Bir Bahadur · Manchikatla Venkat Rajam Leela Sahijram · K.V Krishnamurthy Editors Plant Biology and Biotechnology Volume I: Plant Diversity, Organization, Function and Improvement Tai Lieu Chat Luong Plant Biology and Biotechnology Bir Bahadur • Manchikatla Venkat Rajam Leela Sahijram • K.V Krishnamurthy Editors Plant Biology and Biotechnology Volume I: Plant Diversity, Organization, Function and Improvement Editors Bir Bahadur Sri Biotech Laboratories India Limited Hyderabad, Telangana, India Leela Sahijram Division of Biotechnology Indian Institute of Horticultural Research (IIHR) Bangalore, Karnataka, India Manchikatla Venkat Rajam Department of Genetics University of Delhi New Delhi, India K.V Krishnamurthy Center for Pharmaceutics, Pharmacognosy and Pharmacology, School of Life Sciences Institute of Trans-Disciplinary Health Science and Technology (IHST) Bangalore, Karnataka, India ISBN 978-81-322-2285-9 ISBN 978-81-322-2286-6 DOI 10.1007/978-81-322-2286-6 (eBook) Library of Congress Control Number: 2015941731 Springer New Delhi Heidelberg New York Dordrecht London © Springer India 2015 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Printed on acid-free paper Springer (India) Pvt Ltd is part of Springer Science+Business Media (www.springer.com) Foreword Plants are essential to humanity for food, environmental intensification and personal fulfillment Plants are also the foundations of healthy ecosystems ranging from the Arctic to the tropics Plant biology is a living science dealing with the study of the structure and function of plants as living organisms, ranging from the cellular and molecular to the ecological stage It concerns the scientific study of plants as organisms and deals with the disciplines of cellular and molecular plant biology and the traditional areas of botany, e.g., anatomy, morphology, systematic physiology, mycology, phycology, ecology, as well as evolution The backbone of plant biology resides in its applications and spans from anatomy, plant physiology, and plant ecology to biochemistry, cell biology, and genetics Biotechnology is the use of living systems and organisms to develop or make useful products or “any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use.” Depending on the tools and applications, it often overlaps with bioengineering and biomedical engineering For thousands of years, humankind has exploited biotechnology in agriculture, food production, and medicine It is believed that the term biotechnology was coined in 1919 by Hungarian engineer Károly Ereky During the twentieth and early twenty-first centuries, biotechnology was expanded to include diverse sciences such as genomics, recombinant gene technologies, applied immunology, and development of pharmaceutical therapies and diagnostic tests The past few years have witnessed the establishment of Departments or Institutes of Plant Biology and Biotechnology in different parts of the world As the integration of the two subjects has expanded, undergraduate and postgraduate degrees have been instituted with distinct syllabi Over the years, extraordinary developments have taken place, and significant advances have been made in biotechnology and plant biology Unfortunately, there are not many texts on the confluence of the two subjects; hence, there is a dire need for texts that are pertinent for teaching courses and conducting research in this area The present set of volumes is compiled to fill this gap and is edited by four eminent, talented, and knowledgeable professionals, Profs Bir Bahadur, M V Rajam, Leela Sahijram, and K V Krishnamurthy They have tried v vi to compile and cover major developmental processes to give the student a feel for scientific research Volume contains 33 chapters, describes the past, present, and future of plant biology and the principles and strategies, and summarizes the landmark of research done on various aspects The same authors have also compiled the first five chapters along with other colleagues to set the stage for the reader to comprehend the ensuing chapters One chapter gives a comprehensive description of plant biodiversity; two chapters give an overview of plant– microbe interaction Reproductive strategies of bryophytes, Cycads: an overview constitute the contents of two chapters A single cohesive chapter on AM fungi describes them as potential tools in present-day technologies required for sustainable agriculture and to lessen the dependence on chemical fertilizers The use of AM fungi as biofertilizers and bioprotectors to enhance crop production are well accepted, e.g., mining the nutrients, stimulating growth and yield, and providing resistance against water stress and pathogen challenge The reproduction process by which organisms replicate themselves in a way represents one of the most important concepts in biology Through this, the continuity of the existence of species is ensured At the base level, reproduction is chemical replication and with progressive evolution, cells with complexity have arisen and in angiosperms involving complex organs and elaborate hormonal mechanism Three chapters that exclusively deal with genetics of flower development, pre- and postfertilization growth, and development respectively are written in a masterly way A single chapter on seed biology and technology should be of special interest to crop breeders and geneticists alike The role of apomixis in crop improvement is most striking, and attract the attention of crop breeders wanting to secure pure lines Physiological aspects spanning from photosynthesis to mineral nutrition, which are important aspects of improving yield, have been reviewed pithily Four chapters discuss details of induced mutations, polyploidy, and male sterility in major crops, and the potential of the utilization of these techniques is essential to shaping scientific minds These have been discussed in depth Each chapter is compiled by a distinguished faculty who has taken seriously its commitment to satisfy the intellectual urge of lifelong learners Areas of faculty research interest include cell and molecular biologists, geneticists, environmental biologists, organism biologists, developmental and regenerative biologists, and bioprocess technologists Each chapter provides an authoritative account of the topic intended to be covered and has been compiled by one or more experts in the field Each chapter concludes with carefully selected references that contain further information on the topics covered in that chapter I am privileged to have known some of the authors both professionally and personally and am very excited to see their invaluable contributions For the students wishing to update themselves in the convergence of biology and biotechnology, the present volume not only furnishes the basics of the life sciences but provides plenty of hands-on functional experience, starting with plant diversity, organization, function, and improvement Experienced life scientists, biologists, and biotechnologists have collaborated and pooled their talent and long experience in cross-disciplinary topics centered Foreword Foreword vii on recent research focus areas Interdisciplinary experts have combined their academic talent and strengths to further scientific discoveries in areas such as microbial diversity; divergent roles of microorganisms; overview of bryophytes, cycads, and angiosperms; etc The strength of the volume lies in reproductive biology e.g., genetics of flower development, pre- and postfertilization reproductive growth, and development in angiosperms From finding better ways to deliver crop improvement, perk up the quality of produce, and exploit plant genomics and plant-based technologies to the myriad other ways, the life sciences touch our world, and there has never been a more exciting – or important – time to be a life scientist If you want to learn more about what biology and biotechnology in plants can for you, please pick up this volume and browse in depth This volume is intended for scientists, professionals, and postgraduate students interested in plant biology and biotechnology or life sciences The volume will be indispensible for botanists, plant scientists, agronomists, plant breeders, geneticists, evolutionary biologists, and microbiologists Honorary Scientist of the Indian, National Science Academy, Biotechnology Laboratories Centre for Converging Technologies University of Rajasthan, Jaipur, India Satish C Maheshwari Preface Plant biology has been a fundamental area of biology for many centuries now, but during the last 30 years or so, it has undergone great transformation leading to a better and deeper understanding of many key fundamental processes in plants The idea of preparing these two volumes grew out of a need for a suitable book on plant biology and biotechnology for contemporary needs of students and researchers The present volumes, to the best of our belief and knowledge, cover the most contemporary areas not adequately covered in most, if not all, books currently available on plant biology, plant biotechnology, plant tissue culture and plant molecular biology Every effort has, therefore, been made to integrate classical knowledge with modern developments in these areas covering several new advances and technologies This will definitely enable a better understanding of many aspects of plants: molecular biology of vegetative and reproductive development, genetically engineered plants for biotic and abiotic stress tolerance as well as other useful traits, use of molecular markers in breeding, all the ‘-omics’ and various biotechnological aspects of benefit to mankind to meet challenges of the twenty-first century, to mention just a few These books have been designed to provide advanced course material for post-graduates in plant sciences and plant biotechnology, applied botany, agricultural sciences, horticulture and plant genetics and molecular biology These also serve as a source of reference material to research scholars, teachers and others who need to constantly update their knowledge Volume of the book provides an in-depth analysis on topical areas of plant biology, with focus on Plant Diversity, Organization, Function and Improvement, including mechanisms of growth, differentiation, development and morphogenesis at the morphological, cellular, biochemical, genetic, molecular and genomic levels Contributors to these volumes were selected from a wide range of institutions in order to introduce a diversity of authors, and at the same time, these authors were selected with vast expertise in their specific areas of research to match with the diversity of the topics These authors not only have a deep understanding of the subject of their choice to write critical reviews by integrating available information from classical to modern sources but have also endured an unending series of editorial suggestions and revisions of their manuscripts Needless to say, this is as much their book as ours ix 31 Alien Crop Resources and Underutilized Species for Food and Nutritional Security of India Disclaimer The views and opinions expressed in this book chapter are those of the authors and not necessarily reflect the official policy or position of their affiliated institutions/organization References Alexander J, Coursey DG (1969) The origin of Yam cultivation In: Ucho PJ, Dimbleby GW (eds) The domestication and exploitation of plants and animals Duckworth, London Arora RK (1991) Plant diversity in the Indian gene centre In: Paroda RS, Arora RK (eds) Plant genetic resources conservation and management IBPGR, New Delhi Arora RK, Nayar ER (1984) Wild relatives of crop plants in India NBPGR Sci Mongr, NBPGR, New Delhi Arora RK, Sharma GD, Joshi V, Phogat BS, Bhatt KC, Rana JC (2006) 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N, Thuilleta AC (2011) Biodiversity, evolution and adaptation of cultivated crops C R Biol 334:450–457 Weltzien E, Rattunde H, Clerget B, Siart S, Toure A, Sagnard F (2006) Sorghum diversity and adaptation to drought in West Africa In: Jarvis D, Mar I, Sears L (eds) Enhancing the use of crop genetic diversity to manage abiotic stress in agricultural production systems International Plant Genetic Resources Institute, Rome Whitmore TM, Turner BL II (2002) Cultivated landscapes of middle america on the eve of conquest Oxford University Press, New York Zeven AC, De Wet JMJ (1982) Dictionary of cultivated plants and their regions of diversity Centre of Agricultural Publicity and Documentation, Wageningen Zeven AC, Zhukovsky PM (1975) Dictionary of cultivated plants and their centres of diversity PUDOC, Wageningen Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications 32 Tanmoy Ghosh, Chetan Paliwal, Rahulkumar Maurya, and Sandhya Mishra Abstract Microalgae, one of the largest global primary producers, are a potential source of bioactive compounds They are unique in producing superfine chemicals that can be used in various industrial sectors like pharmaceuticals, nutraceuticals and cosmeceuticals The chapter is intended to provide an insight to two of the most important pigments obtained from them, phycobiliproteins and carotenoids having species specificity which can be used as a chemotaxonomic marker Their unique structural properties play a crucial role in their biological functions The water-soluble phycobiliproteins are used as fluorescent tags in flow cytometry and immunochemistry, while liposoluble carotenoids are potential alternatives to synthetic dyes in the food industry Keywords Microalgae • Phycobiliproteins • Carotenoids • Fluorescence • Applications 32.1 T Ghosh • C Paliwal • R Maurya • S Mishra (*) Academy of Scientific and Innovative Research, New Delhi, India Discipline of Salt & Marine Chemicals, CSIRCentral Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India e-mail: smishra@csmcri.org Introduction Photosynthesis is an important biochemical reaction responsible for meeting our energy demands directly or indirectly The solar energy received on earth is converted into chemical energy by means of photosynthesis, which is then stored in various forms Algae, either unicellular or filamentous, are one of the most primitive photosynthetic organisms found in both freshwater and marine habitats They are subdivided as macroalgae (seaweeds) and microalgae Microalgae are global primary producers which contribute from one-third to more than B Bahadur et al (eds.), Plant Biology and Biotechnology: Volume I: Plant Diversity, Organization, Function and Improvement, DOI 10.1007/978-81-322-2286-6_32, © Springer India 2015 777 T Ghosh et al 778 Table 32.1 General composition of different algae (% of dry matter) Protein Strain 50–56 Scenedesmus obliquus 22 Botryococcus braunii 8–18 Scenedesmus dimorphus 48 Chlamydomonas reinhardtii 51–58 Chlorella vulgaris 10–20 Chlorella protothecoides 6–20 Spirogyra sp 55–65 Dunaliella tertiolecta 57 Dunaliella salina 39–61 Euglena gracilis 28–45 Prymnesium parvum 52 Tetraselmis maculata 28–39 Porphyridium cruentum 46–63 Spirulina platensis 60–71 Spirulina maxima 63 Synechococcus sp 43–56 Anabaena cylindrica Source: Adapted from Spolaore et al (2006) half of the total primary productivity (Van Den Hoek et al 1995; Miyamoto 1997; Guschina and Harwood 2006) Because of their high growth rates and ability to mitigate CO2 from the environment and utilize non-arable land for their cultivation, they are considered as potential energy feedstock for their utilization in biofuel (biodiesel, bioethanol and biogas etc) production Apart from being an energy feedstock, microalgae are a great store of many different biomolecules such as polyunsaturated fatty acids (PUFAs), sterols, pigments, enzymes, vitamins, minerals, proteins and carbohydrates which are beneficial both economically and medically The general composition of the algae in terms of carbohydrates, lipids, nucleic acids and proteins is provided in Table 32.1 They are able to potentially accumulate up to 50 % of their dry weight as carbohydrates, primarily in the form of starch, glucose, cellulose or hemicelluloses or polysaccharides of various kinds (Ho et al 2012; Yen et al 2013) Algal polysaccharides are, to a large extent, sulphated polysaccharides with important medical applications Crude polysaccharide extracts from vari- Carbohydrates 10–17 18 21–52 17 12–17 12–20 33–64 10–15 32 14–18 25–33 15 40–57 8–14 13–16 15 25–30 Lipids 12–14 55–60 16–40 21 14–22 55 11–21 20 14–20 22–38 9–14 4–9 6–7 11 4–7 Nucleic acid 3–6 – – – 4–5 – – – – – 1–2 – – 2–5 3–4.5 – ous microalgae such as Chlorella vulgaris, Chlorella stigmatophora, Scenedesmus quadricauda and Phaeodactylum tricornutum have anti-inflammatory, immunomodulatory and antioxidant properties (Guzman et al 2003; Mohamed 2008) Microalgae synthesize ω-3 and ω-6 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), γ-linoleic acid and arachidonic acid (ARA) essential in maintaining the tissue integrity, which humans are not able to synthesize They have many beneficial properties like they are anti-inflammatory, play a role in brain development, help in functioning of the nervous system and delay ageing Most of these polyunsaturated fatty acids (PUFAs) are used as health supplements, as baby food additives, in therapeutics and as poultry feed (Ahren et al 1983; Cohen and Heimer 1992; Gordon and Ratliff 1992; Borowitzka 1993; Barclay and Zeller 1996; Pulz and Gross 2004; Guedes 2010; De Jesus Raposo et al 2013) Some species of microalgae are also found to be rich in vitamins and industrially important enzymes Porphyridium cruentum is a good source of vitamins C and E as well as provitamin 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications A (Sarrobert and Dermoun 1991) Navicula ostrearia, a diatom, is a rich source of vitamin E (De Jesus Raposo et al 2013) Dunaliella salina, besides being known for β-carotene production, is also a source of thiamine, pyridoxine, riboflavin, nicotinic acid, biotin and tocopherol (Drokova and Popova 1974) Carbonic anhydrase, a crucial enzyme responsible for the conversion of CO2 into bicarbonate ions and carbonic acid, is produced by Isochrysis galbana, Amphidinium carterae and Prorocentrum minimum (Yingying and Changhai 2009; De Jesus Raposo et al 2013) Superoxide dismutase, another enzyme crucial for antioxidant activity in vivo, is produced by Anabaena sp., Porphyridium sp., Phaeodactylum tricornutum and Synechococcus sp (Thepenier et al 1988; Guzman-Murillo et al 2007; De Jesus Raposo et al 2013) We are witnessing a shift of research interests in functional foods obtained from natural sources, which contain additional nutrients and are beneficial to humans There has been felt a need to investigate such potentially important high-value products like antioxidants, anti-inflammatory compounds, natural colouring agents, fluorescent dyes and many others, from natural sources (Eisenreich et al 2004) Microalgae have been exploited for such bioactive compounds for use in pharmaceutical, nutraceutical, food and cosmetic industries From an economic point of view, microalgal cultivation is often preferred for the production of high-value compounds like phycobiliproteins and carotenoids (Spolaore et al 2006; Chu 2012; Markou and Nerantzis 2013) Nevertheless, there still exists a need to improve microalgal cultivation and harvesting technology along with the techniques used for the extraction and purification of the desired molecules (Molina et al 2003) Currently, different food companies are interested in improving their products through substitution of natural products because of larger profit margins compared to conventional food products and their acceptability to the public in general (Hasler 2002; Siro et al 2008) A full description of all these biomolecules would exceed the scope of this chapter which pri- 779 marily details about the various pigments sourced from these organisms Our main focus would be pigments derived from marine microalgae which have potential health and commercial benefits A number of these pigments have antioxidant, antiinflammatory and neuroprotective properties which have been conclusively proved through various in vitro and in vivo studies Apart from these biological properties, they play an important role in diagnostic biosensors and fluorescence analytical techniques Microalgal pigments are broadly classified into two groups: • Water-soluble phycobiliproteins sourced from microalgae as well as macroalgae are used for developing fluorescent markers in conjugation with immunoglobulins and other proteins • Lipid-soluble carotenoids such as astaxanthin, zeaxanthin and ß-carotene from microalgae which serve as provitamins and antioxidants 32.2 Phycobiliproteins Phycobiliproteins are accessory light-harvesting pigments predominantly found in cyanobacteria (blue-green algae), Rhodophyta (red algae), Cryptophyta and Glaucophyta The phycobiliproteins are further classified on the basis of their spectral properties into three major subgroups: phycocyanin, allophycocyanin and phycoerythrin Their composition varies with the species and environmental conditions of the source organism (Chu 2012) Due to their fluorescent properties, they were adopted for use in diverse applications such as fluorescence-activated cell sorting, flow cytometry and histochemistry soon after their introduction as pigmented molecules in 1982 They can also be used as markers for electrophoresis, isoelectric focusing and sizeexclusion chromatography due to their high absorptivity in visible light wavelengths 32.2.1 Structure Phycobiliproteins are composed of apoproteins (α and β subunits) covalently linked to prosthetic 780 T Ghosh et al Fig 32.1 Structure of a phycobilisome groups called phycobilins Phycobilins are openchain, tetrapyrrole chromophores sharing structural similarity with the bile pigment bilirubin (Glazer 1989) The two conserved subunits, α and β, form an (αβ) monomer, which are further aggregated to form trimers (αβ)3 and disc-shaped hexamers (αβ)6 The trimeric and hexameric structures form the functional units of PE and PC In a complete LHC, also termed as a phycobilisome, the central core is occupied with rods of APC joined to disc-shaped hexameric PC and PE which extend outwards as antennae (Fig 32.1) The light energy is captured by PE and is transferred to chlorophyll for further reaction via PC and APC The absorption maxima vary from 562 to 568 nm for C-PE, 615 to 620 nm for C-PC and 650 to 652 nm for APC (MacColl 1998) (Table 32.2) 32.2.2 Extraction and Purification of Phycobiliproteins The extraction of phycobiliproteins chiefly involves cell disruption in a buffered environment after which the crude extract is either centrifuged or filtered to remove cellular debris Cell disruption is done through ultrasonication, freeze-thaw cycles using liquid nitrogen, cavitation using nitrogen gas, osmotic shock, enzymatic treatments or high-pressure homogenization (Table 32.3) Wet biomass is directly utilized for the extraction of these proteins as high-temperature drying usually results in a lower-quality product or a lower yield Usually, 0.05 or 0.1 M phosphate buffer pH 7.0 or 7.2 is used as the extraction buffer although 0.5 M ammonium sulphate is also used Purification of these proteins is usually done using ammonium sulphate precipitation, polyethylene glycol precipitation, ion-exchange or sizeexclusion chromatography, expanded bed chromatography or membrane filtration to get their purified forms More often than not, a combination of these techniques is used to reach the desired purity level and the source organism Drying is usually performed using lyophilization which prevents denaturation of the pigment The measure of purification is determined by calculating the purity ratio, a ratio of the absorbance of the particular phycobiliprotein at its absorption maxima to that of aromatic amino acids in all proteins at 280 nm For example, the purity ratio 32 781 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications Table 32.2 Spectral and physical properties of cyanobacterial phycobiliproteins Phycobiliproteins C-phycocyanin C-phycoerythrin Allophycocyanin Absorbance maxima (nm) 615 566 652 Fluorescence emission (nm) 647 617 660 Molecular weight (kDa) 108 55 100 Absorptivity (L g−1 cm−1) 7.0 8.0 7.3 Molar absorptivity (M-cm)−1 1.54 0.44 0.73 Table 32.3 Different methodologies adopted for the extraction of phycobiliproteins Extraction method Freeze-thaw and sonication Phycobiliprotein Phycocyanin Name of species Spirulina platensis High-pressure homogenization Phycocyanin Spirulina platensis Freeze-thaw Phycoerythrin, phycocyanin, allophycocyanin Spirulina platensis, Phormidium sp A27DM, Lyngbya sp A09DM, Halomicronema sp A32DM, Pseudanabaena tenuis, Spirulina fusiformis, Arthronema africanum, Calothrix sp., Oscillatoria quadripunctulata, Pseudanabaena sp Sonication Phycoerythrin Variable speed stirring Phycocyanin Nitrogen cavitation Phycobiliproteins Cyanosarcina sp SK40, Phormidium sp PD40-1, Scytonema sp TP40, Leptolyngbya sp KC45 Anabaena marina ATCC 33047 Synechococcus sp Lysozyme treatment Phycocyanin Synechococcus sp of C-PE is calculated by A568/A280 and for C-PC using A620/A280 The absorbance values are considered within a range of 0.05–1 at the absorptive maxima of the phycobiliprotein (Bennett and Bogorad 1973) A ratio greater than is generally considered food grade, while a ratio greater than is considered as analytical grade purity The purified forms of the proteins are generally stable in phosphate buffer pH 7.0 or 7.2 or in ammonium sulphate suspensions The latter are usually dialyzed against the corresponding buffer before use They are stored in temperatures 4–10 °C in the dark to reduce the effects of light References Zhang and Chen (1999) Patel et al (2004), Song et al (2013) and Seo et al (2013) Minkova et al (2007), SantiagoSantos et al (2004), Soni et al (2010), Minkova et al (2007), Mishra et al (2008), Su et al (2010), Cano-Europa et al (2010), Parmar et al (2011) and Mishra et al (2011) Pumas et al (2011) Ramos et al (2010) Viskari and Colyer (2003) Gupta and Sainis (2010) 32.2.3 Applications of Phycobiliproteins 32.2.3.1 As Food Colourants Natural colouring agents have always held an upper hand when it comes to the food industry Due to the toxic nature of synthetic colourants and the necessity of colour additives for food processing, there is an increased awareness and curiosity for natural options in this field However, studies are still underway for the stability of such proteins in pH ranges used in commercial food manufacturing industries worldwide PBPs are used as natural food colourants in chewing gums, T Ghosh et al 782 jellies, ice creams and fermented milk products since many of the synthetic dyes used globally are thought to be possible carcinogens Their other advantages include their intense colours and high solubility in water (Santiago-Santos et al 2004; Chakdar et al 2012) A lower stability to temperature and light has not deterred the food processing industry to use C-PC as an alternative to synthetic dyes such as gardenia and indigo A study reported that C-PC was insoluble in acidic solutions (pH 3) and denatures at temperatures above 45 °C (Jespersen et al 2005) Additionally, the fluorescent properties of PE have been exploited to produce transparent lollipops, cake decorations and soft drinks and alcoholic beverages that fluoresce at pH 5–6 These special effects were tried out to increase the marketability of the respective food items (Dufosse et al 2005) Although still not approved for use in the USA and European Union (EU), the US Food and Drug Administration (US FDA) has been approached by Desert Lake Technologies in 2012 for grant of generally recognized as safe (GRAS) status to C-PC (CyaninPlus™) developed by them (FDA, GRAS 2012) 32.2.3.2 As Pharmaceutical Agents PBPs have been recognized as beneficial pharmaceutical agents since many years, and the fact has been reliably established through studies Oriental cuisine and medicine have traditionally been rich in microalgae since ancient times, but it is only now that their beneficial effects are being investigated scientifically (Bocanegra et al 2009) The current total market value of PBP products has been estimated to be US $60 million (Borowitzka 2013) The nutritional and therapeutic aspects of Spirulina, a blue-green algae, have been critically reviewed which have proved that the beneficial aspects of the cyanobacteria can be attributed to its C-PC content among other things (Kay and Barton 1991; Mishra 2006) 32.2.3.3 As Antioxidants The antioxidant properties of PC have been well documented over the years According to published research, C-PC successfully reduced lipid peroxidation and oxidative haemolysis in normal human erythrocytes induced by a free-radical generator, AAPH C-PC extract from Aphanizomenon flos-aquae, a cyanobacterium, was also found to significantly inhibit lipid peroxidation in blood plasma by Cu+2 (Benedetti et al 2004) In vitro studies have established C-PC as an antioxidant, anti-inflammatory, neuroprotective, nephroprotective and hepatoprotective agent (Romay et al 1998; Farooq et al 2004; Mishra 2006; Sekar and Chandramohan 2008) Radical scavenging activity of C-PC includes scavenging peroxyl, peroxynitrite and hydroxyl free radicals while preventing or inhibiting lipid peroxidation and DNA damage (Bhat and Madhyastha 2001; Bermejo et al 2008) C-PC has been demonstrated to significantly reduce hippocampal cell death in gerbils and rats (Thaakur and Sravanthi 2010; Penton-Rol et al 2011), reduces necrosis and inflammation in hepatic cells (Gonzalez et al 2003; Sekar and Chandramohan 2008; Kuriakose and Kurup 2010) and decreases Kupffer cell phagocytosis (Remirez et al 2002) C-PE from Pseudanabaena tenuis was examined for its antioxidant ability in mice model fed with mercury and found to reduce the extent of damage in all animals (Cano-Europa et al 2010) 32.2.3.4 As Anti-inflammatory Molecules C-PC was also analysed as an anti-inflammant in human models where its ability to effectively inhibit the activity of cyclooxygenase-2, an enzyme involved in the process of inflammation, was studied It was found that although C-PC effectively inhibited the said enzyme, reduced PC or the isolated PCBs were poor inhibitors without being selective for the enzyme The results suggest that the apoprotein of C-PC may have an important role to play in the anti-inflammatory activity of C-PC (Reddy et al 2000) Another study analysed the in vivo effect of excessive C-PE dosage in test mice to evaluate the potential risks due to overdosage It was found that C-PE had no deleterious effect on the body weight, food intake or toxicity signs even at a dosage of 2,000 mg kg−1 body weight C-PE This is a significant finding for adopting a C-PE based 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications treatment approach since it indicates no negative health effects through overdosage (Soni et al 2010) 32.2.3.5 As Anticancer Agents The anticancer activity of C-PC was studied using human chronic myeloid leukaemia cell line K562 It was observed that as little as a 50 μM dose decreased the proliferation of K562 cells by 49 % for 48 h (Subhashini et al 2004) Also, C-PC induced apoptosis in prostate cancer (LNCaP) cell line by diminishing the required dosage of topotecan, an anticancer medication which frequently causes adverse side reactions in patients (Gantar et al 2012) In another study involving human hepatoma cell line (HepG2), C-PC led to a reduction in the proliferation of the cells with the highest reduction observed at a concentration of μg/ml C-PC along with a loss of nuclear entities due to fragmentation (Basha et al 2008) A separate study has examined the healing effect of C-PC on workers exposed to nuclear radiations in a nuclear power plant The study was carried out as part of a publication on nuclear power plant operations, safety and environment It was found that C-PC has the ability to influence repair of damaged DNA, essential for the preservation of genomic integrity However, the protein also showed DNA lesion in subjects exposed to high doses of radiation; the lesions were not found to be persistent This may be attributed to the adaptive phenomena due to the chronic adaptation exposure Although the results are promising, the authors categorically state that the study should be treated as pilot one with the need for further experiments to prove conclusively the role played by PBP such as PC in DNA repair mechanisms (Stankova et al 2011) The studies firmly establish the anticancer properties of C-PC that might open up an exciting avenue for medical treatments for various life-threatening cancers Although the findings are rather sporadic instead of being coherently directed towards a particular type of cancer, the promising results are sure to encourage researchers to focus more on specific types of cancer 783 32.2.3.6 As Fluorescing Molecules The fluorescence properties of PBP have played an important role in the development of various fluorescence-based techniques including fluorescence-activated cell sorting (FACS), flow cytometry, protein-protein conjugation and fluorescence immunoassays and fluorescence microscopy (Mishra 2006) There are many reasons which can explain the advantages of using PBP as fluorescent molecules, such as (1) low interference by other molecules due to absorption and emission at far red end of spectrum, (2) a large Stokes shift of 80 nm or more which minimizes noise due to other phenomena, (3) high solubility in water leading to minimal side reactions, (4) quantum yield independent of pH and (5) protection from quenching by other biological molecules (Kronick and Grossman 1983) An excellent overview of the relevant properties of PBPs has been provided in Glazer (1994) The isoelectric points of the PBP range from 4.7 to 5.3 32.2.3.7 Bioconjugates Conjugation of proteins with PBP has attracted much interest due to their highly sensitive detection characteristics and multiparameter detection A recent US patent application has claimed a process to develop fluorescent kits using PBP and chemical dyes attached together to take advantage of the intermolecular energy transfer phenomena (Mao et al 2012) However, the conjugation studies and applications are not new The utility of such conjugated molecules for cell cytometry applications and diagnostic procedures was recognized long time ago (Stryer and Glazer 1985) The only limiting factor has been the molecular weight of the PBP (200 kDa) which hinders their diffusion into cells of interest and hence limits their applicability to antibody conjugates for flow cytometry and enzyme-linked immunosorbent assays (Giepmans et al 2006) Another example of a conjugated PBP with a suitable dye molecule utilizes R-PE and compares the fluorescence of the pair to that of native R-PE to assess energy transfer from the PBP to the dye The transfer efficiency was found to be >99 % The conjugate was used to label streptavidin that retained the fluorescence properties and was T Ghosh et al 784 useful in flow cytometry applications (Diwu et al 2012) The fluorescence properties of B-PE were studied in a nonpolar environment using AOT (sodium bis-(2-ethylhexyl)sulphosuccinate)/ water/isooctane micro-emulsions AOT is an anionic surfactant that can solubilize small quantities of water in various nonpolar solvents Results indicated that the stability of B-PE in water droplets inside the emulsion is enhanced than the protein that is in the aqueous state It may be that the protein inside the water droplet retains the same configuration and hence its fluorescence properties, but the chromophores are more protected inside the emulsified environment (Bermejo et al 2003) PBPs are extremely amenable to bioconjugation and have bright chromophores These factors have together contributed to their being used as fluorophores conjugated to various other molecules using standard chemistry The only drawbacks of the process are that the reactions should be suitable for a biological origin molecule and not disturb the original configuration to avoid a loss of fluorescence Although limited reports are available for PBP as nanomaterial, commercial applications for the few discovered bioconjugates have already been in use for some time Commercial ventures are already manufacturing and marketing PBP-conjugated fluorescent dyes and antibodies for flow cytometry and immunolabelling, respectively (Sapsford et al 2013) 32.3 Carotenoids Carotenoids are composed of more than 600 natural lipid-soluble pigments with their colour ranging from yellow to red and are found predominantly in plants (Takaichi 2000; Kleinegris et al 2010) The structures of carotenoids differ in cyclization (one or both ends of the molecule), hydrogenation level and functional groups (Dutta et al 2005) Some carotenoids are found in both plants and algae, while some are limited only to algae (Takaichi 2011) Chemical synthesis is a low-cost method for obtaining high-purity carotenoids, but its major drawback is the non-biological reaction precur- sors/by-products which may have deleterious health effects, and hence, it is suggested to find economical carotenoid production of biological origin The modern tools of bioprocessing and recombinant DNA technology can significantly increase carotenoid production Also, it is important to know that there are several disadvantages associated with the production of carotenoids from food such as complicated extraction and purification process, season fluctuation, limited resources, etc (Asker et al 2012) Carotenoids are isoprenoid compounds made up of 40-carbon (C40) backbone synthesized via head-to-head condensation of two geranylgeranyl diphosphate (GGDP, C20) molecules Naturally occurring carotenoids are generally trans in nature (Dutta et al 2005) They are synthesized in living systems by carotenogenesis pathways, which have been extensively studied in cyanobacteria (Takaichi 2011) Carotenoids exhibit different properties like singlet oxygen quencher, binding affinity for hydrophobic surfaces, antitumor activity, provitamin A activity, anti-inflammatory activity, hepatoprotective activity and antioxidant activity and are also a part of cellular communication, immune-modulation activity such as decrease in UV-induced immune suppression and increase the activity of natural killer cells (van den Berg 1999; Dutta et al 2005; Vílchez et al 2011; Han et al 2012) Carotenoids are mainly classified into two subgroups (Sergio et al 1999): (a) Carotenes: Hydrocarbons consisting of specific end groups, e.g lycopene and ß-carotene (b) Xanthophylls: Oxygenated carotenoids Xanthophylls are further subdivided depending upon the type of functional groups attached: (i) Containing hydroxyl groups, e.g zeaxanthin and lutein (ii) Containing methoxy group, e.g spirilloxanthin (iii) Containing oxo group, e.g echinenone (iv) Containing epoxy group, e.g antheraxanthin Carotenoids can also be classified as primary and secondary carotenoids Among them, primary 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications carotenoids play a crucial role in the photosynthetic organisms, while the secondary carotenoids are not essential for photosynthesis and are localized either in plastoglobules or in cytosolic lipid droplets; they are produced under stress conditions and can be accumulated to high levels (Sasso et al 2012) 32.3.1 Microalgal Sources Different microalgal strains of research interest are Dunaliella salina, Sarcina maxima, Chlorella protothecoides, Chlorella vulgaris and Haematococcus pluvialis for the commercialization of carotenoid production (Lordan et al 2011) The table below depicts different microalgae as sources of carotenoids (Table 32.4) 32.3.2 Factors Affecting Production of Carotenoids The growth conditions and environmental parameters are important parameters that control carot- 785 enoid accumulation in organisms (Walter and Strack 2011) The factors that affect carotenoid production in marine microalgae are described below: Light There are different theories on which photostimulation of carotenoid synthesis depends; one describes high light intensity, and other focuses on high illumination time to cause a rise in carotenoid concentration (Bhosale 2004) Temperature Temperature plays a crucial role in carotenoid production; a decline in thermal conditions from 34 °C to 17 °C caused a 7.5 times increase in α-carotene content in Dunaliella sp (Bhosale 2004) Nutrients Nannochloropsis gaditana deprived of phosphate/sulphur causes an improvement in zeaxanthin concentration due to rapid inhibition of PSII driven by S-limitation that diminishes the primary photosynthetic product formation, i.e NADPH and Fdred which later on caused insufficient ascorbate supply for the Table 32.4 Microalgal sources of different carotenoids Carotenoids α-carotene ß-carotene Lutein Astaxanthin Zeaxanthin Fucoxanthin Canthaxanthin Sources Dunaliella salina Dunaliella salina, Botryococcus braunii, Spirulina platensis, Chlorococcum sp., Synechocystis sp., Parietochloris incisa Muriellopsis sp., Scenedesmus almeriensis, Chlorella protothecoides, Chlorella zofingiensis, Botryococcus braunii, Neospongiococcus gelatinosum, Chlorococcum citriforme Chlamydomonas acidophila, Diacronema vlkianum Haematococcus pluvialis, Botryococcus braunii, Chlorella zofingiensis, Scotiellopsis oocystiformis, Neochloris wimmeri, Diacronema vlkianum, Euglena rubida Dunaliella salina, Spirulina sp., Microcystis aeruginosa, Botryococcus braunii, Chlamydomonas acidophila Phaeodactylum tricornutum, Cylindrotheca closterium, Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica, Vischeria punctata, Vischeria stellata Anabaena spp Reference Christaki et al (2013) Del Campo et al (2007), Solovchenko et al (2008) and Ranga Rao et al (2010) Fernández-Sevilla et al (2010), Del Campo et al (2007), Ranga Rao et al (2010), Cuaresma et al (2011) and Durmaz et al (2009) Christaki et al (2013), Ranga Rao et al (2010), Del Campo et al (2004), Orosa et al (2000), Zhang and Lee (1997) and Durmaz et al (2009) Christaki et al (2013), Ranga Rao et al (2010), Sajilata et al (2008) and Cuaresma et al (2011) Kim et al (2012) and Li et al (2012a, b) Shahidi and Brown (1998) T Ghosh et al 786 xanthophyll cycle and hence reduced xanthophyll biosynthesis (Forján et al 2007) Metal ions/salts Addition of ferrous salt increases the hydroxyl radical which, in turn, promotes cellular carotenoid synthesis in Haematococcus pluvialis This method can substitute high light illumination which is costlier and an energy-intensive process Chlorococcum spp has provided similar results in the presence of inorganic salts (Bhosale 2004) 32.3.3 Extraction of Carotenoids The major issue in microalgal biotechnology is the downstream processing where microalgal biomass harvesting remains a prominent research area Experience suggests that effective harvesting technology is completely dependent on strain characteristics (Del Campo et al 2007) There is no defined protocol for the extraction of carotenoids as various factors contribute in the transformation or degradation during their extraction; various precautions like dim light, antioxidants need to be taken to prevent photo-damage and oxidation (Oliver and Palou 2000) Extraction can be performed using organic solvents like hexane, methanol and acetone, but they are not recommended due to their toxicity Green solvents like vegetable oils or supercritical CO2 are more suitable for this process (Wiltshire et al 2000; Macías-Sánchez et al 2008; Guedes et al 2011; Christaki et al 2013) 32.3.4.2 Antioxidant Activity Reactive oxygen species (ROS) include both free radicals and non-radical oxidants which are the most reactive molecular species responsible for DNA, protein and lipid degradation (Pérez-Rodríguez et al 2009) Carotenoids have the ability to scavenge singlet molecular oxygen and peroxyl radicals, which makes them strong antioxidants (Sies and Stahl 2004) These properties help them in preventing chronic and degenerative diseases like cancer A study shows that the risk of colon cancer gets reduced due to the inclusion of ß-carotene in the diet (Vílchez et al 2011) High ß-carotene doses show better CD4 to CD8 lymphocyte ratio (Christaki et al 2013) 32.3.4.3 Membrane Stabilization Researchers have reported mechanical stabilization of liposomal membranes by carotenoids such as zeaxanthin at higher temperatures (Hara et al 1999) They form a complex molecular structure with lipid membranes and control the dynamics and physical properties of lipid membranes, protecting lipid peroxidation (Popova and Andreeva 2013) It was also found that incorporation of carotenoids into membrane decreases their permeability, whereas polar carotenoids on phospholipids mimic as cholesterol and play important role in the modulation of membranes which does not contain cholesterol (Gruszecki and Strzalka 2005) 32.3.5 Practical Applications 32.3.4 Properties of Carotenoids 32.3.4.1 Provitamin A Activity Provitamin A activity is the conversion of provitamin A into vitamin A, whose deficiency leads to premature deaths, particularly among children About 10 % of the natural carotenoids have the ability to get converted into retinol which has provitamin A activity ß-carotene has 100 % provitamin A activity (Zeb and Mehmood 2004; Vílchez et al 2011) 32.3.5.1 Molecular Photovoltaic Nanomaterial Precursors The total energy, dipole moment, isotropic polarizability and molecular structure of the carotenoids make them eligible candidates for applications in dye-sensitized solar cells (DSSC) There has been a comparative study on the ionization potential and electron affinity of the carotenoids to validate them for the above purpose (Ruiz-Anchondo et al 2010) 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications 32.3.5.2 Food Industry/Food Colourants Carotenoids are the precursors of various flavouring and odouring agents They also function as colour enhancers and hence have a wide use in the food and feed industry ß-carotene can be of use in food and beverages such as fruit juices, soft drinks and confectionery to improve their appearance and also because of their antioxidant properties (Christaki et al 2013) The application of carotenoids in the food industry is limited due to their poor water solubility and low bioavailability which can be overcome through their encapsulation into nano-emulsions (Qian et al 2012) 32.4 Conclusions Microalgal pigment production is the most significant area of research in the field of blue biotechnology Classically, pigments have been produced synthetically, but a rising demand for natural pigments has promoted large-scale cultivation of microalgae for pigment production The enzymes and genes required for the regulation and control of biosynthesis of pigment production need to be investigated along with their applications to enhance their productivity The extraction process of the pigments can be improved by simultaneous extraction of lipids or other bioactive molecules to offset the single product production cost Further to this, the interaction of pigments with other biological molecules and pigment-based nanostructure is an area which is still unravelled and can be explored in more detail Acknowledgements CP, TG and RM wish to thank AcSIR for Ph.D enrolment and CSIR for Senior Research Fellowship Authors would also like to thank Dr P.K Ghosh, Director, CSIR-CSMCRI, and Prof Bir Bahadur for encouraging and providing an opportunity to gain an in-depth knowledge on the subject while formulating the chapter Sincere thanks are also due to Dr Arvind Kumar (DC, SMC) for providing financial support through SIP Project (CSC-0203) and Dr Basil George (DST Young Scientist) along with all the present and ex-laboratory colleagues for their continuous support 787 References Ahren TJ, Katoh S, Sada E (1983) Arachidonic acid production by the red alga Porphyridium cruentum Biotechnol Bioeng 25:1057–1070 Asker D, Awad TS, Beppu T, Ueda K (2012) Isolation, characterization, and diversity of novel radiotolerant carotenoid-producing bacteria In: José-Luis Barredo (ed) Microbial carotenoids from bacteria and microalgae Humana Press, NY, US, pp 21–60 Barclay W, Zeller S (1996) Nutritional enhancement of n-3 and n-6 fatty acids in rotifers and Artemia nauplii by feeding 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