Survey, Molecular Systematics and Nanobiotechnological Potentials of Marine Cyanobacteria and Diatom

Thesis submitted to Bharathidasan University

For the award of the degree of

DOCTOR OF PHILOSOPHY IN MICROBIOLOGY

By

Mr. D. MUBARAK ALI

(Ref. No. 022323/Ph.D1/Microbio/FT/Oct 2008)

Under the tutelage of

Dr. N. THAJUDDIN

Department of Microbiology School of Life Sciences

Bharathidasan University Tiruchirappalli – 620024

Tamil Nadu, India

May 2012

Dr. N. THAJUDDIN Associate Professor

UNIVERSITY DEPARTMENT OF MICROBIOLOGY

SCHOOL OF LIFE SCIENCES

21.05.2012

Certificate

This is to certify that this thesis entitled “Survey, Molecular Systematics and

Nanobiotechnological Potentials of Marine Cyanobacteria and Diatom” submitted

by Mr. D. Mubarak Ali, for the degree of Doctor of Philosophy in Microbiology, to

the Bharathidasan University is based on the results of studies carried out by him

under my guidance and supervision. This thesis or any part of thereof has not been

submitted anywhere else for the award of degree, diploma, Associateship, fellowship

or other similar titles to any candidates.

(N. THAJUDDIN)

Tiruchirappalli – 620 024, India, Phone: +91 431 2407082; Mobile +91 098423 79719; E-mail: nthaju2002@yahoo.com

Mr. D. MUBARAK ALI

Doctoral Research Scholar & CSIR – SRF

DECLARATION

I do hereby declare that this work has been originally done by me under the tutelage

of Dr. N. Thajuddin, Associate Professor, Department of Microbiology, Bharathidasan

University, Tiruchirappalli and this work has not been submitted elsewhere for any other

degree, diploma or other similar titles.

(D. MUBARAK ALI)

Dedicated this thesis to Almighty…

This is not the end; this is not even beginning of the end; but it perhaps. This

is end of the beginning. - Winston Churchill

ACKNOWLEDGEMENT

“Omnipotent and Omnipresent, in the of name of Allah”

The writing of this thesis has been an incredible journey and a monumental milestone in my academic life. I could not have embarked on this expedition and traveled this far without the passionate and continued support of advisors, colleagues, friends and my family.

This is my respectful commitment to thank Dr. N. Thajuddin, Associate Professor, Department of Microbiology, Bharathidasan University, Tiruchirappalli for his responsibility of guidance and supervision in my research carrier. His independency on my research work, friendly approaches and his kind support are the secret of successful completion of my work on time. He is a great ambassador of real meaning of guide and supervisor and also thank his family for its love and affection.

Dr. Maggy F. Lengke, Professor, Department of Earth Sciences, University of Western Ontario, London, helped me in many ways to enable and shape my work and continued to provide me firm support and constructive feedback along the way. For that, I will always be grateful.

Dr. S. Maruthamuthu, Scientist, Central Electrochemical Research Institute (CECRI), Karaikudi, India. He is the one who initiated my research on this topic at first and I thank him for his moral support.

Dr. K. Jeganathan Associate Professor, Co-ordinator, Centre for Nanoscience and Nanotechnology, BDU. Its not only my doctoral committee member, but also has enlightened me in so many aspects about innovations, ideas and other academic matters. He is a wonderful guide and ally whom one has the good fortune to know.

I wish to express my sincerest thanks to Dr. K. Natarajaseenivasan, Associate Professor and Head, Dr. V. Rajeshkannan, Dr. G. Muralitharan and Dr. D. Dhanasekaran, Assistant Professors, Department of Microbiology and Dr. Joseph Selvin, Professor and Head, Department of Bioinformatics, BDU for their support and encouragement.

This is my duty to thank Mr. Vincent, St. Joseph College, Trichirappalli, Dr. S. Shanthi and Dr. Sivakumar, Annamalai University, Chithambaram, Mr. S. Ravishanker, Central Electro Chemical Research Institute, Karaikudi, Mrs. Dr. Pushpa Viswanathan, Cancer Institute, Chennai for their timely help and providing me instrumentation facility.

I wish to record my heartful thanks to R. Mahesh, Post. Doc. Kyung Hee University, South Korea, Dr. Kasthuri, Post Doc, and Dr. M. I. Mohd Ershath, Post Doc. National University of Singapore, Singapore. Dr. Mr. M. Rahuman Sheriff, Researcher, Max Planck’s Institute of Molecular Physiology, Germany, Mr. K. Harish Nag, Researcher, Institute of Botany and Microbiology, Belgium, Mr. Satish,

Researcher, Delhi University, New Delhi and Mr. S. Chandru, Researcher, New Delhi for their encouragement and timely help whenever I approached.

My sincere gratitude also goes to Professors Dr. C. Thangamuthu and Dr. M. Ponnavaikko, Former Vice Chancellors, Bharathidasan University, Dr. G. Subramanian, Founder and former Director, NFMC, Dr. M. A. Akbersha, Director, Mahatma Gandhi Dorenkamp Centre (MGDC), Dr. M. Gunasekaran, Professor in Biology, Fisk University, USA, Dr. L. Uma, Professor and Director, NFMC, Dr. D. Prabakaran Associate Professor, NFMC, Dr. A. Pannerselvam, Associate Professor, Dept. of Botany and Microbiology, A.V.V.M Sri Pushpam College, Tanjore, Dr. K. Kathiresan, Professor, CAS in Marine Biology, Annamalai University, Chithambaram, Dr. S. Ravikumar, Dept. of Oceanography and CAS, Alagappa University, Karaikudi, Dr. N. Anand, Professor and Dr. Rengasamy, Professor and Director, CAS in Botany, University of Madras, Chennai and Dr. B. G. Raghavan, Chennai without their encouragement I could not have come this far.

I also express my thanks to Dr. M. B. Viswanathan, Professor, Dept. of Plant Science, Dr. S. Ravikumar, Asst Professor, Dept. of Biochemistry, Dr. P. Santhanam Asst. Professor, Dept. of Marine Sciences, BDU, our collaborators Dr. N. Rameshbabu and Dr. C. Velmathi Asst. Professors, National Institute of Technology, Trichirappalli and Dr. Prabakaran, Sri Gowri Biotech, Tanjore for their constant support and encouragement.

I express my heartfelt thanks to my initial advisors, Mr. K. Kandasamy, Mr. A. K. Saravanan, Mr. S. Anbalagan, Mrs. S. Shahitha Begam, Ms. Nashima Begam, Mrs. Stella Baby, Mr. Dhanasekaran and Librarians from Muthayammamal College of Arts and Sciences, Rasipuram, Nammakkal for their encouragement and proper guidance.

I would also like to thank the non-teaching technical community, especially Mrs. M. Umarani, Mr. S. Elangovan, Mr. K. Vijayakumar, Mr. Arumugam, Mr. G. Durairajan, Mr. G. Pazhanivel, Mr. K. Gobikanna, Mr. S. Kathiresan from Department of Microbiology, Mr. Stephen, Mrs. Malathy, Mr. Kaviyarasan, Admn building, BDU, among many others, for their professional and administrative backup and for securing a place where we can rest our fatigued minds.

A book with thousand pages starts from single dot. This is my intense to thank my teacher, Mrs. G. Porselvi Prakasham, G. B. H. S. School, Thammampatti (1999-2003). She was the one, who sowed research seed in my mind during my school hood and I also thank to Dr. N. Geetha Achunathan for her motivation and encouragement from my childhood on wards.

I express my heartfelt thanks to Ms. C. Divya, Mr. M. Jayarajan, Mrs. Mari Nivetha, Mrs. T. Shenbagavalli and Mr. D. Pandiaraj, Researchers, Dept of Microbiology, BDU Mr. V. Gopinath, Researcher, SRM University, Chennai and

Mr. K. Venteshwarlu, Researcher, National Institute of Technology, Trichirappalli for their moral support and care.

I place my heartfelt thanks to Mr. R. Praveen Kumar for his cooperation in research and his love and affection towards me and my work and also I place heartfelt thanks to Mrs. A. Ilavarasi Krishnan for her inevitable support and encouragement.

I cannot overlook the warmth that my labmates gave to me and the humour and smiles of friendship that they brought to me; Dr. C. Nithya, Dr. M. Shatheesh Kumar, R. Arvind Kumar A. Suresh, D. Vijayan, A. Parveez Ahamed, M. Abhijith, N. Reehana, the list is endless…

I place my sincere thanks to juniors M. Leo Antony, K.A. Sheik Syed Ishack, K. Kannan, E. Baldev, F. Lewis Oscar, D. Sanker Ganesh, C. Anchana Devi for their constant encouragement and love and affection towards me and my work.

I wish to record the support from my friends Mr. J Arunkumar, Mr. P. Prabakaran, Mr. M. Sundaravadivel, Mrs. S. Manupriya, Mrs. Sudha, Mr. T. Vinodh Kumar, Mr. V. Purusothaman Mr. K. P. Prakash, Mr. R. Suresh, Mr. R.V., Dr. M. Jaccob, and Mr. S. Karthik for their love cheered me on.

This is my immense pleasure to thank my fine arts masters Mr. M. Saravanan, Dr. C. Lalithambal, Mrs. S. Sri Vidya and Mr. R. Venkatesh for their encouragement and timely help. I place my sincere thanks to Mr. Abdul Azees, Karaikudi for his timely help and love and affection.

As the whole, my heartfelt thanks to all my seniors, juniors, colleagues, Ph.D and M. Phil Scholars of Dept. of Microbiology, Eco-Biotechnology, Env. Management, Biotechnology, Marine Biotechnology, Animal Biotechnology, Plant Biotechnology, Marine Sciences, Bioinformatics, Biochemistry, Biomedical Sciences, Physics, Chemistry, Geography and English BDU for their prayers and wishes which made me complete this thesis well and good.

I gratefully acknowledge the financial support offered by the Council for Scientific and Industrial Research (CSIR-SRF Fellowship), Department of Biotechnology (DBT-JRF Fellowship) and University Grant Commission (UGC-PF), Govt. of India, funded my research work and I also thank Department of Science and Technology (ITS), INSA–CICS (TG) for their travel grant to participate in the overseas conferences at South Korea and Singapore respectively and made my stay in the Bharathidasan University possible and to the Staff at Vaigai, Porunai and Cauvery Hostel, who, though were so willing all the time, to attend to my needs any way.

As personal note, I thank everyone in my family and PG, UG and School friends for their endless support, encouragement, care, love and affection. Because of them only, my work sailed in smooth way.

To all, I say, thank you and God bless!!!

Mubarak Ali, D

Contents Page No.

Survey and Molecular Systematics of marine Cyanobacteria and Diatom

Chapter I

Introduction 1

Review of Literature 6

Materials and Methods 36

Results and Discussion 42

Partial Biochemical Characterization of C-Phycobiliproteins

Chapter II

Introduction 47

Review of Literature 50

Materials and Methods 60

Results and Discussion 64

Nanobiotechnological Potentials of Marine Cyanobacteria and Diatom

Chapter III

Introduction 72

Review of Literature 78

Materials and Methods 101

Results and Discussion 107

Annexure

Summary 124

References 126

List of GenBank Accession (NCBI) 162

Publications

Chapter I

Survey and Molecular Systematics of Marine Cyanobacteria and Diatoms

1

INTRODUCTION

As primary producers in the worlds oceans, they are keystone species in global

nutrient cycling. About 20% of the total carbon and silica sequestered are fixed by less

than a few hundred species in the marine plankton (Guillard and Kilham 1978;

Goldman 1993; Hasle and Syversten 1996; Mann 1999). Knowledge of the geological

age of the diatoms gives palaeoclimatologists a clue as to how long these organisms

have been modifying the biosphere (Kooistra and Medlin 1996; Siever 1991). The cell

wall structure is species specific, demonstrating that diatom silica morphogenesis is

genetically encoded.

Cyanobacteria are prokaryotic oxygenic phototrophs found in almost every

conceivable habitat on earth (Ferris et al. 1996; Ward et al. 1997; Nubel et al. 1999,

2000; Abed and Garcia-Pichel 2001; Garcia-Pichel and Pringault 2001). They exist in

different morphologies including unicellular and filamentous forms (Castenholz 2001).

While unicellular types exist as single cells, suspended or benthic, or aggregates,

filamentous types may be thin or thick, single trichome or bundles either with or

without a sheath. Cyanobacteria are able to perform different modes of metabolism

with the capacity to switch from one mode to another (Stal, 1995).

All cyanobacteria carry out oxygenic photosynthesis but some cyanobacterial

species can switch to the typical bacterial anoxygenic photosynthesis using sulfide as

electron donor (Cohen et al. 1986). Under anoxic conditions and during the dark,

cyanobacteria carry out fermentation (Stal 1997). Some cyanobacteria form heterocysts

and have the ability to fix atmospheric nitrogen (Capone et al. 2005). Phylogenetic

analysis of cyanobacteria based on 16S rDNA genes showed that they are a diverse,

monophyletic phylum of organisms within the bacterial radiation. Research on

cyanobacteria in the last decades focused largely on their ecology, morphology,

physiology and 16S rDNA-based phylogeny but relatively little has been done on their

potential uses in biotechnology. The overwhelming available knowledge on the

diversity and physiology of cyanobacteria serves as an excellent base for exploring

their applications in biotechnology (Thajuddin and Subramanian, 2005).

2

In the last few years, cyanobacteria have gained much attention as a rich source

of bioactive compounds and have been considered as one of the most promising groups

of organisms to produce them (Bhadury et al. 2004; Dahms et al. 2006). These

cyanobacterial metabolites include antibacterial (Jaki et al. 2000 and MubarakAli et al.,

2008), antifungal (Kajiyama et al. 1998), antiviral (Patterson et al. 1994), anticancer

(Gerwick et al. 1994), antiplasmodial (Papendorf et al. 1998), algicide (Papke et al.

1997) and immunosuppressive agents. Screening of cyanobacteria for antibiotics has

opened a new horizon for discovering new drugs. Some cyanobacteria have also been

found to intracellularly accumulate polyhydroxyalkanoates (PHA), which are

comparable in properties to polyethylene and polypropylene (Steinbuchel et al. 1997).

These biodegradable plastics could replace oil-derived thermoplastics in some fields.

Recent research on cyanobacteria has demonstrated that they form ideal consortia with

chemotrophic bacteria and can effectively be used to cleanup oil contaminated

sediments and wastewaters (Abed and Koster 2005).

Cyanobacterial taxonomy has been established based on morphological features,

such as the shape and dimensions of the cells, presence of structurally differentiated

cells, and whether the cells grow as solitary cells or in colonies (Paerl, 1988).

Molecular methods have become an indispensable tool for characterization of

cyanoprokaryotes and the assessment of evolutionary relations among them in recent

decades. The direct sequencing of various genes is the most common method used in

taxonomy of cyanobacteria. However, RFLP (Restriction Fragment Length

Polymorphism) is also widely applied, especially for more detailed examination of the

genetic variability of closely related taxa (Ernst et al., 1995) or to infer the extent of

cyanobacterial diversity in nature. Also random amplified polymorphic DNA (RAPD)

analysis is sometimes used in order to discriminate between genotypes of close

relatives. Much less common are the allozyme or the whole-cell protein analysis

(Neilan, 1995).

Diatoms are single celled photosynthesizing eukaryotic algae that produce

intricately structured cell wall made of nanopatterned silica (SiO2) and are found in

3

almost every aquatic environment including fresh and marine water, soil, in fact almost

anywhere in moist. They are non motile, or capable of only limited movement along a

substrate by secretion of mucilaginous material along a slit like groove or channel

called a raphe, both benthic and planktonic forms exist. Diatoms are formally classified

as belonging to the division Chrysophyta, Class Bacillariophyceae. The Chrysophyta

are algae which form endoplasmic cysts, store oil rather than starch, posses a bipartite

cell wall and secrete silica at some stages of their life cycle. Diatoms are nearly all

autotrophic, heterotrophic members are rare (Li and Volcani 1987).

Diatoms are commonly between 20-200 microns in diameter or length, although

sometimes they can be up to 2 millimeter long first appeared more than 180 million

years ago. First recorded occurence of diatoms are from Jurassic however, these are

uncertain and the earliest recorded well preserved diatoms are centric forms from

Aptian-albian stages of the Cretaceous. At first diatoms are thought to be animals

because what are now called raphid diatom move at a speed of upto 25µms-1 when

attached to surfaces. Chris Bowler (2001) explains that molecular phylogeny and

morphological studies suggest that diatoms originated probably as the result of a

eukaryote being invaded or engulfed by a photosynthetic eukaryote, most probably a

red algae.

Diatoms are phylogenetic similarity to green algae and higher plants is derived

from the primary endosymbiotic event, which is thought to have occurred at least 700

million years ago (Kowallik, 1992). Therefore, diatom cells have a range of features

that make them highly divergent from the classical cellular structure of higher plants,

including:

(a) The use of the brown carotenoid pigment, fucoxanthin for light energy transfer

within the light harvesting complexes of photosystems I and II

(b) The presence of four membranes around their plastids. The inner two

membranes are equivalent to the membranes that normally surround higher

plant chloroplasts, whereas the second membrane (as counted from the outside)

4

is thought to be derived from the endosymbiont’s plasma membrane, and the

outer membrane is continuous with the endoplasmatic reticulum of the host cell.

(c) The presence of a rigid cell wall composed largely of amorphous silica (i.e.

glass). The exquisite lacework-like patterning of diatom cell walls is reproduced

with high fidelity from generation to generation and is species specific. For

these reasons, it has been used since the last century for taxonomic

classification.

Current phylogenetic trees place diatoms close to Alveolata lineages and far

from the green and red lineages of other photosynthetic eukaryotes (Baldauf et al.,

2000). The expressed sequence tags (EST) program has revealed the presence of genes

encoding enzymes involved in cAMP metabolism, as well as genes encoding the

components of the animal extracellular matrix, such as fibronectins, elastins, and

tenascins, none of which appear to be present in higher plants. As a consequence, one

could almost consider diatoms as photosynthetic animals rather than unicellular plants.

The rigid part of the cell wall (frustule) of diatoms is composed of amorphous

silica (Pickett-Heaps et al., 1990; Parkinson and Gordon 1999; Vrieling et al., 2000),

located in the girdle bands and two valves: the epitheca consisting of an epivalve and

epicingulum, and the hypotheca consisting of hypovalve and hypocingulum (Round et

al., 1990). The timing of diatom valve formation has not been directly determined in an

individual cell, however, some estimates were made based on measurements on whole

synchronized cultures. For instance, 2D valve formation in Melosira varians Agardh

takes approximately 8 minutes. Cell wall morphogenesis in Ditylum brightwellii (West)

Grunow, and in Navicula pelliculosa (Kutz) Hilse occurs during respectively a 53 min

and a 2-3 h period of the cell cycle (Sullivan and Volcani, 1981).

Commonly, in pennate diatoms the development of the new hypovalve starts

from the axial area and grows towards the outer cellular edges (Pickett-Heaps et al.,

1990). Subsequently, the frustule thickens in the third dimension and is completed by

coverage with a casing followed by exocytosis of the valve and girdle bands, and

5

finally cell separation (Darley et al. 1976; Darley and Volcani 1971; Pickett-Heaps et

al., 1990).

Application of the M13 PCR fingerprinting method was useful for almost all

forms of cyanobacteria strain and species discrimination. PCR amplification

techniques viz., repetitive DNA element PCR (REP-PCR), short randomly repeated

repetitive PCR (STRR-PCR) and arbitrarily primed PCR (RAPD-PCR) were used for

the taxonomic discrimination among the strains of the unicellular cyanobacterium,

Synechococcus elongatus collected across the coastal regions of the Indian

subcontinent. In comparison with the STRR and RAPD, the REP primer set generates

fingerprints of lower complexity, but still the phenogram clearly differentiated the

strains. In conclusion, described PCR fingerprinting methods can be considered as

promising tools for the differentiation at the strain level of cyanobacteria from the same

species (Muralitharan and Thajuddin, 2010). 151 unique cyanobacterial genes in 8

studied genomes and found a few examples of largely conserved gene order, which

could prove useful for solving problems of cyanobacterial evolution on a larger scale

(Martin et al., 2003).

Molecular approaches have been divided into two classes: PCR independent and

PCR based approaches. Molecular assessment of cyanobacterial biodiversity has been

studied by using markers like 16S rDNA, phycocyanin locus, nif gene, rpo gene, ITS

region etc. A general overview of biodiversity assessment, molecular techniques and

markers used for biodiversity assessment recommends combinatorial approach with

different molecular markers. It is likely to improve the degree of resolution and provide

as possible the broadest picture and in depth information about biodiversity

documentation (Kumari et al., 2009).

Due to innate biotechnological potentials and most abundantly available in

marine ecosystems, both cyanobacteria and diatoms has been selected in the present

study.

6

REVIEW OF LITERATURE

Diatom Biology

Diatoms were first discovered by light microscopy and mistakenly addressed as

unicellular animals due to their brownish color and their capability to move on

substrates (Lind et al., 1997). According to the species concept, organisms mating with

each other belong to the same species. Diatom taxonomy thus was based mainly either

on the identification of ribosomal sequences (Medlin et al., 1996) or more classically

on the morphology and the shape of the frustules, the extracellular silica cell walls.

These frustules, which are formed by two valves that fit together like Petri dishes, often

are highly ornamented and show species-specific structures.

The ability of diatoms to genetically define these structures makes them highly

interesting for nanotechnological applications (Drum and Gordon, 2003). Because of

their siliceous composition frustules were often well preserved in fossil deposits

(Damste et al., 2004) and strong sedimentation of diatom shells in ancient oceans led to

the deposition of siliceous earth or diatomite, a material of high importance for

industrial uses (Harwood, 1999).

Diatoms play a significant ecological role; about half of the global annual net

primary production in the oceans is due to phytoplankton which is dominated by

diatoms (Falkowski et al., 1998). Diatoms thus produce and represent the main input

into the marine food web. They are not only strongly involved in fixation of CO2, but

also in cycling of soluble silicates by integrating them into the shells and by releasing

parts of them after decomposition at the bottom of the oceans (Bidel and Azam, 1999).

It is not yet fully understood why diatoms filled aquatic niches so successfully, even

though they have the capability to grow at a wide range of light intensities (Falkowski

et al., 2004) and the apparent ability to perform C4 photosynthesis (Reinfelder, 2004).

Cell Wall Structure, Cell Cycle and Sexual Reproduction

The diatom cell wall consists of two halves of identical structure, although one

half (epitheca) is slightly larger and overlaps the other half (hypotheca). Together the

7

epitheca and hypotheca completely enclose the protoplast. Each theca is composed of a

valve displaying ornate structures with often elaborate pore patterns, and several girdle

bands exhibiting generally uniform pore patterns. The terminal girdle band of each

theca is termed the pleural band and is located in the region where the epitheca and the

hypotheca overlap (Fig 1).

Fig 1: Schematic overview of the siliceous components diatom cell wall

Diatoms can be divided into three major groups according to their shape and

symmetry of their cell walls: radial centrics, polar centrics, and pennates. Radial

centrics have a petri dish-like architecture with a circular center of symmetry (annulus)

in the middle of the valve, and rows of pores (striae) radiating from the annulus. Polar

centrics have bi or multipolar valves with an elongated or distorted annulus. Pennate

diatoms are bilaterally symmetrical, and instead of an annulus possess a rib (sternum)

running along the longitudinal axis of each valve.

In raphid pennates the sternum contains a slit (raphe) that is interrupted by the

central nodule in the center of the valve. Raphid pennates are able to move on surfaces

by means of traction generated by adhesive mucilage (polysaccharides or

proteoglycans) that is secreted through the raphe (Edgar and Pickett-Heaps, 1984). The

traction force appears to be generated by an actin-myosin based system that is located

8

precisely underneath the raphe on the cytosolic side of the plasma membrane (Poulsen

et al., 1999). Araphid pennates, radial centrics, and polar centrics are nonmotile.

The rigidity and architecture of the silica cell wall imposes restrictions on the

mechanism of cell division and growth. The valves can only be formed during cell

division, and the increase in cell volume during interphase requires the stepwise

synthesis of girdle bands in order to avoid the occurrence of gaps within the cell wall as

the distance between the epitheca and hypotheca increases (Fig 2). Furthermore,

because new valves always develop within the confines of the parental cell wall, the

sibling cell that inherits the parental hypotheca is usually smaller than the parental cell.

Fig 2: Schematic overview of mitotic cell division and hypovalve and girdle band formation. N, Nucleus; MC, microtubule center; SDV, silica deposition vesicle.

The key events occurs during mitotic cell division are as follows: a) the nucleus

of each daughter cell moves to the side of the cell where the hypotheca will be formed.

(b) A microtubule center (MC) positions itself between the nucleus and the plasma

9

membrane above which the hypotheca will eventually be placed. (c) A specialized

vesicle known as the silica deposition vesicle (SDV) forms between the MC and the

plasma membrane in a region that becomes the “pattern center.”(d) The SDV elongates

into a tube and then spreads along one side of the cell. (e) A new valve is formed within

the SDV by the targeted transport of silica, proteins, and polysaccharides. During this

process, the SDV becomes acidic as a result of the silica polymerization process

(Vrieling et al., 1999). Some of the organic components eventually form a coat around

the silica framework, whereas others are involved in silica deposition. (f) Once valve

biogenesis is complete, it is exocytosed by fusion of the SDV membrane (the

silicalemma) with the plasma membrane. As a consequence, the inner face of the

silicalemma is thought to become the new plasma membrane. (g) Following separation,

the daughter cells can expand unidirectionally along the cell division axis by the

biogenesis of girdle bands. These structures are also formed within SDVs in an

analogous way.

As a consequence, the average cell size in a diatom population gradually

decreases with continued vegetative growth (the MacDonald-Pfizer rule). Ultimately,

this size reduction would result in cells too small to be viable and the diatom population

would die. The only way to escape this fate is by sexual reproduction (Chepurnov et al.,

2004). During this process meiotic cell divisions occur, and the resulting gametes shed

their cell walls. Immediately after gamete fusion a specialized zygote (termed

auxospore) is formed, which is covered by an organic cell wall, and undergoes an

enormous increase in volume within a relatively short time (hours to a few days).

Already during the first division of the auxospore, silicified valves are formed

that exhibit the species-specific shape and silica patterns.The molecular details of this

amazing process are almost completely unexplored with the exception of a study by

Armbrust, who discovered a new gene family (Sig) that is expressed during the onset of

sexual reproduction in T. weissflogii (Armbrust, 1999). Sig genes have also been

identified in other Thalassiosira species, yet the function of the Sig-encoded proteins is

still unknown (Armbrust and Galindo, 2001).

10

The size of some of the best-studied diatoms growing in laboratory culture (e.g.,

Cylindrotheca fusiformis, Phaeodactylum tricornutum, Thalassiosira pseudonana)

remains constant, and sexual stages of these organisms have never been observed. The

mechanism by which these species avoid size reduction was not yet fully understood,

but appears to involve the ability to form expandable girdle bands (Hildebrand et al.,

2007).

Organic components of the cell wall

Early light-microscopy studies of diatom cell walls using ruthenium red

presented evidence that organic material is intimately associated with the silica

structures (Pickett-Heaps et al., 1990). Later, imaging of ultrathin-sections after

anhydrous hydrogen fluoride (HF) treatment (i.e., dissolution of silica) followed by

transmission electron microscopy indicated the presence of organic material both on the

silica surface and inside the silica (Pickett-Heaps et al., 1990, Volcani, 1981). Based on

the ruthenium red staining, it was assumed that the silica-associated organic

components are polysaccharides (pectin).

Indeed, to date a plethora of extracellular polysaccharides have been

characterized from diatoms, which appear to play a role in cell adhesion to surfaces,

gliding of pennate diatoms, and protection against desiccation (Hoagland et al., 1993).

While these polysaccharides are relatively loosely attached to the diatom cell wall, and

can often be extracted by treatment with hot water or mildly alkaline solutions,

polysaccharides have recently been characterized that are much more tightly bound to

or even embedded within the diatom silica.

These silica-associated polysaccharides are composed of 3-linked mannans that

are highly polyanionic due to the attachment of numerous uronic acid residues and or

sulfate groups (Chiovitti et al., 2003). It was unclear if the polyanionic mannans are

involved in silica biosynthesis as no functional studies have been performed.

Furthermore, the polyanionic mannans might be protein-linked in vivo, since silica-

embedded highly mannosylated and sulphated glycoproteins have been identified from

11

the diatoms T. pseudonana and C. fusiformis (Poulsen and Kroger, 2003, Poulsen et al.,

2003).

Cyanobacteria

Cyanobacteria or cyanoprokaryota are oxygen producing, photosynthesizing,

gram-negative prokaryotes which had a main part in the evolution of the Earth´s

atmosphere. Cyanobacteria thrive in a wide range of mostly aquatic habitats. The

cellular organization and basic functions regarding growth and photosynthesis have

been comprehensively described (Sandgren, 1988) and since in late 1990s, the

sequencing of entire genomes of or smaller specific regions of within a genome such as

toxin production has allowed more extensive understanding of their unique lifestyle and

metabolic traits (Kaneko et al., 2001).

While cyanobacteria generally have relatively simple basic metabolic

requirements consisting of carbon dioxide, light, water, and inorganic nutrients and

they possess efficient uptake and retention mechanisms for phosphate, nitrate and

bicarbonate. In addition to the above mechanisms, cyanobacteria are capable of

producing high affinity iron chelators, known as siderophores, under iron deficient

conditions to alleviate iron stress and some filamentous cyanobacterial genera possess

heterocysts, cells specialized to fix atmospheric nitrogen. In total, cyanobacteria bear

unique traits that allow them to dominate many phytoplankton communities as a result

of the interactions between their unique physiological traits and the physical and

chemical characteristics of the aquatic system itself (Ritchie et al., 1997).

Biological diversity

Although cyanobacteria probably evolved as a group of organisms about 2,000

million years before the advent of eukaryotes, they comprise fewer taxa than eukaryotic

microalgae (Bisby, 1995). The concept of species in the cyanobacteria has, however, no

distinct boundaries. The situation is similar for most organisms, except for those that

are sexually reproductive depending on the classification system used; the number of

species recognised varies greatly. Based on the International Code of Botanical

Nomenclature the class Cyanophyceae, for example, contains about 150 genera and

12

2,000 species (Hoek et al., 1995). Chemotaxonomic studies include the use of markers,

such as lipid composition, polyamines, carotenoids and special biochemical features.

The resulting data support the more traditional examinations of phenotypic and

ecological characteristics. Physiological parameters are conveniently studied using

laboratory cultures (Packer and Glazer, 1988). The diversity of cyanobacteria can be

seen in the multitude of structural and functional aspects of cell morphology and in

variations in metabolic strategies, motility, cell division, developmental biology, etc.

The production of extracellular substances and cyanotoxins by cyanobacteria illustrates

the diverse nature of their interactions with other organisms (i.e. allelopathy) (Rizvi and

Rizvi, 1992).

A molecular approach to the systematics of cyanobacteria may be most fruitful

for inferring phylogenetic relationships. Macromolecules, such as nucleic acids and

proteins, are copies or translations of genetic information. The methods applied involve

direct studies of the relevant macromolecules by sequencing, or indirectly by

electrophoresis, hybridisation, or immunological procedures (Wilmotte, 1994). Nucleic

acid technologies, especially the polymerase chain reaction (PCR), have advanced to

the point that it is feasible to amplify and sequence genes and other conserved regions

from a single cell. To date, 16S rRNA has given the most detailed information on the

relationships within the cyanobacteria (Rudi et al., 1997). However, the molecular

results obtained should be integrated with other characteristics as the base for a

polyphonic taxonomy (Vandamme, et al., 1996). A considerable morphological, as well

as a genotypical, polymorphy exists in the cyanobacteria, although as data from rRNA

sequencing indicates they are correlated to a high degree.

The phylogenetic relationship of cyanobacteria is the rationale behind the

meaningful systematic groupings. However, it is difficult to set up a system of

classification that serves both the everyday need for practical identification, and offers

an expression of the natural relationship between the organisms in question (Mayr,

1981). Meanwhile, it will be necessary to use the available manuals and reference

books to help in these investigations and with the proper identification of the

cyanobacteria. They are photoautotrophs, cyanobacteria can be grown in simple

13

mineral media. Vitamin B12 is the only growth factor that is known to be required by

some species. Media must be supplemented with the essential nutrients needed to

support cell growth, including sources of nitrogen, phosphorus, trace elements, etc.

Toxigenic strains of cyanobacteria are deposited in international-type culture

collections (Rippka, 1988; Sugawara et al., 1993). Clonal cultures are distributed for

research, taxonomic work and teaching purposes.

Molecular Systematics:

Microbial Systematics has long remained an enigma. Conceptual advances in

microbiology during the twentieth century included the realization that a discontinuity

exists between those cellular organisms that are prokaryotic (i.e. whose cells have no

nucleus) and those that are eukaryotic (i.e. more complexly structured cells with a

nucleus) within the organization of their cells. The microalgae investigated by

phycologists under the International Code of Botanical Nomenclature (ICBN) (Greuter

et al., 1994) included organisms of both eukaryotic and prokaryotic cell types. The

cyanobacteria (Geitler, 1932) constituted the largest group of the latter category. The

prokaryotic nature of these organisms and their fairly close relationship with eubacteria

made work under provisions of the International Code of Nomenclature of Bacteria

(ICNB) (Sneath, 1992) more appropriate (Rippka et al., 1979; Waterbury, 1992).

The prevailing systematic view is that comparative studies of the genetic

constitution of the cyanobacteria will now contribute significantly to the revision of

their taxonomy. Relevant classification should reflect as closely as possible the

phylogenetic. The integration of phenotypic, genotypic and phylogenetic information

renders possible a consensus type of taxonomy known as polyphasic taxonomy

(Vandamme et al., 1996). The names "cyanobacteria" and "blue-green algae"

(Cyanophyceae) are valid and compatible systematic terms. This group of micro-

organisms comprises unicellular to multicellular prokaryotes that possess chlorophyll a

and perform oxygenic photosynthesis associated with photosystems I and II

(Castenholz and Waterbury, 1989).

14

Occurrence in nature

The majority of Cyanobacteria are aerobic photoautotrophs. Their life processes

require only water, carbon dioxide, inorganic substances and light. Photosynthesis is

their principal mode of energy metabolism. In the natural environment, however, it is

known that some species are able to survive long periods in complete darkness.

Furthermore, certain cyanobacteria show a distinct ability for heterotrophic nutrition

(Fay, 1965). Cyanobacteria are often the first plants to colonize bare areas of rock and

soil. Adaptations, such as ultraviolet absorbing sheath pigments, increase their fitness in

the relatively exposed land environment.

Many species are capable of living in the soil and other terrestrial habitats,

where they are important in the functional processes of ecosystems and the cycling of

nutrient elements (Whitton, 1992). The prominent habitats of cyanobacteria are limnic

and marine environments. They flourish in water that is salty, brackish or fresh, in cold

and hot springs, and in environments where no other microalgae can exist. Most marine

forms (Humm and Wicks, 1980) grow along the shore as benthic vegetation in the zone

between the high and low tide marks. The cyanobacteria comprise a large component of

marine plankton with global distribution (Wille, 1904; Gallon et al., 1996).

A number of freshwater species are also able to withstand relatively high

concentrations of sodium chloride. It appears that many cyanobacteria isolated from

coastal environments tolerate saline environments (i.e. are halotolerant) rather than

require salinity (i.e. are halophilic). As frequent colonisers of euryhaline (very saline)

environments, cyanobacteria are found in salt works and salt marshes, and are capable

of growth at combined salt concentrations as high as 3-4 molar mass (Reed et al.,

1984). Freshwater localities with diverse trophic states are the prominent habitats for

cyanobacteria.

Numerous species characteristically inhabit, and can occasionally dominate,

both near surface epilimnic and deep, euphotic, hypolimnic waters of lakes (Whitton,

1973). Others colonize surfaces by attaching to rocks or sediments, sometimes forming

mats that may tear loose and float to the surface. Cyanobacteria have an impressive

15

ability to colonize infertile substrates such as volcanic ash, desert sand and rocks (Jaag,

1945; Dor and Danin, 1996). They are extraordinary excavators, boring hollows into

limestone and special types of sandstone (Weber et al., 1996). Another remarkable

feature is their ability to survive extremely high and low temperatures. Cyanobacteria

are inhabitants of hot springs (Castenholz, 1973), mountain streams (Kann, 1988),

Arctic and Antarctic lakes (Skulberg, 1996a) and snow and ice (Kol, 1968; Laamanen,

1996). The cyanobacteria also include species that run through the entire range of water

types, from polysaprobic zones to katharobic waters (Van Landingham, 1982).

Cyanobacteria also form symbiotic associations with animals and plants.

Symbiotic relations exist with, for example, fungi, bryophytes, pteridophytes,

gymnosperms and angiosperms (Rai, 1990). The hypothesis for the endosymbiotic

origin of chloroplasts and mitochondria should be mentioned in this context. The

evolutionary formation of a photosynthetic eukaryote can be explained by a

cyanobacteria being engulfed and co developed by a phagotrophic host (Douglas,

1994). Fossils of what were almost certainly prokaryotes are present in the 3,450

million year old Warrawoona sedimentary rock of north-western Australia.

Cyanobacteria were among the pioneer organisms of the early earth (Brock

1973; Schopf, 1996). These photosynthetic micro-organisms were, at that time,

probably the chief primary producers of organic matter, and the first organisms to

release elemental oxygen into the primitive atmosphere. Sequencing of

deoxyribonucleic acid (DNA) has given evidence that the earliest organisms were

thermophilic and thus able to survive in oceans that were heated by volcanoes, hot

springs and bolide impacts (Holland, 1997).

Organization, function and behavior:

The structure and organization of cyanobacteria are studied using light and

electron microscopes. The basic morphology comprises unicellular, colonial and

multicellular filamentous forms. Unicellular forms, for example in the order

Chroococcales, have spherical, ovoid or cylindrical cells. They occur singly when the

daughter cells separate after reproduction by binary fission. The cells may aggregate in

16

irregular colonies, being held together by the slimy matrix secreted during the growth

of the colony. By means of a more or less regular series of cell division, combined with

sheath secretions, more ordered colonies may be produced.

A particular mode of reproduction, which may supplement binary fission,

distinguishes cyanobacteria in the order Chamaesiphonales and Pleurocapsales. In the

Chamaesiphonales exospores are budded off from the upper ends of cells. In the second

order, the principal mode of replication is by a series of successive binary fissions

converting a single mother cell into many minute daughter cells (baeocytes or

endospores).

Filamentous morphology is the result of repeated cell divisions occurring in a

single plane at right angles to the main axis of the filament. The multicellular structure

consisting of a chain of cells is called a trichome. The trichome may be straight or

coiled. Cell size and shape show great variability among the filamentous cyanobacteria.

Species in the order Oscillatoriales, with unseriated and unbranched trichomes, are

composed of essentially identical cells. The other orders with a filamentous

organization (orders Nostocales and Stigonematales) are characterized with trichomes

having a heterogeneous cellular composition. Vegetative cells may be differentiated

into heterocysts (having a thick wall and hyaline protoplast, capable of nitrogen

fixation) and akinetes (large thick-walled cells, containing reserve materials, enabling

survival under unfavourable conditions). In the order Stigonematales, the filaments are

often multiseriated, with genuine branching. Both heterocysts and akinetes are present.

The only means of reproduction in cyanobacteria is asexual. Filamentous forms

reproduce by trichome fragmentation, or by formation of special hormogonia.

Hormogonia are distinct reproductive segments of the trichomes.

They exhibit active gliding motion upon their liberation and gradually develop

into new trichomes. In contrast to eukaryotic microalgae, cyanobacteria do not possess

membrane-bound sub-cellular organelles; they have no discrete membrane-bound

nucleus; they possess a wall structure based upon a peptidoglycan layer; and they

contain 70 S rather than 80 S ribosomes (Fay and Van Baalen, 1987; Bryant, 1994).

17

The photosynthetic pigments of cyanobacteria are located in thylakoids that lie free in

the cytoplasm near the cell periphery. Cell colours vary from blue-green to violet-red.

The green of chlorophyll a is usually masked by carotenoids (e.g. beta-carotene)

and accessory pigments such as phycocyanin, allophycocyanin and phycoerythrin

(phycobiliproteins). The pigments are embodied in phycobilisomes, which are found in

rows on the outer surface of the thylakoids (Douglas, 1994). All cyanobacteria contain

chlorophyll a and phycocyanin. The basic features of photosynthesis in cyanobacteria

have been well described (Ormerod, 1992). Cyanobacteria are oxygenic phototrophs

possessing two kinds of reaction centres, PS I and PS II, in their photosynthetic

apparatus. With the accessory pigments mentioned above, they are able to use

effectively that region of the light spectrum between the absorption peaks of

chlorophyll a and the carotenoids.

The ability for continuous photosynthetic growth in the presence of oxygen,

together with having water as their electron donor for CO2 reduction, enables

cyanobacteria to colonize a wide range of ecological niches (Whitton, 1992).

Phycobiliprotein synthesis is particularly susceptible to environmental influences,

especially light quality. Chromatic adaptation is largely attributable to a change in the

ratio between phycocyanin and phycoerythrin in the phycobilisomes. Thus,

cyanobacteria are able to produce the accessory pigment needed to absorb light most

efficiently in the habitat in which they are present.

Cyanobacteria have a remarkable ability to store essential nutrients and

metabolites within their cytoplasm. Prominent cytoplasmic inclusions for this purpose

can be seen with the electron microscope (e.g. glycogen granules, lipid globules,

cyanophycin granules, polyphosphate bodies, carboxysomes) (Fay and Van Baalen,

1987). Reserve products are accumulated under conditions of an excess supply of

particular nutrients. For example, when the synthesis of nitrogenous cell constituents is

halted because of an absence of a usable nitrogen source, the primary products of

photosynthesis are channeled towards the synthesis and accumulation of glycogen and

lipids. Dinitrogen fixation is a fundamental metabolic process of cyanobacteria, giving

18

them the simplest nutritional requirements of all living organisms. By using the enzyme

nitrogenase, they convert N2 directly into ammonium (NH4) (a form through which

nitrogen enters the food chain) and by using solar energy to drive their metabolic and

biosynthetic machinery, only N2, CO2, water and mineral elements are needed for

growth in the light.

Nitrogen-fixing cyanobacteria are widespread among the filamentous,

heterocyst forming genera (e.g. Anabaena, Nostoc) (Stewart, 1973). However, there are

also several well documented examples of dinitrogen fixation among cyanobacteria not

forming heterocysts (e.g. Trichodesmium) (Carpenter et al., 1992). Under

predominantly nitrogen limited conditions, but when other nutrients are available,

nitrogen fixing cyanobacteria may be favored and gain growth and reproductive

success. Mass developments (often referred to as "blooms") of such species in limnic

(e.g. eutrophic lakes) and marine environments (e.g. the Baltic Sea) are common

phenomena world-wide. Many species of cyanobacteria possess gas vesicles. These are

cytoplasmic inclusions that enable buoyancy regulation and are gas-filled, cylindrical

structures. Their function is to give planktonic species an ecologically important

mechanism enabling them to adjust their vertical position in the water column (Walsby,

1987). To optimize their position, and thus to find a suitable niche for survival and

growth, cyanobacteria use different environmental stimuli (e.g. photic, gravitational,

chemical, thermal) as clues.

Gas vesicles become more abundant when light is reduced and the growth rate

slows down. Increases in the turgor pressure of cells, as a result of the accumulation of

photosynthate, cause a decrease in existing gas vesicles and therefore a reduction in

buoyancy. Cyanobacteria can, by such buoyancy regulation, poise themselves within

vertical gradients of physical and chemical factors. Other ecologically significant

mechanisms of movement shown by some cyanobacteria are photomovement by slime

secretion or surface undulations of cells (Häder, 1987; Paerl, 1988).

19

Cyanobacterial Taxonomy:

A characteristic cyanobacterial membrane lipid has been extracted from late

Archean sedimentary rocks dated to 2.65 Ga .The microfossils found at the Apex Chert

in Western Australia, believed to be cyanoprokaryotes, are even 800 million years

older. A minimum date for the evolution of heterocytic forms is set to 1.5 billion years

ago; to the period the oldest fossils interpreted as akinetes have been dated (Summons

et al., 1999).

For more than 150 years cyanobacteria were considered to be eukaryotic algae,

with botanists and phycologists placing them into the Cyanophyceae or cyanobacteria.

Thus, initial classifications followed the International Code of Botanical Nomenclature.

Traditional techniques for cyanobacteria identification and systematics have relied

essentially on the observation of morphological characteristics. The confirmation by

molecular methods that these denominated cyanobacteria, where in fact photosynthetic

bacteria and their transfer from Cyanophyceae to Cyanobacteria were of most

importance (Rasmussen and Svenning, 1998).

Evolutionary Markers:

The assessment of the phylogeny of organisms through gene sequence analysis

has increased dramatically since the advent of PCR and automated sequencing. A

number of genes have been used as evolutionary markers for inferring phylogenetic

relations and delineation of cyanobacterial taxonomy, being the 16S rDNA gene

analyzed most extensively (Nubel et al., 1997).

Phylogenetic studies using this marker helped to clarify the phylogenetic

relationships among cyanobacteria, revealed the structure and intraspecific diversity of

cyanobacterial communities and provide further evidence to the cyanobacterial origin

of chloroplasts and the existence of a divergent evolutionary pathway among bacteria

(Bergsland & Haselkorn, 1991). Bacterial rRNA genes are commonly organized in an

operon in the order 16S rRNA - 3S rRNA - 5S rRNA, each rRNA gene being separated

by an internal transcribed spacer (ITS) region. The amplification of the 16S - 23S

rRNA internal transcribed spacer (ITS) in cyanobacteria have shown to present

20

different sizes and also to be more variable in sequence even within closely related

taxonomic groups than the 16S rRNA (Iteman et al., 2002).

Other Markers

In some cases other sequences have also been used for phylogenetic inferences:

the non-coding intergenic spacer of the phycocyanin operon (PC-IGS); other DNA-

dependent RNA polymerase regions, rpoB and rpoD; the gene encoding a serine-type

of protease with a regulatory role in the differentiation process of heterocysts (hetR);

nitrogen fixation (nif) genes; carbon-fixation-associated gene (RubisCO spacer)

(rbcLX); and the subunit B protein of DNA gyrase (gyrB) (Robertson et al., 2001).

C-Phycocyanin gene:

Cyanobacteria are the only microorganisms to produce significant quantities of

Phycocyanin (PC) and its derivative Allophycocyanin. This distribution of PC in

aquatic microorganisms makes the study of PC gene sequence heterogeneity ideal for

the classification of freshwater cyanobacteria. The entire PC operon contains genes

coding for two bilin subunits and three linker polypeptides. The intergenic spacer (IGS)

between the two bilin subunit genes, designated b (cpcB) and a (cpcA), of the PC

operon was chosen as a potentially highly variable region of DNA sequence useful for

the identification of cyanobacteria to the strain level. C-phycocyanin is one of the major

photosynthetic biliproteins and also one of the important components in the electron

transfer of photosynthesis. It has some functions that are good for human health, such

as antioxidant, radical scavenging, anti-inflammatory and anti-cancer properties, and

also used in the food, biotechnology, and cosmetic industry because of their color,

florescent and antioxidant properties. So in recent years it has been drawn more and

more attention (Reddy et al., 2003, Sekar and Chandramohan, 2007).

The Phycobilisome components (phycobiliproteins) are responsible for the blue-

green pigmentation of most cyanobacteria. All phycobiliproteins are water-soluble and

therefore cannot exist within the membrane as do carotenoids, but aggregate forming

clusters that adhere to the membrane called phycobilisomes. Phycocyanin absorbs

orange and red light, particularly near 620 nm (depending on which specific type it is),

21

and emits fluorescence at about 650 nm (also depending on which type it is).

Allophycocyanin absorbs and emits at longer wavelengths than Phycocyanin. A major

advantage of using molecular techniques instead of microscopic methods is the ability

to enhance the taxonomic resolution from genus-level to genotype-level, which is not

possible to attain through other methods (Ouellette and Wilhelm, 2003).

The C-phycocyanin gene sequence was first reported (Pilot & Fox, 1984), in

which the oligonucleotide synthesis of alpha and beta subunits of cpc gene of a

freshwater cyanobacterum Agmenellum quadruplicatum was described and also

reported the cpcA and cpcB gene sequence in Spirulina platensis. The cpc gene

sequences of some Spirulina strains are used as a site to analyze their phylogenetic

relationships. There is no more detailed information about the regulatory sequence and

cpc operon (Yu et al., 2002).

Molecular characterization of ten marine cyanobacterial isolates belonging to

the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and

the 16S-23S internally transcribed spacer region. DNA sequences from the

phycocyanin operon discriminated ten genotypes, which corresponded to seven

morphotypes identified by traditional microscopic analysis. The cpcB coding region

revealed 17% nucleotide variation, while cpcA exhibited 29% variation across the

studied species. Phylogenetic analyses support the conclusion that the Phormidium and

Leptolyngbya genera are not monophyletic. The nucleotide variations were

heterogeneously distributed with no or minimal informative nucleotides. Our results

suggest that the discriminatory power of the phycocyanin region varies across the

cyanobacterial species and strains. The DNA sequence analysis of the 16S - 23S

internally transcribed spacer region also supports the polyphyletic nature of the studied

Oscillatorian cyanobacteria. This study demonstrated that morphologically very similar

strains might differ genotypically. Thus, molecular approaches comprising different

gene regions in combination with morphological criteria may provide better

taxonomical resolution of the order Oscillatoriales (Premanandh et al., 2006).

22

Cyanobacterial Features:

Cyanobacterial morphology ranges from simple unicellular, colonial and

multicellular filamentous forms. The vegetative cell wall is of gram-negative type but

in some species the peptidoglycan layer is considerably thicker than in other bacteria.

Minute pores are present in regular or scattered order in the wall of all cyanobacteria,

but the arrangement varies greatly. Many unicellular and filamentous cyanobacteria

possess an “envelope” outside the lipopolysaccharide (LPS) “outer membrane”, which

is called: sheath, glycocalyx, or capsule

The photosynthetic apparatus of cyanobacteria contains photosystem I and

photosystem II as found in higher plants with Chlorophyll a and specific accessory

pigments, including Allophycocyanin, Phycocyanin and Phycoerythrin. Cyanobacteria

possess the ability to use low light intensities effectively, since they are able to produce

the accessory pigments needed to adsorb light most efficiently in the habitat in which

they are present, providing them a great advantage for the colonization of their wide

range of ecological niches. Phycobiliprotein synthesis is particularly susceptible to

environmental influences, especially light quality. The chromatic adaptation is largely

attributable to a change in the ratio between Phycocyanin and Phycoerythrin in the

Phycobilisomes. The photosynthetic pigments are located in thylakoids that are free in

the cytoplasm near the cell periphery. Cell colours vary from blue green to violet-red

due to the chlorophyll a masking by the carotenoids and accessory pigments. The

pigments are involved in phycobilisomes, which are found in rows on the outer surface

of the thylakoids.

Cyanobacteria get their name from the bluish pigment phycocyanin, which they

use to capture light for photosynthesis. In some cyanobacteria, the color of light

influences the composition of phycobilisomes. In green light, the cells accumulate more

phycoerythrin, whereas in red light they produce more phycocyanin. Thus the bacteria

appear green in red light and red in green light. This process is known as

complementary chromatic adaptation.

23

Cyanobacteria are also able of storing essential nutrients and metabolites within

their cytoplasm. The occurrence of fimbriae (pili) is abundant in many cyanobacteria

with varying patterns. Some filamentous forms are also able of gliding (sliding), using

mucilaginous excretions as propellant. Some cyanobacteria have evolved specialized

cells for nitrogen fixation (heterocytes), survival in stressed conditions (akinetes), and

dispersion (hormogonia).

Cyanobacteria have many fascinating features such as buoyancy,

photosynthesis, fixation of atmospheric nitrogen and production of a wide variety of

bioactive compounds. In addition, cyanobacteria form symbiosis with several

eukaryotic hosts such as plants, fungi, and protists. Probably owing to their

physiological flexibility and long evolutionary history, cyanobacteria inhabit a large

variety of terrestrial and aquatic habitats from deserts to lakes as well as hot springs and

glaciers. Cyanobacteria form biofilms (microbial mats) on shores and on the surface of

stones, plants, and artificial objects. Cyanobacterial blooms are frequently toxic and

thus pose a health risk for humans and animals, cause an aesthetic problem, and reduce

the recreational value of water (Mur et al., 1999).

Limitations of phenotypic characters in cyanobacterial identification led to the

development of molecular techniques, including DNA base composition, DNA and

RNA hybridizations, gene sequences and PCR fingerprinting methods for

cyanobacteria taxonomy. However, it has been difficult to define taxonomic or

phylogenetic relationships within the cyanobacteria because of the scarcity of distinct,

consistent characters that support a taxonomic scheme. The problems of cyanobacterial

taxonomy are name changes of some strains, besides the misidentification issue of

others. Consequently, an ever changing classification system and a lack of a consensus

phylogeny are the proofs of the unresolved evolutionary relationships among

cyanobacteria.

A molecular approach to the systematic of cyanobacteria may be most fruitful

for inferring phylogenetic relationships. Macromolecules, such as nucleic acids and

proteins, are copies or translations of genetic information. The methods applied involve

24

direct studies of the relevant macromolecules by sequencing, or indirectly by

electrophoresis, hybridization, or immunological procedures (Wilmotte, 1994). Nucleic

acid technologies, especially the polymerase chain reaction (PCR), have advanced to

the point that it is feasible to amplify and sequence genes and other conserved regions

from a single cell. However, the molecular results obtained have integrated with other

characteristics as the base for polyphasic taxonomy (Vandamme et al., 1996).

Rapid developments in genomic and other molecular research technologies and

developments in information technologies have combined to produce a tremendous

amount of information related to molecular biology by the application of

bioinformatics. Common activities in bioinformatics include mapping, analyzing DNA

and protein sequences, then for aligning different DNA and protein sequences to

compare, create and to view 3-D models of protein structures. Major research efforts in

the field include sequence alignment, gene finding, genome assembly, drug design,

drug discovery, protein structure alignment, protein structure prediction, prediction of

gene expression and protein-protein interactions, genome-wide association studies and

the modeling of evolution. Software tools were used for sequence analysis and structure

prediction includes BLAST, protein structure viewing, NCBI, EMBL etc (Mount,

2002).

Protein has three main structures: primary structure which is essentially the

linear amino acid sequence and usually represented by a one letter notation. Alpha

helices, beta sheets, and loops are formed when the sequences of primary structures

tend to arrange themselves into regular conformations; these units are known as

secondary structure. Protein folding is the process that results in a compact structure in

which secondary structure elements are packed against each other in a stable

configuration. This three-dimensional structure of the protein is known as the protein

tertiary structure. However, loops usually serve as connection points between alpha-

helices and beta-sheets, they do not have uniform patterns like alpha-helices and beta-

sheets and they could be any other part of the protein structure rather than helices or

strands (Kendrew et al., 1960).

25

In Bioinformatics, BLAST is an algorithm for comparing primary biological

sequence information, such as the amino-acid sequences of different proteins or the

nucleotides of DNA sequences. A blast search enables a researcher to compare a query

sequence with a library or database of sequences, and identify library sequences that

resemble the query sequence above a certain threshold. Molecular phylogenetics is the

analysis of hereditary molecular differences, mainly in DNA sequences, to gain

information on an organism's evolutionary relationships. The result of a molecular

phylogenetic analysis is expressed in a phylogenetic tree. Molecular phylogenetics is

one aspect of molecular systematics, a broader term that also includes the use of

molecular data in taxonomy and biogeography.

Molecular modelling is used to model the molecules. Both theoretical and

computational techniques are used in molecular modelling. Molecular modelling

methods are used to investigate the structure, dynamics and thermodynamics of

inorganic, biological, and polymeric systems. The types of biological activity that have

been investigated using molecular modelling include protein folding, enzyme catalysis,

protein stability, conformational changes associated with biomolecular function, and

molecular recognition of proteins, DNA, and membrane complexes. Cyanobacteria, our

model organisms, are regarded as an origin of producing phycocyanin pigments;

therefore they are considered as excellent candidates to study molecular techniques to

resolve many of the issues and problems in cyanobacterial taxonomy. Thus, the work is

mainly focused on the following objectives.

Molecular Modeling of C-Phycocyanin:

Molecular modeling is a powerful methodology for analyzing the three

dimensional structure of biological macromolecules. There are many ways in which

molecular modeling methods have been used to address problems in structural biology.

It is not widely appreciated that modeling methods are often an integral component of

structure determination by NMR spectroscopy and X-ray crystallography. The overall

aim of modeling methods will often be to try to relate biological activity to structure.

An important step towards this goal is to be able to compute the potential energy of the

molecule as a function of the positions of the constituent atoms. The common feature of

26

molecular modeling techniques is the atomistic level description of the molecular

systems; the lowest level of information is individual atoms or a small group of atoms.

This is in contrast to quantum chemistry which is also known as electronic structure

calculations, where electrons are considered explicitly. The benefit of molecular

modeling is that it reduces the complexity of the system, allowing many more atoms to

be considered during simulations (Foster, 2002).

Structural modeling of the gene:

Alpha-helix is spiral turns of amino acids while a beta-sheet is flat segments or

strands of amino acids formed usually by a series of hydrogen bonds. As the

polypeptide chain coils in, the CO and NH groups of residues form hydrogen bonds

which stabilize the helix. Most of the residues in a helix are bonded in this way, making

it somewhat a rigid unit of structure with a little free space in its core. A helix and can

have 4 - 50 residues and makes a whole turn every 3.6 residues.

Beta-strands are the most regular form of extended polypeptide chain in protein

structures. Like alpha-helices, beta-sheets are stabilized by hydrogen bonds between

CO and NH groups, but they are distantly separated along the chain. Because of the

geometry of the peptide backbone, the amino acid side chains of beta-strands alternate

on either side of the sheet.

Loops usually serve as connection points between alpha-helices and beta-sheets,

they do not have even patterns like alpha-helices and beta-sheets and they could be any

other part of the protein structure. They are recognized as random coil and not

classified as protein secondary structure. When the polypeptide chain makes very sharp

changes in direction using as few as four residues by means of hydrogen bond, it forms

turns. These secondary structures commonly contain proline or glycine or both residues

(Hutchinson and Thornton, 1994).

Tertiary Structure:

The three-dimensional structure of the protein, which is formed from the

secondary structures as subunits elements, is known as the protein’s tertiary structure.

27

Protein folding is the process that results in a compact structure in which secondary

structure elements are packed against each other in a stable configuration.

Hydrogen bonds, van der Waals forces, and oppositely charged amino acid side-

chains are other interactions that help to stabilize the fold. Folds are considered as sets

of connected secondary structure elements, so they are known as topologies. Longer

polypeptide chains that are usually clearly distinguished by a naked eye as self-

contained units of structure, and have distinct hydrophobic cores, are known as

domains. Homology modeling can produce high-quality structural models when the

target and template are closely related, which has inspired the formation of a structural

genomics consortium dedicated to the production of representative experimental

structures for all classes of protein folds (Dill, 1990).

The Crystal structure of the light-harvesting protein phycocyanin from the

Cyanobacterium Cyanidium caldarium with novel crystal packing has been solved at

1.65-Å resolution. The structure has been refined to an R value of 18.3% with excellent

backbone and side-chain stereochemical parameters. In crystals of phycocyanin used in

this study, the hexamers are offset rather than aligned as in other phycocyanins that

have been crystallized to date. Analysis of this crystal’s unique packing leads to a

proposal for phycobilisome assembly in vivo and for a more prominent role for

chromophore b-155. This new role assigned to chromophore b-155 in phycocyanin

sheds light on the numerical relationships among and function of external

chromophores found in phycoerythrin and phycoerythrocyanin (Stec et al., 1999).

Impact of Molecular Methods on Cyanobacterial Taxonomy:

Molecular methods have had a great impact on every level of cyanobacterial

taxonomy. Prochlorophytes, which were considered a special group of oxyphototroph

prokaryotes for the lack of phycobiliproteins and the presence of chlorophyll b, have

been shown to be polyphyletic according to the 16S rDNA and scattered throughout the

cyanobacterial lineage. But contradictory to the general concept, a phycobiliprotein

gene, similar to that of the marine Synechococcus, has been detected in

Prochlorococcus marinus CCMP 1375 strain (Hess et al., 1996).

28

Another important task solved by 16S rDNA sequencing was the origin of

plastids that were proven to have descended from cyanobacteria. This was later

confirmed also by tufA gene sequence. Plastids form a monophyletic group within

cyanobacterial lineage, but no strong candidate for the sister taxon to plastids exists at

present (Delwiche et al., 1995 and Turner, 1997). Heterocytic cyanobacteria were

confirmed and the orders Chroococcales and Oscillatoriales were shown to be

polyphyletic. The baeocyte-forming order Pleurocapsales seemed to be monophyletic at

first, but it turned out to be polyphyletic too, with Chroococcidiopsis thermalis being a

close relative of heterocytous cyanobacteria. The same may be stated for heterocytous

order Stigonematales - it has been shown recently, on the basis of both 16S rDNA and

nif genes, that it is polyphyletic as well (Giovannoni et al., 1988 and Zehr et al., 1997).

The situation on the generic and subgeneric level is even more confused. The

ecological studies concerning diversity of cyanoprokaryotes in various habitats are

many, but it is often not clear what is meant under the taxonomic designations assigned

to the studied organisms.

Many misidentified strains in culture collections are available and no

morphological data for a bulk of available cyanobacterial sequences. However, some

work has been done on the cytomorphological and polyphasic characterization of

chroococcalian "Synechocystis", "Synechococcus" and a few other unicellular strains as

well as of hetercytous Aphanizomenon/Anabaena/Nostoc strains. The studies on

filamentous "Phormidium" and "Oscillatoria" genera are few. Although there is often

no correlation between morphological and molecular traits, especially for taxa with

very simple morphology, some morphologically well defined genera were shown to be

monophyletic. These include Microcystis, Planktothrix or marine Trichodesmium

species. Notwithstanding the unreliability of traditional morphological criteria, some

cytomorphological and ultra structural characters were found to correlate well with

molecular data. This concerns e.g. the cell division type and especially the thylakoid

arrangement, which seems to have substantial taxonomic value. Some other traits, such

as perforation-patterns in the cell wall of cyanobacteria may prove useful on certain

taxonomic levels (Pffeifer and Palinska, 2002 and Palinska and Krumbein, 2000).

29

Relationship between Genotypic and Phenotypic Characters

Cyanobacteria are morphologically diverse in comparison to the rest of bacteria.

Nevertheless, only a quite restricted number of morphotypes can be recognized.

Molecular methods enabled revelation of cryptic genetic, physiological and ecological

diversity among them. Rather broadly defined species Phormidium retzii was shown to

be quite variable on molecular level. Although there were some morphological

distinctions between individual populations, they did not correlate with genetic

similarity. Strains morphologically corresponding to the genus Geitlerinema sp.

generated very different restriction patterns (Casamatta et al., 2003).

The studies on cyanobacterial communities of both geothermal springs in the

Philippines and Lake Fryxell in Antarctica revealed significantly higher degree of

diversity by molecular methods than by light microscopy. Genetically distinct toxic

Microcystis and Planktothrix populations were found in different parts of the same

lake. However, it is also possible that the strains, closely related on molecular level,

show substantial phenotypic variability, as shown for several Merismopedia isolates.

Co-existing Prochlorococcus ecotypes were detected, almost identical genetically, but

possessing very different light-dependent physiologies (Taton et al., 2003, Palinska et

al., 1996, Moore et al., 1998).

Interesting topic is the correlation between phylogenetic relatedness and

ecological or ecophysiological characters. Unicellular cyanobacteria from hypersaline

habitats in various geographical regions form a well defined cluster on the basis of their

16S rDNA that was denominated Halothece cluster. Moderately halophilic, benthic

strains with very thin trichomes also form a distinct cluster in the phylogenetic tree and

were assigned to a new genus Halomicronema (Garcia-Pichel et al., 1998).

An interesting fact that the only available 16S rDNA sequences of

cyanobacteria from stone surfaces of buildings group with those of desert strains from

distant geographic region, while sequence homology with the strains from other

30

habitats is quite low. The sequence similarity somehow reflects the capacity to survive

in such extreme environments (Crispim and Gaylarde, 2005)

The analysis of the hli gene family, which has to do with adaptation to high

light intensities, revealed that some groups of this gene family are specific either for

marine or freshwater cyanobacteria .A nice example of ecological divergence of

morphologically and phylogenetically closely related, but distinct genera

Trichodesmium and Hydrocoleum (Blennothrix) was documented. The first one is

planktonic, the latter is the most common mat-forming cyanobacterium in tropical

oceans (Bhaya et al., 2002 and Abed et al., 2006).

Biotechnological Applications of cyanobacteria:

Cyanobacteria constitute a resource for several applications such as aquaculture,

food, feed, fuel, fertilizer, medicine, industry and even in combating pollution (WHO

1999).

Cyanobacterial bioactive compounds Cyanobacteria have been identified as a

new and rich source of bioactive compounds (Abarzua et al. 1999; Shimizu 2003;

Bhadury et al. 2004; Dahms et al. 2006). Isolated compounds belong to groups of

polyketides, amides, alkaloids, fatty acids, indoles and lipopeptides (Abarzua et al.

1999; Burja et al. 2001).

The literature review showed that to date up to 19 cyanobacterial strains

produce more than 20 different bioactive compounds. Most of the bioactive compounds

isolated from cyanobacteria tend to be lipopeptides, i.e. they consist of an amino acid

fragment linked to a fatty acid portion. The range of biological activity of secondary

metabolites isolated from cyanobacteria includes antibacterial, antifungal, antialgal,

antiprotozoan, and antiviral activities.

Only few cyanobacteria produce bioactive compounds that show a broad

spectrum of biological activities. For example, the cyanobacterium Phormidium sp. has

been reported to inhibit growth of different Grampositive and Gram-negative bacterial

31

strains, yeasts, and fungi (Bloor and England 1989). Another example is Lyngbya

majuscula (Burja et al. 2001) that produces numerous chemicals including nitrogen-

containing compounds, polyketides, lipopeptides, cyclic peptides and many others

(Shimizu 2003).

The biological activity of these compounds is also diverse and includes protein

kinase C activators and tumour promoters, inhibitors of microtubulin assembly,

antimicrobial and antifungal compounds and sodium-channel blockers. Secondary

metabolites with antibacterial activity are widely produced by cyanobacteria (Dahms et

al. 2006). These compounds are effective against Gram positive and ⁄ or Gram-negative

bacteria. Both toxic and nontoxic strains of cyanobacteria are producers of antibacterial

compounds that are distinct from cyanotoxins. Antifungal compounds include

fisherellin A, hapalindole, carazostatin, phytoalexin, tolytoxin, scytophycin,

toyocamycin, tjipanazole, nostocyclamide and nostodione produced by cyanobacteria

belonging to Stigonematales, Nostocales and Oscillatoriales. Additionally,

cyanobacteria produce a broad spectrum of antialgal compounds, which may be used to

control algal blooms.

Cyanobacteria probably use these compounds in order to out-compete other

microorganisms. Antialgal compounds produced by cyanobacteria inhibit growth of

algae, their photosynthesis, respiration, carbon uptake, enzymatic activity and induce

oxidative stress (Dahms et al. 2006). In contrast to the large amount of antibacterial and

antialgal compounds isolated from cyanobacteria there are only a few compounds that

show antiviral properties, although 2–10% of extracts of different cyanobacterial

species have been shown to have antiviral activity (Cohen 1999). These include

acetylated sulfoglyco-lipids from Oscillatoria raoi (Reshef et al. 1997) and spirulan

from Spirulina platensis (Hayashi et al. 1991). The compounds isolated from Lyngbya

lagerhaimanii and Phormidium tenue has been shown to have anti-HIV activity

(Rajeev and Xu 2004). Gamma linolenic acid (GLA) found rich in S. platensis and

Arthrospira sp. is medically important since it is converted in the human body into

arachidionic acid and then into prostaglandin E2. This compound has a lowering action

on blood pressure and plays an important role in lipid metabolism. Some of the marine

32

cyanobacteria constitute potential sources for large-scale production of vitamins, such

as vitamins B and E (Plavsic et al. 2004).

Piechula et al. (2001) demonstrated that some cyanobacteria can produce

thermostable enzymes. Out of 21 endonucleases from Phormidium, four enzymes that

catalyse the hydrolysis of DNA and RNA were active in a wide range of temperatures

from 15 to 60oC. Phenyltransferases enzymes catalysing the consecutive condensation

of homoallylic diphosphate of isopentenyl diphosphates at temperatures above 60oC

have been isolated from the thermophilic cyanobacterium, Synechococcus elongates

(Ohto et al. 1999). Thermostable polyphosphate kinase from the thermophilic

cyanobacterium Thermosynechococcus elongatus BP-1 was successfully employed in

an ATP regeneration system that could be used at high temperatures for the effective

production of d-amino acid dipeptides (Sato et al. 2007). These enzymes and other heat

stable bioactive compounds are of great interest in biotechnology. So far none of the

isolated compounds have demonstrated anti-larval activity and inhibition of settlement

of larvae and algal spores (Dobretsov et al. 2006).

Cyanobacteria have been used to synthesize isotopically labelled compounds

such as sugars, lipids and amino acids, which are nowadays commercially available

(Patterson 1996). This is achieved by growing the cyanobacetria in photobioreactors

and allowing them to photosynthetically transform simple labelled compounds such as 14CO2,

13CO2, 33H2O and 15NO3 into complex organics cyanobacteria as a healthy food

source Strains of Spirulina, Anabaena and Nostoc are consumed as human food in

many countries including Chile, Mexico, Peru and Philippines. Arthrospira platensis

(misidentified as S. platensis) is grown in large scale using either outdoor ponds or

sophisticated bioreactors but marketed in the form of powder, flakes, tablets and

capsules. It is used as a food supplement because of its richness in nutrients and

digestibility. It contains more than 60% proteins and is rich in beta-carotene, thiamine

and riboflavin and is considered to be one of the richest sources of vitamin B12. Nostoc

commune is rich in fibres and proteins and can play an important physiological and

nutritional role in the human diet. Aphanizomenon sp. is collected from natural blooms

in the Lake Klamath (Oregon, USA) to be used as healthy food. Marine nitrogen-fixing

33

cyanobacteria have also been tested to feed fishes in aquacultures. The Tilapia fish

showed high growth rates when fed with marine cyanobacteria in indoor and outdoor

cultures (Mitsui et al. 1983). Phormidium valderianum has been used in India to serve

as a complete aquaculture feed source based on its nutritional value and nontoxic

nature. In view of the cyanobacterial significance as a food source, very little research

has been performed and published about this.

Cyanobacterial alternative energy sources Cyanobacteria have been used to

produce hydrogen gas that constitutes an alternative future energy source to the limited

fossil fuel resources (Dutta et al. 2005). The advantages of using biological hydrogen

as a fuel are its eco-friendly nature, efficiency, renewability and the absence of carbon

dioxide emission during its production and utilization (Lindbald 1999).

Cyanobacteria produce hydrogen either as a byproduct of nitrogen fixation,

when nitrogenase-containing heterocystous cyanobacteria are grown under nitrogen

limiting conditions, or by the reversible activity of hydrogenases enzymes.

Heterocystous cyanobacteria are thus more efficient in hydrogen production than

nonheterocystous types (Pinzon-Gamez et al. 2005). More than 14 cyanobacterial

genera including Anabaena, Calothrix, Oscillatoria, Cyanothece, Nostoc,

Synechococcus, Microcystis, Gloeobacter, Aphanocapsa, Chroococcidiopsis and

Microcoleus are known for their ability to produce hydrogen gas under various culture

conditions (Lambert and Smith 1977; Sveshnikov et al. 1997; Masukawa et al. 2001).

Anabaena spp. is able to produce significant amounts of hydrogen. Nitrogen- starved

Anabaena cylindrica cells produce the highest amount of hydrogen (30 ml of H2 per lit

culture per hour) (Jefferies et al. 1978). Gloeocapsa alpicola showed increase in

hydrogen production under sulfur starvation (Antal and Lindblad 2005) whereas S.

platensis could produce hydrogen under complete dark and anoxia (Aoyama et al.

1997).

Large-scale hydrogen production by Spirulina and Anabaena spp. has been tried

using different types of bioreactors that included vertical column reactor, tubular type

and flat panel photobioreactor (Dutta et al. 2005). These reactors were designed to

34

make use of solar light for illumination, to maximize the area for incident light (high

surface to volume ratio) and to allow sterilization and hydrogen collection with

convenience and ease. Nevertheless, the reactors are subjected to continuous

modification in order to increase their productivity and to decrease costs of

maintenance and production. So far, these modifications succeeded in bringing down

the cost of biologically produced hydrogen to $25 per m-3 compared to $170 per m-3 for

the hydrogen produced by splitting of water (Block and Melody 1992).

Increased production of hydrogen was also achieved by genetically modifying

the nitrogenase enzyme in hydrogen-producing strains. The ongoing research on

hydrogen production by cyanobacteria focuses on finding new strains with higher

potential to produce hydrogen, optimizing the mass production of hydrogen in

bioreactors and modifying the physiology and the genetic system of H2-producing

cyanobacteria to ensure maximum production of hydrogen Cyanobacteria as

biofertilizers. Heterocystous cyanobacteria and several nonheterocystous cyanobacteria

are known for their ability to fix atmospheric nitrogen (Capone et al. 2005). The

fertility of many tropical rice field soils has been mainly attributed to the activity of

nitrogen-fixing cyanobacteria.

Species of cyanobacteria, namely strains of Spirulina (Arthrospira) platensis,

Nostoc commune and Aphanizomenon flos-aquae, are currently used as food and

supplements due to their amounts of proteins (e.g. up to 70% of Spirulina dry weight),

lipids, chlorophyll, carotenoids, vitamins, minerals, unique pigments and also due to

their potential prebiotic effects. Spirulina is also used as an aquaculture and animal feed

source. As a crop, Spirulina has the advantage of growing well in saline ponds in arid

environments, places that few other crops could support (Pulz and Gross, 2004).

Several fine chemicals such as pigments (carotenoids and phycobiliproteins),

vitamins (B-complex group and vitamin E) and enzymes with varied applications from

cyanobacteria are being commercialized. The pigments are used as natural food

colourants, food additives, and cosmetics and as enhancers of ornamental fish colour.

Isotopically labelled (14C, 15C, 3H and 15N) cyanobacterial metabolites such as sugars,

35

lipids and aminoacids are commercially available. Several endonucleases produced

from cyanobacteria are being marketed, e.g. AcyI (Anabaena cylindrica).

Cyanobacteria produce numerous bioactive compounds. Some secondary

metabolites are potentially of therapeutical importance, such as antiviral compounds,

immunomodulators, inhibitors, or cytostatics. N2-fixing cyanobacteria are used as

biofertilizers, for instance in the rice paddies where nitrogen to support the rice plants

comes from fixation by free-living and symbiotic cyanobacteria, besides being also

used in other tropical and subtropical agriculture.

Reports concerning the advantageous effects of cyanobacteria inoculation on

other crops such as barley, oats, tomato, radish, cotton, sugarcane, maize, chili and

lettuce have been described (Thajuddin and Subramanian, 2005). It has been reported

that the marine cyanobacterium, Oscillatoria formosa NTDM02 was efficiently

decolorized textile dyes and laboratory dye (MubarakAli et al., 2010).

36

MATERIALS AND METHODS

Study area

Fig. 3 Map showing the sample collection site at Mandapam, Kurusadai Island and

Rameshwaram, India

Sample collection

The samples were collected from rock surfaces in Kurusadai Island, during the

month of December 2009 of monsoon season and the diversity of Cyanobacteria were

observed initially by the use of light microscope (Optika, California). Diatom samples

were collected from stagnant water in Palkalaiperur, during the month of December to

February (2010) of monsoon season and the diversity of diatoms were observed by the

use of light microscope (Optika, California).

37

Isolation of diatoms

The diatoms were isolated by using enriched sea water medium F/2 (Guillard

and Ryther, 1962) by serial dilution, capillary or micropipette method and streak plate

method. The cultures were grown under the fluorescent light of 1500 lux, 12:12

light:dark (L:D) condition at 25±2ºC. After incubation the cultures were examined at

regular intervals under light microscope.

Isolation of Cyanobacteria:

The Cyanobacteria were isolated by using enriched sea water medium MN and

ASN-III (Rippika, 1988) by serial dilution and streak plate method. The cultures were

grown under the fluorescent light of 1500 lux, 12:12 light: dark (L: D) condition at

25±2ºC. After incubation the cultures were examined at regular intervals under light

microscope.

Morphological characterization of diatom and marine Cyanobacteria

The isolated diatom and marine Cyanobacteria were examined carefully by using

light microscope and microphotograph was taken (Optika Photomicroscope Unit) to

determine the morphology. The identification of Cyanobacteria in marine water

samples was achieved using standard monograph (Geitler, 1932; Desikachary, 1959).

The diatom strains were genus was identified based on frustules morphology by using

the atlas of diatoms (Desikachary, 1989). The isolated cyanobacterial strains were

maintained as axenic cultures in the germplasm of Department of Microbiology,

Bharathidasan University.

Molecular characterization of diatom

DNA extraction from diatom (Grachev et al., 2006)

A grown culture was centrifuged to obtain about 50µl of packed biomass. Cells

were suspended in 100µl of a lysis buffer (100 mM Tris-HCl, pH 8.0,1.4 M NaCl, 2%

CTAB, 20mM EDTA), homogenized with a pestle at 60ºC for 10 minutes, combined

with 150µl of the lysis buffer, and incubated at 60ºC for 30 minutes with occasional

shaking. Cell debris was removed by centrifugation (3 minutes), and the supernatant

was transferred into another tube. The pellet was washed with 100µl of the lysis buffer,

38

and the supernatant was added to the first one. The pooled supernatant was combined

with 7.5µl of RNase, incubated at 60ºC for 30 minutes, combined with 3µl of

proteinase K, incubated again at 60ºC for 30 minutes, and combined with 1ml of 1%

CTAB (aqueous solution) at room temperature.

The resulting suspension was stirred and frozen-thawed. The pellet was

collected by centrifugation (15 minutes) and washed twice with 300µl of 1% cetavlon

(aqueous solution). The supernatant was carefully removed by overturning the tube on

filter paper. The pellet was extracted twice with 400µl of ethanol at 60ºC for 10

minutes. The supernatant (ethanol solution of the CTAB salt of DNA) was collected by

centrifugation (5 minutes) and combined with 80µl of 3M sodium acetate and kept at -

20ºC overnight. The precipitate (DNA sodium salt) was collected by centrifugation,

washed with ethanol, and air-dried. DNA was dissolved in 50µl of a PCR buffer and

stored at -20°C.

The extracted DNA were estimated directly from ethidium bromide

fluorescence in agarose gel (0.8% in 1X TBE buffer) against a standard marker,

(Finzymes, Finland) by using a gel documentation with associated software.

Oligonucleotide primers and PCR conditions:

All PCR reactions were carried out in a 40µl volume containing (i)20µl of 2x

prime Taq premix ( GENET BIO, Korea) which contains 1unit Taq DNA Polymerase,

20mM Tris-Hcl, 80mM KCl, 4mM MgCl2, enzyme stabilizer, sediment, loading dye,

pH 9.0, 0.5mM of each dATP, dCTP, dGTP, dTTP. (ii) 1µl of upstream primer (10

pmoles/µl) (iii) 1µl of downstream primer (10pmoles/µl) (iv) 1µl (50ng) of extracted

DNA from diatom (v) 7 µl of sterilized distilled water.

PCR amplification of the small subunit of 18S rRNA gene (SSU rDNA) was

done in a thermal cycler gradient (Applied biosystems, Germany) with the primer pairs

1F and 1528R (Medlin et al. 1988). Conditions for PCR were as follows: initial

denaturation at 94ºC for 5 mins, followed by 36 cycles of 94ºC for 2 min, 54ºC for 4

min, and 72ºC for 2 min, and a final extension at 72ºC for 10 min.

39

18S rDNA gene amplification:

A fragment of 18S rDNA gene was amplified as mentioned above (Ulf Karsten

et al., 2006). After the reaction was completed, 10µl of amplified DNA was separated

on 1.2% agarose, stained with ehidium bromide and recorded using a CCD camera in

UVP Imager (UVP Bioimaging, England). A ready to use DNA size standard samples

(Finzymes, Finland) was added in the gel to determine the molecular weight of the

marker.

Primer Sequence 5´→3’ Author

1F AAC CTG GTT GAT CCT GCC AGT

Medlin et al.,1988

1528 R TGA TCC TTC TGC AGG TTC ACC

TAC

Molecular characterization of marine Cyanobacteria:

DNA extraction:

The extraction of genomic DNA from cyanobacterial isolates were carried out

(Smoker and Barnum 1988). Briefly, 1ml of overnight grown cultures was centrifuged

at 10,000 rpm for 5 minutes and the pellet was collected, then collected pellet was

washed using STE buffer (NaCl – 50mM; Tris-HCl - 50mM; EDTA - 5mM) and

suspended in 500μl of STE buffer. Followed by 20μl of lysozyme (10mg/ml) was

added and incubated in water bath at 55°C for 30 minutes. After incubation, 10μl of

proteinase K (10mg/ml) and 20μl of 10% SDS was added and incubated in water bath

at 55°C for 30 minutes. The mixture was then cooled in ice and extracted with equal

volume of phenol: chloroform: Isoamyl alcohol mixture (25:24:1) and centrifuged at

12,000 rpm for 10 minutes. After centrifugation, the supernatant was separated with

care and equal volume of 4M Ammonium acetate and two volumes of Isopropanol

were added. Mixture was again centrifuged at 14,000 rpm for 15 minutes and the

supernatant was decanted and the pellet was washed with 70% ethanol, dried. Finally,

the DNA pellet was dissolved in 100μl of TE buffer and stored at 20°C until further

use. The loading dye was added to each DNA samples and the samples were transferred

to separate wells in 0.8% agarose gel and after loading, the electrophoresis was carried

40

out using 1X TAE buffer at a constant supply of 100 V for 30 minutes. The bands were

documented under transilluminator.

16S rDNA gene amplification:

PCR amplification was performed for the respective two samples of purified

DNA using CYA 106 (5’ - CGG ACG GGT GAG TAA CGC GTGT- 3’) and CYA 781

(5’ - GAC TAC TGG GGT ATC TAA TCC CA T - 3’) primers. The polymerase chain

reactions conditions include initial denaturation of template DNA was achieved at 94oC

for 3 minutes. Further denaturation was carried out at 94oC for 45 sec; annealing at

56oC for 30 sec, elongation at 72oC for 1min and a final elongation at 72oC for 7 min

for 30 cycles. Amplified products were isolated by electrophoresis on 1.2% agarose gel

using 1X TAE buffer at a constant supply of 100 V for 30 minutes. Again, resolved

bands were documented under Uv transilluminator.

C- Phycocyanin (PC) Gene amplification:

PCR amplification of C-Phycocyanin was performed for the respective two

samples of purified DNA using PCβ F (GGC TGC TTG TTT ACG CGA CA) and

PCαR (CCA GTA CCA CCA GCA ACT AA) primers (Neilan, 2002). The polymerase

chain reactions conditions include initial denaturation of template DNA was achieved

at 94oC for 5 minutes. Further denaturation was carried out at 940C for 2 mins;

annealing at 550 C for 1min, elongation at 720C for 3 mins and a final elongation at

720C for 8 min for 40 cycles. The reaction mixture of amplification of PC-IGS region

contains 40µl.

Sequencing and Construction of phylogeny:

Sequencing was done with amplified samples with respective forward and

reverse primers and sequences. The query sequence was uploaded in alignment box and

the query was submitted to BLAST analysis for pair-wise sequence alignment. The

evolutionary distances were computed using the Maximum Composite Likelihood

method (Tamura et al., 2004) and are in the units of the number of base substitutions

per site and it was computed in MEGA 5 software.

41

Molecular modeling of C-Phycocyanin gene:

Prediction of primary, secondary and tertiary structure of C-Phycocyanin and

determination of conserved domains of the C-phycocyanin sequences were analyzed

using online and offline softwares for molecular modeling of the protein.

Tertiary Structure:

Retrieval of target protein sequences from NCBI in Fasta format and save as in

notepad. The template structure was searched through Blastp algorithm against PDB

database. Target sequence alignment and template protein structure were derived for

Structure prediction using spdV software. The modelled structures were subjected to fit

the models and to optimize the stereochemistry atoms of molecules. The final modelled

structure to predict 3D structure profiles and it was visualized.

Conserved Domain determination (CDD):

CDD is a protein annotation resource that consists of a collection of well-

annotated multiple sequence alignment models for ancient domains and full-length

proteins. These are available as position-specific score matrices (PSSMs) for fast

identification of conserved domains in protein sequences via RPS-BLAST. CDD

content includes NCBI-curated domains, which use 3D-structure information to

explicitly define domain boundaries and provide insights into sequence / structure /

function relationships, as well as domain models imported from a number of external

source databases (Pfam, SMART, COG, PRK, TIGRFAM)

Identify the putative function of a protein sequence, identify the amino acids in

a protein sequences that are putatively involved in functions such as binding or

catalysis, as mapped from conserved domain annotations to the query sequences. View

a query protein sequence embedded within the multiple sequence alignment of a

domain model, interactively views the 3D structure of a conserved domain, Find other

proteins with similar domain architecture and interactively view the phylogenetic

sequence tree for a conserved domain model of interest with or without a query

sequence embedded (Marchler-Bauer et al. 2011).

42

RESULTS AND DISCUSSION

Totally, 50 cyanobacterial species and 38 diatom species were recorded. They

were identified taxonomically with standard monographs. The schematic representation

of collection and marine cyanobacterial diversity were shown (Fig. 4). There are five

family from cyanobacterial diversity such as Chroococcaceae (7), Nostacaceae (4),

Oscillatoriaceae (37), Pseudanabaenaceae (1) and Mastigocladaceae (1) respectively,

where as in diatom were found to be in thirteen families such as Achanthaceae (2),

Bacillariaceae (6), Catenulaceae (1), Cymbellaceae (2) Desmidiaceae (1),

Fragilariaceae (4), Gomphonemataceae (1), Mastoglioaceae (2), Naviculaceae (8),

Pinnulariaceae (4), Pleurosigmataceae (4), Stauroneidaceae (1), Stephanodiscaceae

(2) (Table 1a &b).

The morphological evidence of the cyanobacteria and diatom were shown (Fig.

5a & b and 6). Cyanobacterial taxonomy follows the International Code of

Nomenclature of Bacteria (Stainer et al., 1978) and published a taxonomy of the

cyanobacteria that was based on physiological, morphological and some genetic criteria

(Rippka et al., 1979). The revision of the status of the nomenclature of Cyanobacteria

under the Bacteriological Code, only 13 names of cyanobacterial species have been

proposed and since the corresponding genera name had not been validly published

according to the International Code of Nomenclature of Bacteria. Although at the

present there has been an attempt to classify cyanobacteria using both Botanical and

Bacteriological Code of Nomenclature (Tindall, 2006).

The traditional observation of the morphological characters by light microscopy

requires considerable expertise to identify species. Moreover, some phenotypic

features, such as gas vacuoles or akinetes etc. may vary with environmental or growth

conditions and even their features may lost during repeated subcultures (Rudi et al.,

1997). Although cyanobacterial identification at genus level is easier to be reached,

particularly where morphological characteristics are significantly different from other

genera, there are some cases where this separation is not clear, namely the delineation

of Microcystis from Synechocystis, Anabaena from Nostoc, as well as Anabaena from

Aphanizomenon and Nodularia.

Figure 4 Schematic representation of cyanobacterial sample collection and morphological divergence of marine Cyanobacteria

43

Table 1a. Microbial diversity of Cyanobacteria Chroococcaceae

1. Chroococcus minor Kutzing

2. Chroococcus sp. Kutzing

3. Gleocapsa sp. Kutzing

4. Synechococcus elongatus Nageli

5. Synechocystis sp. Sauvageau

6. Microcystis marginata Menedhini

7. Merismopedia convvoluta Brebisson

Nostacaceae

8. Nodularia spumigena Mertens

9. Nodularia sp. Mertens

10. Anabaena spiroides Klebahn

11. Anabaena cylindrica Lemmermann

Oscillatoriaceae

12. Phormidium tenue Gomant

13. Phormidium valderianum Gomant

14. Phormidium chlorinum Kutzing

15. Phormidium tenue Meneghini

16. Phormidium fragile Gomant

17. Phormidium subtilissma Kutzing

18. Phormidium angusitissimum

19. Phormidium corium Agardh

20. Phormidium tenue Gomant

21. Oscillatoria subrevis Schmidle

22. Phormidium willei Gardner

23. Oscillatoria formosa Vincent

24. Oscillatoria boryana Bory

25. Oscillatoria subrevis Schmidle

26. Oscillatoria princeps Rabenhorst

27. Oscillatoria tenius Agardh

28. Oscillatoria amphibium Anagnostidis

29. Oscillatoria laete-viriens Crouan

30. Oscillatoria limosa Agardh

31. Oscillatoria princeps Roberhorst

32. Oscillatoria limosa Agardh

33. Oscillatoria curviceps Agardh

34. Oscillatoria boryana Bory and Gomant

35. Oscillatoria princeps Rabenhorst

36. Oscillatoria rubencens De Candolle

37. Oscillatoria tenue Gomant

38. Oscillatoria sp. Bory and Gomant

39. Oscillatoria chalybya Metens

40. Oscillatoria acuminata Gomant

41. Oscillatoria salina Biswas

42. Jaaginema pseudogeminatum Schmid

43. Spirulina subsalsa Oersted

44. Spirulina princeps West and West

45. Spirulina meneghiniana Zanardini

46. Lyngbya majuscula Harvey

47. Lyngbya allergii Kutzing

48. Lyngbya confervoides Agardh

Pseudanabaenaceae

49. Pseudoanabaena sp Lauterborn

Mastigocladaceae

50. Hapalosiphon welwitschii West and

West

Figure 5a Microphotograph of marine cyanobacteria: 1. Oscillatoria princeps 2. Oscillatoria tenuis 3. Chrococcus minor 4. Oscillatoria brevis 5. Lyngbya masjacula 6. Lyngbya confervoides 7. Phormidium valderianum 8. Gleocapsa sp.

Figure 5b Microphotograph of marine Cyanobacteria: 1. Anabaena spirodes 2. Spirulina subsalsa 3. Oscillatoria subbrevis 4. Oscillatoria formosa 5. Microcystis marginata 6. Oscillatoria boryana 7. Oscillatoria limosa 8. Phormidium willei.

44

Table 1b. Microbial diversity of Diatoms

Achanthaceae

1. Achnanthes gibberula Bory De Saint

2. Achnanthes minutissima Bory

Bacillariaceae

3. Denticula vanheurcki Brun

4. Nitzchia vasnii Smith

5. Nitzchia sp. Smith

6. Nitzchia subtilis Grunow

7. Nitzchia palea Smith

8. Nitzchia sigmoidea Smith

Catenulaceae

9. Amphora sp. Smith

Cymbellaceae

10. Cymbella tumida Agardh

11. Cymbella affinis Agardh

Desmidiaceae

12. Docidium baculum Playfair

Fragilariaceae

13. Fragilaria construens Grunnow

14. Fragilaria sp. Lyngbye

15. Synedra ulna Ehrenberg

16. Synedra acus Ehrenberg

Gomphonemataceae

17. Gomphoneis herculeana Ehrenberg

Mastoglioaceae

18. Mastogloia smithii Smith

19. Matogloia sp. Cleve

Naviculaceae

20. Caloneis silicula Cleve

21. Caloneis lewisii Patrick

22. Navicula andium Muller.

23. Navicula capsidata Kutzing

24. Navicula seminulum Grunow

25. Navicula andium Muller

26. Navicula viridula Ehrenberg

27. Navicula capsidata Kutzing

Pinnulariaceae

28. Pinnularia burkii Ehrenberg

29. Pinnularia subcaptata Gregory

30. Pinnularia burkii Pat

31. Pinnularia sp. Ehrenberg

Pleurosigmataceae

32. Gyrosigma sp. Hassall

33. Pleurosigma acuminatum Grunow

34. Pleurosigma sp. Smith

35. Pleurosigma angulatum Queckett

Stauroneidaceae

36. Stauroneis sp. Ehrenberg

Stephanodiscaceae

37. Cyclotella sp. Kutzing

38. Cyclotella sp. Kutzing

Figure 6 Microphotograph of Diatoms: 1. Stauroneis sp. 2. Navicula palea 3. Synedra ulna 4. Navicula vasni 5. Pinnularia burkii 6. Navicula 7. Navicula andium 8. Gyrosigma 9. Pinnularia subcaptata 10. Gomphonema herculeana 11. Synedra acus 12. Nitzchia subtilis 13. Caloneis silicula 14. Cymbella affinis 15. Caloneis lewisii 16. Cymbella 17. Fragilaria construns 18. Navicula seminulum 19. Achnathes minutissima 20. Navicula virudula 21. Pleurosigma acuminatum 22. Pinnularia sp. 23. Pleurosigma 24. Pleurosigma angulatum

45

In addition, it is difficult to accurately identify cyanobacteria of the order

Oscillatoriales. The identification problems increase further at species level, being of

course impossible to differentiate strains through this approach. Sometimes, even for

someone with experience, misidentification can be obtained, as occurred with an

Aphanizomenon strain, originally classified as Aphanizomenon flos-aquae and later re-

classified as Aph. issatschenkoi with the help of the 16S rRNA phylogenetic

positioning (Neilan, 2002).

Molecular characterization of the marine cyanobacteria and diatoms were done

with 16S rDNA and 18S rDNA sequence. Amplification of 18S rDNA and 16S rDNA

gel picture was shown (Fig. 7), in which clear resolved bands at 2.1kb and 1.6kb were

found in diatom and cyanobacteria respectively. The evolutionary relationships of the

isolates were found out with phylogenetic tree, in which the isolated strain were closely

associated with marine forms of cyanobacteria and some of new reports were found in

case of diatom (Fig 8 & 9).

Molecular phylogenetics is the analysis of hereditary differences in DNA which

gave the information on an organism's evolutionary relationships. The result of a

molecular phylogenetic analysis is expressed in a phylogenetic tree. The first molecular

and phylogenetic characterization of two species of the genus Nostoc - Nostoc linckia

and Nostoc punctiforme based on the cpcB-IGS-cpcA locus of the phycocyanin operon

(Teneva et al., 2005). The phylogenetic position of these two species within order

Nostocales as well as within division Cyanoprokaryota has been determined. The

results indicate that genus Nostoc is heterogeneous. Analysis of the IGS region between

cpcB and cpcA showed that Nostoc and Anabaena are distinct genera, while the

taxonomic status of the genera Aphanizomenon and Anabaena is still unclear, since the

similarity is up to 96.3%. Therefore, additional analyses and revision are required.

Reported molecular and phylogenetic data will be useful to solve other problematic

points in the taxonomy of genera Aphanizomenon, Anabaena and Nostoc.

The ribosomal sequences were deposited to GenBank (NCBI) to record our

isolated diatom and cyanobacterial strains. Accession numbers for the each organism

a)

b)

Figure 7 Amplification of ribosomal rDNA: (a) diatom (18S rDNA) and (b) cyanobacteria (16S rDNA); marker (M); 1-5 represents selected diatom and cyanobacterial isolates.

M 1 2 3 4 5

M 1 2 3 4 5

2500

2000

1000

1500

500

Figure 8 Evolutionary relationship of diatom based on the 18S rDNA sequences. Organisms indicated by red color are the selected isolates while black colored ones are retrieved from GenBank (NCBI). Differences between sequences are indicated by the sum of the horizontal length; the scale bar is in fixed nucleotide substitutions per sequence position.

Figure 9 Evolutionary relationship of marine Cyanobacteria based on the 16S rDNA sequences by Maximum Likelihood Method. Organisms indicated by red color are the selected isolates while black colored ones are retrieved from GenBank (NCBI). Differences between sequences are indicated by the sum of the horizontal length; the scale bar is in fixed nucleotide substitutions per sequence position.

EF654085.1| Y18795.1| Spirulina sp. strain MPI S2 16S rRNA gene

JQ347352.1| Uncultured bacterium clone AP60 16S ribosomal RNA gene partial sequence

EF188837.1| Phormidium boryanum BDU 92181 16S ribosomal RNA gene partial sequence

GU585847.1| Phormidium tenue NTDM05 16S ribosomal RNA gene partial sequence

HQ738569.1| Oscillatoria boryana BRIP02 16S ribosomal RNA gene partial sequence

JQ867393 Oscillatoria boryana NTDM11

DQ845200.1| Oscillatoria boryana BDU92181 16S ribosomal RNA gene partial sequence

Leptolynbya valderiana

GU812858.1| Phormidium willei NTDM01 16S ribosomal RNA gene partial sequence

EF654085.1| Phormidium persicinum SAG 80.79 16S ribosomal RNA gene partial sequence

JQ867392 Leptolynbya valderiana NTDM10

GU186898.1| Leptolyngbya valderiana BDU 41001 16S ribosomal RNA gene partial sequence

Y18792.1| Spirulina sp. strain MPI S4 16S rRNA gene

GU585848.1| Spirulina subsalsa NTDM21 16S ribosomal RNA gene partial sequence77 67

69

35 49

29

24

27 16

0.2

Table 2 Selected cyanobacterial 16S rDNA sequences were submitted to GenBank (NCBI) with Accession numbers mentioned below.

S. No Organisms Sequence Accession Numbers

1. Oscillatoria willei NTDM01 16S rDNA GU812858

2. Phormidium tenue NTDM05 16S rDNA GU585847

3. Leptolyngbya valderianum

NTDM10

16S rDNA JQ867392

4. Oscillatoria boryana NTDM11 16S rDNA JQ867393

5. Spirulina subsalsa NTDM21 16S rDNA GU585848

Table 3 Selected diatom 18S rDNA sequences were deposited to GenBank (NCBI) with Accession numbers mentioned below.

S. No. Organisms Sequence Accession Numbers

1. Synedra sp. NTDMN01 18S rDNA HM635776

2. Synedra sp. NTDMD01 18S rDNA HM635777

3. Amphora sp. NTDMN02 18S rDNA HM635778

4. Stauroneis sp. NTDMD02 18S rDNA HM635779

5. Cymbella tumida NTDMN03 18S rDNA HM585048

46

have received from GenBank were tabulated (Table 2 & 3). In addition, C-phycocyanin

gene sequence also been deposited to GenBank with translated protein sequence

(Table 4).

Protein conformational structures are primary, secondary and tertiary structures

were predicted. Among which tertiary structure gives three-dimensional structures in

different isoforms or different structural elucidation such ribbon model, ball and stick

model, space-filling model and so on. A represenatative of the molecular modeling

studies ribbon model and ball and stick models were elucidated with amino acid

sequence (Fig. 11). In this protein model, sequence start with Met aminoacids and ends

with Ile aminoacids.

Conservation domains were analysed in which the query sequence highly

matched with phycoblisome superfamily (Fig 12). It was further analyzed for the

identity with cyanophyceae and chlorphyceae phycocyanin family. Flow-chart for the

determination of cyanobacterial Phycocyanin gene was shown (Fig 13). Molecular

modelling methods are now routinely used to investigate the structure, dynamics,

surface properties and thermodynamics of inorganic, biological and polymeric systems.

The types of biological activity that have been investigated using molecular modelling

include protein folding, enzyme catalysis, protein stability, conformational changes

associated with biomolecular function, and molecular recognition of proteins, DNA,

and membrane complexes (Foster, 2002). The query sequence was closely identical

with cyanobacterial species with 75 % identity, whereas 37% of identity was observed

chlorphyceae algal groups; hence, perhaps it may be used as genetic marker for

development of DNA barcodes for the Cyanobacteria (Fig 14). After performing the

structure prediction of SPDBV software, the final 3D Structure calculated for modelled

structure reactivity, which can provide valuable information of protein structure and

function. The in silico design of drug formulations, plays crucial roles in the biomedical

research. Computational research on biomedics has generated a lot of information

concerning its protein structure and reactivity.

Table 4 Selected cyanobacterial phycocyanin gene sequences were deposited to GenBank (NCBI) with Accession numbers mentioned below.

S. No. Organisms Sequence Accession Numbers

1. Phormidium tenue NTDM05 C-PC gene JF421685

2. Phormidium animale NTMP03

C-PC gene JF421686

3. Oscillatoria acuminata NTDM04

C-PC gene JF421687

4. Spirulina subsalsa NTDM21

C-PC gene Bankit No 1421025

5. Oscillatoria chlorina

C-PC gene Bankit No 1431046

6. Oscillatoria salina

C-PC gene Bankit No 1431047

Figure 10 Amplification of Cyanobacterial Phycocyanin (C-PC) gene: marker (M); 1-5 represents selected cyanobacterial isolates

M 1 2 3 4 5

2500

2000

1000

1500

500

a)

b)

Figure 11 Three- Dimensional structure of C-Phycocyanin gene: ribbon model (a) and Ball and stick model with 63 amino acid alpha chain (b)

Figure 12 Determination of conserved domains of the cyanobacterial phycocyanin gene sequence from Phycobilisome superfamily

Figure 13 Flowchart showing the multiple sequence alignment and determination of conserved domanins of the cyanobacterial phycocyanin gene sequence

Figure 14 Comparative sequence analysis of cyanobacterial phycocyanin gene with PDB database: 75% similarity with 1JBO (a) and 37% similarity with 3BRP (b)

Chapter II

Partial Biochemical Characterization of Cyanobacterial Phycobiliproteins

47

INTRODUCTION

Cyanobacteria are photosynthetic microorganisms, which carry out

photosynthesis similar to higher plants. However, instead of chloroplasts they contain

membranous structures called thylakoids, where phycobilisomes are found. These

microorganisms only contain chlorophyll a and almost 50% of the light required is

captured by the phycobiliproteins. Phycobiliproteins are antenna pigments, light

harvesting, and consist of chromophores called bilins, which are attached to cysteine

residues of the apoprotein (Samsonoff and MacColl, 2001).

The assembly of the phycobilisome is stabilized by a group of polypeptides

named ‘linker’ polypeptides (L). They induce a face-to-face aggregation of

phycoerythrin or phycoerythrocyanin and phycocyanin trimers. They additionally cause

the tail-to-tail joining of hexameric assemblies of these biliproteins to form larger

aggregates such as peripheral rods and core cylinders. Allophycocyanin assemble into

phycobilisome core with the assistance of three types of linker polypeptides: the large

core-membrane linker polypeptide (LCM) involved in the interaction of PBS core with

the thylakoids, rod-core linker polypeptides (LRC) that mediate the attachment of the

peripheral rods to the PBS core and the small core-linker polypeptides (LC) which

participate in the assembly of core substructure.

The peripheral rods of the phycobilisomes are composed of phycoerythrin or

phycoerythrocyanin and phycocyanin in association with the appropriate rod-linker

polypeptides (LR). Phycoerythrin hexamers of some marine cyanobacteria and red

algae contain a third type of subunit γPE in the central cavity of the hexamer. These

subunits are bifunctional phycobiliproteins that act as light harvesting phycobiliproteins

and linker polypeptides (Sidler 1994). The morphology of phycobilisomes varies with

the organisms. These particles may be ellipsoidal, hemidiscoidal, or bundles of rod

shaped elements. The differences in gross morphology do not reflect fundamental

differences in the placement of the major phycobiliproteins or in the fundamental

properties of the particle (Glazer 1989).

48

In contrast to the proteins of the cyanobacteria and of the red algae, the

cryptomonad phycobiliproteins are unusual in several ways such as (1) phycobilisomes

are lacking due to the formation of tetrameric complexes and therefore not possible to

form phycobilisomes, (2) each species of cryptomonad contains only one type of

phycobiliproteins either phycoerythrin or phycocyanin, (3) the proteins are found on the

lumenal rather than the stromal side of the thylakoid membrane, (4) the proteins form

dimers, rather than trimers or hexamers, and (5) they contain several unusual bilins, for

example in the cryptophycean biliprotein phycocyanin 645, three chemically different

bilins such as Cys-bilin 618, DiCys-bilin 584 and Cys-bilin 584 are present (Becker et

al. 1998; Wedemayer et al. 1991).

Cyanobacterial phycobiliproteins have gained importance in the commercial

sector, as they have several applications. The primary potential of these molecules are

as natural dyes but a number of investigations have shown on their health-promoting

properties and broad range of pharmaceutical applications. Thus, one of the

applications of phycocyanin is to use as food pigment replacing current synthetic

pigments. They are used as a colourant in chewing gum, ice sherberts, popsicles,

candies, soft drinks, dairy products and cosmetics like lipstick and eyeliners. In

addition, phycobiliproteins are widely used in clinical and immunological research

laboratories (Spolaore et al. 2006). They serve as labels for antibodies, receptors and

other biological molecules in a fluorescence-activated cell sorter, and they are used in

immunolabelling experiments, fluorescence microscopy and diagnostics.

The major organisms exploited for production are the cyanobacterium,

Spirulina for phycocyanin and the red alga Phorphyridium for phycoerythrin (Roman et

al. 2002). The purpose of the review is to assess the wealth of knowledge on the

applications of phycobiliproteins and the technological backup available for

commercial venturing. This can be envisaged by analyzing application oriented

research, patents and commercial activities. A compilation of this knowledge and its

analysis will be of immense use for attaining further improvements.

49

The phycocyanins have an apparent molecular mass of 140–210 kDa and two

subunits, α and β. Studies of crystal structure in phycocyanin have shown that all of

these have a very similar three-dimensional structure. The subunits are associated in an

(α β) protomers, which in turn can be associated in trimmers (α β)3 and hexamers (α β)6

(Stec et al., 1999). The chromophores confer a characteristic colour of each

phycobiliprotein: phycocyanin (blue brilliant), phycoerythrin (red) and allophycocyanin

(green–blue). The characteristic coloration of the phycobiliproteins, make them

attractive to food and cosmetic industries as natural pigments (Santos et al., 2004).

Objectives:

In this chapter, work is emphasized on the biochemical characterization of C-

Phycobiliprotein (specifically, C-PE) with following process.

• Selection of cyanobacterial strains for the pigment production

• Extraction of C-Phycobiliproteins from selected Cyanobacteria

• Spectrometric quantification of C-Phycobiliproteins

• Optimization of physical and chemical methods

• Study on effect of preservatives on the C-Phycoerythrin

• Biomedical application of C-Phycoerythrin

50

REVIEW OF LITERATURE

Cyanobacterial Pigments:

Cyanobacteria are the pioneer oxygenic phototrophs on earth whose distribution

around the world is surpassed only by bacteria (Adams et al., 2000). Worldwide

attention is drawn towards cyanobacteria for their possible use in mariculture, food,

feed, biopolymer, biofuel and bio-fertilizer, production of various secondary

metabolites including vitamins, toxins, enzymes, pharmaceuticals, pharmacological

probes and pollution abatement (Thajuddin and Subramanian, 2005 and Shrivastav et

a., 2010). Phycobiliproteins, including phycoerythrin, phycocyanin and

allophycocyanin are a class of chromo-protein bearing covalently bound linear

tetrapyrole chromophore.

Phycobiliproteins are the major photosynthetic accessory pigments in

cyanobacteria (prokaryotic); rhodophytes (red algae, eukaryotic); cryptomonads

(biflagellate unicellular eukaryotic algae) and cyanelles (endosymbiotic plastid-like

organelles). Phycobiliproteins are brilliant-colored and water-soluble antennae-protein

pigments organized in supramolecular complexes, called phycobilisomes (PBSs),

which are assembled on the outer surface of the thylakoid membranes. The colors of

phycobiliproteins originate mainly from covalently bound prosthetic groups that are

open-chain tetrapyrrole chromophores bearing A, B, C and D rings named phycobilins.

There are four main classes of phycobiliproteins – allophycocyanin (APC bluish green),

phycocyanin (PC: deep blue), phycoerythrin (PE: deep red) and phycocyanobilin (PCB:

orange) (Hemlata et al., 2011).

Classification of Phycobiliprotein:

They are classified by their spectral properties as Phycoerythrobilin (PEB,

Amax 560 nm), Phycocyanobilin (PCB, Amax 620–650 nm), Phycobiliviolin (PXB,

Amax 575 nm) and Phycourobilin (PUB, Amax 498 nm). There are three main types of

phycobiliproteins, phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC).

PE is at the tip of the rod-like phycobilisomes, PC is in the middle, while APC forms a

core attached to the reaction and energy transfer proceeds successively from

51

PE→PC→APC→chlorophyll a. Phycobiliproteins are chromo-proteins that have anti-

inflammatory, hepato-protective and antioxidant properties, it can also play important

role in photodynamic action on cancerous tumors and leukemia treatment (Khan et al.,

2005; Kulshreshtha et al., 2008; Vadiraja et al., 1998; Patel et al., 2006 and Paul et al.,

2006).

Cyanobacteria with high levels of specific phycobiliproteins are of commercial

interest. The primary potential of these molecules are as natural dyes in food industry.

A number of investigations have shown their health-promoting properties and

pharmaceutical applications.

Phycocyanin and phycoerythrin are the two currently used natural pigmented

proteins having versatile applications. They are stable at low temperature with some

preservative like citric acid, in acidic and basic solutions and therefore, they could be

utilized as food colorant (chewing gum, jellies), health drinks, beverages and coloring

agent in sweet confectionary and cosmetics (Patel., 2004; Mishra et al., 2008 and

Eriksen, 2008). Of late, more attention is given to the application utilizing its

biochemical and biophysical structural properties. These natural pigments are also used

in fluorescent labeling of antibodies that are applied in diagnostic kits in immunology,

cell biology and biomedical research (Sekar and Chandramohan, 2008).

Nutritional requirements for production of c-Phycobiliproteins:

Macronutrients such as nitrogen and phosphorus and micronutrients such as iron

and molybdenum are known to play a large part in regulating the metabolism and

ultimately the growth of cyanobacteria (Carr and Whitton 1982, Carpenter 1983).

Nitrogen is an essential element for plant growth, required for the synthesis of

amino acids, proteins, nucleotides, nucleic acids, coenzymes, chlorophyll and other

photosynthetic pigments (Raven et al., 1992). Most cyanobacteria are nitrogen fixers,

converting atmospheric nitrogen to ammonia via the enzyme nitrogenase (Postgate,

1987). The resulting ammonia is assimilated into glutamate through the reductive

amination of α-ketoglutarate, by the enzyme glutamate synthase (GOGAT) (Marscher,

52

1995). The glutamate is subsequently catalysed by glutamine synthetase (GS) and

transformed to glutamine, which is then utilised in the synthesis of organic compounds

(Garrett & Grisham, 1999).

Phosphorus is essential for energy-carrying phosphate compounds (ATP, ADP,

NADPH and NADP), nucleic acids, several essential coenzymes and phospholipids

(Marsher 1995). The orthophosphate ion, PO4 3-, is the principal form of phosphorus

taken up by marine plants. Other sources of phosphorus include inorganic

polyphosphates and organic-phosphorus compounds; however, both of these forms

require complex extracellular cleavage and hydrolysis, leaving orthophosphate ions as

the preferred source of phosphorus for uptake (Lobban & Harrison, 1994). Upon

uptake, the orthophosphate ion is esterified with hydroxyl groups of sugars and

alcohols or bound to another phosphate group (Mengel & Kirkby, 1987).

Uses of Phycobiliproteins

As colorant

There is an increasing demand for natural colors which are of use in food,

pharmaceuticals, cosmetics, textiles and as printing dyes. However, their utility is

limited to few of these since the natural dyes have low tinctorial values and persistence.

Due to the toxic effect of several synthetic dyes, there is an increasing preference to use

natural colors for various end uses. Phycobiliproteins are used as a natural protein dye

in the food industry (C-phycocyanin) and in the cosmetic industry (C-phycocyanin and

R-phycoerythrin).

Phycocyanin derived from Spirulina platensis is used as a natural pigment in

food such as chewing gum, dairy products and jellies (Santos et al. 2004). Despite its

lower stability to heat and light, phycocyanin is considered more versatile than gardenia

and indigo, showing a bright blue color in jelly gum and coated soft candies (Lone et

al. 2005). They are also used in coloring of many other food products such as

fermented milk products, ice creams, soft drinks, desserts, sweet cake decoration, milk

shakes and cosmetics. The shade of blue color produced from the red microalga

Phorphyridium aerugineum does not change with pH. The color was stable under light,

53

but sensitive to heat. Within a pH range of 4 to 5, the blue color produced is stable at

60°C for 40 min. This property was important for food uses, since many food items are

acidic, particularly drink and confections. The blue color was added to beverages

without heat application (Pepsi® and Bacardi Brezzer®) which did not lose their color

for at least 1 month at room temperature. The color was very stable in dry preparations.

Sugar flowers for cake decoration maintained their colors for years of storage. Foods

prepared with the phycobiliproteins include gelatin and ice cream. In addition to its

coloring properties, phycoerythrin possesses a yellow fluorescence.

Opportunities for exploiting this property for special effects were studied. A

range of foods that fluoresce under natural light and UV light were prepared and tested.

These include transparent lollipops made from sugar solution, dry sugar-drop candies

for cake decoration (that fluoresce under UV light), and soft drinks and alcoholic

beverages that fluoresce at pH 5–6. Fluorescent color has also been added to alcoholic

beverages containing up to 30% alcohol but the shelf life for such products is short

(Dufosse et al. 2005).

As fluorescent agent

Phycobiliproteins play an important role in fluorescent based detection systems,

particularly for flow cytometry. The spectral properties, such as (1) excitation and

emission at the red end of the spectrum, where interference from biological matrices

tend to be less, (2) a large Stokes shift, so that interference from Rayleigh and Raman

scatter and other fluorescing components is less significant or nonexistent, (3)

immunity from quenching by naturally occurring biological substances, (4) high

solubility in aqueous environment so that non-specific binding effects are minimal, and

(5) fluorescence quantum yield independent of pH of phycobiliproteins, particularly

R-phycoerythrin (R-PE) and allophycocyanin (APC), have made them dominant

reagents in this class of flurochromes (Kronick and Grossman 1983).

Synthesis of conjugates of phycobiliproteins with molecules having biological

specificity, like immunoglobulins, protein A, biotin and avidin, were reported and

showed that phycobiliproteins conjugates are excellent reagents for two color

54

fluorescence analysis of single cells using fluorescence activated cell sorter (FACS) (Oi

et al. 1982). The stabilized phycobilisomes designated PBXL-3L was accessed as a

fluorochrome for flow cytometric immunodetection of surface antigens on immune

cells (Telford et al. 2001b). The lesser-known but potentially important series of low-

molecular weight cryptomonad-derived phycobiliproteins are evaluated for their

applicability to flow cytometry both in extracellular and intracellular labeling

applications (Telford et al. 2001a).

Fluorescent labeling reagents are an essential component of a huge industry

built on sensitive fluorescence detection. This powerful multi-chromophore protein-

labeling reagent has changed the flow cytometry industry and permitted sensitive two-

color lymphocyte subset analysis with a single argon ion laser. Phycoerythrin excites

very strongly at 488 nm but the emission is shifted to 580 nm by energy transfer events

between chromophores within the protein.

Phycoerythrin as a second color works well with fluorescein- labeled

antibodies. They can also be excited at 488 nm but are detected separately at 525 nm to

provide the second colour of detection. HIV monitoring and cancer diagnostics were a

strong driving force for the growth of this reagent. Three- and four-color analyses with

a single laser are now commonplace with phycobiliprotein-based energy transfer

reagents. Low molecular weight fluorescent labels such as cyanine dyes Cy5

(Indodicarbocyanine) and Cy7 (Indotricarbocyanine) are good acceptors from excited

PE and shift emission farther to red and deep red wavelengths and 11- color analyses

are now possible (Waggoner 2006).

PE is also a very important reagent in proteomics and genomics and form the

basis of the detection system in Affymetrix chips (DNA microarrays). Phycoerythrin

labeled streptavidin is added after complete binding and produces a strong signal from

array elements containing the biotin-labeled DNA or protein probes (De Rosa et al.

2003).

55

As pharmaceutical agent

In the last decade, the screening of micro algae, especially the cyanobacteria,

for antibiotics and pharmaceutically active compounds has received ever increasing

interest (Borowitzka 1995). The pharmacological property attributed by phycocyanin

includes antioxidant, anti-inflammatory, neuroprotective and hepatoprotective activity.

When it was evaluated as an antioxidant in vitro, it was able to scavenge alkoxyl,

hydroxyl and peroxyl radicals, and inhibits microsomal lipid peroxidation induced by

Fe+2 –ascorbic acid or the free radicals initiators 2, 2′ Azobis (2- amidinopropane)

dihydrochloride, (AAPH).

They also reduced edema, histamine (Hi) release, myeloperoxide (MPO)

activity and the levels of prostaglandin (PGE2) and leukotriene (LTB4) in the inflamed

tissues. Phycocyanin also reduces the levels of tumor necrosis factor (TNF-α) in the

blood serum of mice-treated with endotoxin and it showed neuroprotective effects in

the rat cerebellar granule cell cultures. Aphanizomenon flos-aquae (AFA) as a source of

phycocyanin have been described as a strong antioxidant (Bhat and Madyastha 2000;

Romay et al. 2003). The involvement of phycocyanin in the antioxidant protection of

the AFA extract against the oxidative damage was demonstrated in vitro (Benedetti et

al. 2004). In red blood cells, oxidative hemolysis and lipid peroxidation induced by

aqueous peroxyl radical generator, AAPH were significantly lowered. In plasma

samples, they inhibited the extent of lipid oxidation induced by pro-oxidant agent

cupric chloride (CuCl2). Such findings about the phycocyanin consider the potential

benefits in the prevention of many pathological disorders associated with oxidative

stress and inflammation. Allophycocyanin was found to inhibit enterovirus 71- induced

apoptosis. At concentration nontoxic to the host cells (0.045±0.012 μM),

allophycocyanin was found to inhibit enterovirus 71-induced cytopathic effects, viral

plaque formation, and viral-induced apoptosis (Shih et al. 2003).

In addition, inhibitory effect of phycocyanin from Spirulina platensis on the

growth of human leukemia K562 cells in a dose- and time-dependent manner was

reported (Liu et al. 2000). R-PE subunits were indicated as an attractive option for

improving the selectivity of photodynamic therapy (PDT). Mouse tumor cells S180 and

56

human liver carcinoma cells SMC 7721 cells was treated with RPE subunits (Bei et al.

2002). C-Phycocyanin derived from Spirulina platensis powerfully influenced serum

cholesterol concentrations and imparted a stronger hypocholesterolemic activity

(Nagaoka et al. 2005). Phycocyanin also exerts hepatoprotective and anti-inflammatory

effects in a human hepatitis animal model. It reduced alanine amino transferase (ALT),

aspartate amino transferase (AST) and malondialdehyde (MDA) in the serum

(Gonzalez et al. 2003).

In addition, at a concentration of 0.25 mg/ml, influence of C-phycocyanin on

hepatocellular parameters related to liver oxidative stress and kupffer cell functioning

indicated that C-phcocyanin elicite a concenteration-depedent inhibition of carbon

phagocytosis and carbon-induced O2 uptake by perfused livers, with a 52% diminution

in the carbon-induced sinusoidal release of lactate dehydrogenase. C-phycocyanin also

suppressed the 3,3′,5-triiodothyronine (T3) induced increase in serum nitrite levels and

in the activity of hepatic nitric oxide synthase (NOS) (Remirez et al. 2002).

Importance of c-phycobiliproteins:

Phycoerythrin is good for human health, having antioxidant, radical scavenging,

anti-inflammatory and anticancer properties. C-PE can be used as nutrient ingredients

and natural dyes for food and cosmetics besides, being potential therapeutic agent in

oxidative stress-induced diseases and as fluorescent marker in biomedical research and

currently used as a natural food colorant in China (Li and Qi, 1997; Romay et al., 1998;

Gonzalez et al., 1999; Olvera-Ramirez et al., 2000; Bhat and Madhyastha, 2000;

Estrada et al., 2001; Tseng, 2001; Soni et al., 2008 and Mishra, 2007). Although,

several methods have been developed for the separation and purification of C-PE from

cyanobacteria, the purity and recovery is relatively low because, it is highly sensitive to

light, oxygen and moisture, hence, it is needed to process it along with efficient

preservatives. Sodium azide (NaN3) and dithiothreitol (DTT) are commonly used as

preservatives in phycoerythrin for analytical purpose, but they are toxic; so for

developing process of food grade C-PE only edible preservatives with unique

properties can be used. It is therefore, desired to develop a simple, but more well-

57

organized method for the separation, purification and stabilization of the food grade C-

PE from cyanobacteria (Mishra et al., 2010).

Extraction of Phycobiliproteins:

Each microorganism has particular characteristics referring to the location of

intracellularly produced proteins, meaning that the molecule of interest might be

located in the cytoplasm, periplasm or even be stored in some cellular organelle, such

as in the mitochondria. Hence, the extraction protocol could vary according to the

desired protein. In general, the extraction method is the key for maximum recovery of

phycobiliproteins in the natural state from algae (Niu et al., 2006).

The extraction of phycobiliproteins involves cell rupture and release of these

proteins from within the cell. The cell walls of cryptophytes are easily disrupted, but

those of cyanobacteria are extremely resistant (Siegelman and Kycia, 1978). Thus, the

use of variations in the osmotic pressure, abrasive conditions, chemical treatment,

freezing-thawing and sonication, amongst other disruption methods, are necessary.

Mechanical cell disintegration methods are currently preferred for large-scale

operations (Gacesa and Hubble, 1990; Kula and Schütte, 1987) since a complete

disintegration of the biomass is desired, with high product and activity yields. Some

papers report C-PC extraction from cyanobacterium. Moraes et al. (2010) and Silveira

et al. (2007) studied the optimization of extraction from dried biomass.

The reported methods to extract C-PC from wet biomass include freezing and

thawing (Sony et al., 2008; Sarada et al., 1999; Abalde et al., 1998; Bermejo et al.,

2006), sonication (Bermejo et al., 2006; Abalde et al., 1998), homogenization (Sarada

et al., 1999), lysozyme treatment (Bermejo et al., 2006; Stewart and Farmer, 1984) and

acid treatment (Sarada et al., 1999; Bermejo et al., 2006).

Preservation of Phycobiliproteins:

Although, several methods have been developed for the separation and

purification of C-PC from cyanobacteria, the purity and recovery is relatively low

because, it is highly sensitive to light, oxygen and moisture, hence, it is needed to

58

process it along with efficient preservatives. Sodium azide (NaN3) and dithiothreitol

(DTT) are commonly used as preservatives in C-PC for analytical purpose, but they are

toxic; so for developing process of food grade C-PC only edible preservatives with

specific properties can be used. It is therefore, desired to develop a simple, but more

efficient method for the separation, purification and stabilization of the food grade C-

PC from cyanobacteria (Patel et al., 2005). C-PC can be used as nutrient ingredients

and natural dyes for food and cosmetics besides, being potential therapeutic agent in

oxidative stress-induced diseases and as fluorescent marker in biomedical research and

currently used as a natural food colorant in China (Li and Qi, 1997; Romay et al., 1998;

Romay et al., 1998; Romay et al., 2001; Gonzalez et al., 1999; Bhat and Madhyastha,

2000; Hirata et al., 2000; Estrada et al., 2001 and Tseng, 2001).

Phycobiliproteins extracted from cyanobacteria have received a great attention

due to its potential in biotechnology and biomedical sciences (therapeutic agent and

immunodiagnostic probes) because of their versatile properties, i.e. its fluorescent

nature and radical scavenger activity, which is suitable in photodynamic therapy for

tumor cells (Paul et al., 2006). Their radical scavenger activity makes them potent

antioxidant and anticancer agent; lipid peroxidase activity as an antidote for toxic

metals like chromium; immunomodulator and bio-enhancer activity makes them anti-

inflammatory and antidiabetic (Mishra, 2007; Mishra et al., 2005; Patel et al., 2006

and Patel et al., 2004). Preservatives are required to ensure that manufactured foods or

additives remain safe and unspoiled for a long time (shelf life). The present study

describes the effect of selected food grade preservatives for C-PE in order to develop a

stable product with long shelf life.

Food additives have been used by mankind for centuries, e.g. salt, sugar and

vinegar were among the first to be used as preservatives in food. In the past 30 years,

however, with the advent of processed foods, there has been a massive explosion in the

chemical adulteration of foods with additives. Considerable controversy has been

associated with the potential threats and possible benefits of food additives. Some food

additives are known to be carcinogenic or toxic. Hyperactivity in children, allergies,

asthma, and migraines are often associated with adverse reactions to food additives.

59

Preservative for the food grade C-phycoerythrin is indispensable for making the

process commercially viable due to its sensitivity.

Preservatives are required to ensure that manufactured foods or additives remain

safe and unspoiled for a long time (self life). The present study describes the effect of

selected food grade preservatives for phycoerythrin in order to develop a stable product

with long shelf life. Food additives have been used by mankind for centuries, e.g. salt;

sugar and vinegar were among the first to be used as preservatives in food.

Phycoerythrin can be used as a food colorant which is not only safe but, has an extra

advantage of being potential antioxidant. Preservative for the food grade phycoerythrin

is indispensable for making the process commercially viable due to its extreme

sensitivity towards light oxygen and temperature under aqueous condition.

60

MATERIALS AND METHODS

Materials:

All the chemicals used in this study were procured from Sigma, Hi-Media and

Qualigens. All buffers and reagents used in this study were prepared in double distilled

water.

Selection of Cyanobacterial Strain:

Among isolated marine cyanobacteria described in Chapter I, preliminary

screening was done for pigment production. Among them, a marine cyanobacterium,

Phormidium tenue NTDM05 has been selected for quantification of phycobiliproteins

and further studies. A marine cyanobacterium was mass cultured raised in ASN-III

Medium (Stanier et al., 1971). Culture was allowed to grow in light intensity provided

by cool-white fluorescent tubes of 50 µmol photons.m-2.s-1 following 12:12 hour, light

and dark regime at 30oC ±1oC.

Extraction and quantification of Phycobiliproteins:

Cyanobacterial cells were harvested by centrifugation (10,000g for 15 min).

Adhered salts were removed by washing with double distilled water and then biomass

was dried (50ºC). Biomass was homogenized with phosphate buffer and repeated

freezing and thawing was done in dark. The mixture was subsequently centrifuged

(10,000g for 20 min, 4ºC) to separate the phycobiliprotein containing clear supernatant.

Absorbance of supernatants was measured at wavelength 620, 652, and 562 nm

(Optizen Scan UV–VIS spectrophotometer) for C-phycocyanin, allophycocyanin and

phycoerythrin respectively (Siegelman and Kycia. 1978).

The amount of C-PC, C-PE and C-APC in the sample was calculated using

simultaneous equations of Bennet and Bogorad (1973) as follows:

Phycocyanin (mgmL−1) = [A620 −0.474 (A652)]/5.34

Allophycocyanin (mgmL−1) = [A652 −0.208 (A620)]/5.09

Phycoerythrin (mgmL−1) = [A568 −2.41 (PC) −0.849 (APC)]/9.62

61

Extraction of C-Phycoerythrin:

Cyanobacterial cells harvested by whatman filter paper (No.1) filtration were

washed with phosphate buffer (pH 7.2) and lyophilized. Two grams of freeze dried

biomass was suspended in 50 ml of sodium–phosphate buffer (0.1 M, pH 7.2,).

Repeated freezing at −20 ◦C and thawing at room temperature 25±5 ◦C was done in the

dark, The mixture was subsequently centrifuged at 10,000×g for 30 min at 0±5 ◦C and a

phycoerythrin containing clear supernatant was collected and precipitated by

ammonium sulphate fractional precipitation (25 and 60%), The pellet was suspended in

water followed by dialysis against distilled water at 0±5 ◦C for 48 h in dark, by

intermittent changing of distilled water at regular intervals and freeze dried. The

complete process for extraction of phycoerythrin is shown in Fig. 1.

Phormidium tenue NTDM05

Grown in ASN and MN-III medium

Harvesting the biomass for extraction

Extraction in Phosphate Buffer system by simple freeze and thaw method

(12h freeze and 12h thawing)

Cell debris removal by centrifugation

Ammonium sulphate precipitation

Dialysis

Stored at -4oC

Lyophilized

Figure 1 Process for extraction of C-Phycoerythrin from Phormidium tenue NTDM05

62

Ammonium sulfate fractionation:

The crude extract to obtain 20% saturation of finely powdered ammonium

sulfate was gradually added with continuous stirring for 1 h. The resulting solution was

kept overnight and centrifuged at 17,000 x g for 20 min. The supernatant was pooled

and subjected to 70% ammonium sulfate saturation in a manner similar to that of 20%

saturation. After overnight incubation, the solution was centrifuged at 10,000 rpm for

20 min. The pellets were resuspended in a small quantity of 20 mM Tris–Cl buffer (pH

8.1) and subjected to dialysis for 48 h against 100 times volume of the same buffer,

with a change of buffer three times.

Optimization of the production of C-Phycobiliproteins:

Physical and chemical methods were adopted for the higher production of c-

phycobiliproteins in selected cyanobacteria.

Physical methods:

In this method, different methods were adopted, firstly, extraction methods such

as effect of different cell disruption methods like homogenization (Oranda et al. 1978),

sonication (Furuki et al. 2003), heat shock (Soni et al. 2006) and freezing and thawing

(Soni et al. 2006) on phycobiliprotein yield. Secondly, temperature, where 20 – 60oC

were adopted.

Chemical methods:

In this method, micro nutrients such as Magnesium sulphate (4, 4.5, 5, 5.5,

6mg/l), Sodium carbonate (20, 40, 60, 80, 100mg/l), Potassium phosphate (20, 40, 60,

80, 100mg/l), Sodium Nitrate (1.5, 2.25, 3., 3.75, 4.5, 5.25, 6mg/l) are important

nutrients for the production of phycobiliproteins specially, C-phycoerythrin, in buffer

system Sodium phosphate buffer (pH-7.0) of 0.1M, Tris buffer (pH-7.2), Phosphate

buffered saline, HEPES and double distilled water was used. Finally, pH (6.0, 6.5, 7.0,

7.5 & 8.0) of medium was checked on phycobiliprotein yield.

63

Experimental selection of effective preservatives:

The stability of C-phycoerythrin in aqueous forms was checked by adding

different preservatives viz. Sucrose, Calcium chloride, Sodium chloride and Citric acid,

kept in two different conditions,

(1) At 0±5 ◦C in the refrigerator

(2) At room temperature 35±5oC

For checking efficacy of preservatives sucrose (0.1 g), Calcium chloride (0.1 g),

Citric acid (0.1 g), Sodium chloride (0.1 g) was added in 25 ml phycoerythrin solution

[5mg of freeze dried c-phycoerythrin was dissolved in 25 ml of sodium phosphate

buffer (0.1 M, pH 7.2)] separately and kept at 0±5 ◦C and 35±5 ◦C for study.

The absorbance of C-phycoerythrin solution containing preservative along with

the control without preservative was measured after regular interval, up to 30 days, on a

Optizen Scan UV-Vis, NIR spectrophotometer at wavelength 250–700nm for

calculating the concentration of C-Phycoerythrin using appropriate equation (Bennetts

and Bogorad, 1973).

Antibacterial activity of extracted C-Phycobiliprotein:

Antibacterial activity of extracted C-Phycobiliprotein was analyzed by well –

diffusion method with human pathogens such as E. coli, Enterobacter sp.,

Pseudomonas sp., Vibrio cholerae, Klebsiella pneumoniae. 24h cultures of human

pathogens were streaked on Muller Hinton Agar (MHA) and wells were formed in

15mm depth on the agar plate. Extracted phycobiliprotein was inoculated on the well in

different concentration such as 1mg/ml, 10mg/ml and 100mg/ml. The plates were

incubated at 37oC for 24h and extended to 48h. The antibacterial effect of extracted

phycobiliprotein were documented and discussed.

Determination of fluorescence property:

Fluorescence property of extracted C-Phycobiliprotein was analyzed in

Fluorescence spectroscopy and Uv-Transilluminator. An aliquot of extracted C-

phycobiliprotein from Phormidium tenue NTDM05 was tested for fluorescence

property for the application biolabelling purpose.

64

RESULTS AND DISCUSSION

One of the most important requirements for obtaining phycobiliproteins from

Cyanobacteria is the selection of extraction and purification protocol. An extraction

and purification procedure that works well for a phycobiliprotein from one organism

may not be the method of choice for the corresponding phycobiliprotein from another

organism. In this study, this reasons the phycobiliprotein extraction and

optimization and preservation methods were compared in Phormidium tenue

NTDM05. Hemlata et al., (2011) also described these extraction and purification

methods in Anabaena.

Uv – Vis Spectrum of phycobiliprotein showed peaks at 540 nm in higher

percentage (120mg/g), which indicates that the phycoerythrin is a major constituents

among phycobiliproteins that produced by Phormidium tenue NTDM05 (Fig 2).

Whereas other phycobiliproteins like phycocyanin and allophycocyanin present in the

meager amount of 70mg/g and 20mg/g respectively. Hence, the works have focused on

the extraction, production and optimization of C-Phycoerythrin.

The data of quantitative evaluation of phycobiliproteins in the selected

cyanobacterial species revealed C-PE as the major component with respect to other

phycobiliproteins, i.e., PC and APC, which makes the purification process easy. Since

APC is present in very low amount, this molecule does not interfere in the purification

process. This may be due to both the strain specificity in the phycobiliprotein content

and the culture conditions of each cyanobacterial strains.

In the first set of experiments for the optimization of nutrients, culture was

grown under increasing concentration of carbonate. The results showed an increase in

the biomass coupled with an increase in photosynthetic activity, indicated by increased

photosynthetic pigments like chlorophyll-a, carotenoids, and phycobilins.

A culture was grown under increasing concentration of Magnesium sulphate for

the optimization of production (Fig 3). The highest biomass was observed in the culture

a)

b)

Figure 2 Small–scale culturing of Phormidium tenue NTDM05 for pigment production (a) and microphotograph of Phormidium tenue NTDM05 with red color pigmentation (b)

Figure 3 Optimization of Phycobiliproteins from Phormidium tenue NTDM05 by chemical methods. Various concentration of Magnesium sulphate (4.0 – 6.0 mg/l).

Table 1 Influence of Magnesium sulphate concentration on pigment production in Phormidium tenue NTDM05

Concentration of MgSO4 (mg/g)

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

4.0

4.5

5.0

5.5

6.0

71.21± 0.52

110.13 ± 1.41

95.33 ± 0.72

82.26 ± 1.50

65.26 ± 1.81

23.45 ± 0.63

42.21 ± 0.93

39.63 ± 1.21

32.12 ± 0.36

27.36 ± 1.31

121.03 ± 1.23

201.12 ± 0.72

190.12 ± 1.81

160 .32± 1.75

121.021± 0.63

Values are mean ± SD of three values

4.0 4.5 5.0 5.5 6.0

6.5

Figure 4 Optimization of Phycobiliproteins from Phormidium tenue NTDM05 by chemical methods. Various concentration of Sodium phosphate (20-100mg/l)

Table 2 Influence of Phosphate concentration on pigment production in Phormidium tenue NTDM05

Concentration of Na2PO4 (mg/g)

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

20

40

60

80

100

95 .51± 1.31

87.23 ± 1.21

81.02 ± 0.93

76.21 ± 1.81

55.33 ± 0.72

39.03 ± 0.52

35.36 ± 1.23

32.23 ± 0.63

29.12 ± 1.50

19.50 ± 0.36

181.12 ± 1.21

161.03 ± 1.75

155.50 ± 0.72

143.41 ± 0.63

101.05 ± 1.75

Values are mean ± SD of three values

100 80 60 40 20 C

65

containing 4.5mg/l of Magnesium sulphate, which was increased concentration than the

control (4mg/l) and the concentration of C-phycoerythrin was found to be 201.12mg/g

(Table 1). The same type of observation was also found in Sodium phosphate, in which

various concentration of Sodium phosphate (20-100 mg/l) (Fig 4) amended in the

culture medium, where 20mg/l of sodium phosphate showed significantly increased the

production of C-phycoerythrin maximum of 181.12mg/g of biomass. In other hands,

when increase the concentration of sodium carbonate and potassium phosphate from

the control concentration, there was slow growth compared with control culture

(Table 2).

The highest biomass was observed in the culture containing 20mg/l of Sodium

carbonate, which was normal concentration as control (20mg/l) and the concentration

of C-phycoerythrin was found to be 303.26mg/g (Table 3). Eiler et al., (2003) showed

that bacterial biomass production was limited by supply of carbon. These results

showed that there was a correlation between substrate carbon and growth. However,

there was a stagnation of biomass production at a carbon concentration that range from

20-100 mg/l.

On optimization of one of the nutrients was Sodium nitrate on the production of

phycoerythrin also been investigated in which 3.75mg/l of NaNO3 showed significantly

increased production of C-Phycoerythrin. It was increased concentration than the

control (3mg/l) and the concentration of C-Phycoerythrin was found to be 261.61mg/g

(Table 4)

There are several reports on the growth limitations of microbial communities.

Bacterial biomass, production, and rate of growth of all responded to organic

enrichments, indicating that bacterial growth was constrained primarily by the

availability of dissolved organic matter (Church et al., 2000) Carbonate and

bicarbonate ions are the most easily available forms of inorganic carbon to the marine

Cyanobacteria. In the present study, sodium carbonate (Na2CO3) was used as the source

of carbon in the medium. Farjala et al., (2006) have suggested that the low availability

of phosphate and nitrogen coupled with excess organic carbon was the main factor

Table 3 Influence of carbonate concentration on pigment production in Phormidium tenue NTDM05

Concentration of Na2CO3 (mg/g)

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

20

40

60

80

100

164.01 ± 1.50

155.25 ± 0.93

131.03 ± 0.72

111.37 ± 1.81

103.04 ± 0.52

71.32 ± 1.21

63.41 ± 0.52

59.04 ± 1.41

51.62 ± 1.75

45.81 ± 0.63

303.26 ± 1.31

291.67 ± 0.36

249.24± 1.75

201.13 ± 1.21

195.35 ± 1.23

Values are mean ± SD of three values

Table 4 Influence of nitrate concentration on pigment production in Phormidium tenue NTDM05

Concentration of NaNO3 (mg/g)

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

1.5

2.25

3.0

3.75

4.5

5.25

6.0

71.21 ± 1.75

73.35 ± 0.63

85.19 ± 0.52

138.43 ± 0.72

120.18 ± 0.36

115.21 ± 1.50

95.36 ± 1.81

29.16± 1.21

31.27 ± 0.93

35.36 ± 1.81

55.15 ± 1.23

53.67 ± 1.12

49.23 ± 1.31

45.21 ± 0.63

121.36 ± 0.52

132.21± 1.23

163.15 ± 0.36

261.61 ± 1.41

221.57 ± 1.75

211.12± 1.21

191.02 ± 0.93

Values are mean ± SD of three values

66

responsible for the relatively low bacterial utilization of DOC. Badger and Andrews

(1982) stated that during steady-state photosynthesis in seawater equilibrated with air,

HCO- uptake into cell is the primary source of internal inorganic carbon for

cyanobacteria. Sultemeyer et al., (1998) have stated the presence of inducible high

affinity HCO- and CO2 transporter at the chloroplast envelope membrane of

microalgae. Use of Chlorophyll-a fluorescence measurements for ascertaining the

photosynthetic condition of entire phytoplankton communities across large temporal

(Suggett et al., 2006a) and spatial (Behrenfeld et al., 1996; Suzuki et al., 2002; Moore

et al., 2005, 2006a,b and Suggett et al., 2006b). Chlorophyll-fluorescence was used to

study the relation between increased carbon substrate and growth of the cyanobacteria.

Fox (1996) has observed the presence of carbon supplement enhancing chlorophyll-a

content ranging from 6.1 to 7.6 mg/l in Spirulina sp.

The present study also analyzed the effect of carbon on other pigments such as

phycocyanin and allophycocyanin. Fox (1996) has observed the influence of carbon

content level of carotenoids in Spirulina and reported that it’s ranged from 2.9 to

4.0mg/g. In the present study when 400mg/l carbonate was provided, the carotenoids

production was 12.7µg/l, which is far higher than the earlier reports. Whereas in

Phycocyanin content was found to be highest (7.2 µg/l) at 450mg/l which lesser than

the reports of Palaniselvam et al., (1998), in which they observed 13ug/l level of

phycocyanin in the Phormidium tenue NTDM05 at carbonate concentration of 20mg/l.

the allophycocyanin and phycoerythrin showed the maximum concentration of 11.2

µg/l and 3.3 µg/l at 400mg/l of carobonate respectively. In earlier studies Fatima et al.,

(1998) have recorded the phycocyanin level of 13mg/g in Phormidium tenue in the

presence of carbonate. Sakthivel (2004) reported that the level of phycocyanin varies

from 2.99 to 14.63 mg/g respectively in Oscillatoria cortiana and Phormidium tenue.

It was observed that there is no significant increased production of

phycobiliprotein was found in pH optimization study (Table 5). At pH 8 showed

comparatively higher amount of phycoerythrin (110.22mg/g). When pH increase

production of pigments also decreased and after the incubation period, pH of the

medium has been changed into neutral in all the experiment. The cellular mechanism of

Table 5 Influence of pH on pigment production in Phormidium tenue NTDM05

pH

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

4.0 ± 0.5

6.0 ± 0.5

8.0 ± 0.5

10.0 ± 0.5

12.0 ± 0.5

22.03 ± 1.75

37.63 ± 1.21

65.33 ± 0.63

61.22 ± 0.93

45.36 ± 1.81

9.0.12 ± 0.72

13.03 ± 0.52

29.23 ± 1.41

24.11 ± 1.23

19.01± 0.36

39.33 ± 1.31

71.45 ± 1.21

110.22 ± 1.50

103.63 ± 1.81

81.03 ± 0.63

Values are mean ± SD of three values

Table 6 Influence of temperature on the production of pigment from Phormidium tenue NTDM05

Temperature (oC)

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

15

25

35

45

55

41.11 ± 1.50

55.36 ± 1.31

73.33 ± 1.23

71.22 ± 1.75

69.12 ± 0.72

17.01 ± 1.23

21.03 ± 1.81

29.36 ± 0.93

27.33 ± 0.63

28.30 ± 1.21

71.36 ± 0.52

90.33 ± 0.72

125.02 ± 1.23

123.45 ± 1.21

118.61 ± 0.52

Values are mean ± SD of three values

67

the cyanobacteria have played significant role in the alteration of pH in the medium. In

temperature study 35oC showed significant results in the production of pigments

(125.02mg/g) of phycoerythrin. Both pH and temperature of the medium not showed

tremendous changes in the production. Concentration of the pigments was same that of

pigments produced in normal condition and no significant on the production of the

biomass (Table 6).

During the process of extraction, cyanobacterial cells were lysed by freezing at

20oC and thawing at room temperature, which also allows APC and PE with other

proteins and nucleic acids to be extracted along with CPC. The absorption spectra of

crude extract of Phormidium tenue NTDM05, showed the presence of C-PC, APC,

other proteins, and nucleic acids corresponding to their maximum absorption at 620,

652, 280, and 260 nm wavelengths, respectively (Fig 5).

Extraction was considered to be important method to extract C-Phycoerythrin

very effectively. Among tested methods, freeze thawing process was considered to be

the best extraction method (Table 7). It extracts nearly 120.56mg/g of C-Phycoerythrin

pigment. Whereas in other methods, there was considerable amount of pigment loss and

needs skilled personnel to extract them. During sonication process for extraction, the

temperature was raised about 50oC; in this temperature all pigment protein gets

precipitated and denatured. It extracts 51.22mg/g of c-phycoerythrin. In grinding

method, phycobiliprotein losses its color and it was very difficult to purify the C-

Phycoerythrin and other cellular protein. It gives 49.32 mg/g of C-Phycoerythrin (Fig

6).

Selection of suitable buffer for maximum phycobiliproteins extraction is very

crucial (Viskari and Colyer, 2003) as these ensure that proteins are not denatured due

to shift in pH (Roe, 2000). During comparison of various buffers, sodium

phosphate buffer was found as best buffer with 130.68mg/g phycobiliprotein. Whereas,

extraction in double distilled water was found to be 115 .02mg/g. The lowest

yield in distilled water may be due to its incapability to lyse the cell wall. Hemlata

et al., (2011) reported that sodium phosphate buffer was best for extraction of 100 mg/g

Figure 5. Different extracion methods. A) Grinding B) Sonication C) Osmosis D) Freeze thawing methods were adopted for extraction of C-Phycoerythrin of phycobiliproteins from Phormidium tenue NTDM05. E) Uv-Vis spectrum of extracted C-Phycoerythrin after optimization of different methods.

Table 7 Influence of methods for extraction of Phycobiliprotein from Phormidium tenue NTDM05

Methods

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

Sonication

Osmosis

Freeze-thawing

Grinding

29.01 ± 0.52

63.03 ± 0.93

71.67 ± 0.63

27.03 ± 0.72

11.03 ± 1.21

15.25 ± 1.75

29.23 ± 0.36

9.05 ± 1.81

51.22 ± 1.50

92.01 ± 1.31

120.56 ± 1.21

49.32 ± 1.50

Values are mean ± SD of three values

A-D

68

of phycobiliprotein. Soni et al., (2006) a l so found ou t the p resence o f

Sod ium phospha te showed h igher phycocyan in in phycocyanin in

Oscillatoria and Spirulina Sodium phosphate buffer is also being reported to have

inhibition property to metabolic enzymes. Cyanobacterial cell walls are thick and

extremely resistant t o e x t r e me c o n d i t i o ns (Stewart & Farmer, 1984). It acts as

physical barrier fo r the release of phycobiliprotein into the extracting

medium. So, optimization of cell disruption method is also very important for the

maximum extraction (Table 8).

Fourier transform infrared spectra (FT-IR spectra) IR spectra of freeze-dried C-

PE (Fig. 7) of Phormidium tenue NTDM05 showed the protein specific amide I band at

1637 cm-1 (C=O stretching) and amide II at 1365 cm-1. Position and shape of amide I

band are employed for the analysis of protein secondary structure. The sharp amide I

band at 1637 cm-1 for C-PC indicates the a-helix as the major element of its secondary

structure. It was found that IR spectrum of C-PC of Phormidium, Spirulina and

Lyngbya sp showed same type peak pattern. Besides their experiment, IR spectra of

freeze-dried C-PC further ensure its purity by the absence of inorganic sulfates and

phosphates (represent intense band at 1043 and 1015 cm-1) (Patel et al., 2005).

The absorption spectrum was recorded with preservative and without

preservative at 4oC and 35oC. It was found that the loss of C-PE content in aqueous

solution (i.e. buffer of pH 7.0) at 0±5oC is less than 35±5oC. Loss of C-PE content

observed after addition of different preservatives at 0±5oC is very less, ranging from

2.5 to 40 mg/g while at 35±5oC. C-PE content loss is 5-10mg/g after 45 days as shown

in Table 9. C-Phycoerythrin in aqueous phase was found to be more stable with citric

acid (1 mg/ml) as preservative at 35±5oC, as observed through the spectral analysis, no

discoloration of the C-Phycoerythrin was observed after 45 days both at 0±5 ◦C and

35±5oC with citric acid as preservative and showing only 9.55mg/g loss of C-PE

content at 35±5oC after 45 days, while only 5mg/g C-PE loss was observed at 0±5oC

after 45 days. Even after 45 days C-PE in aqueous solution with citric acid at 35±5oC

retained its color as shown in Fig. 8 C-PE solution without any preservative (control)

showed discoloration after 5 days with 25mg/g loss of C-PE content and completely

Figure 6 Display of phycobiliprotein extracted from Phormidium tenue NTDM05 after optimized production: phycoerythrin (red) and Phycocyanin (blue) extracted from Spirulina subsalsa NTDM21

Table 8 Influence of buffers for extraction of Phycobiliprotein from Phormidium tenue NTDM05

Buffers

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

HEPES

Phosphate Buffer

Tris – Buffer

D. Distilled water

49.23 ± 0.93

75.33 ± 1.75

45.51 ± 1.21

69.36 ± 1.50

21.11 ± 0.52

29.36 ± 1.31

19.01 ± 1.75

25.02 ± 1.81

92.33 ± 0.72

130.68 ± 1.21

85.84 ± 0.63

115.02 ± 0.52

Values are mean ± SD of three values

Table 9 Optimization of preservatives for preservation of C-Phycoerythrin extracted from Phormidium tenue NTDM05 (initial concentration of C-PE- 50mg/g)

Preservatives

Amount of Phycobiliproteins (mg/g)

Phycocyanin Allophycocyanin Phycoerythrin

Calcium chloride

Citric acid

Fructose

Sucrose

Control

06.05 ± 0.31

22.31 ± 1.50

15.02 ± 0.72

17.45 ± 1.21

07.31 ± 0.52

2.5.01 ± 0.63

08.02 ± 1.21

6.5.05 ± 1.31

07.12 ± 0.93

2.9.23 ± 0.63

10.32± 0.36

40.45 ± 0.52

29.63 ± 1.41

31.67 ± 1.81

11.5.05 ± 0.72

Values are mean ± SD of three values

Frequency (cm-1) Bond Assigned groups

3398.2 N-H Pyrroles

2074.1 NH3 Amino acids

1637.0 N=N Azo compounds

1365.8 C-H Alkanes

662.5 C-H Alkynes

Figure 7 Fourier transform infrared spectra (FT-IR spectra) IR spectra of freeze-dried C-PE of Phormidium tenue NTDM05 and table showing the bonding pattern and assigned groups.

69

discolored after 45 days showing 47.5mg/g loss of C-PE content at 35±5 ◦C, while no

discoloration was observed in C-PE sample even after 45 days at 0±5 oC and C-PE

content loss observed was only 39mg/g, because C-PE is heat sensitive at pH 7.2.

C- Phycoerythrin was found to be sensitive to heat and light in aqueous

solution; C-PE is insoluble in acidic solution (pH 3.0) and is highly soluble and stable

at pH 7.0 which is the reason for its efficient extraction in buffer of pH 7.0. Hence,

after extraction and purification of the product, immediate addition of preservative is

crucial prior to lyophilization and further packing in the amber/dark colored bottles to

protect from light. It denatures at temperature above 45 ◦C at pH 7.0, leading to

discoloring.

The stability of food grade C-PE with citric acid as preservative was found to be

more than sodium chloride, calcium chloride and sucrose at 35±5 ◦C which is

comparable to its stability at 0±5 ◦C; the pH 7.0 is found to be most suitable for

maintaining its unique properties; where it is in hexameric form citric acid acts as a

chelator and lowers the pH preventing the degradation of protein (Jespersen et al., 2005

and Edwards et al., 1997). The present work leads to the conclusion that out of the

three tested edible preservatives-(citric acid, sucrose, sodium chloride and calcium

chloride). Citric acid (conc. 1mg/ml) is one of the most effective preservative for

maintaining the stability of C-Phycoerythrin in aqueous phase at 35±5 ◦C even for 45

days with minimum loss at room temperature 35±5 ◦C.

There was no antibacterial property was observed in extracted phycobiliprotein

against tested organisms. This gives another way to exploit the protein for food industry

for colorant. It has been reported that the effect of various concentrations of C-PE on

three human cell lines using MTS assay which measures the cell viability on the basis

of mitochondrial dehydrogenase activity. In this assay, the C-PE concentrations used

did not adversely affect formazan formation.

Based on the previous hypothesis, phycoerythrin has an impact on the defense

against oxidative stress and to examine the relationship between intracellular ROS-

Figure 8 Effect of preservatives on the stability of C-Phycoerythrin. A) 7th Day incubation at 370C B) 14th Day incubation at 370C C) 30th Day Incubation at 370C

70

inducing action of permanganate and oxidative DNA damage, and further assessed the

protective actions of C-PE. The oxidative DNA damage generated by KMnO4 was

measured by comet assay in fibroblast cells. Various concentrations of KmnO4 were

checked for generation of fibroblast DNA damage (data not shown). The 25 mM

KMnO4 treatment at 4 °C was found to generate consistent comets and was kept

constant throughout the experiment. As compared with the positive control, the

increased tail movements caused by permanganate-induced oxidative DNA damage

were not observed in the cells treated with C-PE.

The fluorescence property of C-phycoerythrin was observed and the absorption

and emission spectra of the purified PE from growth phases of a culture was recorded.

The absorption spectrum of the purified PE from a young culture was same with a

sharp peak at 562 nm but it varied for PE from old culture, whereas the emission

wavelength for purified PE was found to be 580 nm (Fig. 9).

In PE purified from young culture of 25 days of Phormidium tenue NTDM05,

a sharp absorption peak was observed at 562 nm whereas for PE purified from 45 days

old culture a shoulder peak at 542 nm along with an absorption peak at 562 nm was

observed. However, when the same proteins were compared for their emission

wavelength, no difference was found as both the proteins emitted light at 580 nm. This

proved that PE with single subunit was equally functional as PE with two subunits.

It has been reported that the purified PE from young culture of Halomicronema

sp. A32DM showed an absorption peak at 562 nm whereas for PE from old culture, the

absorption peak shifted to 542 nm along with a shoulder peak at 562 nm. On comparing

for their emission wavelength, a similar result to that of Phormidium sp. A27DM was

observed thus proving PE with single subunit to be functional in this culture also (Rigbi

et al., 1980). On the other hand, PE purified, from both young and old cultures

of Lyngbya sp. A09DM showed an absorption peak at 562 nm. However, there was an

increase in the shoulder peak at 620 nm for the PE from old culture. It was suggested

that the culture might have started producing phycoerythrocyanin (PEC) for its survival

in nutrient depletion conditions rather than cleavage of PE observed in the other two

(A)

(B)

Figure 9 Fluorescence property of C-Phycoerythrin extracted from Phormidium tenue NTDM05. A) C-Phycocyanin and C-Phycoerythrin illuminated with Uv-Transilluminator showing bright orange color fluorescence B) fluorescence emission overlay spectra of purified C-Phycoerythrin showing fluorescence property.

Intensity

Wavelength

71

cultures. When compared for emission wavelength, both the proteins emitted light at

580 nm proving both of them to be functional. Phycoerythrin generally emits light at

580 nm (Rigbi et al., 1980). PE purified from all the cultures at all growth phases

emitted light at around 580 nm, thus proving them to be functional.

The results indicated the successful development of an efficient method for the

extraction of C-phycoerythrin from an isolate Phormidium tenue NTDM05. The culture

has the ability to produce high amount of C-PE when grown under normal white light,

suggesting it to be a good candidate for the production and extraction of phycoerythrin.

Freezing–thawing is a mild, nondenaturing condition for the extraction of

phycobiliproteins as compared to other methods we studied. This extraction process

may need to be studied for each strain of cyanobacteria depending upon the cell wall

rigidity, but once the required freezing and thawing temperature are standardized, it

could give very good reproducibility in each batch of extraction.

On the whole, in this chapter a cyanobacterial species was selected among

isolated species such as Phormidium tenue NTDM05 based on the pigment production.

It was found that micronutrients are played an important role in the production of

phycobiliprotein in general and phycoerythrin in particular. The extraction methods are

crucial step to extract the pigmented protein from the cell, in which freeze-thawing was

the best method of extraction. Regarding the preservation of the pigments, citric acid

was found to be best preservatives, which preserves upto one month without losing its

color at 4oC and minimum loss of colors at 37oC. It was found that there was no

antibacterial activity observed in the protein; it can be used for the food additives as

colorant and protein supplements.

Chapter III

Nanobiotechnological Potentials of Marine Cyanobacteria and Diatoms

72

INTRODUCTION

Study of Nanobiotechnology:

Nanotechnology is enabling technology that deals with nano-meter sized

objects. It is expected that nanotechnology will be developed at several levels such as

materials, devices and systems. Synthesis of nanoparticles using biological entities has

great interest due to their unique shape dependent optical, electrical and chemical

properties have potential application in nanobiotechnology. In addition,

nanobiotechnology allows fabrication, synthesis and polymerization of nano and micro

scale structure and architecture of various biomolecules including protein and nucleic

acids with and without organic and inorganic materials.

One key aspects of nanotechnology concerns the development of reliable

experimental protocols for the synthesis of nanomaterials over a range of chemical

composition size and high monodispersity. In the context of the current drive to

develop green technologies in materials synthesis, this aspect of nanotechnology

assumes considerable importance. In utilizing is microorganism for the synthesis of

nanoparticles.

Nanoparticles are now considered much more important due to physical,

chemical and biological properties than that is exhibit in the bulk material. Synthesis of

nanomaterials by physical and chemical ways were studied past decade with impact of

environmental pollution, later researchers developed their synthesis by biological

entities is much simpler and eco-friendly. Most of the prokaryotic and eukaryotic

microorganisms were studied in this direction. Microbial cells are highly organized

units, regarding morphology and metabolic pathways, capable of synthesizing

reproducible particles with well defined size and structure, more importantly; biogenic

nanoparticles often exhibit water-soluble and biocompatible properties, which are

essential for many applications.

Nature has devised various processes for the synthesis of nano and micro scaled

inorganic materials which have contributed to the development of relatively new and

73

largely unexplored area of research based on the biosynthesis of nanomaterials (Sastry

et al., 2004). The synthesis and assembly of nanoparticle would benefit from the

development of clean, non-toxic and environmentally acceptable “green chemistry”

procedures, probably involving organism ranging from bacteria to fungi and even

plants (Bhattacharya and Rajinder 2005 and Sastry et al., 2004). Hence both unicellular

and multicellular organisms are known to produce inorganic material either intra of

extracellularly (Mann, 1996).

Biosynthesis of Nanoparticles:

The development of simple and versatile methods for the preparation of

nanoparticles in a anisotropic size or shape selected and controlled manner has been a

challenging but intellectually rewarding task (Xia et al., 2009 and Sau et al., 2010). The

nanoparticles morphology often emerges as a result of a competitive growth of different

crystallographic surfaces. This is typically achieved by altering the relative growth rates

of different facets by the selective localization of surface-modifying or capping agents,

but also by the modulation of nucleation and reaction parameters such as time,

temperature, reagent concentration, and pH (Sohn et al., 2009).

There are several reports in the literature on the cell-associated biosynthesis of

metallic nanoparticles using several microorganisms. Also, for the past few years,

various rapid chemical methods have been developed for the synthesis of metallic

nanoparticles in general and silver nanoparticles in particular. The synthesis of silver

nanoparticles has attracted much attention because their unique shape-dependent

optical, electrical and chemical properties have potential applications in

Nanotechnology. Silver nanoparticles are used in photographic reactions, catalysis,

chemical analysis, exhibit strong toxicity to a wide range of micro organism and

antibacterial applications, therefore development of silver nanoparticles are expected to

open avenues to fight and prevent diseases (MubarakAli et al., 2011)

Silver nanoparticles production as the nanoparticles possess more surface atom

than a micro particle which greatly improve its characteristics. The biosynthesis of

silver nanoparticles of different sizes ranging from 1- 70nm and shapes, including

74

spherical, triangular and hexagonal has been conducted using bacteria, fungi, plants and

plant extracts.

Metal nanoparticles, particularly gold, are being considered in wide ranging

applications, such as photonics, information storage, electronic and optical detection

systems, therapeutics, diagnostics, photovoltaics, and catalysis (Murphy et al., 2005;

Burda et al., 2005; Sau et al., 2010; Talapin et al., 2010; Cuenya, 2010; Daniel and

Astuc, 2004; Eustis and El-Sayed, 2006; Sardar et al., 2009; Zhou et al., 2009; Rosi

and Mirkin, 2005 and Giljohama et al., 2010). While the chemical composition of a

nanoparticle is important, even more important is the morphology (size and shape) of

the nanoparticles and its surface / colloidal properties.

Biosynthesis of Nanocomposites:

The unicellular eukaryotic algae known as diatoms make cell walls out of silica

with an amazing diversity of structures on the nanoscale (Round et al., 1990). Because

the complexity of diatom silica structures can exceed that possible using synthetic

material production approaches, and diatoms reproduce these structures faithfully, in

enormous numbers, and inexpensively, diatoms are being developed as a source of

nanostructured materials (Sandhage et al., 2005). Facilitating this techniques diatoms

can be utilizing as templates for functionalized coatings (Payne et al., 2005; Rosi et

al., 2004; Weatherspoon et al., 2007), or convert their silica entirely to other molecules

while maintaining nanoscale features (Bao et al., 2007; Kusari et al., 2007; Sandhage

K.H. et al., 2002; Sandhage et al., 2005; Unocic R.R. et al., 2004; Weatherspoon et al.,

2006; Zhao J.P. et al., 2005). In addition, the development of diatoms as a source of

nanostructured materials, it would be beneficial to develop approaches whereby

structure could be genetically tailored to suit particular applications (Hildebrand, 2005;

Sandhage et al., 2005). This requires better understanding of the silicification process.

Understanding the process of diatom silica cell wall synthesis requires elucidation of

the underlying cellular and molecular biology, especially, important is to know how

genes and organic molecules (proteins, polyamines, carbohydrates, and lipids) are

involved in formation of specific mineral substructures.

75

The cell wall structure is a species-specific characteristics demonstrating that

diatom silica morphogenesis is genetically encoded. It is expected that these structures

exhibit superior properties in a wide range of applications. The fabricated and self

assembled of semiconductor nanostructures that possess unique optical and electronic

properties (Rorrer, 2005). The supramolecular chemistry and catalysis have led to novel

surface and size dependent chemistry such as enation selective catalysis at the surface.

The ability of diatoms to make complex, nanoscaled, three dimensional silica shells

called “frustules” offers attractive possibilities for their application to

nanobiotechnology (Hildebrand. 2005).

Bioengineered nanosystem is extremely difficult to realize on the basis of

current scientific knowledge and bottom up assembly is very powerful in creating of

identical structures with atomic precision, such as supramolecular functional entities in

living organisms. In this regards diatoms, a prolific class of single celled algae that

make micro scale biosilica or frustules with intricate submicron features dominated by

two dimensional pore arrays, have been advertized as a paradigm for the controlled

production of nanostructured silica with interesting properties (Sumper, 2006). The

fabrication of novel biomaterials through molecular self assembly is studied currently

(Zhang, 2003).

Biosynthesis of Semiconductor:

CdS is an important semiconductor owing to its unique electronic and optical

properties, and its potential applications in solar energy conversion, nonlinear optical,

photo electrochemical cells and heterogeneous photocatalysis (Hu et al., 1998 and

Weller et al., 1993). The synthesis of CdS nanoparticles has been tried by various

methods such as the direct reaction of metals with sulfur powders under high

temperature (Kaito et al., 1989), the thermal decomposition of molecular precursors

containing M–S bonds (Milhemy et al., 1984 and Ludoph and Malik, 1998) or the use

of poisonous H2S as the sulphur source at higher temperature (Pickeet, 1996) and

chemical precipitation method involving the precipitation of metal ions with Na2S as

the source of S2- ions (Herron, 1990 and Chen, 1996), in which the homogeneity at an

early stage results in a broadening of the product particle size distributions.

76

Developing reliable protocols for the synthesis of nanometer scale

semiconductor particles is a problem of great importance, (Shipway, 2000 and

Alivisatos, 1996) resulting in researchers seeking inspiration from biological systems

where biominerals are routinely synthesized (Addadi, 1992 and Mann, 1996). Both

prokaryotic (bacteria) (Labrenz, et al., 2000 and Holmes, et al., 1995) and eukaryotic

organisms such as yeast (Dameron, et al., 1989) have been found to produce

semiconductor nanoparticles within the cell wall of the microorganisms.

Cadmium sulfide nanoparticles (CdS NPs) are one of the widely studied

semiconductor nanoparticles that have unique photochemical and photo physical

properties. The semiconductor nanoparticles were often referred as Quantum Dots

(QDs), these particles when embedded within an appropriate matrix act as potential

wells that confine and stabilize electrons in discrete energy levels. The technologically

useful properties of CdS QDs are due in part to the fact that the band-gap is tunable

over a range of 1.5 to 3.5eV (Flenniken, et al., 2004).

Biological synthesis of CdS NPs has been studied effectively by wide range of

organisms for instance algae (Hosea, 1986 and Scarano and Morelli, 2003), fungi

(Ahmed, 2002), yeast (Dameron, et al., 1989 and Prasad and Anal Jha, 2010) and

bacteria (Bai, et al., 2009 and Prasad and Anal Jha. 2010). More lately, apart from the

synthesis of such CdS NPs, the stabilization of synthesized particles were mediated by

extracted and purified active compounds from microbes is efficient method to

understand the stabilizing agent present in the microorganism.

A deserve mention that the cyanobacteria and diatom species selected for this

purpose is easily and abundantly available in eutrophic ponds and marine ecosystem of

India as well as in different parts of the world. Some other characteristics of these

organisms are efficient and rapid biosorption of metals, low production cost, ease in

separation of compounds from the culture media. In order to utilizing the efficiency of

nanomaterials synthesis in cyanobacteria and diatom, the work have framed with the

following objectives:

77

To synthesis silver and gold nanoparticles using selected marine

Cyanobacteria

To synthesis and stabilize CdS nanoparticles by extracted c-phycoerythrin

from Cyanobacteria

To synthesis silicon-germanium oxide (Si-Ge) nanocomposites using

Diatom

To characterize the synthesized nanocomposites and nanoparticles by TEM,

HRSEM, EDAX, EDS, FTIR, FS, Uv-Vis spectroscopic studies

To evaluate the antimicrobial properties towards multi drug resistant (MDR)

clinical human pathogens.

To evaluate the antioxidant property and fluorescence property of

synthesized nanoparticles.

78

REVIEW OF LITERATURE

The study of nanometer size crystallites provides an opportunity to observe the

evolution of material properties with size. The study of intermediate size regime gives

the information about the collective behavior of bulk materials which emerges from the

discrete nature of molecular properties.

Physical and Chemical Properties of Nanoparticles:

The principal parameter of nanoparticles is their shape (including aspect ratios

where appropriate), size and the morphological sub-structure of the substance.

Nanoparticles are presented as an aerosol (mostly solid or liquid phase in air), a

suspension (mostly solids in liquids) or an emulsion (two liquid phases). In the

presence of chemical agents (surfactant), the surface and interfacial properties may be

modified. Indirectly such agents can stabilize against coagulation or aggregation by

conserving particle charge and by modifying the out most layer of the particle.

Depending on the growth history and the lifetime of nanoparticles, very

complex compositions, possibly with complex mixtures of adsorbents, have to be

expected. In the typical history of combustion nanoparticles, for example, many

different agents are prone to condensation on the particle while of processes are to be

expected and have been identified only for a small number of particulate model

systems. At the nanoparticles – liquid interface, polyelectrolyte have been utilized to

modify surface properties and the interaction between particle and their environment.

They have been used in a wide range of technologies, including adhesion, lubrication,

stabilization, and controlled flocculation of colloidal dispersions (Liufu et al., 2004).

At some point between the Angstrom level and the micrometer scale, the simple

picture of nanoparticles as a ball or droplet changes. Both physical and chemical

properties are derived from atomic and molecular origin in a complex way. For

example, the electronic and optical properties and chemical reactivity of small cluster

are completely different from better known property of each component in the bulk or

at extended surface. Complex quantum mechanical models are required to predict the

79

evolution of such properties with particle size, and typically very well defined

conditions are needed to compare experiments and theoretical predictions.

Nanoparticle – Nanoparticle Interaction:

At the nanoscale, particle-particle interaction is either dominated by weak van

der walls forces, stronger polar and electrostatic interaction or covalent interactions.

Depending on the viscosity and polarisability of the fluid, particle aggregation is

determained by the interparticle interaction. By the modification of the surface layer,

the tendency of a colloid to coagulate can be enhanced or hindered. For nanoparticles

suspended in air, charges can be accumulated by physical processes such as glow

discharge or photoemission. In liquids, particle charge can be stabilized by

electrochemical processes at surfaces. Fluid interactions are of key importance to

describe physical and chemical processes, and the temporal evolution of free

nanoparticles. They remain difficult to characterize due to the small amount of

molecules involved in the surface layer. Surface energy, charge and salvation are

relevant parameters to be considered due to the crucial role of the nanoparticles.

Nanoparticles interaction and the nanoparticles are fluid interaction; the term

free nanoparticles can be easily misunderstood. The interaction forces either attractive

or repulsive, crucially determine the fate of individual and collective nanoparticles

resulting in aggregates and or agglomerates may influence on their behavior. In gas

suspensions, aggregation is crucially determined by the size and diffusion, and

coagulation typically occurs in fasters then in the liquid phase as the sticking

coefficient is closer to unity than in liquids 3.5 sources of free nanoparticles.

Nanoparticles are formed through the natural or human mediated disintegration

of larger structure or by controlled assembly processes. The associated processes occur

either in the gas phase, in a plasma, in a vacuum phase or in the liquid phase, eventually

followed by the intentional or unintentional transfer into one or more relevant fluid

media and then to an individual receptor in an exposure setting.

80

Formation of Nanoparticles in Liquid phase:

Defined bottom up production of nanoparticles on the liquid phase with respect

to particle size, chemical composition, surface and change properties occurs mainly

through controlled chemical reactions (Frens, 1973), and self assembly processes have

evolved by controlling growth conditions. In view of the ecological cycling of

nanomaterials, some emphasis has to be given to the corrosion and disintegration of

bulk material, where little knowledge is currently available (Oberdorster, et al., 2005).

Naturally occurring processes generating nanosized structures in the liquid

phase include erosion and chemical disintegration of organic (plant and micro organism

debris) or geological (clays) parent materials. In all these types of disintegration

process, the surface properties and their change through chemical reaction are critical in

determining whether individual nanoparticles will be formed in the respective medium

(Boyle et al., 2005). Schematic representation of formation of silver nanoparticles in

liquid phase by chemical process (Fig. 1).

Figure 1 Schematic representation of formation of silver nanoparticles in liquid phase by chemical process

Formation of Nanoparticles in the Gas phase:

The main route of bottom- up formation of nanoparticle in the gas-phase is by a

chemical reaction leading to a non-volatile product, which undergoes homogeneous

nucleation followed by condensation and growth. Recently, this has become an

81

important pathway for the industrial production of nanoparticle powders, which may be

of metal, oxides, semiconductors, polymers and various forms of carbon, and which

may be in the form of spheres, wires, needles, tubes, platelets, or other shapes. This is

also the unintentional pathway by which nanoparticles are formed following the

oxidation of gas-phase precursors in the atmosphere, in volcanic plumes, in natural and

man-made combustion processes, or in fumes associated with any man-made process

involving volatilizable material at elevated temperature, such as welding or smelting,

polymer fabrication, or even cooking .

As with the liquid phase case, disintegration processes of parent materials

provide a pathway which only leads to nanoparticles suspended in the gas phase under

special conditions. While in the liquid phase the presence of emulsifying agents

accompanying an erosion or chemical disintegration process could support the

suspension process, the dispersion of nanoparticles into a gas from liquid emulsions or

dry powders is severely limited by the strong adhesive forces between individual

nanoparticles. Therefore, any mechanically induced stress on the parent material mostly

leads to particles in the micrometer range and above. Only under accidental conditions,

example in the case of uncontrolled release of a powder or an emulsion from a highly

pressurized vessel could strong shear forces overcome these adhesive forces (Reeks and

Hall, 2001). In contrast, the spraying of liquids containing nanoparticles or soluble

material at very low concentrations, followed by drying of the solvent, can lead to the

resuspension of nanoparticles or to the formation of new nanoparticles from the solutes.

This can lead to redistribution of nanoparticles, biological material or toxic substances

into nanoparticulate airborne form.

Environmental Sources of Airborne Nanoparticles:

The amount of nanoparticles in the air can be surprisingly similar in urban and

rural areas, with as much as 106 to 108 nanoparticles per liter of air depending on

conditions. In rural areas; nanoparticles mostly originate from the oxidation of volatile

compounds of biogenic or anthropogenic origin, including secondary organic aerosols.

In urban areas, the primary sources of these particles are diesel engines (Schneider et al

2005) or cars with defective or cold catalytic converters (Zhiqiang et al., 2000). Photo-

82

oxidation processes also lead to significant numbers of nanoparticles in urban areas.

Real-time measurements show that exhaust aerosol concentrations range between104 to

106 particles cm-3 with the majority of the particles by number being less than 50nm in

diameter. The highest particle number concentrations and smallest particle size are

associated with high-speed road traffic, presumably due to the subtle conditions during

concomitant cooling and dilution of the exhaust gases. Emission factors for gasoline

vehicles ranged from 1.9 to 9.9x1014 particles.km-1 and 2.2x1015 to 1.1x1016

particles km-1 fuel (Kittelson et al., 2003a,b).

The awareness that combustion processes significantly contribute to the

nanoparticles load by number has been rising recently and has provided a new

motivation for airborne particle research (Donaldson et al., 2001a). Wieser and

Gaegauf (2000) have evaluated different wood combustion systems with respect to

emissions. Particle sizes were mainly in the range of 30 to 300 nm. Particles of less

than 300 nm did not add much to the total particle count in flue gas. The particle

distribution of manually operated appliances varied during a burn cycle, while

continuous fed wood combustion systems show a fairly constant particle size

distribution. Total particle numbers for automatically fed burners were smaller than

with manual operation. Around 95% of the particles were smaller than 400 nm. The

most frequent size of the particle number concentrations for batch operated appliances

is approximately 110 nm, whereas the particle distribution changes significantly during

the burn cycle. Silver nanoparticles array on Track Etch membrane support as flow-

through optical sensors for quality control (Julian and et al., 2007).

Biosynthesis of Nanoparticles using Cyanobacteria:

Morphological control over the shape of gold nanoparticles has been achieved

by using Plectonema boryanum UTEX 485, a filamentous cyanobacterium. When it

was reacted with aqueous Au(S2O3)23- and AuCl4

- solutions at 25-100oC for up to 1

month and at 200oC for 1 day resulted in the precipitation of cubic gold nanoparticles

and octahedral gold platelets, respectively (Lengke et al., 2006).

83

The mechanism of gold bioaccumulation by cyanobacteia (Plectonema

boryanum UTEX 485) from gold (III) chloride solutions have documented that

interaction of cyanobacteria with aqueous gold (III)-chloride initially promoted the

precipitation of nanoparticles of amorphous gold(I)- sulfide at the cell walls, and finally

deposited metallic gold in the form of octahedral(III) platelets near cell surfaces and in

solutions (Lenkge et al., 2006).

Adding further to the mechanism, a sulfate reducing bacterial enrichment was

used to destabilize gold(I)- thiosulfate complex to elemental gold and proposed that this

could occur by three possible mechanisms involving iron sulfide, localized reducing

conditions, and metabolism (Lengke and Southam, 2006) (Table 1).

Table 1 Biosynthesis of nanoparticles using Cyanobacteria:

S. No.

Cyanobateria used Nanoparticles produced

Size References

1. Plectonema boryanum UTEX 485

Ag,

Au,

Pd,

Pt

200nm

<10nm

30nm

300nm

Lengke et al. 2005, 2006, 2007

2. Anabaena sp. Au, Ag, Pd, Pt <70 nm Brayner et al., 2007

3. Calothrix sp. Au, Ag, Pd, Pt <70 nm Brayner et al. 2007

4. Leptolyngbya sp. Au, Ag, Pd, Pt <70 nm Brayner et al., 2007

5. Spirulina platensis Ag,

Au and Bimetallic

7-16nm

6-10nm

17-25nm

Govindraju et al., 2008

6. Oscillatoria earlei NTDM03

Colloidal Ag 150 nm Mubarak Ali and Thajuddin, 2009

7. Spirulina subsalsa Au <20nm Chakroborty et al, 2009

8. Lyngbya majuscula Au <20nm Chakroborty et al, 2009

9. Oscillatoria willei NTDM01

Ag 150nm Mubarak Ali et al., 2011

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Synthesis of metallic and other semiconductor nanoparticles by variety of

microbial entity to plant materials. Synthesis of nanoparticles from bacteria (Table 2),

actinomycetes (Table 3), fungi (Table 4), yeast (Table 5), microalgae (Table 6), plants

(Table 7) and biomolecules (Table 8). All are listed in separate tabular form.

Table 2 Biosynthesis of Nanoparticles using Bacteria:

S.No Biological entity Nanoparticles

Produced Size Reference

1 Bacillus subtilis 168 Au 5-25 nm

Beveridge and Murray 1980

2 Pseudomonas stutzeri AG259 Ag 35-46 nm Slawson et al., 1992

3 Clostridium thermoaceticum CdS -

Cunningham and Lundie 1993

4 Pseudomonas stutzeri Ag 200 nm Klaus et al., 1999

5 Pseudomonas stutzeri Ag 200 nm Joerger et al., 2000

6 Desulfobacteriaceae ZnS 2-5 nm Labrenz et al., 2000

7 Magnetotactic bacteria Fe3O4 & FeSO4 - Roh et al., 2001

8 Desulfovibrio desulfuricans Pd - Yong et al., 2002

9 Lactobacillus Strains Au-Ag alloy - Nair and Pradeep 2002

10 E. coli CdS - Sweeney et al., 2004

11 Pseudomonas aeruginosa Au 15-30 nm Husseiny et al., 2007

Table 3 Biosynthesis of Nanoparticles using Actinomycetes

S.No Biological entity (Actinomycetes)

Nanoparticles Produced

Size Reference

1 Rhodococcus. Sp Au 5-15 nm Ahmad et al., 2003

2 Thermomonospora. Sp Au 8 nm Ahmad et al., 2003

3 Thermomonospora. Sp Au - Sastry et al., 2003

85

Table 4 Biosynthesis of Nanoparticles using fungi

S.No Biological entity Nanoparticles

produced Size Reference

1 Verticillium sp. Au 20 nm Mukherjee et al., 2001

2 Verticillium sp. Ag 25 ±12 nm Mukherjee et al., 2001

3 Fusarium oxysporium Au 20-40 nm Mukherjee et al., 2002

4 Fusarium oxysporum CdS 5-20 nm Ahmad et al., 2002

5 Colletotrichum sp. Au 20-40 nm Shankar et al., 2003

6 Fusarium oxysporium Ag 5-15 nm Ahmad et al., 2003

7 Fusarium oxysporum ZrO2 3-11 nm Bansal et al., 2004

8 Verticillium sp. Ag 25 ± 12 nm Senapati et al., 2004

9 Fusarium oxysporum BaTiO3 4-5 nm Bansal et al., 2004

10 Trichothecium sp. Au - Ahmad et al., 2005

11 Fusarium oxysporum Si and Ti 5-15 nm Bansal et al., 2005

12 Fusarium oxysporum and Verticillium sp.

Fe3O4 20-50 nm Bharde et al., 2006

13 Verticillium luteoalbum Au 100 nm Gericke and Pinches 2006b

14 Aspergillus fumigatus Ag 5-25 nm Bhainsa and D’Souza 2006

15 Fusarium oxysporum CdS - Kumar et al., 2007

Table 5 Biosynthesis of Nanoparticles using Yeast

S.No Biological entity Nanoparticles Produced

Size Reference

1 Candida glabrata CdS 20 Ǻ Dameron et al., 1989

2 Schizosaccharomyces pombe CdS 20 Ǻ Dameron et al., 1989

3 Schizosaccharomyces pombe CdS 1-1.5 nm Kowshik et al., 2002

4 MKY3 Ag 2-5 nm Kowshik et al., 2003

5 P. jadinii Au 100 nm Gericke and Pinches 2006

86

Table 6 Biosynthesis of Nanoparticles using Microalgae:

S.No Biological entity Nanoparticles Produced

Size Reference

1 Chlorella vulgaris Au - Hosea et al., 1986

2. Phaeodactylum tricornutum CdS - Scarano and Morelli, 2003

3. Shewanella algae Au 10-20

nm Konishi et al., 2004

4. Rhizoglonium hieroglyphum Au <20nm Chakroborty et al, 2009

5. Navicula minima Au <20nm Chakroborty et al, 2009

6. Rhizoglonium riparium Au <20nm Chakroborty et al, 2009

7. Chlorella sp Al2O3 Sadiq et al., 2011

8. Chlorella vulgaris Au 40-60nm Tiyaporn et al., 2011

9. Stauroneis sp. Si-Ge 200 nm Mubarak Ali et al., 2011

Table 7 Biosynthesis of Nanoparticles using Plants:

S. No Plant materials used Nanoparticles

produced Size References

1. Cinnamomum camphora Ag and Au 55 - 80nm Huang et al., 2006

2. Medicago sativa Ag 2-3nm Gardea-Torresdey et

al., 2003

3. Pelargonium graveolens Ag 16-40nm Shankar et al., 2003

4. Avena sativa Au 5-20nm Armendariz et al.,

2004

5. Azadirachta indica Ag, Au and Ag-

Au 50-100nm Shanker et al., 2004

6. Tamarindus indica Au 20 to 40 nm Shanker et al., 2004

7. Emblica officinalis Ag and Au

(10-20nm) &

(15-25nm) Ankamwar et al.,

2005b

8. Medicago sativa Ti/Ni bimetallic 1-4nm Schabes-Retchkiman

et al., 2006

9. Aloe vera Ag and Au 15.2 ± 4.2nm Chandran et al., 2006

10. Coriandrum sativum Au 6.75-7.91nm Badrinarayanan and

Sakthivel, 2008

11. Carica papaya Ag 60 to 80 nm Mude et al., 2009

12. Parthenium hysterophorus Ag 52-80nm Parashar et al., 2009

13. Avena sativa and Tritium

vulgare Au <20nm

Armendariz et al., 2009

14. Mentha piperita Ag and Au 90nm and

150nm MubarakAli et al.,

2011

87

Table 8 Biosynthesis of Nanoparticles using Biomolecules

S.No Biomolecules Nanoparticles

Produced Size

Reference

1 R-Phycoerythrin CdS 15 nm Brekhovskikh et al., 2005

2 Soluble Starch Ag 10-34 nm Vigneshwaran, et al., 2006

3 Starch CdS 15nm Rodriguez, et al., 2008

4. C-Phycoerythrin CdS 5nm MubarakAli et al., 2012

Diatom biology:

Diatoms have played a decisive role in the ecosystem for millions of years as

one of the foremost set of oxygen synthesizers on earth and as one of the most

important source of biomass in oceans. Estimates indicate that these photosynthetically

active organisms are responsible for 20-25% of total terrestrial primary production

(Field et al., 1998) and approximately 40% of annual marine biomass production

(Falkowski et al., 1998), making them the most dominant group of organisms

sequestering carbon from the atmosphere.

Diatoms have fascinated botanists since they were first discovered by light

microscopy and mistakenly addressed as unicellular animals due to their brownish

color and their capability to move on substrates (Lind et al., 1997). According to the

species concept, organisms mating with each other belong to the same species. Diatom

taxonomy thus was based mainly either on the identification of ribosomal sequences

(Medlin et al., 1996) or more classically on the morphology and the shape of the

frustules, the extracellular silica cell walls. These frustules, which are formed by two

valves that fit together like Petri dishes, often are highly ornamented and show species-

specific structures.

Diatoms play a significant ecological role; about half of the global annual net

primary production in the oceans is due to phytoplankton which is dominated by

diatoms (Falkowski et al., 1998). Diatoms thus produce and represent the main input

into the marine food web. They are not only strongly involved in fixation of CO2, but

88

also in cycling of soluble silicates by integrating them into the shells and by releasing

parts of them after decomposition at the bottom of the oceans (Bidel and Azam, 1999).

It is not yet fully understood why diatoms filled aquatic niches so successfully, even

though they have the capability to grow at a wide range of light intensities (Falkowski

et al., 2004) and the apparent ability to perform C4 photosynthesis (Reinfelder, 2004).

Diatom nanotechnology:

The ability of diatoms to genetically define these structures makes them highly

interesting for nanotechnological applications (Drum and Gordon, 2003). Because of

their siliceous composition frustules were often well preserved in fossil deposits

(Damste et al., 2004) and strong sedimentation of diatom shells in ancient oceans led to

the deposition of siliceous earth or diatomite, a material of high importance for

industrial uses (Harwood, 1999).

There is a tremendous research interest in the area of nanotechnology to develop

reliable processes for the synthesis of nanomaterials over a range of sizes and chemical

compositions. Although the conventional methods of synthesis of metal sols, known

since the times of Michael Faraday, continue to be used for generating metal

nanoparticles, there have been several improvements and modifications in the methods

which provide a better control over the size, shape, and other characteristics of the

nanoparticles (Burda, et al., 2005 and Sun and Xia, 2002). These developments have

enabled studies of quantum confinement as well as other properties dependent on size,

shape, and composition (Rao, et al., 2002).

Ligating nanoparticles with organic molecules and assembling these in one,

two, or three-dimensional mesostructures have added another dimension to this field

where in collective properties of nanoparticles have been of special interest

(Kakkassery et al., 2004). The exciting potential of nanomaterials can be routed to nano

device applications, only with a combination of nano building units and strategies for

assembling them.

89

Synthesis and assembly strategies of nanoparticles mostly accommodate

precursors from liquid, solid or gas phase; employ chemical or physical deposition

approaches and similarly rely on either chemical reactivity or physical compaction to

integrate the nanostructure building blocks within the final material structure.

Bottom up approach:

The bottom up approach of nanomaterials synthesis first forms the

nanostructured building blocks (nanoparticles) and then assembles these into the final

material. An example of this approach is the formation of powder components through

aerosol and sol-gel techniques and then the compaction of the components into the final

material. Nanoparticles with diameters ranging from 1 nm to 10 nm with consistent

crystal structure, surface derivatisation, and a high degree of monodispersity can be

processed by these techniques (Murray et al., 1993).

Top-down approach

The top-down approach begins with a suitable starting material and then sculpts

the functionality from the material. This technique is similar to the approach used by

the semiconductor industry in forming devices out of an electronic substrate (silicon),

utilizing pattern formation (such as electron beam lithography) and pattern-transfer

processes (such as reactive-ion etching) that have the requisite spatial resolution to

achieve creation of structures at the nanoscale. This particular area of nanostructure

formation has tremendous scope and is a driving issue for the electronics industry.

90

Figure 2 Schematic representation of top down and bottom up process

Diatom Nanocomposite:

Nanotechnology, the presumed key technology of the twenty-first century, aims

to control the structure of matter on a scale between 1–100 nanometers, as this allows

91

exploitation of new physical phenomena (Poole and Owens, 2003). The establishment

of an industry that produces nanotechnology based devices critically depends on

methods for inexpensive manufacturing of nanoscale materials on a massively parallel

scale; yet current nanomanufacturing methods are slow and require expensive

equipment.

It has recently been suggested that the silica structures produced by diatoms

may be particularly useful for nanotechnological applications, because they exhibit

highly controlled nanopatterns that can be produced by biological self-assembly in

large quantities and at low cost (Parkinson and Gordon, 1999 and Sandhage et al.,

2005). A variety of applications have been suggested for diatom silica, including

membranes for biomolecule separations (Losic et al., 2006; Parkinson and Gordon,

1999), size-selective biosensors (Parkinson and Gordon, 1999), microelectromechanical

systems (Drum and Gordon, 2003), and masks for microfabrication (Parkinson and

Gordon, 1999).

To fully exploit the potential of diatom silica, methods have been recently

developed to introduce additional functionalities. The chemical attachment of single-

stranded DNA (ssDNA) to cleaned diatom silica enabled the diatom silica to be

covered with a dense layer of gold particles or the fluorescent dye Cy3 through

hybridization with Au-labeled and Cy3-labeled complementary ssDNA. The modified

diatom silica may be useful for catalysis and optics, specifically as substrates for

Surface Enhanced Raman Scattering (SERS) (Rosi et al., 2004).

Recently, an alternative method for biomolecule attachment to diatom silica has

been established by Kroger and coworkers. This method is based on molecular genetic

engineering of T. pseudonana and enables immobilization of the enzyme

hydroxylaminobenzene mutase (HabB) within the diatom silica by expressing a tpSil3-

HabB fusion protein. Due to the presence of the silaffin domain, the fusion protein

became incorporated into diatom silica in vivo, remained attached to the silica after

92

isolation of the cell wall, and the HabB moiety was catalytically active (Poulsen et al.,

2007).

The method is likely applicable to a large variety of enzymes or receptor

proteins, and represents a new paradigm for utilizing biomineralization for the

sustainable production of nanomaterials with tailored functionalities. Because of the

excellent mechanical (Hamm et al., 2003) and mass transport properties of diatom

silica (Losic et al., 2006), enzyme-functionalized diatom cell walls may be attractive

materials for microfluidic-based devices.

The applications for diatom cell walls are limited by the chemical and physical

properties of silica. Therefore, it would be desirable to convert diatom silica into other

materials without changing the morphology. Indeed, the pioneering work of Sandhage

and co-workers has resulted in a number of protocols that yielded exact replicas of

diatom cell walls consisting entirely of MgO, TiO2, BaTiO3, elemental Si, and several

other materials.

These materials have a wide variety of properties and potential applications. For

example the fluorine-doped TiO2 replicas of diatom silica were highly efficient

catalysts for the rapid hydrolysis of organophosphate-based pesticides (Lee et al.,

2007), and the Si replicas represented ultra rapid, low-voltage NO sensors (Bao et al.,

2007).In a different approach, Mirkin and co-workers have used diatom silica as

sacrificial template to generate 30-nm thick silver microshells that preserved the sub-

100-nm topological features of diatom silica and could be used as SERS substrates for

the sensitive detection of organic molecules (Payne et al., 2005).

Nanofabrication:

Currently, there is enormous interest in bioinspired processes for the fabrication

and self-assembly of semiconductor nanostructures that possess unique optical and

electronic Although biomimetic approaches typically focus on biomolecule-mediated

93

processes, living cells also have the potential to direct the fabrication of nanostructured

materials, particularly for photonic device applications (Parker and Townley, 2007).

Diatoms are nature’s priceless manufactures of 3D intricate nanocomposites

(Sandhage et al., 2002). It has been reported that the fabrication of a photosynthetic

biodevice using living diatoms (Leabeau et al., 2002). They revealed that the diatom

cell can be cultured on agar films that are prepared on a glass surface, and these cells

can perform photosynthesis. Although this was an important study on developing

biodevices by using living diatoms, no microscopic characterization of the device was

included. To date no other study has reported the development of biodevices by using

living diatoms.

Biological processes have been developed to incorporate atoms other than

silicon into the silica diatom frustules while maintaining the frustules detailed

morphology. This process has allowed us to fabricate silicon-germanium (Si-Ge) oxide

nanocomposites that possess optoelectronic properties (Rorrer et al., 2005). Diatoms

carry out this process under ambient conditions of temperature and pressure and

produce no environmental pollutants. In that study, several electron microscopy and

microanalysis techniques were used to characterize the morphologies and elemental

composition of the processed diatoms and more importantly to assess and direct the

optimization of the fabrication of the nanocomposites.

The electron microscopy characterization and microanalysis results suggest that

germanium doped into the silica frustules of the diatom has an upper threshold that if

exceeded introduces structural aberrations and the assimilated germanium is randomly

distributed within a diatom frustules. Soluble germanium can be metabolically inserted

into the biosilica of the pennate diatom Pinnularia sp., by a two stage bioreactor

cultivation process, where the incorporation of Ge into the frustules biosilica reduced

the frustule pore diameter but otherwise did not affect the frustules morphology

(Jeffreys et al., 2006).

94

Nanostructured metal oxide semiconductor materials possess unique optical and

electronic properties, and have future applications as photoluminescent materials for

thin film displays, nanoscale electronic devices, or solar cells. Many biological routes

had been tried for the fabrication of nanostructured metal oxide semiconductor

materials that also possess microscale design elements (Rorrer et al., 2005).

Figure 3 Schematic representation of synthesis of Si-Ge oxide formation in diatom cell

95

Applications of Nanoparticles:

Figure 4 Diagramatic representation of application of nanoparticles in different fields

Nanomaterials have been blossoming in the field of nanotechnology. Their

unique size-dependent properties make these materials superior and in dispensable in

many areas of human activity (Murray et al., 2000).

The nanomaterials level is the most advanced at present, both in scientific

knowledge and in commercial applications. The fact that the nanoparticles exist in the

same size domain as proteins makes nanoparticles suitable for biotagging or labeling.

However, size is just one of many characteristics of nanoparticles that it is rarely

sufficient if one is to use nanoparticles as biological tags. The bioinorganic interface

should be attached to the nanoparticles for the good interactions with the biological

target (Bruchez et al., 1998).

96

Nanoparticles usually form the core of nanobiomaterial. It can be used as a

convenient surface for molecular assembly and may be composed of inorganic or

polymeric materials. It can also be in the form of nanovesicle surrounded by a

membrane or a layer. The shape is more spherical but cylindrical, plate-like and other

shapes are possible. The size and size distribution might be important in some cases, for

example, if penetration through a pore structure of a cellular membrane is required. The

size and size distribution are becoming extremely critical when quantum sized effects

are used to control material properties. A tight control of the average particle size and a

narrow distribution of sizes allow creating very efficient fluorescent probes that emit

narrow light in a very wide range of wavelengths. This helps with creating biomarkers

with many and well distinguished colors. The core itself might have several layers and

be multi functional.

Tissue Engineering:

Natural bone surface is quite often contains features that are about 100 nm

across. If the surface of an artificial bone implant were left smooth, the body would try

to reject it. Because of that smooth surface is likely to cause production of a fibrous

tissue covering the surface of implant. This layer reduces the bone-implant contact,

which may result in loosening of the implant and further inflammation (De La et al.,

2003).

In order to reduce the bone implant rejection as well as to induce the production

of osteoblast, the nanosized features can be created on the surface of hip or knee

prosthesis. The osteoblasts are the cells responsible for the growth of the bone matrix

and are found on the advancing surface of the developing bone. More than 90% of the

human bone cells from suspension adhere to the nanostructured metal surface. It was

shown that biomimetic approach a slow growth of nanostructured appetite film from

the stimulated body fluid-resulted in the formation of a strongly adherent, uniform

nanoporous layer. The layer was found to be built of 60 nm crystallites and posses a

stable nanoporous structure and activity (Ma et al., 2003).

97

Cancer Therapy:

Photo dynamic cancer therapy is based on the destruction of the cancer cells by

laser generated atomic oxygen, which is cytotoxic. A greater quantity of a special dye

that is used to generate atomic oxygen is taken in by cancer cells when compared with

the healthy tissue. Hence, only the cancer cells are destroyed then exposed to a laser

radiation. Unfortunately the remaining molecules migrate to the skin and the eyes and

make the patient very sensitive to the day light exposure. This effect can last for up to

six weeks (Yoshida et al., 1999).

To avoid these side effects, the hydrophobic version of the dye molecule was

enclosed inside a porous nanoparticle (Roy et al., 2003). The dye stayed trapped inside

the ormosil nanoparticle and didn’t spread to the other parts of the body. At the same

time its oxygen generating ability has not been affected and the pore size of about 1nm

freely allowed for the oxygen diffuse out.

Multicolor Optical Coding For Biological Assays:

The ever increasing research in proteomics and genomics generates escalating

number of sequence data and requires development of high throughputs screening

technologies. Realistically various array technologies that are currently used in parallel

analysis are likely to reach saturation when a number of array elements exceed several

millions.

The selection of nanoparticles used in those experiments had six different colors

as well as ten intensities. It is enough to encode over 1 million combinations. The

uniformity and reproducibility of beads was high letting for the bead identification

accuracies of 99.99 % (Han et al., 2001).

Manipulation of Cells and Bio Molecules:

Functionalized magnetic nanoparticles have found many applications including

cell separation and probing. For example, prophyrins with thiol or carboxyl linkers

were simultaneously attached to the gold or nickel segments respectively. Thus it is

possible to produce magnetic nanowires with spatially segregated fluorescent parts. In

98

addition because of the large aspect ratios the residual magnetization of these

nanowires can be high. Hence weaker magnetic fields can be used to drive them. It has

been show that a self assembly of magnetic nanowires in suspension can be controlled

by weak external magnetic fields. This would potentially allow controlling cell

assembly in different shapes and forms (Reich et al., 2003).

Protein Detection:

Gold nanoparticles are widely used in immune histochemistry to identify

protein-protein interactions. The group of Mirkin has designed a sophisticated multi

functional probe that is built around a 13 nm gold nanoparticle. The nanoparticles are

coated with hydrophilic oligo nucleotides containing a Raman dye at one end and

terminally capped with a small molecule recognition element (e.g. biotin).

Moreover this molecule is catalytically active and will be coated with silver in

the solution of Silver (I) and hydroquinine. After the probe is attached to a small

molecule or an antigen it is designed to detect the substrate which is exposed to silver

and hydroquinine solutions. The protein can be detected by modifying the probe to

contain antibodies on the surface (Nam et al., 2003).

Electrochemical Application:

The bioreduction of aqueous gold chloride ions by the bacterium E.coli DH5α

assist the formation of gold nanoparticles. The nanoparticles are bound to the surface of

the bacteria and this composite may be used for application in realizing the direct

electrochemistry of hemoglobin. The bacteria-mediated production of gold

nanoparticles is a green chemistry synthesis procedure and also provides a new

approach to achieve the direct electrochemistry of proteins (Du et al., 2007).

Nanoparticles as ROS Scavenger:

Major problem with T2DM patients is delayed healing of wound. Several

literatures show this is because of increase in oxidative stress and decreased

antioxidants level content leads to increased inflammation, which results huge amount

of ROS production that leads to premature apoptosis of inflammatory cells.

99

Recently, nanotechnology and nanoparticle field is a fast-growing research area.

Research has already led to significant breakthrough and several products are available

commercially. Recently, role of silver nanoparticle in diabetic wound healing therapy

has also been considered. However, the complete mechanisms of silver nanoparticle in

bacteriostatic phenomena in wound healing remain unknown.

It has been reported a novel Ag+-loaded zirconium phosphate nanoparticle

plays crucial role in diabetic wound healing (Manish Mishra et al., 2008). Some of the

nanoparticle now a day are preparing in a manner that act as free radical scavenger

(Wasrheit, 2004). Moreover, reports reveal Cerium oxide (CeO2, ceria) plays major

active role because of its excellent free radical scavenging potentials (Chung, 2003).

This metal oxide is monodisperse particles with single crystals and few twin boundaries

(Zhang et al., 2002) as well as expanded lattice parameter (Perebeinos et al., 2002).

Moreover, CeO2 tends to be a nonstoichiometric compound. Cerium atom

characterized by both +4 and +3 oxidation states.

Literatures shows X-ray photoelectron spectroscopy and X-ray absorption near

edge spectroscopy suggests that the concentration of Ce3+ relative to Ce4+ increases as

particle size decreases with a conservative [Ce3+] minimum of 6% in 6 nm

nanoparticles and 1% in 10 nm particles (Zhang et al., 2004). This dual oxidation state

means that these nanoparticles have oxygen vacancies (Robinson et al., 2002). The loss

of oxygen and the reduction of Ce4+ to Ce3+ is accompanied by creation of an oxygen

vacancy. This property is responsible for the interesting redox chemistry exhibited by

CeO2 nanoparticles and makes them attractive for wound healing process followed by

scavenging properties.

Nanoparticles made of other metal oxides were also considered for its potential

scavenger behavior. These included particles aluminum oxide (Al2O3), commonly

known as alumina. That differs in crystal structure and yttrium oxide (Y2O3, yttria).

Moreover, Alumina is also thermodynamically stable at all temperatures and has a

corundum like structures with oxygen atoms adopting hexagonal close-packing and

100

Al3+ ions, filling 2/3 of the octahedral sites in the lattice. However, yttria has a cubical

structure (Kingery et al., 1976). Yttrium oxide is now a day considered most significant

due to its highest free energy of oxide formation from elemental yttrium among known

metal oxides (Kilboun et al., 1994).

It is characterized by only small changes from stoichiometry under normal

conditions of temperature and pressure (Kofstad, 1972) and by atmospheric absorption

of H2O and CO2 (Kilbourn et al., 1986). The particular polymorph of it is the

monoclinic B form, which is closely related to it’s A - form having hexagonal close-

packing (hcp). Unlikely the A form which has only sevenfold coordination states, it has

six fold (Kofstad, 1972). The B form structure is slightly less dense than the A form

variant and has the yttrium cations in nonequivalent sites in the crystal (Atou et al.,

1990). These groups of nanoparticles are relatively nontoxic to neutrophiles and

macrophages, and that CeO2 and Y2O3 particles protect cells from death due to

oxidative stress. This protection is due to the direct antioxidant properties of the

nanoparticles.

Cerium and yttrium oxide nanoparticles are able to rescue cells from oxidative

stress-induced cell death in a manner that appears to be dependent upon the structure of

the particle but independent of its size within the range of 6–1000 nm. This might be

useful for the diabetic wound healing. Furthermore, three alternative explanations were

demonstrated that the cerium oxide and yttrium oxide particles protect from oxidative

stress. They may act as direct antioxidants, block ROS production, which inhibit

programmed cell death pathway and they may directly cause a low level of ROS

production, which rapidly induces a ROS defense system before the glutamate-induced

cell death program is complete. The latter is a form of preconditioning that could be

caused by the exposure of cells to particulate material known to induce low levels of

ROS (Becker et al., 2002). Therefore it could be suggested that CeO2, alumina and

yttrium may be useful for diabetic wound healing mediated be ROS scavenging

potentials of above nanoparticles and have diabetic wound healing therapy.

101

MATERIALS AND METHODS

Selection of Cyanobacteria and diatom

Cyanobacteria culture:

The filamentous marine cyanobacteria, Phormidium willei NTDM01 and

Phormidium tenue NTDM05 were selected for this study and were grown in batch

culture in ASN - III. The pH of the medium was adjusted to 8.0 using a 1M NaOH

solution before experimentation; the culture were transferred and grown for

approximately 6-8 weeks to reach a stationary growth phase. It was then centrifuged

and washed three times with sterile deionized water to remove the salts and trace metals

from the medium before being used for the cyanobacterial experiments.

Diatom Culture:

A diatom species such as Stauroneis sp. has been chosen for the study. It was

grown in F/2 medium. The pH of the medium was adjusted to 8.0 using a 1M NaOH

solution before experimentation; the culture was transferred and grown for

approximately 2-3 weeks to reach a stationary growth phase. It was then centrifuged

and washed three times with sterile distilled water to remove the salts and trace metals

from the medium before being used for the diatom experiments.

These strains were selected to explore nanobiotechnological potentials. In this

regards, Phormidium willei NTDM01 has been chosen for the synthesis of silver and

gold nanoparticles and Phormidium tenue NTDM05 has been selected for the synthesis

of CdS nanoparticles and finally, a diatom Stauroneis sp. also been selected to adopt for

the synthesis of Si-Ge oxide nanocomposites. The detailed experimentation for the

synthesis of nanomaterials have described below.

Biosynthesis of Nanoparticles:

Synthesis of silver nanoparticles using cyanobacteria have adopted a method

that described earlier by Lengke et al., (2005) such as biotic and abiotic experiment and

for the gold nanoparticles modified method of Lengke et al., (2006) have been adopted.

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Silver nanoparticles:

a. Biotic experiment:

The cyanobacterial experiments were conducted to examine the role of

cyanobacteria in the synthesis of silver nanoparticles from aqueous solution of AgNO3.

The experiments were conducted using washed cyanobacteria and without the presence

of the growth medium.

To initiate the experiment 5mL of the silver solution (~560mg/l) was added to

5mL of washed Cyanobacteria culture. The experiment were conducted at 25 oC for 28

days after the incubation period with silver solution and maintained in the dark, and

cyanobacteria populations were measured with time. pH, cell viability and precipitation

were measured with time.

b. Abiotic experiments:

Abiotic control experiments consisting of ~560mg/l in silver were conducted

using AgNO3 solution without addition of cyanobacteria.

Gold nanoparticles:

a. Biotic experiment:

The cyanobacterial experiments were conducted to examine the role of

cyanobacteria in the synthesis of gold nanoparticles from aqueous solution of HAuCl4.

The experiments were conducted using washed cyanobacteria and without the presence

of the growth medium.

To initiate the experiment 100mL of the Chloroauric acid (1mM) was added to

5g of washed cyanobacteria culture. The experiment were conducted at 25oC for 5 days

and maintained in the dark, and cyanobacteria populations were measured with time

and the pH, cell viability and precipitation were measured with time.

b. Abiotic experiments:

Abiotic control experiments consisting of Au2+ (1mM) were conducted using

HAuCl4 solution without addition of the presence of Cyanobacteria.

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Characterization of silver and gold nanoparticles:

Uv-Vis spectrum analysis:

Aqueous silver nitrate solution (10-3M) taken in a clean conical flask inoculated

with isolated cyanobacteria. The reduction of the Ag+ ions by biological species in the

solution was monitored by sampling the aqueous component and measuring the Uv -

Vis Spectrum of solutions. The Uv - Visible spectra of this solution was recorded in

Lambda 35 in absorbance mode from 300 to 900nm.

Fourier Transform Infra Red analysis:

The experimental system after incubation of AgNO3 solution to form slime

sediment (brown color). This sediment characterization analysis by employing FTIR

(Fourier Transform Infra Red). The silver nanoparticles prepared as described above

were analyzed by Spectrum RX1. The FTIR spectrum was taken in the mid IR region

of 400-4000 cm-1. The spectrum was recorded using ATR (Attenuated Total

Reflectance) technique. The sample was directly placed in the Potassium bromide

crystal and the spectrum was recorded in the transmittance mode.

Scanning Electron Microscope with Energy Dispersive Spectrum analysis:

A drop of aqueous solution containing the silver nanomaterials (brown color)

was placed on the sterile stains steel coupon and it air dried to applied gold sputtering

(model). These plates were examined at different magnifications (10,000X, 40,000X)

by scanning electron microscope (Model – JEOL Instrument JSM-5610). The natures

of elements were identified by EDX.

Transmission Electron Microscopic study:

A drop of the aqueous solution of silver and gold nanoparticles were separately

placed onto a carbon coated copper grid, and air-dried and then analyzed in a

transmission electron microscope (TEM).

Biosynthesis of Nanocomposites:

Si-Ge Oxide Nanocomposites:

The two stage cultivation methods are followed for the synthesis of silicon-

germanium nanocomposites as described (Qin et al., 2008 and MubarakAli et al., 2011)

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1. In first stage, the cells were grown in nutrient medium containing sufficient

silicon concentration for its growth. The silicon concentration (30mg/l) and cell

density is monitored at regular intervals. When all the silicon was used up and

the cell density had become constant, the cells were then considered to be

starved of silicon, this condition prevails one week.

2. In the second stage, the silicon and germanium (1:0.5) is added to the liquid

medium for the biosynthesis of Silicon –Germanium nanocomposites. After 7

days the cells were harvested from the medium and washed thrice in deionized

water in order to remove surface adsorbed germanium and other trace elements.

Then the sample was centrifuged at 2000rpm for 5mins and the pellet was

collected.

Characterization of Si-Ge Oxide Nanocomposites:

The collected pellet was dispersed on the thin slide and allowed to dry in hot air

oven. The small portion of slide was placed onto carbon tape affixed to a stub, the gold

was coated before introduction to the Scanning Electron Microscopy equipped with

EDS. (Model – JEOL Instrument JSM-5610). The natures of elements were identified

by EDS.

Biosynthesis of Semiconductor:

Synthesis of CdS nanoparticles:

CdS nanoparticles were synthesized by reacting Cd2+ with extracted c-

phycoerythrin. The experiment were conducted wherein aqueous solution containing a

mixture of the salt CdCl2 and Na2S in the concentration of 0.25mM and 1mM

respectively.

The reaction volume was kept for incubation and regular monitoring of color

changes; temperature and pH of the solutions were analyzed periodically for 5days

(MubarakAli et al., 2012).

Characterization of CdS nanoparticles:

The appearance of nanoparticles was gauzed in a UV- vis spectrophotometer, in

the wavelength range 200 to 900nm, periodically. Once the nanoparticles were formed,

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the preparation was lyophilized and analyzed adopting FTIR and HRSEM and EDAX,

with Li drift Si detector (Thermo elution corporation, USA). Finally, a drop of the

sample was placed onto a carbon coated copper grid, and air-dried and then analyzed in

a transmission electron microscope (TEM).

Biomedical application:

The synthesized nanoparticles were analyzed for antibacterial and antioxidant

property with standard protocols.

Antibacterial activity

The multi drug resistant (MDR) pathogens were isolated from hospitalized

patients. Following pathogens were selected for the antibacterial property such as

E.coli, Staphyloccocus aureus, Proteus vulgaris, Klebsiella pneumoniae and

Pseudomonas aeruginosa by well–diffusion method.

The pathogenic cultures were bringing into broth culture for antibacterial assay.

Approximately 7-mm diameter of well was made on Muller Hinton Agar plate with the

help of gel puncture. The cultures were swabbed on test media with sterile cotton swab.

25μl of synthesized particles were inoculated to the well, and then the plates were

incubated in incubator for 37 °C for 24 h, the zones of inhibition was tabulated and

discussed.

Antioxidant property

The effect of extracts on DPPH radical was estimated by DPPH radical

scavenging assay (Pathiranan and Shahidi, 2005). A solution of 0.135 mM DPPH in

methanol was prepared and 1.0 ml of this solution was mixed with 1.0 ml (1:1 ratio) of

nanoparticle suspension in methanol containing (50, 100, 150, 200, 250 µg/ml) of the

nanoparticle. The reaction mixture was vortexed thoroughly and kept in the dark at

room temperature for 30 min. The absorbance of the mixture was measured

spectrophotometrically at 517 nm. Citric acid was used as reference. The ability to

scavenge DPPH radical was calculated by the following equation:

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DPPH radical scavenging activity (%) = [(Abs*control – Abs*sample)] × 100

(Abs*control)]

Where.,

Abs*control is DPPH solution + methanol (1:1 ratio)

Abs*sample is the absorbance of DPPH radical + synthesized nanoparticles

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RESULTS AND DISCUSSION

Biosynthesis of nanoparticles:

Silver nanoparticles:

In cyanobacterial experiment with Phormidium willei NTDM01, soluble silver

was completely precipitated from solutions within 28days. A grayish- black silver

precipitate on cyanobacteria was observed in experiment macroscopically. The pH was

constant at 5.0 and 4.7. In abiotic experiments using silver nitrate, total soluble silver

concentration and pH were constant until the completion of experiment (Fig. 4a & b).

The UV-vis Spectrum reveals that the band observed at 450 nm due to the

plasmon resonance of the silver nanoparticles and low wavelength region recorded

from the reaction medium exhibited an absorption band at ca.265 nm and it was

attributed to aromatic amino acids of proteins (Fig. 4c). It is well known that the

absorption band at ca 265 nm arises due to electronic excitation in tryptophan and

tyrosine residue in the protein.

The FTIR spectrum was recorded from the film of silver nanoparticles, formed

after several days of incubation with the bacteria (Fig.5). The bands seen at 3280 cm−1

and 2924 cm−1 were assigned to the stretching vibrations of primary and secondary

amines respectively. The corresponding bending vibrations were seen at 1651 cm−1 and

1548 cm−1, respectively. The two bands observed at 1379 cm−1 and 1033 cm−1 can be

assigned to the C–N stretching vibrations of aromatic and aliphatic amines,

respectively. The presence of protein as the stabilizing agent surrounded the silver

nanoparticles (Muherjee et al., 2001). The silver ions were reduced in the presence of

nitrate reductase, leading to the formation of a stable silver hydrosol 10-25 nm in

diameter and stabilized by the capping peptide.

SEM observation on products of marine cyanobacterium, Phormdium willei

NTDM01 in AgNO3 experiment is presented (Fig.6a). At room temperature, the

addition of AgNO3 to the cyanobacteria caused the precipitation of silver nanoparticles

at cell surfaces. Small spherical silver nanoparticles with size ranging from 100nm to

a & b

c)

Figure 4 Biosynthesis of Silver Nanoparticles: a) AgNO3 solution (560mg/l) as abiotic experiment b) synthesized silver nanoparticles showing brown color solution after synthesis in biotic experiment. c) Uv-Vis Spectrum of Silver nanoparticles, which shows Plasmon resonance of silver nanoparticles at 450nm.

Figure 5 FTIR spectrum of silver nanoparticles using marine cyanobacterium, Phormidium willei NTDM01

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200nm (extracellularly) were also precipitated in solution silver nanoparticles were

deposited at cell surfaces. EDS showed the occurrence of silver particle in higher

amount with trace of magnesium, calcium and chloride. In this analysis by EDS of the

silver nanoparticles was confirmed the presence of elemental silver signal (Fig.6b).

TEM micrograph of a thin section of cyanobacteria showed nanoparticles of

silver deposited inside the cell in spherical shaped nanoparticles size is about 20 nm.

Octahedral, spherical, and anhedral nanoparticles of silver precipitated in solution (Fig.

7).

The reaction of cyanobacteria with AgNO3 solutions results in the formation of

silver nanoparticles. The presence of spherical silver nanoparticles was observed in

experiment. The precipitation of silver nanoparticles was not observed for abiotic

experiment that were run under similar condition and duration, that suggesting that

cyanobacteria were required for silver precipitation and the precipitation processes

were not controlled by inorganic chemical reactions.

A brief overview of the use of micro organisms such as cyanobacteria, yeast,

algae, fungi and actinomycetes in the biosynthesis of metal nanoparticles has been

described. In the present study, spectrum, UV-vis observation indicated the release of

protein into the culture medium indicting the electronic excitation of aromatic amino

acids. A similar observation has been made in Fusarium oxysporum and suggests a

possible mechanism for the reduction of the metal ions present in the solution. The

overall peaks from FTIR observation confirm the presence of protein in the samples of

silver nanoparticles. It can be assumed that nitrate reducing bacteria to produce nitrate

reductase enzyme (protein). This reduces to silver nitrate solution form silver

nanoparticles. The synthesis of silver nanoparticles using ß-NADPH-dependent nitrate

reductase and phytochelatin in vitro has been demonstrated (Shanker, 2003).

In this study, cyanobacterial cells were encrusted with silver nanoparticles and

separation of some filaments into their constituent cells were still observed. The

nanoparticles were not in direct contact even within the aggregates, indicating

a)

b)

Figure 6 SEM photograph of silver nanoparticles encrusted on the surface of Phormidium willei NTDM01 in the range of 100 nm -200 nm. EDS Spectrum of silver nanoparticles in the images showed elemental signal in higher percentage.

Figure 7 TEM photograph of silver nanoparticles showed spherical shaped nanoparticles in the size range is about 20 nm.

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stabilization of the nanoparticles by capping agent. This corroborates with the previous

observation in F. oxysporum (Shanker, 2003). Cyanobacteria commonly use nitrate as

the major source of nitrogen for three different purposes including growth, generation

of metabolic energy, and redox balancing (Suzuki et al., 1995). The presence of silver

nanoparticles scattered throughout the cells suggests that Ag+ and NO3 entered in the

cyanobacterial cells through a transport system and degraded in solution. The presence

of silver nanoparticles inside the cytoplasm, Ag+ is presumably reduced to Ago Because

AgNO3, a toxic reagent, was used in metabolic processes, it ultimately killed the cells.

During the death of cyanobacteria, nanoparticles of silver produced inside the

cells were release through the cell membrane into solution, as indicated by the

precipitation of silver nanoparticles around the cells. The dead cyanobacteria also

released organics (protein, and other biochemical) that caused further precipitation of

silver from solution outside the cells. The protein molecules act as reducing agent for

silver nanoparticles. The protein molecule made up of different functional group in

amino acid sequences such as amino, carboxyl, sulfate groups present in the

cyanobacterial protein favors the formation of extremely small-sized silver

nanoparticles with narrow size distribution. Hydroxyl and sulfonic groups are

beneficial to synthesis of silver nanoparticle with a slightly larger particle size in a

weak reducing environment. It has already been reported that the silver nanoparticles

can be stabilized by functional groups, such as carboxyl, hydroxyl, and amido and thiol

group, except the steric effect arising from the large molecular structures of the organic

modifier (protein) on the surface of the particles (Temgire, 2004 and Vigneshwaran et

al., 2007).

The microorganisms used in the synthesis of nanomaterials as a possible viable

alternative them, the more popular physical and chemical methods. The use of

cyanobacteria as a source of enzymes that can catalyze specific reactions leading to

inorganic nanoparticles is a new and rational biosynthesis strategy that is being

developed. Extracellular secretion of enzymes offers the advantages of obtaining large

quantities in a relatively pure-state, free from other cellular proteins associated with the

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organism and can be easily processed by filtering of the cells and isolating the enzyme

for nanoparticles synthesis from cell-free filtrate.

Gold nanoparticles:

Synthesis of gold nanoparticles was confirmed by changes of pale yellow color

chloroauric acid into ruby red colored solution, which indicates that formation of gold

nanoparticles by the action of cyanobacterial proteins present in the reaction vessel

(Fig. 9a). The UV-Vis spectrum reveals that the band observed at 540nm due to the

plasmon resonance of the gold nanoparticles and low wavelength region recorded from

the reaction medium exhibited an absorption band at ca.265nm and it was attributed to

aromatic amino acids of proteins. It is well known that the absorption band at ca 265nm

arises due to electronic excitation in tryptophan and tyrosine residue in the protein. It is

also gave an idea for the synthesized nanoparticles due to the action of protein that

released from cyanobacteria and the nanoparticles surrounded by the extracellular or

intracellular protein (Fig. 9b).

In this study, cyanobacterial and chlorophycean biomass treated with 1mM of

HAuCl4 solution started to turn purple within 72 h of exposure, in contrast to the

control sets which remained the same (Fig. 8a & b). The UV-visible spectra of the

extracted purple solutions of gold nanoparticles are shown in Fig 9. It showed

absorbance at about 525 nm, it was reported that the UV–visible spectroscopy of

nanoparticle solutions obtained from P. tenue and Microcoleus biomass. The

characteristic absorption bands at around 530nm at all pHs. In the case of P. tenue,

maximum absorption was observed at 529nm (pH 5 and pH 7) or 532nm (pH 9),

whereas Microcoleus showed absorption maxima at 534nm (pH 5 and pH 7) and at

530nm (pH 9). Ulva showed absorbance peaks at 542 nm, 541nm and 542nm at pHs 5,

7 and 9, respectively and Rhizoclonium showed prominent peaks at 537 nm, 529nm and

517 nm. No absorption band beyond 600nm was visible in any of these cases (Parial et

al., 2012).

In this study, FTIR spectrums of gold nanoparticles were performed, in which

after the experimentation; powdered samples was analyzed for FTIR spectrum to gold

a)

b)

Figure 8 Biosynthesis of Gold Nanoparticles: a) microscopic image of Phormidium willei NTDM01 b) microscopic image of Phormidium willei NTDM01 in biotic experiment, in which inside the filaments colored ruby red due to the synthesis of gold nanoparticles.

a & b

c)

Figure 9 Biosynthesis of Gold Nanoparticles: a) gold chloride solution (1mM) b) extracted gold nanoparticles by sonication showed ruby red color and c) Uv-Vis Spectrum of gold nanoparticles, which shows Plasmon resonance of gold nanoparticles at 540nm.

a)

b)

Figure 10 FTIR spectrum of gold nanoparticles using marine cyanobacterium, Phormidium willei NTDM01: a) infra red spectrum of cyanobacterial species and b) infra red spectrum of gold nanoparticles, which depicts functional group of the protein residue may responsible for the possible reduction of Au2+ to metallic Auo.

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nanoparticles. The peaks were observed at 3394.11 cm-1, 2079.07 cm-1, 1607.93 cm-1,

1404.90 cm-1, 1091.61 cm-1 and 677.84 cm-1 in pure cyanobacterial sample. Whereas in

gold nanoparticles experiments, the peak was observed at 3436.89 cm-1, 2358.58 cm-1,

2076.07 cm-1, 1638.32 cm-1, 666.69 cm-1. The presence and absence of peaks and

formation of peaks, which reveals that chemical modification was happened in order to

formation of nanoparticles by the action of cyanobacterial protein and the metallic

nanoparticles were combined with functional group of the protein and stabilized the

synthesized nanoparticles (Fig. 10a & b).

The results of HRSEM of gold nanoparticles showed that spherical shaped

uniform sizes of nanoparticles were observed. The size of the particles was found to be

10 nm. The gold nanoparticles encrusted on surface of the cyanobacterial cells. EDAX

peaks also confirmed that presence of gold signal in the sample in higher percentage

(Fig. 11a & b). The results obtained from TEM gave clear indication about the size and

shape of the nanoparticles. The solution extracted from gold-loaded biomass of P.

willei treated showed the presence of spherical particles of 10 nm diameter, along with

nanotriangles of approximately 15 nm (Fig. 12). It has already been reported that the

colour of gold nanoparticles can vary from red to blue, depending upon the shape and

size of the particles (Sharma et al., 2009).

The gold nanoparticles solutions extracted in the present study were purple,

because of the excitation of surface plasmon vibrations in the gold nanoparticles

(Mulvaney, 1996). After Au (III) exposure, the purple, ruby red or blue colorations of

microorganisms are due to the reduction of Au (III) to Au (0) and subsequent formation

of Au nanoparticles intra- and/or extracellularly (Druff et al., 1987; Chow & Zukoski,

1994; Lujan et al., 1994; Chakraborty et al., 2009; Parial et al., 2012). The present

study showed that the Phormidium willei NTDM01 have potential as bionano-

factories, and this is evident from the visual transformation of the cyanobacteria from

green to deep purple. The biosynthesized gold nanoparticles were extracted

successfully by sonicating the gold loaded biomass of the experimental taxa.

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It is highly desirable to synthesize nanorods in a clean and non-toxic way,

because a toxic chemical CTAB (cetyl trimethylammonium bromide) is used in most of

the conventional chemical methods of nanorod synthesis (Johnson et al., 2002); this

chemical could lead to undesirable functional aberrations in target cells, when

nanoparticles are used in photothermal therapy or in a drug delivery system. CTAB

helps synthesis in two ways: firstly, it can give a directional template promoting

formation of rods and secondly, it is attached to the surface of the nanoparticles and

gives the nanoparticles colloidal stability in solution. Therefore, complete wash-out of

CTAB from the surface of nanoparticles results in the loss of colloidal stability. It has

been recently demonstrated a green chemical approach for the controlled biosynthesis

of gold nanorods by Nostoc ellipsosporum without any shape anisotropy (Parial et al.,

2012).

The surface plasmon band for gold nanoparticles usually has a range of 510–

560nm in aqueous solutions, depending on the nanoparticle morphology. Non-spherical

gold nanoparticles exhibit multiple absorption bands, correlated with their multiple

axes, in comparison to the single one for isodiametric particles. In the present study two

discrete surface plasmon bands were observed in the case of P. willei indicating

possible synthesis of non-spherical gold nanoparticles and it was later confirmed that

these were nanotriangles by TEM study.

Other studies have also indicated that pH plays a key role in controlling particle

morphology. Brown et al. (2000) reported changes in the shape of gold crystals with

changes in pH while studying the role of polypeptides on the resulting gold crystals.

Gericke & Pinches (2006) observed that the pH influences the reduction rate which

regulates the synthesis of different shapes and sizes of gold nanoparticles and further

suggested that spherical particle formation is favoured at low reduction rates whereas

high reduction rates resulted in the formation of nanorods and platelets.

The present study, suggests that a high reduction rate at lower pH may lead to

the formation of nanotriangles. TEM analysis of extracted nanoparticle solutions of P.

willei showed the production of mostly spherical particles. In algae, both processes

Figure 11 SEM photograph of gold nanoparticles encrusted on the surface of Phormidium willei NTDM01 in the range of 90 nm. EDAX Spectrum of gold nanoparticles in the images showed elemental signal in higher percentage.

Figure 12 TEM photograph of gold nanoparticles showed different shaped such as spherical (5 nm), hexagonal (10 nm) and triangles (15 nm).

113

occur, as gold nanoparticles are recorded either intracellularly or extracellularly in the

medium, as in Lyngbya majuscule and Spirulina subsalsa (Chakraborty et al., 2009).

Interestingly, it has been observed that gold solution incubated with dried,

powdered algal biomass did not turn purple over time (one week), which confirms that

the reduction of gold is associated with cellular metabolism and presumably involves

reducing enzymes or synthesis of other metabolites. In the experiment, cyanobacterial

cells were poisoned and died after converting Au (III) to Au (0). In conclusion, it seems

that synthesis of fairly mono-dispersed gold nanoparticles by the reaction of aqueous

chloroaurate ions with cyanobacterial biomass represents a more eco-friendly and cost-

effective method than the conventional approach using toxic chemicals. The aqueous

gold(III)-chloride solutions at different concentrations investigated were stable for one

week in abiotic experiments (without the presence of cyanobacteria) under similar

conditions.

On the addition of HAuCl4 to the cyanobacteria, precipitation of nanoparticles

(<20 nm) containing gold was observed by HRSEM-EDS at the cell surfaces and at the

interface between the cell wall and solution within several minutes. These nanoparticles

were amorphous to electron diffraction. After 17 h, the dead cyanobacteria released

cytoplasmic material around the cells, causing further precipitation of metallic gold

nanoparticles, with octahedral and sub-octahedral shapes and diameters up to 6 nm. A

tendency for the frequency of larger octahedral platelets to increase with run duration

indicated a weak Ostwald ripening effect (Parial et al., 2012).

Gold concentrations in river and marine waters range up to about 50 ppb, while

near gold deposits; soluble gold concentrations can be up to 1 ppm (Korobushkina et

al., 1983 and McHugh, 1988). Thus, the gold concentrations in natural environments

are 2-3 orders of magnitude lower than those used in this study. This study showed that

the gold reduction process from HAuCl4 to metallic gold by cyanobacteria was

facilitated by higher gold concentrations. In natural environments where gold

concentrations were relatively low, the reduction of HAuCl4 to metallic gold by

cyanobacteria is presumably a slow process. In addition, at low gold concentrations (at

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ppb level), gold toxicity would not kill all viable bacterial cells; therefore, the

remaining cells would be able to grow and probably adapt to the presence of gold

(Reith, 2006). Passive gold accumulation via organic sulfur present in the

cyanobacterial outer membrane or periplasma may explain the mechanisms of gold

bioaccumulation by cyanobacteria.

The recovery of gold from solutions has been actively studied for industrial

purposes (Nakajima et al., 2003; Pethkar, et al., 1998 and Romero-Gonzalez, 2003).

Mining, mineral processing, and metallurgical industries generate billions of tons of

metal waste every year (Eccles, 1995). Conventional methods for removing metals

from ore processing solutions, including chemical precipitation, chemical oxidation or

reduction, ion exchange, filtration, electrochemical treatment, membrane technologies,

and evaporation, have been used for gold recovery (Volesky, 1990). Because these

processes are extremely expensive, alternative methods for metal recovery using living

or dead microorganisms have been actively investigated. The present study indicate that

the interaction of HAuCl4 with cyanobacteria can be used as a promising method for

recovery of gold particles from ores.

Biosynthesis of Semiconductor:

CdS nanoparticles:

In the earlier study, cytotoxicity of C-PE was evaluated in human cell lines by

assessment of cell proliferation and neutral red uptake; it was found that the C-PE is not

cytotoxic (Soni et al., 2010). Among various concentration of CdCl2 and Na2S, CaCl2

at 0.25mM and Na2S at 1mM proved to be most optimum concentration for the

synthesis of CdS nanoparticles. The first step in this reaction sequence in the

nanoparticles synthesis was formation of C-PE- Cd2+ complex, then this complex

reacted with Na2S to produce CdS nanoparticles, when the color reaction mixture

changed to yellow to orange (Fig. 13a). CdS nanoparticles thus synthesized measured

about 5 nm and, this size remains unchanged well over 8 months of storage. In an

earlier investigation using R-PE, the solution was contains only coarse CdS

nanoparticles.

a & b

Figure 13 Biosynthesis of CdS Nanoparticles: a) extracted c-phycoerythrin from Phormidium tenue NTDM05 b) biosynthesized CdS nanoparticles showing orange color solution and c) Uv-Vis Spectrum of CdS nanoparticles: where, CdS nanoparticles coupled with c-phycoerythrin showed maximum absorbance at 390 nm (a) whereas c-phycoerythrin showed maximum absorbance at 560 nm (b).

Abs

Wavelength

a

b

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It also found that R-PE prevents the growth and aggregation of nanoparticles

(Brekhovskikh and Bekasova., 2005). A similar experiment in higher pH the charged

capping agent found to be very high, which might be the basic of the colloidal stability

of QDs. Also, the size of the Cd-R-PE complex was primarily depending on the

concentration of salts, pH of the reaction mix and the concentration of the pigments.

(Liji Shobhana, et al., 2011).

Soluble starch has also been also used as a capping agent in the synthesis of

CdS and CdSe nanoparticles, ranging from 8-15nm (Wei et al., 2004 and Li et al.,

2007). The hydroxyl group of the starch polymer acts as passivation contacts for the

stabilization of the CdS nanoparticles in aqueous solution (Rodriguez, 2008). The

synthesis of CdS nanoparticles occurs due to pH sensitive membrane-bound oxido-

reductase and carbon source-dependent rH2 in the culture solution (Prasad et al., 2010).

This work also emphasizes that the fluorescent protein in the pigment is mainly due to

tyrosine, phenylalanine and tryptophan moieties, which would be the basis of the

formation of CdS nanoparticles very efficiently and stabilizing it to prevent

agglomeration.

The presence of CdS nanoparticles was confirmed by the absorption peak at

470nm. The maximum absorption peak position of hollow nanoparticle chains is at

487nm with the band gap at 2.56 eV (Liu et al., 2007). Compared with that of bulk

material (515nm, 2.42 eV), the absorption peak was a blue shift at 28 nm and its band

gap enlarged about 0.14eV. This is indication of quantum size effect (Gao et al., 2002).

It was reported that the band gap value 2.57 eV is shifted compared with the bulk value

and this could be a consequence of a size quantization effect in the sample (Castanon et

al., 2005) and the absorption peak of c-PE was 550 nm (Fig. 13b).

The FTIR spectrum of the c- phycoerthrin is shown in Fig. 14a. The

characteristic bands due to S-H stretching, appearing at 3398, 2074, 1637, 1365 cm-1

for pure C-PE were not seen in capped CdS, which is due to the interaction of thiol

group of the capping agent with CdS. The characteristics band such as S-H stretching

was found in the entire capping agent that was not found in all capped CdS. It could be

a & b

Figure 14 FTIR spectrum recorded for CdS nanoparticles stabilized by C-PE. (a) spectrum of C-PE (b) the bands seen at cm-1 were assigned to the C-N stretching vibrations of the aromatic amino amines.

Figure 15 HRSEM photograph of CdS nanoparticles encrusted on the surface of Phormidium willei NTDM01 in the range of 5 nm - 30 nm. EDAX Spectrum of CdS nanoparticles in the images showed elemental signal in higher percentage.

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formed due to bonding with CdS NPs (Liji Shobhana, et al., 2011). In the interaction

between carboxymethyl chitosan (CMCH) and CdS NPs, there were two kinds of

bonding; carboxymethyl group was strongly bound by coordination with Cd ions on to

the particle surface and the unsubstituted other groups were linked to the particle

surface via hydrogen bonding (Li et al., 2003). From this observation, it was suggested

that the biological molecules can possibly perform the function for formation as well as

stabilization of the CdS nanoparticles in aqueous medium (Bai et al., 2009).

The peak at 1543, 1364, 1223 cm-1 appeared only in CdS-CPE complex (Fig.

14b). This new band was not observed in unbound CdS, which indicates a new

vibration from the CPE due to C-S band at another possible scheme of bonding

between CPE and CdS. The interaction mechanism is mainly due to hydrogen bonding.

Generally CdS QDs grow well in a solution that may has a large amount of hydroxyl

groups and in water-bound surface, which are active sites for the formation of hydrogen

bonding with the surrounding molecules (Ladizhansky, et al., 1998).

The morphology and size of the CdS nanoparticles were observed adopting

HRSEM. It was found that CdS nanoparticles encrusted on the surface of the cell in the

range of 5 nm to 30nm (Fig 15). The morphology and size of the CdS nanoparticles

were observed adopting TEM. It was found that the nanoparticles were of uniform size,

at about 5 nm. The EDAX revealed the presence of both Cd and S in the nanoparticles

synthesized (Fig. 16). The lifetime of capped CdS QDs was higher than that of

uncapped CdS QDs. The results also indicated that the lifetime of the particles

increased with increase in size, which may be due to the uniform passivation caused

MSA on the surface of CdS particles (Liji Shobhana et al., 2011). Based on the studies

carried out hypothetical mechanism was proposed for the formation of CdS

nanoparticles by the action of functional amino acids present in the C-Phycoerythrin

protein extracted from cyanobacteria (Fig. 17).

In the present study the particle size was 5.1±0.2nm. This size range may

possibly be due to the formation of nanoparticles over a time period. In support of this

inference, an earlier report also found difference in size of the particles over the

Figure 16 CdS nanoparticle. (a) TEM image, spherical shaped CdS nanoparticles in the range of 5 nm in size and EDAX of the CdS nanoparticles confirming the presence of CdS signals in higher percentage.

Figure 17 Hypothetical mechanisms on synthesis of CdS nanoparticle: Aminoacids in the chromoproteins binds with Cd2+ ions for the formation of CdS nanoparticles and also which stabilize them to avoid agglomeration.

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incubation time (Prasad et al., 2010). Since CdS nanoparticles, synthesized in aqueous

phase, could be a novel fluorescence probe for ultrasensitive detection of DNA as a

biolabel (Wang et al., 2010), the enhanced synthesis of CdS nanoparticles using C-PE,

are the unique properties of the particles might prove to the particles that prove to be of

great advantage.

Biosynthesis of Nanocomposits:

Si-Ge Oxide Nanocomposites:

A diatom, Stauroneis sp. culture was treated with germanium in two different

ways and they were periodically monitored for structural changes in their frustules

morphology. The pH was also monitored and changes were not observed. The

fabrications of germanium in diatom frustules were examined by electron microscopy

characterization.

The ability of diatoms to make complex, nanoscaled, three-dimensional silica

shells called “frustules” offers attractive possibilities for their applications to

nanobiotechnology (Hildebrand, 2005) and synthesis of new materials. For example,

silicon in the shell can be replaced with other elements by chemical processes while

preserving the intricate patterns of the frustule (Sandhage et al., 2002).

Scanning Electron Microscopy (SEM) and Energy Dispersive X ray

Spectroscopy (EDS) was used to study the integrity of the sample. SEM was to study

the surface morphology of the sample and EDS was used to study the structural

properties and chemical composition of the diatom frustules. Here the EDS spectrum

scan was used to locate and determine the distribution of Si-Ge nanocomposites in the

frustules.

The microscopy results showed that the Stauroneis sp. grown in an enriched sea

water medium, normal growth different view of the diatoms was documented for this

study (Fig. 18). The microscopy results showed that the Stauroneis sp. grown in an

enriched sea water medium along with germanium and the same culture that grown in a

silica and germanium alone did not showed any structural or morphological changes

a)

b)

c)

Figure 18 Scanning Electron Microscopic view of a diatom, Stauroneis sp. a) regularly arranged pores in the frustules b) showing both gridle and valve view and c) showing three dimensional view very clearly.

118

(Fig. 19). In Stauroneis, the cultures that grown in nutrients along with germanium did

not showed any structural changes (Fig. 19a) and the nanostructures such as pores and

raphe were observed clearly on the frustules. But with the same culture that grown in

germanium and silicate alone shows some aberrations in their frustules morphology

(Fig. 19b). The concentration of silicon and germanium used in all those cultures was

0.5mM: 0.1mM.

The incorporation of germanium into the diatom frustules was analysed by EDS

spectrum analysis. Characteristic energy peaks of germanium and silicate were

observed at 1 to 22 keV. In EDS spectrum of Stauroneis that grown in enriched sea

water medium and cultures with germanium and silicate alone, have no incorporation

of germanium and they are not fabricated with germanium and they could not used as

nanocomposite.

The EDS spectrum of Stauroneis culture that grown in enriched seawater

medium along with germanium showed that the germanium was incorporated into the

diatom frustules and the amount was very low and the distribution profile of

germanium in the diatom frustules is similar to the silicate although the intensity was

quite different. The maximum peak was obtained in silicate signal than germanium

signal. The results indicate that the Si-Ge nanocomposites were not homogenously

distributed within the diatom frustules (Fig. 20).

In this present study, scanning electron microscopy and microanalysis

techniques were used to characterize the morphologies and elemental composition of

the processed and unprocessed diatoms. Microscopic results show that the diatom cells

suffered varying degrees of morphological changes, probably from the incorporation of

germanium.

It is also possible that the morphological changes could have been caused by the

availability of insufficient silicon for the normal growth of the structural organs. The

samples with high germanium levels have been found to consist of more broken pieces

of diatom frustules than samples with no germanium. The difference in bond lengths

a)

b)

c)

Figure 19 SEM images of Stauroneis sp., grown in enriched medium (F/2) along with germanium (1a). It showed clear image of nanopore structures (1b) EDS spectrum of germanium doped culture Stauroneis sp., grown in enriched medium with germanium.

Figure 20 SEM images of Stauroneis sp., grown in enriched medium (F/2) along with germanium in molar ratio of 1:0.5, which showed nanometer sized pores ranges about 150 nm -200 nm (a), whereas Stauroneis sp., grown in enriched medium (F/2) along with germanium in molar ratio of 1:1. It showed aberrated diatom structure (b).

119

between silicon oxide and germanium oxide probably weakens the diatom frustules and

cause them to break easily during the processing of the cells or to collapse when

exposed to a high energy beam of electrons (Gutu and Jiao, 2006).

The results indicate that the Si-Ge nanocomposite was not distributed within the

diatom frustules. This was because the amount of germanium incorporated into the

diatom cells was very low. Experimental parameters such as molar ratio of silicon to

germanium determine amount of germanium assimilated by diatom cells.

Biomedical application:

Antibacterial property against multidrug resistant clinical pathogens:

Synthesized silver nanoparticles active against clinically isolated multi drug

resistant bacterial pathogens and a fungal pathogen namely, Aspergillus flavus. Zone of

inhibition of the isolates were tabulated (Table 8). All isolated pathogens are multi drug

resistant for the 15 antibiotics tested. It was quite interesting that the bacterial and

fungal isolates which were originally sensitive towards synthesized silver nanoparticles.

More specifically, E.coli and Pseudomonas aeruginosa showed good antibacterial

activity after 24 h and 48 h incubation respectively multi drug resistant. In contrast,

Proteus mirabilis and Streptococcus pyogens had antibacterial activity but not like

other organism, which maintain the zone pattern until 24 h and 48 h incubation. The

enhanced antibacterial effects of silver nanoparticles are characterized and also stated

that once inside the cell, nanoparticles would interfere with the bacterial growth

signaling pathway by modulating tyrosine phosphorylation of putative peptides

substrate critical for cell viability and division and the nanoparticles were not in direct

contact even within the aggregates, indicating stabilization of the nanoparticles by a

capping agent (Shrivasatava et al., 2007 and Duran et al., 2005).

Shahverdi et al. (2007) reported that the silver nanoparticles have an

antimicrobial effect on S. aureus and E. coli. The oligodynamic effect of silver has

antibacterial activity against microorganisms. The changes in the local electronic

structure on the surface of the smaller sized particles lead to the enhancement of their

chemical reactivity leading to bactericidal effect (Thiel et al., 2004). It was suggested

Table 8 Antibacterial and Antifungal activity of synthesized silver nanoparticles against multi drug resistant clinically isolated pathogens.

Organisms Zone of Inhibition (mm)

24h 48h

Escherichia coli 8 9

Staphylococcus aureus 7 8

Proteus mirabilis 7 7

Pseudomonas aeruginosa 8 10

Salmonella typhi 10 11

Streptococcus pyogens 8 8

Organisms Zone of Inhibition (mm)

24h 48h

Aspergillus flavus 5 7

120

that the pre and post washed silver nanoparticles delayed bacteria growth at the

interface of silver nanoparticles as reported by Morones et al. (2005) and direct usage

of the silver nanoparticles from the native reaction solution warranted more effective

(Sondi et al., 2004).

In fact, it is well known that Ag ions and Ag-based compounds are highly toxic

to microorganisms, showing strong biocidal effects on 12 species of bacteria including

E. coli (Zhao, 1998). Recently, it has been showed that hybrids of Ag nanoparticles

with amphiphilic hyperbranched macromolecules exhibited effective antimicrobial

surface coating agents (Aymonier et al., 2002). Reducing the particle size of materials

is an efficient and reliable tool for improving their biocompatibility. In fact,

nanotechnology helps in overcoming the limitations of size and can change the outlook

of the world regarding science (Mirkin, 2000). Furthermore, nanomaterials can be

modified for better efficiency to facilitate their applications in different fields such as

bioscience and medicine.

It is well known that Ag ions and Ag-based compounds have strong

antimicrobial effects (Furno et al., 2004), and many investigators are interested in using

other inorganic nanoparticles as antibacterial agents (Furno et al., 2004; Abuskhuna et

al., 2004 and Hamouda et al., 2000). These inorganic nanoparticles have a distinct

advantage over conventional chemical antimicrobial agents. The most important

problem caused by the chemical antimicrobial agents is multidrug resistance.

Generally, the antimicrobial mechanism of chemical agents depends on the specific

binding with surface and metabolism of agents into the microorganism.

Various microorganisms have evolved drug resistance over many generations.

Thus far, these antimicrobial agents based on chemicals have been effective for

therapy; however, they have been limited to use for medical devices and in prophylaxis

in antimicrobial facilities. Therefore, an alternative way to overcome the drug

resistance of various microorganisms is needed desperately, especially in medical

devices, etc. Ag ions and Ag salts have been used for decades as antimicrobial agents in

various fields because of their growth-inhibitory capacity against microorganisms.

121

Also, many other researchers have tried to measure the activity of metal ions against

microorganisms.

Studies of Russel and Hugo (1994) on the antimicrobial properties of Ag and

Cu and Marsh (2002) on Zn were reported. However, Ag ions or salts has only limited

usefulness as an antimicrobial agent for several reasons, including the interfering

effects of salts and the antimicrobial mechanism of [the continuous release of enough

concentration of Ag ion from the metal form.] In contrast, these kinds of limitations can

be overcome by the use of Ag nanoparticles. However, to use Ag in various fields

against microorganisms, it is essential to prepare the Ag with cost-effective methods

and to know the mechanism of the antimicrobial effect. Besides, it is important to

enhance the antimicrobial effect.

When compares to the chemical and physical synthesis, biological synthesis of

Ag nanoparticles can be prepared cost effectively and thus synthesized. These Ag

nanoparticles are homogeneous and stable. The nanosize allowed expansion of the

contact surface of Ag with the microorganisms, and this nanoscale has applicability for

medical devices by surface coating agents. In this study, to evaluate the antimicrobial

effects against various microorganisms, we used seven representative microorganisms,

E. coli and S. aureus. P.mirabilis, P. aeruginosa, S. typhi and S. pyogens. There were

distinct differences among them. When Ag nanoparticles were tested in yeast and E.

coli, they effectively inhibited bacterial growth. Gram-positive and gram-negative

bacteria have differences in their membrane structure, the most distinctive of which is

the thickness of the peptidoglycan layer. It is concluded that the lower efficacy of the

Ag nanoparticles against S. aureus may derive from the difference as a point of

membrane structure.

In contrast, Sondi and Salopek-Sondi (2004) reported that the antimicrobial

activity of silver nanoparticles on Gram-negative bacteria was dependent on the

concentration of Ag nanoparticle, and was closely associated with the formation of

dpitsT in the cell wall of bacteria. Then, Ag nanoparticles accumulated in the bacterial

membrane increased the permeability, resulting in cell death. The experimental

122

evidences are insufficient to explain the antimicrobial mechanism of positively charged

Ag nanoparticles.

Amro et al. (2000) suggested that metal depletion may cause the formation of

irregularly shaped pits in the outer membrane and change membrane permeability,

which is caused by progressive release of lipopolysaccharide molecules and membrane

proteins. Also, Sondi and Salopek-Sondi (2004) speculate that a similar mechanism

may cause the degradation of the membrane structure of E. coli during treatment with

Ag nanoparticles (Sondi and Salopek-Sondi, 2004). Although their interference

involved some sort of binding mechanism, still unclear is the mechanism of the

interaction between Ag nanoparticles and molecules of the outer membrane. Recently,

Danilczuk and co-workers (2006) reported Ag-generated free radicals through the ESR

study of Ag nanoparticles.

It is expected that the antimicrobial mechanism of Ag nanoparticles is related to

the formation of free radicals and subsequent free radical–induced membrane damage.

In conclusion, Ag nanoparticles prepared by the cost effective reduction method

described here have great promise as antimicrobial agents. Applications of Ag

nanoparticles based on these findings may lead to valuable discoveries in various fields

such as medical devices and antimicrobial systems. Silver and silver-based compounds

have been in use for centuries in the treatment of burns and chronic wounds (Castellano

et al., 2007).

Antioxidant property of synthesized nanoparticles:

The synthesized silver and gold nanoparticles showed antioxidant property,

when increase concentration of nanoparticles then the scavenging activity also is

increased. This gradual increase was observed in both of the nanoparticles (Fig 21). It is

known that gold nanoparticles exhibit high catalytic activity in some oxidation

reactions, such as low temperature CO oxidation, whereas gold as a compact metal is

chemically inactive because of both the size effect and the character of interaction of

the metal nanoparticles and support accompanied by the charge transfer between them.

a)

b)

Figure 21 Antioxidant property of synthesized Silver and Gold Nanoparticles: a) antioxidant property of silver nanoparticles (AG (NP)) taken in different concentration such as 20, 40, 60, 80 and 100µg/ml for scavenging activity b) antioxidant property of gold nanoparticles (AU (NP)) taken in different concentration such as 20, 40, 60, 80 and 100µg/ml for acavenging activity.

123

The antioxidant activity of the gold nanoparticles in chitosan oligomer solutions

was studied by ESR spectroscopy from their ability to deactivate hydroxy radicals. The

•OH radicals were generated in a hydrogen peroxide ascorbic acid redox system (molar

ratio 1: 1). It was impossible to detect the PBN adduct directly with the hydroxy radical

because of its short lifetime. For instance, at pH 7.4 in an aqueous medium the halved

lifetime (1/2) of the PBN-OH adduct is 38s (Kotake and Janzen, 1991).

Therefore, we used the secondary radical spintrapping technique. Dimethyl

sulfoxide was added to the system, and its interaction with the hydroxy radicals

afforded Me• radicals. The Me• radicals were detected by their stable adduct with PBN

characterized (Takeshita et al., 2004) by the following HFC constants: aN = 1.6 mT

and а H = 0.36 mT. This method finds wide use in studies of the formation of hydroxyl

radicals (Kadiidka and Mason, 2002 and Burkitt and Mason, 1991).

Annexure

Summary, References, List of GenBank Accessions and Publications

124

SUMMARY

On the whole, the research work on survey, molecular systematics and

nanobiotechnological potentials of diatom and marine cyanobacteria has been

summarized in three chapters.

Chapter I

Totally, 50 cyanobacterial species and 38 diatom species were recorded. They

were identified taxonomically with standard monographs.

Five cyanobacterial and five diatoms were phylogenetically characterized using

16S and 18S rDNA sequencing.

16S and 18S rDNA sequences were deposited in GenBank (NCBI) with

accession numbers. Molecular modeling was done with C-phycocyanin (PC) gene

sequence isolated from cyanobacteria.

Chapter II

A marine cyanobacterium Phormidium tenue NTDM05 was selected for the

production of phycobiliproteins. Extraction and quantification of C-Phycobiliprotein

was done from selected Cyanobacteria

Optimum conditions were found to the production of C-phycoerythrin by

physical and chemical methods such as temperature, pH, extraction methods, buffer

system and nutrients.

Citric acid was found to the best preservative to preserve C-phycoerythrin and

antimicrobial property and biochemical composition of the pigments were studied for

food additive

125

Chapter III

Silver and Gold nanoparticles has been synthesized using marine

cyanobacterium, Phormidium willei NTDM01 and characterized with TEM, SEM,

EDS, EDAX, FS, FTIR and Uv-vis spectrum.

It was found to be 20nm range of silver nanoparticles have synthesized

intracellularly and 150nm size of particles synthesized extracellularly. Anisotropic gold

nanoparticles synthesized by the action intracellular proteins in the cyanobacteria.

Antimicrobial activity of silver nanoparticles was analyzed against multidrug

resistant clinical pathogens. In vitro antioxidant property and Fluorescence property

were studied for application of biolabelling process.

Diatom cell was selected for the nanofabrication of Si-Ge nanocomposite in

ambient condition. By bottom up process, the Si-Ge nanocomposite formed in the

diatom, Stauroneis sp., was characterized by SEM and EDS.

CdS nanoparticles were synthesized by biological technique, using c-

phycoerythrin as capping agent. This present study average particle come out to be

5.1±0.2nm for C-PE assisted CdS nanoparticles after the statistical analysis. The

difference in size may possibly due to the fact that nanoparticles are being formed at

different times. The earlier report also found that difference size of particles due to the

incubation time.

This simplest and possible procedure may apply to the commercial production

of uniform and stable CdS nanoparticles effectively. Hypothetical mechanism for the

synthesis of CdS nanoparticle has been confirmed with previous reports.

This work emphasizes on the synthesis and application of nanomaterials using

green nanotechnology procedure for the development of eco-friendly, less-laborious

and inexpensive materials without affecting their properties and with increased

efficiency.

126

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S. No

Accession No Culture Detail Sequencing Detail

1. GU186910 Oscillatoria boryana NTAS01 16S rDNA gene sequence

2. GU186909 Chlorella sp. NTIA01 18S rDNA gene sequence

3. GU585845 Trichoderma viride NTDMF01 28S rDNA gene sequence

4. GU585846 Trichoderma viride NTDMF02 28S rDNA gene sequence

5. GU585847 Phormidium tenue NTDM05 16S rDNA gene sequence

6. GU585848 Spirulina subsalsa NTDM21 16S rDNA gene sequence

7. GU646678 Trichoderma harzianum PSMT1 28S rDNA gene sequence

8. GU812859 Phormidium animale NTMP03 16S rDNA gene sequence

9. GU812860 Oscillatoria acuminata NTDM04 16S rDNA gene sequence

10. GU812858 Oscillatoria willei NTDM01 16S rDNA gene sequence

11. GU812856 Phormidium chlorium NTDMP01 16S rDNA gene sequence

12. GU812857 Jaagniema pseudogeminatum NTMP02 16S rDNA gene sequence

13. HM217756 Trichoderma viride NTDMF01 ITS gene sequence

14. HM217757 Trichoderma viride NTDMF02 ITS gene sequence

15. HM585048 Cymbella tumida NTDMN03 18S rDNA gene sequence

16. HM635776 Synedra sp. NTDMN01 18S rDNA gene sequence

17. HM635777 Synedra sp. NTDMD01 18S rDNA gene sequence

18. HM635778 Amphora sp. NTDMN02 18S rDNA gene sequence

19. HM635779 Stauroneis sp. NTDMD02 18S rDNA gene sequence

20. HM635780 Trichoderma viride NTDMF01 Calmodulin gene (CaM) sequence

21. HM768826 Monoraphidium sp. NTAI03 18S rDNA sequence

22. HQ123322 Chlorella vulgaris 18S rDNA sequence

23. HQ123323 Chlorella vulgaris NTMP06 18S rDNA sequence

24. HQ141712 Aspergillus fumigatus NTLF06 ITS Sequence

25. HQ219673 Aspergillus terreus NTLF02 ITS Sequence

26. HQ245157 Penicillium citrinum NTLF05 ITS Sequence

27. HQ141713 Fusarium Sp. NTLF07 ITS Sequence

28. HQ219695 Fusarium sp. NTLF08 ITS Sequence

29. HQ141714 Trichoderma longibrachiatum NTLF01 ITS Sequence

30. HQ219674 Nodulosporium sp NTLF04 ITS Sequence

31. HQ141711 Emericella nidulans NTLF03 ITS Sequence

32. HQ219686 Chlorella sp. NTMP01 Calmodulin (CaM) gene sequence

33. HQ245158 Trichoderma koningii NTLF11 ITS Sequence

34. HQ537428 Trichophyton rubrum NTDMF20 ITS Sequence

35. HQ537429 Trichophyton mentagraphytes NTDMF21 ITS Sequence

GENBANK ACCESSIONS

163

36. HQ537430 Epidermophyton floccosum NTDMF22 ITS Sequence

37. ADR65079 Chlorella sp. NTMP01 Calmodulin (CaM) protein sequence

38. JF421687 Oscillatoria acuminata NTDM04 Phycocyanin gene Sequence

39. JF421685 Phormidium tenue NTDM05 Phycocyanin gene Sequence

40. JF421686 Phormidium animale NTMP03 Phycocyanin gene Sequence

41. Bankit No 1431047 Oscillatoria salina Phycocyanin gene Sequence

42. Bankit No 1431046 Oscillatoria chlorina Phycocyanin gene Sequence

43. Bankit No 1421025 Spirulina subsalsa NTDM21 Phycocyanin gene Sequence

44. Bankit No 1448107 Phormidium tenue NTSM01 16S rDNA gene sequence

45. JN402964 Phormidium tenue NTSM02 16S rDNA gene sequence

46. Bankit No 1448113 Aspergillus sydowii NTSMP01 ITS Sequence

47. Bankit No 1448113 Penicillium cinnamopurpureum NTSMP02

ITS Sequence

48. Bankit No 1448113 Cladosporium sp. ITS Sequence

49. JN402974 Anabaena variabilis NTVMK01 16S rDNA gene sequence

50. JN402975 Anabaena sp. NTVMK02 16S rDNA gene sequence

51. JN402976 Westiellopsis Sp. NTVMK03 16S rDNA gene sequence

52. JN402980 Nocardiopsis sp. NTRJM01 16S rDNA gene sequence

53. JN402977 Nocardiopsis sp. NTRJM03 16S rDNA gene sequence

54. JN402979 Streptomyces sp. NTRJM05 16S rDNA gene sequence

55. JN216833 Aspergillus niger NTGMP01 ITS Sequence

56. JN216834 Aspergillus fumigatus NTGMP02 ITS Sequence

57. JN216835 Penicillium sp. NTGMP03 ITS Sequence

58. JN216836 Fusarium sp. NTGMP04 ITS Sequence

59. JN216837 Aspergillus terreus NTGMP05 ITS Sequence

60. JN66080 Trichoderma viride NTSA01 ITS sequence

61. JN66081 Trichoderma viride NTSA02 ITS sequence

62. JN66082 Trichoderma viride NTSA03 ITS sequence

63. JN402978 Rhodococcus sp NTRJM04 16S rDNA gene sequence

64. JN402981 Rhodococcus sp NTRJM04 16S rDNA gene sequence

65. JN402965 Poterioochromonas sp. NTNV01 18S rDNA gene sequence

66. JN402966 Poterioochromonas sp. NTNV02 18S rDNA gene sequence

67. JN402967 Poterioochromonas sp. NTNV03 18S rDNA gene sequence

68. JN402968 Poterioochromonas sp. NTNV04 18S rDNA gene sequence

69. JN402969 Poterioochromonas sp. NTNV07 18S rDNA gene sequence

70. JN402970 Botrycoccus sp NTNV08 18S rDNA gene sequence

71. JN402971 Hartmannella vermiformis NTNV09 18S rDNA gene sequence

72. JN402972 Poterioochromonas sp. NTNV04 18S rDNA gene sequence

73. JN402973 Poterioochromonas sp. NTNV05 18S rDNA gene sequence

164

74. JN601431 Staphyloccocus aureus NTMUR01 16S rDNA gene sequence

75. BankIt 1474793 Klebsiella pneumoniae NTMUR02 16S rDNA gene sequence

76. BankIt 1474793 Moraxella cattarhalis NTMUR03 16S rDNA gene sequence

77. JN601432 Candida albicans NTMUR04 18S rDNA gene sequence

78. JQ731679 Coelastrella sp. NTEB01 18S rDNA gene sequence

79. JQ796863 Botryococcus sp. NTAI06 18S rDNA gene sequence

80. JQ796861 Acharochaet rupens NTAI07 18S rDNA gene sequence

81. JQ796860 Tetradesmus sp NTAI05 18S rDNA gene sequence

82. JQ796862 Tetradesmus sp NTAI04 18S rDNA gene sequence

83. JQ867392 Leptolynbya valderiana NTDM10 16S rDNA gene sequence

84. JQ867393 Oscillatoria boryana NTDM11 16S rDNA gene sequence

85. JQ867394 Planktothrix pseudoadardhii NTRD01 16S rDNA gene sequence

http://www.ncbi.nlm.nih.gov/

Digest Journal of Nanomaterials and Biostructures Vol. 6, No 2, April-June 2011, p. 385-390

BIOSYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES USING MARINE CYANOBACTERIUM, OSCILLATORIA WILLEI NTDM01

D. MUBARAK ALI., M. SASIKALA., M. GUNASEKARAN, N. THAJUDDIN* Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620024, India Department of Biology, Fisk University, Nashville, TN 37208, USA Pioneering of reliable and eco-friendly process for synthesis of metallic nanoparticles biologically is an important step in the field of application of nanobiotechnology. This paper reports the extracellular biosynthesis of silver nanoparticles using marine cyanobacterium, Oscillatoria willei NTDM01 which reduces silver ions and stabilizes the silver nanoparticles by a secreted protein. The silver nitrate solution incubated with washed marine cyanobacteria changed to a yellow color from 72h onwards, indicating the formation of silver nanoparticles. The characteristics of the protein shell at 265nm were observed in Ultra violet spectrum for the silver nanoparticles in solution. While Fourier Transform Infra Red (FTIR) confirmed the presence of a protein shell which are responsible for the nanoparticles biosynthesis. Scanning Electron Microscopy (SEM) studies showed that the formation of agglomerated silver nanoparticles due to the capping agent in the range of 100 - 200nm. EDS spectrum of the silver nanoparticles was confirmed the presence of elemental silver signal in high percentage. Apart from Eco-friendliness and easy availability and low cost cyanobacterial biomass production will be more advantageous when compared to other classes of micro organism. Biosynthesis nanoparticles would have greater commercial viability if the nanoparticles could be synthesized more rapidly in the reaction vessel. (Received January 10, 2011; accepted February 2, 2011) Keywords: Marine cyanobacteria, silver nanoparticles, Oscillatoria willei NTDM01, FTIR, EDS

1. Introduction Nanotechnology is enabling technology that deals with nano-meter sized objects. It is

expected that nanotechnology will be developed at several levels: materials, devices and systems. Synthesis of nanoparticles using biological entities has great interest due to their unique shape dependent optical, electrical and chemical properties have potential application in nanobiotechnology. The synthesis and assembly of nanoparticle would benefit from the development of clean, nontoxic and environmentally acceptable "green chemistry" approaches for nanoparticles.

The biosynthesis of silver nanoparticles of different sizes, ranging from 1- 70nm, and shapes, including spherical, triangular and hexagonal has been conducted using bacteria, fungi, plant extracts (1-4). The mechanisms for the bioreduction of silver by bacteria involve reducing and other proteins in which sulphur and carboxylate group from cell wall (2-4). The silver nitrate caused the reduction of silver through a nitrate dependent reductase and synthesized the nanosized materials in Fusarium oxysporum (3). In this study, the formation of silver nanoparticles was investigated using silver nitrate in the presence of the marine cyanobacterium, Oscillatoria willei NTDM01. Marine cyanobacteria are one of the largest, photoautotrophic bacteria in marine ecosystem and are known to have high affinity transport system for nitrate.

*Corresponding author: nthaju2002@yahoo.com

386

2. Materials and methods The marine cyanobacterial samples was collected and isolated from Kurusadai Island (Lat.

9° 18�N & Long 79°10�E) at Gulf of Mannar, Tamilnadu, India. The culture was grown in MN- III medium at pH 8 (6) and made them axenic using different combination of antibiotics. The culture was transferred and grown for approximately 4-6 weeks to reach a stationary 'growth' phase. It was then centrifuged and washed several times with distilled water, deionized water to remove the trace metals from the surface of the cyanobacteria.

Table 1. FTIR analysis of O. willei NTDM01, which shows the functional group of the protein

Frequencycm-1 Type Vibration Mode

3437.05 Sec.amides, freeNH(trans) N-H Structure 2926.52 Alkanes(-CH2-) C-H Structure 2857.58 Alkanes(-CH2-) C-H Structure 2378.14 Charged amines(C=NH+) NH- Structure 2096.19 Organo-silicon compounds Si-H Structure 1639.24 Alkenes(CHR1=CHR1-cis) C=C Structure 1394.59 Alkanes, tery.butyl C-H deformation 1224.56 Aliphatic esters CH3COOR 1102.71 Secondary alcohols C-OH Structure

To initiate the experiments, 5mL of silver solution (~560mg/L) was added to 5mL of

washed cyanobacterial cultures. The experiment were incubated at 25° C for 28 days and maintained in the dark. Abiotic experiments consisting of ~560mg/L silver were conducted using silver nitrate solution without the presence of cyanobacteria. The pH and cell viability were monitored during the course of the study. The sample have been characterized for the development of silver nanoparticles by using Ultra Violet �Visible Spectrum (Uv-Vis) , Fourier Transform Infra Red (FTIR), Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopic (EDS) investigation (3,7).

3. Results The axenic strain was identified as Oscillatoria willei NTDM01 (Fig.1). In cyanobacterial

experiment, soluble silver was completely precipitated from solutions within 28days. A grayish- black silver precipitate on cyanobacteria was observed in experiment macroscopically. The pH was constant at 5.0 and 4.7. In abiotic experiments using silver nitrate, total soluble silver concentration and pH were constant until the completion of experiment.

387

Fig. 1. Microphotography of marine cyanobacterium, Oscillatoria willei NTDM01.

The UV-vis Spectrum reveals that the band observed at 450 nm due to the plasmon

resonance of the silver nanoparticles and low wavelength region recorded from the reaction medium exhibited an absorption band at ca.265 nm and it was attributed to aromatic amino acids of proteins (Fig.2). It is well known that the absorption band at ca 265 nm arises due to electronic excitation in tryptophan and tyrosine residue in the protein.

a

b Fig. 2. UV-Vis spectrum of plasmon resonance of silver nanoparticles synthesized by O.willei NTDM01 (2a), low wavelength region recorded, where the reducing protein

present in O. willei NTDM01.

388

The FTIR spectrum was recorded from the film of silver nanoparticles, formed after several days of incubation with the bacteria (Fig.3). The bands seen at 3280 cm 1 and 2924 cm 1 were assigned to the stretching vibrations of primary and secondary amines respectively. The corresponding bending vibrations were seen at 1651 cm 1 and 1548 cm 1, respectively. The two bands observed at 1379 cm 1 and 1033 cm 1 can be assigned to the C�N stretching vibrations of aromatic and aliphatic amines, respectively. The presence of protein as the stabilizing agent surrounded the silver nanoparticles (7). The silver ions were reduced in the presence of nitrate reductase, leading to the formation of a stable silver hydrosol 10-25 nm in diameter and stabilized by the capping peptide.

SEM observation on products of marine cyanobacterium, Oscillatoria willei NTDM01 in AgNO3 experiment is presented (Fig.4). At room temperature, the addition of AgNO3 to the cyanobacteria caused the precipitation of silver nanoparticles at cell surfaces (Fig.4a). Small spherical silver nanoparticles with size ranging from 100nm to 200nm (extracellularly) were also precipitated in solution silver nanoparticles were deposited at cell surfaces (Fig.4b). EDS showed the occurrence of silver particle in higher amount with trace of magnesium, calcium and chloride. In this analysis by EDS of the silver nanoparticles was confirmed the presence of elemental silver signal (Fig.4c).

100

90

Fig. 3. FTIR analysis of O. willei NTDM01 shows the presence of protein shell for the

reduction of silver ions.

4000 3000 200 150 100 4000

1

2

3

4

5

6

7

8

cm-1

%T

3437

2926

2857

23782096

1224465

1102

1026 604

389

a

b

Fig. 4a. Precipitation of silver nanoparticles on the cyanobacterial surfaces. b. SEM micrograph of cyanobacteria cells with nanoparticles silver deposited outside the cell with size

range of 150nm c. EDS of the silver nanoparticles was confirmed the presence of elemental silver signal in high percentage.

The reaction of cyanobacteria with AgNO3 solutions results in the formation of silver

nanoparticles. The presence of spherical silver nanoparticles was observed in experiment. The precipitation of silver nanoparticles was not observed for abiotic experiment that were run under similar condition and duration, that suggesting that cyanobacteria were required for silver precipitation and the precipitation processes were not controlled by inorganic chemical reactions.

4. Discussion A brief overview of the use of micro organisms such as cyanobacteria, yeast, algae, fungi

and actinomycetes in the biosynthesis of metal nanoparticles has been described. Our UV-vis observation indicated the release of protein into the culture medium indicting the electronic excitation of aromatic amino acids. A similar observation has been made in Fusarium oxysporum and suggests a possible mechanism for the reduction of the metal ions present in the solution (8). The overall peaks from FTIR observation confirm the presence of protein in the samples of silver nanoparticles. It can be assumed that nitrate reducing bacteria to produce nitrate reductase enzyme (protein). This reduces to silver nitrate solution form silver nanoparticles. The synthesis of silver nanoparticles using ß-NADPH-dependent nitrate reductase and phytochelatin in vitro has been demonstrated for the first time (9). The cyanobacterial cells were encrusted with silver nanoparticles and separation of some filaments into their constituent cells were still observed. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by capping agent. This corroborates with the previous observation in F. oxysporum (8). Cyanobacteria commonly

390

use nitrate as the major source of nitrogen for three different purposes including growth, generation of metabolic energy, and redox balancing (10). The presence of silver nanoparticles throughout the cells suggests that Ag+ and NO3 entered the cyanobacteria cells through a transport system and degraded in solution. The presence of silver nanoparticles inside the cytoplasm, Ag+ is presumably reduced to Ago Because AgNO3, a toxic reagent, was used in metabolic processes, it ultimately killed the cells. During the death of cyanobacteria, nanoparticles of silver produced inside the cells were release through the cell membrane into solution, as indicated by the precipitation of silver nanoparticles around the cells. The dead cyanobacteria also released organics (protein, and other biochemical) that caused further precipitation of silver from solution outside the cells. The protein molecules act as reducing agent for silver nanoparticles. The protein molecule made up of different functional group in amino acid sequences such as amino, carboxyl, sulfate groups present in the cyanobacterial protein favor the formation of extremely small-sized silver nanoparticulates with narrow particle size distribution and hydroxyl and sulfonic groups are beneficial to synthesis of silver nanoparticle with a slightly larger particle size in a weak reducing environment. It has already been certified that the silver nanoparticles can be stabilized and are well functional groups, such as carboxyl, hydroxyl, and amido and thiol group, except the steric effect arising from the large molecular structures of the organic modifier (protein) on the surface of the particles (11,12). The micro organisms in the synthesis of nanomaterials as a possible viable alternative to the more popular physical and chemical methods, currently in vogue. The use of cyanobacteria as a source of enzymes that can catalyze specific reactions leading to inorganic nanoparticles is a new and rational biosynthesis strategy that is being developed. Extracellular secretion of enzymes offers the advantages of obtaining large quantities in a relatively pure-state, free from other cellular proteins associated with the organism and can be easily processed by filtering of the cells and isolating the enzyme for nanoparticles synthesis from cell-free filtrate.

Acknowledgments The authors wish to acknowledge support from University Grant commission (UGC) for

their financial assistance. References

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Digest Journal of Nanomaterials and Biostructures Vol. 6, No 1, January-March 2011 p. 117 - 120

BIOSYNTHESIS AND CHARACTERIZATION OF SILICON-GERMANIUM OXIDE NANOCOMPOSITE BY DIATOM

D. MUBARAK ALI, C. DIVYA, M. GUNASEKARANa, N. THAJUDDIN Department of Microbiology, School of Lifescience Bharathidasan University, Tiruchirappalli, Tamil Nadu, IndiaaDepartment of Biology, Fisk University, Nashville Tennessee, USA

Diatoms are single cell photosynthesizing eukaryotic algae that produce intricately structured cell wall made of nano-patterned silica. The biologically fabricated nanostructures offer substantially different properties related adhesion, tribology, optic and electronic behavior. In this study biosynthesis and characterization of Silicon- Germanium (Si-Ge) oxide nanocomposite in the diatom, Stauroneis sp., by two stage cultivation process. The exponential growth of Stauroneis sp., inoculated with enrichment medium amended with Germanium. The silicon to germanium concentration molar ratio was 1:0.5. Scanning electron microscopic studies reveals that no structural change is observed when germanium concentration is low, the increased concentration of germanium led to structural changes of the diatoms. The Si-Ge oxide nanocomposite was characterized by Electron Microscopy equipped with energy dispersive spectroscopy to evaluate the chemical composition and structural properties of the diatoms.

(Received December 1, 2010; accepted January 14, 2011)

Keywords: Nanocomposites, silicon, germanium, diatom, Stauronesis sp., EDS

1. Introduction

The cell wall structure is a species-specific characteristics demonstrating that diatom silica morphogenesis is genetically encoded. It is expected that these structures exhibit superior properties in a wide range of applications. The fabricated and self assembled of semiconductor nanostructures that possess unique optical and electronic properties [1]. The supramolecular chemistry and catalysis have led to novel surface and size dependent chemistry such as enation selective catalysis at the surface. The ability of diatoms to make complex, nanoscaled, three dimensional silica shells called “frustules” offers attractive possibilities for their application to nanobiotechnology [2]. Bioengineered nanosystem is extremely difficult to realize on the basis of current scientific knowledge and bottom up assembly is very powerful in creating of identical structures with atomic precision, such as supramolecular functional entities in living organisms. In this regards diatoms, a prolific class of single celled algae that make micro scale biosilica or frustules with intricate submicron features dominated by two dimensional pore arrays, have been advertized as a paradigm for the controlled production of nanostructured silica with interesting properties [3]. The fabrication of novel biomaterials through molecular self assembly is studied currently [4]. In this study we report that the fabrication of Silicon- Germanium oxide nanocomposites characterized by Electron Microscopy and EDS.

______________________ *Corresponding author: nthaju2002@yahoo.com

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2. Materials and methods The photosynthetic freshwater diatom, Stauroneis sp., was isolated from the freshwater

ponds from Bharathidasan University Campus and maintained in F/2 medium and cultivated in appropriate growth conditions such as light (2000lux), pH (8.0±0.2). The two stage cultivation methods is follwed for the synthesis of silicon-germanium nanocomposites as described [5]. In first stage, the cells were grown in nutrient medium containing sufficient silicon concentration for its growth. The silicon concentration (30mg/l) and cell density is monitored at regular intervals. When all the silicon was used up and the cell density had become constant, the cells were then considered to be starved of silicon, this condition prevails one week. In the second stage, the silicon and germanium (1:0.5) is added to the liquid medium for the biosynthesis of Silicon –Germanium nanocomposites. After 7 days the cells were harvested from the medium and washed thrice in deionized water in order to remove surface adsorbed germanium and other trace elements. Then the sample was centrifuged at 2000rpm for 5mins. The pellet was collected and dispersed on the thin slide and allowed to dry in hot air oven. The small portion of slide was placed onto carbon tape affixed to a stub, the gold was coated before introduction to the Scanning Electron Microscopy equipped with EDS.

3. Results and discussion

The incorporation of the Germanium into diatom cells was achieved and nanostructured pores could be found without morphological aberrations (Fig. 1). In this study the freshwater diatom, Stauroneis sp., was used to fabricate Silicon- Germanium by its metabolic activity. The SEM was used to assess and validate the integrity of the diatoms. The diatom, Stauroneis sp., frustules is observed in different growth conditions. After separation of diatom frustules from the organic matter, the sample was dispersed in alcohol, and then placed on the stub [6]. The characteristics peaks of Germanium were observed at 1.22 keV, 9.8 keV and 10. 4keV. The maximum peak of silicon was observed at 1.9keV (Fig. 2). It has been reported that the distribution profile of germanium in the diatom frustules is similar to that of silicon intensity is quite different [6]. It has been reported that silica from bioreactor cultured Nitzschia frustulumcells possessed blue photoluminescence, where the luminescence intensity and wavelength were dependent on the change in frustules nanosturucture as the cell culture moved from the exponential to the stationary phase of growth [5]. The soluble Germanium can be metabolically inserted into biosilica of the pennate diatom, Pinnularia sp., by two stage bioreactor process [7]. The metabolic incorporation of germanium into the biosilica of the diatom Nitzschia frustulum is described for the biological fabrication.

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a

bFig. 1 SEM images of Stauroneis sp., grown in enriched medium (F/2) along with

germanium(1a). It showed clear image of nanopore structures (1b).

Fig 2. EDS spectrum of germanium doped culture Stauroneis sp., grown in enriched medium with germanium.

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Fig 3. SEM images of Stauroneis sp., grown in enriched medium (F/2) along with germanium in molar ratio of 1:1. It showed aberrated diatom structure.

Fig. 3 shows that due to the equal volume of Silicon- Germanium, the diatom cells are fully aberrated. An Electron Microscopic study reveals that due to the incorporation of germanium, the diatom cells lead to various degrees of aberrations and also unavailability of sufficient silicon for the cell wall biosynthesis. The Stauroneis sp., with high percentage of germanium concentration has been found to cause cell aberrations than with the low percentage. The difference in bond length between silicon oxide and germanium oxide probably weakens the diatom frustules and cause them to break easy during the processing of the cells [6].

In this study incorporation of germanium into diatom cells were achieved. Several workers also reported that there was no Silicon- Germanium homogeneity in the distribution of the diatom cells. The experimental protocol should be standardized to achieve the homogeneity intricate Silicon-Germanium nanocomposites. These optoelectronic properties of the nanocomposites, which depend on the amount of Germanium assimilation can therefore be controlled.

Acknowledgement

The authors are grateful thanks to the University Grant Commission (UGC) for their grant.

References

[1] G. L. Rorrer, C-H. Chang, S.H. Liu, C. Jeffryes, J. Jiao, J. A. Hedberg, J. Nanosci. Nanotechnol. 5, 41 (2005). [2] M. Hildebrand. J. Nanosci. Nanotechnol. 5, 146 (2005). [3] M. Sumper, E. Brunner, Adv. Funct. Mater. 16, 17 (2006). [4] S. Zhang, Nat. Biotechnol. 21, 1171 (2003). [5] T. Qin, T. Gutu, J. Jiao, C-H. Chang, G.L. Rorrer, J. Nanosci. Nanotechnol. 8, 2392 (2008). [6] T. Gutu, L. Dong, J. Jiao, G.L. Rorrer, C-H. Chang, C. Jeffryes, Q. Tian. Microc Microanal.

11, 1958 (2005). [7] C. Jeffryes, T. Gutu, J. Jiao, G. L. Rorrer. Mater. Sci. Eng. C. 28, 107 (2008).

African Journal of Basic & Applied Sciences 3 (1): 09-13, 2011ISSN 2079-2034© IDOSI Publications, 2011

Corresponding Author: Dr. N. Thajuddin, Department of Microbiology, Bharathidasan University, Tiruchirappalli-620 024, India, Tel: +91-431-2407028, E-mail:-nthaju2002@yahoo.com.

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Efficiency of Textile Dye Decolorization by Marine Cyanobacterium, Oscillatoria formosa NTDM02

D. Mubarak Ali, A. Suresh, R. Praveen Kumar, M. Gunasekaran and N. Thajuddin1 1 1 2 1

Department of Microbiology, School of Life sciences, Bharathidasan university, Tiruchirappalli-620 024, India

Department of Biology, Fisk University, Nashville Tennessee, USA 1

Abstract: Cyanobacteria are the Gram negative, oxygen evolving photosynthetic prokaryotes andhave wide variety of application in biotechnological research. Releasing of textile dye effluents intogeneral water bodies is a major environmental and health problem. Micro algae and cyanobacteria areconsidered as an important source for decolorizing dye and textile effluent. The dye Amido Black and textiledye effluent is chosen for this investigation and the marine cyanobacterium, Oscillatoria formosa NTDM02was used for the decolorization process. Chlorophyll-a content of this organism was tested before and after thetreatment. The experimental results showed that the marine cyanobacterium, Oscillatoria formosa NTDM02,which decolorize the textile effluent efficiently in short period of time. This can be used for the bioremediationof dye effluents.

Key words: Oscillatoria % Effluent % Dye % Chlorophyll-a % Cyanobacteria % Bioremediation

INTRODUCTION MATERIALS AND METHODS

Microorganism, as bioremediation agents in the Culture Conditions: The marine cyanobacterium,treatment of waste water containing textile dyes [1]. Oscillatoria formosa NTDM02 (obtained from theCyanobacteria have a ubiquitous distribution [2-5] but Microbial Culture Collections of Microbiologytheir role in functioning of ecosystems, including Department at Bharathidasan University, Tamilnadu,degradation of recalcitrant compounds including India) was maintained at 25°C in MN III [15] medium withdyes [6-9]. Azo-aromatic dyes are the major group of light/dark cycle of 16/8 h under 6000 Lux illumination. Fortextile dye stuffs. When these colored effluents enter the experiment, 7-day-old logarithmic growing cells ofwater bodies they may either inhibit the biological activity Oscillatoria formosa NTDM02 was used.or cause water-borne disorders such as nausea,hemorrhage, ulceration of skin and mucous membrane, Analysis of [H ] concentration (pH): pH of the mediumdermatitis, perforation of nasal septum, severe irritation of and effluent were monitored at every stages of the studyrespiratory tract or cancer [10]. These structures can be using pH meter (Elico) to analyze the variation during thereductively cleaved into colorless amines by several decolorization process. bacterial species [11, 12]. The cleavage of azo bonds is agratuitous process which can occur when the micro Estimation of Chlorophyll-a: Chlorophyll a was measuredorganisms using a reduced carbon compounds as thegrowth substrate [13]. The marine cyanobacterium,Phormidium valderianum produces hydrogen and canremove dyes from the solution [14]. Microbialdecolorization processes have the advantage of beingsimple in design and low cost compared withconventional treatments.

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spectrophotometrically at 665nm from the cell mass beforeand after the treatment to compare the growth [16].

Study of Dye Decolorization: The dye effluent wascollected from textile industry at Karur, Tamilnadu, Indiaand Amido Black G200 was used for the study ofdecolorization control. For this study five set of

Phykos 42 (1): 10 14 (2012) Ilavarasi, et al., Production of FAME from freshwater microalgae

©Phycological Society, India

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Production of FAME from freshwater microalgae and profiling of fatty acids for biodiesel feedstock

Ilavarasi, A., MubarakAli, D., Parveez Ahamed, A and N. Thajuddin*

Department of Microbiology, School of Life Sciences, Bharathidasan University,

Tiruchirappalli 620024, Tamil Nadu, India.

*Corresponding author: E. mail: nthaju2002@yahoo.com

Abstract

Due to diminishing fossil fuel reserves, it is necessary to shift for an alternative fuel source that could be sustainable and eco-friendly. Microalgae produce different ratios of lipids, proteins and carbohydrates. The lipid composition of microalgae is similar to vegetable oil which has already been used as biodiesel so that it can potentially be employed for biodiesel production. In present study has demonstrated the total lipid content, fatty acid profile and biodiesel production from a naturally isolated fresh water strain of Chlorella sp. and the total lipid content was found to be 8% under normal nutrient conditions. Gas Chromatography of FAME was analyzed, it was found to be the major fatty acids were palmitic acid, stearic acid, oleic acid, linoleic acid and alpha linolenic acid and pH of the biodiesel was found to be 8.1.

Keywords: Microalgae, Chlorella sp., BG-11, Lipid, FAME, GC, Biodiesel.

Introduction:

Robust population explosion, receding fossil fuel reserve, enhanced GHG, larger emission of flue gases etc have brought the world scientists and biotechnologists to the threshold of exploring alternative energy sources especially biodiesel. Biodiesel is characterized as a renewable, biodegradable and eco-friendly fuel which has attracted wide attention. Most recently, research effort has been aimed at identifying suitable biomass species which can provide high energy outputs to replace the fossil fuels. Many of the researchers have attempted to produce biodiesel from non-edible sources like frying oil, greases, tallow, jatropha, mahua and soy bean oils. Besides these non-edible sources we can use microalgae as feed stock for biodiesel.

Microalgae are oxygenic photosynthetic organisms which inhabit almost all moist and lighted environments. They are veritable miniature biochemical factories, appeared to be more photosynthetically efficient than terrestrial plants and are efficient CO2 fixers. Many of them are exceedingly rich in oil, fast growing and so they are used in biodiesel production. They have the ability to replace the fossil fuel (Chisti, 2007). Even, there is a pressing need to improve the biodiesel production from microalgae and that too on selection of suitable biomass species. The nutrients deprivations would be influence on the lipid accumulation in a dominant indigenenous microalga, Chlorella sp., BUM11008 for biodiesel production (Praveenkumar et al., 2012). In the present study, fresh water microalga, Chlorella sp. NTAI01 was

isolated and identified. The total lipid was characterized both quantitatively and qualitatively in biofuels aspects.

Materials and Methods: Samples:

The microalgal samples were collected from stagnant fresh water body in Bharathidasan University campus, Tiruchirappalli, India.

Isolation, identification and growth condition of microalgae:

The collected samples were enriched initially in BG -11broth in conical flasks (250ml) at 24±2°C under 37.5µmol-1m2s-1 intensity with 16:8 hours photoperiod for 10 days. Then the enriched culture samples were spread on BG 11 agar plates and incubated at the above said conditions (Ilavarasi et al., 2011). After incubation, individual colonies were picked and transferred to the same media for purification in 250ml conical flask. The culture broth was shaken manually for five to six times a day. The purity of the culture was monitored by regular observation under microscope. The isolated microalgae were identified microscopically using light microscope with standard manual for algae (Shashikant and Gupta, 1998).

Measurement of growth:

The growth of the algal biomass was assessed by means of optical density with five days intervals from 5th