Contents
Chapter I .................................................................................1
Introduction ............................................................................1
1.2 Photosynthesis in Microalgae ......................................................................... 2
1.2.1 The light reactions of photosynthesis ...................................................... 2
1.2.2 The dark reactions of photosynthesis ...................................................... 3
1.3 Application of microalgae .............................................................................. 4
1.3.1 CO2 sequestration .................................................................................... 4
1.3.2 Biofuel production ................................................................................... 5
1.3.3 Wastewater treatment .............................................................................. 7
1.3.4 Food and niche products.......................................................................... 7
1.4 Inorganic nutrients for microalgae ................................................................. 7
1.4.1 Nitrogen ................................................................................................... 8
1.4.2 Phosphorus .............................................................................................. 8
1.4.3 Sulphur .................................................................................................... 9
1.4.4 Calcium and Magnesium ....................................................................... 10
1.4.5 Sodium, Potassium and Chlorine .......................................................... 10
1.4.6 Silicon .................................................................................................... 10
1.4.7 Iron ........................................................................................................ 10
1.4.8 Trace Elements ...................................................................................... 11
1.4.9 Carbon source ........................................................................................ 11
1.5 Lipids in Microalgae .................................................................................... 11
1.6 Lipid Induction in microalgae ...................................................................... 12
1.6.1 Nitrogen limitation ................................................................................ 13
1.6.1 Phosphorus limitation ............................................................................ 13
1.7 Growth Kinetics and Measurement Methods ............................................... 14
1.7.1 Growth ................................................................................................... 14
1.7.2 Measurement Methods .......................................................................... 14
1.8 Lipid extraction method ............................................................................... 15
1.9 Feasibility of microalgae study in Nepal ...................................................... 16
1.10 Objectives of Study .................................................................................... 17
1.11 Research problems ..................................................................................... 18
iii
1.12 Research questions ..................................................................................... 18
Literature Review .................................................................19
2.1 Introduction .................................................................................................. 20
2.1 Chlorella vulgaris ......................................................................................... 20
2.3 R & D for lipid induction in microalgae ...................................................... 23
Material and Methods ..........................................................26
3.1. Introduction ................................................................................................. 27
3.2 Isolation ........................................................................................................ 27
3.3 Transfer to growth media ............................................................................. 27
3.4 Growth Kinetics ........................................................................................... 28
3.5 Determination of Growth. ............................................................................ 28
3.6 Lipid Extraction ............................................................................................ 29
3.7 Selection of potential species ....................................................................... 30
3.8 Preparation of inoculum ............................................................................... 30
3.9 Optimization of media composition ............................................................. 31
3.9.1 Nitrogen concentration .......................................................................... 31
3.9.2 Phosphorus concentration...................................................................... 33
3.9.3 Carbon concentration ............................................................................ 34
3.9.4 Growth Kinetics .................................................................................... 35
3.9.5 Lipid Extraction ..................................................................................... 36
3.9.6 Kinetics and Yield parameters .............................................................. 36
3.9.6.1 Growth evaluation .......................................................................... 36
3.9.6.2 Estimation of lipid content ............................................................. 37
3.9.6.3 Estimation of biomass productivity ................................................ 37
3.9.6.4 Estimation of lipid productivity ..................................................... 37
3.9.6.4 Determination of the CO2 Consumption rate ................................. 37
Results ....................................................................................38
4.1 Growth Kinetics of different microalgal species .......................................... 39
4.2 Seasonal variation on the growth of 3 potential algal species ...................... 39
4.3 Determination of Growth ............................................................................. 40
4.4 Optimization of Nitrogen ............................................................................. 41
4.4.1 Growth Kinetics .................................................................................... 41
iv
4.4.2 Growth Rate .......................................................................................... 41
4.4.3 Lipid content .......................................................................................... 43
4.4.4 Biomass Productivity ............................................................................ 44
4.4.5 Lipid Productivity.................................................................................. 44
4.4.6
Effect of Nitrogen on chlorophyll content........................................ 45
4.5 Optimization of Phosphorus ......................................................................... 46
4.5.1 Growth Kinetics .................................................................................... 46
4.5.2 Growth Rate .......................................................................................... 46
4.5.3 Lipid content .......................................................................................... 49
4.5.4 Biomass Productivity ............................................................................ 49
4.5.5 Lipid Productivity.................................................................................. 50
4.6 Effect of carbon concentration ..................................................................... 50
4.6.1 Effect of optimized media ..................................................................... 50
4.6.1.1 Growth Kinetics ............................................................................. 50
4.6.1.2 Growth Rate ................................................................................... 51
4.6.2 Effect on normal media ......................................................................... 53
4.6.2.1 Growth Kinetics ................................................................................. 53
4.6.2.2 Growth Rate ................................................................................... 54
4.6.3 Lipid Content ......................................................................................... 56
4.6.4 Biomass productivity ............................................................................. 56
4.6.5 Lipid productivity .................................................................................. 57
4.6.6 CO2 consumption rate ........................................................................... 57
Discussion ..............................................................................58
5.1 Effect of Nitrogen ......................................................................................... 59
5.2 Effect of phosphorus .................................................................................... 60
5.3 Effect of carbon ............................................................................................ 62
Conclusion .............................................................................65
References .............................................................................68
Appendix ...............................................................................73
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Chapter I
Introduction
1
1.1 Biology of Microalga
Algae are one of the oldest life-forms microscopic organisms and are the primary
synthesizers of organic matter in aquatic environments. They have high suface area-tovolume ratios, enabling the rapid uptake of nutrient and carbon dioxide (CO2) and much
faster cell growth rate than land-based plants. They are primitive plants (thallophytes), i.e.
lacking roots, stems and leaves, have no sterile covering of cells around the reproductive
cells and have chlorophyll a as their primary photosynthetic pigment. Algae structures are
primarily for energy conversion without any development beyond cells, and their simple
development allows them to adapt to prevailing environmental conditions and prosper in
the long term.
1.2 Photosynthesis in Microalgae
1.2.1 The light reactions of photosynthesis
The main role of the light reactions is to provide the biochemical reductant (NADPH2) and
the chemical energy (ATP) for the assimilation of inorganic carbon. The light energy is
trapped in two photoreactions carried out by two pigment–protein complexes, PS I and PS
II. The photosystems operate in series connected by a chain of electron carriers usually
visualised in a so-called ‘Z’ scheme
Upon illumination, two electrons are extracted from water (O2 is evolved) and transferred
through a chain of electron carriers to produce one molecule of NADPH2. Simultaneously,
protons are transported from an external space (stroma) into the intrathylakoid space
(lumen) forming a pH gradient. According to Mitchel’s chemiosmotic hypothesis, the
gradient drives ATP synthesis, which is catalysed by the protein complex called ATPase
or ATP synthase. This reaction is called photophosphorylation and can be expressed as:
2
1.2.2 The dark reactions of photosynthesis
Carbon assimilation
The fixation of carbon dioxide happens in the dark reaction using the NADPH2 and ATP
produced in the light reaction of photosynthesis. The reaction can be expressed as:
In order to fix one molecule of CO2, two molecules of NADPH2 and three molecules of
ATP are required (representing an energy of 5.2 X 104 J, about 13 kcal). As concern the
quantum efficiency of CO2 fixation, it was found that at minimum ten quanta of absorbed
light are required for each molecule of CO2 fixed or O2 evolved.
The reaction mechanism of carbon fixation was worked out by Calvin and Benson in the
1940s and early 1950s using
14
C radiolabelling technique (Nobel Prize, 1961). The
conversion of CO2 to sugar (or other compounds) occurs in four distinct phases (Fig 1)
forming the so-called Calvin–Benson cycle:
Figure 1: Calvin-Benson cycle
3
1. Carboxylation phase: The reaction whereby CO2 is added to the 5-carbon sugar,
ribulose bisphosphate (Ribulose-bis-P), to form two molecules of phosphoglycerate
(Glycerate-P). This reaction is catalysed by the enzyme ribulose bisphospate
carboxylase/oxygenase (Rubisco).
2. Reduction phase: In order to convert phosphoglycerate to 3-carbon products (TrioseP) the energy must be added in the form of ATP and NADPH2 in two steps:
phosphorylation of phosphoglycerate to form diphosphoglycerate and ADP, and
secondly,
reduction
of
diphosphoglycerate
(Glycerate-bis-P)
to
phosphoglyceraldehyde (Glyceraldehyde-P) by NADPH2.
3. Regeneration phase: Ribulose phosphate (Ribulose-P) is regenerated for further CO2
fixation in a complex series of reactions combining 3-, 4-, 5-, 6- and 7-carbon sugar
phosphates. The task of generating 5-carbon sugars from 6-carbon and 3-carbon
sugars is accomplished by the action of the transketolase and aldolase enzymes.
4. Production phase: Primary end-products of photosynthesis are considered to be
carbohydrates, but fatty acids, amino acids and organic acids are also synthesized in
photosynthetic CO2 fixation. Various end products can be formed under different
conditions of light intensity, CO2 and O2 concentrations, and nutrition.
1.3 Application of microalgae
1.3.1 CO2 sequestration
Global warming (GLW) is due to rising atmospheric concentrations of carbon dioxide,
nitrogen- and sulfur-oxides, methane and other greenhouse gases. Increasing
concentrations of gases will rise the average surface temperature of the Planet by up to
60C during the 21st century. Carbon dioxide, the major GLW gas, is produced by both
mobile and stationary sources. Instead using various adaptation strategies by accepting the
consequences of global warming, time has come to find an appropriate technology could
sequester CO2 in an efficient manner. Application of microalgae for the purpose of CO2
sequestration is found to be an environmentally friendly technology that can assimilate
CO2 within various ranges of concentration from ambient (0.04%) to 100% v/v CO2 by
selecting adequate species. In theory microalgae can use up to 9% of the incoming solar
energy to produce 280 tons of dry biomass per ha-1 y-1 while consuming/ sequestering
roughly 513 tons of CO2. This shows that microalgae are able to fix CO2 efficiently from
different sources, including atmosphere and industrial exhaust gases. Industrial exhaust
4
gases provide a CO2-rich source for microalgae cultivation and a potentially more efficient
route for CO2 bio-fixation.
One of the limitations in CO2 bio-fixation by microalgae is the low solubility of CO2 in
water. Free CO2 molecules are easily lost to atmosphere and hence, continuous pumping
of air is necessary to ensure sufficient carbon source is available for microalgae to utilize
during photosynthesis. On the other hand, bicarbonate ion (HCO3−) from sodium
bicarbonate (NaHCO3) can become an alternative carbon source to grow phototrophic
microalgae, mainly due to its high solubility in water compared to CO2 gas. In fact,
NaHCO3 or generally known as baking soda is widely available in the market with at a
very low price, making it a feasible carbon source if successfully implemented in
microalgae cultivation system. Microalgae species have the capacity to use carbonate such
as Na2CO3 and NaHCO3 for cell growth. Some of these species typically have a high
extracellular carbon hydrase activity, which is responsible for the conversion of carbonate
to free CO2 and thereby facilitate the assimilation. Thus some species of microalgae can
fix CO2 released from the carbonates and may be advantageous in many aspects: a) CO2
released from industrial facilities could be converted to carbonate salt by chemical
reactions, b) a limited number of microalgae species growth on media containing high
concentrations of carbonate, therefore the control of other invading species is relatively
simple and, c) a majority of these species have high optimum pH (9 -11).
The bicarbonate–carbonate buffer system can provide CO2 for photosynthesis through the
following reactions:
These reactions imply that during photosynthetic CO2 fixation, OH - accumulates in the
growth solution leading to a gradual rise in pH. It is not uncommon to measure pH’s as
high as 11 in high algal density production systems where no additional CO2 has been
supplied
1.3.2 Biofuel production
Algae can be used to generate energy in several ways. One of the most efficient ways is
through the utilization of the algal oils to produce biodiesel. Some algae can produce
5
hydrogen gas under specialized growth conditions. The biomass from alga can also be
burned, similar to wood, to generate heat and electricity. Compared with terrestrial crops,
which take an entire season to grow and contain a maximum of about 5% dry weight of
oil, microalgae grow quickly and contain high oil content efficiently in the range of 20–
50% dry weight of biomass. Some algal species, during their exponential growth, can
double their biomass in periods as short as 3.5 hours. At this moment of growing energy
crisis, microalgae can proved to be a natural remedy for current situation of fuel drought
as well as environmental disastrous impact caused by climate change. Although the
microalgae oil yield is strain-dependent it is generally much greater than other vegetable
oil crops, as shown in Table 1 that compares the biodiesel production efficiencies and land
use of microalgae and other vegetable oil crops, including the amount of oil content in a
dry weight basis and the oil yield per hectare, per year. Table 2 shows that although the oil
contents are similar between seed plants and microalgae there are significant variations in
the overall biomass productivity and resulting oil yield and biodiesel productivity with a
clear advantage for microalgae. In terms of land use, microalgae followed by palm oil
biodiesel are clearly advantageous because of their higher biomass productivity and oil
yield.
Table 1: compares the biodiesel production efficiencies
6
1.3.3 Wastewater treatment
The quick development of human activities has greatly increased the input of nitrogen and
phosphorus into the bodies of water. This input induces eutrophication and causes
deterioration in natural quality of water. Removal of nitrogen and phosphorus from waste
water is a fundamental way to prevent the eutrophication and water bloom. Microalgae
have attracted a significant amount of attention due to their ability to remove nitrogen and
phosphorus from wastewater and their great potential in producing biodiesel. Thus, the
coupling of advanced wastewater treatment and biodiesel production based on microalgae
is a promising technology. Microalgae remove nitrogen and phosphorus from water
mainly by uptake into algal cells. The most widely studied microalgae species for nitrogen
and phosphorus removal are Scenedesmus, Chlorella, and Spirulina. The secondary
effluents of domestic wastewater usually contain relatively low inorganic nutrients
(around 10–15 mg/L nitrogen and 0.5–1 mg/L phosphorus). So, microalgae species could
be utilize for removing inorganic nutrients efficiently accumulating high lipid content in
algal cells. As a novel ‘‘green technology,” microalgae have many advantages in the
removal of nitrogen and phosphorus.
1.3.4 Food and niche products
Microalgae are often used as food, both for people and livestock. Algae are also rich in
iodine, potassium, iron, magnesium, and calcium. Many types of algae are also rich in
omega-3 fatty acids, and as such are used as diet supplements and as a component of
livestock feed. The algal biomass may contain high levels of vitamins such as B 12 and
carotene, minerals, and protein. It may also contain essential amino acids such as DHA
and EPA. The high growth rates also make microalgae an attractive option as a food
supplement.
1.4 Inorganic nutrients for microalgae
The great taxonomic diversity of the algae is also reflected in their nutritional
requirements, and for most algal species we do not yet know what their exact nutrient
requirements are. This state of ignorance is well reflected in the wide range of nutrient
media used. Nutrients can, however, be classified into macronutrients (i.e. those required
at g.L-1 concentrations) and micronutrients (i.e. those required at mg.L-1 or μg.L-1
concentrations).
7
1.4.1 Nitrogen
Nitrogen is a fundamental element for the formation of proteins and nucleic acids. Being
an integral part of essential molecules such as ATP, the energy carrier in cells . The usual
nitrogen sources in algal media are (1) nitrate; (2) ammonium; or (3) urea. Some of the
prokaryotic algae, the heterocystous blue-green algae (cyanobacteria), can also fix
atmospheric N2. Most algae can use nitrate (NO3-), nitrite (NO2-) or ammonium (NH4+) as
an N source, with similar growth rates achieved irrespective of the nitrogen source.
When ammonium is used as the sole N source the pH of the medium may fall sharply,
especially in dense cultures at high temperatures, often leading to a rapid decline in
growth and even death of the culture. High concentrations of >1 mM ammonium may
inhibit growth, especially at high temperatures. If both ammonium and nitrate are supplied
the cultures generally do not take up the nitrate until the ammonium has been used up.
This is because ammonium is the end product of nitrate reduction and therefore causes
feedback-inhibition and repression of the nitrate uptake and reduction system.
Urea is also potentially a good nitrogen source for almost all algal species. Urea is usually
hydrolysed before its N is incorporated into the algal cells. This occurs by the action of
either the enzyme urease, or the enzyme urea amidolyase (UALase). Algae that metabolise
urea have either urease or UALase, but not both.
Nitrogen limitation would cause three changes: decreasing of the cellular content of
thylakoid membrane, activation of acylhydrolase and stimulation of the phospholipid
hydrolysis. These changes may increase the intracellular content of fatty acid acyl-CoA.
Meanwhile, nitrogen limitation could activate diacylglycerol acyltransferase, which
converts acyl-CoA to triglyceride (TAG). Therefore nitrogen limitation could both
increase lipid and TAG content in microalgal cells.
1.4.2 Phosphorus
Phosphorous is another major nutrient for algae. Phosphorus is an important component
required for normal growth and development of algal cells. Phosphate is a part of the
backbone of DNA and RNA, which are essential macromolecules for all living cells.
Phosphorus is also a key component of phospholipids. It is not unusual for algae to
become nutrient-limited (i.e., nitrogen- and phosphorus-limited) in the natural
environment The major form in which algae take up phosphorous is as inorganic
phosphate (H2PO4-and HPO42-). Those algae which can utilise organic phosphate
8
compounds hydrolyse these extracellularly by the action of phosphoesterase or
phosphatase enzymes and then take up the inorganic phosphorus produced. High
concentrations of phosphorous may actually inhibit the growth of some algae.
It has been shown that phosphorus, rather than nitrogen, is the primary limiting nutrient
for microalgae in many natural environments. Phosphorus typically constitutes 1% of dry
weight of algae, but it may be required in significant excess since not all added phosphate
is bioavailable due to formation of complexes with metal ions. Immediate effects of
phosphorus limitation include a reduction in the synthesis and regeneration of substrates in
the Calvin-Benson cycle and a consequential reduction in the rate of light utilization
required for carbon fixation.
Phosphorus limitation also leads to accumulation of lipids. Total lipid content in
Scenedesmus sp. was observed to increase from 23% to 53% with a reduction in initial
total phosphorus (as phosphate) concentration of 0.1 from 2.0 mg/l. Phosphatidylglycerol
(PG), which is one of four major glycerolipids constituting membrane lipids in
chloroplasts, was observed to decrease with phosphorus limitation in Chlamydomonas
reinhartdtii. PG is essential for cell growth, the maintenance of chlorophyll-protein
complex levels, and normal structure-function of the PSII complex. Total acidic lipid
(such as sulphoquinovosyldiacylglycerol and PG) content of the chloroplast did not
change significantly since a decrease in one acidic lipid was accompanied by an increase
in another acidic lipid. Phosphate limitation also reduces the synthesis of n-3 PUFA .
Similar to the effects of nitrogen deficiency, phosphorus starvation reduces chlorophyll a
and protein content thereby increasing the relative carbohydrate content in algal cells.
Phosphate deficiency has been demonstrated to result in accumulation of astaxanthin and
an overall reduction in cell growth. A decrease in cellular phycobilisome under conditions
of phosphorus deficiency (due to cell division and the cessation of phycobilisomes
synthesis) has also be shown. Theodorou et al. observed that phosphorus starvation in
Selenastrum minutum reduces respiration rate.
1.4.3 Sulphur
Sulphur is required to form the sulphur-containing amino acids methionine, cystine,
cysteine as well as biotin, pantothenic acid, thiamine, lipoic acid, sulphur lipids and
sulphated polysaccharides etc. Most algae obtain the sulphur from inorganic sulphate,
9
although a few species have also been shown to be able to take up organic sulphur
compounds.
1.4.4 Calcium and Magnesium
Calcium is required for maximum growth by many algae although its exact function is
unknown. Calcium does play a role in membrane stability and is incorporated into scales
in some species.
Magnesium is essential for all algae. It is required for ribosome stability and for the
function of chlorophyll.
1.4.5 Sodium, Potassium and Chlorine
Sodium is required by some algae, but apparently not by others. Marine species generally
require some sodium.
Potassium is a cofactor for a variety of enzymes and is probably required by all algae.
Chloride ions also appear to be universally required, in part because of the role they play
in potassium and sodium uptake.
1.4.6 Silicon
Most algae have a very low requirement for silicon which is probably met by silicon
contamination of other media components, especially in seawater-based media. Diatoms,
however, have a larger silicon requirement to form their siliceous valves. Lack of silicon
can reduce growth, and in some species totally inhibit DNA replication. Silicon is only
taken up as silicic acid (H4SiO4).
Germanium is taken up by diatoms in place of silicon but cannot be incorporated into the
valve. If germanium is added at a molar ratio of Ge/Si of 0.1 to 0.2, the growth of almost
all diatoms is inhibited. This can be used to remove diatom contamination from cultures
during isolation.
1.4.7 Iron
Iron is essential for all algae. It is required for nitrogen assimilation, in photosynthesis and
for the synthesis of cytochromes. In order to be taken up by algae it usually has to be
available in a chelated form, usually with EDTA or with citrate. High concentrations of
iron are inhibitory to growth.
10
1.4.8 Trace Elements
A range of trace elements are added to algal cultures. Specific requirements for some of
these have been demonstrated in some algae, but much more work needs to be done on
their functions and on the actual requirements. Trace elements usually added are boron
(especially important to diatoms and blue-green algae), manganese, copper, zinc,
molybdenum, vanadium, cobalt, nickel and selenium (especially important for some
dinoflagellates).
1.4.9 Carbon source
Carbon is one of the other major nutrients that must be supplied. It is essential for
photosynthesis and hence algal growth and reproduction. Carbon fixed by the algae can
end up in three destinations; it will either be used: (a) for respiration; (b) as an energy
source; or, (c) as a raw material in the formation of additional cells. Reduced carbon
fixation rate implies a reduction in algal growth rate. Algae require an inorganic carbon
source to perform photosynthesis. Carbon can be utilized in the form of CO2, carbonate, or
bicarbonate for autotrophic growth and in form of acetate or glucose for heterotrophic
growth. CO2 in water may be present in any of these forms depending upon pH,
temperature and nutrient content:
CO2 + H2 ↔ H2CO3 ↔ H + + HCO3− ↔ 2H+ + CO32− (1)
With an increase in pH, carbonate increases while molecular CO2 and bicarbonate
decrease. At the average pH of 8.2, 90% of the total CO2 is present in the form of HCO 3−;
only 1% exists as molecular CO2 and the rest is bicarbonate.
Under environmental pressure the cessation of microalgal cell division is observed and the
synthesis of CO2 is switched to lipid as storage of energy, so the lipid content per
microalgal biomass is increased.
1.5 Lipids in Microalgae
Microalgae produce different kinds of lipid which can be can be grouped into two
categories, storage lipids (non-polar lipids) and structural lipids (polar lipids). Storage
lipids are mainly in the form of TAG made of predominately saturated FAs and some
unsaturated FAs which is transesterified to produce biodiesel. On the other hand,
structural lipids typically have a high content of polyunsaturated fatty acids (PUFAs),
which are also essential nutrients for aquatic animals and humans. Polar lipids
(phospholipids) and sterols are important structural components of cell membranes which
11
maintain specific membrane functions, providing the matrix for a wide variety of
metabolic processes and participate directly in membrane fusion events. In addition to
this, some polar lipids may act as key intermediates in cell signaling pathways and play a
role in responding to changes in the environment.
Among non-polar lipids, TAGs are an abundant storage product which is catabolized to
provide metabolic energy. TAGs are mostly synthesized in the light, stored in cytosolic
lipid bodies which are then reutilized for polar lipid synthesis in the dark. Microalgal
TAGs are generally characterized by both, saturated and monounsaturated FAs. However,
some oil-rich species have demonstrated a capacity to accumulate high levels of longchain polyunsaturated fatty acids (PUFA) as TAG. PUFA-rich TAGs are metabolically
active and are suggested to act as a reservoir for specific fatty acids.
1.6 Lipid Induction in microalgae
The ability of microalgae to survive in diverse and extreme conditions is reflected in the
tremendous diversity and sometimes unusual pattern of cellular lipids obtained from these
microalgae. Moreover, some of these microalgae can also modify lipid metabolism
efficiently in response to changes in environmental conditions. Under optimal growth
conditions, large amounts of algal biomass are produced but with relatively low lipid
contents, which constitute about 5–20% of their dry cell weight (DCW), including
glycerol-based membrane lipids. Essentially, microalgae biomass and TAGs compete for
photosynthetic assimilate and a reprogramming of physiological pathways is required to
stimulate lipid biosynthesis. Unfavorable environmental or stress conditions shift lipid
metabolism from membrane lipid synthesis to neutral lipid storage (20–50% DCW),
mainly in the form of TAG, enabling microalgae to endure these adverse conditions. This,
in turn, increases the total lipid content of green algae.
Nutrient availability has a significant impact on growth and propagation of microalgae and
broad effects on their lipid and FA composition. Under normal growth conditions, ATP
and NADPH produced by photosynthesis are consumed by generating biomass, with ADP
and NADP+ eventually being available again as acceptor molecules in photosynthesis.
When cell growth and proliferation is impaired due to the lack of nutrients, the pool of the
major electron acceptor for photosynthesis, NADP+, can become depleted. Since
photosynthesis is mainly controlled by the abundance of light, and cannot be shut down
completely, this can lead to a potentially dangerous situation for the cell, damaging cell
12
components. NADPH is consumed in FA biosynthesis; therefore, increased FAs
productions (which in turn are stored in TAGs) replenish the pool of NADP+ under
growth-limiting conditions.
Environmental stress condition when nutrients are limited, invariably cause a steadily
declining cell division rate. Surprisingly, active biosynthesis of fatty acids is maintained in
some algae species under such conditions, provided there is enough light and CO2
available for photosynthesis. When algal growth slows down and there is no requirement
for the synthesis of new membrane compounds, the cells instead divert and deposit fatty
acids into TAG. Under these conditions, TAG production might serve as a protective
mechanism.
1.6.1 Nitrogen limitation
Nitrogen is an essential constituent of all structural and functional proteins in the algal
cells and accounts for 7%–20% of cell dry weight. Inorganic nitrogen taken up by algae is
rapidly assimilated into biochemically active compounds and recycled within cells to meet
changing physiological needs. Major effects of nitrogen deficiency in algal culture include
the enhanced biosynthesis and accumulation of lipids and triglycerides with a concomitant
reduction in protein content. This, in turn, results in a higher lipid/protein ratio at the
expense of growth rate. Algae grown in nitrogen-depleted cultures also tend to divert their
photosynthetically fixed carbon to carbohydrate synthesis; however, the physical
significance of this is not clear. Other effects of nitrogen reduction include decrease in
oxygen evolution, carbon dioxide fixation, chlorophyll content, and tissue production.
1.6.1 Phosphorus limitation
Phosphorus is an important component required for normal growth and development of
algal cells. It has been shown that phosphorus, rather than nitrogen, is the primary limiting
nutrient for microalgae in many natural environments. Phosphorus typically constitutes
1% of dry weight of algae, but it may be required in significant excess since not all added
phosphate is bioavailable due to formation of complexes with metal ions. Immediate
effects of phosphorus limitation include a reduction in the synthesis and regeneration of
substrates in the Calvin-Benson cycle and a consequential reduction in the rate of light
utilization required for carbon fixation. Phosphorus limitation also leads to accumulation
of lipids. Similar to the effects of nitrogen deficiency, phosphorus starvation reduces
13
chlorophyll a and protein content thereby increasing the relative carbohydrate content in
algal cells.
1.7 Growth Kinetics and Measurement Methods
1.7.1 Growth
Growth can be identified as any form of biomass accumulation in the algal culture.
Typically, for unicellular algae, growth is estimated from the culture with an
understanding that the growth parameter being followed increases as a fixed percentage of
the total unit time. When the parameter of interest is cell number or a proxy measure
(fluorescence, biomass dry weight, and optical density) that is directly proportional to cell
number, these methods provide an estimate of the population growth rate when they can
be shown to be linearly correlated with cell number or biomass. However, linear
correlation is only satisfied when the algal culture exists in its balanced or exponential
growth phase. For every culture, there is a period of acclimation that exists for the species
where growth rate is quite variable. In a closed system, where food is limited, all algae
progress through several different phases:
Phase
Description
1
Adaptation/Lag Ph ase
2
Accelerating Growth Phase
3
Exponential/Balanced Growth Phase
4
Decreasing Log/Linear Growth Phase
5
Stationary Phase
6
Accelerating Death Phase
7
Log Death Phase
1.7.2 Measurement Methods
To determine growth rates of the algal suspension, calculations are made while the culture
is in the exponential growth phase. Absorbance and Dry Weight estimations are two of the
more prominent methods used by researchers. Both methods were used in this research.
For determining cell growth rates, Becker suggests creating a standard curve correlating
absorption of the suspension versus the dry weight at different concentrations. As Becker
showed, and as many others have demonstrated biomass concentration can be related to
suspension absorbance. In fact, the amount of light that passes through the suspension will
14
be inversely proportional to the concentration of organisms, in accordance with Beer’s
Law relating absorption to concentration. To avoid interference with absorbance by
chlorophyll or other photosynthetic pigments, the usual wave lengths used are 550 or 750
nm (i.e. wavelengths where the absorption by chlorophyll and most other pigments is at a
minimum). Considering the wavelengths where Chlorophyll-a and -b absorb, an
Absorbance at 750 nm is recommended and will be used to construct the standard curve.
Use of the standard curve yields a linear equation that compares absorbance of the
suspension at 750 nm to the cell concentration at that particular time. The equation, in its
generic form, will appear as follows:
y = Ax
where A is the linear regression line fit variable, x is the A750 reading and y is the cell
concentration of the suspension at the particular absorbance x expressed in gm/l.
Dry weight estimation is one of the more common and easier methods to use for the
determination of algal growth. Aliquots of algal suspension are measured over different
time intervals. This method provides an estimate for the productivity of the culture in
suspension and is usually used when determining volumes of CO2 sequestered or in
determining the amount of lipids produced per unit of biomass.
1.8 Lipid extraction method
The total lipid content of microalgae is an important parameter for determining the
potential of microalgae in biofuel production. Thus, a reliable method for the quantitative
extraction of lipids from microalgae biomass is of critical importance. Natural lipids
generally comprise mixtures of nonpolar components such as glycerides (primarily
triacylglycerol) and cholesterol, as well as some free fatty acids and more polar lipids.
Some microalgal lipids are already valued ingredients in aquaculture feeds and as
nutritional supplements for humans. However, microalgae are known to synthesize a
diversity of unusual lipid compounds which may be commercially exploitable and have
been proposed as a suitable biorefinery feedstock for value added co-production of fine
chemicals and fuels. While specific analytical methods may exist for selected lipid
compounds, total lipid extraction is favourable when screening for a variety of lipids.
Isolation, or extraction, of lipid from microalgae is performed with the use of various
organic solvents. The extraction methods devised by Bligh and Dyer have found general
acceptance as standard procedures for recovery of total lipids. This method relies on
15
chloroform and methanol to form a monophasic solvent system to extract and dissolve the
lipids. A biphasic system is then produced in a purification step by the addition of water,
leading to the separation of polar and non-polar compounds into an upper and lower phase
respectively.
Advantage
- simple
- standard method, well established
- determines total lipids
- Samples can be analysed directly with no pre-drying necessary
- lipids can be used for further determinations
Disadvantage
- adverse effects of chloroform on the environment (EU regulation controlling chlorinated
solvents)
- laborious
1.9 Feasibility of microalgae study in Nepal
For developing country like Nepal, wind and solar power are expensive alternatives.
Further, biodiesel from the vegetable oil competes with the food resources of the country.
As such, the challenge for this generation of scientists and engineers is to develop cost
effective, environmentally-friendly sources of energy. Biofuels have received wide
attention in recent years, especially algae-derived biofuels. Algae reproduce quickly,
produce oils more efficiently than crop plants, and require relatively few nutrients for
growth. These nutrients can potentially be derived from inexpensive waste sources such as
flue gas and wastewater, which provides a mutual benefit in helping to mitigate carbon
dioxide waste. Sunlight is considered as a major light energy source for growth of
photoautotrophic algae and Nepal receives very pleasant sunshine, i.e. 6.8 hours per day
with the intensity of solar insolation ranging from 3.6 to 5.9 kW/sq.m./day, which make
algae as possible candidate of energy source. One of the main factors impeding the ability
of algae-derived biodiesel to become widely used is its high cost relative to current
petroleum-sourced fuels. In order to reduce cost, ways to optimize the growth and lipid
productivity of algae species have been explored. The optimization of algae growth is a
key step in increasing lipid yield and making algae-derived biodiesel an economically
viable alternative to traditional fuels. While research using monocultures of algae looks
16
extremely promising, there is a dire need for simple, cheap technology for alternate fuel
for developing country like Nepal. While the climate over here is conducive for
microalgae, it would be advantageous for Nepal to initiate research on microalgae. As
such, this project mainly focus on optimization of lipid productivity by manipulating the
composition of nitrogen, phosphorus and carbon sources compare to that of standard BBM
media.
Algae species used for this study is Chlorella vulgaris. This species were chosen due to
their common occurrence around the world, easy growth characteristics, and significant
lipid content. Chlorella vulgaris has a distinct morphology and high lipid content. In
addition, it can also be grown readily under a variety of environmental conditions. In
addition to the species selection, a nutritional requirement for culture optimization is
another essential factor that significantly affects the growth rate and the yield of products.
Providing optimum conditions some microalgae can double their biomass in less than 24
hours. Such high yield and high density biomass is ideal for intensive cultivation and can
provide an excellent biomass source of biofuels. Numerous studies have been conducted
on enhancement of the lipid content in a number of microalgae by applying various stress
condition during cultivation such as nitrogen deprivation, high light intensity, high
salinity, phosphate limitation. Significant increase in lipid contents in microalgae was
reported after being subjected to such stress conditions. Oil levels of 20-50% in dry
weight biomass are quite common and some can exceed 80%. However, stress conditions
also have a negative influence on growth resulting in low overall biomass production.
Therefore, it is more appropriate to apply stress condition in order to increase lipid content
and at the same time not affecting its growth rate.
1.10 Objectives of Study

To develop the basic knowledge and tools for isolating and purifying microalgae and
establishing laboratory scale culture systems.

To understand the principles of algal culture and nutritional and environmental
requirements of microalgae.

To optimize the biomass productivity and lipid productivity by altering the
concentration of nitrogen, phosphorus and carbon.

To define the nitrogen and phosphorus requirements of cultured Chlorella vulgaris in
order to efficiently grow the algae in a carbon sequestering scheme
17

To provide sufficient information about the role of microalgal species in sequestering
CO2 more efficiently

To develop a guideline for its commercialization.
1.11 Research problems

Development of techniques for single species cultivation, evaporation reduction, and
CO2 diffusion losses.

Maintaining a single species culture as there may be a risk of contamination from
other microalgal species as well as other microorganisms like bacteria, fungi and
viruses.

Harvesting, dewatering from microalgal biomass are challenging issues because they
consume large amounts of energy – mainly because of the small cell size and
relatively low biomass levels of microalgal cultures.

Lack of fundamental information and resources required to rationally optimize the
culture conditions which results in high cost of algae-derived fuel compared to that
of petroleum-sourced fuels.
1.12 Research questions

How to isolate and culture a single microalgae species?

On what basis potential species of microalgae species are selected?

How to measure growth rate and lipid content of selected microalgae species?

How do the growths of microalgae cells accelerate by supplying CO2?

How does microalgae cultivation help to mitigate CO2?

What are the required conditions to cultivate algae on an economically and
environmentally sustainable basis?
18
Chapter II
Literature Review
19
2.1 Introduction
Algae are a large and diverse group of simple typically autotrophic organisms, ranging
from unicellular to multicellular forms. Microalgae are rich in lipid and proteins and can
function as cell factories, specifically as photoautotrophic cultivation systems that utilize
sunlight and carbon dioxide to produce high-value materials including lipid, bioactives
products and fertilizer. Like any other photosynthetic entity, microalgae utilize the energy
of the sun to increase their growth. Biomass is produced according to the following
reversible reaction:
6 CO2 + 6 H2O + hν
C6H12O6 + 6 O2
Due to shortages of fossil fuels and the recent interest in Greenhouse Gas (GHG)
emissions, microalgae are being researched with greater frequency for its role in several
remediation processes and for bio-fuels. Current sources of bio-diesel include soybean oil,
rapeseed oil, palm oil, corn oil, jatropha, animal fats, and waste cooking oil. However,
considering the scope of the world’s energy uses, these sources cannot possibly replace
the fossil fuels currently in use. Use of microalgae offers a lot of advantages over other
available feedstock of biodiesel which are as following:
1. rapid growth rate and productivity (microalgae can produce 50 times more
biomass compared to higher plants)
2. no competition for land with crops (different types of microalgae are able to grow
in a variety of environmental conditions, even on the limited areas of land)
3. no competition with food market;
4. high oil content (oil yield in microalgae can exceed 75% by weight of dry
biomass;
5. when used for biodiesel fuel production algae can simultaneously reduce CO2
content in exhaust gases, minimize contamination – wastewater treatment from
inorganic salts, such NH4 +, NO3-, PO43-, using them as nutrient materials
These benefits are two-fold, reduction in GHG emissions, and an increase in algal biomass
for bio-fuels production.
2.1 Chlorella vulgaris
The unicellular photosynthetic microalga C. vulgaris is a member of the Class
Trebouxiophycea of the Phylum Chlorophyta. It is spherical in shape, and ranges from 210 μm in diameter. A green alga, it contains the green photosynthetic pigments
20
chlorophyll-a and chlorophyll-b within its chloroplast. While capable of autotrophic
growth, it is routinely cultured with a small amount of nutrients. In fact, some researchers
have grown C. vulgaris heterotrophically and have achieved interesting results. Chlorella
is a cosmopolitan genus with small, unicellular, ovoidal nonmotile cells; it does not
produce zoospores. Cells have a thin cell wall, and cup-shaped chloroplast. Pyrenoid may
be present. The accumulation of starch occurs within the chloroplast. Chlorella reproduces
by forming daughter cells or autospores (4–8–16) of the same shape as the parent cell. It
grows in autotrophic, heterotrophic and mixotrophic conditions. Besides autotrophic
strains, heterotrophic strains are also cultivated. Chlorella is the most important species in
the microalgal industry; it is cultivated and sold essentially as health food.
Typical growth observed can reach as high as 0.99 day-1 and achieve between four and
six doublings per day, if given sufficient nutrient conditions. Interest in C. vulgaris began
in the early 1950s when it was recognized first by the Japanese as an adequate protein
source. Later, investigation by the U.S. regarding its use as a food supplement for the
space program and for alternative fuels during the oil crisis of the 1970s was initiated.
However, the first large-scale production began in the 1960s in Japan. By 1980, after the
U.S. had largely forgotten about mass production (mostly because the oil crisis of the mid1970s had ended), there were 46 large scale factories in Asia (mostly Japan) producing
more than 1000 kg of algae (mainly Chlorella) species per month.
C. vulgaris has a nutrient composition of 51-58% protein, 12-17% carbohydrate, and 14 22% lipid. From a protein and lipid perspective, these values compare favorably to those
other traditional sources for milk and soy. Because of these values, and because they can
be manipulated to maximize certain components, C. vulgaris is a widely used nutritional
supplement. Additionally, research has been conducted regarding the utility of C. vulgaris
as a bio-fuels option. Specifically, full fatty acid profiles have been published regarding its
use as a bio-fuel substitute. While there are better options for bio-fuels, C. vulgaris
presents itself as an algal species that is not only robust in its tolerance of various
environmental factors, but as already mentioned, has utility across many industries. When
its oil is blended with other algal species’ oil or diesel fuel itself, it presents an adequate
bio-fuel substitute. It is for these aforementioned reasons and because of its ubiquitous
nature that C. vulgaris was investigated in this study.
21
Figure 2: Biosynthetic routine of biochemical components for Chlorella vulgaris.
The biosynthesis of lipids along with carbohydrates and proteins was involved with many
enzymatic reactions, which could be divided into four parts:
1. The photosynthetic reactions including the light and dark reactions: during the
process of illumination, chlorophyll could transform light energy to ATP and
NADPH, which could then be used to convert CO2 into glyceraldehydes-3phosphate (G3P) in the dark reaction.
2. The carbohydrate biosynthesis: part of G3P was utilized to produce
carbohydrates, such as glucose and starch through series of reactions.
3. The protein formation process: part of G3P was transformed into pyruvate and
then acetyl-CoA via the action of glycolysis and the further catalysis of pyruvate
dehydrogenase.
4. The lipid accumulation: catalyzed by acetyl-CoA carboxylase (ACCase), the
acetyl-CoA was converted to malonyl-CoA followed by fatty acids after
continuous cycles, and the lipid accumulation was attributed to both fatty acids
and G3P.
22
2.3 R & D for lipid induction in microalgae
The chemical composition of the algae is not a constant factor but varies over a range of
nutrient conditions. Several factors influence the proportion of chemical constituents
within the algal biomass. Most notable among the environmental factors are light and dark
cycles and the nutrients carbon, nitrogen and phosphorus. When the objective of algal
cultivation is oil production, it is must to maximize lipid content within the algal cell. For
this, one must effectively stress the algae which will retards algal reproduction rates and
focuses cell energy toward life sustaining processes within the cell. Considering these
reasons, Becker suggests cultivating algae for bio-fuels production in two stages; first,
algae are grown under normal conditions to first maximize biomass growth rate, and then
second, nitrogen is removed or the algae are otherwise stressed in an effort to force the
algae species to convert carbohydrates into lipids.
Many microalgae grown (stressed) under nitrogen limiting conditions show increased
lipids production within their cells. For instance, Converti et al. studied the effects of
temperature and nitrogen concentration on cell growth and lipid content in two strains of
algae—Chlorella vulgaris and Nannochloropsis oculata. Reducing the nitrate
concentrations in the growth media by 75% (1.5 to 0.375 g L−1 for Chlorella vulgaris and
0.3 to 0.075 L−1 for Nannochloropsis oculata), lipid accumulation tripled and doubled
respectively, with only a small reduction in growth rate at optimal growth temperature.
This result indicates that it may be possible to achieve higher lipid productivity for
biofuels production by employing nitrogen limitation with fine temperature control. They
observed increase in lipid synthesis from 5.90% to 16.41% and from 7.90% to 15.31% in
Chlorella vulgaris and Nannochloropsis oculata, respectively when nitrogen is deprived
by 75%. Additionally, it appears to be clear across the literature that lipids are maximized
through nitrogen deprivation. However, the results reported by Converti et al. regarding
growth rate seem to be the exception vice the rule. Most observers see reduced growth
rates coupled with nitrogen deprivation. In fact, Illman et al. observed lower growth rates
with increased lipid content from 18 to 40% under nitrogen limiting conditions.
Chlorella is a good choice for biodiesel production is a conclusion reached by Mata et al.
(2010) in their extensive review of microalgae and biodiesel production. They found lipid
content measured as percent dry weight biomass ranged from 5.0% to 58.0%, lipid
productivity as mg/L/day from 11.2 to 40.0, and biomass productivity as g/L/day from
0.02 to 0.20 for C. vulgaris. C. vulgaris is also reported to grow in heterotrophic and
23
mixotrophic (combining auto- and heterotrophic) conditions as well as the typical
autotrophic condition.
Decrease in NaNO3 concentrations in the medium and increase in algal metabolism
products led to a slight decrease in the biomass amount. Hsieh and Wu et al. and Yeesang
et al. found that increase in N source concentration leads to decreasing of lipid content in
the cells. According Hsieh and Wu the critical urea concentration was observed at 0.1 g l -1
when the biomass grew intensively and had higher lipid content compared to that
cultivated with a sufficient nitrogen amount. they also found out that nitrogen deprivation
results in impairment of photosynthesis, where carbon fixation is greatly reduced via
glyoxylate cycle leading to increments in the intracellular content of fatty acid acyl Co-A
and activate diacylglycerol acyl transferase, which converts fatty acid acyl Co-A to
triglyceride.
Moreover, Becker in 1994 reported that urea is the best nitrogen source of culturing
Chlorella vulgaris. Consequently, an optimized supply of urea or deficiency of NaNO3 is
considered to be a cultivation strategy for microalgae lipid production. Pruvost et al. in
2011 found that nitrate starvation trigger the lipid accumulation in freshwater algae,
Chlorella vulgaris. The highest total lipid content was reported to be 11-14% of dry cell
weight in Chlorella vulgaris.
Deng et al. in 2011 found that phosphorus deficient condition tends to promote the
initiation of intracellular lipid content through TAGs formation due to the impairment of
phospholipid synthesis. Kozlowska-Szerenos et al. in 2000 found that C. vulgaris grown
in medium with 45.5 mg/L P used five to 17% of the P, while those grown in medium
with 4.5 mg/L used it all.
Gianluca Belotti et al in Sapienza University of Rome found that nitrogen starvations
only slightly improve lipids productivity. But by means of nitrogen and phosphorus costarvation, total lipids productivity increased significantly. They concluded that during costarvation nitrogen and phosphorus, lipid composition of Chlorella vulgaris switch in
favour of nonpolar lipids in all the trophic regimes tested.
Most studies that have examined the effect of inorganic carbon addition and lipid
production or indeed biochemical composition in microalgae cultures have focused on the
addition of gaseous CO2. But Wiebe et al in 1940 reveal lower solubility of CO2 in water
for which inorganic carbon sources like NaHCO3 could be utilize as an alternative method
for determining impact of CO2 on growth and lipid productivity indirectly. Furthermore,
24
bicarbonate has greater solubility than CO2, thus reducing issues associated with low
retention times. Indeed, sodium bicarbonate has been used as a carbon source for the study
of growth and biochemical composition in a range of microalgae species and has been
shown to stimulate triacylglycerol accumulation in microalgal species. Jeong et al, 2003
found that 15.3 mg/L bicarbonate salt is equivalent to 243 mg/L CO 2 gas. Yeh et al. in
2010 demonstrate that Chlorella vulgaris has an optimum level of sodium bicarbonate
addition of 1,000 mg L−1 for biomass production above which reduced cell
densities/biomass are recorded.
25
Chapter III
Material and Methods
26
3.1. Introduction
This research is mainly focused on the optimization of three vital nutrients, namely,
nitrogen, phosphorus and carbon in the Bold Basal Media. These three nutrients were
selected as the parameters to be manipulated so as to yield a combination that promote
growth as well as lipid accumulation in Chlorella vulgaris. Effects of these three factors
were systematically investigated on both the cell growth and lipid content of C. vulgaris
for enhancement of lipid productivity. As dominating the photosynthesis of cells and
nitrogen as a significant part of chlorophyll, Chlorophyll content was also quantified in
response to different nitrogen concentration conditions.
3.2 Isolation
During isolation, serial dilutions of all algal samples were performed up to 109 to reduce
the number of algal cell from original sample. Streak plate technique was carried out in
1X BBM media containing 2% bacteriological agar to obtain single species from a culture
containing different species of microalgae.
Procedure
1. Bacteriological agar media of 2% was prepared in standard BBM and autoclaved
along with petriplates.
2. After autoclaving, media is then cooled to a certain extent and poured in
petriplates inside biosafety cabinet.
3. The inoculating loop was sterilized on flame and allowed to cool. The loop was
dipped into the samples/culture and a loopful of culture was taken and quadrant
streak method was applied.
4. Plates were then covered and incubated in growth chamber at 26-28 0C and 4000
lux in inverted position for 2 weeks.
5. After 2 week of streaking, pure single colony was isolated from quadrant streaked
petriplates and transferred in 20ml test tube containing 10ml 1X BBM allowing it
to grow for 2 weeks. Here algal sample of different species are separated in
different tubes.
3.3 Transfer to growth media
Prior to transferring to growth media, it was ensure that the final culture isolated in test
tube contain a single species of microalgae. Therefore, a loopful of all the culture of
microalgae cultures were first viewed under microscope by preparing slides of those
27
cultures. After confirmation of single species culture were transferred to growth media to
identify growth kinetics of the isolated algae.
Procedure
1. Initially, BBM media of concentration 1X was prepared by mixing all the stock
solutions pre-prepared in required amounts according to the given protocol.
2. All algae culture from the test tube were taken and washed properly in 1X BBM
by centrifuging for 10 mins at 2000 rpm for three times.
3. After third centrifugation, supernatant was removed and cell pellet was cultured
into new sterile culture flask containing 500 ml of media for analysis of growth
kinetics.
4. Aeration was provided to culture through silicon pipes whose one end is inserted
into the culture flask and other end to the aerator which helped to provide aeration
for proper mixing of cells in the culture flasks.
5. For analyzing growth and lipid content, algal culture flasks were then placed in
growth chamber under 12 h light and 12 h dark photoperiod at 25 °C so that the
growing algae could get abundant amount of light for their growth cultures.
3.4 Growth Kinetics
Growth kinetic of all algal samples was determined by spectrophotometer.
Procedure
1. After transferring all algae samples to growth media, 1ml of sample were
retrieved from each of the culture flasks from the very first day of culture and
dispense into clean cuvette.
2. 1ml of 1X BBM was taken as blank and placed in clean cuvette.
3. These cuvette were then put inside spectrophotometer.
4. The optical densities of each of the samples were taken every day at two
wavelengths 750 nm.
5. The obtained absorbances were used to plot the growth curve of respective algae
cultures.
3.5 Determination of Growth.
For each pure culture, a regression equation was prepared, as Becker discussed, during
the culture’s exponential growth phase. The dry weight of algal cells was measured by
28
centrifuging an aliquot of culture suspension on pre-weighed 15ml centrifuge tube. The
pellet were rinsed with distilled water, dried for 24 hours at 70°C, and re-weighed. A750
measurements were determined by Spectrophotometer. The absorbance was compared
with each suspension’s respective dry weight. Direct correlation between absorbance and
dry weight for each pure culture were examined and each was expressed by a function.
Procedure
1. Initially, Chlorella vulgaris was culture in 500ml standard Bold Basal Media until
the exponential phase is reached i.e 5-6 days.
2. After 6 days, dilution of algal solution was done in following manner:
Volume of Algal solution (ml)
Volume of media(ml)
0
30
5
25
10
20
15
15
20
10
25
5
30
0
3. After dilution, absorbances of all these 7 samples were taken at 750nm and 10ml
of each of these samples were transferred into different pre-weight centrifuge
tubes.
4. Each of these samples is centrifuged and washed with distilled water.
5. After washing all the samples were centrifuged and dried in hot air oven
temperature maintained at 700C for 24 hrs.
6. After 24hrs weight of pre-weight centrifuge containing pellet of cell was
determined.
3.6 Lipid Extraction
Cellular or fluid lipid was separated from other constituent of cell through extraction for
further analysis of lipid content.
Procedure
1. Microalgal cells are harvested by allowing the cell suspension culture to settle
overnight.
2. Harvested cells were then filtered through filter paper
29
3. Cells were then kept overnight in oven at for drying and also to achieve cell
disrution.
4. Dried cells were then weighed and crushed in mortar and pestle.
5. Lipid extraction was then done by following Bligh and Dyer protocol. Here, lipid
was extracted using methanol: Chloroform: water in ratio of 1:1:0.8.
6. Initially, 500ul of methanol was premixed with dried biomass.
7. Further 500ul of chloroform and 300ul of distilled water was added to the mixture
and vortex for few minutes.
8. Solvent mixture was then centrifuged for 5 min at 3000 rpm.
9. The upper (methanol-water) phase was aspirated and the lower (chloroform) lipid
phase was pipette out and kept in clean eppendorf tube.
10. Remaining solids were re-extracted using steps 4,5,6,7 and 8.
11. Solvent from lower phase was evaporated in hot air oven maintained at
temperature 450C for 24hrs.
12. After 24hrs, methanol was added into dried lower phase of lipid to remove trace
amount of water present in it.
13. The dried lipid samples were weighed gravimetrically for determining their lipid
content.
3.7 Selection of potential species
On the basis of growth rate and lipid content Chlorella vulgaris was selected for further
studied on optimization of growth kinetics and enhancing lipid accumulation in microalgal
cell.
3.8 Preparation of inoculum
The culture of microalgae was done in autoclaved Bold Basal Media (BBM) whose initial
pH was adjusted to 6.8. For inoculum preparation, the culture was grown in 250ml conical
flask containing 250ml of sterile medium, aerated with compressed air, surrounding
temperature of 25–280 C and illuminated with cool-white fluorescent light in a light: dark
ratio of 12:12. When inoculum of microalgae reached to exponential phase, 15ml of algal
inoculums were centrifuged (4000rpm x 10min). Deposited algae were washed with sterile
Bold Basal Media for three times and used for culture during optimization process of
nitrogen, phosphorus and carbon.
30
Procedure
1. Initially, standard BBM media of concentration 1X was prepared by mixing all
the stock solutions pre-prepared in required amounts according to the given
protocol.
2. Pure culture of Chlorella vulgaris was first examined for the presence of single
species. Thereafter, algae culture from the test tube was taken and washed
properly with 1X BBM by centrifuging for 10 mins at 2000 rpm for three times.
3. After third centrifugation, supernatant was removed and cell pellet was cultured
into new sterile 100ml culture flask containing 50ml of media for inoculum
preparation.
4. Culture flask is then incubated in growth chamber for 10-12 days without aeration
for preparation of 1st inoculum culture.
5. After 10 days, microscopic examination was done to ensure the presence of single
species of Chlorella vulgaris. Thereafter, 15 ml of culture from the 1 st inoculum
culture was taken and washed properly with sterile 1X BBM by centrifuging for
10 mins at 2000 rpm for three times.
6. 2nd inoculum culture was then prepared by culturing into 250ml conical flask
providing aeration for 6-7 days and subsequently used it for optimization
procedure.
3.9 Optimization of media composition
3.9.1 Nitrogen concentration
Nitrogen is a essential component of microalgae nutrition. Limitation of nitrogen
concentration in algal nutrition has been reported to increase overall lipid content. Thus,
this research tends to optimize the growth kinetics and lipid content of Chlorella vulgaris
by culturing microalgae in BBM with different nitrogen concentration. NaNO3 is source
of nitrogen in Bold Basal Media and original concentration of nitrate in standard BBM is
0.25 gm L-1. For optimization of nitrogen, experiment was performed on bold basal media
with original nitrate concentration along in a media containing nitrate concentration ½, ¼
and double times that of the standard media.
Procedure
Preparation of media
31
1. Four different media with different concentration of nitrate (NaNO3) were
prepared in order to optimize the lipid content of microalgae compared to
standard.
2. For this, four different media with nitrate concentration of 0.5 gm/l, 0.25 gm/l,
0.125gm/l and 0.0625 gm/l were prepared simultaneously.
3. All the media was then autoclaved maintaining pH of all media at 6.8.
4. After autoclave, culture of chlorella vulgaris was done in these media.
Transfer into growth media
Procedure
1. 15 ml of pure culture of Chlorella vulgaris from inoculum culture was taken into 4
different centrifuge tube and washed properly with 1X BBM by centrifuging for
10 mins at 2000 rpm for three times.
2. After third centrifugation, supernatant was removed and cell pellet was cultured
into 4 sterile culture flask containing 250ml media of different nitrate
concentration.
3. Aeration was provided to all cultures through sterile silicon pipes whose one end
is inserted into the culture flask and other end to the aerator which helped to
provide aeration for proper mixing of cells in the culture flasks.
4. For analyzing growth and lipid content, algal culture flasks were then placed in
growth chamber under 12 h light and 12 h dark photoperiod at surrounding
temperature of 26-28 °C so that the growing algae could get abundant amount of
light for their growth cultures.
Estimation of Chlorophyll
Chlorophyll level is largely influenced by nitrogen concentration. Pigment levels of four
different culture conditions at different nitrogen concentration were determined by
spectrophotometric method using solvent like acetone.
Procedure
1. Dry algal biomass was put separately in 100% acetone in a concentration of 50ml
for each gram.
2. The mixture is then crushed in mortar and pestle for 2-3 minutes.
3. The mixture is then centrifuged at 2500 rpm for 10 minutes.
4. The supernatant was separated and absorbance were read at 662nm and 645nm.
32
5. It was recorded that Chlorophyll a showed the maximum absorbance at 662nm
and Chlorophyll b at 646nm.
6. The amount of these pigments was calculated according to the formulas of
Lichtentaler and wellburn (1985).
7. The formulas were showed below:
Chla= 11.75 A662 -2.350 A645
Chlb= 18.61 A645 – 3.960 A662
Where, Chla = Chlorophyll a
Chlb = Chlorophyll b
3.9.2 Phosphorus concentration
Like nitrogen, phosphorus is also one of the essential nutrients for microalgae. And after
culture in different nitrate concentration, appropriate nitrate concentration was chosen
which was further studies on different phosphate concentration were conducted. K 2HPO4
and KH2PO4 are the source of phosphate in Bold Basal Media and culture was performed
by varying their concentration by ½, ¼ and double times with respect to that of the
standard media including standard one.
Procedure
Preparation of media
1. Four different media with different concentration of phosphate were prepared in
order to optimize the lipid content of microalgae compared to standard.
2. For this, the concentration of K2HPO4 and KH2PO4 were varied by ½, ¼ and
double times to that of the concentration in standard media.
3. Four different media with different phosphate concentration were prepared which
contain optimized concentration of nitrate. Along with this, culture was also
performed in standard Bold Basal Media which was taken as control.
4. pH of all media were maintained at 6.8 and autoclaved at 1210C for 15 minutes.
5. After autoclave, culture of Chlorella vulgaris was done in these media.
Optimized
Bold
Basal
Nitrogen
different
Media
with KH2PO4
phosphorus
33
K2HPO4.
concentration
concentration
125mg/l
2X
350mg/l
150mg/l
125mg/l
1X
175mg/l
75mg/l
125mg/l
1/2X
87.5mg/l
37.5mg/l
125mg/l
1/4X
43.75mg/l
18.75mg/l
250mg/l
Control
175mg/l
75mg/l
Transfer into growth media
Procedure
1. 15 ml of pure culture of Chlorella vulgaris from inoculum culture was taken into 5
different sterile centrifuge tube and washed properly with autoclaved 1X BBM by
centrifuging for 10 mins at 2000 rpm for three times.
2. After third centrifugation, supernatant was removed and cell pellet was cultured
into 5 sterile culture flask containing 250ml media of different nitrate
concentration.
3. Aeration was provided to all cultures through sterile silicon pipes whose one end
is inserted into the culture flask and other end to the aerator which helped to
provide aeration for proper mixing of cells in the culture flasks.
4. For analyzing growth and lipid content, algal culture flasks were then placed in
growth chamber under 12 h light and 12 h dark photoperiod at surrounding
temperature of 26-28 °C so that the growing algae could get abundant amount of
light for their growth cultures.
3.9.3 Carbon concentration
After nitrogen and phosphorus, study of the growth kinetics and lipid content were done
on the optimized media as well as standard media of Bold Basal Media. Due to lower
solubility of CO2 in water NaHCO3 was chosen as alternative a carbon source in Bold
Basal Media. For this, culture was performed in three different concentration of NaHCO3
i.e. 0.12 gm/l, 0.24 gm/l and 0.36 gm/l.
Procedure
Preparation of media
1. Effect of carbon was observed in both optimized media and Standard Bold Basal
Media. Here, optimized media contain the optimum concentration of nitrate and
34
phosphate obtains from above study whereas standard media contain specified
concentration of nitrate and phosphate.
2. The study was performed on three different concentration of NaHCO3 i.e. 0.12
gm/l, 0.24 gm/l and 0.36 gm/l.
3. Hence, eight different media were prepared out of which 4 consist of optimized
media while other 4 consist of standard Bold Basal media which contain NaHCO3
in 0 gm/l, 0.12 gm/l, 0.24 gm/l and 0.36 gm/l, respectively.
4. pH of all media was maintained at 6.8 and autoclaved at 1210C for 15 minutes.
5. After autoclave, culture of Chlorella vulgaris was done in these media.
Transfer into growth media
Procedure
1.
15 ml of pure culture of Chlorella vulgaris from inoculum culture was taken into 8
different sterile centrifuge tube and washed properly with autoclaved 1X BBM by
centrifuging for 10 mins at 2000 rpm for three times.
2.
After third centrifugation, supernatant was removed and cell pellet was cultured
into 8 sterile culture flask containing 250ml media. Out of 8 culture flask, four
culture flask contain different concentration of NaHCO3 in optimized media
while other four contain different concentration of NaHCO3 in standard media
3.
Aeration was provided to all cultures through sterile silicon pipes whose one end
is inserted into the culture flask and other end to the aerator which helped to
provide aeration for proper mixing of cells in the culture flasks.
4.
For analyzing growth and lipid content, algal culture flasks were then placed in
growth chamber under 12 h light and 12 h dark photoperiod at 26-28 °C so that
the growing algae could get abundant amount of light for their growth cultures.
3.9.4 Growth Kinetics
Growth kinetic of all algal samples was determined by spectrophotometer.
Procedure
1.
After transferring all algae samples to growth media, 1ml of
sample were retrieved from each of the culture flasks from the very first day of
culture and dispense into clean cuvette.
2.
1ml of 1X BBM was taken as blank and placed in clean cuvette.
3.
These cuvette were then put inside spectrophotometer.
35
4. The optical density of each of the samples was taken every day at wavelengths:
750 nm.
5. The obtained absorbances were used to plot the growth curve of respective algae
cultures.
3.9.5 Lipid Extraction
Cellular or fluid lipid was separated from other constituent of cell through extraction for
further analysis of lipid profile.
Procedure
1. Microalgal cells are harvested by gravimetric separation.
2. Harvested cells were then centrifuged.
3. Cells were then kept overnight in oven at for drying and also to achieve cell
disrution.
4. Dried cells were then weighed and grind in mortar and pestle.
5. 30mg of dried biomass was kept in eppendorf tube.
6. 500ul of methanol was premixed with dried biomass.
7. Further 500ul of chloroform and 300ul of distilled water was added to the mixture
and vortex for few minutes.
8. Solvent mixture was then centrifuged for 5 min at 3000 rpm.
9. The upper (methanol-water) phase was aspirated and the lower (chloroform) lipid
phase was pipette out and kept in clean eppendorf tube.
10. Remaining solids were re-extracted using steps 4,5,6,7 and 8.
11. Solvent from lower phase was evaporated at by keeping it in oven for 24 hrs at
450C
12. 100ul of methanol is then added in dried sample and evaporate it again in oven to
remove the trace amount of water.
13. The dried lipid samples were weighed gravimetrically for determination of lipid
content.
3.9.6 Kinetics and Yield parameters
3.9.6.1 Growth evaluation
Growth rate (GR) was calculated using the cell density (g/L). Growth rate (d -1) was
calculated as follows:
36
GR= ln(X2 /X1)
t2 - t1
where X1 and X2 were the biomass concentration (g l-1) on days t1 and t2, respectively
3.9.6.2 Estimation of lipid content
The lipids content L (%) was calculated by the equation:
L (%) = WL *100
WB
Where, WL and WB are the weight of extracted lipids and of dry biomass, respectively.
3.9.6.3 Estimation of biomass productivity
The biomass productivity PB (mg L-1d-1) was calculated by equation:
PB = ( WBF –WB0)
t
where WB0 and WBF are the weights of dry biomass at the begin and the end of a batch run
and t is the overall culture time.
3.9.6.4 Estimation of lipid productivity
The lipid productivity PL (mg L-1d-1) was calculated as the product of biomass productivity
and lipid content according to the following equation:
PL = PB * L (%)
3.9.6.4 Determination of the CO2 Consumption rate
The CO2 consumption rate (PCO2, mg/l/d) was derived with equation:
PCO2 = 1.88 * PB
37
Chapter IV
Results
38
4.1 Growth Kinetics of different microalgal species
Figure 3: Growth kinetics of 8 different species of algae at 750nm
4.2 Seasonal variation on the growth of 3 potential algal species
Figure 4: Effect of seasonal variation on the growth kinetics of 3 microalgal species
39
4.3 Determination of Growth
For each pure culture, a regression equation was prepared, as Becker discussed, during the
culture’s exponential growth phase. The absorbance was compared with each suspension’s
respective dry weight. There was a direct correlation between absorbance and dry weight
for each pure culture examined and each was expressed by a function:
y= 0.2893 * x
R2 = 0.9682
Where, x is the algal suspension absorbance at 750nm and y is the cell concentration (g/L).
Figure 5: Regression Equation for Standard Curve
40
4.4 Optimization of Nitrogen
4.4.1 Growth Kinetics
Figure 6: Growth Curve of Chlorella vulgaris at different nitrate concentration
4.4.2 Growth Rate
Figure 7: Growth rate of microalgae culture in Standard Bold Basal Media
Growth Rate
0.5657676 d-1
41
Day 2
Figure 8: Growth rate of microalgae culture in Bold Basal Media with 2X
concentration of Nitrogen
Growth Rate
0.607021 d-1
Day 2
Figure 9: Growth rate of microalgae culture in Bold Basal Media with 1/ 2 X
concentration of Nitrogen
Growth Rate
0.490416 d-1
42
Day2
Figure 10: Growth rate of microalgae culture in Bold Basal Media with 1/ 4 X
concentration of Nitrogen
Growth Rate
0.54071 d-1
Day2
4.4.3 Lipid content
Figure 11: Lipid content of Chlorella vulgaris at different Nitrate concentration
43
4.4.4 Biomass Productivity
Figure 12: Biomass Productivity of Chlorella vulgaris at different Nitrate
concentration
4.4.5 Lipid Productivity
Figure 13: Lipid Productivity of Chlorella vulgaris at different Nitrate concentration
44
4.4.6
Effect of Nitrogen on chlorophyll content
Figure 14: Chlorophyll concentration at different nitrogen concentration
45
4.5 Optimization of Phosphorus
4.5.1 Growth Kinetics
Figure 15: Growth Curve of Chlorella vulgaris at different phosphate concentration
4.5.2 Growth Rate
Figure 16: Growth rate of microalgae culture in Standard Bold Basal Media
Growth Rate
0.5657676 d-1
46
Day 2
Figure 17: Growth rate of microalgae culture in Optimized Bold Basal Media with
2X concentration of phosphate
Growth Rate
0.419995 d-1
Day 2
Figure 18: Growth rate of microalgae culture in Optimized Bold Basal Media with
1X concentration of phosphate
Growth Rate
0.490416 d-1
47
Day 2
Figure 19: Growth rate of microalgae culture in Optimized Bold Basal Media with
1/ 2 X concentration of phosphate
Growth Rate
0.483733 d-1
Day 2
Figure 20: Growth rate of microalgae culture in Optimized Bold Basal Media with
1/ 4 X concentration of phosphate
Growth Rate
0.5243 d-1
48
Day2
4.5.3 Lipid content
Figure 21: Lipid content of Chlorella vulgaris at different Phosphate concentration
4.5.4 Biomass Productivity
Figure 22: Biomass Productivity of Chlorella vulgaris at different Phosphate
concentration
49
4.5.5 Lipid Productivity
Figure 23: Lipid productivity of Chlorella vulgaris at different Phosphate
concentration
4.6 Effect of carbon concentration
Two experimental approaches have been taken. The first approach was to assess the
effects of bicarbonate addition on the lipid accumulation of Chlorella vulgaris on the
normal bold basal media, and the second, to specifically examine the effects of
bicarbonate addition on the rates of lipid production Chlorella vulgaris in optimized media
with limited amount of nitrogen and phosphorus.
4.6.1 Effect of optimized media
4.6.1.1 Growth Kinetics
50
Figure 24: Growth Curve of Chlorella vulgaris at different sodium bicarbonate
concentration in optimized media
4.6.1.2 Growth Rate
Figure 25: Growth rate of microalgae culture in Optimized Bold Basal Media with
0mg/l sodium bicarbonate
Growth Rate
0.483733 d-1
51
Day 2
Figure 26: Growth rate of microalgae culture in Optimized Bold Basal Media with
120mg/l sodium bicarbonate
Growth Rate
0.512014 d-1
Day 2
Figure 27: Growth rate of microalgae culture in Optimized Bold Basal Media with
240mg/l sodium bicarbonate
Growth Rate 0.63933726 d-1 Day 2
52
Figure 28: Growth rate of microalgae culture in Optimized Bold Basal Media with
360mg/l sodium bicarbonate
Growth Rate
0.715801 d-1 Day 2
4.6.2 Effect on normal media
4.6.2.1 Growth Kinetics
Figure 29: Growth Curve of Chlorella vulgaris at different sodium bicarbonate concentration
in normal media
53
4.6.2.2 Growth Rate
Figure 30: Growth rate of microalgae culture in Normal Bold Basal Media with
0mg/l sodium bicarbonate
Growth Rate
0.565768 d-1
Day 2
Figure 31:Growth rate of microalgae culture in Normal Bold Basal Media with
120mg/l sodium bicarbonate
Growth Rate
0.637733 d-1
54
Day 2
Figure 32: Growth rate of microalgae culture in Normal Bold Basal Media with
240mg/l sodium bicarbonate
Growth Rate
0.700506 d-1
Day 2
Figure 33: Growth rate of microalgae culture in Normal Bold Basal Media with
360mg/l sodium bicarbonate
Growth Rate
0.757394 d-1
55
Day 2
4.6.3 Lipid Content
Figure 34:Biomass Productivity of Chlorella vulgaris at different sodium bicarbonate
concentration in optimized and normal BBM media
4.6.4 Biomass productivity
Figure 35: Biomass Productivity of Chlorella vulgaris at different sodium
bicarbonate concentration in optimized and normal BBM media
56
4.6.5 Lipid productivity
Figure 36: Lipid Productivity of Chlorella vulgaris at different sodium bicarbonate
concentration in optimized and normal BBM media
4.6.6 CO2 consumption rate
Figure 37: CO2 consumption rate of Chlorella vulgaris at different sodium
bicarbonate concentration in optimized and normal BBM media
57
Chapter V
Discussion
58
Nutrient limitation is an efficient trigger to increase lipid content per algal biomass, and it
has been reported by many other researchers. Generally microalgae accumulate lipid
under nutrient limitation when energy source (light) and carbon source (CO2) are available
and when the cellular mechanisms for the photosynthesis are active. The onset of
significant lipid storage production in microalgae is promoted by stress conditions of
nitrogen and phosphorus.
5.1 Effect of Nitrogen
Nitrogen ions are important nutrients for microalgae which support both growth and
biomass synthesis. It is considered to be one of the limiting factors in microalgae
productivity. Most of the microalgae species are able to utilize a wide range of both
organic and inorganic nitrogen sources. The main source of the nitrogen used in this
experimental research to study the effect of nitrogen ions on growth and lipid productivity
is nitrate salt i.e NaNO3. The results obtain from study illustrate that increase in nitrogen
ions concentrations influenced positively in microalgal growth rate and dry weight yield
during the culturing period. When the microalgae were grown at limited nitrogen
concentration of 62.5 mg/l nitrogen ions, there was an initial rapid growth in response to
the presence of the nitrogen which was comparable to that of media containing 500mg/l of
nitrogen concentration. However, from Fig.6. it can be seen that optical density of media
containing 1/4th times concentration of nitrogen to that of control slow down from day 3
because of the depletion of nitrogen ions in the medium. A maximum growth rate
(0.607021d-1) and dry weight yield (45.8409mg/l/d) was achieved at a nitrogen ion
concentration of 500mg/l. Biomass productivity was found to be increased with increase
in nitrogen concentration. Hence, this concludes that nitrogen is one of the limiting factors
for the growth of microalgae. Lowest biomass productivity of 32.647 mg/l/d was observed
in a 1/4X media whereas biomass productivity in1/2X media and control (1X was
recorded as 40.0535 and 45.8405, respectively.
Simultaneously, study on the lipid content and lipid productivity of Chlorella vulgaris was
performed to determine the optimum concentration of nitrogen for maximum lipid
productivity. In contrast to that of growth rate and biomass productivity, an increasing
trend is observed in lipid content as the concentration of nitrate was decreased. Lipid
content in control media of BBM was found to be 24% of dry weight. But, when nitrate
concentration was double to that of the standard Bold Basal Media (control), lipid content
59
was found to be 19% of dry weight. On contrary, when nitrogen concentration was
decreased by ½ and ¼ th to that of control, lipid content was recorded to be 28% and 29%
of dry weight, respectively. Hence, nitrogen plays a vital role for lipid induction in
Chlorella vulgaris. However, this doesn't conclude that nitrate concentration of 1/4th of the
original is optimum concentration microalgae culture. To be an optimum concentration, it
is equally important that lipid productivity also increases with lipid content. Fig.13.
clearly shows that lipid productivity is maximum (11.33 mg/L/d) when nitrate
concentration was ½ times that of control. At this concentration, a sharp enhancement in
lipid content was found which ultimately results in increase in lipid productivity. Despite
of maximum biomass productivity observed in 2X media, lipid productivity was observed
to be low attributing to the effective involvement of nitrogen in cell growth and
physiological function rather than lipid synthesis. In addition, study on variation in
Chlorophyll concentration which confirms that with decrease in nitrogen concentration
Chlorophyll concentration also decreases due to which impairment in photosynthesis
occur. From the morphological study and from Fig. 40, we can see the change in color of
microalgae culture. At lower concentration of nitrate, the cells were conspicuously
chlorotic, having progressed from dark green to yellow green in color.
Various researchers have demonstrated that nitrogen limitation would cause three
changes: 1. decreasing of the cellular content of thylakoid membrane, 2. activation of
acylhydrolase and 3.stimulation of the phospholipid hydrolysis. These changes may
increase the intracellular content of fatty acid acyl-CoA. Meanwhile, nitrogen limitation
could activate diacylglycerol acyltransferase, which converts acyl-CoA to triglyceride
(TAG). Therefore nitrogen limitation could both increase lipid and TAG content in
microalgal cells. The concentrations used in the experiments described in this dissertation
are very high compared with those of natural levels. This proved that microalgae can be
used both for nitrate remediation in wastewater and for absorbing CO2.
5.2 Effect of phosphorus
The requirement of phosphate for optimal growth differs considerably from species to
species in microalgae. Phosphate ions are required by microalgae for most of their cellular
activities, especially those involved in generating and transforming metabolic energy.
Polyphosphate, which participates in metabolism, is also stored in the biomass and can be
used when the external source of phosphates is insufficient. Grobbelaar (2004) suggested
60
that the approximate molecular formula for microalgae is CO0.48H1.83N0.11P0.01 and that
consequently phosphate ions are required in minute quantities to make biomass.
Similar to nitrogen, phosphorus concentration are also reported to have inverse relation in
microalgae growth and lipid. After the study of the growth of Chlorella vulgaris at
different nitrogen concentration, NaNO3 with ½ times that concentration of standard BBM
was found to be optimum in term of growth and lipid content. Further, optimization of
phosphorus was also done in the media containing ½X of nitrogen concentration to
maximize the lipid productivity. For this, Chlorella vulgaris was cultured in the media
containing phosphorus same as that in control and by manipulating the concentration of
phosphorus by 2, ½ and ¼ times that of the control media which was denoted as 2X, 1/2X
and 1/4X. In this study we observe that increasing the phosphorus 2 times that of the
standard media was not favorable in terms of growth, lipid content and lipid productivity.
But when the concentration of phosphorus is decreased by ½ and ¼ times compare to that
of the standard media, lipid content get increased which was recorded as 29.05% and
28.94%, respectively. In addition, lipid productivity was also found to be high
i.e.12.26mg/l/d in media containing ½ times concentration of nitrogen and phosphorus
compare to that of the control media i.e. 10.75mg/l/d. For this reason it can be conclude
that phosphorus limitation also play some vital role during lipid production. Comparisons
of the data presented in Figs. 11 and 21 indicated that the contribution of P-starvation to
lipid accumulation under N-starvation was not as significant as N-starvation. Hence, costarvation of essential nutrients like nitrogen and phosphorus enriches the content of lipid
and lipid productivity although biomass productivity was not satisfactory. Higher lipid
productivity in nutrient limited condition might be due to the decrement in the protein
synthesis rate, resulting in feedback inhibition in citric acid cycle. For this reason, the
composition of media with ½ concentrations of nitrogen and phosphorus compare to that
of the control media is considered to be favorable in microalgae culture for higher lipid
accumulation.
For the production of biodiesel from microalgae too, the presence of higher phosphorus
results in lower efficiency as the amount of phospholipids is more than that of TAG.
Hence, environment stresses caused by co-limitation of essential nutrients like nitrogen
and phosphorus is beneficial for lipid accumulation in microalgae cell especially for
biodiesel production.
61
5.3 Effect of carbon
An adequate supply of inorganic carbon is essential for regular photosynthesis and growth
in photoautotrophic microalgae. This may be achieved through the supply of gaseous CO2
to the media in which the microalgae are growing. To sustain a stable growth and higher
biomass production, CO2 must be added into the culture medium from external sources.
However, due to very low solubility of CO2 in water (1.45 g L
-1
at 250C, 100kPa), the
majority of carbon dioxide tends to be lost in the air. So it is convenient to use bicarbonate
form instead of CO2 gas as NaHCO3 are highly soluble in water, which can serve as a
better source of CO2 for growing microalgae with minimum efforts. When NaHCO3
dissolves in water, it dissociates to produce HCO3- ions that serve for microalgae as
readily available dissolved inorganic carbon. However, the optimal upper limit of
NaHCO3 concentrations in the growth medium vary accordingly with varying microalgae
species/strains. Many microalgae species can actively take up HCO3− from the external
environment via transport across the plasma membrane into the cytosol and derive CO 2
from HCO3− via the action of carbonic anhydrase maintaining a steady state flux to
ribulose-1,5-bisphosphate carboxylase oxygenase for photosynthesis. Alternatively,
extracellular carbonic anhydrase can catalyse the inter-conversion of HCO3− and CO2.
In the application of algae to sequester carbon dioxide and energy production, tolerance to
CO2, is of great importance. High levels of CO2 are considered to be favorable for
accumulation of total lipids and polyunsaturated fatty acids.
This study has been carried out to assess the effect of bicarbonate addition on the growth
with the focus on lipid synthesis which is relevant for biodiesel production. This study
builds to investigate the effects of different levels of sodium bicarbonate on the rate of
lipid production in microalgae under nutrient-deplete conditions. Two experimental
approaches have been taken. The first approach was to assess the effects of bicarbonate
addition on the lipid accumulation of Chlorella vulgaris on the normal bold basal media,
and the second, to specifically examine the effects of bicarbonate addition on the rates of
lipid production Chlorella vulgaris in optimized media with limited amount of nitrogen
and phosphorus.
A study on concentration of 120mg/l, 240mg/l and 360mg/l of sodium bicarbonate was
performed in both optimized as well as normal media. Jeong et al in 2003 found that 15.3
mg/L bicarbonate salt is equivalent to 243 mg/L CO2 gas. So, during research
62
concentration of NaHCO3 was correlated with the concentration of CO2 to determine the
CO2 fixation capability. Increase in growth rate was found with increase in concentration
of sodium bicarbonate. Maximum growth rate of 0.7158 d-1 and 0.7574d-1 was observed in
optimized and normal BBM medium supplemented with 360 mgL -1 of NaHCO3,
respectively. With the addition of NaHCO3 we can observe highest growth during the
exponential phase compare to that in control. From this we can also conclude that with
addition of carbon sources like sodium bicarbonate or with the supply of CO2 we can
shorten the culture period of microalgae which will ultimately reduce the cost of
production. By comparing growth rate Chlorella vulgaris supplemented with NaHCO3 in
both optimized media and normal media, we can see that growth rate of normal media is
higher than that of the optimized one which is due to the nutrient limitation condition.
Similar like in the case of growth rate, NaHCO3 also enhances biomass productivity and
lipid productivity of Chlorella vulgaris. Additionally, CO2 consumption rate of Chlorella
vulgaris grown on optimized and normal BBM was also determined. And, this study
illustrate that CO2 consumption rate of optimized media is comparatively less than that of
normal media. It was found that CO2 consumption rate in normal media supplemented
with 360mg/l NaHCO3 is 92.3613 mg/l/d while in optimized media it was found to be
84.8706mg/l/d. The difference in CO2 consumption is due to the impairment of
photosynthesis in optimized media, where carbon fixation is greatly reduced via
glyoxylate cycle leading to increments in conversion of fatty acid acyl Co-A to
triglyceride. The fixation of carbon dioxide happens in the dark reaction using the
NADPH2 and ATP produced in the light reaction of photosynthesis. And CO2
consumption rate of optimized media may be less due to limited amount of nitrogen and
phosphorus concentration which results in lower rate of photosynthesis.
In summary, the findings suggest that bicarbonate addition can significantly increase the
growth rate and lipid productivity of Chlorella vulgaris. Furthermore, the rates and levels
of lipid synthesis and/or turnover under nutrient-deplete conditions can be promoted with
bicarbonate addition, although CO2 consumption is minimum in it. Under nutrient limited
conditions, the production of cellular storage lipid comes at the expense of regular growth
and cell division, which is arrested owing to a reduction in photosynthetic activity and
diverted metabolic demand. The nitrogen and phosphorus starvations divert the inorganic
carbon fixation from DNA and proteins synthesis toward lipids synthesis. This synergic
effect can be explained because although nitrogen is considered the single most critical
63
nutrient to channel metabolic flux to lipid biosynthesis, even phosphorus is known to
affect lipids metabolism in algae. The absence of phosphorus in the cultural medium in
fact causes the photosynthesis repression. In this way the photosynthesis derived
precursors of starch cannot be synthetized and the energetic surplus induced by the
limitation of cellular duplication is largely stored in form of lipids. Therefore, the
combining CO2 mitigation and nitrogen limitation as a strategy for lipid accumulation in
microalgae may provide an innovative alternative to current carbon-reduction and biofuelproduction strategies.
64
Chapter VI
Conclusion
65
Optimizing the growth media is a critical step to develop an economical route for
sustainable algal biomass production. In 1994 Becker wrote, “The successful growth of
algae is more or less an art and a daily tightrope act with the aim of keeping the necessary
prerequisites and various unpredictable events involved in algal mass cultivation in a sort
of balance.” The goal of this study was to define the nitrogen and phosphorus
requirements of cultured Chlorella vulgaris in order to efficiently grow the algae in a
carbon sequestering scheme by growing C. vulgaris. The outcomes from this study have
shown that a small amount of nitrogen and phosphate and carbon supply were helpful for
increasing lipid productivity compare to that of the control.
Nutritional mode variations documented marked influence on both biomass growth and
lipid productivity of microalgae. Intergrating stress condition and carbon supply has
significantly enhanced lipid synthesis and its productivity. This shows that microalgae
hold the prominent position in the field of biofuel production and CO2 sequestration
process. Algae have a stoichiometric composition of C:N:P ratios of 50:8:1 while
domestic wastewater has a composition of (C:N:P) 20:8:1. With addition of carbon source
(CO2) from industries effluent, wastewater can serve as excellent medium for algal
growth. Secondary effluents of domestic wastewater usually contain relatively low
inorganic nutrients (around 10-15 mg/l nitrogen and 0.5-1 mg/l phosphorus), so
microalgae can be also utilized in wastewater treatment. Since the wastewater has
adequate nutrient composition, algae growth using wastewater may completely eliminate
the ecological issues like eutropication and climate change. In addition, it may alleviate
the cultivation costs to some extent. Besides minimizing environmental issues it also solve
the current problem of scarcity of fossils fuel producing biofuel which is also nowadays
considered as environmentally friendly technology. Most of the world’s fossil fuel and
industrial carbon emissions have little value at best, and will take on large costs in the
future, both environmentally and monetarily. Algal biotechnology is a technological field
that is suited to sequester this carbon dioxide.
For the success of any sustainable biofuel, there are three principal considerations:
technical feasibility; economic viability; and, resource sustainability. Algal-based biofuel
is technically feasible. However, to date, economic viability has not been achieved.
Furthermore, resource sustainability, in terms of land, water, nutrient and energy
utilization, must be meticulously quantified for each type of production system in order
for the feedstock to be considered truly “sustainable”. With large-scale biofuel production
66
processes, this water-energy-nutrient nexus is the subject of significant consideration and
debate. Potential benefits of microalgae cultivation are enormous, and research into algae
production using wastewater and CO2 emissions are inviting a highly prioritized
proposition in sustainable and economic development. The ability to expand algal oil
harvests depends mostly on: (a) isolation of new strains of algae that produce large
amounts of desirable lipids, and (b) identification of environmental conditions that
promote rapid growth of the oil-producing algae.
Algae can play an important role in the biobased economy. Algae are efficiently cultivated
in places that are unsuitable for agriculture and where nature is not harmed. Sustainable
production of biodiesel, but also many other products such as proteins, colorants and raw
material for bioplastics is achievable. To achieve profitable cultivation of algae, the
production efficiency must be increased three times and costs must be reduced ten times.
In addition, besides oil for biofuel, other useful substances such as proteins must also be
extracted from the algae. Results of this investigation have revealed the potentialities of
the microalgae strain Chlorella vulgaris to be utilized as a renewable biomass feedstock of
biofuels. These experimental results may be helpful towards a large scale algae cultivation
of the studied microalgae strain, which could be potentially used for sustainable biofuel
production.
67
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Appendix
73
Appendix 1: Composition of Bold Basal Media Used
BOLD’S BASAL MEDIUM (BBM)
Reference: Stein, J. (ED.) Handbook of Phycological methods. Culture methods and
growth measurements. Cambridge University Press. 448 pp.
This medium is highly enriched and is used for many of the green algae.
STOCK
1. KH2PO4
2. CaCl2.2H2O
3. MgSO4.7H2O
4. NaNO3
5. K2HPO4
6. NaCl
7. Na2EDTA
KOH
8. FeSO4.7H2O
H2SO4 (conc.)
9. Trace Metal Solution
10. H3BO3
11. Soil extract
*Trace Metal Solution:
STOCK SOLUTION
8.75 g/500 ml
1.25 g/500 ml
3.75 g/500 ml
12.5 g/500 ml
3.75 g/500 ml
1.25 g/500 ml
10 g/L
6.2 g/L
4.98 g/L
1 ml/L
See below*
5.75 g/500 ml
50gm/500ml
Substance
g/Litre
1. H3BO3
2. MnCl2.4H2O
3. ZnSO4.7H2O
4. Na MoO4.5H2O
5. CuSO4.5H2O
6. Co(NO3)2.6H2O
2.86 g
1.81 g
0.222 g
0.390 g
0.079 g
0.0494 g
ml/Litre
10 ml
10 ml
10 ml
10 ml
10 ml
10 ml
1 ml
1 ml
1 ml
0.7 ml
10ml
Each of the above substances for the Trace Metal solution is dissolve separately, prior to
adding the next on the list. pH of the medium is adjusted to 6.8 with NaOH or HCl and
autoclave.
Preparation of soil extract
1. Fine soil sample are collected from a barren land and sundry for 1 day.
2. After 1 day 50 gm of fine soil is mixed in 500ml of distilled water.
3. The mixture is then boiled for 45 minutes and cooled.
4. After cooling the mixture is then filtered.
5. Filtrate is then autoclaved for 2 times maintaining a gap of 1 day
74
Appendix 2 : Standard Curve
Table 2: Coorelation between Absorbance and dry weight
Absorbance
wt(gm/l)
0
0
0.243
0.03
0.438
0.09
0.589
0.15
0.733
0.21
0.879
0.25
1.044
0.32
1.246
0.38
Appendix 3: Experimental Conditions
Species
Photoperiod
C. vulgaris
12:12
Irradiance(Lux) Temp (°C)
4000
Water Source
26± 2
Distilled H2O
Appendix 4: Experimental Data
Table 3: Optical density at different nitrogen concentration
Days
2X
Control
1/2X
1/4X
0
0.203
0.173
0.190833
0.196
1
0.287333
0.277833
0.293333
0.323667
2
0.529333
0.487667
0.478667
0.553
3
0.851
0.7
0.706667
0.827667
4
1.073333
0.953667
0.874833
0.948
5
1.26
1.1295
1.076833
1.044333
6
1.459333
1.302333
1.257167
1.168
7
1.580333
1.452
1.355167
1.215667
8
1.722667
1.570833
1.457833
1.282667
9
1.776667
1.661667
1.550833
1.318667
10
1.871333
1.741333
1.597167
1.41
11
1.946
1.7855
1.66
1.437333
75
Table 3: Lipid content at different nitrogen concentration
nitrogen
lipid
conc.
content(%)
2X
19.88889
Control
24.38889
1/2X
28.33333
1/4X
29.66667
Table 4: Biomass Productivity at different nitrogen concentration
nitrogen
Biomass
conc.
productivity(mg/l/d)
Control
44.16252
2X
45.8409
1/2X
40.05359
1/4X
32.64707
Table 5: Lipid Productivity at different nitrogen concentration
nitrogen
Lipid
conc.
productivity(mg/l/d)
Control
10.75396
2X
9.104943
1/2X
11.33286
1/4X
9.683397
76
Table 6: Chlorophyll concentration at different nitrogen concentration
Experimental
Average
variation
value
1XChla
27.5749
1XChlb
13.32365
2XChla
30.88997
2XChlb
16.64903
1/2XChla
16.03092
1/2XChlb
8.67346
1/4XChla
9.269183
1/4XChlb
3.480627
Table 7: Optical density at different phosphorus concentration
S.N(P)
Control
2X
1X
1/2X
1/4X
0
0.173
0.182333
0.190833
0.2124
0.188
1
0.277833
0.270333
0.293333
0.3154
0.286
2
0.487667
0.405333
0.478667
0.5112
0.477
3
0.7
0.606
0.706667
0.748
0.703667
4
0.953667
0.743667
0.874833
0.9446
0.919333
5
1.1295
0.869333
1.076833
1.1318
1.144333
6
1.302333
1.040667
1.257167
1.285
1.276333
7
1.452
1.150667
1.355167
1.4066
1.406667
8
1.570833
1.264333
1.457833
1.538
1.479
9
1.661667
1.334333
1.550833
1.6304
1.539667
10
1.741333
1.4
1.597167
1.6906
1.619333
11
1.7855
1.415
1.66
1.762
1.657
77
Table 8: Lipid content at different phosphorus concentration
Phosphorus
Lipid
conc.
content
(%)
2X
21.38889
control
24.3889
1X
28.3333
1/2X
29.33333
1/4X
28.94444
Table 9: Lipid Productivity at different phosphorus concentration
Phosphorus
Lipid
conc.
productivity(mg/l/d)
Control
10.75396
2X
7.332276
1X
11.33286
1/2X
12.26089
1/4X
11.73869
Table 10: Biomass Productivity at different phosphorus concentration
Phosphorus
Biomass
conc.
productivity(mg/l/d)
Control
44.16252
2X
34.15493
1X
40.05359
1/2X
41.53349
1/4X
40.60282
78
Table 11: Optical density at different NaHCO 3 concentration in normal BBM
Days
NB
N1
N2
N3
0
0.173
0.211
0.21
0.208
1
0.277833
0.322
0.3325
0.394
2
0.487667
0.608
0.6695
0.756
3
0.7
0.8395
0.972
1.148
4
0.953667
1.066
1.197
1.4345
5
1.1295
1.252
1.391
1.6195
6
1.302333
1.452
1.5655
1.721
7
1.452
1.5545
1.6695
1.82
8
1.570833
1.642
1.762
1.8725
9
1.661667
1.721
1.8065
1.9165
10
1.741333
1.805
1.8565
2.03
11
1.7855
1.905
1.9435
2.076
Table 12: Optical density at different NaHCO3 concentration in optimized BBM
Days
OB
O1
O2
O3
0
0.2124
0.22
0.226
0.217
1
0.3154
0.3385
0.3435
0.36
2
0.5112
0.564
0.65
0.7365
3
0.748
0.792
0.9275
1.0565
4
0.9446
0.9965
1.1005
1.295
5
1.1318
1.1885
1.2825
1.429
6
1.285
1.3135
1.415
1.5385
7
1.4066
1.438
1.5215
1.6085
8
1.538
1.546
1.615
1.7
9
1.6304
1.6515
1.7015
1.798
10
1.6906
1.741
1.789
1.876
11
1.762
1.8285
1.905
1.9335
79
Table 13: Lipid content at different NaHCO3 concentration
Lipid
Carbon
content
concentration
(%)
NB
24.3889
N1
25.33333
N2
26.41667
N3
27.25
OB
29.33333
O1
29.33333
O2
29.58333
O3
31.25
Table 14: Biomass Productivity at different NaHCO 3 concentration in normal and optimized
media
Carbon
Biomass
concentration productivity(mg/l/d)
NB
44.16252
N1
44.5522
N2
45.59105
N3
49.1284
OB
41.53349
O1
42.30355
O2
44.1577
O3
45.14395
80
Table 15: Lipid Productivity at different NaHCO 3 concentration in normal and optimized
media
Carbon
Lipid
concentration productivity(mg/l/d)
NB
10.75396
N1
11.2955
N2
12.05046
N3
13.39582
OB
12.26089
O1
12.4111
O2
13.06617
O3
14.12108
Table 16: CO2 consumption rate at different NaHCO 3 concentration in normal and optimized
media
CO2
Carbon
Consumption
concentration
rate (mg/l/d)
NB
83.02554
N1
83.75814
N2
85.71117
N3
92.36139
OB
78.08295
O1
79.53067
O2
83.01648
O3
84.87063
81
Appendix 3: Photo Gallery
Figure 38: Isolation of microalgae
Figure 39: Single colony isolation
Figure 40: Microalgae culture at different
Figure 41: Microalgae culture at different
concentration of nitrogen
concentration of phosphorus
Figure 42: Initial day of culture of Chlorella
Figure 43: Initial day of culture of Chlorella
vulgaris in normal media with NaHCO3
vulgaris in optimize media with NaHCO3
82
Figure 44:Final day of culture in normal
Figure 45: Final day of culture in optimize
media with NaHCO3
Figure 46: Final day of culture in different
media with NaHCO3
Figure 47: Difference in chlorophyll amount in
phosphorus concentration
normal and optimized media
Figure 48:Gravimetric separation of algal
Figure 49: Crushing of dried biomass
83
Figure 42: Lipid extraction by Bligh
Figure 43: Chloroform layer containing lipid
and Dyer method
Figure 50: Algal Biomass after extraction
84