1

“DESIGN OF ALGAL PHOTOBIOREACTOR USING RECYCLED PET BOTTLES”

A Dissertation-I
Submitted in the partial fulfilment of the requirement for the award of Degree of
Master of Technology in Chemical Engineering (Specialization in Computer Aided
Chemical Process Design) Submitted to

RAJIV GANDHI PROUDYOGIKI VISHWAVIDYALAYA, BHOPAL, [M.P.]

By
Rushal Ughade

Under the supervision of:

Mr. Nitesh Parmar
Asst. Prof.
(Department of chemical Engineering)

IPS ACADEMY, INDORE

INSTITUTE OF ENGINEERING & SCIENCE
DEPARTMENT OF CHEMICAL ENGINEERING
SESSION 2019-2020

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 IPS ACADEMY, INDORE
INSTITUTE OF ENGINEERING & SCIENCE
DEPARTMENT OF CHEMICAL ENGINEERING

CERTIFICATE OF APPROVAL
This dissertation entitled “Design of algal photobioreactor using recycled PET bottles” being

submitted by Rushal Ughade, Enrollment no. 0808CM18MT03 has been examined by us and is

here by approved for the award of the degree of Master of Technology ([Link].) in Chemical

Engineering with the specialization in Computer Aided Chemical Process Design for which it has

been submitted. It is understood that by this approval the under signed do not necessarily endorse

or approve any statement made, opinion expressed or conclusion drawn there in, but approve the

dissertation only for the purpose for which it has been submitted.

(Internal Examiner) (External Examiner)

Date: Date:

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 IPS ACADEMY, INDORE
INSTITUTE OF ENGINEERING & SCIENCE
DEPARTMENT OF CHEMICAL ENGINEERING

RECOMMENDATION
It is to recommended that dissertation entitled “Design of algal photobioreactor using recycled

PET bottles” is submitted to Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal (M.P.), in the

faculty of Engineering by Rushal Ughade, 0808CM18MT03, in the partial fulfillment of the

requirement of the award of the Degree of Master of Technology in Chemical Engineering

with Specialization in Computer Aided Chemical Process Design.

Mr. Nitesh Parmar Dr. Rajesh Kumar Kaushal

Supervisor Head

Dr. Archana Keerti Chowdhary

principle

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 IPS ACADEMY, INDORE
INSTITUTE OF ENGINEERING & SCIENCE
DEPARTMENT OF CHEMICAL ENGINEERING

DECLARATION
I hereby declare that the work, which is being presented in the dissertation, entitled “Design of

algal photobioreactor using recycled PET bottles” partial fulfillment of the requirements for

the award of degree of Master of Technology in Chemical Engineering IES IPS Academy,

Indore [M.P.] is an authentic record of my own work carried under the guidance of Mr. Nitesh

Parmar Asst. Professor. I have not submitted the matter embodied in this report for award of

any other degree.

I also declare that’ “A check for plagiarism has been carried out on the thesis and is found with

in the acceptable limit and report of which is enclosed herewith.

Name: Rushal Ughade

Enrolment No- 0808CM18MT03

Supervisor check for plagiarism done by

principle

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 ACKNOWLEDGEMENT

First and foremost, I express sincere gratitude towards the abiding presence and a bounding grace
of God through the execution of the project.

I consider myself as very fortunate to work under the guidance of Mr. Nitesh Parmar, Asst. Prof.
Department of Chemical Engineering, Institute of Engineering & Science, IPS Academy and I
express my sincere gratitude and obligation to him with pleasure and contentment for his
unstinted support, valuable guidance and facilities provided.

I also express my heartfelt thanks to Dr. Archana Keerti Choudhary Principal, Institute of
Engineering & Science, IPS Academy for their support and encouragement during the span of my
course and during my thesis work.

I also express my profound gratitude to Prof. Rajesh Kaushal, Head of the Department of
Chemical Engineering, Institute of Engineering & Science, IPS Academy for whole hearted co-
operation during this project work.

It gives me immense pleasure to express my special thanks to Supporting Staff of Department of

Chemical Engineering, Institute of Engineering & Science, IPS Academy, who always took keen
interest in supporting me during my work.

Finally I wish to express my wholehearted thanks to all who have helped me directly and
indirectly in the successful completion of this project.

Rushal Ughade
Enrollment No:- 0808CM18MT03

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CONTENT

LIST OF FIGURES

LIST OF ABBREVIATION

ABSTRACT

CHAPTER 1 INTRODUCTION 15

1.1 Algal photo bioreactor 15

1.2 Algal basics 16

1.3 Methods of cultivation of algae 16

1.3.1 Open pond system 17

1.3.2 Closed system 18

1.3.3 Raceway ponds 18

1.4 Types of algae photobioreactor 19

1.4.1 Plate photo bioreactor 19

1.4.2 Tubular photo bioreactor 20

1.4.3 Bubble column photo bioreactor 21

1.5 Photo bioreactors key systems 21

1.6 Requirements to develop a high-performance photo bioreactor for algal cultivation 22

1.7 Sources of CO2 22

1.8 CO2 fixation using algae 23

1.9 Objectives 23

CHAPTER 2 LITERATURE REVIEW 23

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CHAPTER 3 FACTORS AFFECTING GROWTH RATE OF ALGAE 30

3.1 Effect of temperature 30

3.2 Effect of pH 31

3.3 Effect of Nitrogen & NOx 32

3.4 Effect of Light 34

3.5 Effect of Proper Mixing 35

3.6 Effect of Culture density 35

3.7 Effect of CO2 concentration 36

3.8 Effect of O2 accumulation 36

3.9 Nutrients requirement and their effect on the growth 37

3.10 Effect of Carbon 38

3.11 Effect of phosphorous 38

3.12 Effects by some of other element’s as nutrients

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CHAPTER 4 PAST USES OF ALGAE 40

CHAPTER 5 METHODOLOGY 41

5.1 Experimental setup 41

5.2 Chemicals 41

5.3 Preparation of feed stock 41

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 5.3.1 Nutrient media 1: Ammonium Sulphate and Urea 41

5.3.2 Nutrient media 2: Minimum Salt Media (MSM) 41

5.4 Algae culture Conditions 42

5.5 Kinetic study 43

5.6 Effect of nutrient media 43

5.6.1 Nutrient media 1: Ammonium sulphate and Urea 43

5.6.2 Nutrient media 2: MSM 43

5.6.3 Growth analysis 43

CHAPTER 6 RESULTS AND DISCUSSION 43

6.1 Kinetic study analysis 43

6.2 Growth curve Phases 44

6.3 Specific growth rate determination 46

6.4 CO2 Fixation rate determination 48

CHAPTER 7 CONCLUSION AND FUTURE SCOPE 50

7.1 Algal oil production 50

7.2 Biodiesel production 52

CHAPTER 8 REFRENCES 53

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10
 LIST OF FIGURE

S. No. Figure no. Description Page no.

1 1.1 Algal photo-bioreactor 15

2 1.2 Green algae 16

3 1.3 Open pond system 17

4 1.4 Closed system 18

5 1.5 Raceway pond 19

6 1.6 Plate photo-bioreactor 20

7 1.7 Tubular photo-bioreactor 20

8 1.8 Bubble column photo-bioreactor 21

9 2.1 Comparison of biodiesel produced per unit area per year 25

10 5.1 MSM Composition 42

11 5.2 Algae Cultivation Day 1 42

12 5.3 Algae Cultivation Day 2 42

13 6.1 Biomass growth curve for Media 1 (Ammonium sulphate-Urea) 45

14 6.2 Biomass growth curve for Media 1 (MSM) 46

15 6.3 Ammonium Sulphate-Urea Graph 47

16 6.4 MSM Graph 48

17 6.5 CO2 fixation rate ammonium sulphate-urea 49

18 6.6 CO2 fixation rate MSM 49

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LIST OF ABBREVIATION

PBRs Photo-Bio Reactors

ATP Adenosine Triphosphate
NADPH Nicotinamide Adenine Dinucleotide Phosphate

TRCs Transparent Rectangular Chambers

PET Polyethylene Terephthalate
MSM Minimum Salt Media
FAME Fatty Acid Methyl Esters

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 ABSTRACT

The cultivation of algae provides better option for renewable energy production and as the source of

green products. Algae cultivation can thus contribute to a reduction of CO 2 emissions. This report

describes that systems used to cultivate algae from recycled water bottle for biofuel production. For

the better quantity of algae cultivation it required a treatment of Nutrients. In this report we discussed

about the construction of algae bioreactor from recycled water bottle and treatment of nutrients like

Ammonium sulphate, Urea which required for cultivation of Algae. Result shows that the Algae

cultivated from this Algae bioreactor from recycle water bottle is containing large amount of oil then

the Algae grow in river, wells, ponds etc.

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1. INTRODUCTION
1.1. Algal Photo bioreactor

An algal photo-bioreactor or bioreactor is used for cultivating algae on purpose to fix CO2 or
produce biomass. Specifically, algal bioreactors can be used to produce fuels such as biodiesel
and bioethanol, to generate animal feed, or to reduce pollutants such as NO 2 and CO2 in flue gases
of power plants. Fundamentally, this kind of bioreactor is based on the photosynthetic reaction
which is performed by the chlorophyll containing algae itself using dissolved carbon dioxide and
sunlight energy. The carbon dioxide is dispersed into the reactor fluid to make it accessible for the
algae. The bioreactor has to be made out of transparent material. The algae are photoautotroph
organisms which perform oxygenic photosynthesis.

The equation for photosynthesis:

𝐾
𝐽
∆H = +2870 𝑚𝑜𝑙
0
6 CO2 + 6 H2O C6H12O6 + 6 O2 ………….. (1)

Fig.1.1: Algal photo-bioreactor

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1.2. Algal basics

Algae are an informal term for a large, diverse group of photosynthetic organisms which are not
necessarily closely related and are thus polyphyletic. Included organisms range from unicellular
genera, such as Chlorella and the diatoms, to multi-cellular forms, such as the giant kelp, a large
brown alga which may grow up to 50 meters in length. Most are aquatic and autotrophic and lack
many of the distinct cell and tissue types, such as stomata, xylem and phloem, which are found
inland plants. The largest and most complex marine algae are called seaweeds, while the most
complex freshwater forms are the Charophyta, a division of green algae which includes, for
example, Spirogyra and the stonewort’s. Algae are simple plants that range from algae to large
seaweeds, such as giant kelp. Algae can be grown using brackish, sea, and wastewater unsuitable
for cultivating agricultural crops. Most algae grow through photosynthesis by converting sunlight,
CO2, and a few nutrients, including nitrogen and phosphorous, into biomass . Other algae can
grow in the dark using sugar or starch.

Fig.1.2: Green algae

1.3. Methods of Cultivation of Algae

Algae is a eukaryotic photosynthetic organism which has various environmental engineering

applications including sewage treatment, eutrophication prevention and fertilizer recovery, CO 2
scrubbing and can also be used as a fertilizer, a food source for people and animals and a source
of biofuel. Algae are phototrophic microorganisms. Wastewater can be used to supply nitrogen
and phosphorus, the main nutrients needed for the cultivation of algae. The use of residual algal

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biomass is then refined to other usable products such as biofuels. The biomass accumulated can
be fermented to produce methane or ethanol.

1.3.1 Open pond system

Open pond system is the furthermost usually applied for large scale algae cultivation due to its
low cost and ease in operation and maintenance. It is commonly used for engineering application,
to produce significant number of products for commercial purposes at relatively low cost. The
open pond is commonly designed as 0.25 m in size, with area of 0.2–0.5 ha for commercial
resolve algal productions. Open pond system possesses high surface area per volume ratio
allowing the high CO2 mitigation. Additionally, if the nutrient sources used is waste-water,
incorporated with CO2 supplied from flue gas, the open pond system is usually applied. It
provides effective wastewater treatment concurrently. Yet, open pond system suffered some
disadvantages whereby it requires significant land area, the culture is subjected to high risk of
pollution or predators and the evaporative water lost can be noteworthy due to the open structure.
Thus, some ponds are covered with transparent material to pro-mote rising period of algae,
prevent disappearance loss and facilitate CO2 distribution.

Fig.1.3: Open pond system

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1.3.2 Closed system

Closed photobioreactors (PBRs) have gained much interest by researchers due to better control of
cultivation limitations and capability to satisfy carbon necessity. It was also evident that PBR
cultivation has accomplished high photosynthetic effectiveness and biomass productions
associated to open pond system .These advantages are even more important if the desired algae
are used for pharmaceutical purposes or highly selective products applications’ are designed in
configuration to maximize photosynthetic efficiency and CO 2 mass transfer efficiency; minimize
cultivation dark zone and power ingesting. The PBRs used are brief in the following sections.

Fig.1.4: Closed system

1.3.3. Raceway ponds

Raceway pond, an open pond system looks like race track, frequently narrow with 15–25 cm in
deepness. It is prepared with paddle wheel agitation, so as to ensure good movement and nutrients
homogenization. In adding, the flow of culture is controlled and guided with baffles placed in the
flow channel. This encourages the liquid velocity of the ponds are to be functioned with more
than

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30 cm per second. Raceway ponds are currently the most commonly used large scale cultivation
system for commercial scale. If allied to closed photo bioreactor system, raceway cultivation
produced low biomass productivity of Chlorella sp. and Spiralling due to carbon limitation, as
only 5% of carbon is directly transported by atmospheric air. In order to accomplish outstanding
carbon sequestration, the algal farms can be placed immediate the industrial plant, so as to utilize
CO2 in the flue gas efficiently.

Fig.1.5: Raceway pond

1.4 Typs of Algae Photobioreactors

There are three basic types of reactors-

1.4.1 Plate photo bioreactor

A plate reactor simply consists of perpendicularly arranged or inclined rectangular boxes which
are often separated in two parts to effect an agitation of the reactor fluid. usually these boxes are
arranged to a system by linking them. Those connections are also used for making the process of
filling/emptying, introduction of gas and transport of nutritive substances, easier. The introduction
of the flue gas mostly occur at the bottom of the box to ensure that the carbon dioxide has enough
time to interrelate with algae in the reactor fluid.

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 Fig.1.6: Plate photo-bioreactor

1.4.2 Tubular photo bioreactor

A tubular reactor consists of vertical or horizontal arranged tubes, associated together to a pipe
system. The algae-suspended fluid is able to pass in this tubing. The tubes are usually made out of
transparent plastics or borosilicate glass and the constant circulation is kept up by a pump at the
end of the system. The introduction of gas takes place at the end/beginning of the tube system.
This way of introducing gas causes the problem of deficiency of carbon dioxide, high
concentration of oxygen at the end of the unit during the circulation, and bad efficiency.

Fig.1.7: Tubular photo bioreactor

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1.4.3 Bubble column photo bioreactor
A bubble column photo reactor consists of vertical arranged cylindrical column, made out of
transparent material. The preface of gas takes place at the bottom of the column and causes a
turbulent stream to enable an optimum gas exchange. At current these types of reactors are built
with a maximum diameter of 20 cm to 30 cm in order to make sure the required supply of
sunlight energy. The biggest problem with the sunlight resolute construction is the limited size of
the diameter. Feuerman invented a method to collect sunlight with a cone shaped collector and
transfer it with some fibreglass cables which are adapted to the reactor in order to enable
constructions of a column reactor with wider diameters. On this scale the energy consumption due
to pumps etc. and the CO2 cost of manufacture may outweigh the CO2 captured by the reactor.

Fig.1.8: Bubble column photo bioreactor

1.5 Photo bioreactors key systems :-

i Optical transmission system

ii. Air handling & gas exchange systems
iii. Mixing system
iv. Nutrient system
v. Instrumentation system

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1.6 Requirements to develop a high-performance photo bioreactor for algal cultivation

i. In order to attain high productivity, the volume of the non-illuminated parts of the reactor
should be minimized.
ii. In order to ensure a high efficiency of light use by the culture, the design must provide for
the uniform illumination of the culture surface and the fast mass transfer of CO2 and O2.
iii. To prevent rapid fouling of light-transmitting surfaces of reactors, photo bioreactors must
be frequently shut down for their mechanical cleaning and sterilization
iv. High rates of mass transfer must be attained by means that neither damage cultured cells
nor suppress their growth.

For the industrial-scale production of biomass, the energy utilization required for mass transfer
and the preparation of the light-receiving surface of the algal suspension must be reduced to its
minimum possible.

1.7 Sources of CO2

Incineration of fossil fuel such as coal, oil, and gas is the largest source of CO 2 emissions
globally. Flue gas emitted from these sources mostly contains nitrogen (N 2), carbon-di-oxide
(CO2), oxygen (O2), and water vapour. It also contains minor amounts of CO, NO x, SOx and
particulate matters. description of flue gas , India shows that percentage of CO 2 in the flue gas
was 11.2% while SOx and NOx were 672.0, 610.08 mg/Nm3, respectively. Flue gases data from
fossil fuel power plants also consisted of 4–14% of CO 2 and up to 200 ppm of NOx and SOx
depending on the type of fuel and its type of combustion process. Coal fired plants generally have
higher percentage of CO2 emissions. Power plants can use electrostatic precipitator for removing
dust particles. Some of them also have desulfurization and gentrification facility for the removal
of oxides of sulphur and nitrogen, respectively. The composition of the flue gas of coal fired
thermal power plants after passing through electro-static precipitator and desulfurization facility
was 13% CO2, 5% O2, 10 ppm or below SOx, 150 ppm or below NO x, and 10 mg/Nm3 or below
dust. NOx present in the flue gas generally poses no problem for algal growth while the main
impact is due to SOx which decreases the pH due to formation of sulphurous acids. Finally the
choice of CO2 point source depends on the cost associated with it which varies from point source
to point source. It is approximately $6–$12, $5–$70, $20–$95 , $30– $145 per ton CO 2 from

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ethanol facilities, hydrogen and ammonia production or gas-processing plants , fossil power
plants and other industrial sources, respectively .

1.8 CO2 fixation using algae

Micro algae are a promising alternative to CO 2 mitigation by CO2 fixation, bio fuel production
and wastewater treatments. CO2 fixation by photoautotrophic algal cultures has the potential to
diminish the release of CO2 into the atmosphere. CO2 can be fixed from different sources which
can be characterized as:-

i. CO2 from the atmosphere

ii. CO2 from industrial exhaust gases (e.g., flue gas and flaring gas)
iii. Fixed CO2 in the form of soluble carbonates (e.g., NaHCO3 and Na2CO3).

1.9 Objectives

The objective of present work defines as follows:

i. Kinetic study was conducted over different media.

ii. Determination of specific growth rate of algal species in PET bottles
iii. Determination of CO2 fixation rate for different media.

2. LITERATURE REVIEW
2.1 Review
Jean U and Hokemen, 2017 chemicals provide an alternative to conservative petroleum-based
feedstock’s owing to their larger energy security, compact environmental impacts, foreign
exchange savings, and socioeconomic benefits. In the past two decades, there no. of research
has been growing and technological development of bio fuels and bio energy by ace- demia,
industry, and other association. In view of increasing efforts on algae biomass production and
its exchange into energy and high-value products, this research topic covers significant aspects
of algal strain selection, culture systems, inorganic carbon utilization, lipid metabolism and
quality, biomass harvesting, Taking out of lipids and proteins, and thermo chemical conversion
of algal feedstock’s into bio crude.

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Adrianus Jan Hagendijk 2015, before designing a photo bioreactor, it is vital to understand the
best conditions for the production of algae because these have a direct influence on the needs. In
order to produce algae biomass under the specific conditions, one has to examine current photo
bioreactors that have been designed in order to set up whether they are capable of optimum
production under the production conditions; determine possible factors that could influence the
production negatively and how they could be prohibited; and undertake a cost analysis to
determine whether the production of algae is an economically viable process using the specific
reactor. All of these criteria have to be met for a photo bioreactor to be viable in the production of
algae biomass.

Mata T. 2012 analyzed the factors that may affect the production of biomass and the conduct of
brewery waste water. Many parameters were studied to reach the highest biomass production and
the most suitable conditions for cultivating algae. The utmost biomass obtained was 0.9 g of dry
biomass per litre of growth on the 9th day.

Cheirsilp B 2012, for the production of advanced biomass amount with high lipid content the
growth was cultivated under stepwise increasing concentration as a fed-batch. This procedure
insured approximately twice lipid production than conservative batch cultivation. The
composition of the main fatty acid was appropriate for biodiesel production.

Khoeyi Z 2011, used three algae sample placed in unlike light conditions (photoperiod, intensity),
there was a huge disparity in the growing concentration between them as the utmost biomass was
recorded between 0.1 g and 2.05 g when the algae culture exposed to 62.5 μmol photons m- 1 s-1
for a 16:8 h light/dark photoperiod duration.

Demirbas and Faith Demirbas, 2011, Algae are some of the most essential photosynthetic
organisms on earth. They can live in either saline or fresh water and need mainly light energy,
water and carbon dioxide for survival. Algae are part of a varied group of organisms which can be
establish in simple unicellular to complex multicellular forms and can be found growing within
most biotopes, due to their ecological diversity and physiological flexibility to the specific
environments .

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 Plant Source Biodiesel (l/ha/yr) Area to produce global Area as percent global
oil demand arable land

(hectare x 106)

Cotton 325 15002 756.9

Soyabean 426 10932 551.6
Mustard seed 572 8524 430.1
Sunflower 952 5121 258.4
Rapseed/ canola 1190 4097 206.4
Jatropha 1892 2577 130
Oil palm 5950 819 41.3
Algae 12000 406 20.5

Fig.2.1: Comparison of biodiesel produced per unit area per year

Posten C. 2009, review the different parameters for designing photo bioreactors that consequence
the growth of algal mass. There are several suggestions to increase light distribution along with
enough agitation, aeration and energy demand for higher performance. The suggested way to add
to light rather than increasing the transparent surface and bringing the algal growth to the light is
to use alternative ways to bring light to the biomass layers by using milli and micro scaled multi
structures – fleece of glass fibers can be used – or a build in light conductive structure can be used
to guide light into a compact closed reactor. Another way is to use lenses effect or LEDs light to
distribute the light uniformly inside the reactor.
Jacob-lopes E. 2009, evaluate growing algae under dissimilar light cycles, and 24:0 (night: day)
in that order. A reduction in biomass production was experiential in parallel with the reduction in
light period duration.

Hsieh C. 2009 built-up an open PBR tank with rectangular see-through chambers to increase the
photosynthetic competence of micro algal growth. The transparent rectangular chambers made of
high light conducting translucent acrylic. This structure allows deeper light conductance into PBR
even at high cell concentration. The TRCs provides a high effective operation of light for the
growth with high rate production. The biomass obtained using this design was 56% higher than
PBRs without TRCs.

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Kitaya Y. 2005 investigated the belongings of temperature; CO 2/O2 concentrations and light
intensity were examined on cellular development of algae. Algae were cultured under five levels
of temperatures from 25˚C to 33˚C, three levels of CO 2 concentrations from 10% to 30 %, and six
levels of photosynthetic photon flux from 20 to 200 μ mol m- 2 [Link] results demonstrated that
the highest multiplication rate of the algae cells was at temperature of 27-31˚C, CO 2 concentration
of 4%, O2 concentration of 20% and light flux of about 100 μ mol m-2 s-1.

Maryam Al-Qasmi 2004 said that algae arrange their own food by photosynthesis. Algae can be
growth our self by the help of light, enough nutrients and some certain condition of temperature.
Many researchers worked on special type of PBRs to increase the rate of photosynthesis. Light
conditions have an effect on directly the growing and photosynthesis of .Algae needs a light/dark
command for productive photosynthesis, it needs light for a photochemical phase to produce
ATP, NADPH and also needs dark for biochemical phase manufacture essential molecules for
growth. Experimental investigations reveal that the increase in light duration is directly
proportion to increase in number of cultivated algae as well as the increase in the light intensity.

Choi, Suh and Lee, 2003; noticed that due to their rapid growth rate, algae show potential to
produce biofuels, food and high-value molecules from a renewable source and supply the demand
for specific natural and renewable products. Algal are cell factories that are sunlight-driven and
could potentially produce an array of compounds which include polysaccharides, lipids, proteins,
carotenoids, pigments, vitamins, steroids, pharmaceuticals and Biofuels.

Rochet M., et. al., 1986, studied certain conditions in southern Hudson Bay from March to May
1983 to observe the response of sea-ice algae to different changes in light intensity and quality.
When algal growth was incubated under blue light it doubled as compared to the growth under
white light. According to the availability of blue green light environment ice algae had adaptive
response to it and showed high concentration of chlorophyll. On the other hand the same algae
responded to light spectrum when in incubated under white illumination by increasing their
chlorophyll. "The ability to chromatically adapt may become a critical factor in species
competition".

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Currently a Bubble column reactor is considered to be the best design for a photo bioreactor and
also the most scalable. Due to the limited information available, testing was conducted to
determine the effect of:

1) Different manufacturing materials

2) The gas dispersion unit

3) The diameters of the tubing

4) The density

Bubble column reactors were used to test the effects of the four variables and were considered to
be the most important aspects in the design. For testing these variables and their interaction, It is
one of the most popular algae species used for production currently. As temperature and the
availability of light play a large role in the production of algae, all testing was done in a
laboratory environment to ensure small temperature changes and the constant availability of light.

It was found that the 50 mm and 90 mm reactors showed a better performance with acrylic tubing
while the 110 mm reactors showed a better performance with PVC tubing. The gas dispersion unit
is affected by the gas flow rate, the density, the diameter of the tubing and the material that is
used.
The gas dispersion units’ effect is dependent on the diameter of the reactor seeing that the 50 mm
reactor shows better performance with the small unit, while the 110 mm reactor shows better
performance with the large unit, due to the gas flow rate that is required in the reactors. Because
the gas flow rate and gas dispersion unit directly affect the agitation, the optimal density is
affected directly due to the availability of light and therefore the tubing material. The gas
dispersion units should fit properly into the reactor and be capable of handling the gas flow rate
that is required. The diameter of the tubing does not show any effect but could have an effect
under different testing conditions and could not be conclusively eliminated. The density of algae
does have an effect, although most reactors showed a better production rate at a higher culture
density.

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A cost analysis shows that the bubble column reactors under the tested conditions are not
financially viable. A large component of the cost is carbon dioxide and medium, which is a
composition of nutrients. This could be removed if a free source were obtained, which would
make the system financially viable. These sources could include waste water and flue gas from
industrial processes.

Research on algae production only started in Stanford in the USA, Essen in Germany and Tokyo
after 1948. Hereafter, the commercial production of algae biomass began in Japan during the
early 1960s with Chlorella and during the 1970s research on Spirulina followed Borowitzka,
1999. The first attempts to grow algae occurred during World War II; algae were grown in open-
pond systems by the Germans as a food supplement

Algae production on an industrial scale is currently expensive when compared to fossil fuels: the
estimated production cost of algae oil is between $1.40 and $1.81 per litre and this cost needs to
be reduced to around $0.45 per litre in order to compete with petrol and diesel.

One of the main driving forces behind the research on algae is to find a replacement for the
current fossil fuels. Because the use of algae-based biofuels would create a closed carbon system,
it would thus have no net effect on the environmental levels of carbon dioxide or possibly even
reduce the levels of carbon dioxide in the atmosphere.

The biggest benefit of algae biomass production is the ability to produce algae biomass on
nonarable land. This does not directly threaten current food production, while energy crops
compete directly with food production for arable land. Waste created by a specific processing
method could possibly be reprocessed to obtain another product, thus increasing the economical
and processing viability of producing algae biomass, e.g. if the desired product from the biomass
is the lipid content, the waste could be processed to obtain protein, carbohydrates or any other
product that is produced by the specific strain of algae, making it more economical and
environmentally friendly. Due to the possibilities for algae biomass, the design of a
photobioreactor will play an important role.

Goldman, 1981, stated that Culturing requirements are species specific, but some media are

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“Broad” with respect to meeting the nutritional/culturing needs of groups of algae. Successful
culturing entails formulating the medium and environmental conditions to meet the target algae’s
requirements for optimal growth. Temperature, light, pH, salinity and mixing, as well as nutrient
quantity and quality are the parameters of interest to obtain optimal growth.

Moving from the laboratory to large scale is not just “doubling” the batch. It does not work for
brewing and it does not work for growing algae. One problem is that the laboratory algae may
have been grown under “unbalanced growth” conditions. It is essential to develop standards or
standardized ranges that parallel the conditions that will exist in the larger scale cultivation unit in
the laboratory. Range requirements for nitrogen, phosphorus and carbon, quality and quantity of
light, temperature, salinity, and mixing or turbulence with respect to a particular species must be
carefully established before moving out of the laboratory.

CO2 bubbling can physically damage cells and, unless filtered with a 0.2 μm filter unit there is a
chance of bacterial or viral contamination. Bubbling does increase the surface area exposure to
CO2 and removes the excess O2 produced. If there is not sufficient algae biomass to utilize the
CO2, the higher concentration of CO2 can lower the pH.

Optimal growth requires optimal nutrient availability, temperature and light intensity. Optimal in
this case means most advantageous to the specific algae, since each species has specific growth
requirements.

According to Moroney and Somanchi, 1999, the maximum value of dissolved inorganic carbon
till which it is active depends upon strain, pH, light availability, pre adaptation of cells etc. For
example, in cyanobacteria CO2 is 200 µM as against approximately 10 µM dissolved CO 2 in
water in equilibrium with air.
Matsuda and Coleman, 1995 said that CO 2 concentrating mechanisms catalyzes the
interconversion of CO2 and HCO3- and is an important component in the survival of the algae.
Fenton and Ó hUallacháin, 2012, explained that with the expansion of the commercial
algaegrowing industry, competition with the agricultural sector for inorganic fertilizers is
expected to increase. Coupled with the fact that fossil fuel prices are likely to increase, this could

30
mean that inorganic fertilizers may be an economically unviable source of nutrients for algal
production systems. Approximately 50% of the required fossil energy input for the growth of
algae for biofuel is linked to inorganic fertilizers. The nutrient content of agriculturally derived
organic fertilizers, runoff and drainage water have the potential ability to facilitate the growth of
algal biomass. Agricultural waste water characteristically contains a large amount of both organic
matter and nutrients. Wetlands have a finite ability for nutrients to be recovered and stored by
aquatic plants, sedimentation, detritus, microbes and fauna. Removal of both N and P was more
efficient here than in unplanted wetlands, when using waste water from a dairy farm. Studies
which included unplanted control wetlands found that planted wetlands removed more nitrogen
from waste water which was rich in ammonia. The biggest potential problem when using
agricultural runoff water is the inclusion of pest-control poisons that could kill the algae.

Yun et al., 1999, states that flue gas from thermal power stations and steel-making plants has a
carbon dioxide concentration that is approximately 500 times higher than that of atmospheric
levels.
The use of waste water from the steel-making plants requires the addition of phosphates, while
allowing a calculated rate of 23,100 kg of CO 2 to be fixed daily and the production of 12,430 kg
of algal biomass daily. The use of steel-making waste water is likely to cause the algal biomass to
be contaminated by heavy metals. The contamination could have no or little effect on the
commercial value of the algae but this would depend on the intended use of the specific algae
biomass that was produced.

3. FACTORS AFFECTING GROWTH RATE OF ALGAE

3.1 Effect of Temperature
Temperature of flue gas emitted from power plants and other sources are around 120 0C.
Feasibility of sequestering CO2 from flue gas depends on either installing heat exchanger system
or using thermopile species. Several species have been identified which can tolerate high
temperature up to 600C.

algae may be able to grow at a variety of temperatures, different species show different optimal
temperatures which are specific to each. For example, the optimum temperature range for species
were found to be 19-21, 19-21 and 24-26°C. For many species, optimal growth temperatures of

31
15–26°C have been observed with maximum cell densities obtained around 23°C. However,
optimal temperatures are also influenced by other environmental parameters, such as light
intensity and the distance between cultivation apparatus and artificial illumination system.

Munoz, 2008, explains about the higher temperature effects. Higher temperatures generally
accelerate the metabolic rates of algae, whereas low temperatures lead to inhibition of algal
growth.
In suitable temperature condition, the enzymes in algal cells possess the highest activity.

Dauta,1990, states that Temperature is one of the major factors that regulate cellular,
morphological and physiological responses of algae.

Torzillo,1986, also explain that the control of temperature is a key factor for culturing algae
outdoors. Actually, temperature can vary depending on the geographic region of cultivation
Seasonal and even daily fluctuations in temperature can interfere with algae production.
Temperatures can reach as high as 30°C higher than ambient temperature in a closed photo
bioreactor without temperature control equipment. For this reason, evaporate cooling or shading
techniques are employed frequently to inhibit temperatures of that magnitude.

3.2 Effect of pH
The pH of the medium is linked to the concentration of CO 2 and pH increases steadily in the
medium as CO2 is consumed during flow downstream in a cultivation system. The pH affects
mainly the availability of nutrients such as iron, organic acids and even CO2.

The pH of the culture medium can be influenced by dissolving CO 2 and SOx from the flue gas.
With elevated CO2 concentrations, pH drops down to pH 5, and with higher SO x concentrations
even down to pH 2.6 have been reported.

According to Maeda, 1996 whereas the pH change due to the CO 2 had just minor influence on the
algal growth, the strong pH change caused by the SO x inhibited all growth. With buffered
medium the pH drop could be prevented and almost no changes in growth rates compared to

32
lower SOx concentrations were observed. This indicates that the influence of SO x on the growth
is, in certain limits mainly due to pH changes than sulphate concentrations in the medium, which
can be prevented by buffering or active pH control.

Oswald,1988 also worked on the same and came to a conclusion that the Increasing CO 2
concentrations can lead to higher biomass productivity, but will also decrease pH, which can have
an adverse effect upon algal physiology. By contrast, algae have been shown to cause a rise in pH
to 10–11 because of CO2 uptake. This increase in pH can be beneficial in open ponds for instance
for neutralization of pathogens in algal wastewater treatment, but can also inhibit algal growth.

There is a complex relationship between CO 2 concentration and pH in algal photo bioreactor

related to the chemical equilibrium among chemical species such as CO 2, H2CO3, HCO3- and
CO32-. The chemical equilibrium between these forms is pH dependent with CO 2 the predominant
form at lower pH below 7 and CO32- predominant above pH 10. Rapid growth of algae can, with
the assimilation of CO2 as the C source, cause the pH to rise.

The pH is a fundamental parameter which regulates cell metabolism and biomass formation.
Each strain of algae has a narrow optimal range of pH and most algal species are favoured by
neutral pH. However, there are some extremophilic species which dwell in environments that are
characterized by very low or high pH-values.

3.3 Effect of Nitrogen & NOx

Besides high amount of CO2 and high temperature, NOx influence the growth of the algae.
Tolerance of algae to NOx varies widely among the various species.

Griffiths et al., 2011, It has been reported that, under nitrogen deficient conditions, many other
strains show increase in their lipid content and modification on fatty acids composition.
Scott et al., 2010, explained that the general principle is that when there is insufficient nitrogen
for the protein synthesis required by growth, excess carbon from photosynthesis is channelled into
such storage molecules as triacylglycerols or starch.

33
 In general, lipid productivity and content are inversely correlated with each other; and stress
conditions, e.g. deprivation or limitation of nitrogen or of phosphate, to a lesser extent, limit cell
growth while increasing lipid content. Nitrogen limitation has been observed to result in lipid
content increase in many of the species.

Ammonia, urea and nitrate are often selected as the nitrogen source for the mass cultivation of
algae. The choice of the suitable source of nitrogen depends on strain considered since metabolic
pathways related to nitrogen are species-specific. Although ammonium and urea are often used in
mass cultivation owing to the relatively low-cost, selecting proper nitrogen source for each algal
species is important in improving biomass and oil productivity.

Urea and nitrate were found to be better than ammonia for the growth and lipid accumulation in
some of the species. In contrast, for Ellipsoidion sp. the ammonium has been demonstrated to
produce higher biomass and lipid content than those of urea and nitrate.

It has also been reported that the triglycerides accumulated in Nannochloris sp. under nitrogen
deficient conditions could be 2.2 times of that in nitrogen sufficient cultures.

the tolerance of this species under 20 ppm SOx and 60 ppm NOx. Chlorella was found to have a
higher tolerance capacity than Chlorella sp. T-1 as it can tolerate up to 100 ppm of NO x along
with 10–15% CO2. However, Chlorella sp. HA-1 could not grow under higher combination of
SOx and NOx because of a drastic decrease in pH. Adding certain amounts of sodium sulphite
instead of gaseous SO2 to the medium resulted in a slower growth at 50 mM and cell death at 250
and 500 mM.

Nannchloris can grow below 100 ppm of NO x on the other hand species like Dunaliella tertiolecta
can grow in up to 1000 ppm of NO x. Tetraselmis species can grow in the combination of 125 ppm
NOx .

34
3.4 Effect of Light
The light energy is used by the cells either for maintenance purposes or formation of new
biomass. Consequently, the biomass productivity and the cell growth rate are directly linked to
the light energy.

Cepak,2013 gave a theory on better light requirements. Studies have shown that green algae grow
better in blue and red light because they contain chlorophyll a and b which is major light
harvesting pigments that is sensitive to these wavelengths. In one study, for several cell
concentrations of Chlorella vulgaris the absorption of light was in the 400-500 nm (blue) and
625675 nm (red) range while the rest of the light was scattered amongst the cells. In another
study, it was found that in red light (625-675 nm) Scenedesmus oblique increased significantly in
cell volume and the division of nuclei occurred earlier.

the effect of circular and plan geometry in light penetration. For similar algae cell concentrations,
circular geometry allows a better light penetration, than the plane geometry allowing a higher
volume fraction of the reactor to receive sufficient amounts of light however, plan geometry helps
in uniform distribution of light.

A patent is filed claiming 10 times higher aerial productivity in open ponds using Spirulina as a
model microorganism. This strategy can also be used in achieving higher aerial CO 2
sequestration, biomass formation and e.g. hydrogen production in photo-bioreactors.

the CO2 fixation and biomass production optimum light intensity is necessary. Below the
optimum light intensity, light becomes the limiting factor for the algal productivity. The exposure
of cells to long period with high light intensity causes photo inhibition due to damage of repair
mechanism. Light intensity at the depth of dense algal suspension is greatly reduced because of
the absorption and scattering of light. Attenuation of light intensity is dependent of its
wavelength, cell concentration, penetration distance of light and the geometry of photo bioreactor.
Since the blue and red light are mostly consumed by the algae, it penetrates little in algae
suspension than green light. This effect is more pronounced in the dense culture. In the

35
engineering point of view geometry of reactor can reduce the attenuation of light in micro algal
suspension.

in outdoor condition, light availability is the dominant factor determining the productivity. Light
saturation imposes serious problem for the growth of algae at the central daylight hours.

Uniform and impartial distribution of incident light to every layer of cells can be achieved by
using the algal strains having small antenna size. In normal case first layer of cells gets maximum
light and decreases drastically to the consecutive further layers of cells. A small antenna size
strain reduces this wastage of light and prevents the cells from photo inhibition.

the sunlight can be provided better. Reducing the chlorophyll concentration is another such
method. Basic science behind this idea is that by limiting the cells from excess chlorophyll
concentration, higher cell density can be used which in turn results in higher productivity.

3.5 Effect of Proper Mixing

Proper mixing not only helps in the uniform mixing of nutrient but also in the better distribution
of light over cells. It minimizes the intensity and also takes advantage of flashing light effect. This
effect increases the productivity in tubular photo-bioreactor up to 40%.
Carlozzi, 2008 also tried to take advantage of mixing by constructing strongly curved tubular
photobioreactor allowing cells exposed to higher light at the surface to go inner region reactor and
hence limiting cells from taking higher value of intensity.

3.6 Effect of Culture density

Productivity and light utilization efficiency value are the functions of the cell density. It is crucial
to select the optimum cell concentration for the efficient CO 2 sequestration. Below the optimum
cell concentration, not all the light energy is captured by the cells while at above the optimum cell
concentration, a larger proportion of the cell are in the dark due to self-shading. However, highly
dense culture also makes cells more tolerant to high percentage of CO2 concentration.

36
3.7 Effect of CO2 concentration
A flow rate of 0.25 vvm reports that 2% (v/v) of CO 2 is optimum for the growth of a certain
species of algae while at 10% (v/v) specific growth rate becomes insignificant.
in aqueous environment dissolved CO2 always exist in equilibrium with H2CO3, HCO3- and CO32-
which concentration depends upon pH and temperature. Due to fast inter-convertible reaction
among them, consumption of any of inorganic carbon does not affect the equilibrium. Algal cells
preferentially uptake HCO3- over CO2 despite of the fact that former is a poor source of carbon
than later. The reactions given were as follows:

CO2 + H2O↔ H2CO3

H2CO3 ↔ H+ + HCO3
HCO3- ↔ H+ + CO32-

Algal cells can tolerate CO2 only up to a certain level after which it becomes detrimental for the
growth of the cells because of the two reasons. Firstly environmental stress induced by the higher
CO2 concentration which causes biological reduction in the capacity of algal cells for CO 2
sequestration. Secondly at higher CO2 concentration, the culture pH decreases due to the
formation of high amount of bicarbonate buffer. The biomass productivity increases with increase
in CO2% (v/v) in the gas mixture up to certain percentage beyond which productivity decreases.

However, the sequestration of CO2 from flue gas emitted by coal fired thermal power plant
confirms that the same species of algae can tolerate up to 100% CO 2 concentration but the
maximum growth rate was obtained when using 10% CO 2 with no significant decrease in growth
rate up to 50% CO2 concentration. They also concluded that preadaptation of cells with lower
percentage of CO2 concentration leads the tolerability of cells in higher percentage of CO2.

3.8 Effect of O2 accumulation

Trapped oxygen in the liquid culture causes toxic effects like photo bleaching and reduces the
photosynthetic efficiency. An efficient degassing system is required in order to remove formed
O2. Accumulation of O2 is a serious problem in reactors with poor gas exchange like horizontal
tubular reactors, especially when continuous run tubing increases. The problem of accumulation

37
of O2 increases when a helical tubular reactor is scaled-up by increasing the light harvesting unit.
Hence it is necessary to have a separate degassing unit in which the distance between the entrance
and exit is such that even smallest bubbles can disengage. It is not of major concern in reactors
which have an open gas transfer area as in stirred tank and vertical reactors.

3.9 Nutrients requirement and their effect on the growth

The average elemental composition of freshwater algae is CH 1.7O0.4N0.15P0.0094, with the N content
being particularly sensitive to its environment. To achieve the best growth rate, these nutrients
have to be supplied in an optimum quantity in order not to limit the growth rate in any way. If
saline water is used, the addition of other salts may be required for maximum algal growth.
A minimum requirement for balanced nutrition of CH 1.83O0.48N0.11P0.01 is necessary for the
production of algae.

There are three main different situations of nutrient supply: nutrient-sufficient, nutrient-limited
and nutrient-deficient. The first case should be evident, whereas the difference between the latter
two cases may be subtle. Nutrient limitation occurs when cells are grown in an environment of a
constant, but insufficient, supply of a limiting nutrient, to which the cells generally adapt.
Nutrient deficiency is characterized by the culture’s reliance on endogenous reserves because
there are no nutrients in the environment.

some nutrients need to be present in excess. For example, phosphorus must be supplied in excess
because the phosphates react with metal ions.

Phosphorous is most often limited in nature because it is effectively bound in sediment. This
element, in the form of orthophosphate, is generally considered the main limiting nutrient in
freshwater aquatic ecosystems: that is, if the entire phosphorous are used, autotrophic growth will
cease, no matter how much nitrogen is available.

On the other hand, in nature nitrogen is not necessarily limiting because bacteria are fixing
nitrogen and supplying the algae with a constant nitrogen source. Nutrient deficiencies and excess
nutrients, both, can cause physiological and morphological changes in algae since they can inhibit
some of the vital metabolic pathways.

38
Algae need some essential macronutrients such as carbon, nitrogen, phosphorus, sulphur, calcium,
magnesium for their growth while micronutrients requirement is limited to small amount of some
elements such as iron, boron, manganese, copper, molybdenum, vanadium, cobalt, nickel,
selenium and in same case silicon. Some of these nutrients can be easily found in nature bound in
minerals while others are supplied by bacteria metabolism.

It is important to develop a balanced medium for optimum algae cultivation and CO 2 fixation.

Nitrogen and phosphorous are considered elements key to algal metabolism then they must be
found in the media in which algae are grown. The researchers asserted that balancing the nutrients
based on the elemental composition of the biomass should be the basis for effective medium
design.

3.10 Effect of Carbon

The direct uptake of bicarbonate by an active transport system has been found in several species.
Adoption of carbonate-utilizing strains for CO2 fixation could be advantageous in two aspects:

1) As only a limited number of algal species thrive in media containing high concentration of
carbonate salts, species control is relatively simple;

2) Most of these species have high pH optimum in the range of 9 to 11 further simplifying
species control.

A number of algal species have been shown to be able to utilize carbonates such as Na 2CO3 and
NaHCO3 for cell growth, which is responsible for the conversion of carbonate to free CO 2 to
facilitate CO2 assimilation.

3.11 Effect of phosphorous

Similar to oil, phosphorus is a non-renewable resource, which is currently extracted at such rates
that the global phosphate reserves will be depleted in as little as 50 – 100 years. Algae production

39
would therefore be in direct competition with the phosphorous fertilizers that are required for
food production. This increases the importance of using phosphorous efficiently in algae
production by recycling the nutrients in algae production systems wherever possible and possibly
using algae to recover phosphorous from run-off water.

Phosphorus-starved cells take longer to start their growth cycle, as the reparation of damaged
functional macromolecules has to be completed first. Phosphorus stored intracellular can mainly
be found in the form of a polyphosphate as it forms part of the main energy transporter in living
cells.

Recent reviews on polyphosphate metabolism in photosynthetic organisms Phosphorus is an

important limiting nutrient in many ecosystems such as lakes, rivers, and estuaries, and also one
of the most likely to limit the rate of phytoplankton production. Algae use phosphorus for their
metabolism in form of polyphosphate.
Phosphorus is one of the most important nutrients as it regulates both growth and metabolism in
cells. It plays a significant role in energy transport, biosynthesis, photosynthesis and respiration.
Phosphorus is also a key factor for algae to accumulate lipids. Only a few studies have been done,
although no common conclusion could be drawn since the process appears to be species
dependant.

Like all living organisms, algae require phosphorous for growth and development. Inorganic
phosphate is the preferred method for the addition of phosphorus to algae culture, although many
algae can utilize polyphosphates and organic phosphorous sources. It is possible to use
phosphorous from waste water, but reliable growth is harder to achieve due to variations in waste
water composition and the generally high ammonia concentrations.

Even if with a little extent compared to the larger results on nitrogen, phosphorus starvation can
also enhance algal biomass and lipid productivity, as reported for a certain species.

Alternative sources of phosphorous could include waste water, manure or even ashes containing
phosphorous from the combustion of biomass. Bone meal, which is rich in both phosphorous and
calcium, has been used successfully in cultures.

40
3.12 Effects by some of other element’s as nutrients
Iron, sulphur, magnesium, and other elements are also indispensable for the growth of algae.
Iron is involved in electron flow from H 2O to nicotinamide adenine dinucleotide phosphate. Also
some investigations have been addressed to the effect of iron on algae growth. High iron
concentrations have been show to enhance cell growth some metabolic pathways may be
modified upon exposure to high levels of that mineral element in the medium.

4. PAST USES OF ALGAE

Sulphur is an essential component of two amino-acids, cysteine and methionine. In its absence,
protein biosynthesis is impeded and the photosynthetic system PSII repair cycle is blocked.

Magnesium is required as essential element in the core of the tetrapyrrolic ring which is the base
of the chlorophyll molecule.

The large-scale production, wastewater treatment has been using algae for years. The algae are
used to remove nitrogen and phosphorus, while providing oxygen to bacteria. Algae can also
remove heavy metals such as cadmium zinc, nickel, and lead. This process is considered
environmentally sound, recycles nutrients more efficiently, does not lead to and secondary
pollution, produces biomass that can be harvested unless the algae removes heavy metals and
produces oxygen.

Another large-scale production of algae is food production. It can be used for human consumption
or animal consumption. Algae can contain high amounts of protein β-carotin, and omega-3.
Algae improved immune reaction and reproduction of animals, since they are a good source of
vitamins, minerals and fatty acids. It can also help with stomach ulcers and wounds. Algae show
great potential for diabetes and cancer treatment. Raven et al., 1999, stated that manganese is
essential for O2 evolution, and calcium has an important role in the thylakoid lumen in facilitating
H2O dehydrogenation and O2 evolution.

41
5. METHODOLOGY
5.1 Experimental setup

The experiments basically consist of a simple setup consisting of the following:

1. Recycled PET bottles

2. Algae
3. Nutrients
4. Water
5. Stand
6. Tube light

5.2 Chemicals

The chemical compounds used are:

NaHCO3, NH2CONH2, (NH4)2SO4, KH2PO4, K2HPO4, CaSO4.2H2O, MgSO4.7H2O and Distilled
water.

5.3 Preparation of the stock solution

5.3.1 Nutrient media 1: Ammonium Sulphate and Urea

Stock solution of 1400 ml of nutrients is prepared using ammonium sulphate ((NH 4)2SO4);
molecular weight 132.14 and urea (NH2CONH2); molecular weight 60.06. For 1400 ml of stock
solution of nutrients, 14 g of ammonium sulphate and 14 g urea is dissolved in 1400 ml of
distilled water. With this stock solution at various concentrations of sodium bicarbonate
(NaHCO3) i.e. 20 mM, 30mM, 40mM, 50mM, 60mM, 100mM and 150mM, 200 ml of each of
the solution is prepared in seven PET bottles. Further 2 g of culture i.e. algae from fresh pond is
added in each of the bottles.

5.3.2 Nutrient media 2: Minimum Salt Media (MSM)

Same as Nutrient media 1, another media with stock solution of 1000 ml of MSM nutrient is
prepared using the following composition.

42
 S. No. Constituents Quantity (g/L)
1 (NH4)2SO4 1
2 MgSO4.7H2O 0.5
3 CaSO4.2H2O 0.05
4 K2HPO4 0.8
5 KH2PO4 0.3

Fig.5.1: MSM Composition

This time only two samples were prepared by dissolving the above components in table 2 in
1000ml of distilled water. With this stock solution two solutions at concentrations of 40mM and
50mM of NaHCO3 are prepared with 200 ml solution of each. Further 2 g algae are added to two
of the solutions in the PET bottles.

5.4 Algae culture Conditions

The samples of algae were collected from Regional Park, Indore. After the preparation of stock
solution and adding algae in different concentrations of NaHCO 3, the temperature was maintained
at round 260C and providing light source through tube light.

Fig.5.2: Algae Cultivation Day 1 Fig.5.3: Algae Cultivation Day 9

5.5 Kinetic study

43
The kinetic study is carried out for 12 days with an interval of 24 hrs. The study is performed at
different concentrations of NaHCO3. With nutrient media 1, 20mM, 30mM, 40mM, 50mM,
60mM, 100mM and 150mM concentration of NaHCO 3 are prepared from the stock solution of
exactly 200 ml each. And similarly with nutrient media 2, 40mM and 50mM concentrated
solutions are prepared. The experiment is conducted at approximately 26 0C. It is observed that the
samples of 100mM and 150mM could not survive the test. So study for the CO 2 fixation of other
remaining bottles was carried for 12 days.

5.6 Effect of nutrient media

5.6.1 Nutrient media 1: Ammonium sulphate and Urea

After the complete observation of 2 days it was found that the samples with 100mM and 150mM
didn’t show an appreciable growth and were found to be dead. While the other samples are found
to be growing.

5.6.2 Nutrient media 2: MSM

The samples in this media tend to show better growth that the nutrient media 1. The values are
found to be much higher.

5.6.3 Growth Analysis

Growth analysis is done in order to check the growth of the samples on daily basis. Dry weight
was taken in a time interval of 24 hrs in order to check the biomass. Further a growth rate curve is
plotted and specific growth rate, maximum productivity, biomass productivity per day and CO 2
fixation rate are determined for the species at different concentrations.

6. RESULTS AND DISCUSSION

6.1 Kinetic study analysis
The research was conducted for 12 days for both the media. The analysis of kinetic study is
carried out by formative biomass of algae cultivated at two different nutrient media. Hence
specific growth rate is estimated through the plot between the no. of cell vs time, Finally CO 2
fixation rate was calculated at different concentration of NaHCO3 for both the media.

44
6.2 Growth curve Phases
There are four phases in growth curve.
i. Lag phase: During this phase species adapt themselves to growth conditions.
ii. Exponential phase or log phase: It is also known as Log phase. It is a period characterized
by cell doubling. The number of new species appearing per unit time is proportional to the
present population.
iii. Stationary phase: Stationary phase results from a situation in which growth rate and death
rate are equal. Due to a growth-limiting factor such as the depletion of an essential
nutrient, and/or the formation of an inhibitory product such as an organic acid.
iv. Death phase: species run out of nutrients and die.

Biomass vs. time plot is shown in below table for nutrient media as ammonium sulphate and urea
and for MSM media correspondingly. In case of Ammonium sulphate and urea the most
favourable growth was found to be in case of 50mM NaHCO 3 as compared to other
concentrations till 6th day. After that the growth ceases and death phase starts. In case of MSM
the growth occurs till 8th day for both 40mM and 50mM NaHCO3 and the decrement in growth
was experiential.

45
 0.020
20 mM
0.018 30 mM
40 mM
0.016 50 mM
60 mM
0.014
Biomass (g/mL)

0.012

0.010

0.008

0.006

0.004

0.002

0 1 2 3 4 5 6 7

Days

Fig.6.1: Biomass growth curve for Media 1 (Ammonium sulphate-Urea)

40 mM
0.010 50 mM

0.008
Biomass (g/mL)

0.006

0.004

0 1 2 3 4 5 6 7 8 9
Days

46
 Fig.6.2: Biomass growth curve for Media 2 (MSM)

6.3 Specific growth rate determination

Specific growth rate is the increase in cell mass per unit time. Is is expressed in time -1. It provide
information of how fast the cells are multiply in a culture. It is mathematically expressed as:-

dX/dt = μX

Where X is concentration of cell mass, μ is growth rate constant and t is time .

Further integrate this equation between the limits 0 and t and X and X0 following equation is
obtained

ln X2/X1= μ( t2 −t1)

The plot of ln(X/X0) vs time is plotted and linearly fit to conclude specific growth rate. The slope
of the curve will define μ value. Linear fit curve for two different media are shown below in
Figure 13 and 14 correspondingly.

47
 Days
Fig.6.3: Ammonium Sulphate-Urea Graph

48
 Fig.6.4: MSM Graph

6.4 CO2 Fixation rate determination

CO2 fixation is distinct as conversion of carbon dioxide into some other organic compounds that
are requisite for growth. As for this case NaHCO3 is converted into HCO3-.

CO2 Fixation rate

= (Carbon content in algae × Mol. Wt. of CO2 × Biomass productivity per day)/ Mol. Wt. of carbon

= 1.87 × Biomass productivity per day

49
  Ammonium Sulphate-Urea
Samples Specific Adj. R Max. Biomass CO2 Fixation
Productivit Productivity/Da Rate(g/mL/Day)
Growth Square
y (g/mL) y (g/mL/Day)
Rate
20mM 4.80E-04 0.8424 0.002 0.00033 0.00062
NaHCO3
9
30mM 0.00204 0.4356 0.0068 0.00170 0.00318
NaHCO3
1
40mM 0.36366 0.7221 0.0016 0.00320 0.00598
NaHCO3
1
50mM 0.32135 0.8881 0.0098 0.00163 0.00305
NaHCO3
9
60mM 0.37716 0.8923 0.00898 0.00180 0.00336
NaHCO3
4

Fig.6.5: CO2 Fixation Rate

Ammonium Sulphate-Urea

 MSM

Samples Specific Adj. R Max. Biomass CO2 Fixation

Growth Square Productivity Productivity/Day Rate(g/mL/Day)
Rate (g/mL) (g/mL/Day)
40mM 0.13722 0.74165 0.00642 0.00080 0.001501
NaHCO3
50mM 0.13063 0.89998 0.00662 0.00083 0.001547
NaHCO3

Fig.6.6: CO2 Fixation Rate MSM

7. CONCLUSION AND FUTURE SCOPE

50
7.1 Algal oil production

The fall of fossil fuel minerals has caused an rise the need and cost of diesel. The implausibility in
their accessibility is measured to be the important set off for researchers to investigate the option
sources of energy, which can enhancement or return fossil fuels. Presently, biodiesel is formed
from different crops, such as, soybean, rapeseed, sunflower, palm, coconut, jatropha, karanja, used
fried oil and animal fats. There will be certain margins in the use of these oils as alternate fuels
because of its food demand lifetime, lower yield, higher land usage and higher cost inter alia.
Among the various biomass candidates for bio fuel production, microalgae are being measured as
a more feasible feedstock because microalgae are aquatic, non-edible, highly genetically
changeable and fastgrowing with efficiency 3–35 times higher than global plants in terms of
energy content. Also, cultivation of microalgae is less water demanding than terrestrial plants.
Algal Samples A total of three fresh water algae samples were collected. Algal biomass was
collected from fresh water bodies by mesh net and instantly after collection the laboratory, air
dried for two days later on dried at 40 C in an oven for 2-3 days till the dry weight was steady.
Apart fresh algae samples were observed for detection and conserved in 4% formaldehyde and
deposited at Phycology. The algal samples collected and analyzed were identified as - Spirogyra,
Hydrodictyon, Pithophora. Dried algal biomass (5g) was taken in solvent mixture (100 ml) of
chloroform and methanol (2:1, vol./vol.) and the substance were refluxed for 4 hrs. After the
extraction, the contents were cooled and filtered (or centrifuged) to break up the biomass and
washed the biomass with 25 ml of chloroform twice to extract the residual lipids here in the
biomass. The extracts were pooled and taken in a separating funnel and washed with 1% aqueous
sodium chloride solution (50 ml) two times. The solvent layer was passed through anhydrous
sodium sulphate (sodium sulphate was taken in a glass funnel with cotton plug) and separated the
solvent using rota-evaporator under vacuum to get the algal oil. The weight of algal oil was taken
to conclude the oil content in biomass. Algal oil characters were compared with bio fuel standards.
Biodiesel is typically a mixture of fatty acid alkyl esters, acquired by transesterification of oils or
fats. The algal oil has been transformed to FAME (Fatty Acid Methyl Esters) by the
transesterification process. The extracted algal oil is preheated to 60ºc and added to a mixture of
methanol and sodium hydroxide. It was then mixed with a blender for an hour. The methanol used
was 20% of the amount of algal oil used. 3.5 g of sodium hydroxide was used for a jumble of oil.
After the tranesterification process the mixture get is collected and kept for 8 hours or more to

51
allow the glycerine to settle at the bottom and the Fatty Acid Methyl Ester (Biodiesel) will form
the top layer. The biodiesel is detached and washed with water 4-5 times and was heated or dried
in air to evaporate the enduring water. It is important to remove most of the water in the oil as any
remaining water may affect the combustion process and hence affects the engine operation. The
water content was found to be less than 0.20% in the current methyl ester which is typical for
biodiesels. While humanity essentially needs energy for its existence, it must seem for the sources
which are renewable and limitless. In recent years, biomass-imitative fuels have received growing
attention as a solution to our nation’s continued and increasing dependence on imported oil, which
exposes the nation to the risk of crucial Break-down in fuel supply, creates economic and social
worries for businesses and persons, and effect our national security. Algae are a very assorted
group of mainly aquatic photosynthetic organisms that report for almost 50 % of the
photosynthesis that takes place on Earth. Algae have a wide range of antenna pigments to produce
light energy for photosynthesis giving different types of 88 algae their attribute colour. Algae are
anticipated to play a role in the global carbon cycle by helping remove over carbon dioxide from
the environment. Recently, algae are known as a capable biodiesel source due to their proficient
absorption and change of solar energy into chemical energy. Depending on size, algae are
classified as microalgae or seaweed. Microalgae are unicellular micro-organisms. Microalgae are
considered as prokaryotes and eukaryotes. Organelles are the major variation between prokaryotes
and eukaryotes. Prokaryotes do not have chloroplast, mitochondria and nuclei but they have
chlorophyll a and high protein contents. Microalgae are further divided into different groups based
on their taxonomy, as well as blue-green, green, yellow-green, red, brown, and golden algae.
There are more than 50,000 variety of microalgae. Microalgae can also be characterized based
upon carbon supply. Some microalgae use inorganic carbon such as CO2, are well-known as
autotrophs. Autotrophs carry out photosynthesis using light as energy source while heterotrophic
microalgae utilize organic carbon as sugars. There are some type which can use both, organic and
inorganic carbon sources are called mixotrophs. Biodiesel production due to quicker growth and
easier cultivation.

7.2 Biodiesel production

52
Biodiesel can be received from edible oil seed crops such as sunflower, palm, rapeseed, soybean,
coconut, etc. which are measured as the first generation biodiesel feedstocks. However, use of
such feedstocks for biodiesel production has certain problems as they disturb the worldwide
balance of food reserves and safety. The non-edible seed crops of jatropha, karanja, jojoba, mahua
and waste cooking oil, grease, animal fats, etc. have gained value in the last few years as the
second generation feedstocks for biodiesel production. However, these second generation
feedstocks are not enough to entirely replacement the present transportation requirements. Recent
focus is on microalgae using as the third generation feedstock . Using microalgae has many
advantages, like microalgae have capability to fix atmospheric CO2 and change it into sugars,
which are then change into fuel after biochemical processing , microalgae do not race for land and
can grow up anywhere - freshwater, brackish water, and also in wastewater , they require less
water and nutrients for their 89 growth as compared to terrestrial crops and microalgae have high
growth rate and increase lipids up to 70 % in their cell body - potential oil yields from assured
algae strains are projected to be at least 60 times higher than from soybeans, about 15 times more
constructive than jatropha, and about to 5 times that of oil palm per acre of land on an annual
basis. although these advantages, the scale-up applications of microalgae biofuels have some
technical boundaries . A process of biodiesel production from microalgae can consist of
cultivation, harvesting/dewatering, extraction, and adaptation to biodiesel.

As this study mention, carbon dioxide emission might be the cause of global warming, and one
way to decrease the emission is by algae. Like all living belongings, an algae requirements the
accurate environment in order for it to do at its best, and, for this case, capturing carbon dioxide.
In this study, the specific growth rate of algae, CO 2 fixation amount, biomass productivity is
resolute at different concentration of two different media. For nutrient media 1 i.e. with
ammonium sulphate and urea as nutrient , the concentrations tested are 20mM, 30mM, 40mM,
50mM, 60mM, 100mM and 150mM of NaHCO3, where 60mM is found to show the best growth
rate. The growth rate was found to be 0.3376 cells/day.

For nutrients media 2 with MSM as nutrient, the concentration tested is 40mM and 50mM.
Where, 40mM of NaHCO3 was found to be optimum for the growth of algae. The growth rate is
found to be 0.13722 cells/day.

53
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