introduction & review of literature -...

Introduction & Review of Literature



1. Introduction and review of literature

1.1. Backdrop of algae

Phytoplanktons are the most important biomass producers in global aquatic

ecosystems. The organisms populate the top layers of the oceans and freshwater

habitats where they receive sufficient solar radiation for photosynthesis (Hader et al.,

1998). Algae produce approximately 52,000,000,000 tons of organic carbon per year,

which is almost 50% of the total organic carbon produced on earth each year (Field et

al., 1998). Therefore it appears to be an important biomass producer using

atmospheric carbon dioxide and solar energy. Hence there is an increasing quest all

over the world to explore microalgae to address various applications in

pharmaceutical, nutraceutical and food, textile, aquaculture, biofuel, and carbon

dioxide mitigation, bioremediation of heavy metals and for many such prospective

industrial and commercial applications.

Algae are the simple photosynthetic aquatic organisms belong to both

Eukaryota and Prokaryota (Gupta, 1981). They exist as single-celled organisms to

multicellular organisms with fairly complex differentiated from than the single celled

ones. These complex forms are distinguished as macroalgae which includes the

marine forms such as seaweeds. They are devoid of well differentiated structures such

as leaves, roots, flowers, and other organ structures that characterize higher plants

(Dawson, 1966; Fritsch, 1977).

The history of algae is as old as human civilization, people collected macro

algae and seaweeds for food around 2,500 years ago in China (Tseng, 1981). The use

of microalgae by humans dates back 2000 years to the Chinese, who used Nostoc to

survive during famine (Spolaore et al., 2006). Japanese also found macroalgae as a

food source during the 4th

century (McHugh, 2003). Europeans started using

seaweeds in their diet from the past 500 years (McCoy, 1987). During the mid- 17th

century Japanese have started cultivating seaweeds for food supplement (Pulz and

Gross 2004). Nostoc, Spirulina, and Aphanizomenon species have been exploited for

many centuries in Asia, Africa and Mexico as a nutrient-dense food (Jensen et al.,

2001; Olaizola 2003). In the 1940s, microalgae became popular as live feeds in

aquaculture. After 1950s, algal biotechnology developed rapidly, starting in Germany

and extending into the USA, Israel, Japan, and Italy for producing protein and fat as a

nutrition source from algal biomass (Burlew, 1953). Simultaneously the use of

microalgae in wastewater treatment and the systematic examination of algae for


biologically active substances, particularly for antibiotics was also initiated

(Borowitzka, 1995). During 1960s, the commercial production of Chlorella as a

novel health food supplement was a big success in Japan and Taiwan (Kawaguchi,

1980). In USA, interest grew in developing algae as photosynthetic gas exchangers

for long term space travel as well (Borowitzka, 1999). Energy crises during 1970s

energized the idea of using microalgal biomass as renewable fuels and fertilizers with

an eco-friendly process (Pulz and Scheibenbogen, 1998; Spolaore et al., 2006). At the

same time first large-scale Spirulina production plant was established in Mexico

(Borowitzka, 1999) and later during 1980s and onwards there were more than 46

large-scale algae production plants in Asia particularly in India, and large commercial

production facilities in the USA and Israel were started to operate for microalgae

production for protein, fat and for other nutraceutical and pharmaceutical molecules

(Spolaore et al., 2006).

Particularly during the last two to three decades, algal biotechnology grew

progressively and occupied a key position in scientific world to address various

aspects of environment and mankind. Scientific and technological information on

algae is accumulating day by day and thus various algologists, ecologists,

environmentalists, policy makers and industrialists look forward to explore these tiny

wonderful plants for diversified applications in agricultural, food, medicine and for

environmental prospective. This radical shift in phycological research has initiated

various groups of researchers across the globe to explore the potentials of algae to

address various environmental, food and biofuel issues. Currently almost all countries

across the continents are using microalgae and macroalgae for various foods, health

and other benefits. Today, more than 40,000 algal species are known (Hallmann,

2007). But so far only few algal forms have been commercially exploited. In this

context, isolating newer strains and improving the existing domestic flora, through

modern biotechnological approaches, will certainly enhance the algal potential and

algae based business.

In India, algae have been primarily studied for over a century as a curriculum

at Universities and only in the last few decades some research institutes and

Universities of repute have taken algal research and development as a matter of

serious concern. The focus on algae as a source of chemicals for food,

pharmaceutical, nutraceutical and biofuel uses have changed Indian phycological

research from taxonomy & physiological studies to chemical aspects. As a result, The


Central Marine Chemical Research Institute (CSMCRI), Bhavnagar, has pioneered

the utilization of seaweed for production of polysaccharides having potential use in

Industry. Indian Agricultural Research Institute (IARI), New Delhi and Madurai

Kamaraja University of Madurai, Tamil Nadu Agricultural University (TNAU),

Coimbatore worked on algal biofertilizers. Central Food Technological Research

Institute (CFTRI), Mysore and Murugappa Chettair Research Centre of Chennai have

worked extensively on Food applications of Spirulina and Scenedesmus. However

CFTRI later continued with research on several microalgae viz. Dunaliella,

Haematococcus, Botryococcus and Porphyridium. National Facility on Bluegreen

Algae at IARI, New Delhi and National Facility for Marine Cayanobacteria at

Triuchinapalli has brought to focus the need in conserving algal biodiversity and

ecology. Microalgae are of particular interest because, they can grow rapidly and it is

possible to cultivate them in sea or brackish or sewage water resources in marginal

lands to produce reasonably high biomass per unit area than by any other higher

plants.

1.2. Botryococcus

Botryococcus is a green unicellular colonial microalgae and its species are known to

produce large amounts of lipids ranging from 10 to 60% (w/w). However, lipid

productivities depends on the physiological conditions and also the chemical race to

which they belong (Fang et al., 2004; Metzger and Largeau, 2005; Guschina and

Harwood, 2006). Palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic acids

(18:3) are the major constituent fatty acids (Zhila et al., 2005; Kalacheva et al., 2001;

Volova et al., 2003) of Botryococcus lipids. Nearly 13 species of the genus

Botryococcus are being recognized and among them the species Botryococcus braunii

is well worked out and is widespread in freshwater and brackish lakes, reservoirs,

ponds and is recognized as one of the potent resource for the production of lipids and

hydrocarbons. B. braunii is classified into three chemical races A, B and L depending

on the type of hydrocarbons synthesized (Metzger and Largeau, 2005; Achitouv et

al., 2004).The existence of B. braunii in USA, Ivory Coast, Portugal, Bolivia,

Morocco, Philippines, Thailand, France and West Indies has confirmed its wide

distribution (Wolf et al., 1985; Metzger et al., 1985; Okada et al., 2000). Further,

these geographical regions belong to different climatic zones like continental,

temperate, tropical and alpine indicating its adoptability to grow in varied climatic

conditions as well (Tyson, 1995).


1.3. Morphology and phylogenetic position

Komarek and Marvan (1992) proposed the existence of at least 13 species in the

genus Botryococcus. Morphological heterogeneity was found to exist within an alga

examined after water sampling from the isolated area and the same strain cultivated in

the laboratory conditions with respect to size and shape of the cells and colonies. It

was also reported that, there is variation in each chemical race and for the same strain;

there are differences in the morphological features in relation to culture age and

culture conditions (Plain et al., 1993). Botryococcus colonies exhibit a typical

morphology characterized by a botryoid to spherical organization of individual

pyriform-shaped cells (generally cell body is ovoid, 6-10 µm long, 3-6 µm width) held

together by a refringent matrix containing lipids, which some times links two or more

distinct clumps of cells. They reproduce asexually by autospores and sexually by

fertilization of egg and sperm (Largeau et al., 1980).

Botryococcus was first reported as a member of chlorophyceae and then it was

placed in Xanthophyceae based on the structure of its plastids and starch granules.

However, its ultrtrastructural studies have again referred it as a member of

Chlorococcales (Chlorophyceae, Chlorophyta). Analysis of 18s rRNA sequence data

is facilitating the ease of finding a phylogenetic relationship among the microalgae.

However, phylogenetic analyses of several strains of the species B. braunii were

carried out using 18s rRNA sequence analyses by Senousy et al., (2004). Based on

their 18s rRNA sequence analysis they have demonstrated that the four strains of B.

braunii belong to three existing chemical races (A, B and L) (Figure 1) which form a

monophyletic group and whose closest relatives are in the genus Choricystis in the

trebouxiophyceae, whereas the previously reported B. braunii (Berkely strain)

sequence form a member of chlamydomonadales in the chlorophyceae (Gavrilescu

and Chisti, 2005). However, still there is debate on whether the numerous strains of

B. braunii either belong to a single species or three species or to several sub species

(Metzger and Largeau, 2005).


Figure 1. Types of hydrocarbons produced by three different chemical races of

Botryococcus braunii.

1.4. Metabolites of Botryococcus

1.4. 1. Hydrocarbons of Botryococcus

Botryococcus braunii is classified into A, B and L races based on the type of

hydrocarbons synthesized (Figure 1). The race - A produces C23 to C33 odd numbered

n-alkadienes, mono-, tri-, tetra-, and pentaenes, derived from fatty acids (Gelpi et al.,

1970; Metzger and Casadevall, 1987; Metzger et al., 1990; Metzger, 1999; Okada et

al., 2000). These linear olefins can constitute upto 61% of the dry cell mass of the

green active state colonies. The L race produces a single hydrocarbon C40H78, a

tetraterpene known as lycopadiene, which constitutes up to, 2-8% of the dry biomass

(Huang and Poulter, 1989 Metzger et al., 1990;). Whereas B race produce

triterpenoid hydrocarbons, C30–C37terpenoid hydrocarbons referred to as

botryococcenes and C31–C34 methylated squalenes accounting for its capacity of

accumulating high levels of hydrocarbons (20 – 86% on dry weight basis) in natural

populations (Brown et al., 1969; Achitouv et al., 2004).Certain strains of the B race

also biosynthesise cyclobotryococcenes (David et al., 1988; Audino et al., 2002).

C33 Botryococcene

Squalene

Tetramethylsqualene

B race

vii

viii

ix

C33 Botryococcene

Squalene

Tetramethylsqualene

B race

vii

viii

ix

Lycopadiene L racexLycopadiene L raceLycopadiene L racex

Pentacosa-1, 16-diene

Heptacosa-1, 18-diene

Nonacosa-1, 20-diene

Hentriaconta-1, 22-diene

Heptacosa-1, 17,19-triene

Nonacosa-1, 20, 22-triene

Alkadienes

Alkatrienes

A race

i

ii

iii

iv

vi

v

Pentacosa-1, 16-diene

Heptacosa-1, 18-diene

Nonacosa-1, 20-diene

Hentriaconta-1, 22-diene

Heptacosa-1, 17,19-triene

Nonacosa-1, 20, 22-triene

Alkadienes

Alkatrienes

A race

i

ii

iii

iv

vi

v


1.4.2. Alkanes

Saturated hydrocarbons are synthesized from methylated aldehydes by the activity of

an enzyme called decarboxylase. These methylated aldehydes are generated from

fatty acids via methylation by S- adenosyl methionine and by fatty acyl reductase. B.

braunii produces saturated straight-chain, branched-chain (C14–C28) and long-chain

linear aliphatic (C20–C27) hydrocarbons in the range of C12–C32 (Volova et al., 2003).

Dennies and Kolattukudy (1991) have demonstrated the alkane biosynthesis by

decarbonylation of aldehyde catalyzed reactions in the absence of oxygen in the

microsomal preparations of B. braunii. Yang et al., (2004) also reported the

production of saturated hydrocarbons (C21–C31) from B. braunii and C27 as major

hydrocarbon. Audino et al. (2001) identified the macrocyclic alkanes (ranging from

C15 to C34) and their methylated analogues (ranging from C17 to C26) in B. braunii rich

sediment (torbanite). Dayananda et al. (2007b) reported that Indian isolate producing

alkanes in the range of C21 to C33. Audino et al. (2002) reported that macrocyclic

alkanes are specific to the highly resistant algaenan of B. braunii therefore these

macrocyclic alkanes can be used as biomarkers for B. braunii. Apart from the

hydrocarbons, B. braunii has also been reported to produce certain compounds, which

possess growth-promoting activity on roots at very low concentrations (Murakami,

2000).

1.4.3. Lipids

Botryococcus braunii produces large amounts of fatty acids, comprising mainly the

palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic acids (18:3). The lipid

productivities by this algae ranges from 10 to 60% (w/w) and it depends mainly on

the physiological conditions and also the race (different chemical races) to which they

belong (Audino et al., 2001). Methyl branched fatty acids are also reported in B.

braunii (Dayananda et al., 2006a) and are known to inhibit endothelial cell and

leukocyte proliferation (Krishnan and Collin, 2003; Krishnan, 2003).

The algae B. braunii is also known to produce large amounts (5 - 42% on dry

weight) of ether lipids which are not glycerol derivatives, but they are closely related

to hydrocarbons. Alkadienyl-o-alkatrienyl ether, alkenyl-o- botryalyl ether and

resorcinolic ether lipid are the characteristic ether lipids of the race A (for details see

the review Metzger and Largeau, 2005). They can constitute up to 5 - 42% on dry

weight basis, however, their concentrations varies with the growth phase of the algae

and also with the individual strains. Rosales et al. (1992) have reported higher levels


of ether lipids in exponential growth phase and decreased levels in stationary phase.

There are about twelve series of ether lipids called as lycopanerols, which were

isolated from L race strains and they accumulate up to 10% on dry weight basis. On

the other hand B race strains produce few ether lipids such as diepoxy- tetramethyl

squalene, botyolin-A and braunixanthin-1 and are also called by the general name

botryolins, and are present in only minor amounts (Metzger and Largeau, 2005).

1.4.4. Exopolysaccharides

Casadevall et al. (1985) reported for the first time that B. braunii also produces exo-

polysaccharides (EPS) which are released into the medium. The races A, B and L are

known to produce up to 250 g m3

and 1kg m3, respectively. A strain (B. braunii UC

58) isolated in Portugal has been reported to produce large amounts ranging from 4 to

5.51 kg m-3. The exopolysaccharides produced by B. braunii are heterogeneous

polymers of galactose, glucose, arabinose, rhamnose, fucose, uronic acids and also

contains unusual sugars such as 3-O-methyl fucose and 3-O-methyl rhamnose. These

polysaccharides have extensive utility in laundry products, and some industries such

as adhesives, paper, paint, textile and food. Though, many algae are known to produce

exopolysaccharides, only few are reported to have commercial utility based on their

ability to alter rheological characteristics of solutions (Banerjee et al., 2002).

1.4.5. Carotenoids

Botryococcus species also produces appreciable amounts of carotenoids and

chlorophylls. Carotenoids are more predominant in B and L races as compared to the

A race strains. Some of the major carotenoids produced by B. braunii include

canthaxanthin, β-carotene, lutein, violaxanthin, echinenone, 3-OH-echinenone,

loroxanthin, adonixanthin and neoxanthin. Botryoxanthin-A, botryoxanthin-B, α-

botryoxanthin-A, braunixanthin 1 and braunixanthin 2 (Grung, et al.,1989; Grung et

al.,1994; Okada et al., 1996; Okada et al., 1997; Okada et al., 1998; Banerjee et al.,

2002). These carotenoids have the potential application in pharmaceutical industry as

nutraceuticals.

1.5. Commercial importance of microalgae

Microalgae are being considered as a goldmine for various potential applications

(Figure 2) in foods, feeds, biofuels and high value bioactives (Metting and Pyne,

1986; Schwartz, 1990; Kay, 1991; Shimizu, 1996, 2003; Borowitzka, 1999;

Akkerman et al., 2002; Banerjee et al., 2002; Melis, 2002; Lorenz and Cysewski,


2003; Singh et al., 2005; Walker et al., 2005; Spolaore et al., 2006; Rastogi and

Sinha, 2009; Santoyo et al., 2009). They are also being recognized for various other

uses including sequestration of carbon dioxide, bioremediation of heavy metals,

biotransformation and treatment of sewage and municipal waste as well (Mallick,

2002; Suresh and Ravishankar, 2004; Kalin et al., 2005; Munoz and Guieysse, 2006;

Debelius et al., 2009). At present microalgae are commercially cultivated for human

nutritional products around world in several dozen small- to medium-scale production

systems which account for several ten to hundred tons of algal biomass production

annually (Becker, 1986; Becker and Venkataraman, 1982). A leading algologist

Benemann estimates total world commercial microalgal biomass production at about

10,000 tons per year. Spirulina, Chlorella, Dunaliella and Haematococcus are the

major microalgae currently cultivated photosynthetically for nutritional and

pharmaceutical use. About half of this worldwide microalgal biomass is commercially

produced in China and the rest is produced by Japan, Taiwan, USA, India and

Australia, with smaller producers in other countries as well. More than 99% of

microalgal biomass is commercially produced in open air ponds (Benemann, 2009).

Microalgae are also being grown for live aquaculture feeds in hundreds of systems

around the world that individually produce from a few kilos to at most a few tons of

biomass annually and in these systems small-scale photobioreactors are often used

(Fernandez and Southgate, 2007). Microalgae are even produced commercially by

using dark fermentations wherein starch or sugar is used as a major ingredient of algal

medium, with this route a few tons of algal biomass is produced mainly in the Far

East for Chlorella for nutritional supplement. In the US the same method is used for

production of algal oil which is rich in the omega-3 fatty acids like DHA and GLA for

infant formula ingredient (Benemann, 2009). Some of the important industrially

potential microalgal species with their importance as a valuable product have been

summarized in Table 1.


Table 1. Industrially potential microalgal species and their commercial applications

Microalgae Commercial applications References

Chlamydomonas reinhardtii

Biomass for animal health and feed;

environmental monitoring; hydrogen

production; bioremediation

Walker et al., 2005

Dunaliella salina Dietary supplements; cosmetics; feed;

β-carotene and other carotenoids for

health food

Pulz and Gross, 2004

Murthy et al., 2005

Dunaliella bardawil β-carotene for health food, dietary supplements and cosmetics

Walker et al., 2005

Vanitha et al., 2007

Haematococcus pluvialis Astaxanthin for health food,

pharmaceuticals, aquaculture feed


Sarada et al., 2006

Chlorella vulgaris Biomass for health food, dietary

supplements, aquaculture feed and

extracts for cosmetics


Chlorella sp. Polysaccharides for dietary

supplements

Walker et al., 2005

Spirulina platensis Phycocyanin, phycoerythrin, and

biomass for health food,

pharmaceuticals, feed, and cosmetics

Becker, 2004

Spirulina pacifica Biomass and extracts for nutrition, food coloring, feed, immunological

diagnostics, and dietary supplements

Walker et al,.2005

Lyngbya majuscule Immune modulators for

pharmaceuticals and nutrition


Scenedesmus sp. Protein, Essential amino acids Becker and Venkataraman,

1982

Porphyridium sp. Biomass, phycoerythrin, PUFA Dufosse et al, 2005;

Kathiresan et al.,2007

Botryococcus brauni Biomass; hydrocarbons; Lipids Banerjee, 2002; Dayananda

et al., 2005


Figure 2. Microalgae applications in various fields

1.6. Cultivation of microalgae

Microalgae are cultivated either in open or closed systems. Open microalgal

cultivation systems include raceway ponds, circular tanks, lagoons, lakes and ditches.

Photobioreactors represents the closed cultivation systems and are of mainly two

types horizontal and vertical with a range of dimensions and designs (Tredici et al.,

1992, 1998; Chisti, 2007; Fernandez et al., 1998, 2000). They offer the controlled

cultivation parameters like pH, light, temperature, dissolved oxygen, carbon dioxide

levels, and online replenishment of algal nutrition etc which are very vital in guiding

microalgal growth and production of desired compounds. It does not mean that

photobioreactors are free from disadvantages they do have certain limitations like

they are more expensive than ponds, and pose major challenges in design and

operating (overheating, fouling, gas exchange limitations, harvesting, etc.). Therefore,

photobioreactors are not being extensively used for commercial production of low

value compounds like proteins and other applications like biofuel. Owing to their


huge investments, cleaning and power in puts etc. For example biofuels production

requires systems of hundreds of acres, this would mean thousands of such units, at

high capital and even greater operating costs. However, the exploitation of

photobioreactors powered by sun light for both photosynthesis and to run the process

can often works out to be better and cheaper for high value metabolite production

from microalgae.

Open ponds, specifically shallow, mixed, raceway ponds, are much cheaper to

build and operate, can be scaled upto several acres, and are the method of choice for

commercial microalgae production around the world. Engineering designs of open

ponds, including mixing systems (typically paddlewheels) and carbon dioxide supply

and transfer, is rather well understood. However, the biology of algae mass culture

pertains to newer strains of commercially important would be of greater challenge

rather than the engineering aspects of design and operation of open pond cultivation

systems. Open pond cultures generally suffer from various limitations, including more

rapid contamination and invasions by other algae and algae grazers, fungi, amoeba,

etc. Closed systems have advantages in colder climates, though, on the other hand,

they would require cooling most days, with water spray the only practical cooling

method, at high water use. Almost all commercial algal biomass production is

currently produced with open ponds (Benemann, 2009).

The commercial cultivation of microalgae began with the cultivation of

Chlorella in Japan in the 1960s followed by the cultivation of Spirulina in Mexico,

the USA and China in the 1970s. During the last four decades the biotechnological

industry of photosynthetic microorganisms has grown and become much diversified.

The most important commercially produced microalgae are Spirulina, Chlorella and

Dunaliella. This is achieved because of their growth in highly selective media and

hence they remain relatively free from contamination by other microorganisms

(Borowitzka, 1999). In contrast, promising research and development is being taken

up by various academic and government laboratories to understand the algae

cultivation with small-scale closed photobioreactors and scale up studies in open

ponds as well (Moreno et al., 2003; Radmann et al., 2007; Reichert et al., 2006;

Chisti, 2007).

The areas of the world that are highly suitable for agriculture is dark green in

the Figure 3 and the unsuitable areas are light green. These agriculture unsuitable

areas are possibly having a new option in the production of algae farms. Highly


productive areas for agriculture and the development of algae should remain mutually

exclusive categories for best land use. In addition, high insulation and warm

temperature is required for high algal productivity and therefore, latitudes within a

certain distance of the equator are best for locating algae farms has been proposed by

Lindsay McGraw (2009).

Figure 3. Suitable areas of the globe for algal cultivation (Adopted from Lindsay

McGraw, 2009).

1.7. Microalgal growth requirements

Microalgae require light, carbon dioxide, water and inorganic salts for their growth

and majority of the microalgae show their optimal growth at temperature between

20°C to 30°C (Soeder and Stengel, 1974; Brand and Guillard, 1981; Becker and

Venkataraman, 1982; Ben-Amotz et al., 1989; Grobbelaar et al., 1984; Lee and Bazin,

1990; Boussiba and Vonshak 1991; Volkman, 1991; Chanawongse et al., 1994;

Banerjee et al., 2002; Chisti, 2007). Algal medium comprise to provide the inorganic

elements that constitute the algal cell. Apart from carbon, hydrogen and oxygen

microalgae require nitrogen, phosphorus, iron, sulphur and in few cases silicon as

their essential inorganic components. Sea water is often supplemented with salts of

nitrate and phosphate fertilizers and a few other micronutrients for growing

microalgae (Grima et al., 1999). Algal growth media are generally inexpensive

although its cultivation appears to be costlier than the other agricultural crops. This is


mostly because of designing cultivation ponds and their maintenance and other

operational costs. The main advantage of microalgae is that their biomass contains

approximately 50% of carbon by dry weight (Sanchez-Miron et al., 2003) and this

carbon is typically assimilated from carbon dioxide. Production 100 tons of

microalgal biomass in open ponds or in closed photobioreactor systems supplied with

carbon dioxide would approximately fix 183 tons of carbon dioxide (Sawayama et al.,

1995; Yun et al., 1997). Therefore microalgae are being considered as a potential

organism in mitigating carbon dioxide.

Physiological and biochemical studies on various commercially exploited

microalgae have revealed that the nutrients, light, temperatures have significant

influence over the growth and secondary metabolite flux of microalgae (Boussiba and

Vonshak, 1991; Becker and Venkataraman, 1982). Hence it is an important criterion

to provide adequate nutrients and optimal growth conditions to achieve optimal

growth (Kaplan et al., 1986; Borowitzka, 1988).In the other way, microalgae can be

exploited for high value metabolite production by imposing certain stress to induce

various metabolic fluxes (Droop, 1954; Pringshiem, 1966).

For heterotrophic cultures of microalgae several various organic compounds

like glycine, alanine, asparagine, aspartic acid, glutamine, succinamide, citrulline,

arginine, ornithine, glucose, acetate and ethanol have been adopted (Sivasankar and

Oaks, 1996; Arnow et al., 1953; Richardson et al., 1969; Parslow et al., 1984; Snoog

1980; Chen and Johns, 1994; Martinez and Drus, 1991).

1.8. Use of Response surface methodology in algal studies

Response surface methodology (RSM) is a collection of mathematical and statistical

techniques widely used to determine the effects of several variables. RSM is

important in designing, formulating, developing, and analyzing new scientific

studying and products. It is also efficient in the improvement of existing studies and

products. It has also been used to optimize different bioconversion processes (Rao et

al., 2000). This method has been successfully applied to the optimization of medium

composition for growth and metabolite production from algal cultures as well (Sarada

et al., 2002). Experimental designs are more efficient for this purpose than the

classical method of studying one variable at a time. In the present study, a central

composite experimental design combined with RSM has been used to determine the

influence of medium constituents to identify the optimum of culture conditions for

enhanced growth.


1.9. Screening and isolation of microalgae

Morphological identification coupled with genetic and chemical marker analysis is

being widely used to identify microalgal species (Auinger et al., 2008). The rDNA of

the 18s rRNA of the small subunit of the eukaryotic ribosome is usually sequenced to

determine the phylogenetic relationships of eukaryotic algae. While the rDNA for the

5S rRNA has also been used in phylogenetic studies, however the data have been

suspect because of large deviations in nucleotides (Ragan, 1994). Though nucleotides

coding for all ribosomal subunits are encompassed within a single operon and

transcribed by a single RNA polymerase (Kawai et al., 1997). The DNA coding for

other molecules such as ribulose biphosphate carboxylase, ribulose biphosphate

oxygenase and actin have also been used in determining phylogeny (Freshwater et al.,

1994; Fujiwara et al., 1994; Bhattacharya and Ehlting, 1995). Additionally chemical

marker analysis and other molecular markers like RAPD, ISSR and RFLP are also

being routinely used to identify the diversity among the species sub-species and even

in closely related organisms such as two near isogenics lines. Gene sequencing is

becoming the most fascinating field of phycological systematics in the last decade and

has provided vital information on the phylogenetic relationships between the algae.

However molecular data alone is not adequate in revealing the species identity hence

morphological and chemical markers are also being used in phylogenetic studies.

1.10. Genetic transformation of microalgae

During the last two decades, there were more than 25 successful genetic

transformation of algal species reported and most of these were achieved by nuclear

transformation. Stable transformations were achieved for various species of green

algae, brown algae, red algae, diatoms, dinoflagellates and cyanobacteria (Table 2).

However there were few transient transformations reported for few species of green

algae, red algae and for diatoms as well. Genetic transformation has been achieved in

microalgae by using Agrobacterium infection, electroporation, agitation of a cell

suspension in the presence of DNA and glass beads, conjugation, agitation in the

presence of DNA and silicon carbide whiskers and by gene gun methods

(Schiedlmeier et al., 1994, Minoda et al., 2004, Cheney et al., 2001, Qin et al., 2003,

Koksharova and Wolk, 2002, Debuchy et al., 1989, Dunahay 1993, Shimogawara et

al., 1998, Kumar et al., 2004, Purton and Rochaix, 1995).


Table 2. Reported genetic transformation of algal species

Algal species Transformation References

Chlamydomonas reinhardtii nuclear Kindle et al., 1989

Volvox carteri nuclear Schiedlmeier et al., 1994

Dunaliella salina nuclear Geng et al,. 2003, 2004

Dunaliella viridis nuclear Sun et al., 2006

Chlorella sorokiniana nuclear Dawson et al., 1997

'Chlorella kessleri nuclear El-Sheekh, 1999

Cyanidioschyzon merolae nuclear Minoda et al., 2004

Porphyra yezoensis nuclear Cheney et al., 2001

Porphyridium sp. chloroplast Lapidot et al., 2002

Laminaria japonica nuclear Qin et al., 1999

Undaria pinnatifida nuclear Qin et al,. 2003

Phaeodactylum tricornutum nuclear Zaslavskaia et al., 2001 2001

Synechocystis sp. nuclear Dzelzkalns and Bogorad,1986

Haematococcus pluvialis nuclear Kathiresan et al., 2009

Anabaena sp. nuclear Thiel and Poo, 1989

Spirulina platensis nuclear Kawata et al., 2004

Symbiodinium microadriaticum nuclear Ten Lohuis and Miller, 1998

Amphidinium sp. nuclear Ten Lohuis and Miller, 1998

Euglena gracilis chloroplast Doetsch et al., 2001

Cyclotella cryptica nuclear Dunahay et al., 1995

Cylindrotheca fusiformis nuclear Fischer et al., 1999

Navicula saprophila nuclear Dunahay et al., 1995

1.11. PUFA and their uses

The distribution of lipids in algae is affected by different factors. Generally it is

determined by systematic position of algae. All most all of the algal divisions are

unique in their lipid quality and quantity even. At present, the production of lipids in

general and polyunsaturated fatty acids (PUFA) in particular by marine and

freshwater microalgae is the subject of intensive research and increasing commercial

attention as well (Wen and Chen, 2003; Sijtsma and de Swaaf, 2004). Currently fish

oil is being considered as a major source for the commercial production of PUFAs.

Since there is an increasing demand for purified PUFAs, some other alternative

sources are being explored and also due to the process of purification of PUFAs from

complex crude fish oils is an expensive and relative difficult technique (Wen and

Chen, 2003). Hence freshwater and marine algal forms which contain large amounts


of high-quality PUFAs can grow heterotrophically or autotrophically on cheap

organic and inorganic substrates and thus grown algae are widely used at the moment

to produce PUFAs for aquaculture operations. Furthermore the improvement of

strains by genetic manipulation, optimization of culture conditions and development

of efficient cultivation systems are also being considered to cut down the cost of

production of desired metabolites of commercial importance.

Polyunsaturated fatty acids (PUFAs) are the fatty acids having 18 carbons or

more in length with two or more methylene interrupted double bonds in the cis

position. PUFAs are grouped into two categories as ω-6(or n-6) fatty acids and ω-3(or

n-3) fatty acids. The nutritionally essential PUFAs include linoleic acid (LA), γ-

linolenic acid (GLA) and α-linolenic acid (ALA), whereas the very long chain

polyunsaturated fatty acids (LCPUFA) have 20 or 22 carbon atoms and 4, 5 or 6

methylene interrupted cis double bonds in ω-3 or ω-6 arrangements) includes

arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid

(DHA) (Alonso and Maroto, 2000). Polyunsaturated fatty acids (PUFA)

supplemented through diet have effects on diverse physiological processes impacting

normal health attributes such as the regulation of plasma lipid levels, cardiovascular

and immune function, insulin action, neuronal development and visual function etc.

Ingestion of PUFA will lead to their distribution to virtually every cell in the body

with effects on membrane composition and function, eicosanoid synthesis and

signaling as well as the regulation of gene expression. Their usefulness is not just

limited for the maintenance of good health but they were also reported to be helpful in

prevention and curing of various chronic diseases and disorders (Belarbi et al., 2000;

Simonopoulos, 1991; Wingmore et al., 1996).

GLA plays an important role in the prevention of skin diseases, diabetes and

reproductive disorders (Gunstone, 1992, 1998; Horrobin, 1992). AA and DHA are

found in high proportions in neuronal tissues such as brain, retina and testis and are

essential in the normal development and functions of these tissues (Singh and

Chandra, 1988; Innis, 1991; Brown, 1994). Use of AA and DHA in infant formula has

also been shown to be beneficial to the growth and visual development in pre-term

infants (Gill and Valivety, 1997). EPA in the proper functioning of the circulatory

system and cardiovascular diseases is now well understood (Dyerberg, 1986; Iacono

and Dougherty, 1993; Simonopoulos, 1991; Wingmore et al., 1996). PUFAs also

serve as a precursor for several of biologically active molecules such as eicosanoids,


growth regulators and hormones (Gill and Valivety, 1997; Innis, 1991; Parent et al.,

1992; Peck, 1994). Because of their various health attributes the market demand for

GLA, DHA and EPA is ever increasing (Belarbi et al., 2000).

1.12. Transgenic production of GLA

Gamma-linolenic acid (C18:3) is an essential fatty acid (Gill and Valivety, 1997;

Broun et al., 1999) synthesized by the action of delta 6-desaturase on linoleic acid and

is the intermediate precursor for arachidonic acid biosynthesis. The desaturation

process is a rate determining step due to the impairment of the desaturase activity or

the imbalance in the intake of w-6 and w-3 fatty acids in mammals. Dietary γ-

linolenic acid (GLA) can effectively overcome this problem and hence there is

increasing interest in this acid and its rich biological resources as well. Biosynthetic

production of GLA is limited only to few plants species (Borage, Evening primrose

and Black currant etc.), cyanobacteria (Spirulina maxima, Spirulina platensis and

Synechocystis Sp.) and fungal (Mortierella ramannia, Mortierella isabellina, Mucor

rouxii, Mucor circinelloides, Mucor mucedo, Rhizopus nigricans, Cunninghamella sp.

Cunninghamella echinulata, etc.) sources (Kennedy et al.,1993; Hiruta et al., 1996;

Preez et al., 1997; Certik et al., 1997; Bandyopadhyay et al., 2001; Xian et al., 2001;

Conti et al., 2001; Mamatha et al., 2008; Muhid et al., 2008).

However, lipid biotechnology is up till now a very young discipline. Despite

the fact that breathtaking progress has been made in genetic engineering for alteration

of composition of oils of oil rich organisms and oil crops as well. The genetic

modification of fatty acid composition was manipulated successfully by genetic

engineering of biosynthetic pathways of various oil seed crops and in other oil rich

organisms by desaturase genes (Table 3). In particular GLA was produced in an

oilseed crop by introducing an enzyme called delta 6-desaturase from its native

sources. The enzyme encoding for delta 6-desaturase has been successfully identified

and isolated from borage (Sayanova et al., 1997), Mortierella alpina (Huang et al.,

1999), Mucor rouxii (Laoten et al., 2000), Pythium irregulare (Hong et al., 2002),

Physcomitrella patens (Girke et al., 1998), Echium sp. (Marotoa et al., 2002),

Primula sp. (Sayanova et al., 2003) and Synechocystis (Reddy et al., 1993) etc. And

was successfully cloned and functionally expressed in yeast, tobacco and some have

also been tested in oilseed crop plants (Reddy and Thomas, 1996; Sayanova et al.,

1997; Huang et al., 2001; Hong et al., 2002; Qiu et al., 2001; Sato et al., 2004; Knauf


et al., 2006; Das et al., 2006). However the commercial applications of biotechnology

for the production and modification of fats and oils are restricted so far only to a few.

Table 3. List of desaturase genes that are cloned to alter the fatty acid profiles

Genes Biological sources References

Delta 6-desaturase Arabidopsis thaliana

Rosa hybrida

Anabaena variabilis

Synechocystis sp.

Fukuchi et al., 1995

Fukuchi et al., 1995

Sakamoto et al., 1994

Sakamoto et al., 1994

n-3 desaturase

(plastidial)

Arabidopsis thaliana

Glycine max

Brassica napus

Nicotiana tabaccum

Triticum aestivum

Gibson et al., 1994

Yadav et al., 1993

Yadav et al., 1993

Hamada et al., 1994

Horiguchi et al., 1998

n-3 desaturase

(microsomal)


Glycine max

Brassica napus

Limnanthes douglasii

Nicotiana tobaccum

Triticum aestivum

Perilla frutescens

Yadav et al., 1993

Yadav et al., 1993

Yadav et al., 1993 Bhella and Mackenzie, 1995

Hamada et al., 1994

Horiguchi et al., 1998

Chung et al., 1999

Delta 12-desaturase

(microsomal)


Glycine max

Okuley et al., 1994

Heppard et al., 1996

1.13. Importance of the work in the present scenario

Phytoplanktons are the most important biomass producers in global aquatic

ecosystems. The organisms populate the top layers of the oceans and freshwater

habitats where they receive sufficient solar radiation for photosynthesis (Hader et al.,

1998). It has been reported by many researchers that microalgae can be used as a

potential source of food, bioactive molecules and pharmaceutically significant

compounds (Benemann et al., 1986; Mayer and Gustafson, 2004). Botryococcus, a

green colonial microalgae widespread in freshwater and brackish lakes, reservoirs,

ponds and is recognized as one of the potent resource for the production of lipids.

Botryococcus species are known to produce large amounts of lipids ranging from 10

to 60 % (w/w) and palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic acids

(18:3) as its major fatty acids (Sushchik et al., 2003; Kalacheva et al., 2001; Volova

et al., 2003). Botryococcus species are an untapped yet a potential resource with great

prospect for the production of lipids (Metzger and Largeau, 2005). In light of this,

the biotechnological exploitation for lipids needs to be explored. Though

Botryococcus is known to produce large amounts of lipids it is possible to improve its

fatty acids compositions through biotechnological approaches to produce nutritionally

superior specialty lipids. Polyunsaturated fatty acids (PUFA) are essential for cellular


metabolism and have been involved in a wide range of medical applications (Deluca

et al., 1995). Gamma-linoleic acid (GLA) is an important conditionally essential fatty

acid (Horrobin, 1990, 1992). GLA is an omega-6 polyunsaturated fatty acid (PUFA).

GLA is found naturally in the fatty acid fractions of some plant seed oils. Most

notably, sources of GLA include evening primrose oil, borage oil, black currant oil,

and hemp seed oil (Lawson and Hughes 1988). Conversion of linoleic acid to GLA is

catalyzed by the enzyme delta-6-desaturase. Linoleic acid is converted first to GLA

then to arachidonic acid by an alternating sequence of delta-6-desaturation, chain

elongation, and delta-5-desaturation (Johnson et al., 1997). GLA and its related fatty

acids are known to be crucial in combating and preventing various diseases viz

diabetes, osteoporosis, cancer, dry eye syndrome, ulcerative colitis, premenstrual

syndrome, asthma and cardiovascular diseases etc. (Deluca et al., 1995; Kernoff et

al.,1977; Rakesh et al., 2006; Gadek et al., 1999; Whitehouse et al., 2003).

So, the present study focused on the, isolation and characterization of

indigenous strains of Botryococcus for lipids and screening for specialty lipids.

Botryococcus species are known to produce large amounts of lipids rich in linoleic

acid which may be further enriched for its oil quality by introducing delta 6-

desaturase, which is a key enzyme in omega 6-fatty acid and PUFA production.

Therefore, in the present Ph.D. research we attempted to introduce delta 6-desaturase

enzyme from cyanobacterial source to get the desired fatty acids composition.

Evaluation of safety of Botryococcus, to determine the influence of specialty lipids of

microalgal source on serum and tissue lipids in experimental animals was

investigated. Towards these studies the following objectives were outlined.

1.14. Objectives

• Isolation and characterization of indigenous strains of Botryococcus for lipids and

studies on lipid production.

• Determination of safety of Botryococcus and influence of specialty lipids on

serum and tissue lipids in experimental animals.

• Enrichment of algal oil by cloning of delta 6-desaturase gene from cyanobacterial

source.

introduction & review of literature -...

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