhlet Method

4.8.2 Modified- Bligh and Dryers Method

4.9 Lipid Profiling

4.10 Trans Esterification Process

5. Results and Discussion

5.1 Lipid Extaction

5.1.1 Modified Bligh and Dryer Method

5.1.2 Soxhlet Method

5.2. Comparative Study

5.2.1 Oedogonium sp.

5.2.2 Botryococcus braunii

5.3 Lipid Extraction and Trans Esterification

5.4 Economic Feasibility

6. Conclusion

7. Summary

8. References
 1. INTRODUCTION:

The rapid diminishing of fossil fuel reserves and their contribution to increased

concentrations of carbon dioxide in the atmosphere has led man to make use of

biofuels derived from plant and microalgae. Biofuels have the potential to revolutionize

energy industry and also fight against the global warming, which has brought

considerable changes over the past few decades. Biodiesel is an alternative fuel for

diesel engines that is gaining attention in the United States and India,

after reaching a considerable level of success in Europe. Its primary advantages are

that it is one

of the most renewable fuels currently available and it is also non-toxic and

biodegradable. It can

also be used directly in most diesel engines without requiring extensive engine

modifications.

Micro algae are microscopic algae, found in freshwater and marine systems, are like

sunlight driven cell factories that convert carbon dioxide to potential biofuels, foods,

feeds and high value bio actives and are also used in bioremediation techniques.

Microalgae is known to provide a wide range of biofuels, such as methane, bio

hydrogen, bio ethanol. Though the production of biofuel from microalgae is yet not

economical to compete with the cost of fossil fuels without additional support from the

government. Therefore, research is being done to make the process viable.

Recent researches such as metabolic engineering and genetic methods have been used

to develop microorganisms for high productivity, having high energy value.

Microalgae represents a group of organisms much simpler from plants, having no cell

differentiation, therefore genetic manipulations can be done to increase their oil

content.

Microalgae having suitable amounts of fatty acids along with better oil extraction

techniques can be used to decrease the overall cost production.

2. Aim and Objective

The aim of the project is comparative study of biomass potential and lipid

profiling of Botryococcus, Oedogonium and an unknown sample of

cyanobacteria.

1. Study of biomass potential obtained from Dhaniakhali pond and comparative

study of growth kinetics of the respective samples.

2. Lipid extraction from the obtained biomass using 2 methods:

a) Using soxhlet apparatus.

b) Using modern bligh and dryer method.

3. Lipid Profiling of the extract obtained from soxhlet extraction of biomass

using gas chromatography.

4.The latest technology adopted on Technical Feasibility with respect to other

article.
 3. LITERATURE REVIEW:

 Brown et al (1969) conducted the analysis of hydrocarbon content and its

relationship to physiological state in the green alga Botryococcus braunii and
concluded that three physiological states:green active state colonies, brown
resting state colonies and large green cells.
 Y Dote et al (1994) performed a test on recovery of liquid fuel from
hydrocarbon-rich microalgae by thermochemical liquefaction. A greater amount
of oil than the content of hydrocarbons in Botryococcus braunii (50 wt% db) was
obtained, in a yield of 57–64 wt% at 300 °C. The oil was equivalent in quality to
petroleum oil. The recovery of hydrocarbons was a maximum (>95%) at 300°C.
 Demirbas et al., (2011) reported in this paper that Algae containing 35-70% of
lipids is called as oilgae. Micro algae are the only source of renewable biodiesel
capable of meeting global demand for transport fuel. It is Degradable, nontoxic,
and a solution to global warming via reducing emission gases. Algae are rich in
oil and can grow in wide range of conditions. It can create fuels (with existing
feed stocks). Aim – Microalgae biofixation of Carbon-di-oxide, to operate large
scale systems that are able to convert a significant fraction of Carbon-di-oxide
output from a power plant into biofuels.
 Anoop Singh et al., (2011) the energy security under reduction in climate
change effect generates the opportunity to explore new biomass sources. Algae
sequester a significant quantity of carbon from atmosphere, industrial gases,
nutrient from industrial effluents and municipal waste water.
Algal biofuels has dual advantages-
 Supply biomass for befouls production
 Protect our environment from air and water pollution
 Algal biofuel is environmentally sound but not economically so attractive.
 Campbell et al. (2011) the following paper elucidated two things Potential
environmental consequences and economically. Cost effectiveness of the biodiesel
production from microalgae basically grew up in open ponds. Biodiesel can be
produced using three sources (i) Algae (ii) Calona (iii) ULS (ultralow sulfur) diesel.
 Comparison of greenhouse gas (GHC) emission and costs are studied. Process of
production of biodiesel using algae is attractive, but economically not so attractive.
 E.B. et al.,(2010) This article reports the results of the screening of microalgae
capable of removing nitrogen and phosphorus while accumulating lipids in
effluents from secondary domestic wastewater treatment. From the 20 strains of
microalgae tested for growth in secondary effluent from treated domestic
wastewaters, B. braunii LEM 14 produced the best combined results. B. braunii
LEM 14 showed a considerable amount of CO2 uptake (144.91 mgCO2 g-1 biomass
L-1 day-1) and the ability to accumulate lipids without the need to control nitrogen
concentration. These results indicate that B. braunii LEM 14 is a strain with
potential uses in the development of a large-scale process of wastewater tertiary
treatment that can be coupled with bio fuels production and atmospheric carbon
dioxide mitigation.
 Ahmad A.L et al., (2010) this paper presents a comparison between the use of
microalgae and palm oil as biodiesel feed stocks. It was found that microalgae are
the more sustainable source of biodiesel in terms of food security and
environmental impact compared to palm oil. Theoretically, microalgae have been
shown to be a potential source to produce third generation biodiesel because of
their many advantages as a sustainable feedstock for biodiesel production.
However their production needs more research to identify the most suitable
microalgae species and improve their oil yield. No other potential sources of
biodiesel come close to microalgae in being realistic production for biodiesel
because there were perform poorly in many environmental impacts.
 4. Materials and Methods:

4.1 COLLECTION OF SPECIES:

The species is collected from natural sources(pond).

Morphological Characterization of the unknown algae species used:

1.One loop full of given culture of algae is taken in aseptic conditions in front of

Bunsen burner.

2.Smear is prepared on the slide with the loop full of culture.

3.One drop of methylene blue dye is added on the smear.

4.The dye is kept for 1-2 min for fixation.

5.The culture stained with dye (methylene blue) is observed under microscope to note

down the morphological features.

4.2 Media preparation:

COLLECTION OF NUTRIENTS:

The nutrients are collected from CIT, CIPT, IIEST(Shibpur).

Figure 1: Microscopic View of the Algae Sample Figure 2: Dyeing with Methylene Blue

Figure 3: Compound Microscope with Sample Slide (CIPT)

HOW TO PREPARE THE MEDIA?

The nutrients are taken as mentioned in the following table. Then the nutrients are

mixed with 100 ml distilled water. Then we prepare a homogeneous solution which is

further used as a culture media.

The soil media is prepared by adding soil sample to distilled water. Then the solution

is filtered and finally two types of media is prepared.

Sl. No. Chemical Name g/100ml H2O

1. Na2EDTA 0.74

2. Ammonium Molybdate 0.61

3. NaHCO3 0.82

4. Boric Acid 0.27

5. CuSO4 0.25

6. NaNO3 0.77

7. MgSO4 0.43

8. KCl 0.41

9. KI 0.67

10. Cholesterol 0.10

11. FeCl3 0.72

12. Zinc Acetate 0.42

Table 1: Nutrients for Culturing of Unknown Sample

4.3 STERILIZATION OF MEDIA:

The media is then sterilized in an autoclave. This sterilization is moist heat

sterilization. The required temperature is 121 celcius and when the autoclaves

pressure reaches to 15 lb/sq inch then we have to sterilize for 15 min. And with this 8

test tubes,the media is also sterilized.

4.4 INNOCULATION OF SAMPLE:

The term inoculation means introduction of culture into a given nutrient media for

subsequent incubation at 20-30 deg Celsius for 4-5 days for algal growth.

After sterilizing the salt media and soil media are taken in 4 test tubes each.5 ml of

media is taken in each test tube.Then the species sample are taken in an aseptic

chamber there the innoculum is added to the media.

4.5 INCUBATION:

The test tubes (plugged by cotton) are then taken into incubator where the samples

are left for 5 days at 20-30° Celsius temperature. The species are incubated.

4.6 CENTRIFUGE:

Four equal volumes of samples are taken and is centrifuged so as to get the solid mass

of algae separated from the media liquid. The centrifuge works at 100 rpm for 20 min.
After centrifuging the weight are taken to compare the two biomasses obtained from

different media i.e soil and nutrient media .The weight is taken after regular intervals.

4.7 STUDY OF GROWTH KINETICS:

Algal growth is the division or increase in number of cells i.e the increase in biomass.

The algal growth can be divided into 4 phases:

Phase 1: Lag or Acclimatization phase: In the beginning of the growth curve the growth

of cells is not much and the number of cells remain constant. It is the preparatory

time needed to sense the different nutrient sources available and develop new

machinery for undergoing cell division (also known as the halting time).

Phase 2: Log or Tropho phase: Once the algal cells are adapted to the new

environment they start growing rapidly, shown but the higher optical density results

using spectrophotometer.1 cell->2 cells->4 cells->8 cells->16 cells is the growth

pattern in the log phase and the number of cells after every division is 2^n, where n

signifies the number of divisions. Log or the exponential phase is divided into early

and late log phase. The late log phase is significantly an important phase for bacteria

produces important by products using different nutrient available.

Generation time (G):is the time taken for one cell to divide into two.

G=t/n
G=t/n
However, this equation is not valid for the log phase and have to be changed into:

𝑡
G=
(𝑙𝑜𝑔𝑛1 −𝑙𝑜𝑔𝑛2 )

Where ‘t’ is the total time taken for division, and ‘n’ is the number of bacterial change.

Specific growth rate: is the number of cell division of each cell per unit time.

Phase 3: Stationary or idiophase: It is the horizontal line after the log phase, since the

environment is same even after so many divisions, there is a shortage of nutrients.

Due to this limiting condition algae reaches a stationary and stabilizing growth and

another fact id the accumulation of oxygen (released during photosynthesis) and other

byproducts produced by the algae becomes toxic the algae itself.

Phase 4: Death phase: Where functionality of the cells decreases.

Figure 4: Autoclave Figure 5: Media and Algae sample

Figure 6: Centrifuge Figure 7: Inoculated Algae

Figure 8: The Cell Growth Phases

Figure 9: The Cell Cycle

4.8 LIPID EXTRACTION:

The lipid was extracted by two methods:

4.8.1 Soxhlet apparatus:

Description

A Soxhlet Extractor has three main sections: A percolator, boiler and reflux, which

circulates the solvent, a thimble (usually made of thick filter paper) which retains the

solid to be laved, and a siphon mechanism, which periodically empties the thimble.

Assembly

1. The biomass sample to be extracted is placed inside the thimble.

2. The thimble which consists of filter paper is loaded into the main chamber of

the Soxhlet extractor.

3. 20 ml hexane to be used as the solvent for the extraction is placed in a

distillation flask.

4. The flask is placed on the heating element.

5. The Soxhlet extractor is placed atop the flask.

6. A reflux condenser is placed atop the extractor.

Operation

The solvent hexane is heated to reflux. The solvent vapor travels up a distillation arm,

and floods into the chamber housing the thimble of biomass. The condenser ensures

that any solvent vapor cools, and drips back down into the chamber housing the semi-

solid biomass. The chamber containing the semi- solid biomass slowly fills with warm

solvent. Some of the desired lipid dissolves in the warm solvent. When the Soxhlet

chamber is almost full, the chamber is emptied by the siphon. The solvent is returned

to the distillation flask. The thimble ensures that the rapid motion of the solvent does

not transport any solid material to the still pot. This cycle may be allowed to repeat

many times, over hours or days. The soxhlet apparatus is placed in a water bath or

flameless heater.

During each cycle, a portion of the non-volatile compound dissolves in the solvent.

After many cycles the desired compound is concentrated in the distillation flask. The

advantage of this system is that instead of many portions of warm solvent being

passed through the sample, just one batch of solvent is recycled.

After extraction the solvent is removed, typically by means of a vacuum evaporator,

yielding the extracted compound. The non-soluble portion of the extracted solid

remains in the thimble, and is usually discarded.

 4.8.2 Modified Bligh and Dryer method:

At first the biomass sample was dry pressed followed by hot air oven drying at a

temperature of 105° Celsius for 1 hour.

The Petri dish was then weighed with and without the sample and a 250 ml conical

flask was weighed to store the extract.

The sample was transferred in a homogenizer using solvent, which is a mixture of

chloroform and methanol used in the following ratios:

1)1:2

2)1:1

3)2:1

The solvent is used for 3 times for each of the ratios,

First the 1:2 ratios are used, and using the homogenizer the mixture was homogenized

uniformly. then it was transferred equally into 4 test tubes. Then the test tubes were

centrifuged at 100 rpm for 20 min, so as to separate the clear liquid extract from the

algal biomass and the extract was poured into the flask. The procedure was repeated

two more times followed by the next batch using 1:1 ratio of solvent. Then the

extracted lipid was filtered through sodium sulphate placed in a filter paper. The

collected extract was then finally vacuum dried using vacuum evaporator.
Figure 10: Soxhlet Extraction Apparatus Figure 11: Homogenizer (For Bligh and Dryer)
4.9 Lipid profiling:

The fatty acid composition of the extracted lipid is analyzed using gas

chromatography. Fatty acid methyl esters(FAMES) are prepared following the AOCS

method Ce 2-66(AOCS,1989e). Analysis of fatty acids is performed using a gas

chromatography (Agilent 6890N) equipped with a DB-Wax capillary column (30 m x

0.32 mm x 0.25 μm) and a flame ionization detector, according to the AOCS method

Ce 1e-91. N2, H2 and air flow rate was maintained at 1,30 and 300 ml/min

respectively. Inlet and detector temperatures are kept at 250° Celsius and the oven

temperature is programmed to increase from 150 to 190° Celsius at a rate of 15°

Celsius/min, then to hold for 5 min and then it is increased to 230° Celsius at a rate

of 4° Celsius/min and hold it for 10 min.

4.10 TRANS ESTERFICATION PROCESS:

Chemically it is a mixture of fatty acids methyl esters(FAMES). Microalgae has high

ability to accumulate lipids and has photosynthetic rate of 3-8% in comparison to

0.5% of that of terrestrial plants.

The lipids produced by micro algae can be grouped into two categories, storage

lipids(non-polar lipids) and structural lipids(polar lipids).Storage lipids are in the form

of TAG’s, which are predominantly made of saturated fatty acids and some

unsaturated fatty acids, which can be transesterified to produce biodiesel. Structural

lipids typically have a high content of poly unsaturated fatty acids(PUFA). TAG’s are

metabolically active and act as reservoir for specific fatty acids.

The relationship for prediction of biodiesel from fats and oils.

100 lbs of oil + 10 lbs of methanol → 100 lbs of biodiesel + 10 lbs of glycerol

This equation is a simplified form of the following transesterfication reaction.

triglyceride methanol mixture of fatty esters glycerol

Triglyceride + Methanol -> Mixture of fatty esters + Glycerol

CH2-COO-R1 CH3-COO-R1 CH20H

CH-COO-R2 + 3CH3OH + Catalyst -> CH3-COO-R2 + CHOH

CH2-COO-R3 CH3-COO-R3 CH2OH

Figure 3. Transesterification Reaction

where R1, R2, and R3 are long chains of carbons and hydrogen atoms, sometimes

called fatty acid

chains.
There are five types of chains that are common in soybean oil and animal fats (others

are

present in small amounts:

1) Palmitic: R = - (CH2)14 – CH3 16 carbons, (including the one that

R is attached to.) (16:0)

2) Stearic: R = - (CH2)16 – CH3 18 carbons, 0 double bonds (18:0)

3) Oleic: R = - (CH2)7 CH=CH(CH2)7CH3 18 carbons, 1 double bond (18:1)

4) Linoleic: R = - (CH2)7 CH=CH-CH2-CH=CH(CH2)4CH3

18 carbons, 2 double bonds (18:2)

5) Linolenic: R = - (CH2)7 CH=CH-CH2-CH=CH-CH2-CH=CH-CH2-CH3

18 carbons, 3 double bonds (18:3)

These chains are designated by two numbers separated by a colon. The first number

designates the number of carbon atoms in the chain and the second number

designates the number of double bonds. Note that the number of carbon atoms

includes the carbon that is double bonded to the oxygen atom at one end of the fatty

acid (called the carboxylic carbon). This is the end that the methanol attaches to when

methyl esters are produced.

 5. Result and Discussion:

5.1 RESULTS OF LIPID EXTRACTION:

5.1.1 Modified Bligh and dryer method:

Day 1 experiment:

Weight of the canister=13.28gms

Weight of biomass the sample=2.37gms

Weight of the canister +biomass sample after hot air oven drying=13.621gms

Therefore, weight of the dry biomass sample =13.621-13.28 = 0.34gms

% weight of dry biomass sample = (0.34/2.37) *100=14.34% ... (1)

Weight of the conical flask = 114.1 gms

Weight of the extracted lipid + solvent = 7.14 gm

After vacuum evaporation weight of the lipid was = 0.33 gm

5.1.2 Soxhlet method:

Weight of the dry biomass sample = 19.044 gm

Weight of filter paper + cotton + thread = 2.458 gm

Weight of filter paper + cotton + thread + sample = 13.846 gm

Weight of conical flask = 224.18 gm

Weight of the residue oil + conical flask = 225.18 gm

Therefore, weight of the extracted oil = 1 gm

Yield was 1 gm of oil from 19.044 gm of sample.

Therefore % yield in wet basis = (1/19.044) * 100 = 5.25%

From (1) Moisture in the sample = (19.044 * 14.34)/100 = 2.73 gm

Therefore, dry sample = 19.044-2.73 = 16.314 gm

And % yield in dry basis = (1/16.314) * 100 = 6.129%

Fatty acid composition:

1) Take a little sample with the help of a capillary tube.

2) Add diethyl ether and shake.

3) Add 0.5(N) methanolic KOH and shake.

4) Add 1 ml of BF3.CH3OH and do a vapor distillation for minutes.

5) Then adding 2 drops of petroleum ether and it generates 2 layers.

6) Separate the first clean layer and boil until the solvent evaporates.
5.2 Comparative study:

The species used:

5.2.1 Oedogonium sp.:

Figure 12: Oedogonium sp.

Oedogonium is a genus of filamentous green algae, with unbranched filaments

that are one cell thick. Oedogonium can be free-floating, though it is usually

attached to aquatic plant by a holdfast. It appears greenish and inhabits

calm, fresh Oedogonium can reproduce asexually by fragmentation of the

 filaments, through some other types of non-motile spores, and also through

zoospores, which have many flagella. These develop in a zoosporangium cell,

one zoospore per zoosporangium. After settling and losing its flagella, a

zoospore grows into a filament.[ water. The life cycle of Oedogonium is haplontic,

i.e., meiosis is zygotic. Antheridia which produce sperm, and oogonia which

produce an egg, release the sperm and egg. The egg and sperm then fuse and

form a zygote which is diploid (2n). The zygote then produces the filamentous

green alga which is haploid (1n).

5.2.2 Botryococcus braunii:

Figure 13: Botryococcus braunii

Botryococcus braunii (Bb) is a green, pyramid-shaped planktonic microalga that is of

potentially great importance in the field of biotechnology. Colonies held together by a

lipid biofilm matrix can be found in temperate or tropical oligotrophic lakes and

estuaries, and will bloom when in the presence of elevated levels of dissolved inorganic

phosphorus. The species is notable for its ability to produce high amounts

of hydrocarbons, especially oils in the form of Triterpenes, that are typically around

30–40% of their dry weight. Compared to other green algae species it has a relatively

thick cell wall that is accumulated from previous cellular divisions; making extraction

of cytoplasmic components rather difficult. Fortunately, much of the useful

hydrocarbon oil is outside of the cell.

5.3 Lipid Extraction and transesterification

Our main aim was to extract the lipid content by the extraction method to

produce the biodiesel

There are different methods such as:

 Soxhlet method

 Modified Bligh and Dryer method

Soxhlet method

We used soxhlet apparatus for lipid extraction process the apparatus could

accommodate 12gms of Oedogonium Sp. On which experiment was conducted for 18

hrs based on the standard protocol. We removed the content from soxhlet apparatus

after 18 hrs. In this process 12gms of Oedogonium sp of dry weight was used and the

lipid extracted was 0.32gms & efficiency of 33.33% was obtained. Oedogonium sp.
contains approximately 8% of lipid dry weight. Biomass may contain more lipids

reproduced by the addition of chloroform and recovered for use.

12 gram of sample was accurately weighed and transferred to extraction tube. The

soxhlet flasks were dried in the oven at 105ºC for an hour and then cooled in the

desiccators for 30min and tarred with and without the stopper. The sample was

covered with cotton wool in the extraction tube and placed the tube in the soxhlet

apparatus with a tarred flask. Approx. 100 ml chloroform was added to the soxhlet

flask, the sample was extracted over night with a condensation rate of 2-3 drops per

second. The soxhlet flask was weighed with glass stopper again. The chloroform was

distilled off; the flask was dried in the oven for 2 hours at 105±1ºC. The soxhlet flask

was cooled in the desiccators and weighed. The two reaction tubes were subjected to

methylation procedure.

12g dry weight used; 0.32g of lipid extracted;8% lipid content = (0.96g) (0.32 X

100)/0.96 = 33.33% efficient

Figure 14: Extraction using Soxhlet Method

5.3.1 Modified Bligh and Dyer Method:

For Oedogonium:

We obtained 0.083g of lipid extract by this method.

25 g wet weight = 2.1g dry weight

0.083 g of lipid is extracted

From literature: 8% lipid content is present in Oedogonium sp. = 0.96g

Lipid extraction efficiency: (0.083 X 100)/ 0.96 = 51.83%.

After calculating, the efficiency was found to be 51.83%. Hence by observing the

results of both Soxhlet and Modified Bligh & Dyer methods, the modified Bligh & dyer

method is more efficient.

The above study shows that 12gms of Oedogonium sp of dry weight was used and the

lipid extracted was 0.32gms & efficiency of 33.33% was obtained. Oedogonium sp.

contains approximately 8% of lipid dry weight. Whereas we obtained about 1 gm of

lipid from 19.044 gm of the unknown specie used, containing 6.129% of lipid in dry

basis.

For Botryoccoccus:

To 1.797 gram dry weight of sample 40 ml methanol and 20 ml chloroform was added

and the sample was homogenized for 10 min on the homogenizer (motar and pestle).

To the mixture was then added Additional 20 ml chloroform and 20 ml water was
added and homogenized using ultra sonication for another 10 mins. The sample was

filtered through black band filter paper (150 mm, Whatman, Dassel, Germany) in a

glass funnel. The solvent was collected in a cylinder. The funnel was covered with a

watch glass to reduce solvent evaporation during the filtration. After the filtration, the

solvent from the cylinder was allowed to settle in separating funnel and after about 15

mins two layers were seen one the dark green biomass rich chloroform layer at the

bottom and one clear methanol/ water layer on top. The bottom layer was transferred

into the original flask from the separating funnel, 20ml chloroform was added into the

flask. After homogenization for 5 min the sample was filtered again and the solvent

extract mixture was collected in the same cylinder. The methanol water layer was

discarded.

Treatment of extract (chloroform phase):

The chloroform phase in the conical flask was transferred to a tarred tray for

evaporation using a volumetric pipette. The solvent was evaporated under an infrared

lamp at 550- 600 Celsius. After the tray had cooled down, the flask with biomass was

weighed. Initially empty weight of the conical flask was noted down. The difference in

the two weights gives the weight of biomass extracted.

Treatment of water phase:

Treatment of water phase was neglected due to negligible amount of biomass.

5.4 Economic feasibility:

a. Cost in Rs.

1. Procurement of Seedling (7 kg-Culture ) 500

2. Transport (Rail & Road) 100

3. Preservation (Refrigerated) 200

4. Culture development (Lab scale & Bench Scale) 750

5. Growth of culture (Nutrients) 50

6. Collection and transfer of grown culture (Same lab) 0

7. Cost of light, fan and electric consumption(Electric power) 150

8. Analysis of culture

 Isolation of friendly culture (active species) 1000

9. Equipment cost

 Media cost 500

 Petri dish (8 nos.) 400

 Incubation cost 100

 Heating cost (Electric power) -15 kwh 100

10. Lipid extraction cost

 Soxhlate extractor 1000

 Flasks and beaker 200

 Solvent 100

 MISC. 500

11. Quality of lipid per unit culture 450

 12. Labor cost 100

13. Infrastructure including testing from other lab 12000

Total cost 22,200

----------------------------------------------------------------------------------------------------------

B.

1. Sale value of lipid 20000

2. Sale value of biomass 10000

3. Sale of waste biomass 10000

----------------------------------------------------------------------------------------------------------

Total value receivable 40000

Gross profit= B-A= Rs. 17,800

Compound Interest (I) = 20,000(1+12/100)3 for 3 years [ having interest =12%]

= P (1 + r / 100) n

= 28098.56 in Rs.
Net profit=G.P-(Interest+ Principal payable+ Taxes) Taxes 5% of net sale value

= 17,800 – (9366.186 +2,000)

= Rs. 6433.82

R. O. I=Net profit/ Total cost

= 3,256 /22,200 x100

= 14.667

Pay Back period

Year Net Cash flows Cumulative Cash

flow

1st 0 0

2nd +6.433.82 +6,433.82

3rd +6,433.82 +12,867.64

4th +6,433.82 +19,301.46

5th +3,256 +25,735.28

Loan amount from Bank = Rs. 20,000.00

Pay-back period over 7 years = Rs. 19,301.46.00

Rs. 698.54

Rs. 6,433.82 in 12 months

1 month = Rs. 6433.82/12= Rs. 536.1516

Rs. 698.54 = Rs. 536.151 + Rs. 162.39

Hence pay-back period = 3 years 1.2 months

Thus for economical feasibility studies it appears that for 1 kg lipid per day the pay-

back period is 7 yrs 2 months. But with increase in production capacity from 1 kg to

10 kg per day, the costs at ‘a’ will be drastically reduced in labor cost , material and

equipment utilization costs to almost half and thus the cost factor of ‘a’ will become

multiplied by 10.

Thus for 10 kg lipid production per day

The gross profit = 4,00,000-17,800 x 10 = Rs. 2,22,000

 6. Conclusion

Foreign techniques:

1) OMEGA (offshore membrane enclosure for growing algae).

2) Green triangles of microalgae through which flue gas rich in nitrous oxide is passed.

3) Bioprocess of algae integrated with corn ethanol production in Ihowa.

With 33% efficiency oedogonium and bottyroccocus algae have proved to have more

lipid content than the unknown species collected from Dhaniakhali.

The future scopes of our work would involve charachterization of other algal species

and make comparisons with the oedogonium and bottyrrococcus species.And thus

find species with highest lipid content.

The biodiesel produced cost about half of what diesel costs and can be used with little

or no modification in the vehicular engines since they have similar chemical

structures.

In order for biodiesel to become a major due source:

1) The technology involved in the manufacturing process must be improved.

2) More land space will need to be allocated to grow and manufacture biofuels.

3) The cost of manufacturing the fuel will need to become significantly lower in order

to compete with fossil fuels.

4) All engines will need to be converted to diesel engines.

The process of manufacturing biodiesel may involve high initial cost but if it is

integrated with other processes such as corn ethanol plant and production of green

manure from the used algal biomass and cow dung or organic waste. The solution to

making biodiesel an alternative solution to the existing fossil fuels will be integrated

solutions with marine aquaculture, tapping of wind energy and tidal energy.

Biodiesel being a renewable source of energy has great scope of future work. With

time and innovations, it will be used to solve a great range of problems from absorbing

flue gas, rich in poisonous nitrous oxide gas to production of green manure.
 7. Summary

The rapid diminishing of fossil fuel has led to the research of alternative sources.

Biodiesel is a revolutionary renewable source of energy, derived from plants and

microalgae.

Biodiesel having similar structure as diesel can be used in diesel engine with little or

no modification.

Microalgae having fair amount of lipid can be solvent extracted followed by

transesterfication process.

In this project we have prepared two media, one of soil media and the other of nutrient

media for growth of microalgae, followed by sterilization, inoculation and incubation.

Centrifugation process was carried after that to settle down the solid biomass from the

liquid media. The oil was then extracted from the biomass using soxhlet and modified

bligh and dryer method. The unknown sample was found to have 6 % lipid on dry

weight basis.

Then the lipid collected was run through gas chromatography and it was found that

the lipid was a mixture of oleic, stearic, palmitic, linoleic, linolenic acid.

The lipid was then trans esterified using methanol in 1:1 ratio and biodiesel as the

product along with glycerol as the byproduct was obtained.

The project here undertaken proved that the unknown sample collected from the dried

fields of Dhaniakhali have proved to be a good source of lipid but contains less lipid

when compared to botyrococcus and oedogonium species.

 8. References

[1] A.C. Brown, B. A. Knights (1969) Hydrocarbon content and its relationship to
physiological state in the green alga Botryococcus braunii, Phytochemistry, 8 (3): 543-
547.

[2] Y Dote, S Sawayama, S Inoue, T Minowa, S Yokoyama (1994) Recovery of liquid fuel

from hydrocarbon-rich microalgae by thermochemical liquefaction, 73 (12):1855-

1857.

[3] Ayhans Demirbas et al.,( 2011) Bio diesel from oilgae, biofixation of carbon dioxide

by microalgae: A solution to pollution problems17(6):551-556 vol. 88, issue 10, pages

3541-3547

[4] Anoop Singh (2011), Key issues to consider in microalgae based biodiesel
production Volume 29(1):687-700.

[5] Peter K. Campbell et al. (2011) Greenhouse gas sequestration by algae-Energy and

greenhouse gas life cycle studies 102(1):50-6.

[6] Screening of microalgae with potential for biodiesel production and nutrient removal from
treated domestic sewage/Sydney E.B. et al.,, Applied Energy
DOI: 10.1016/j.apenergy.2010.11.024 Volume 88, Issue 10, October 2011, Pages 3291–3294

[7] Microalgae as a sustainable energy source for biodiesel production:

A review/Ahmad A.L et al., Renewable and Sustainable Energy Reviews 2010 15
(2011) 584–593.

[8] Microalgae for biodiesel production and other applications: A review Teresa M.

Mata et al., Renewable and Sustainable Energy Reviews 14 (2010) 217–232.