Project Report on

“INFLUENCE OF DIFFERENT NITROGEN, PHOSPHORUS AND

CARBON SOURCES ON GROWTH OF Chlorella vulgaris AND
PROTEIN ESTIMATION”

TO BE SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE AWARD OF THE DEGREE OF

BACHELORS OF TECHNOLOGY
IN
BIOTECHNOLOGY

Submitted by:

RAHUL CHOUDHARY
2K15/BT/020
Under the supervision of

Dr. NAVNEETA BHARADVAJA

(ASSISTANT PROFESSOR)

Department of Biotechnology

Delhi Technological University

(Formerly Delhi College of Engineering)

 Delhi Technological University
Formerly Delhi College of Engineering
Bawana Road, Delhi-110042

CANDIDATE’S DECLARATION

I, RAHUL CHOUDHARY, 2K15/BT/020 student of B.Tech BioTechnology,

hereby declare that the project Dissertation titled “INFLUENCE OF

DIFFERENT NITROGEN, PHOSPHORUS AND CARBON SOURCES ON

GROWTH OF Chlorella vulgaris AND PROTEIN ESTIMATION” which is

submitted by me to the Department of Biotechnology, Delhi Technological

University, Delhi in partial fulfillment of the requirement for the award of the

degree of BACHELOR of Technology, is original and not copied from any source

without paper citation. The work has not previously formed the basis for the

award of any Degree, Diploma Associateship, Fellowship or other similar title or

recognition.

Place: Delhi RAHUL CHOUDHARY

 Department of Biotechnology
DELHI TECHNOLOGICAL UNIVERSITY
(Formerly Delhi College of Engineering)
Bawana Road, Delhi-110042

CERTIFICATE

I hereby certify that the Project Dissertation titled “INFLUENCE OF

DIFFERENT NITROGEN, PHOSPHORUS AND CARBON SOURCES ON

GROWTH OF Chlorella vulgaris AND PROTEIN ESTIMATION” which is

submitted by RAHUL CHOUDHARY, 2K15/BT/020, Department of

Biotechnology, Delhi Technological University, Delhi in partial fulfillment of the

requirement for the award of the degree of Bachelors of Technology, is a record of

the project work carried out by the student under my supervision. To the best of my

knowledge this work has not been submitted in part of full for any Degree or

Diploma to this University or elsewhere.

Place: Delhi

Date: Dr. Navneeta Bharadvaja

(Assistant Professor)

3
 ACKNOWLEDGEMENT

At the time of submission of my B. Tech Dissertation, I would first like to thank

GOD for giving me patience, strength, capability, and willpower to complete my
work. Apart from our efforts, the success of this project depends largely on the
encouragement and guidelines of many others. I, therefore, take this opportunity to
express my gratitude to the people who have been instrumental in the successful
completion of this project.

My initial thank is addressed to my mentor Dr. Navneeta Bharadvaja, Assistant

Professor, Department of Biotechnology, Delhi Technological University, who gave
me this opportunity to work in a project under him. It was her enigmatic
supervision, constant encouragement and expert guidance which have enabled me to
complete this work. I humbly seize this opportunity to express my gratitude to him.

I would also like to extend my sincere gratitude to Professor Jai Gopal Sharma for
providing basic infrastructure and facilities.

I am highly indebted to Mr. Lakhan Kumar for their guidance and constant
supervision as well as for providing necessary information regarding the instruments
and experiments and also for their support in completing the reports.

I extend my thanks to technical staff Mr. Jitender Singh and Mr. Chhail Bihari who
had been an aid whenever required. At last but never the least, words are small
trophies to express my deep sense of gratitude and affection to my loving friend and
my parents who give me infinite love to go for this achievement.

Rahul Choudhary

2K15/BT/020

4
 CONTENTS

S.No. NAME PAGE No.

1 Candidate’s Declaration 2

2 Certificate 3

3 Acknowledgement 4

4 Contents 5

5 List of Figures and Tables 6

6 Abstract 8

7 Introduction 9

8 Materials and Methods 12

9 Result and Discussion 23

10 Future prospects 38

11 Conclusion 39

12 References 40

5
 LIST OF FIGURES
S.No. NAME PAGE
No.
1. Microscopic view of Chlorella vulgaris 12
2. BG11 media stock solution in 5L conical flask. 13
3. Measuring pH(7.4) in pH meter. 16
4. Chemical reaction in Lowry method. 21
5. Different conical flasks with different source and concentration 24
in BG11 media.
6. Light source setup for proper conditioning of Chlorella vulgaris 24
growth.
7. Spectrophotometer. 25
8. Falcon tubes inside the Centrifuge. 26
9. Dry biomass in Falcon tubes. 26
10. Vortexer. 27
11. Liquid biomass. 27
12. Microcentrifuge. 28
13. Pellet formation in eppendorf tube. 28
14. Eppendorf tube rack. 28
15. Sonictor 29
16. Lowry method. 30

17. Different Carbon Sources Optical Density. 31

18. Different Nitrogen Sources Optical Density. 32

19. Different Phosphorus Sources Optical Density. 33

20. Lowry Method Graph. 36

6
 LIST OF TABLES

S.No. NAME PAGE

No.
1 Different sp. Strain and their biomolecules. 10

2 Protein content (% dry weight basis), class, and kingdom of 11

different microalgae.

3 BG11 MEDIUM 14

4 Different Carbon compounds Concentration. 31

5 Different Nitrogen compounds Concentration. 32

6 Different Phosphorus compounds Concentration. 33

7 Different sources and concentration with Optical 34

Density.

7
1. ABSTRACT

Microalgae play a very important role as living food for aquatic organisms. However, out of the
80,000 species of microalgae, only 50-60 species are commercially important as living food and
for other nutritional purposes. Moreover, these microalgae are available in freshwater as well as
marine water which should be isolated first and then used for monoculture or production for use
as live food or for other marketable purposes.

Initially, microalgae drew the attention of the scientific community as a renewable source of
biofuels due to its high productivity over a short period of time and potential of substantial lipid
accumulation. The dual role of microalgae—i.e., phytoremediation coupled with energy
production—is well established, however, commercially, algal biofuel production is not yet
supportable due to high energy inputs. Efforts are being made to make the algal biofuel economy
through adjustment in the cultivation conditions, harvesting, and extraction of value added
products. Recent studies have established algal biomass production with various types of
wastewater and industrial effluents.

Different sources of water were taken like waste water from industrial effluent, lake water from
Hauzkhas Lake. The collected water samples were stored in transparent white bottles for a day
and next day the beside media preparation, culture was separated. The media used were basic
BBM media and BG11 media. The cultures were inoculated and kept under stringency. The cell
lines were repopulated and were then stored as samples to be sent for further classification and
molecular analysis to Chandigarh Centre. The whole sub-culturing process lasted for about 21
days and the untouched bottles were found to have started collected slime.

8
2. INTRODUCTION

Microalgae grow in almost every habitat in every part of the world. They can be found on very
different natural substrates, from animals such as snails, crabs, and turtles to plants such as tree
trunks, branches and leaves, aquatic plants, and microalgae, from springs and rivers to hyper
saline lagoons and salt lakes. They also colonize artificial habitats, such as dams and reservoirs,
fountains and pools, but cans, bottles, plant pots, or dishes allow algae to extend their natural
range. The ubiquity of these organisms together with the flexibility of their metabolic
requirements makes many algal species easily available for investigation, collection, or simple
observation.

Algae are aquatic, autotrophic organisms that mainly contain three important biopolymers;
protein, carbohydrates and lipids. Algae have been shown to be potential organisms for fuel
production since they have higher photosynthetic efficacy, higher biomass production, higher
lipid containing and higher growth rate. Algae can be compared to sunlight focused factories
which can produce large biomass and this biomass can be harvested throughout the year. Algae
have a marvellous capacity of fixing atmospheric CO2 into hydrocarbons which finally lead to
higher lipids and thus result in giving higher Biodiesel yields. This conversion rate is higher than
utmost of the energy crops and algae yield more oil per hectare as compared to any crop,
allowing a fast turnaround for biofuel production. Even though hydrocarbon valuation studies
have been done in some specific studies there is still a requirement of identification and isolation
of new algal cultures which could be potential biofuel producers. Thus, the present study
involves isolation and characterization of commonly found fresh water algae and their
potentiality and sustainability as biofuel fabricators.

Freshly, microalgae are common in both industrial and scientific cultivation. There are different
fields of application for microalgae includes food, biofuels, fish food and pharmaceutical
products. Recently, various applications were found for Chlorella vulgaris (C. vulgaris) such as
a health food, fish food and nutrition supplements for human consumption, as well as for lipid
and biodiesel production. Algae formed its own food by autotrophic nutrition. The food
produced is stored as proteins, carbohydrates (mostly as starch) and lipid.

9
The search for new protein sources to supplement the remaining conventional sources in order to
fill the so called “protein gap” has been the route of inspiration for many scientists for years.
Single-cell protein (SCP) refers to protein extracted from pure or mixed cultures of algae, yeast,
fungi or bacteria used as a auxiliary for the conventional protein sources exploited for human and
animal consumption. Proteins are macromolecules with a multifaceted chemical structure, and
this complexity is the basis of their multiple physiological, morphological, and technological
uses. Proteins can be used as sole protein concentrates or may be combined into processed foods.
In the latter case, each component of the processed food product plays a specific role, be it
nutritional, technological or functional. Knowledge of the techno-functional and nutritional
properties of proteins is therefore a requirement for their proper utilization in the food industry.

Sp. Strain Protein (%) Carbohydrates (%) Lipid (%)

Chlorella pyrenoidosa 57 26 2
Chlorella vulgaris 41–58 12–17 10–22
Spriogyra sp. 6–20 33–64 11–21
Spirulina maxima 60–71 13–16 6–7
Spirulina platensis 42–63 8–14 4–11
Synechoccus sp. 63 15 11
Chlorella emersonii 9.03 37.9 29.3
Chlorella zofingiensis 11.2 11.5 56.7
Chlorella FC2 IITG 10.4 24.5 37.3

Table 1 –Different sp. Strain and their biomolecules

Microalgae have been identified as one of the most reliable sources of protein and were a source
of interest to the mainstream of those involved in agricultural and food domains during the
second half of the twentieth century.

10
Microalgae have the ability to synthesize all essential amino acids within their cell, thus holds
high levels of proteins. The amino acid configuration of the algae compares positively with that
of other Proteins. Moreover, proteins also have both structural and metabolic functions, and the
cellular proteins are the major component of the photosynthetic apparatus, cell growth machinery
andCO2 fixation pathways. .Very high protein content has been stated in some of microalgae
species such as Spirulina maxima, (60%–71%), Synechoccus sp. (63%), Anabaena cylindrical
(43%–56%), and Chlorella vulgaris (41%–58%).

Table 2. Protein content (% dry weight basis), class, and kingdom of different microalgae.

Alga Protein Class Domain Reference

content (kingdom)
Anabaena 43-56 Cyanophycea Procaryota Becker, 2007
Aphanizomenon 62 Cyanophycea Procaryota Becker, 2007
Arthrospira 56-77 Cyanophyceae Procaryota Paoletti et al., 1980
Chlorella 42.2 Trebouxiophyceae Eukaryota Servaites et al. 2012
Chlorella ovalis 10.97 Trebouxiophyceae Eukaryota Slocombe et al. 2013
Chlorella 57 Trebouxiophyceae Eukaryota Becker, 2007
Chlorella spaerckii 6.87 Trebouxiophyceae Eukaryota Slocombe et al.
(Plantae)
Chlorella vulgaris 51-58 Trebouxiophyceae Eukaryota Becker,2007 Sean et
(Plantae) al., 2015
Dunaliella 12.26 Chlorophyceae Eukaryota Slocombe et al.,
Dunaliella salina 57 Chlorophyceae Eukaryota Becker, 2007
Dunaliella 11.4 Chlorophyceae Eukaryota Barbarino et al.,
Porphyridium 31.6 Porphyridiophycea Eukaryota Sean et al., 2015
Porphyridium 35 Porphyridiophycea Eukaryota Gonzàles López et
cruentum 28-39 (Plantae) al., 2010 Becker,
Scenedesmus 41.8 Chlorophyceae Eukaryota Romero et al., 2012
Scenedesmus 48 Chlorophyceae Eukaryota Gonzàles López et
obliquus 50-55 (Plantae) al., 2010 Becker,
Spirulina platensis 60-71 Cyanophyceae Procaryota 2007
Paoletti et al., 1980
55.8 (Bacteria) Sean et al., 2015
Tetraselmis 36 Prasinophyceae Eukaryota Schwenzfeier et al.,
Tetraselmis chuii 31 Prasinophyceae Eukaryota Brown, 1991 Sean et
46.5 (Plantae) al., 2015

11
3. Materials And Methods

• Sample collection

The microalgae strain used in this study was C. vulgaris. Microalgae strain of sp. Chlorella
vulgaris is taken from the Plant biotechnology lab.

The sample was transferred to the laboratory immediately and stored at room temperature- 37°C
to avoid any physical-chemical changes in the sample. Floating microalgae can be collected with
a mesh net e.g., with 25–30 μm pores or, if in sufficient quantity i.e., coloring the water, by
simply scooping a jar through the water. A small amount of the bottom sediments will also
provide many of the algal species that live in or on these sediments. Some algae live attached to
other types of substrate, such as dead leaves, twigs, and any underwater plants, which may be
growing in the water. Macroalgae and the attached microalgae can be collected by hand,
including part or all of the substrate such as rock, plant, wood, etc. if possible. Algae growing on
soil are difficult to collect and study, many requiring culturing before sufficient and suitable
material are available for identification.

12
• Labeling
Any sample should be labeled with standard information such as the locality, date of collection,
and as many of the following features as possible: whether the water is saline, brackish or fresh,
whether the collection site is terrestrial, a river, a stream, or a lake, whether the alga is
submerged during water level fluctuations or floods; whether the water is muddy or polluted,
whether the alga is free floating or attached, and if the latter, the type of substrate to which it is
attached, and the color, texture and size of the alga.

• Storage
Algae can be stored initially in a glass jar, plastic bottle or bag, or in a vial with some water from
the collecting site. The container should be left open or only half filled with liquid and wide
shallow containers are better than narrow deep jars. If refrigerated or kept on ice soon after
collecting most algae can be kept alive for short periods (a day or two). If relatively sparse in the
sample, some algae can continue to grow in an open dish stored in a cool place with reduced
light.

• Media Preparation

Fig.2 BG11 media stock solution in 5L conical flask.

Standard stock solution of BG11 was prepared for further use in large glass conical flask with a
capacity of 5 litres. A total of 4 litres of media is prepared.

13
BG11 MEDIUM

• Physiochemical condition for microalgae culture

1. Optimisation of photoperiod

Duration of photoperiod is an influential factor in the production of biomass. to study the effect of
various photoperiods on biomass productivity of Chlorella vulgaris, various cultures were
carried out with different photoperiods. the various photoperiods chosen for the study were
14:10 and 18:6 (light: dark) cycles.

14
2. Optimisation of light intensity

Light intensity plays a significant role in the culture of photosynthetic organisms. hence the
effect of this parameter on the biomass yield of C. vulgaris was studied by growing the strain
under three different light intensities viz., 2000, 6000 and 10000 lx.

As with all plants, micro-algae photosynthesize, i.e. they assimilate inorganic carbon for
conversion into organic matter. Light is the source of energy which drives this reaction and in
this regard intensity, spectral quality and photoperiod need to be considered. Light intensity
plays an important role, but the requirements vary greatly with the culture depth and the density
of the algal culture: at higher depths and cell concentrations the light intensity must be increased
to penetrate through the culture (e.g. 1,000 lux is suitable for Erlenmeyer flasks, 5,000-10,000 is
required for larger volumes). Light may be natural or supplied by fluorescent tubes. Too high
light intensity (e.g. direct sun light, small container close to artificial light) may result in photo-
inhibition. Also, overheating due to both natural and artificial illumination should be avoided.
Fluorescent tubes emitting either in the blue or the red light spectrum should be preferred as
these are the most active portions of the light spectrum for photosynthesis. The duration of
artificial illumination should be minimum 18 h of light per day, although cultivated
phytoplankton develop normally under constant illumination.

3. Temperature

The optimal temperature for phytoplankton cultures is generally between 20 and 24°C, although
this may vary with the composition of the culture medium, the species and strain cultured. Most
commonly cultured species of micro-algae tolerate temperatures between 16 and 27°C.
Temperatures lower than 16°C will slow down growth, whereas those higher than 35°C are lethal
for a number of species.

15
4. Optimisation of initial pH

Fig.3 Measuring pH(7.4) in pH meter.

Medium pH is an important factor which significantly affects the growth of the algae. the
variation in pH affects the solubility and availability of nutrients, enzyme activity, and transport
of substrates across plasma membrane and electron transport in respiration and photosynthesis.
So, pH was optimized for the microalga C. vulgaris. The growth patterns under various pH
regimes, varying from 4 to 8.But we take pH=7.4. The pH range for most cultured algal species
is between 7 and 9, with the optimum range being 8.2-8.7. Complete culture collapse due to the
disruption of many cellular processes can result from a failure to maintain an acceptable pH.

• POPULATION DYNAMICS

Batch culture systems are highly dynamic which can be described by different phases. The
different phases reflect changes in the biomass and in its environment. Algal population shows a
typical pattern of growth according to a sigmoid curve, consisting of a succession of six phases,
characterized by variations in the growth rate.

16
1. Lag phase

An initial lag or induction phase, where, just after the inoculum. In this phase the cells begin to
absorb the nutrients supplied with the culture medium and increase in size, but not in number,
and the specific growth rate is at the sub-maximum level. The growth lag could be due to the
presence of non-viable cells or spores in the inoculum or may be viable, but not in condition to
divide. The lag in growth could be also attributed to the physiological adaptation of the cell
metabolism to growth, such as the increase of the levels of enzymes and metabolites involved in
cell division and carbon fixation. Cultures inoculated with exponentially growing algae have
short lag phases, which can seriously reduce the time required for upscaling.

2. Acceleration growth phase

At the late lag phase, the cells have adjusted to the new environment and begin to grow and
multiply in accelerating growth phase.

3. Exponential phase

After the accelerating phase, the cells enter the exponential (or logarithmic) growth phase.
At this phase, cells grow and divide as a function of time according to the exponential
function:

where N2 and N1 are the number of cells at two successive times and µ is the growth rate.
During this phase, the growth rate reached is kept constant. The growth rate is mainly
dependent on algal species and cultivation parameters, such as light intensity, temperature, and
nutrient availability. Because cells start to shade each other as their concentration increases,
the culture enters the phase of retardation.

17
4. Retardation phase
In retardation phase, cell growth rate decreases because mainly light, but also nutrients, pH,
carbon dioxide, and other physical and chemical factors begin to limit growth. Following this
phase, the cell population continues to increase, but the growth rate decreases until it reaches
zero, at which point the culture enters the stationary phase.

5. The stationary phase

During which the cell concentration remains constant at its maximum value.

6. Death phase

The final stage of the culture is the death or “crash” phase, characterized by a negative growth
rate; during this phase water quality deteriorates, mainly due to catabolite accumulation, and
nutrients are depleted to a level incapable to sustain growth. Cell density decreases rapidly
and the culture eventually collapses.
In practice, culture crashes can be caused by a variety of reasons, including the depletion of
a nutrient, oxygen deficiency, overheating, pH disturbance, or contamination.
The key to the success of algal production is maintaining all cultures in the exponential phase of
growth. Also, the nutritional value of the produced algae is inferior once the culture is beyond
Phase 4 due to reduced digestibility, deficient composition, and possible production of toxic
metabolites.

• Harvesting of Microalgal Biomass

Microalgal harvesting refers to the Centrifugation of a process recovery of algal biomass by

using centrifugal force to accelerate the rate of sedimentation. This separation process is based
on algal cell size and density difference between the algal biomass and the medium. The main
advantage of this process is that it is easy to apply to all strains and able to recover/concentrate at

18
a high rate, and the harvested biomass is free from flocculants or any other chemical
contamination due to the absence of chemical addition process. Although it has some
advantages, this process is energy intensive and requires higher maintenance costs, which are
major disadvantages of this process.

• Dry and wet mass

The determination of dry weight requires cell separation, washing steps and drying to constant
weight. Representative aliquots of algal cultures are taken and the cells are separated by
membrane filtration or centrifugation. The filter membrane or centrifuge tubes should be pre-
weighed. The cells are normally washed with diluted medium or buffer several times, followed
by rinsing with distilled water. The lowest feasible drying temperature range is 60 degree celsius
to 100 degree celsius should be used to prevent loss of volatile components. Lower drying
temperature could be employed under reduced pressure (e.g. in vacuum oven).

Wet weight is measured using the same procedures described above, but without drying.
Although faster, this determination method is by far less accurate since defined water content is
seldom obtained.

• Cell Disruption by Sonicator

Ultra sound waves of frequencies greater than 20 kHz rupture the cell walls by a
phenomenon known as cavitation. The passage of ultrasound waves in a liquid medium
creates alternating areas of compression and rarefaction which change rapidly. The cavities
formed in the areas of rarefaction rapidly collapse as the area changes to one of
compression. The bubbles produced in the cavities are compressed to several thousand
atmospheres. The collapse of bubbles creates shock waves which disrupt the cell walls in the
surrounding region. The efficiency of the method depends on various factors such as the
biological condition of the cells, pH, temperature, ionic strength and time of exposure.
Ultrasonication leads to a rapid increase in the temperature and to avoid heat denaturation of
the product it is necessary to cool the medium and also to limit the time of exposure.

19
• Phosphate Buffer preparation

1. Make solutions A and B first (using a 500 ml volumetric flask).

2. Stock Solution A (0.2 M Sodium Phosphate Monobasic):

Add 13.8g NaH2PO4*H2O in 500 ml distilled water and stir well.

3. Stock Solution B (0.2 M Sodium Phosphate Dibasic):

Add 26.81g Na2HPO4*7H2O in 500 ml distilled water and stir well.

4. To make 0.1 M Phosphate Buffer, pH 7.4 (1L PB)

1 part of Solution A (100ml)

4 parts of Solution B (400ml)
5 parts of distilled water (500ml)

(0.02 M Sodium Phosphate Monobasic, 0.08 M Sodium Phosphate Dibasic)

• Lowry method

Principle:
The method combines the reactions of Cu++ with the peptide bonds under alkaline conditions
with the oxidation of aromatic protein residues.
The Lowry method based on the reaction of Cu, produced by the oxidation of peptide bonds, with
Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid). The
reaction mechanism is not well understood, but involves reduction of the Folin reagent and
oxidation of aromatic residues (mainly tryptophan, also tyrosine).

20
 Fig4. Chemical reaction in lowry method
Chemicals Required:

1. Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.

Dissolve 1gm NaOH IN 250 ml distilled water. Now dissolve 2gm of sodium carbonate
in 100 ml of 0.1N NaOH solution.

2. Reagent B: 0.5% copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartarate.

Prepare fresh by mixing stock solutions.
3. Alkaline copper solution (Reagent C): Mix 50mL of reagent A and 1 mL of reagent B
prior to use.
4. Diluted Folin’s reagent (Reagent D): Dilute Folin-Ciocalteau reagent with an equal
volume of 0.1 N NaOH
5. Standard Protein solution: Dissolve 50mg BSA in 50mL of distilled water in a
volumetric flask. Take 10mL of this stock standard and dilute to 20 mL in another flask
for working standard solution. One mL of this solution contains 500 µg protein.

The Lowry method was used to measure the protein content of the pretreated biomass. For
the control pretreatment, 20 mg aliquots of the freeze-dried biomass were suspended for
20-min in 10 mL of lysis buffer in a Falcon tube to facilitate the extraction of proteins. An
aliquot of this well-mixed suspension was diluted with the lysis buffer such that the

21
protein concentration in the diluted mixture was in the range of 0 and 1000 mg L-1.

Reagent C (1 mL) was added to the eppendorf tube. The tube was vortexed and after 10
min, 0.1 mL of Folin reagent were added. This was immediately followed by vortex
mixing. After 30 min the absorbance of the sample was measured at a wavelength
of 660 nm in a spectrophotometer. The spectrophotometer had been zeroed using a
blank that had been prepared in exactly the same way as the sample, except that it
lacked the protein solution. Interfering substances generally increase the absorbance of
the reagent blank , but this is easily addressed by preparing the blank in exactly the
same way as the sample, but without the protein, as in this study. Dilute protein
extracts of algal biomass are extremely unlikely to contain interfering substances at
concentrations that are above the acceptable threshold for the Lowry assay.
Sample processing was carried out in the dark to prevent degradation of the Folins
reagent. The spectrophotometric absorbance was converted to protein concentration
using a calibration curve established with BSA dissolved in lysis buffer. The protein
content of the biomass was calculated using the following equation:

Protein (%W/W) = C*V*D X 100

m

where C is the protein concentration (mg L-1) obtained from the calibration curve, V
is the volume (L) of the lysis buffer used to resuspend the biomass, D is the dilution
factor and m is the amount biomass (mg).

 Concentration of protein should range from 0.10-2 mg of protein per ml. Because this
method is sensitive to low concentration of protein.

 If protein concentration of sample is high (Above 500 mg/ml) then measure at 550 nm.

22
4. RESULTS AND DISCUSSION

STAGE 1:

After the media preparation poured the media in 36 Erlenmeyer flask or conical flask. For these tests,
thirty six 250 mL Erlenmeyer Flasks, each filled with 100 mL of BG11 growth media and
different concentrations of carbon, nitrogen and phosphorus were added and used as algae
growth reactors.
By selecting the inoculum from the algae strain which involves obtaining the algae strain in an
optimal state that is compatible with inoculation into the thirty six different culture source
medium flasks with different source and concentration.
The inoculated cultures were maintained at 18–25 degree Celsius with a 16:8 hrs light and
dark cycle.

Carbon sources are -

1. Glucose
2. Mannitol
3. Sodium acetate
4. Sucrose

Nitrogen sources are –

1. Potassium nitrate
2. Sodium nitrate
3. Calcium nitrate
4. Urea(CH4N2O)

Phosphorus sources are –

1. Sodium hydrogen phosphate
2. Potassium hydrogen phosphate
3. Ammonium phosphate
4. Sodium di-hydrogen phosphate

23
Fig.5 Different conical flasks with different source and concentration in BG11 media.

Fig.6 Light source setup for proper conditioning of Chlorella vulgaris growth.

24
STAGE 2:
Algae growth was monitored by measuring the Optical density (OD) of the algal medium with
spectrophotometer at a wavelength of 670 nm. Measurements were taken in gap of 3 days.

1. For OD, first take blank solution (distilled water).

2. Take 3 ml of solution from each flask by the help of 1ml micro-pipette in test tubes.
3. Take OD reading of samples in spectrophotometer.
4. Save the Data for further analysis.

Fig.7 Spectrophotometer

When using spectrophotometer for OD, first check baseline reading. For baseline reading we add
distilled water in cuvette for calibration of our instrument reading.
Always take OD from lower concentration to higher concentration in spectrophotometer for
better and correct readings.

25
STAGE 3:

On last day of OD reading, take out the solution from each flask and shift them into falcon tubes.
Then centrifuge it at 10000 rpm for 5 minutes or 12000 rpm for 2 minutes.
Harvesting process start from next day.

Fig.8 Falcon tubes inside the Centrifuge.

Fig.9 Dry biomass in Falcon tubes

26
STAGE 4:
Cell Harvesting Process

1. First take the falcon tubes which are filled with sample solution.
2. By the help of vortexer we can remove the pellet.
3. Then transfer it to microcentrifuge tubes.
4. Centrifuge it in microcentrifuge at 10000 rpm for 5 minutes.
5. Again centrifuge it. So that we have the pellet and we use biomass effectively.
6. Then dry it for a day in centrifuge tube.

STAGE 5:

Biomass Pre-treatment

For this we used to disrupt the cell by the help of sonicator, which is very essential for initial
preparative step for the purification of intracellular protein.

1. Take 2 ml of sample in 5 eppendorf tubes and microcentrifuge at 10000 rpm for 5

minutes.

27
2. Discard the supernatant and dissolve the pellet in 1ml of distilled water.
3. Transfer the eppendorf tube pellet into 50ml beaker, add 5 ml phosphate buffer in it.
4. Keep the beaker in ultrasonic wave generator tip for a time interval of 3 min half cycle.
5. Set the amplitude to 70%, controller power to 50% and frequency 25 KHz.

28
 6. After sonication, centrifuge the eppendorf tube at 10000 rpm for 5 minutes.
7. Take 3 ml of supernatant and determine the total amount of protein in sample by Lowry’s
Method.

Fig. Sonictor

STAGE 6:
Protein Estimation by Lowry’s Method
1. Use supernatant for protein estimation.
2. Preparation of BSA (Bovine Serum Albumin) stock solution.
3. Weight 50 mg of BSA, make up the volume to 50 ml by adding distilled water in it.

29
4. Dilute 10 ml BSA stock solution to make it to 20 ml by adding distilled water in it. This
is our working standard protein.
5. Add BSA standard solution to seven test tubes with different concentrations.
(0.2 ml,0.4 ml,0.6 ml,0.8 ml,1 ml,1.2 ml,1.4 ml)
6. Pipette out 0.2 ml of three different optimum samples of carbon, phosphorus and nitrogen
in other test tubes.
7. Make up the volume to 1 ml in all test tubes by adding distilled water.
8. And 1ml of distilled water as blank solution in test tube.
9. Add 5 ml of Reagent C to all test tubes.
10. After adding Reagent C incubates for 10 minutes.
11. After incubation add 0.5 ml of Reagent D to each tube and mix well.
12. Incubate the test tubes for 30 minutes in dark.
13. Measure the absorbance in spectrophotometer at 660 nm and plot the standard graph.
14. Estimate and calculate the amount of protein present in the given three samples from the
standard graph.

Fig. Lowry method.

30
 Fig.17 Different Carbon Sources Optical Density
2.5

2
Optical density at 660nn

CARBON
SOURCE
1.5
COMPOUNDS
OD1
1
OD2
OD3
0.5 OD4
OD5

0
1 2 3 4 5 6 7 8 9 10 11 12
Concentration(g/l)

COMPOUNDS Conc.(g/l)
1 0.5
GLUCOSE 2 1
3 1.25
4 0.5
MANNITOL 5 1
6 1.25
7 0.5
SODIUM
8 0.75
ACTETATE
9 0.9
10 0.5
SUCROSE 11 0.75
12 0.9

Table.4 Different Carbon compounds Concentration.

Maximum growth seen in sodium acetate (Carbon source) concentration= 0.75g/l.

31
 Fig.18 Different Nitrogen Source Optical Density
1.8

1.6
Optical Density at 660nm

1.4

1.2
OD1
1
OD2
0.8
OD3
0.6 OD4
0.4 OD5

0.2

0
1 2 3 4 5 6 7 8 9 10 11 12
Concentration(g/l)

COMPOUNDS Conc.(g/l)
1 0.1
KNO3 2 0.25
3 0.32
4 0.1
NaNO3 5 0.25
6 0.32
7 0.05
Ca(NO3) 8 0.1
9 0.15
10 0.1
UREA 11 0.25
12 0.3

Table.5 Different Nitrogen compounds Concentration.

Maximum growth seen in potassium nitrate (Nitrogen source) concentration= 0.32g/l.

32
 Fig.19 Different Phosphorus Sources Optical Density
2

1.8

1.6

1.4
Optical Density at 660nm

1.2 OD1
1 OD2

0.8 OD3

0.6 OD4
OD5
0.4

0.2

0
1 2 3 4 5 6 7 8 9 10 11 12
Concentration(g/l)

COMPOUNDS Conc.(g/l)
1 0.005
Na2HPO4 2 0.01
3 0.015
4 0.005
K2HPO4 5 0.01
6 0.015
7 0.005
(NH4)3PO4 8 0.01
9 0.015
10 0.005
NaH2PO4 11 0.01
12 0.015

Table.6 Different Phosphorus compounds Concentration.

Maximum growth seen in sodium dihydrogen phosphate(Phosphorus source) concentration=

0.015g/l.

33
Based on the data presented in Fig.18, Fig. 19 and Fig.20 it was found that at the initial stage
of the culture algae cells were being adapted to the new environmental conditions, although they
earlier stayed at low metabolism state. This led to inhibition of the cell division, which was
associated with photo inhibition, particularly during the first three days.
At the time, the optical density as well as carbon, nitrogen and phosphorous contents varied
very slightly. The next step involved the acceleration of metabolic processes. The increase in
optical density until the 6th day of the culture was relatively low.
Th e m i croal gae gi ve best growt h i n foll owi ng carbon, ni t rogen and phosph orus
co m po un ds i n our experi m ent wi th i ts opti mum concent ration.

S ou rce Con cen trati on (g/l ) O pti cal Den si ty(660 n m)

S od ium Acet at e 0.75 g/l 1.0895
P ot ass ium Nitrate 0.32 g/l 0.7922
Sodium Dihydrogen Phosphate 0.015 g/ l 0.8796

Ta bl e.7 Differen t s ou rces and con centration wi th O pti cal Dens i ty.

• Total Biomass calculation

Formula for biomass calculation is:
Total Biomass = W2-W1

W1 = weight of empty eppendorf tube.

W2 = weight of dry biomass in eppendorf tube.

Biomass Per day = Total biomass

Total no. of days

34
1. Total biomass of Sodium acetate(0.75g/l):

W1 =1.20519 g W2 =1.36167 g
Total biomass = 0.15648 g or 156.48 mg

Biomass Per day = 156.48 mg

20 days

Biomass Per day =7.824 mg/day

2. Total biomass of Potassium nitrate(0.32g/l):

W1 =1.20039 g W2 =1.22251 g
Total biomass = 0.02212 g or 22.12 mg

Biomass Per day = 22.12 mg

20 days

Biomass Per day =1.106 mg/day

3. Total biomass of Sodium dihydrogen phosphate(0.015g/l):

W1 =1.20776 g W2 =1.33197 g
Total biomass = 0.12421 g or 124.21 mg

Biomass Per day = 124.21 mg

20 days

Biomass Per day =6.2105 mg/day

35
 Fig.20 Lowry Method Graph
1.2
y = -2E-08x3 + 7E-06x2 + 0.0019x + 0.5728
R² = 0.9939 1.10423
1 1.105
1.051
0.9975 0.97
Absorbance at 660nm

0.8
0.846
0.742 0.745
0.6 0.686

0.563
0.4

0.2

0
0 50 100 150 200 250 300
BSA Concentration(ug/ml)

Protein concentration
C1=245 ug/ml
N1=96 ug/ml
P1=160ug/ml

• Calculation for Protein Percentage:

Protein % = Protein (mg) X Dilution X 100

Reagent D(ml)

36
1. For Carbon source(C1),

Protein % = 0.245 mg X 1 X 100

0.5 ml
Protein =49%

2. For Nitrogen source(N1),

Protein % = 0.096 mg X 3 X 100

0.5 ml
Protein % =57.6%

3. For Phosphorus source(P1),

Protein % = 0.160 mg X 2 X 100

0.5 ml
Protein % =64%

37
5. Future Prospects

Microalgae have tremendous potential for food, fodder, and fuel production. The holdup of the
algal technologies is the economic production of biomass due to the high periodic cost of
nutrient media required for the cultivation of microalgae and energy intensive methods of
harvesting and oil extraction. Extensive inventiveness is required in various areas of algal
technologies. The photosynthetic efficiency of microalgae can be improved by genetic,
molecular, and metabolic engineering. Improvements in the cost effective mechanical mixing of
avoiding photo-inhibition will also improve the economic production of algal biomass in open
cultivation systems.

Similarly, the existing design of photo bioreactors also needs more improvements for utilization
of solar energy and high biomass yields. The CO2 supplementation and utilization depend on
algae species and ranges from 1% to 20%, however, this process itself is energy intensive and
consequently uneconomic for pilot operations. Therefore, the way forward is to set up the algae
cultivation systems in close proximity to the industries and use flue gasses to overcome the cost
of CO2 procurement and transport. However, as the industrial flue gasses contain various other
toxic gasses, therefore, the selection of the registrant species and optimization of their growth is
required. More importantly, the CO2 diffusion in the culture is also energy intensive therefore,
this needs mechanical improvisations’. Microalgae are an excellent sequester of micronutrients
from the culture media, therefore, the requirement of the growth media and nutrients are high
which add cost to the produced biomass. The use of various types of wastewater and industrial
effluents as culture media will substantially reduce the cost of biomass production, however, this
needs elaborated research and optimization studies for commercial applications. This has the
dual advantages of wastewater treatment and biomass production. Such biomass can be used for
the production of various types of biofuels such as bioethanol, biodiesel, bio methane, and bio
hydrogen etc. Being comparatively economic, such concepts have elaborated the application of
algal technologies from wastewater Treatments to biofuels.

38
6. Conclusions
In the present study, the growth and protein content of Chlorella vulgaris was compared in
different nitrogen, phosphorus and carbon sources. The study aimed at to find species with the
potential to produce a range of useful product, particularly proteins and biofuel. In the present
study, we have identified a potential for reducing the nutrient requirements by optimizing the
growth medium as well as the concentrations of nitrogen , carbon and phosphorus.
In the past few decades, tremendous advances have been made in the field of algal technologies
for combating numerous techno-economic hurdles and improving biomass production. The
major limitations with the use of algal biomass as an alternative feedstock for biofuels are the
cost involved in its cultivation and harvesting as well as extraction of value added products. Most
of the Sustainability technologies—i.e., cultivation, harvesting, and extraction—have their own
pro and cons.

Majorly, two techniques such as photo bioreactors and open ponds are commonly used for
cultivation; however the initial cost, requirement of suitable growth media, and energy inputs
substantially add cost. Moreover, even with most advances harvesting, lipid extraction, and its
conversion to biofuels, present techniques are not economically beneficial. In the current
scenario, the major challenge of algal biofuels is not yet met due to higher production cost
compared to the low market price of fossil fuels. In this regard, the need of the day is to invent an
economical and sustainable biofuel production technique for wide acceptability. The introduction
of genetically modified microalgae for increasing the energy efficiency with the utilization
limited nutrients and avoiding field contamination with resistant genes could be an option.

Similarly, economical harvesting and ecofriendly optimum extractions also need the attention of
researchers and the scientific community. Though the economic viability of algal metabolites,
byproducts, and biofuel production depends on various human and environmental variables. The
global energy requirement in 2035 is expected to be 812 quadrillion kJ and as per estimates.
Therefore, algal biofuels could be the most prolific photosynthetic biomass as an alternative
renewable resource which can be produced with limited natural resources with the added
advantages of phytoremediation along with CO2 appropriation.

39
7. References

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freshwater and seawater media. In: Andersen, R., Burlington, A. (Eds.), Algal Culture
Techniques. Elsevier Academic Press, USA, pp. 429–539.
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mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch
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