DESIGN AND FABRICATION OF ECONOMICALLY VIABLE HYBRID

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 ANJALI TIWARI AMULYA GRACE

1NH12BT004 1NH12BT009 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, ANJALI TIWARI,

1NH12BT004 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 1.1 Present Scenario 1.2 Introduction to the area of work Chapter 2 Literature Review 2.1 Introduction 2.2 Rationale 2.3 Lacunae 2.4 Objective Chapter 3 Material and Methods 3.1 Materials 3.1.1 Lab Instruments 3.1.2 Glassware 3.1.3 Materials for fabrication 3.1.4 Chemicals 3.2 Methods 3.2.1 Overall Methodology of the Project 3.2.2 Design of photobioreactor 3.2.2.1 Flat plate photobioreactor 3.2.2.2 Tubular Photobioreactor 3.2.2.3 Hybrid photobioreactor 3.2.2.4 Continuous hybrid Photobioreactor 3.2.3 Subculturing and revival of Mother Culture 3.2.4 Morphology analysis of microalgae 3.2.5 Molecular and Phylogenetic analysis 3.2.6 Inoculum in PBR and Flask culture and optical Density at different time intervals 3.2.7 Lipid Estimation at different time intervals 3.2.8 FTIR Analysis 3.2.9 Batch and Continuous kinetics 3.2.1.1 Batch Kinetics 3.2.9.2 Continuous kinetics (Turbidostat) 3.2.10 Second Batch of Culturing in PBR and Flask Chapter 4 Results 4.1 Design of Photobioreactor 4.2 Morphological Analysis of microalgae 4.3 Molecular and Phylogenetic analysis 4.4 Inoculum in PBR and Flask culture and optical Density at different time intervals 4.5 Lipid Estimation at different time intervals 4.6 FTIR analysis 4.7 Batch and Continuous Kinetics 4.8 Second Batch of Culturing In PBR and Flask Chapter 5 Discussion Chapter 6 Conclusion 6.1 summary of the work done 6.2 Summary of overall outcome of project Chapter 7 References Chapter 8 Annexure

01

02 02 05 06 11 13 13 14 15 15 15 15 16 17 17 18 18 19 20 22 23 23 24 25 26 26 27 27 28 29 30 31 31 32 33 34 37 38 39 44 45 45 45 46 48

8.1 Chemical composition 8.2 Abbreviations

48 53

List of Tables Table no.

Title of table

Page no.

1.1

Generation of biofuels

2

2.1 2.2 4.1

Review table Biofuel sources Comparision Morphology Table

9 12 31

4.2

Interpretation of FTIR Analysis

37

5.1

Yield comparison with other papers

44

8.1

51

8.2

Optimized Bolds Basal Media components concentration Lugols Solution Components

8.3

Sulpho-phosphovanillin Reagent Components

52

8.4

Extraction Buffer components for Isolation of DNA PCR components for Amphlification of DNA sequence

52

8.5

51

52

List of Figures Figure no.

Title of figure

Page no.

1.1 3.1

Pie chart showing World utilization percentage of biofuels A ) Helical tube fabrication B ) Acrylic sheet C ) Blue LED light

4 16

3.2.1

CAED design of Flat plate Photobioreactor

19

3.2.2 3.2.3 3.2.4 3.2.5 4.1 4.2 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12

CAED design of Tubular Photobioreactor CAED design of Hybrid Photobioreactor CAED design of Continuous Hybrid Photobioreactor Growth curve of microalgae showing growth phases Completed hybrid Photobioreactor Chlorella rotunda at 100x Gel analysis Phylogenetic tree Growth in first week Growth in second week Growth in third week Growth in first week Growth in second week Growth in third week Graoh of optical density camparision between flask culture and PBR

20 21 22 28 31 32 33 33 34 34 34 34 34 34 35

4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20

Lipid comparision Lipid comparision in bar chart Std. FTIR algal graph Sample FTIR graph Design of PBR Growth after 4 days Growth after 8days Biomass estimation graph comparing absorbance if batch 1PBR and batch 2 PBR

36 36 37 37 40 41 42 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

CHAPTER – 1 INTRODUCTION

1

Department of Biotechnology, NHCE

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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 nonrenewable 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 st

1 2nd 3rd 4th 2

TYPE Agricultural food crops based (e.g. Wheat, sugar etc) Agricultural non-food crops (e.g. Wood etc) Algal species used for oil production 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-tovolume ratio of the photobioreactor and the photosynthetic efficiency by enriched CO2sequestration 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 semirigid, 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 pondphotobioreactor 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 pondphotobioreactor. 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 climatecontrolled 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 Author Year of Journal n publicati o on 1. Velea et.al 2014 Revista de Chimie

Reactor type

Highlights

Lacunae

Hybrid

Hybrid photobioreactor designed

Lacked economic studies of the reactor

Analysis of biomass with kinetics studied

2.

Naqqiuddina

2014

Algal biomass utilization

Closed

A simple floating photobioreactor

Only floating bioreactor discussed Economical aspects not mentioned

Two different designs to compare the results

3.

Sawdon et.al

9

2014

Journal of chemical technology

Closed


A new method studied to give optima growth

Novel method found effective to only tubular type

Design and fabrication of economically viable Hybrid Photobioreactor (closed bubble column) prototype for cultivation of elite microalgae for enhanced lipid (biodiesel)l yield and biotechnolog y Algal Bio refinery

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of microalgae

photobioreactor

Hybrid

Designing of novel low cost photobioreactor

Artificial light source not used

Tubular

Design of a photobioreactor using tubes and solar panel Article mentions about the different systems used for microalgae growth

Temperature control not included

4.

Mottahedeh et.al

2012

5.

Csanyi et.al

2012

Springer

6.

Kunjapur et.al

2012

Industrial and Open Engineering and Chemistry closed research

Economical considerations not mentioned

Less research on Hybrid type systems Design considerations

7.

Ling Xu et.al

2009

Life Sciences

All types

8.

Willson et.al

2008

Research paper

Closed

9.

Grobbelaar et.al

2003

Applied Phycology

Closed

10 .

Masojidek et.al

2003

Applied Phycology

Closed

10


This review outlines different bioreactors, its advantages and disadvantages. Discussion of a photobioreactor optimal for the growth of microalgae Concepts of new Photobioreactors is studied

Novel tubular photobioreactor called

This review outlines different bioreactors, it’s advantages and disadvantages Compromised purification

Due to the mutlicompartment reactor the contamination risk is high Climate conditions not considered


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“Penthouse roof reactor”

11 .

Molina et.al

2000

Journal of biotechnolog y

Closed

Supra high solar conditions taken Fluid mechanics method integrated into the design of tubular photobioreactor

Not very conclusive results were found

Kinetics of the reactor was done

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 (O 2). 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 the indigenously designed photobioreactor.

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species

(Chlorella rotunda)

in


CHAPTER-3 MATERIALS AND METHODS

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CHAPTER – 3 MATERIALS AND MATERIALS 3.1 Materials 3.1.1 Lab instruments 1) 2) 3) 4) 5) 6) 7)

Measuring device, Model no. BL -220H Autoclave Laminar air flow Shaking incubator, Model no. BT-ISI-E Water bath Cooling Centrifuge Colorimeter

3.1.2 Glassware 1. 2. 3. 4. 5. 6.

Conical flasks (100ml , 500ml) Glass rod Beaker (100ml, 500ml) Measuring cylinders (500ml, 100ml) Pipettes Petri dishes

3.1.3 Materials for fabrication 1. 2. 3. 4. 5. 6. 7.

Acrylic sheets LED lights Plastic fittings Pump Aerator CO2 cylinder Metal fittings

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A

B

Fig 3.1: A) Helical tube fabrication B) Acrylic sheet C) Blue LED lights

3.1.4 Chemicals 1. 2. 3. 4. 5. 6.

Salts for Bold's Basal Media Concentrated sulphuric acid Concentrated phosphoric acid Vanillin Absolute ethanol Distilled water

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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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A

BATCH AND CONTINUOUS KINETICS

SECOND BATCH OF CULTURING IN PHOTOBIOREACTOR AND FLASK

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.

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Fig 3.2.1: CAED design of a Flat Plate Photobioreactor 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.

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Fig 3.2.2: CAED design of Tubular Photobioreactor

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

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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.

Fig 3.2.3: CAED design of Hybrid Photobioreactor 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

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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.

Fig 3.2.4: CAED design of Continuous Hybrid Photobioreactor Dimensions: Tanks, Length = 0.3 m Breath = 0.2 m Width = 0.15 m Volume = 2 L x B x W = 18 liters

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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:

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 10ml mlofofmother mother culture added under sterile Mediawas wascooled cooled and and 10 culture waswas added under conditions

sterile conditions

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

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

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

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. The sequence similarity searching was done using BLAST software

Isolation of total genomic DNA using the CTAB method

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A

The genomic DNA was then amplified using PCR

The Phylogenetic analysis was done using CLUSTAL software for oligonucleotide primers

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:

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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 sulphophosphovanillin reagent. The sulpho-phosphovanillin was freshly prepared using vanillin and conc. phosphoric acid.

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

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.

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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,

   

27

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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Fig 3.2.5: Growth curve of Microalgae showing growth phases

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:

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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.

Fig 4.1: Completed Hybrid Photobioreactor

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

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Morphology

Chlorella rotunda

Colour

Yellow

Shape

Flower

Length

10µm

Width

6.5mm



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Fig 4.2: Chlorella rotunda at 100x magnification

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 4.3: Sequential analysis of Chlorella rotunda

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Fig 4.4: Gel Analysis

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Fig 4.5: Phylogenetic tree

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 4.6: growth in first week

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Fig 4.7: Growth in second week


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.

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

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.

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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)

Fig 4.15: Std. FTIR algal graph

Fig 4.16: Sample FTIR graph

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

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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 38 Department of Biotechnology, NHCE


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µ= 0.24 hr-1 td= 2.88 hr Where,     


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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Design and fabrication of economically viable Hybrid Photobioreactor (closed bubble column) prototype for cultivation of elite microalgae for enhanced lipid (biodiesel)l yield td = log 2 µ = 0.693 0.462 td =1.4 hr-1 Where,     


Fig 4.17: Design of second Batch of PBR with additional helical tube and carbon dioxide cylinder

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Fig 4.18: Growth in 4 days

Fig 4.19: Growth in 8 days

Fig 4.20: Biomass estimation graph comparing the biomass absorbance of batch 1 PBR and batch 2 PBR

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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

1

Chlorella homosphaera

Velea et.al

2

Asthrospira plantens

0.781 mg/ml

3

Asthrospira plantens

Naquiddin et.al Masojidk et.al

4

Phaeodactylun tricornutum

Molina et.al

2.5 mg/ml

44


Lipid content Papers 0.68 mg/ml

2.2 mg/ml

Lipid content PBR 1

2.4 mg/ml

Lipid content PBR 2

3.7 mg/ml


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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Design and fabrication of economically viable Hybrid Photobioreactor (closed bubble column) prototype for cultivation of elite microalgae for enhanced lipid (biodiesel)l yield 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 1.4 M NaCl

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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Design and fabrication of economically viable Hybrid Photobioreactor (closed bubble column) prototype for cultivation of elite microalgae for enhanced lipid (biodiesel)l yield Taq DNA polymerase (5U)

0.25

Template DNA (50ng/ μl)

1.00

Total volume

20.0

8.2 Abbreviations 1. 2. 3. 4.

PBR : Photobioreactor LED : Light Emitting Diode CAED : Computer Aided Drawing PCR : Polymeric Chain Reaction

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DESIGN AND FABRICATION OF ECONOMICALLY VIABLE HYBRID

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