BY

Name: Faiza Maroof

Registration No: 2015-UKT-00973

University of Kotli Azad Jammu & Kashmir

Faculty of Sciences
Department of Biotechnology
 Dedication
This dedication is paid to Allah Almighty my creator, my strong pillar, my source of inspiration,
wisdom, knowledge and understanding and our last Holy Prophet (S.A.W.W).

This work is also lovingly dedicated to my parents Mr. and Mrs. Muhammad Maroof with all
love and respect, to my brothers and sister and to my family and friends.
This dedication is also to my teachers;
Who are always great source of inspiration and motivation to me. However, my teacher’s
remained beacon of light for me.

And most especially to best and adorable teacher and my advisor

Dr. Simab Kanwal

i
 Acknowledgments

Faiza Maroof obligations to Department of Biotechnology, Faculty of Sciences and

University of Kotli Azad Jammu and Kashmir for providing research opportunity. Thanks

to Dr. Simab Kanwal for supervising, guiding and supporting during the entire research.

Thanks to Staff members of Department of Biotechnology for guiding. Thanks to M.Phil.

Students for helping. In last I am greatly thankful to my parents who provided me support

and put me on this track.

ii
 Abstract

The aim of study to determine the industrially important bioproducts accumulation in thermophilic

green alga Pediastrum sp. such as carotenoids, chlorophyll and biosurfactants and the

determination of optical density of Pediastrum sp. These all contents are measured at diverse

growth phases of Pediastrum sp. at different temperatures. Optical density measured at mid log

phase, late log phase and stationary phase on both temperatures at the wavelength range of 730

nm. Carotenoids and chlorophyll were measured at different growth phases at different

temperatures i.e. 28°C and 40°C and results of present study are highly significant and applicable.

Biosurfactant from naturally occurring microbes are much better rather chemical surfactants and

in Pediastrum sp. biosurfactants are present in valuable range. All the results are highly significant

and economically import.

iii
Contents
1 Introduction ......................................................................................................................................... 1

2 Review of Literature ........................................................................................................................... 7

3. Materials and Methods ..................................................................................................................... 12

3.1 Sampling and cell cultivation ....................................................................................................... 12

3.2 Determination of Optical Density ................................................................................................ 13

3.3 Determination of Chlorophyll a and Carotenoid Contents ....................................................... 13

3.4 Bio-surfactant Production ............................................................................................................ 14

3.5 Results and Discussion .................................................................................................................. 14

References .................................................................................................................................................. 22

iv
List of Figures

Figure 1 Pediastrum sp. (Michael Plewka, 2011) ....................................................................... 3

Figure 2 Pediastrum sp. SEM ...................................................................................................... 3
Figure 3 Carotenoid metabolism in microalgae. (Jin Liu et. al., 2014) .................................... 5
Figure 4 chemical structure of (a) chlorophyll a and (b) is chlorophyll b ............................... 6
Figure 5 Volvox aureus (algae) under light microscope (Becker, E.W. 2007). ....................... 7
Figure 6 Growth of Pediastrum sp. at 28 °C and 40 ºC. ......................................................... 15
Figure 7 Carotenoids and chlorophyll a content in Pediastrum sp. at different growth
phases and different temperatures. ........................................................................................... 16
Figure 8 Oil spreading method to produce biosurfactant in Pediastrum sp. ........................ 18

v
1
 Industrially Important Bioproducts Accumulation in
Thermophilic Green Alga Pediastrum sp.

1 Introduction
Algae are photosynthetic organisms and these organisms are found everywhere on the

earth, such as in the sea, rivers, lakes, soil, in animals and plants (as symbiotic partners

collaborating). Algae vary from small-single celled types to complex multicellular ones.

There are two main types of algae: the macro algae (are present in littoral zone, which

includes red algae, green algae and brown algae); and microalgae (unicellular species with

no roots) which occupy both benthic and littoral habitats as well as the ocean waters as

phytoplankton. These include organisms such as green and yellow brown flagellates and

blue green algae (Kuruppalil Z., 2011 and Pathak.S et.al., 2014). Around 1-10 million

species of algae are estimated and most of them are microalgae recognized as one of the

oldest residing micro-organisms on earth ( Song.D et.al., 2008). Microalgae are currently

used as an important source of valuable, natural/biologically active molecules such as

carotenoids, chlorophyll, lipids, starch, cellulose, pigments, antioxidants and

pharmaceuticals. Significant advances were achieved in microalgae biotechnology over

last decade (Yan.N et. al., 2016). Importance of bioactive compounds of microalgae

(Pediastrum sp.) are widely used at industrial level and these compounds having

biotechnological importance. Chlorophyll is a natural food colouring agent. Chlorophyll

and its derivatives are also used widely in pharmaceutical industries for example it

accelerates wound healing more than 25%. Since chlorophyll stimulates tissue growth, it

prevents the advancement of bacteria and speeds up the wound healing process. The
1
application of ointments containing chlorophyll derivatives was found not only to eliminate

pain after several days but also to improve the appearance of the affected tissues

(Hosikian.A et. al., 2010).

Chlorophyll and its derivatives also have profound antioxidant properties and the most

significant activity of chlorophyll derivatives in the prevention of cancer is the trapping of

mutagens in the gastrointestinal tract (Hosikian.A et. al., 2010). Carotenoids are bio-

products having industrial importance and beneficial tool in biotechnology industries.

Carotenoids are used in food, pharmaceuticals, cosmetics, and animal feed industries due

to the coloring properties. Carotenoids are also widely used as colorants, used in food

fortification due to their activity as provitamin A and their biological functions to health

benefit, such as strengthening the immune system, reducing the risk of degenerative

diseases, antioxidant properties and hypolipidemic activities (Mezzomo.N and F. S. R.

Sandra, 2016).

Thermophilic micro-organisms are those organisms that live at high temperatures of at least

60°C. These organisms give knowledge to understand how life can thrive under high

temperatures, as well as the potential of thermophiles in biotechnology and whether they

contain information regarding the early evolutionary life forms on earth. Generally,

microorganisms with an optimal growth temperature (OGT) between 60 and 80°C are

designated as thermophiles, whereas those growing optimally at temperatures of more than

80°C are referred to as hyperthermophiles, which are found in the three domains of life,

archaea, bacteria, and eukarya, but the majority are archaea and bacteria (Wang. Q. et. al.,

2013).

2
Pediastrum sp. are is green thermophilic algae belonging to the Division Chlorophyta,

Class Chlorophyceae, Order Chlorococcales, Family Hydrodictyaceae. Pediastrum sp.

have only one chloroplast per cells. The published scientific data is very rare on Pediastrum

sp. taken from blackish and salty water. Preliminary data has indicated that Pediastrum sp.

grows significantly faster than other algae and has high protein content up to 46%

(Prasertsin. T et. al., 2014).

Figure 1 Pediastrum sp. (Michael Plewka, 2011)

Figure 2 Pediastrum sp. SEM

Numerous industrially application of micro-algae can be used such as; to improve the

nutritional value in food, in cosmetics, in biofuels, in food colorants and in pharmaceutical

3
industry. Various industrially important Bioproducts are present in Pediastrum sp. which

are natural and cost effective and used in industry. Structurally defined carotenoids

reported from nature i.e. land plants, algae, photosynthetic bacteria, fungus and animals.

are more than 750. These organisms can produce many types of carotenoids, among them

around 30 types may have functions in photosynthesis and all others which are synthesized

from miscellaneous carotenogenesis pathways. The pathway of carotenoids and

carotenogenesis can be used as chemotaxonomic markers (Shinichi Takaichi, 2011).

Different types of microalgae sech as Euglenophyta, Chlorarachniophyta and Chlorophyta

having the similar carotenoids, such as β-carotene, violaxanthin, 9′-cis neoxanthin and

lutein, plus chlorophyll a and b with land plants. Some classes have additional carotenoids,

which are class specific and lutein’s derivatives such as loroxanthin, siphonaxanthin and

prasinoxanthin. Under a stressful environment, such as high light, UV irradiation and

nutrition stress, some Chlorophyceae, such as Haematococcus, Chlorella and

Scenedesmus, accumulate ketocarotenoids, canthaxanthin and astaxanthin, which are

synthesized by combining CrtR-b and β-carotene ketolase (CrtW, BKT), they are CrtW-

type not CrtO-type from amino acid sequences ( Shinichi Takaichi, 2011).

4
 Figure 3 Carotenoid metabolism in microalgae. (Jin Liu et. al., 2014)

Chlorophyll is one of the valuable bioactive compounds that can be extracted from

microalgal biomass which have wide applications in pharmaceuticals, food and colorant

industries. There are two types of chlorophyll pigments Chlorophyll a and Chlorophyll b

however excessive environmental conditions such as heat, light or can destabilize the

product. This destabilization can degrade the chlorophyll product. The structure of

chlorophyll is porphyrin macrocycle with four pyrrole rings, while presence of single

isocyclic with pyrrole ring built phorbin structure. There are four carbons and a nitrogen

atom in pyrrole ring. The position of nitrogen atoms easily attracts Mg+2 ions for binding.

The position of formyl group which take over the methyl group in ring in chlorophyll-b,

differentiate chlorophyll-b from chlorophyll-a. This structure difference between two

classes of chlorophyll makes identification process easier by their peaks at respective

wavelength and region (665 for Ch.-a, and 652 for Ch.-b) (Hosikian.A et. al., 2010).

5
 a b

Figure 4 chemical structure of (a) chlorophyll a and (b) is chlorophyll b

Biosurfactants are class of surface-active molecules that are synthesized by

microorganisms. Biosurfactants are also regarded as chemical compounds these

compounds are produced extracellular by microorganisms as the cell membrane.

Biosurfactants are those amphiphilic compounds produced on the living surface on

microbial surfaces. They contain both hydrophobic and hydrophilic moieties which

partitions preferentially at the interfaces such as liquid/liquid, solid/liquid, and gas/liquid

interfaces. Biosurfactants are more effective, selective, stable and environmentally friendly

as compare to the surfactants (chemical) and also biosurfactants are compounds that are

safe for environment and used in the process of bioremediation to clean the environment.

Surfactants are substances widely utilized for cleaning in general, removing undesirable

particles or dirtiness by a process called emulsification. These biosurfactants work as

backbone of the biological membranes which transport and exchange the various important

materials and also play a vital role in living system. Different bacteria produced high

molecular weight biosurfactants and comprise lipoproteins, proteins, polysaccharides,

6
 lipopolysaccharides, or complexes having numerous of these structural types, many of

which have yet to be fully characterized (Reddy.S et. al., 2016).

In the present study, quantification of carotenoids, chlorophyll and biosurfactant was done

in thermophilic green alga; Pediastrum sp. isolated from Tattapani hot water spring at AJK.

The objective of present research work was to analyze the accumulation of industrially

important compounds in thermophilic green alga; Pediastrum sp.

2 Review of Literature
Algae are small photosynthetic organisms found everywhere on earth and vary from small

single-celled types to multicellular ones having various shapes with different diameter and

length· There are two main types of algae macro algae and micro algae. These types having

further subtypes.

Figure 5 Volvox aureus (algae) under light microscope (Becker, E.W. 2007).

7
Microalgae a type of algae; are unicellular, microscopic photosynthetic organisms and have

wide range of physiological and biochemical characteristics and contain high quantity of

protein, lipids, vitamins, glycerol, carotenoids and chlorophyll contents etc. Microalgae

having important raw material for amino acids, vitamins and produce pharmaceuticals

products and having wide range of applications in biotechnological industries

(Priyadarshani. I. and Rath.B, 2012) because the products which are obtained from the

microalgae are environmentally friendly and economically viable because the production

of cells, organisms in controlled environment. The bioactive compounds which derived

from microalgae have anti-inflammatory, antimicrobial and antioxidant activities among

others. Also, these micro-organisms are capable to promote the health and reduce the risk

of development of the degenerative diseases (Michele Greque et. al., 2015).

Chlorophyll is present in nature profusely mostly in plants and also in photosynthetic

micro-organisms, and it is vital for survival of both plants and animal kingdoms due to its

ability of light harvesting role in photosynthesis. Chlorophyll emit green light by

selectively absorbing light in red and blue region. Photosynthesis is a process which

renovates the solar energy into chemical energy and also uses the light together which

harvested by chlorophyll compounds; with carbon dioxide and water to produce

carbohydrates and oxygen. The products which are produced in the result of photosynthesis

are necessary for life of animals and plants; carbohydrates for survival of plants and oxygen

for survival of animals. The significance of photosynthesis for life on earth is further

emphasized by plants forming the basis of all food chains. It is assessed that 1.2 billion

tons of chlorophyll are produced annually in the planet. In pharmaceuticals products

chlorophyll and its derivatives are extensively used. Chlorophyll stimulates tissue growth,

8
prevents the advancements of bacteria and speeds up the wound healing process.

Chlorophyll and hemoglobin chemical structures are similar. Photosynthesis carried out

by all plants and photosynthetic microorganisms· Plants are viable to capture CO2 but it is

by no means efficient owing to its slow growth rate. On the other hand, microalgae capture

the solar energy and CO2 with an efficiency of 10 to 50 times greater than that of higher

plants (Reddy et. al., 2010). Microalgae have speedy growth rates and higher productions

than any other plant systems. Microalgae can grow in variable environmental conditions

(Hosikian.A et. al., 2010).

Carotenoids are pigments and these pigments are widely distributed in nature and play vital

role in the protection of cells and organisms against the harmful effects of light, singlet

oxygen and photosensitizing. Carotenoids reported from nature are more than 750;

carotenoids present in land plants, algae, cyanobacteria and photosynthetic bacteria,

archaea, fungus and animals (Shinichi Takaichi, 2011). Only 50 carotenoids are

significantly used as biological active compound from several hundred naturally occurring

carotenoids and these active compounds divides into two groups, with or without

provitamin A. Vitamin A is important for biological functions of body such as vitamin A

is important for growth, development, maintenance of epithelial tissues, reproduction,

immune system, and, in particular, visual cycle acting in the regeneration of photoreceptors

, and its deficiency cause serious health problems in organisms. On the other hand,

excessive intake of vitamin A is toxic, and it cause congenital malformation during

pregnancy, cause bone disease with chronic renal failure in patients, xerophthalmia

blindness, and death. Carotenoids, along with vitamins, acting as antioxidants in biological

systems are the substances most investigated as chemo preventive agents. Antioxidants can

9
act directly in the neutralization of free radicals, preventing or reducing damage caused by

these compounds in cells, or indirectly involved in enzyme systems that have antioxidant

activity. Studies show the relationship between increased feeding of foods rich in

carotenoids and the risk decrease of various diseases. According to Olson (J. A. Olson,

1999), carotenoids reduce singlet oxygen, remove peroxy radicals, control carcinogen

metabolism, inhibit cell spread, arouse communication between cells (gap junctions), also

by increasing immune response. In vitro and in vivo tests suggest that carotenoids are

outstanding antioxidants, scavenging and disabling free radicals. The trans-isomerization

of carotenoids caused by factors such as heat, light, and acids, which is the steadiest form

in nature, for the cis-form, indorsing a slight loss of color and provitamin activity because

of more unsaturation rate. Carotenoids are also susceptible to enzymatic or nonenzymatic

oxidation, which depends on the carotenoid structure (Mezzomo.N and Sandra, 2016).

The surfactants produced from biological organisms such as microorganisms

(fungi,bacteria,yeast) are called biosurfactants (Santos et. al., 2018). Biosurfactants are

amphiphilic compounds produced extracellularly by different microbes such as bacteria,

yeast, filamentous fungi from fungi, from various substances including sugars, oils and

wastes (Sharma. A et. al., 2014). The biosurfactants can also be produced by animals

(Santos et. al., 2018). Surfactants or surface-active agents are those micelles that form from

amphiphiles can be potentially used for surface chemical. Soaps and detergents having

similar characteristics like surfactants. The extraction and emulsification potency of the

surfactant was performed in order to use as bioemulsifier in food (Sharma. A et. al., 2014).

Recently biosurfactants become an important product of biotechnology for industrial and

medical applications. The classification of biosurfactants are based on their chemical

10
structures that include glycolipids, lipopeptides, polysaccharide-protein complexes,

phospholipids, fatty acids and neutral lipids. Also surfactants can be classified on the base

of ionization anionic, cationic, nonionic and amphoteric. Biosurfactants which are

produced from microorganisms have exhibited antimicrobial activity and anti-adhesive

activity against other microorganisms.

The biosurfactants are important and used because of their properties like low toxicity,

biodegradability, and ecological acceptability; furthermore because of low cost raw

materials, such as agricultural and industrial waste can be used as substrates to

biosurfactant production.

Biosurfactants also used in various fields and having a lot of applications because of their

properties such a bioremediation, biodegradation· enhanced oil recovery, pharmaceutics,

food processing among many others. There are many examples of microorganisms which

are used in the production of biosurfactants in literature such as Streptomyces have been

used to produce the biosurfactants especially the bacteria of Actinomycetes group (Santos

et. al., 2018). Microalgae are also used for biosurfactants production because these

organisms are major producers of glycolipids, phospholipids and neutral lipids.

Furthermore, these organisms also produce the biocompounds and these compounds have

various applications in pharmaceuticals and food industries, like biopigments and essential

fatty acids, direct application of biomass in animal and human feed, biofuel production

(H2, biodiesel, bioethanol and biogas), and carbon dioxide biofixation. A major advantage

of cultivating microalgae for biosurfactant production is that many of these

microorganisms fall into the generally recognized as safe (GRAS) category. Such certified

11
 organisms have no risk of toxicity or pathogenicity and can be used for applications in the

food and pharmaceutical industries (Radmann. M. et. al., 2015).

Many species of microalgae are used for the production of biosurfactants such as

cyanobacteria strains Arthrospira sp. For the production of biosurfactants in autotrophic

and mixotrophic cultivation LEB 18 and Synechococcus nidulans LEB 25 and the

chlorophyte strains Chlorella minutissima LEB 108, Chlorella vulgaris LEB 106 and

Chlorella homosphaera in autotrophic and mixotrophic cultivation (Radmann. M. et. al.,

2015). Application of bio-surfactant from algae as bioremediation is more environmentally

compatible and more economical than using modified clay complexes or metal chelators

(Plociniczak et al. 2011). Biodegradative properties of biosurfactant producing

Brevibacterium sp. Reddy et al. (2010) tested a strain PDM-3. They reported that this strain

could degrade 93.92% of the phenanthrene and also had ability to degrade other

polyaromatic hydrocarbons such as anthracene and fluorene. (Moldes et al. 2011) found

that biosurfactant from Lactobacillus pentosus a bacteria which grown in synthetic media,

and it also reduced the concentration of octane in the soil to 58.6% and 62.8%, for soil

charged with 700 and 70,000 mg. kg−1 of hydrocarbon, respectively (Chekroun. B et. al.,

2015).

3. Materials and Methods

3.1 Sampling and cell cultivation

The strain Pediastrum sp. was obtained from Molecular Biology and Biotechnology
Research Laboratory, Department of Biotechnology University of Kotli Azad Jammu
and Kashmir. The strain was originally isolated from Tattapani hot water springs at
AJK (data not published yet). Pediastrum sp. was grown in BG-11 medium at two
different temperatures i.e. 28 °C and 40 °C, with a total volume of 100 ml in cotton

12
 plugged 250 ml conical flasks by using rotatory shaker at 160 rpm (Kanwal. S &
Incharoensakdi. A (2019)).

3.2 Determination of Optical Density

Optical density (OD) analyses by spectrophotometer allowed the calculation of the

biomass growth of microalgae Pediastrum sp. in laboratory conditions by absorption

wavelength at 730 nm. OD was measured after every two days (0 day, 2nd day, 4th

day, 6th day, 8th day, 10th, 12th day), until the stationary phase of growth.

3.3 Determination of Chlorophyll a and Carotenoid Contents

Pediastrum sp· cells were collected at 5000 rpm for 10 min. After centrifugation the

supernatant was removed, and cell pellet was suspended in 1 ml of dimethylformamide

(DMF) followed by incubation in dark for 10 min. After incubation, the DMF extract

was centrifuged at 1,000 rpm for 5min. The pellet was discarded, and absorbance of

supernatant was measured at 461 nm, 664 nm and 625 nm. Chlorophyll a and

carotenoid contents were determined by using following formula: ( Jantaro. S. et. al.,

2010, Chamovitz, D et. al., 1993, Moran, R. 1982).

Carotenoids:

µg/cell × 108 = (OD461-(0·046 × A664)) ×4

total cells

Chlorophyll a:

µg/cell × 108 = (12·1 × OD664) – (0·17 × OD625)

total cells

Total cells (cell/ml) = (A730/0·25) × 108

13
 3.4 Bio-surfactant Production
Cells were removed from different growth phases at 28°C by centrifugation at 7000

rpm for 20 min and filtered. The obtained cell free supernatant was used in the

preliminary test for identifying the presence of bio-surfactant (Sharma Ruchika, 2016).

a) Oil Spreading Method

A thin oil film was made by adding 10 µl of crude oil over 40 ml of distil water in petri

dish· 10µl of cell free supernatant was added carefully in the middle of the oil film· A

clear zone formed in the film showed the presence of bio-surfactant.

b) Emulsification Index

Emulsification index (E24) was used to determine the bio-surfactant property of the

isolates· Emulsification index was obtained by adding 1ml the cell free broth with 4ml

water and 6 ml of kerosene oil in a test tube. The mixture was subjected to vortex for 2

min at high speed· After that it was allowed to stand for 24 h. The emulsification index

is given as percentage of the height of emulsified layer divided by the total height of

the liquid column· Following equation was used to determine the percentage of

emulsification index (Sharma Ruchika, 2016).

E24= Height of emulsion formed (cm) × 100

Total height of solution (cm)

3.5 Results and Discussion

1. Growth analysis

14
The figure. 7 shows the growth of Pediastrum sp. at different phases. Cells were

collected for optical density measurement at different phases of growth i.e. at lag phase,

log phase, mid log phase, late log phase and stationary phase.

1.6

28°C 40°C
1.2

0.8
OD

0.4

0
0 2 4 6 8 10 12

Days

Figure 6 Growth of Pediastrum sp. at 28 °C and 40 ºC.

The cells growth at 28 °C and 40 °C have different results because Pediastrum sp.

is thermophilic sp. and grows better at 40 °C rather 28 °C. Growth of organism was

different at both temperatures i.e. at 40°C growth rate was higher as compare to

28°C. Because at lower temperatures molecules move slower, enzymes cannot

mediate in chemical reactions, and eventually the viscosity of the cell interior brings

all activity to a halt.

With the increase of temperature, molecules move faster, metabolism speed up by

enzymes and cells rapidly increase in size. But, above a certain value all of these

15
 activities are proceeding at such high rates, enzymes start to denature, and the total

effect is detrimental. Cellular growth ceases. These boundary morals describe the

maximum and minimum temperature in life existence (and grow). Each microbial

specie having unique upper and lower limit, which is a defining distinctive for that

species.

2. Determination of Chlorophyll a and Carotenoid Contents

Chlorophyll and carotenoids are naturally occurring bioproducts found in Pediastrum

sp. Chlorophyll a and carotenoids contents were measured at 28°C and 40 °C on

different growth phases i.e. at lag phase, log phase, mid log phase, late log phase and

stationary phase. The contents were higher at 28°C than those at 40°C.

Carotenoids 28°C Chlorophyll 28°C

Carotenoids 40°C Chlorophyll 40°C
2.5

1.5

0.5

0
Mid log Late log Stationary
Growth Phases

Figure 7 Carotenoids and chlorophyll a content in Pediastrum sp. at different growth phases and
different temperatures.

16
Determination of chlorophyll a and carotenoids content at 28 °C on different growth stages is

shown in figure. 8. The concentration of carotenoids is higher at late log phase as compare to mid

log and stationary phase of cell growth 40 °C. And chlorophyll a is present in higher concentration

at mid log phase at 40°C than other growth phases and also chlorophyll a is present in higher

concentration at stationary phase at the 28°C. And also present in high concentration at late log

phase than mid log phase. So results show that the bio-products are present in higher concentration

in Pediastrum sp.

Carotenoids were present in higher concentration at late log phase and also carotenoids are present

in higher concentration at 40°C as compare to 28°C. Chlorophyll content at 40°C are more than

28°C and at mid log phase concentration of chlorophyll is highest than late log and stationary

phase.

Temperature has significant effect on the formation of carotenoids and chlorophyll contents.

Carotenoids absorb light energy for use in photosynthesis and also, they protect chlorophyll from

photodamage. Carotenoid accumulation in algal species increases with temperature because of the

increased oxidative and photodamaging effects noted at elevated temperatures. Tjahjono et al.

1994 reported a three-fold increase in astaxanthin formation in the green alga Haematococcus

pluvialis with an increase in cultivation temperature from 20 to 30 °C. Another study on green alga

Chlorococcum sp. inveterate these results in which carotenoids content was observed and results

increase two-fold in total underneath conditions of nitrogen deficiency by levitation the

temperature from 20 to 35 °C. (Liu et. al., 2000). Production of carotenoids increases with

increasing temperature is generally recognized cellular reaction to improved activity of

biosynthetic enzymes (Juneja. A. et. al., 2013).

3. Analyses of Biosurfactant production

17
 a) Oil Spreading Method

In oil spreading method a zone on the surface of petri plate showed the bio-

surfactant production in strain. In figure. 9, it is indicated on the petri plate

that the bio-surfactant production occurred in Pediastrum grown at 28 °C

and cells were collected from late log phase.

Figure 8 Oil spreading method to produce biosurfactant in Pediastrum sp.

For Oil spreading test, the culture Lactobacillus and Bacillus sp. was

centrifuged and supernatant was added in to oil containing plate. The

biosurfactant producing organism would displace oil and form a clear zone

in the center of the plate which indicates the ability of isolated organism to

displace the oil (Sharma Ruchika, 2016).

b) Emulsification Index

In Pediastrum sp. emulsification index was measured and results of

emulsifiers were shown the presence of biosurfactant. In present study the

18
concentration of emulsification index was 3.44 cm and this value represents

the existence of biosurfactant.

No. Growth Phases Emulsification Index

1 Mid Log 3.57

2 Late Log 3

3 Stationary 3.75

Almost all thermophilic Actinomycetes are screened for the presence of

extracellular enzymes and bio-surfactant production. Actinomycetes found

to produce many kinds of metabolites, including antibiotics, pigments,

enzymes and bio-surfactants (Sharma Ruchika, 2016). Biosurfactants are

mostly used and their applications in cosmetic industry, therapeutic agent

and in oil industries. In oil recovery bio surfactants are also used and these

biosurfactants isolated from microbes are more selective and required in

small quantity rather chemical surfactants.

19
 Conclusion

This study shows the industrially important bioproducts accumulation such as carotenoids,

chlorophyll a and biosurfactants in thermophilic green alga Pediastrum sp. These products are

produced in high concentration in Pediastrum sp. grown at 40°C. These bioproducts are very

important in in pharmaceuticals and food industry. The Pediastrum sp. is also an important

organism in biotechnology and research field because of its potential ability of the production of

plethora of thermostable enzymes.

20
 Future Prospects

Thermophilic microalgae Pediastrum sp. is a novel sp. and an important tool in the field of

biotechnology. It contains various industrially important bio-products in high concentration and

can be further identified in detail. Furthermore, the thermostable enzymes responsible for the

production of naturally occurring bioproducts in Pediastrum sp. could be isolated and

characterized in future studies.

21
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