project report viii semester (2015-16) design and

Project Report – VIII Semester (2015-16)

“Design and fabrication of economically viable Hybrid

Photobioreactor (closed bubble column) prototype for the

cultivation of elite Microalgae for enhanced lipid (biodiesel) yield”

This project has been supported by

Karnataka State Council for Science and Technology, Indian Institute of Science,

& Karnataka State Bio energy Development Board, Government of Karnataka.

KSCST_KSBDB_39S_B _BE_005

AAKRUTI RUIA 1NH12BT004

ANJALI TIWARI 1NH12BT009

AMULYA GRACE 1NH12BT034

Under the guidance of

(Dr.) R. S. UPENDRA

Senior Assistant Professor, Department of Biotechnology

Dr. PRATIMA KHANDEWAL

Prof & Head, Dept. of Biotechnology

DEPARTMENT OF BIOTECHNOLOGY

CERTIFICATE

Certified that the project work entitled “Design and fabrication of Photobioreactors for the

mass cultivation of Microalgae and other value added products” has been carried out by Ms.

Aakruti Ruia, Ms.Anjali Tiwari, Ms.Amulya Grace respectively bearing USN 1NH12BT004,

1NH12BT009, 1NH12BT034, bonafide students of New Horizon College of Engineering.

Signature of the Guide Signature of the HOD Signature of the Principal

ACKNOWLEDGEMENT

“This gratification and euphoria that accompany the successful completion

would be incomplete without the mention of the people who made it possible,

whose constant guidance and encouragement served as a beacon of light and

crowned our efforts with success”

We would like to profoundly thank our Management, New Horizon

College of Engineering for providing such a healthy environment for

successful completion of project work. We would like to express our sincere

thanks to Principal, Dr. MANJUNATHA for his encouragement that

motivated us for successful completion of project work.

We wish to express our gratitude to Research Head of Biotechnology

Department, Dr. PRATIMA KHANDEWAL for providing a good working

environment and for their constant support and encouragement.

We are extremely thankful to our internal guide (Dr.) R.S. UPENDRA for

his constant support, inspiration and valuable guidance throughout the period of

the project.

We are very thankful to KSCST for funding this project and embarking its

completion

Finally we thank all the staff of NHCE, Biotechnology Department and all

those who have helped us and contributed directly and indirectly towards the

successful completion of the project work and also our parents for providing

unconditional support and encouragement for carrying out the project work.

AAKRUTI RUIA, 1NH12BT004

ANJALI TIWARI, 1NH12BT009

AMULYA GRACE, 1NH12BT034

ABSTRACT

Combustion of the fossil fuels is the main source of green house gases and the major cause of

global warming today. Greenhouse gases mitigation is one of the most important methods to

reduce the harmful effects of greenhouse gases hence global warming. At the present

scenario, world is looking for alternative renewable energy resources to substitute fossil

fuels. Microalgae are unicellular organisms that assimilate lipids which can be utilized for

biodiesel production. Microalgae as a feedstock for biodiesel production minimizes the

damages caused to the eco system. Scanty research was documented on using Microalgae as

feedstock for Biodiesel production. Photobioreactor (PBR) is specially designed for effective

cultivation of microalgae, however hybrid PBR have been meagerly researched. Scanty

research has been done on tubular type PBR and the various light source tested being of less

impact on the growth of microalgae. With the lacunae discussed the present investigation

aimed in designing a hybrid PBR for mass cultivation of a newly isolated microalgae species

and also to enhance the yield of biomass and lipid content. The present study designed a

hybrid PBR (flat plate and tubular) based on both batch and continuous kinetic modules.

Study utilized LED as the source of artificial blue light to support the growth of microalgae.

Initially preserved microalgae culture was revived on BBM plate. The purity and metabolic

stability of the revived microalgae was accessed through morphological and microscopic

(both light and SEM) observations. The designed and fabricated hybrid PBR was tested for

microalgae cultures, in both batch and continuous cultivation process (Turbidostat).

Optimized modified Bolds Basal media (BBM) was used in the present investigation. Further

the study compared the growth of microalgae and its biomass yield at both optimal (PBR

cultures) and non-optimal (Flask cultures) conditions. Further growth kinetics of the

microalgae was studied measuring the absorbance at visible range (550nm). Finally lipid

estimation was carried out considering certain time interval for both the media. The results

reported that the indigenously designed photobioreactor successfully grew microalgae in

optimal conditions. The molecular and Phylogenetic analysis revealed that the microalgae

spp is Chlorella rotunda that is not till date has been used for biofuel production. The lipid

estimation carried out revealed that the lipid concentration of PBR cultures was 2.4 mg/ml is

4 folds higher than the flask cultures which was o.24 mg/ml. Also the doubling time was

reduced from 63 hours to 2.88 fours using PBR. A second batch was done to further reduce

the doubling time in PBR providing carbon dioxide source to the PBR. The doubling time

was reduced in the second batch to 1.5 hours and the lipid content increased further to 3.7

mg/ml which is 1.5 folds higher than the previous batch.

TABLE OF CONTENTS

Certificate

Acknowledgement

Abstract

Chapter 1 Introduction 01

1.1 Present Scenario 02

1.2 Introduction to the area of work 02

Chapter 2 Literature Review 05

2.1 Introduction 06

2.2 Rationale 11

2.3 Lacunae 13

2.4 Objective 13

Chapter 3 Material and Methods 14

3.1 Materials 15

3.1.1 Lab Instruments 15

3.1.2 Glassware 15

3.1.3 Materials for fabrication 15

3.1.4 Chemicals 16

3.2 Methods 17

3.2.1 Overall Methodology of the Project 17

3.2.2 Design of photobioreactor 18

3.2.2.1 Flat plate photobioreactor 18

3.2.2.2 Tubular Photobioreactor 19

3.2.2.3 Hybrid photobioreactor 20

3.2.2.4 Continuous hybrid Photobioreactor 22

3.2.3 Subculturing and revival of Mother Culture 23

3.2.4 Morphology analysis of microalgae 23

3.2.5 Molecular and Phylogenetic analysis 24

3.2.6 Inoculum in PBR and Flask culture and optical Density

at different time intervals 25

3.2.7 Lipid Estimation at different time intervals 26

3.2.8 FTIR Analysis 26

3.2.9 Batch and Continuous kinetics 27

3.2.1.1 Batch Kinetics 27

3.2.9.2 Continuous kinetics (Turbidostat) 28

3.2.10 Second Batch of Culturing in PBR and Flask 29

Chapter 4 Results 30

4.1 Design of Photobioreactor 31

4.2 Morphological Analysis of microalgae 31

4.3 Molecular and Phylogenetic analysis 32

4.4 Inoculum in PBR and Flask culture and optical Density

at different time intervals 33

4.5 Lipid Estimation at different time intervals 34

4.6 FTIR analysis 37

4.7 Batch and Continuous Kinetics 38

4.8 Second Batch of Culturing In PBR and Flask 39

Chapter 5 Discussion 44

Chapter 6 Conclusion 45

6.1 summary of the work done 45

6.2 Summary of overall outcome of project 45

Chapter 7 References 46

Chapter 8 Annexure 48

8.1 Chemical composition 48

8.2 Abbreviations 53

List of Tables

Table no. Title of table Page no.

1.1 Generation of biofuels 2

2.1 Review table 9

2.2 Biofuel sources Comparision 12

4.1 Morphology Table 31

4.2 Interpretation of FTIR Analysis 37

5.1 Yield comparison with other papers 44

8.1 Optimized Bolds Basal Media components

concentration

51

8.2 Lugols Solution Components 51

8.3 Sulpho-phosphovanillin Reagent Components 52

8.4 Extraction Buffer components for Isolation of

DNA

52

8.5 PCR components for Amphlification of DNA

sequence

52

List of Figures

Figure no. Title of figure Page no.

1.1 Pie chart showing World utilization percentage of biofuels 4

3.1 A ) Helical tube fabrication

B ) Acrylic sheet

C ) Blue LED light

16

3.2.1 CAED design of Flat plate Photobioreactor 19

3.2.2 CAED design of Tubular Photobioreactor 20

3.2.3 CAED design of Hybrid Photobioreactor 21

3.2.4 CAED design of Continuous Hybrid Photobioreactor 22

3.2.5 Growth curve of microalgae showing growth phases 28

4.1 Completed hybrid Photobioreactor 31

4.2 Chlorella rotunda at 100x 32

4.4 Gel analysis 33

4.5 Phylogenetic tree 33

4.6 Growth in first week 34

4.7 Growth in second week 34

4.8 Growth in third week 34

4.9 Growth in first week 34

4.10 Growth in second week 34

4.11 Growth in third week 34

4.12 Graoh of optical density camparision between flask culture

and PBR

35

4.13 Lipid comparision 36

4.14 Lipid comparision in bar chart 36

4.15 Std. FTIR algal graph 37

4.16 Sample FTIR graph 37

4.17 Design of PBR 40

4.18 Growth after 4 days 41

4.19 Growth after 8days 42

4.20 Biomass estimation graph comparing absorbance if batch

1PBR and batch 2 PBR

42

4.21 Comparisionof lipid concentration of batch 1 PBR and batch 2

PBR

42

4.22 Comparision of Lipid concentration 42

Design and fabrication of economically viable Hybrid Photobioreactor (closed

bubble column) prototype for cultivation of elite microalgae for enhanced lipid

(biodiesel)l yield

2016

1 Department of Biotechnology, NHCE

CHAPTER – 1

INTRODUCTION



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CHAPTER-1

Introduction

1.1 Present scenario

The present era relies on fossil fuel combustion to produce their fuels. Fossil fuels are non-

renewable sources of energy, and are rapidly depleting. The main source of fuels is by coal at an

average 49% of fuels are obtained from burning coal, but the present studies being conducted by

scientists’ estimates that by the year 2051 coal will be depleted (Bauer et.al, 2015). However,

fossil fuels combustion leads to the emission of carbon in the environment, the increasing

amount of carbon is leading to global warming, because carbon dioxide has the ability to

increase the temperature of the atmosphere by trapping the heat it’s a major cause of global

warming. Due to these reasons scientists are now looking for new ways to produce fuels in an

eco-friendly manner.

1.2 Introduction to the area of work

Biofuels are fuels that can be produced by utilizing organic matter derived from plants, animals

or microorganisms (Rudolf, 1926). Biofuels are a key factor that can reduce the global warming

issue and also meet the rising demand of fuels. Biofuels have been researched upon for a long

time and have been modified, thus there are 4 generations of biofuels. First generation of

Biofuels was basic agricultural crops like wheat and sugar were abstracted to give oils or

bioethanol. The second generation was non-food crops related such as wood, crop waste etc. The

third generation used algal based oil production. Fourth generation which are most researched

upon and utilized are engineered microalgae for lipid extraction based biofuels (Yafei et.al,

2014)

Table 1.1: Generations of Biofuels

GENERATION

TYPE

1st Agricultural food crops based (e.g. Wheat, sugar etc)

2nd Agricultural non-food crops (e.g. Wood etc)

3rd Algal species used for oil production

4th Engineered microalgae for lipid based biofuels extraction



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Microalgae are unicellular photosynthetic organisms that assimilate lipids, which can be

extracted for biofuel production (Clayton et.al, 2010). Microalgae help to mitigate carbon

dioxide, because of their photosynthetic properties consume carbon dioxide and utilizes it to

produce lipids. Microalgae are broadly classified into many types the classification is based on

its characteristics. Microalgae classification includes:

I. Dunaliella

II. Pleurochrysis carteroe

III. Chlorella

IV. Brotyococcus braunii

Microalgae biomass is a zero waste generation, as every part of the biomass is utilized to

generate other value added products. Microalgae are entirely an eco-friendly process for the

production of biofuels. The various value added products that can be generated using Microalgal

biomass are:

a) Bioethanol

b) Nutraceuticals

c) Biofertilizer

d) Biodiesel

e) Gasoline

Biofuels are being adapted in various countries as alternative fuels. The natural fuels are being

researched upon for their beneficial properties and environmental friendly nature. Based on

surveys conducted 42% of biofuels utilization is seen in the US, followed by 29% adaptation in

Brazil and 18% utilization by Europe, other countries like Thailand, China and Indonesia are just

starting to use biofuels as an alternative energy resource (Herve et.al, 2011). Photobioreactors

(PBR) are specially designed bioreactors to provide optimal conditions for enhanced growth of

microalgae. There are three basic types of PBR namely, open type, closed type and hybrid type

(Monlina et.al, 2000). The present study is based on hybrid type photobioreactor.



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Fig 1.1: Pie Chart showing World utilization percentage of Biofuels



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CHAPTER – 2

REVIEW OF LITERATURE



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CHAPTER- 2

LITERATURE REVIEW

1.1 Introduction

A wide range of review has been done by studying different research papers from the year 1995

to 2014. This was done to derive the lacunae and set our objectives and design

an indigenous project based on the lacunae derived from review papers

Velea et.al, studied a new hybrid photobioreactor, which combines the advantages of an open

system with those of a flat-plate photobioreactor was developed to improve high surface-to-

volume ratio of the photobioreactor and the photosynthetic efficiency by enriched CO2-

sequestration via bubbling of CO2 into the culture medium to achieve high biomass

productivities. To evaluate the performance of this photobioreactor, we performed a case study

assessing its biomass productivity and the efficiency parameters associated with the

conversion of carbon dioxide in the algal photosynthesis process for cultures of Chlorella

homosphaera.

Naqqiuddin et.al, studied a simple floating photobioreactor (PBR) experiments that were placed

on water bodies without any facilities of computerized controlled systems. The idea is to study

the effects of different photobioreactors shape and different aeration placement on the

productivity of Arthrospira platensis (Spirulina). In this study, simple floating PBRs were

designed in two different shape form using water container, Polyethylene terephthalate (PET)

materials. Simple land PBR was prepared with High-density Polyethylene (HDPE) plastic bag,

(25cm x 50cm). All PBRs were aerated from both top and bottom either with or without air stone

for 10 days of A. platensis cultivation with daily monitoring of growth parameters.

Sawdon studied the internal deoxygenating of tubular photobioreactor for mass production of

microalgae by perfluorocarbon emulsions. In industrial scale-up of closed tubular

photobioreactors, hypercritical oxygen concentration is one of the dominating detrimental factors

limiting the mass culture of microalgae in tubular systems. However, published accounts on

alleviating oxygen stress imposed on large-scale tubular photobioreactors are scarce. In order to

tackle the problem of high concentrations of dissolved oxygen strongly inhibiting microalgal

biomass production, an innovative methodology has been developed which involves gradually



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supplying carbon dioxide and removing the accumulated oxygen through the use of PFC

emulsions

Mottahedeh et.al, constructed a A low-cost, durable, high surface-to-volume integrated

translucent pond-photobioreactor (PBR) rapidly assembled by enclosing a particularly long semi-

rigid, rollable fiber glass sheet into a repeating pattern of height-adjustable shape-sustaining

supports. The elevated pond-PBR includes a low-cost temperature control, gas mixing and

underside solar reflector. The entire system is fully collapsible. A low-cost integrated pond-

photobioreactor for biomass production comprising an elongate thin, flat, bendable, rollable,

transparent/translucent semi-rigid plastic sheet a repeating pattern of shape-sustaining supports

such as C-shaped brackets and sustaining a C-shape configuration along said plastic sheet length;

the plastic sheet C-shape configuration defining a bioreactor chamber two water tanks a

repeating pattern of load-bearing structures for supporting in an elevated position said chamber

and exposing said chamber to sunlight from all directions including from underside, said

structures including, but not limited to, reverse U-shaped structures, accurate structures,

greenhouse structures, warehouse structures, and a combination there of the combination

enclosed plastic sheet, brackets, water tanks and support structures defining an integrated pond-

photobioreactor.

Csányi et.al, constructed a photobioreactor system that comprises a bioreactor including at least

two bioreactor tubes, each having an end and a hollow interior, the ends being connectively

joined by one or more connector units having a hollow portion defined by a circumference, a

solar concentrator configured to collect and concentrate solar power, at least one light guide

associated with the solar concentrator to illuminate the hollow portion of the one or more

connector units, and at least one LED illuminating the one or more connector units.

Kunjapur et.al, studied the general design considerations pertaining to reactors that use natural

light and photosynthetic growth mechanisms, with an emphasis on large-scale reactors.

Important design aspects include lighting, mixing, water consumption, CO2 consumption, O2

removal, nutrient supply, temperature, and pH. Though open pond reactors are the most

affordable option, they provide insufficient control of nearly all growth conditions. In contrast, a

variety of closed reactors offer substantial control, but few feature the likelihood for levels of

productivity that offset their high cost. One of the greatest challenges of closed photobioreactor

design is how to increase reactor size in order to benefit from economy of scale and produce

meaningful quantities of biofuel. This paper also highlights the concept of combining open and

closed systems and concludes with a discussion regarding a possible optimal

reactor configuration.



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Ling Xu et.al,studied Cultivating and harvesting of products from microalgae has led

to increasing commercial interest in their use for producing valuable substances for food, feed,

cosmetics, pharmaceuticals, and biodiesel, as well as for mitigation of pollution and rising

CO2 in the environment. This review outlines different bioreactors and their current status, and

points out their advantages and disadvantages. Compared with open-air systems, there are

distinct advantages to using closed systems, but technical challenges still remain. In view of

potential applications, development of a more controllable, economical, and efficient closed

culturing system is needed. Further developments still depend on continued research in the

design of photobioreactors and breakthroughs in microalgal culturing technologies.

Willson et.al, scalable photobioreactor system for efficient production of photosynthetic

microorganisms such as microalgae and cyanobacteria is described. In various embodiments,

this system may include the use of extended surface area to reduce light intensity and increase

photosynthetic efficiency, an external Water basin to provide structure and thermal regulation

at low cost, flexible plastic or composite panels that are joined together make triangular or other

shapes in cross section When partially submerged in Water, use of positive gas buoyancy and

pressure to maintain the structural integrity of the photobioreactor chambers and use of structure

to optimize distribution of diffuse light.

Grobbelaar et.al, studied the concept of a completely new and novel photobioreactor consisting

of various compartments each with a specific light regime is described. This is in response to the

debate and development which have taken place in recent years concerning photobioreactor

design and closed systems. It is well known that algae can photo-acclimate to various light

intensities. At the extremes, they can be high light (HL) or low light (LL) acclimated. Both HL

and LL acclimated algae typically have very specific characteristics indicating the plasticity of

the organisms, which have developed specific strategies during evolution to cope with

continuous and dynamic light fields. Not only are these considerations important in

photobioreactor design, but also for the production of certain biocompounds, whose synthesis

has specific light requirements.

Masojídek et.al, studied a novel type of closed tubular photobioreactor. This penthouse-roof

photobioreactor was based on solar concentrators (linear Fresnel lenses) mounted in a climate-

controlled greenhouse on top of the laboratory complex combining features of indoor and

outdoor cultivation units. The dual-purpose system was designed for algal biomass production in

temperate climate zone under well-controlled cultivation conditions and with surplus solar

energy being used for heating service water.

Molina et.al, studied the Principles of fluid mechanics, gas–liquid mass transfer, and irradiance

controlled algal growth are integrated into a method for designing tubular photobioreactors in

which the culture is circulated by an airlift pump. A 0.2 m3 photobioreactor designed using the



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proposed approach was proved in continuous outdoor culture of the microalga Phaeodactylum

tricornutum.The culture performance was assessed under various conditions of irradiance,

dilution rates and liquid velocities through the tubular solar collector.

Kun Lee et.al, constructed an α-shape tubular photobioreactor based on knowledge of algal

growth physiology using sunlight. The algal culture is lifted 5 m by air to a receiver tank. From

the receiver tank, the culture flows down parallel polyvinyl-chloride tubes of 25 m length and

2.5 cm internal diameter, placed at an angle of 25 with the horizontal to reach another set of air

riser tubes. Again the culture is lifted 5 m to another receiver tank, and then flows down parallel

tubes connected to the base of the first set of riser tubes. Thus, the bioreactor system looks

like the symbol α. As there is no change in the direction of the liquid flow, high liquid flow rate

and Reynolds Number can be achieved at relatively low air flow rate in the riser tubes.

Table 2.1: Review table

Sl

n

o

Author Year of

publicati

on

Journal Reactor

type

Highlights Lacunae

1.

Velea et.al 2014 Revista de

Chimie

Hybrid Hybrid

photobioreactor

designed

Analysis of

biomass with

kinetics studied

Lacked economic

studies of the

reactor

2.

Naqqiuddina 2014 Algal

biomass

utilization

Closed A simple

floating

photobioreactor

Two different

designs to

compare the

results

Only floating

bioreactor

discussed

Economical

aspects not

mentioned

3.

Sawdon

et.al

2014 Journal of

chemical

technology

Closed A new method

studied to give

optima growth

Novel method

found effective to

only tubular type



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and

biotechnolog

y

of microalgae photobioreactor

4. Mottahedeh

et.al

2012 Algal Bio

refinery

Hybrid Designing of

novel low cost

photobioreactor

Artificial light

source not used

5. Csanyi et.al 2012 Springer Tubular

Design of a

photobioreactor

using tubes and

solar panel

Temperature

control not

included

6. Kunjapur

et.al

2012 Industrial and

Engineering

Chemistry

research

Open

and

closed

Article mentions

about the

different systems

used for

microalgae

growth

Design

considerations

Economical

considerations not

mentioned

Less research on

Hybrid type

systems

7. Ling Xu

et.al

2009 Life

Sciences

All

types

This review

outlines different

bioreactors, its

advantages and

disadvantages.

This review

outlines different

bioreactors, it’s

advantages and

disadvantages 8. Willson

et.al

2008 Research

paper

Closed Discussion of a

photobioreactor

optimal for the

growth of

microalgae

Compromised

purification

9. Grobbelaar

et.al

2003 Applied

Phycology

Closed Concepts of new

Photobioreactors

is studied

Due to the mutli-

compartment

reactor the

contamination risk

is high 10

. Masojidek

et.al

2003 Applied

Phycology

Closed Novel tubular

photobioreactor

called

Climate conditions

not considered



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“Penthouse roof

reactor”

Supra high solar

conditions

taken 11

. Molina et.al 2000 Journal of

biotechnolog

y

Closed Fluid mechanics

method

integrated into

the design of

tubular

photobioreactor

Kinetics of the

reactor was

done

Not very

conclusive results

were found

12

. Kun Lee

et.al

1995 Applied

phycology

Closed

tubular

Design and

construction of

alpha tubular

reactor

Anti-foaming

agents not used

2.2 RATIONALE

The present study considered a photobioreactor for the enhanced growth of microalgae, as

photobioreactors provide optimal conditions that are required for microalgae growth.

Photobioreactors provide a platform to control various growth factors like temperature, pressure

and volume (Kunjapur et.al, 2010). Microalgae being photosynthetic organisms require good

amount of surface area and ample light, which is provided by a

photobioreactor. Hybrid photobioreactor is the highlight of this present study as Hybrid

photobioreactors combine the advantages of both open and closed photobioreactors and are

hence, capable of utilizing natural and artificial light source which can increase the microalgal

biomass and lipid concentration. LED has been used as a source of artificial light as it is more

efficient compared to the other sources utilized in the past and also helps to produce larger cells

of microalgae when compared to other sources of light. Chlorophyll b absorbs light most

strongly in the blue portion of the visible spectrum, hence blue LED light has been used which

can the wavelength ranging from 460nm to 660nm.There are various sources for producing

biofuels. The most commonly used sources are corn, soybean, canola, jatropha, coconut oil palm



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and microalgae (Chisti, 2007). The oil yield produced by corn is 172 L/ha, soybean is 446 L/ha,

coconut is 2689 L/ha, oil palm is 5950 L/ha and microalgae is 136900 L/ha. Hence microalgae

produces high amount of oil yield and the arable land required for microalgae production is

significantly less when compared to other sources.

Table 2.2: Biofuel sources comparison (Chisti, 2007).

Microalgae have many advantages when compared to other crop sources. Microalgae grows at a

faster rate i.e., they can double their numbers in very few hours, can be harvested daily, and have

the potential to produce a large volume of biomass and biofuel many times greater than that

of most productive crops. Like any other plant, algae, when grown using sunlight, consume (or

absorb) carbon dioxide (CO2) as they grow, releasing oxygen (O2). For high productivity, algae

require more CO2, which can be supplied by emissions sources such as power plants, ethanol

facilities, and other sources. Microalgae cultivation uses both land that in many cases is

unsuitable for traditional agriculture, as well as water sources that are not usable for other crops,

such as sea-, brackish- and wastewater. As such, algae-based fuels complement biofuels made

from traditional agricultural processes it can be cultivated to have a high protein and oil content,

for example, which can be used to produce either biofuels or animal feeds, or both. In addition,

microalgal biomass, which is rich in micronutrients, is already used for dietary supplements to

advance human health. Microalgae have grown both in seawater and freshwater. After oil

extraction, the remaining algal biomass can be used as fuel that is burned in industrial boilers and

other power generation sources (Mata et.al, 2009).Microalgae has wide range of application in

various industries. It is used in food industry for manufacture of food

additives, emulsifiers and thickeners, in pharmaceuticals for production of

antibiotics, antibacterial agents, cover of capsules and also used in manufacturing of cosmetics

and bioplastics. It is used as food, feedstock and animal feed. (Jeffrey Funk, 2012)



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2.3 LACUNAE

A detailed study of research papers led to a set of derived lacunae. It was found that very

less research was done on hybrid photobioreactors. Most papers studied flat plate or closed

tubular photobioreactors. Comparison of bio yield from different photobioreactors considering

both optimized and un-optimized conditions was not done. Chlorella vulgaris is the usually used

microalgae for biodiesel production. Chlorella rotunda has never been used till date for the mass

production of biodiesel. Chlorella rotunda is found to be euryhaline in nature i.e., they can grow

both at low salinity like freshwater and at high salinity like seawater.

2.4 OBJECTIVES

From the know rationale and derived lacunae objectives were set. The objectives for the present

study are:

1. Design and fabrication of hybrid photobioreactor (combing flat plate and tubular type

of photobioreactor).

2. Mass cultivation of newly isolated species (Chlorella rotunda) in

the indigenously designed photobioreactor.



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CHAPTER-3

MATERIALS AND

METHODS



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CHAPTER – 3

MATERIALS AND MATERIALS

3.1 Materials

3.1.1 Lab instruments

1) Measuring device, Model no. BL -220H 2) Autoclave 3) Laminar air flow 4) Shaking incubator, Model no. BT-ISI-E 5) Water bath 6) Cooling Centrifuge 7) Colorimeter

3.1.2 Glassware

1. Conical flasks (100ml , 500ml) 2. Glass rod 3. Beaker (100ml, 500ml) 4. Measuring cylinders (500ml, 100ml) 5. Pipettes 6. Petri dishes

3.1.3 Materials for fabrication

1. Acrylic sheets 2. LED lights 3. Plastic fittings 4. Pump 5. Aerator 6. CO2 cylinder 7. Metal fittings



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3.1.4 Chemicals

1. Salts for Bold's Basal Media 2. Concentrated sulphuric acid 3. Concentrated phosphoric acid 4. Vanillin 5. Absolute ethanol 6. Distilled water

A B C

Fig 3.1: A) Helical tube fabrication

B) Acrylic sheet

C) Blue LED lights



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3.2 Methods

3.2.1 Overall methodology of the project

DESIGN OF PHOTOBIOREACTOR

SUB-CULTIVATION AND REVIVAL OF MOTHER

CULTURE

MORPHOLOGY ANALYSIS OF MICROALGAE

MOLECULAR AND PHYLOGENETIC ANALYSIS OF

MICROALGAE

INOCULUM OF CULTURE IN PHOTOBIOREACTOR

AND FLASK AND OPTICAL DENSITY AT

DIFFERENT TIME INTERVALS

LIPID ESTIMATION AT DIFFERENT TIME

INTERVALS

FTIR ANALYSIS

A



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3.2.2 Design of Photobioreactor

The designing of photobioreactor was done on the basis of literature review studied. The present

study designed PBR with general considerations such as, width, length, area and volume. PBR

was designed to provide optimal area required for algal growth and also the conditions required

for the enhanced growth of microalgae.

The designs of PBR include the following:

3.2.2.1 Flat Plate Photobioreactor

A flat plate PBR was designed in order to increase the light utilization and reduce shadowing

effect of light. The study used acrylic sheets for transparency and economical aspects. The

design provided aeration via aerator to the tank, and an array of LED light was provided as the

artificial light source also an opening for harvesting was given.

A

BATCH AND CONTINUOUS KINETICS

SECOND BATCH OF CULTURING IN

PHOTOBIOREACTOR AND FLASK



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Dimensions include:

Length = 0.3 m

Breath = 0.2 m

Width = 0.15 m

Volume = L x B x W = 9 liters

Highlights:

a) Better light utilization

b) Higher yield

c) High biomass productivity due to better aeration

d) More economical

3.2.2.2 Tubular Photobioreactor

This reactor was designed for surface area utilization and was configured with a helical tube

connected to the mother tank via a pump. This design could be operated at both continuous and

batch conditions. The pump provided as a medium to supply the culture to the helical tubes. The

tubes were designed to be helical to avoid any type of shadowing effect and also to make it more

compact and efficient. A LED tube was positioned between the spirals for artificial light.

Fig 3.2.1: CAED design of a Flat Plate Photobioreactor



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Dimensions include:

Tank dimensions,

Length = 0.3 m

Breath = 0.2 m

Width = 0.15 m

Volume = L x B x W = 9 liters

Helical Tube = 1 feet

Volume = 1L

Highlights:

a) Better process control

b) Less contamination

c) Cell damage is less

** Complex design

3.2.2.3 Hybrid Photobioreactor

Fig 3.2.2: CAED design of Tubular Photobioreactor



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This design is completely a hypothetical design. A bubble column was considered and provided

with a spray ball for dispersion of air through the column. A set of tubes where designed to be in

a wheel type configuration and provided with a rotator motor for mixing and aeration. Though

this design had better surface to volume ratio it was very complex to be considered for

fabrication.

Dimensions:

Bubble column,

Height = 0.3 m

Diameter = 0.2 m

Volume = π r2 h = 9.42 litre

No. Of tubes = 4

Length of tubes = 0.2 m

Diameter of tubes = 0.04 m

Highlights:

a) Better agitation

b) Better dispersion of air

c) Higher surface to volume ratio

Fig 3.2.3: CAED design of Hybrid Photobioreactor



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**Hypothetical design

3.2.2.4 Continuous Hybrid Photobioreactor

To combine the advantages of both Flat plate and Tubular designs, the present study

indigenously designed a continuous process hybrid photobioreactor. This design had more

volume, was compact and had better light utilization. The LED array was arranged to run the

whole length of the PBR, hence every single component of the PBR was incident by the light. A

pump was given to provide media to the whole reactor. Aeration was provided via aerator.

Dimensions:

Tanks,

Length = 0.3 m

Breath = 0.2 m

Width = 0.15 m

Volume = 2 L x B x W = 18 liters

Fig 3.2.4: CAED design of Continuous Hybrid Photobioreactor



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Helical Tubes= 1 feet (2)

Volume = 2L

Total capacity = 20 liters

Highlights:

a) Collapsible

b) Operated at both kinetic modules ( Batch and Continuous)

3.2.3 Sub-Culturing and revival of Mother Culture

The mother culture used was optimized by seniors through artificial neural network (Mounisha

et.al, 2015). This sample culture was derived from Varthur Lake and Microalgal growth had

been processed.

The study used this media for sub-culturing and reviving it.

Sub-culturing was carried out by:

3.2.4 Morphology Analysis of Microalgae

To determine the purity of the mother culture Morphological characteristics were

determined and where done by microscopy analysis. The method involved the use of

Key chemicals weighed and added to a 1L conical flask

Distilled water added to dissolve the chemicals and make up the volume upto 1L

Autoclaved for 15-20mins

Media was cooled and 10 ml of mother culture was added under sterile conditions

Media was cooled and 10 ml of mother culture was added under

sterile conditions



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lugol’s solution (Gorgino et.al, 2011) and the methodology was carried out in the

following manner:

3.2.5 Molecular and Phylogenetic Analysis of Microalgae

Molecular analysis was carried out to determine the strain of microalgae spp as well as

the phylogeny of the species. This analysis was done by using bioinformatics software

and electrophoresis. The overall procedure that was carried out was as mentioned below.

1 drop lugoul's solution + 4ml of water + 1ml sample

this was added to the slide and spread, cover with cover slip

Visualised under microscope

The sequence similarity searching was done using BLAST software

Isolation of total genomic DNA using the CTAB method

A



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3.2.6 Inoculums in PBR and Flask cultures and Optical Density at different

time Intervals

After the sub-culturing was done the media was inoculated into the photobioreactor and conical

flask. The media was maintained in both optimal (PBR) and un-optimal (Conical Flask)

conditions. This was done to compare and establish the efficiency of the designed

photobioreactor under suitable conditions. For the photobioreactor 9L of media was prepared

using Optimized Bolds Basal Media (Mounisha et.al, 2015), the sub-culture was inoculated and

the overall volume maintained in the Photobioreactor was 10 L. At the same time 1L of media

with inoculums was kept in conical flask. The PBR was provided aeration and the LED lights

where switched on. To calculate the biomass and growth curve optical density was taken at

regular time intervals.

Optical density was taken after every 2-3days. The absorbance was measured using colorimeter

at 550 nm, where two samples were taken i.e., PBR samples and Conical flask samples and water

was taken as blank.

3.2.7 Lipid Estimation at different time intervals (Mishra et.al, 2014)

Since in microalgae the lipids are converted to biofuels, determining the concentration of lipids

present in microalgae is crucial. Lipid estimation was done using colorimetric method, where the

samples were taken from both PBR and conical flask. The present study also performed lipid

estimation to compare the yield efficiency of designed photobioreactor.

Firstly the sample was prepared:

A

The genomic DNA was then amplified using PCR

The Phylogenetic analysis was done using CLUSTAL software for oligonucleotide

primers



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200µl of sample in 100µl of water

Centrifuge at 4000 rpm for 5mins

Harvest the cells

Followed by sample preparation the lipid was estimated using colorimeter and sulpho-

phosphovanillin reagent. The sulpho-phosphovanillin was freshly prepared using vanillin and

conc. phosphoric acid.

3.2.8 FTIR Analysis

FTIR was done as a qualitative analysis. The analysis was done to determine the bonds present in

microalgae.FTIR were done following the general protocol in the analysis. The specifics

included reference as membrane lipids by OPUS Version.

Take 3ml of sample in labeled test tubes (PBR and OPT). Take 3ml of water as blank.

Add 2ml of conc. H2SO4

Keep at 100 C for 5 mins and cool it and add 5ml of phophovanillin

Incubate at 37 C in shaking incubator for 15 mins at 200 rpm

Take OD at 530 nm



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3.2.9 Batch and Continuous Kinetics

The study utilized both batch and continuous kinetics modules. Where PBR was kept at

continuous conditions at Turbidostat, and the Flask cultures were kept at Batch conditions.

3.2.9.1 Batch Kinetics (Andersen, 2005; Becker, 1994)

Eukaryotic microorganisms have 5 phases in a basic growth curve. The adaptation phase where

the cells adapt to the environment called the lag phase. The acceleration phase where the growth

start’s taking place. Exponential growth phase or the log phase where rapid growth takes place

after adaptation of the cells in the media and primary metabolites are produced. The stationary

phase were nutrients depletion causes the cells to stop growing and here secondary metabolites

are synthesized by the microorganisms. And finally the death phase where the microorganisms

die due nutrients depletion and toxicity of the media.

Using these phases equations were derived as follows:

Where,

n = concentration of cells (mg/ml) n0 = initial cell concentration

µ = net specific growth rate (hr-1)

t = time interval (hours) t0 = initial time interval

td = doubling time (hours)



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3.2.9.2 Continuous Kinetics (Turbidostat) (Sasca et.al, 2013)

The PBR was considered as a Turbidostat in the present study. The main important factor in

continuous conditions is the dilution rate or dilution factor, dilution factor is the ratio of flow to

volume and gives the rate at which the culture was diluted. At steady state the dilution rate is

equal to the growth rate. Following the same phases as the batch kinetics, a series of equations

were derived. The equations include:

Fig 3.2.5: Growth curve of Microalgae showing growth phases



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Where,

D = dilution factor

V = total volume of PBR ( L)

f = flow rate (L/m2)

rx = rate of cell formation

x = cell concentration (mg/ml) xo = initial cell concentration (mg/ml)

3.2.10 Second batch of culturing in PBR and Flask

Through Kinetics calculations the present study determined the doubling time of both PBR and

flask cultures, though the doubling time was greatly reduced in PBR, a second batch of culture

was done to enhance the growth further and reduce the doubling time in PBR.

The second batch of culturing was done by preparing 10L of Optimized Bolds Basal Media

(Mounisha et.al, 2015) and adding 1L of the first batch culture. This batch was also provided

with a carbon dioxide cylinder to give greater conditions of growth to the PBR.

The optical density was determined using colorimeter and taken at regular 2-3 days time, also

lipid estimation was done in the same manner as before using the same time intervals (Mishra

et.al, 2014)



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CHAPTER- 4

RESULTS



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CHAPTER 4

RESULT

4.1 Design of Photobioreactor

The present study indigenously designed and fabricated a Continuous Hybrid Photobioreactor

this consists of 2 tanks and 2 helical tubes, the tanks and helical tubes are made of acrylic sheets

and acrylic rods respectively. Blue LED lights are arranged in the form of array. The

photobioreactor also consists of a pump and aerator for better dispersion of air.

4.2 Morphological analysis of microalgae

In this present study morphological analysis was done to determine the purity of the mother

culture. Bold's Basal Media was optimized using Artificial Neural Network(ANN). Mother

culture was grown in the optimized media. The isolated microalgae was found to be yellow in

colour and flower shaped. The microalgae has a length of 10µm and width of 6.5mm.

Table 4.1: Morphology table

Morphology Chlorella rotunda

Colour Yellow

Shape Flower

Length 10µm

Width 6.5mm

Fig 4.1: Completed Hybrid Photobioreactor



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4.3 Molecular and phylogenetic analysis of microalgae

The present study gave the microalgae for molecular and phylogenetic analysis. The isolated

microalgae was found to be Chlorella rotunda. Sequential analysis was done using BLAST. The

bandwidth was found to be 1190 bp, gel analysis confirmed the bandwidth. Phylogenetic

analysis was done using CLUSTAL software, phylogenetic tree was obtained. Results concluded

that the Chlorella rotunda had 97% similarity with other Chlorella species.

Fig 4.2: Chlorella rotunda at 100x

magnification

Fig 4.3: Sequential analysis of Chlorella rotunda



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4.4 Inoculums in PBR and Flask cultures and Optical density at different

time

Microalgae was inoculated in PBR and Conical flask. Growth was seen in both PBR and conical

flask. On the first week not much growth was seen in PBR. On the second week very light

growth was seen and on the third week thick green colour had developed. The growth in PBR

was significantly very high was compared to growth in conical flask.

Fig 4.4: Gel Analysis Fig 4.5: Phylogenetic tree

Fig 4.6: growth in first week Fig 4.7: Growth in second week



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Fig 4.8: Growth in third week

Fig 4.9: growth in first week Fig 4.10: growth in second week

Fig 4.11: Growth in third week



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Optical density was taken every 2-3 day at 550 nm for both PBR culture and flask culture.

Optical density in PBR was found to be much higher than that of conical flask. A graph was

plotted by taking time on x-axis and absorbance at 550nm on y-axis.

4.5 Lipid estimation at different time intervals

In this study lipid content in the biomass was estimated for every 2-3 days, at both optimized

(PBR) and unoptimized (Conical Flask) condition. Lipid content calculated was compared and

graph was plotted by taking time on x-axis and concentration on y-axis. The result showed that

the lipid content in PBR was 2.5 mg/ml and in conical flask was 0.24mg/ml. Thus the lipid

content in PBR was 4 folds higher than conical flask.

Fig 4.12: Graph of optical density comparison between the flask cultures and PBR



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Fig 4.13: Lipid comparison

. Fig 4.14: Lipid comparison in Bar chart



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4.6 FTIR Analysis

In the present study Fourier transform infrared spectroscopy (FTIR) was done to verify the

presence of lipids in the biomass. The FTIR graph of the sample was compared with the standard

FTIR to determine the bonds present. The sample was found to have Lipid hydrocarbon chain,

esters and amides.

(Mahapatra et.al, 2011)

Table 4.2: Interpretation of FTIR analysis

Sl . No Wavenumber (cm-1) Bond representation Inference

1 429.05 Aromatic bending Alkanes are present

2 1638.81 C=O bonds Lipids are present

3 3254.64 OH bonds OH Stretch

4 3800.68 OH bonds OH Stretch

Fig 4.15: Std. FTIR algal graph Fig 4.16: Sample FTIR graph



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4.7 Batch and continuous kinetics

In this study kinetics was calculated for both batch( unoptimized) and continuous(optimized )

process. The net specific growth rate for batch process was 0.011 hr-1 and for continuous process

was 0.24 hr-1. The doubling time under unoptimized condition was 63 hr and under optimized

condition was 2.88 hr. Thus the doubling time was decreased significantly.

Calculation for batch kinetics:

µ = log ( X2 – X1) 2.303

t2-t1

µ = 2.303 (-1.301+ 2)

216-72

µ = 0.011 hr-1

td = log 2

µ

td= 63 hr

Where,

n = concentration of cells (mg/ml) n0 = initial cell concentration

µ = net specific growth rate (hr-1)

t = time interval (hours) t0 = initial time interval

td = doubling time (hours)

Calculation for continuous kinetics:

D = F/V

F= QxA

Total area= 0.12 m2

Total Volume= 18 L

F = 0.12 x 18= 2.16

D = 2.16/ 9 = 0.24



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µ= 0.24 hr-1

td= 2.88 hr

Where,

D = dilution factor





4.8 Second batch of culturing in PBR and Flask

In this study a second batch of culture was done. A helical tube was added to increase the area

and CO2 cylinder was provided to decrease the doubling time.

Calculation of continuous kinetics for second batch:

D = F/V

F= QxA

A= 0.22 m2 ( addition of helical tube)

Q= 21 L

F= 0.22 x 21 = 4.62

D= F/V

D=4.62/10

D= 0.462

µ= D

Therefore,

µ= 0.462 hr-1



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td = log 2

µ

= 0.693

0.462

td =1.4 hr-1

Where,

D = dilution factor





Fig 4.17: Design of second Batch of PBR with additional helical tube

and carbon dioxide cylinder



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Fig 4.20: Biomass estimation graph comparing the biomass

absorbance of batch 1 PBR and batch 2 PBR

Fig 4.18: Growth in 4 days Fig 4.19: Growth in 8

days



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Fig 4.21: Comparison of lipid concentration of batch 1 PBR and

batch 2 PBR

Fig 4.22: Comparison of lipid concentration under optimized and

un-optimized conditions



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CHAPTER-5

DISCUSSION



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CHAPTER-5

DISCUSSION

The present study designed and fabricated a hybrid photobioreactor that utilized blue LED lights

as artificial light source and the design consisted of an aerator that provided optimal mixing of

nutrients within the reactor. For the better utilization of space the tubes in the reactor were given

a spiral configuration. The study calculated growth kinetics in both batch and continuous

conditions to conclusively determine the growth rate and generation time. For the comparison of

biomass and lipid yield the study estimated the lipid and absorbance at different time intervals.

For the betterment of the generation time the study re-cultured a second batch for the PBR with

the addition of carbon source and extra helical tube.

In the study conducted by Velea et.al, the experiment considered a hybrid photobioreactor which

was given a carbon source and higher surface to volume ratio. The experiments were conducted

under different conditions; the volume of the study was 66 L. The resultant productivity was

found to be 0.68 mg/ml on the 11th day with the volume of 20 L, in comparison with the present

study the lipid yield was 3 times lower than batch 1 and 5 times lower than batch 2 cultures.

In the study conducted by Naquiddin et.al, the study considered a floating photobioreactor which

was a closed type reactor. The experiments were done under different aeration conditions such

as, top and bottom aeration. Also the experiments were carried out in different mixtures time.

The results were 0.781 mg/ml, whereas the present study gave 2.5 mg/ml in the first batch and

3.7 mg/ml in the second batch.

In study Masojidk et.al the study constructed a penthouse roof photobioreactor which measured

the irradiance of algae under super high solar power. It can be mounted on rooftops of houses. It

measured the photons intake in algae. This study gave results of 2.2 mg/ml of algal productivity,

this in comparison with the present study was low as the results of this study was 2.4 mg/ml in

batch 1 and 3.7 mg/ml in batch 2.

In the study Molina et.al, experiments were based on tubular solar collectors; the different

considerations were velocity, volume, liquid density and dilution rate. The results were found to

be 2.5 mg/ml which was same as the batch 1 yield but however less than batch 2 yields.

Table 5.1: Yields Comparison with other papers

Sl.No Organism Author Lipid content

Papers

Lipid content

PBR 1

Lipid content

PBR 2

1 Chlorella homosphaera Velea et.al 0.68 mg/ml

2.4 mg/ml

3.7 mg/ml

2 Asthrospira plantens Naquiddin

et.al

0.781 mg/ml

3 Asthrospira plantens Masojidk et.al 2.2 mg/ml

4 Phaeodactylun tricornutum

Molina et.al 2.5 mg/ml



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CHAPTER- 6

CONCLUSION



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CHAPTER- 6

CONCLUSION

6.1 Summary of the work done

The rising need for fuels and fast depletion of fossil fuels has led to the use of biofuels. Carbon

released in burning of fossil fuels is the main reason for Global warming; hence it’s highly

important to reduce carbon release in air. One of the major ways to mitigate carbon is through

the utilization of Microalgae as they consume carbon to give lipids which are extracted for the

production of biofuels. The present study successfully designed and fabricated a Hybrid

Photobioreactor that gave enhanced growth of chlorella rotunda with higher productivity in lipid

and biomass content. The indigenously designed photobioreactor was provided with an acrylic

sheet tanks, an aerator and also an array of blue lights which gave optimal conditions to the

reactor. As chlorella rotunda have thin cell wall the blue light intensity was properly absorbed.

The helical tubes provided good surface to volume ratio and the area was optimally utilized.

6.2 Summary of the overall Outcome of project

The molecular and Phylogenetic analysis revealed that the strain was relatively new having 95

% similarity and this was submitted in NCBI. The project calculated the growth kinetics in both

batch and continuous conditions to effectively compare the efficiency of the PBR. The study

calculated biomass and lipid in both flask and PBR cultures and compared the results. The

biomass yield in the flask cultures and PBR batch 1 was found to 0.08 and 0.57 respectively and

in PBR batch 2 the biomass yield was 0.95, thus it can be conclusively established that the

biomass yield in PBR in both the cases was higher than un-optimized conditions (flask

cultures).The lipid estimation revealed that the yield in flask cultures was 0.24 mg/ml, whereas

the content in PBR batch 1 was 21.8 % in volume of media and in PBR batch 2 it was 33.1 % in

culture volume of 10 L. The kinetics calculations done in the present study showed that the

doubling time was greatly reduced from 63 hours to 2.88 hours, which was further reduced to 1.4

hours in the second batch of PBR. The increase in surface area with an addition of helical tubes

successfully reduced the doubling time. The present study is also highly adaptable to pilot scale

and biodiesel can be extracted easily due to the presence of lipids.



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CHAPTER – 7

REFERENCES



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Chapter – 7

References

Aditya M. Kunjapur and R. Bruce Eldridge (2010): Photobioreactor Design

for Commercial Biofuel Production from Microalgae Industrial and Engineering Chemistry Research , 49: 3516–3526.

Alicia Sawdon and Ching-An Peng (2014): Internal deoxygenating of tubular

photobioreactor for mass production of microalgae by perfluorocarbon

emulsions. Journal of chemical technology and biotechnology, 90: 1426-1432

Bryan willson, Guy Babbitt, Peter letvin, Nicholas rancis, James Murphy

(2008): Diffuse light extended surface area water-supported

photobioreactor.

PUB. NO.: US 2008/0160591 A1

Istvan Csanyi, Laszlo Balazs, Janos Sneider, Erazmus Gerencser (2010) :

Solar hybrid photobioreactor PUB.NO.: US 8716010 B2

J. Masojídek, Š. Papáček, M. Sergejevová, V. Jirka, J. Červený, J. Kunc,

J. Korečko, O. Verbovikova, J. Kopecký (2003): A closed solar

photobioreactor for cultivation of microalgae under supra-high irradiance:

basic design and performance. Applied phycology, 15: 239-248

Johan U.Grobbelaar and N.Kurano (2003) : Use of photoacclimation in the

design of a novel photobioreactor to achieve high yields in algal mass

cultivation

Applied phycology,15:121-126

Ling Xu, Pamela J. Weathers, Chun-Zhao Liu (2009): Microalgal bioreactors:

Challenges and opportunities. Life Sciences, 9: 178-189



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Megan sumiko fulleringer, Edouard michaux, Derick R.poirier (2009) : Design

of a small scale cultivation system to produce biodiesel.

Mohamed Amar Naqqiuddina, Norsalwani Muhamad Nora, Hishamuddin Omara

& Ahmad Ismaila(2014): Development of simple floating photobioreactor

design for mass culture of Arthrospira platensis in outdoor conditions. Algal Biomass Utilization, 5: 46- 58

Molina E., J. Ferna´ndez, F.G. Acie´n, Y. Chisti (2000):

Tubular photobioreactor design for algal cultures.

Journal of Biotechnology , 92 : 113–131

Sanda velea, Lucia ilie, Emil stepan, Ruxandra chiurtu (2014): New

Photobioreactor Design for Enhancing the Photosynthetic Productivity of

Chlorella homosphaera Culture .

Revista de Chimie, 1:65

Yuan-kun lee, Sun-Yeun Ding, Chin-Seng Low, Yoon-Ching Chang, Wayne

L.Forday, Poo-Chin Chew (1995): Design and performance of an α-type tubular

photobioreactor for mass cultivation of microalgae. Applied phycology , 7: 47-51



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CHAPTER- 8

ANNEXURE



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CHAPTER-8

ANNEXURE

8.1 Chemical Compositions

Table 8.1: Optimized Bolds Basal Media Components Concentration

COMPONENTS WEIGHT(mg)

NaNO3 25

KH2PO4 17.5

K2 HPO4 10

MgSO4.7H2O 7.5

Cacl2.2H2O 2.5

NaCl 2.5

KOH 3.1

FeSO4.7H2O 0.5

H3BO3 1.114

ZnSO4. 7H2O 0.88

MnCl2. 7H2O 0.14

MoO3 0.07

CuSO4. 5H2O 0.15

Co(NO3)2. 6H2O 0.05

(Havarasi et.al, 2011)

Table 8.2: Lugols Solution Components

Components Weight

Potassium iodide (KI) 10g

Distilled Water

100ml



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Iodine 5g

Table 8.3: Sulpho-phosphovanillin Reagent Components

Components Weight

Vanillin 0.6g

Absolute Ethanol 10ml

Deionised water 90ml

These were stirred and added to 400ml of conc. phosphoric acid

Table8.4: Extraction Buffer components for Isolation of DNA

Stock Solution Buffer composition

1 M Tris HCl 100 mM Tris HCl

1M EDTA 100 mM EDTA

4 M NaCl 1.4 M NaCl

1% CTAB

Proteinase K - 0.03μg/μl

SDS 20% w/v

Chloroform: isoamyl alcohol (24:1)

Isopropanol

Ethyl alcohol 70% v/v

Table 8.5:PCR components for Amphlification of DNA sequence

PCR components Volume (μl)

Nuclease free water 10.75

10X reaction buffer with MgCl2 (1.5mM) 2.00

dNTP mix (2.5mM) 2.00

Primer 18S ALG FP (10picomoles/ μl) 2.00

Primer 18S ALG RP (10picomoles/ μl) 2.00



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Taq DNA polymerase (5U) 0.25

Template DNA (50ng/ μl) 1.00

Total volume 20.0

8.2 Abbreviations

1. PBR : Photobioreactor

2. LED : Light Emitting Diode

3. CAED : Computer Aided Drawing

4. PCR : Polymeric Chain Reaction

project report viii semester (2015-16) design and

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