Nuevas Luces en La Biodiversidad y Aplicaciones de Cianobacterias Gupta Et Al 2013

8/10/2019 Nuevas Luces en La Biodiversidad y Aplicaciones de Cianobacterias Gupta Et Al 2013

1/19

Review article

New insights into the biodiversity and applications of cyanobacteria(blue-green algae) Prospects and challenges

Vishal Gupta a, Sachitra Kumar Ratha a, Anjuli Sood b , Vidhi Chaudhary a, Radha Prasanna a ,a Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, Indiab Department of Botany, University of Delhi, Delhi 110007, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 31 December 2011Received in revised form 16 December 2012Accepted 29 January 2013Available online 7 March 2013

Keywords:Antifungal enzymesBioactive compoundsCyanobacteriaDiversityPolyphasic approaches

Cyanobacteria (blue-green algae) are Gram-negative oxygenic photosynthetic prokaryotes with a long evo-lutionary history. They have potential applications in diverse areas, especially in agriculture, as nutrient sup-plements in agriculture and industry (as biofertilizer, plant growth promoting rhizobacteria and as biocontrolagents). Their role as food supplements/nutraceuticals and in bioremediation and wastewater treatment isan emerging area of interest. In addition, they are known to produce wide array of bioactive compounds(secondary metabolites) with diverse biological activities including antiviral, antibacterial, antifungal,antimalarial, antitumoral and anti-in ammatory properties, having therapeutic, industrial and agriculturalsigni cance. One of the major problems has been regarding their classi cation being incongruent with thephylogeny, because the phenotype of cyanobacterial strains is known to be altered under different environ-mental/nutritional conditions. However, because of their simple growth needs, they are the favourite modelorganisms for deeper understanding of several metabolic processes and for the production of recombinantcompounds of medicinal and commercial value. In recent years, cyanobacteria have gained interest for pro-ducing third generation biofuels (both biomass and H 2 production). With the recent advances in metabolicengineering techniques and availability of genome sequences, novel approaches are being explored forrealising the potential of cyanobacteria. Our review provides an overview of the polyphasic approachesused in the analyses of cyanobacterial biodiversity and the potential of these organisms in providing viable

solutions to global problems of food, energy and environmental degradation, which need further impetusthrough adoption of multidisciplinary collaborative programs. 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. Classi cation of cyanobacteria based on morphological characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803. Molecular methods for studying cyanobacterial diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

3.1. DNA ngerprinting based methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Polyphasic approaches and metagenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.3. Non-PCR-based methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. The species concept of cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Cyanobacteria prospects as energy sources and speciality lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856. Cyanobacteria their promise in aquatic bioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 867. Cyanobacteria as valuable sources of bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

7.1. Allelochemicals produced by cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Types of cyanotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Nodularin (NODLN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.4. Saxitoxin (STX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.5. Cylindrospermopsin (CYN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.6. Anatoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.7. UV absorbing pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Algal Research 2 (2013) 79 97

Corresponding author at: Division of Microbiology, Indian Agricultural Research Institute (IARI), New Delhi 110012, India. Tel.: +91 11 25848431; fax: +91 11 25846420.E-mail address: [email protected] (R. Prasanna).

2211-9264/$ see front matter 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.algal.2013.01.006

Contents lists available at SciVerse ScienceDirect

Algal Research

j o u rn a l h o mep ag e : ww w. e l sev i er . co m/ lo ca t e / a lg a l
http://dx.doi.org/10.1016/j.algal.2013.01.006http://dx.doi.org/10.1016/j.algal.2013.01.006http://dx.doi.org/10.1016/j.algal.2013.01.006mailto:[email protected]://dx.doi.org/10.1016/j.algal.2013.01.006http://www.sciencedirect.com/science/journal/http://www.sciencedirect.com/science/journal/http://dx.doi.org/10.1016/j.algal.2013.01.006mailto:[email protected]://dx.doi.org/10.1016/j.algal.2013.01.006


2/19

8. Other value added products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9. Agricultural implications of cyanobacterial diversity and their role in integrated nutrient and pest management . . . . . . . . . . . . . . . . 9110. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Cyanobacteria are Gram negative autotrophic bacteria, exhibitinga repertoire of metabolic capabilities and adaptive mechanisms, includ-ing nitrogen xation (occurs in specialised cells called heterocytes/under microaerobic conditions/through temporal spatiation), chromaticadaptation and the ability to form symbiotic associations with severaleukaryotic hosts such as plants, fungi, and protists [1]. They mainlypossess chlorophyll a , besides accessory pigments such as phycobilins,carotenoids and xanthophylls. They appeared approximately 2600 3500 million years ago, based on fossil records, biomarker analyses andphylogeneticrelationshipswithother living forms [2]. An endosymbioticevent between a cyanobacteriumand a eukaryote is understood to havegiven rise to photosynthetic organelles or plastids. Consequently, algaeand plants photosynthesize and possess chlorophyll a. Cyanobacteriaaremorphologicallydiverse,occurring as lamentous,unicellular, plank-tonic or benthic and colonial (coccoid) forms [3,4]. In colonial forms,cells and laments may be arranged in different arrangements, suchas branched, coiled, or straight/radially in strict planes, or irregularly.Cyanobacteria have evolved specialised cells, such as heterocytes fornitrogen xation, akinetes for survival in stressed conditions and hormo-gonia for dispersal and multiplication.

Among all photosynthetic organisms, cyanobacteria inhabit thewidest range of ecological habitats. They are found in cold and hot,alkaline and acidic, marine, freshwater, saline, terrestrial, and symbi-otic environments. This broad habitat range is due to the presence of a PSII reaction centre that can extract electrons from water and thusare not limited to environments with other scarcer reduced electrondonors, as are other non-oxygenic photosynthetic prokaryotes. In fact,cyanobacteria are able to establish competitive growth in almost anyenvironment that has, at least temporarily, liquid water and sunlight.The temporal and spatial variations taking place during the evolutionand acquisition of many genes and physiological properties haveenabled cyanobacteria to successfullygrow in the diverse range of envi-ronments [5,6].

Cyanobacteria have ability to form blooms, suchas those in temper-ate eutrophic lakes during the warm periods of summer. These bloomsare commonly formed by gas-vacuolated genera such as Anabaena , Aphanizomenon , Microcystis , and Planktothrix . All these genera areknown to contain toxic strains [7]. The unicellular (e.g., Synechococcus )and colonial picocyanobacteria (e.g., Snowella and Merismopedia ) are

abundant in freshwater bodies, although they do not commonly formblooms. Some cyanobacteria such as Prochlorothrix hollandica arerestricted to certain environments [8]. This is the only Prochlorales(Prochlorophyta) species that has been reported to occur in freshwaterlake, while other Prochlorales, including Prochlorococcus , are abundantin marine environments [9]. Zwart et al. [10] identi ed severalcyanobacterial clusters of Microcystis , Aphanizomenon os-aquae , andPlanktothrix agardhii , based on 16S rRNA gene sequences. However,one genotype of Aphanizomenon os-aquae is known to be abundantin the brackish Baltic Sea [11].

Cyanobacteria have been divided into different groups calledas ecostrategists, ecotypes, or functionalgroups based on thephysiolog-ical characteristics, mainly buoyancy, colony formation, and nitrogen

xation. Planktic cyanobacteria are classi ed into bloom-forming,

homogenouslydispersed,stratifying, nitrogen- xing, andsmall colonial

ecostrategists [12]. Hayes et al. [13] have studied the genetic populationstructure of Planktothrix in Lake Zurich and Nodularia populations in theBaltic Sea by diagnostic PCR and concluded that these populations werenot clonal, but showed spatial and temporal variation in their geneticcommunity structure and that genotypes having different alleles forexample, gas vesicle coding genes, were adapted to different environ-mental conditions. A morphologically similar freshwater Synechococcus(Cyanobium ) population contained several genotypes, which respondeddifferently to factors such as nutrient deprivation, light intensity andpredation [9].Their physiological exibility renders their proliferationin diverse habitats terrestrial, aerial and aquatic, from deserts tolakes as well as hot springs and glaciers. Cyanobacteria have the abilityto form bio lms(microbial mats)on shores andon thesurface of stones,plants, besides existing in the rhizosphere of several crops and arti cialstructures/monuments [5,6]. Cyanobacteria can form toxic blooms inwater, which poses a health risk for humans and animals, besidesdiminishing the aesthetic value and utility of water bodies. The hetero-cystous, nitrogen- xing and lamentous cyanobacteria belonging to thegenus Anabaena are among the dominant components of the planktonin freshwater lakes, ponds and reservoirs, as well as in brackish waterbodies worldwide. Several Anabaena spp. isolated worldwide commonlyproduce hepatotoxins such as microcystins, and neurotoxins such asanatoxin-a, anatoxin-a(S) and saxitoxins in freshwater environments[7]. Halinen et al. [14,15] isolated a number of hepatotoxic Anabaenastrains and analysed the genetic diversity from the Gulf of Finland, BalticSea.

One of the major issues which are widely investigated in the globalscenario relates to the classi cation and identi cation of these ubiqui-tous prokaryotes.

2. Classi cation of cyanobacteria based on morphologicalcharacters

Classi cation provides a speci c identi cation and taxonomic nameof the organism to microbiologists [16]. The two most commonlyadopted classi cation systems include the Bacteriological approach asgiven in Bergey's Manual of Systematic Bacteriology [17] and the tradi-tional Botanical approach, which hasbeen more recently improvised byAnagnostidisand Komarek [18]. Traditionally, cyanobacterial identi ca-tion was done on the basis of morphological characters and they wereclassi ed as blue-green algae (Cyanophyta) along with the eukaryotic

algae under the Botanical Code of Nomenclature. Anagnostidis andKomarek [18] divided cyanobacteria (Cyanoprokaryota) into fourorders Nostocales, Stigonematales, Chroococcales, and Oscillatoriales which arefurther divided into families,subfamilies,genera, andspecies.This classi cation system was based on morphological identi cation of species in natural samples and useful in the analyses of cyanobacterialdiversity. In the 1960s, cyanobacteria were found to have cellularfeatures/characteristics of prokaryotes, and consequently, Stanier et al.[19] included cyanobacteria in the Bacteriological code, and Rippkaand co-workers [20] created the Bacteriological classi cation. This wasadopted and revised in the Bergey's Manual of Systematic Bacteriology[17], which represents the recognised authoritative treatise for bacte-riological classi cation. The bacteriological approach is based ongenetic and phenotypic information about cyanobacteria available as

pure cultures/axenic strains and cyanobacteria are divided into four

80 V. Gupta et al. / Algal Research 2 (2013) 79 97


3/19

subsections and further, into subgroups and genera [21,22] . The sub-sections and generic descriptions are still solely based on morphology,due to the paucity of information on genetic aspects for all the pheno-typically characterized isolates [22]. This has complicated the classi -cation of cyanobacteria [8]. Hoffmann et al. [23] proposed a revisedversion of cyanobacterial classi cation, based mainly on 16S rRNAgene sequences, morphology and thylakoid arrangements. Three majorchanges were proposed: heterocytous cyanobacteria were uni ed into

one subclass, Prochlorophytawereincludedinto the cyanobacterial clas-si cation system and the distinction between coccoid and lamentousforms was no longer followed at the highest subclass level. Instead, thedivision into subclasses was based on arrangements of thylakoids andthe presence of differentiated cells. The coccoid and lamentous formswere separated at the order level [23]. However, the robustness of such a classi cation system is not without problems, especially asarrangements of thylakoids and the presence of differentiated cells arein uenced by abiotic and biotic factors. Komarek and Anagnostidis [8]estimated that a large number of the cyanobacterial strains in culturecollections have been misidenti ed. Simple cyanobacteria such asSynechoccous and Cyanoothece are especially dif cult to identify andclassify [24]. Application of molecular techniques to amplify some por-tions of the genome in order to characterize and deduce phylogeneticrelationships of cyanobacteria has increased considerably in the recentyears and helped to solve some of the problems [25].

3. Molecular methods for studying cyanobacterial diversity

A number of researchers have developed probes for the detectionof paralytic shell sh poisons (PSPs) producing Anabaena circinalisstrains and microcystins/nodularins producing Anabaena , Microcystis ,Planktothrix , Nostoc and Nodularia . These probes have high speci cityand sensitivity; thereby lower the detection limit to 1 to 5 fmol of thePCR product [26]. Rudi et al. [27] developed ten speci c 16S rDNAoligonucleotide probes, for the qualitative estimation of the presenceor absence of the various cyanobacterial genera. Baker et al. [28] usedthe intergenic spacer region in the phycocyanin operon for identi ca-tion of several potentially-toxic cyanobacterial species in environmen-tal samples. The rpoC1 gene was successfully tested for the speci cdetection of Cylindrospermopsis raciborskii [29], and Saker et al. [30]identi ed a fragment of the 16S rRNAgeneuseful for the early detectionof Microcystis spp. The various molecular methods such as real-timePCR, uorescence in situ hybridization, microarray based methods,etc., and their utility in the identi cation of toxic cyanobacterial speciesin environmental samples have been discussed in recent reviews[31 35].

New bacterial lineages and greater microbial diversity were ob-served using whole-genome shotgun sequencing of samples isolatedfrom natural environments compared to that of cultivation-basedmethods [32]. Traditional methods of identi cation such as microscopicanalyses arenot suited for recognition of differentgenotypes, for exam-ple, separation of toxic strains from non-toxic ones [7]. Cyanobacterial

speci c primers have been designed for the most commonly usedmarker, 16S rRNA gene [33], and a large database (currently over6000 sequences) allows comparison of the newly obtained 16S rRNAgene sequences. In addition to the 16S rRNA gene, other genes andintergenic spacer (ITS) regions have also been used to study differentaspects of cyanobacterial diversity. The choice of primers/probes andthemethod applieddeterminethe resolutionat thedifferent taxonomiclevels, from strain to domain level. Most of the molecular methods arebased on extraction of total DNA or RNA from environmental samplesand PCR ampli cation of target genes. However, DNA extraction andPCR ampli cation-related biases, such as the success of DNA extractionamong variable species as well as differential ampli cation, the pres-ence of PCR inhibitors, PCR artefacts, and primer speci city and ef -ciency, can all skew the results of the community composition [34].

The cloning of 16S rRNA genes and the subsequent sequencing of the

clones are recent improvements applied to planktic cyanobacterialcommunities, e.g., in the Sargasso Sea, freshwater lakes as well as tobenthic cyanobacteria [35]. Therefore, the choice of technique, sampletype (metagenomic, laboratory grown cultures/mixtures) besides thesequence and speci city of primers can play a major role in enhancingthe discriminatory power of PCR based studies.

The 16S rRNA gene has a universal distribution in prokaryotes andits functional consistency in both the variable and conserved regions

and rather highinformationcontent represent important characteristicsneeded for a good phylogenetic marker gene [36]. In addition, the 16SrRNA gene sequences are relatively easy to align, and a large databasehas accumulated (currently over 6000 cyanobacterial sequences), pro-viding extensive comparative information between strains. However,the 23S rRNA gene is longer than the 16S rRNA gene and consequently,contains more informative sites and leads to a better resolution. But, themain drawback associated with sequencing of 23S rRNA gene is that itsdatabase is small in comparison to the 16S rRNA gene [36].

Horizontal gene transfer (HGT) and the presence of multiple het-erogeneous rRNA gene copies have led to problems in predictingthe reliability and relationships among bacterial strains determinedon the basis of the 16S rRNA genes. Although intragenomic diver-gence of the 16S rRNA genes can be as high as 11.6%, generally itseems to be low, less than 1% [37]. Among cyanobacteria, the observedintragenomic divergence of the 16S rRNA genes has been rather low( b 1.3%) especially related to heterocystous cyanobacteria such as Anabaena PCC9302 (AY038037), Anabaena ATCC 29413 (NC007413),Nostoc punctiforme PCC73102 (NZAAAY000), Nostoc sp. PCC 7120(NC003272). Such heterocytous cyanobacterial strains, for which eitherinformation or whole genomes were available, contain 4 5 copiesof the 16S rRNA gene, whereas unicellular cyanobacteria such asGloeobacter violaceus PCC 7421 (NC005125), Synechocystis sp. PCC 6803(NC000911) and several Prochlorococcus marinus and Synechococcuselongatus strains have only one to two identical copies. HGT of theparts of the 16S rRNA gene has been reported in several closely relatedbacterial strains [38] . In addition, Miller et al. [39] found that twochloro-phyll d-containing cyanobacterial strains obtained a small part (14 18nt) of the 16S rRNA gene from -proteobacteria, which is only distantlyrelated to cyanobacteria. However, recent reports reveal that the impactof HGT on the phylogeny based on these genes is limited and insigni -cant [40].

Cyanobacteria comprise a monophyletic cluster among eubacteria,which also contain the plastids of eukaryotes. The 16S rRNA, sequencedeitherpartially throughgene ampli cation using PCR followed by directsequencing or sequencing after cloning of the PCR products usingdideoxynucleotide method can provide better resolution. The compari-son of 16SrRNA sequences is a powerful tool for deducing phylogeneticand evolutionary relationships among bacteria, archaebacteria andeukaryotic organisms. The 16S rDNA sequence analyses revealed closerelationships among cyanobacteria, indicating that the diversi cationof cyanobacteria happened within a short period of time. Based onthe phylogenetic analyses of the 16S rRNA genes, chlorophyll- a/b

containing prochlorales (Prochlorophyta) were found to be poly-phyletic and clustered with cyanobacteria [41,42] , showing thatProchlorales shared a common ancestor with cyanobacteria. However,the cyanobacterial orders/subsections have not been supported by the16S rRNA gene sequence analysis [43], except for a few heterocystouscyanobacteria [43,44] . The phylogenetic clustering of strains of severalcyanobacterial genera seems to be incongruent with the cyanobacterialmorphologyand does not followtheir current classi catione.g., Anabaenaand Aphanizomenon [45,46] , Oscillatoria [47] and picocyanobacterialgenera such as Synechococcus and Synechocystis [44]. In some cases, themorphologically distinguished Microcystis species, P. agardhii andNodularia were found to be genetically very closely related to each other[45]. Thus, the current classi cation of cyanobacteria does not followtheir genetic relationships, and revisions are needed, as proposed by

Castenholz [22].

81V. Gupta et al. / Algal Research 2 (2013) 79 97


4/19

On the basis of 16S rRNA analyses, Ward [48] suggested that mor-phologically similar unicellular cyanobacteria were phylogeneticallyvery different. Nubel et al. [33] developed and tested a set of oligonu-cleotide primers for the speci c ampli cation of 16S rRNA genesegments from cyanobacteria andplastids by PCR. Rudi et al. [49] devel-oped a diagnosticsystemusing the DNAsequence polymorphismin the16S rRNA regions V6 to V8 for individual strain characterization andidenti cation. Rasmussen and Svenning [50] characterized symbiotic

Nostoc strains isolated from

ve species of Gunnera by genotypicmethods. The strains were analysed using molecular methods with dif-ferent taxonomic resolutions including RFLP of the PCR ampli ed 16SrRNA gene and 16S 23S internal transcribed spacer (ITS) region, com-bined with computer assisted analyses. Lyra et al. [45] examined toxicand non-toxic cyanobacterial strains from Anabaena , Aphanizomenon ,Calothrix , Cylindrospermum , Nostoc , Microcystis , Planktothrix (Oscillatoriaagardhii ), Oscillatoria and Synechococcus genera by RFLP of PCR-ampli ed 16S rRNA genes and 16S rRNA gene sequencing. Iteman etal. [51] studied the taxonomic coherence and phylogenetic relation-ships of 11 planktonic heterocystous cyanobacterial isolates by investi-gating two areas of the rRNA operon, the 16S rRNA gene ( rrnS ) and theinternal transcribed spacer (ITS) located between the16SrRNA and23SrRNA genes. Gugger et al. [52] identi ed 26 Anabaena strains and 14 Aphanizomenon strains based on phenotypic characteristics and 16SrRNA gene sequencing. Svenning et al. [53] found that most of thesym-biotic and a mixture of symbiotic and free-living isolates fell into wellseparated clades in the phylogram.

3.1. DNA ngerprinting based methods

DNA based ngerprinting methods can be used to estimate diver-sity changes or similarities of community structure between samplesor for rapid screening of a large number of samples. Many ngerprint-ing methods based on PCR ampli ed length variations (RAPD, T-RFLP,LH-PCR and ARISA), melting (DGGE/TGGE), or conformational (SSCP)differences are employed [54,55] . Casamayor et al. [56] evaluated thata subpopulation contributing more than 1% to the total populationcan be detected by DGGE. The combination of DGGE and SSCP allowsthe relationships of bands with bacterial sequences in the databasesby band excision, hybridization or sequencing [57,58] . However, acommon limitation of all the ngerprinting methods is that differentsequences of same length can migrate to the same position in a gel[54]. Crosby and Criddle [59] suggested that ITS-based ARISA causedoverestimation of the bacterial diversity as compared to the other16S rRNA-based ngerprint methods. In some cases, T-RFLP wasfound more suitable for samples with low bacterial diversity; however,reports on its use are limited [60]. Liu and Stahl [54] discussed in detailthe limitations associated with the various ngerprintingmethods suchas SSCP, RFLP and LH-PCR and ARISA. Ryskov et al.[61] suggested a M13PCR based ngerprinting method, in which a single primer speci c to a15-bp repeat motif of bacteriophage M13 DNA is employed to amplifyhypervariable genomic DNA sequences. M13 ngerprinting method

proved to bea veryreliabletoolfor the identi cationand discriminationof cyanobacterial strains, but very few reports are available [62].Nishihara and coworkers [63] were the rst to utilize RAPD analysesfor cyanobacteria. Prabina et al. [64] analysed the electrophoretic pat-terns for 17 different cyanobacterial cultures derived, using 6 differentdecamer primers to provide diagnostic ngerprints for each culturebased on RAPD markers.

Repetitive sequences constitute an important part of thecyanobacterial genome. In cyanobacteria, distinct families of repetitivesequences (STRR Short Tandemly Repetitive Repeat) have been de-scribed, in genomes of Microcystis and Calothrix strains [65]. Mazel etal. [65] characterized and discussed the role of the STRR (three distinctfamilies of repeated sequences) in the genome of the cyanobacteriumCalothrix sp. strain PCC 7601. These repeat sequences were present

at a level of about 100 copies per Calothrix genome and consisted

of tandemly ampli ed heptanucleotides. In addition, a 37 bp longtandemly repeated repetitive (LTRR) sequence has been identi ed in Anabaena strainPCC 7120, and also in non-heterocystous cyanobacteria[66]. Rasmussen and Svenning [67] developed a ngerprinting tech-nique based on STRR and LTRR sequences which was useful in revealingheterogeneity among symbiotic isolates from Gunnera species besidesdistinguishing them from free living isolates. Gupta et al. [68] observedthe abundance of HIP1 in the genomes of many cyanobacteria and the

functional signi cance of smt B deletion in Synechococcus strains andthe possible role of HIP1 (estimated to occur at an average frequencyof once every 664 bp) in genome plasticity and adaptation incyanobacteria. Robinson et al. [69] investigated the propagation of anoctameric palindrome (5 -GCGATCGC-3 ), which is known to be abun-dant in cyanobacterial sequences within databases (GenBank, EMBL)and designated as HIP1 (highly iterated palindrome).They observedthat it is polyphyletic in its distribution and propagates by nucleotidesubstitutions rather than insertion. Delaye and Moya [70] analysed theabundance and distribution of HIP1 sequence in prokaryotes ad theirutility as a typing technique for cyanobacteria and suggested that itcan be a useful molecular water-mark to identify horizontally trans-ferredgenes from cyanobacteria to other species. Thenovelty andutilityof HIP and other sequences open up interesting avenues for under-standing cyanobacterial lineages and understanding the evolution of cyanobacteria, in relation to other bacteria.

Our investigations based on the morphological, physiological andgenetic diversity suggested a wide diversity among a set of Anabaenastrains, isolated from diverse regions of India [71 73]. The diversityanalyses using individual data sets (morphological attributes, nger-prints based on repeat sequences HIP, STRR or 16SrDNA-RFLP) andtheir combinations were found to be useful in categorizing the strains.The strains identi ed as belonging to Anabaena fertilissima , Anabaena os-aquae , Anabaena naviculoides , Anabaena circinalis , Anabaenaaphanizomenoides , Anabaena anomala and Anabaena doliolum clusteredtogether emphasizing the importance of components of traditionaltaxonomy, such as morphological data sets representing useful charac-ters with tremendous signi cance in the development of polyphasictaxonomy.

The importance of comparing several regions of the genome hasoften been stressed for understanding phylogenetic and evolutionaryrelationships among the isolates belonging to a genus [74]. DiagnosticPCR, introduced by Hayes and Barker [75] involves a PCR of the targetregions by picking laments or colonies of cyanobacteria followed bydirect cell lysis, instead of using DNA template. Diagnostic PCR hasbeen successfully used to study genetic diversity of Aphanizomenon ,Planktothrix , Microcystis and Nodularia populations [11,76] . Thismethod makes analyses of a large number of samples and quantita-tive measurements of different genotypes, much easier and more eco-nomical. However, the drawbacks associated with this method arethat it is labour intensive and not suited for non colonial cyanobacteria.Quantitative PCR(real-time PCR) is a sensitivemethod that allowsrapidanalysis of a large number of samples, which has been used to investi-

gate toxic cyanobacteria, by quantifying mcy-genes [77]. In contrast toother detection methods, microarrays allow rapid detection of largenumber of samples using species speci c probes. This technology forcyanobacterial studies is less optimized,mainly dueto the limited avail-ability of chips forthese organisms, and need for re nement in terms of sensitivity, speci city and quanti cation [78].

3.2. Polyphasic approaches and metagenomics

Polyphasic approaches are one of the most promising tools forevaluating diversity analyses in the large microbial populations. Anapproach gaining much signi cance is metagenomics which involvescloning of the large DNA fragments, sequencing and then screeningfor speci c genetic markers. This method circumvents the limitations

of PCR and culturing [79]. The major advantage of the metagenomic



5/19

method is the opportunity to assemble whole genomes directly fromDNA extracted from environmental samples, thereby providing in-sight into the diversity of uncultivable organisms, but requires addi-tional molecular tools for analyses. This technique has been appliedto microbial communities in several extreme environments, includingPolar regions, acid mine drainage bio lm, soil and marine samples[80] . Microbial mats, especially those dominated by cyanobacteria,which represent the base of food chain as producers, are commonly

observed in extreme environments, such as geothermal springs,hypersaline basins, ultraoligotrophic ponds, and hot and cold desertsoils. HPLC based pigment signature analysis has been used to con-

rm the dominance of cyanobacteria populations, associated withchlorophytes and chromophytes in the phototrophic communities of Arctic microbial mats [81]. Microscopic analyses revealed a diverseassemblage of morphospecies grouping into orders Oscillatoriales,Nostocales and Chroococcales, while the 16S rRNA gene sequencesgrouped into a total of 24 ribotypes. However, several researchers[82 84] reported that cyanobacteria isolated from both the Arcticand Antarctica are psychrotolerant rather than psychrophilic, withgrowth optima at temperatures that are well above those of the am-bient environment. Metagenomes of cyanobacterial mats from Arcticand Antarctic ice shelves, using high-throughput pyrosequencing andstatistical comparisons of the protein-coding genes showed similari-ties between the mats from the two poles, with the majority of genes derived from Proteobacteria and Cyanobacteria; however, therelative proportions differed, with cyanobacterial genes more preva-lent in the Antarctic mat metagenome. Shotgun metagenomic se-quencing of phototrophic microbial mat communities from ef uentchannels of Mushroom and Octopus Springs (Yellowstone NationalPark, WY, USA) and direct comparison of unassembled metagenomicsequences withgenomes of representative isolates revealednot only ex-tensive genetic diversity and genomic rearrangements, but novel physi-ological attributes in the native populations. Metagenomic sequencesalso showed a high degree of synteny with the reference genomes of Synechococcus spp. strains A and B [85], but synteny declined with de-creasing sequence relatedness to these references. There was evidenceof horizontal gene transfer among native populations, but the frequencyof these events was inversely proportional to phylogenetic relatedness.Such studies can lead to the identi cation of environment-speci cgenes through a gene-centric comparative analysis, besides providingnew opportunities for interpreting and grouping of environmental spe-ci c cyanobacteria.

Suda et al. [47] in their study used a polyphasic approach to clarifythe taxonomy of the water-bloom-forming oscillatorioid cyanobacteria.Seventy- ve strains of oscillatorioid cyanobacteria were characterizedby 16S rDNA sequence analysis, DNA base composition, DNA DNAhybridization, fatty acid composition, phycobilin pigment composition,complementary chromaticadaptation,morphological characters, growthtemperatureandsalinity tolerance. Pfeiffer andPalinska [86] used a com-binationof different molecular methodsand characterize six Phormidiumlike strains. Thacker and Paul [87] compared morphological characteris-

tics, secondary metabolite composition and 16S ribosomal sequencesamong tropical marine Lyngbya spp. and Symploca spp. and found thatgenetic divergence did not correlate with the morphological and chemi-cal variability among the studied strains. Rajaniemi et al. [88] investigat-ed the taxonomy of the genera Anabaena , Aphanizomenon , Nostoc andTrichormus belonging to the family Nostocaceae by morphological andphylogenetic analyses of 16S rRNA gene, rpoB and rbcLX sequences.Prasanna et al. [71] characterized a set of 30 Anabaena strains, isolatedfrom diverse geographical regions of India, using morphological andphysiochemical attributes as well as molecular marker pro les of thir-teenstrains,which provideda foundation for developing polyphasic tax-onomy for this genus. Gupta et al. [89] analysed set of Anabaena strainsisolated from India based on 16S rDNA and ITS sequences, and foundthem genetically diverse from the other reference strains belonging to

the NCBI data base. Interestingly, six strains belonging to ve different

species (based on taxonomic keys) found to produce microcystin toxins(con rmed by PCR ampli cation of microcystin synthase gene ( mcyA)and ELISA based method) and exhibit antifungal traits formed a sepa-rate group in both the trees. It can therefore be hypothesized thatthe antifungal trait may lead to an evolutionary divergence amongthese strains. Comte et al. [90] evaluated a set of strains of lamentousPhormidium -like cyanobacteria for their study using a polyphasicapproach including phenotypic observations of morphological fea-

tures andgenotypicanalyses (restrictionfragmentlength polymorphismof 16S rRNA gene, internal transcribed space, 16S rRNA gene sequenceanalysis). Marquardt and Palinska [91] studied 30 strains of lamentous,non-heterocystous cyanobacteria from different habitats and differentgeographical regions assigned to diverse Oscillatorian genera, using apolyphasic approach, including comparison of phenotypic and molecularcharacteristics. A set of 70 Anabaena strains, which had been character-izedusing morphologicaland biochemicalmarkers,was analysedfurtherusing PCR ampli cation of STRR1A, which generated 23 bands, ampli -cation with STRR mod resulted in 21 amplicons and HipTG generated atotal of 21 amplicons whichwere allpolymorphic [72] . The 70 Anabaenastrains analysed using the molecular pro les of these three primers ledto a total of 65 polymorphic bands and further analysed using Jaccard'ssimilarity coef cient to generatea dendrogram that depicted thegeneticdiversity and phylogenetic relationships.

With the progressive decrease in sequencing costs, phylogeneticanalyses are increasingly based on sequence information to estimatethe evolutionary relationships of cyanobacteria. The phylogenetictree was made by alignment of the sequences followed by testingthe reliability of the constructed tree with bootstrapping [92]. Severalcomputer programs e.g. ClustalW [92] and ARB [93] have been createdfor aligning the sequences. The relationships of the aligned sequencesare usually shown as a tree, in which the branching pattern of the tree(topology) displays the evolutionary relationships of the strains. Themost commonly used tree construction methods are distance, maxi-mum parsimony (MP), and maximum likelihood (ML) [94]. Distancemethods such as neighbour joining (NJ) ( Fig. 1A) [95] usepair-wisedis-tances (i.e. the number of base differences between two sequences),calculated from aligned sequences and usually corrected to evolution-ary distances within a substitution model. The sequences with theshortest distances areclustered together in a tree, where the tree lengthis optimized to correspond to the distance matrix. The MP method usesthe actual sequence data instead of distances and searches for thetree(s) with minimum length, i.e., topology of the tree can be explainedwith a minimum numberof transformations from onecharacterstate toanother. ML method estimates the likelihood for tree topology thatcould have resulted in the sequence alignment under the given modelof evolution and searches for the tree with maximum likelihood [94].Several earlier reports have used similar methods for depicting evolu-tionary relationships among cyanobacterial genera [14,15,46,88] . Anillustrativerepresentation of these methodsis given in Fig. 1A. The phy-logenetic tree was constructed using 16S rDNA sequences (GQ466493 GQ466520) of the 28 Anabaena strains isolated from India [72,73] and

other reference sequences and it was found to be similar in both NJand MP trees ( Fig. 1B and C). Such approaches have shown to provideuseful information which can be validated by workers worldwide ascompared to the use of single marker. Thus, taxonomic problems incyanobacteria can be resolved with the application of modern tools incombination with traditional criteria (physiological, ecological, mor-phological and biochemical), which can provide a morereliable pictureof the evolutionary relationships among themembers of cyanobacteria.

3.3. Non-PCR-based methods

Bacterial cells in complex samples can be identi ed without culti-vation by uorescence in situ hybridisation (FISH). In FISH, the cellsare made permeable to the uorescent-labelled probes by a xative,

the probes are hybridised under stringent conditions to ribosomal



6/19

Distance-basedmethods

Optimality-based

Clustering-based

Character-basedmethods

UPGMA

Neighbor Joining

Fitch-Margoliash

Minimum evolution

Maximum Likelihood

Maximum Parsimony

Phylogenetictree

A

B C A. aphanizoenoides RPAN25 A.doliolum RPAN53 A. laxa RPAN14 A. flosa-quae RPAN52 A. laxa RPAN56 A. laxa RPAN63

A. vaginicola RPAN22 A. variabilis RPAN45 A.sphaerica RPAN12 A. variabilis RPAN16 A. spiroides RPAN58 A. spiroides RPAN57 A. oscillarioides RPAN4 A. spiroides RPAN20 A. ballyganglii RPRPAN35

Anabaena sp. X59559 A. iyengarii RPAN6 A. aphanizomenoides RPAN68 A. oscillarioides RPAN69 Cultured cyanobiont A7 RPAN71 A. laxa RPAN8 A. spiroides RPAN50

A. iyengarii RPAN17 A. fertilissima RPAN47 A. iyengarii RPAN49 A. anomala RPAN34 A. iyengarii RPAN3 A. iyengarii RPAN11 A. iyengarii RPAN70 Anabaena doliolum HM573455

Anabaena variabilis CP000117 Anabaena variabilis DQ234828

Anabaenopsis circularis AF247595 Nostoc sp. PCC 7423 DQ185242

Calothrix sp. AB325535 Anabaena sp. PCC 7108 AJ133162

Anabaena reniformis FM161348 Anabaena kisseleviana AY701558

Aphanizomenon aphanizomenoides FM161350 Nodularia sp. PCC 9350 AY038034

Anabaena bergii EF529485 Anabaena planctonica AJ293108 Anabaena oscillarioides BECID32 AJ630427

Anabaena solitaria AF247594 Anabaena circinalis EU780159 Anabaena planktonica AY701547

Anabaena ellipsoides AY701560 Anabaena smithii AJ630436

Anabaena compacta FM161347 Anabaena compacta AY701569 Anabaena sp. BIR246 EF547190

Aphanizomenon sp. PCC 7905 AJ133154 Anabaena lemmermannii FM242086

A. flos-aquae 1tu30s4 AJ630422 Anabaena sp. BIR257 EF547192 Anabaena sp. BIR258 EF547193 Anabaena sp. BIR260 EF547194 Anabaena sp. BIR250A EF547191

Nostoc sp. PCC 7906 AB325908

100

100

100

100

100

99

98

60

96

100

93

96

51

61

95

70

90

84

88

74

78

80

54

74

100100

0.005

A. spiroides RPAN58 A. spiroides RPAN57 A. spiroides RPAN20 A. ballyganglii RPAN35 A. oscillarioides RPAN4

Anabaena sp. X59559 A. variabilis RPAN45 A. sphaerica RPAN12 A. variabilis RPAN16 A. aphanizomenoides RPAN25 A. laxa RPAN14 A. doliolum RPAN53 A. vaginicola RPAN22 A. laxa RPAN63 A. flosa-quae RPAN52 A. laxa RPAN56 A. iyengarii RPAN17 A. fertilissima RPAN47 A. anomala RPAN34 A. iyengarii RPAN3 A. iyengarii RPAN49 A. iyengarii RPAN11 A. iyengarii RPAN70

A.laxa RPAN8 A. aphanizomenoides RPAN68 A. oscillarioides RPAN69 Culturedcyanobiont A7 RPAN71 A. spiroides RPAN50 A. iyengarii RPAN6 Anabaena doliolum HM573455

Anabaena variabilis CP000117 Anabaena variabilis DQ234828

Anabaenopsis circularis AF247595 Nostoc sp. PCC 7423 DQ185242

Calothrix sp. PCC 7101 AB325535 Anabaena sp. PCC 7108 AJ133162

Anabaena renif ormis FM161348 Anabaena kisseleviana AY701558

Aphanizomenon aphanizomenoides FM161350 Anabaena planctonica AJ293108

Nodularia sp. PCC 9350 AY038034 Anabaena bergii EF529485

Anabaena oscillarioides BECID32 AJ630427 Anabaena sp. BIR257 EF547192 Anabaena sp. BIR260 EF547194 Anabaena sp. BIR258 EF547193 Anabaena sp. BIR250A EF547191

Anabaena lemmermannii FM242086 A. flos-aquae AJ630422

Anabaena sp. BIR246 EF547190 Aphanizomenon sp. PCC 7905 AJ133154 Anabaena compacta FM161347 Anabaena compacta AY701569 Anabaena planktonica AY701547 Anabaena solitar ia AF247594 Anabaena circinalis EU780159

Anabaena ellipsoides AY701560 Anabaena smithii AJ630436

Nostoc sp. PCC 7906 AB325908

100

100

100

100

99

98

71

54

35

89

43

67

69

29

27

25

87

89

85

15

70

48

70

66

76

65

50

42

45

60

99100

10

Fig. 1. A Schematic picture depicting different phylogenetic methods for identifying evolutionary relationships; B and C, phylogram generated using nearly complete 16S-rDNAsequences (1456 bp) using fD1 rD1 primers from 28 Anabaena strains and available NCBI sequences, constructed by Neighbour Joining (NJ) and Maximum parsimony (MP)methods. Bootstrap values are indicated at the corresponding nodes. The evolutionary distances were computed using the Tajima-Nei method and the bar indicates number of base substitutions per site. Filled and un lled triangles indicate microcystin positive Anabaena strains isolated from India (GQ466577 GQ466582) and earlier studies [8,9,45,85] ,respectively.



7/19

RNA, and uorescent signals are detected by an epi uorescencemicroscope or ow-cytometry. The main advantage of FISH is that itallows detection and quanti cation of intact cells. Probes detectingall cyanobacteria and speci cally certain cyanobacterial groups havebeen designed [96]. One of the major limitations of the standardFISH has been its applicability only for ribosomal rRNA and dif cultiesof optimizing thepermeability protocol for diversebacterial populations.In addition, low signal intensity, lowribosomal content of a cell, or auto-

uorescenceof organisms decreasethe sensitivityof FISH.Many of theselimitations may be overcome by recent developments of FISH such as insitu recognition of low copynumber genes by FISH (RING-FISH), increas-ing signal intensity by, e.g. CARD-FISH, and new permeability protocols[97] , but published reports are scarce on this aspect for cyanobacteria.

4. The species concept of cyanobacteria

The species name provides information regarding the phenotypiccharacteristics of an organism and its relationships with other organ-isms. In general, species concept is a very controversial issue forcyanobacteria and prokaryotes and no general agreement exists [48].Previously, the relatedness between two bacterial species was deter-mined by measuringDNA:DNA relatedness (in terms of relative bindingratio) or the difference in the thermal denaturation midpoint ( Tm)between DNAs from two organisms (heteroduplex DNAs). Above 70%relative binding ratio (RBR) and less than 5 C Tm was considered asthetwo species areclosely related [98]. However, reports on suchinves-tigations are becoming scanty, as DNA:DNA relatedness determinationsare time-consuming and allow only pair-wise comparisons of closelyrelated organisms [47,99] . In the current scenario, morphological datais compared with themolecular dataobtained from differentpolyphasicapproaches for species designation of a particular strain. The 16S rRNAgene sequencing is probably the method most commonly used tostudy genetic relationships of bacteria. Bacterial species having a 16SrRNA gene similarity of more than 97.5% might have either low orhigh DNA:DNA relatedness and could belong either to the same or todifferent species [100] . Due to the limited resolution between closelyrelated species, species de nition cannot be based solely on the 16SrRNA gene sequences [100 102] . Therefore, for correct ordering,species concept should be based on next generation sequencing,metagenomic studies, understanding the protein coding genes and itscomparative relatedness with other organisms, which can provide acorrect passport of any microbial data set.

5. Cyanobacteria prospects as energy sources and speciality lipids

The growing concern regarding the depleting status of fossil fuelsand global warming have provided a strong incentive towards devel-opment of carbon neutral alternatives, as supplements/replacementsto fossil fuels. It has been realized that biofuels produced using oilcrops and waste oils cannot alone meet the existing demand for fueldue to their high level of land requirements, which make photosyn-

thetic oxygen evolving microorganisms a more promising feedstockoption [103,104] . Photosynthetic oxygen evolving microorganisms,including microscopic eukaryotic algae as well as cyanobacteria couldprovide substantially more biodiesel than existing oilseed crops whileusing less water and land, and harvesting solar energy via photosynthe-siswhich represents oneof Nature'sremarkableachievements.Microbialbiofuels are produced from the lipid content of the microbial cells inthe form of triacylglycerols (TAGs) which are produced by a simpletransesteri cation reaction to formbiodiesel [105] . Currently,oleaginousalgae are most popular in the microbial biofuel eld, because of theirability to produce substantial amounts of triacylglycerols (TAG) as astorage lipid [106]. However, production of TAG by microalgae requiresenvironmental stress, which makes the process complicated and costly[107] . But, the inadequate supply of TGA is the bottleneck for current

biodiesel production.

In this context, cyanobacteria are promising organisms for biofuelproduction. A list of cyanobacterial strains with their oil content ispresented in Table 1 . Compared to general photosynthetic plantsand eukaryotic microalgae, cyanobacteria are more amenable to ge-netic manipulation for the introduction of genes involved in biofuelproducing chemical pathways or biochemical engineering. One of themajor advantages of cyanobacteria is that over thirty- ve genomeshave been sequenced and transcriptomes/metabolomes characterized,

making these organisms compliant to highly innovative biotechnologyapproaches [108]. Angermayr et al. [103] have envisaged a technologybased on synthetic biology involving a metabolic network that mergesphototrophic and fermentative metabolism, using solar energy, H 2Oand CO2 as inputs. This forms the essence of the photanol concept,which involves photofermentation or the conversion of CO 2 to biofuel.However, diverting the carbon ow by genetic modi cation can lowerthe tness of the organism; therefore, there is a need to understandthe physiology of the organism for successful development as biofuelsources. This is especially applicable for unicellular organisms such asSynechocystis /Synechococcus , in which information at the moleculargenetics and biochemical level is available. Synechocystis PCC6803 isnow being explored as a model system to study phototrophic biosyn-thesis of free fatty acids (FAs) and liquid fuels such as alkyl esters,alkanes and isoprenoids. As many cyanobacteria synthesize alkanes, Jansson [109] proposed that they can be used as drop-in fuel similarto gasoline and jet fuel; by optimizing the expression of the alkane bio-synthesis genes and enhancing the carbon ux through the fatty acidand alkane biosynthesis pathways. This can be a promising option tothe accumulation and/or secretion of notable amounts of alkanes.

Fu [110] undertook genome-scale analyses of the Synechocystis sp.PCC 6803 metabolic network, which included 633 genes, 704 metab-olites and 831 metabolic reactions. This was reconstructed for thestudy of optimal Synechocystis growth, network capacity and func-tions. Heterotrophic, photoautotrophic and mixotrophic growth con-ditions were simulated. The Synechocystis model was used for in silicopredictions for the insertion of ethanol fermentation pathway, whichis a novel approach for bioenergy and biofuel production. In fact, thegenetic engineering platform for cyanobacteria is well establishedand cyanobacteria have been shown to be highly tolerant to the intro-duction of foreign genes [111] . The genetics and metabolic regulationmechanisms of cyanobacteriaarewell understood. Liu andCurtiss [112]have developed an inducible lysis system through genetic engineeringand addition of nickel sulphate, which facilitates the extractionof lipidsfor biofuel production. This information should facilitate the use of cyanobacteria for biofuel production.

Biohydrogen, bioethanol and biodiesel (fatty acid methyl esters, orethyl esters) are the dominant forms of biofuels currently in use, foraddressing the prevailing challenges imposed by high energy de-mand, global warming and climate change. Hydrogen has long been

Table 1Cyanobacterial strains with their oil content.

Microalgae Oil content(% dry wt.)

Anabaena cylindrica 4 7 Anacystis nidulans b 1Chlorogloea fritschii b 1Microcoleus chthonoplastes b 1Nostoc muscorum b 1Oscillatoria williamsii b 1Phormidium huridum b 1Plectonema terebrans b 1Spirulina maxima 6 7Spirulina platensis 4 17Synechocystis sp. PCC 6803 18Synechococcus elongatus 18Synechococcus sp. 11Trichodesmium erythraeum 0.05 0.12



8/19

known to be produced as a nal end product of formation or a sideproduct in photosynthesis in multiple groups of microorganisms.Cyanobacterial strainsbelonging to genera suchas Gloeocapsa , Anabaena , Arthrospira /Spirulina , Cyanothece , Nostoc have been reported to producelarge quantities of hydrogen [113] . This is mainly facilitated by twotypes of hydrogenases, uptakeand bidirectional types which can be pro-duced by diverse methods including steam reformation of oils, photol-ysis of water or dark/photofermentative means. Ananyev et al. [114]

found that environmental and nutritional conditions which increaseanaerobic ATP production and the intracellular reduction potential(NADH+ /NAD+ ratio) represent key variables for enhanced H 2 produc-tion in Arthrospira (Spirulina maxima ). Tamagnini et al. [115] , in theirreview on diversity of cyanobacterial hydrogenases, remarked thatalter-native nitrogenases (containing V, instead of Mo) may be more ef cientH2 producers. However,a major problemis the extremeoxygen sensitiv-ity of hydrogenases involved in hydrogen production. Therefore the twoprocesses (photolysis and hydrogen production) need to be temporarilyuncoupled. This crucial problem is not yet been solved, and no commer-cial application of this process has been reported. With recent advancesin synthetic biology, an interesting option is the construction of mutantswith blocked electron transfer in selected key pathways.

Cyanobacteria are more ef cient in converting solar energy andcarbon dioxide into polysaccharides than plants, and microbes tomake biofuels from glucose produced from the polysaccharidesthrough fermentation [103] . Deng et al. [116] reported that ethanolproduction in genetically engineered S. elongatus PCC7942 occurs witha yield of 54 nmol OD 730 nm /unit lday. Dexter et al. [117] also reportedbioethanol yield of 5.2 mmol OD 730 nm /unit lday from Synechocystis sp.PCC6803.

Ethanol, however, is far from the ideal biofuel for several reasons.Speci cally, it has a low energy density, is volatile and dif cult totransport via carrier pipelines owing to its corrosive properties.Moreover, ethanol is toxic to microbes and its high solubility in theaqueous fermentation culture broth is not advisable [118] . Fortmanet al. [119] reviewed biofuel alternatives to ethanol, with specialfocus on the development of biofuels based on genetic engineeringmicrobes based fatty acids. It was concluded that the fatty acidbased biofuel from cyanobacteria is more suitable and viable thantheir other organisms [108] .

When algal cells are actively growing, polar membrane lipids(phospholipids and glycolipids) generally predominate, but as thecell enters stationary phase many species accumulate triacylglycerols(TAGs) in the range of 20 50% dry weight [107] . Triglyceride biosyn-thesis is conveniently divided into three steps: (i) formation of acetylCoA, (ii) acyl chain elongation and (iii) triacylglycerol formation. Themajority of algal TAGs include saturated or mono-saturated C14 C18fatty acids, but long-chain polyunsaturated fatty acids (PUFAs) areoften present [120] . The fatty acid composition of microalgae variesfrom species to species. Microalgae contain approximately fty fattyacids (excluding positional isomers), ranging from octanoic acids,C8 H16 O2 to C28 H40 O2 [121] . Biodiesel is produced by reacting metha-

nol with biologically derived oils that consist of fatty acids (FAs)ester-linked to a glycerol molecule. During the resultant trans-esteri cation reaction, the FAs are cleaved from the glycerol moleculeand each is bonded to a methyl group, producing biodiesel.

Metabolic engineering of lipid production pathways is anotherfruitful area of research, either through enhancing yields of TAGs orengineering pathways for novel biofuel molecules [122]. One of theoptions involves redirecting the cyanobacterial intermediary metabo-lismthrough channellingof Calvin Cycle intermediates into fermentativemetabolic pathways ( Fig. 2). It has been shown that overexpression of thioesterases can aid in the excretion of fatty acids, for the synthesis of TAGs. Cyanobacterial strains like Spirulina / Arthrospira grow luxuriantlyin high alkaline pH (9.5 11) conditions leading to high cell densities inopen pond reactors with low microbial contamination [123] . Lu [108]

pointed out that the successful overproduction of fatty acids and de

novo biosynthesis of FAEE (fatty acid ethyl esters) and fatty alcoholsin a genetically engineered Escherichia coli strain and enhancing theproduction and secretion of fatty acids in genetically engineeredcyanobacteria, as recent breakthroughs which need to be optimizedfurther at commercial scale. Such strains need to be explored as modelsystems for enhancing lipid/ethanol production through genetic engi-neering or biochemical interventions [124] .

Several researchers have reported that algae produced more oil in

stressed condition as comparedto their optimalgrowth conditions [107] .The major environment stimuli that govern mass multiplication/growthconcomitant with oil production include carbon dioxide, nutrientde cit,light intensity, pH and temperature [125]. The optimized ambiencemakes best use of these miniature factories to maximize the oil pro-duction irrespective of cultivation system. Dismukes et al. [106] havedeveloped a new concept of AMOPs (aquatic microbial oxygenicphototrophs), which includes cyanobacteria, microalgae, and diatoms,as they offer a wide range of alternatives for designer biomass and bio-fuel precursors.

The recent advancements in synthetic biology, computer algorithmsbased modelling and simulation and genetic manipulation provideimmense opportunities for engineering of cyanobacteria for biofuelsor their intermediates. This can be achieved using holistic approachesthrough involvement of microbiologists, biochemists, engineers, com-puter programmers and molecular biologists with suf cient fundingand provision and public private partnerships.

6. Cyanobacteria their promise in aquatic bioremediation

Cyanobacteria constitute an important component of wastewaterecosystem and tend to dominate in eutrophic environments, character-ized by high levels of inorganic nutrients. Their proliferation in suchenvironments is mediated by the production of exopolysaccharidesand occulants, which can also serve in phytoremediation. In wastewa-ter treatment plant (WWTP), their contribution for the total phyto-plankton density attains maximum values of 99.8% [126]. Furtado etal. [127] reported that the abundance of cyanobacteria (91.7% in sum-mer and 96.4% in autumn) was always higher than the abundance of other algae (8.3% in summer and 3.6% in autumn), indicating theirstrong contribution to phytoplankton communities in these systems.Martin et al. [128] isolated fty-one cyanobacterial strains, belongingto Phormidium autumnale , Planktothrix mougeotii , Limnotrix sp. andSynechocystis sp. species from a WWTP located in the north of Portugal,while Lyngbya and Oscillatoria were the most frequent species found inaerobic treatment tanks from an agro-food industry WWTP [126] .Renuka et al. [129] reported the predominance of microalgae belongingto Cyanophyta (58%) followedby Chlorophyta (25%) and Bacillariophyta(17%) in sewage wastewater. Therefore, cyanobacteria can be a propi-tious alternative for wastewater treatment, being an indigenous in-habitant and needing to be only enriched for ful lling their role inbioremediation.

Cyanobacteria are also promising tools for the removal of heavy

metals from single as well as from multi-metal containing wastewa-ters [130,131] . Most studies have been restricted to the laboratory,involving different immobilizing agents and different cynaobacterialgenera ( Table 2 ). Mohapatra and Gupta [131] reported the biosorptionof Zn (II), Cu (II) and Co (II) in Osillatoria angustissima biomass fromsingle, binary and ternary metal solutions, a function of pH and metalconcentrations. The biosorption capacities for single metals were inthe order: Zn(II)>Co(II)>Cu(II) and in binary system, the tolerancewas in the order: Cu(II)>Zn(II), Cu(II)>Co(II) and Zn(II)>Co(II), dueto which the biosorbent exhibited net preference/af nity for Cu(II)sorption over Zn(II) or Co(II). Pre-treatment of cyanobacterial biomassis also known to increase their binding capacity of heavy metals. InS. maxima , pretreatment with CaCl 2 enhanced Pb 2+ sorption capacityby 84 92% [132] . Similarly, the introduction of phosphate group in

Lyngbya taylori increased its capacity to sorp Cd 2+

[133] . Nagase et al.



9/19


10/19

Emulcyan, a sulphated heteropolysaccharide from Phormidium in-creases light availability in water column by occulating suspendedclay particles [148] . Choi et al. [146] reported a polysaccharidal oc-culant containing neutral sugars, uronic acids and proteins from Anabaena . The broad substrate speci city, rapid occulating activityand thermal stability of these occulants can extend their applica-tions in wastewater treatment.

The use of cyanobacteria alone or in consortia in wastewater treat-ment is well established, but so far, no technology has been devel-oped to scale up to commercial level. The selection of cyanobacterialgenera and their tolerance optima to pollutant(s) needs in-depth re-search, before this technology can be fully exploited. Cyanobacteriaalone or their consortia with bacteria or their immobilized formula-tions can have immense promise, as several different contaminantscan be treated simultaneously and same consortia will have widerapplicability at different sites, hence improving the overall cost effec-tiveness of the technology. The biomass produced which may berich in heavy metals or nutrients or carbohydrates and/or proteins,depending upon the contaminant at a particular site. This can addi-tionally contribute as a substrate for the production of biofuels or uti-lized as bio-ore for re-incineration and recovery of precious heavymetals or used as a source of fertilizer (N and P) for cereal/foragecrops.

7. Cyanobacteria as valuable sources of bioactive compounds

Cyanobacteria constitute a rich source of novel bioactive com-pounds exhibiting anti-cancer, anti-viral, antibacterial, antifungal,anti-in ammatory and cytotoxic activities.

7.1. Allelochemicals produced by cyanobacteria

Cyanobacteria and eukaryotic algae are also known to excrete bio-active compounds into the environment, which are important deter-minants of allelopathic activity in water and soil. Allelochemicals aresecondary metabolites that in uence the structure and dynamics of populations or communities of either plants or animals or microbes.Toxic blooms found in eutrophic polluted water bodies lead to severalenvironmental implications, because of their allelopathic interactions.Srivastava [149] reported antagonism between species of Anabaenaand Hapalosiphon intricatus . Both positive and negative allelochemicalincidents are known to be involved in the control of freshwater bloomsequence [150]. Wiegand and P ugmacher [151] in their reviewdiscussed the wide range of toxic secondary metabolites producedby cyanobacteria. Allelopathic compounds include alkaloids, cyclic pep-

tides, terpenesand volatileorganic compounds. Allelopathiccompounds

havevariousmodes of action, frominhibitionof photosynthesis to oxida-tive stresses or cellular paralysis. Suikkanen et al. [152] investigated theallelopathiceffects of three cyanobacterial species ( Nodularia spumigena , Aphanizomenon os-aquae and Anabaena lemmermannii ) that frequentlydevelop intomassoccurrences in Baltic Sea.Theyexposed monoculturesof three phytoplankton species ( Thalassiosina weiss ogii, Rhodomonasspp. and Prymnesium parvum ) to cell free ltrates of the threecyanobacteria and quanti ed allelopathic effects with cell counts. Allthe tested cyanobacteria inhibited the growth of Rhodomonas spp., butnone of them affected P. parvum . Gross et al. [153] investigated theallelopathic activity of two submerged macrophytes, Ceratophyllumdemersum and Najasmarina ssp. intermedia , which inhibited phytoplank-ton and epiphytes.

Doan et al. [154] isolated the alkaloids 12-epi-hapalindole E isonitrilefrom Fischerella spp. and the indolophenanthridine Calothrixin A fromCalothrix spp. and characterized in terms of their ability to kill severalorganisms/cell types, and their biochemical modes of action. Bothcompounds inhibited RNA synthesis and consequently protein synthe-sis. Calothrixin A also inhibited DNA replication in Bacillus subtilis .Casamatta and Wickstrom [155] studied the sensitivity of two disjunctbacterioplankton communities to the exudates from cyanobacterium,Microcystis aeruginosa . Repka et al. [156] studied the association of cyanobacterial toxin, nodularin with some zooplankton species in theBaltic Sea. They reported that among the two cyanobacteria studied, Aphanizomenon showed positive interaction with the abundance of zooplankton and Nodularia did not show any such interaction. Thus, itindicates that zooplankton also plays a crucial role in development of cyanotoxin producing Aphanizomenon .

Asthana et al. [157] reported that the methanolic extract of Fischerella spp. was active against Mycobacterium tuberculosis ,Enterobacter aerogens , Staphylococcus aureus , Pseudomonas aeruginosa ,

Salmonella typhi , E. coli as well as 3 multi drug resistant E. coli strainsin in-vitro assays. They also found that the active principle wasHapalindole T, based on MS, UV, IR and H-NMR analyses, witha molec-ular weight of 386 and a melting point range of 179 182 C. Asthana etal. [158] extracted pharmaceutically important -linolenic acid fromFischerella spp. using chloroform and methanol (1:2) which alsoexhibited allelopathic activity.

7.2. Types of cyanotoxins

Microcystins are the cyclicheptapeptides produced by cyanobacterialgenera such as Microcystis , Anabaena , Planktothrix (Oscillatoria ), Anabaenopsis , Nostoc and Hapalosiphon . Most of the identi ed microcystinvariants are composed of seven amino acids and constituted a general

structural formula [cyclo(-D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha-)],

Table 2Bioremediation of different types of wastewater by cyanobacteria.

Type of wastewater Cyanobacteria Compound

Synthetic heavy metal solution Aphanothece halophytica ZnSynthetic heavy metal solution Lyngbya taylorii Cd, Pb, Ni, ZnSynthetic heavy metal solution Phormidium laminosum Cu, Ni, ZnIndustrial sewage water Nostoc linckia Zn, CdIndustrial ef uent (salt and soda company) and sewage water Anabaena subcylindrica Cu, Co, Pb, MnIndustrial waste water (Cr(VI) plating industry) Nostoc PCC7936 Cr(VI)

Synthetic heavy metal solution Oscillatoria anguistissima Zn, Cu, CoSynthetic wastewater Anabaena doliolium Nutrients, Cu and FeSynthetic wastewater Spirulina platensis NO3

, NH3 , PO4

Domestic sewage ef uent Oscillatoria sp. NO3 and orthophosphate

Ground water Synechococcus sp. strain PCC 7942 NO3

Synthetic heavy metal solution Phormidium laminosum N and PUrban wastewater Phormidium sp. NO3

and PO 4

Synthetic wastewater Phormidium laminosum NO3 and PO 4

Synthetic wastewater Anabaena CH3 NO3 and NH 3

Swine-wastewater Spirulina maxima NH3 N and total phosphorusSynthetic polyphosphonate water Spirulina spp. Hexamethylenediamine-N,N,N_,N_-tetrakis(methylphosphonic acid)Re nery wastewater Aphanothece microscopica CO2 bio xation



11/19

where X and Z are two variable L-amino acids, D-MeAsp is D-erythro- -methylspartic acid and Mdha is N-methyldehydroalanine [159] .Microcystinsare potent inhibitors of two major eukaryoticsignal trans-duction, serine phosphatase 1 and 2A enzymes. Microcystins haveseveral targets (liver, hyperphosphorylation of proteins and -subunitof ATP synthase) inside the body which can ultimately lead to thedeath of the organism [160]. Among different variants of MC, MC-LR was found to be the most potent hepatotoxin. The biosynthesis of

microcystin occurs through genes encoding non-ribosomal peptidesynthetases (NRPS), polyketide synthases (PKS) and hybrid NRPS PKSsystem [159,161] . The size of the entire operon responsible for thebiosynthesis of microcystin varies in cyanobacterial genera. In M.aeruginosa , there is a 55 kb microcystin gene cluster arranged in twobi-directionally arranged operons. They comprise peptide synthetasesgenes ( mcyA, mcyB, mcyC ) polyketide synthases genes ( mcy D), hybridenzymes genes ( mcyE mcyG), methylation gene ( mcy J ), epimerizationgene ( mcyF ), dehydration gene ( mcyI ) and transporter gene ( mcyH ). InPlanktothrix , the 55.8 kb microcystin gene cluster contains genes forpeptide synthetases ( mcyA mcyB mcyC ), polyketide synthases gene(mcyD), hybrid enzymes genes ( mcyE mcyG), a putative thioesterasegene ( mcyT ), a putative ABC transporter gene ( mcyH ) and a putativepeptide-modifying enzyme ( mcyJ ) [161]. In Anabaena strain 90, the55.4 kb microcystin gene clustercontains three operons. The rst oper-on ( mcyA mcyB mcyC ) is transcribed in theopposite direction from thesecond ( mcyG mcyD mcyJ mcyE mcy F mcyI ) and the third operon(mcyH ). The peptide synthetase genes are mcyA, mcyB and mcyC . Inthe second operon, the PKS genes are mcyD, mcyJ , mcyF and mcyI ,whereas, mcyG and mcyE are hybrid genes [159]. The variation inmicrocystins structure is attributed in part to the recombination be-tween mcyC and the rst module of mcyB [162]. Ampli cation of mcyBwas considered as a rapid tool for identi cation of toxic and nontoxiccyanobacteria [163] .

7.3. Nodularin (NODLN)

Nodularin, a cyclic pentapeptide, structurally similar to microcystinis among the most commonly isolated toxin from the lamentous,planktonic cyanobacterium, N. spumigena . The toxin has been reportedto have serious effects on numerous organisms within the ecosystem,including invertebrates and sh, but with no effect on other organisms[164] . Mof tt and Neilan [165] characterized and sequenced thenodularin biosynthesis gene cluster ndaS , from N. spumigena NSOR10.The 48 kb operon constitutes nine ORFs ( ndaAI ) transcribed from abidirectional regulatory promoter region. Most of the ndaS encodedgenes have homologs in the mcyS cluster. Two NRPS enzymes, NdaAand B, complete the cyclic pentapeptide by adding the nal aminoacid residues, L -Thr, D-MeAsp and L -Arg.

7.4. Saxitoxin (STX)

Saxitoxin or paralytic shell sh poisons (PSPs) are highly potent

neurotoxins produced by several freshwater species of cyanobacteria A. circinalis, Aphanizomenon sp., Aphanizomenon gracile , C. raciborskiiand Lyngbya wollei and marine dino agellates genera Alexandrium ,Pyrodinium and Gymnodinium [166]. These toxins are considered a seri-ous toxicological health risk that may affect humans, animals, and eco-systems worldwide. Most of the data associated with STX have beenreportedfrommarineorganisms andonlyscanty information is availablefor STX produced by freshwater cyanobacteria. Using a reverse geneticapproach, Kellmannet al. [167] identi ed the gene cluster putatively re-sponsible for the biosynthesis of saxitoxin in C. raciborskii T3 (sxt ). Thesaxitoxin gene cluster is more than 35 kb, and comparative sequenceanalysis assigns 26 proteins. STX biosynthesis is initiated with arginine,S -adenosylmethionine,and acetateby a newtypeof polyketidesynthase,which can putatively perform methylation of acetate, and Claisen con-

densation reaction between propionate and arginine. Further steps

involve enzymes catalyzing three heterocyclizations and various tailor-ing reactions that result in the numerous isoforms of saxitoxin. The dis-tribution of these genes also supports the idea of the involvement of similar gene homologs for STX production in various cyanobacteria.

7.5. Cylindrospermopsin (CYN)

Eight cyanobacterial species have so far been identi ed

as cylindrospermopsin producers

C. raciborskii , Aphanizomenonovalisporum , Aphanizomenon osaquae , Umezakia natans , Raphdiopsiscurvata , Anabaena bergii , Anabaena lapponica and L. wollei [168] .Cylindrospermopsin has hepatotoxic, besides general cytotoxic[169] , and neurotoxic [170] effects and is a potential carcinogen[171] . Its toxicity is due to the inhibition of glutathione and proteinsynthesis as well as the inhibition of cytochrome P450. The chief toxin producer, C. raciborskii, presents a major problem for watermanagement, on a global scale [172] . The cylindrospermopsin biosyn-thesis ( cyr ) gene cluster from C. raciborskii AWT205 was recentlysequenced [173] . The cluster spans 43 kb and contains 15 ORFs,which encode all the functions required for the biosynthesis, regulationand export of the toxin. Biosynthesis is initiated via an amidinotransferonto glycine followed by ve polyketide extensions and subsequentreductions, rings are formed via Michael additions in a step-wise man-ner. The uracil ring is formed by a novel pyrimidinebiosynthesis mech-anism and tailoring reactions, including sulfation and hydroxylationthat complete biosynthesis.

7.6. Anatoxins

Anatoxins are a group of alkaloids which are highly toxic to birdsand mammals and produced among a number of cyanobacterialspecies including Anabaena os-aquae , Aphanizomenon os-aquae ,Cylindrospermum , Raphidiopsis and Planktothrix sp. (formerly Oscillatoriasp.) [174] . Two analogs of anatoxins have been described: anatoxin-aand homoanatoxin-a, both are secondary amines. Anatoxin-a andhomoanatoxin-a are potent agonistsof the nicotinic acetylcholine recep-tor [175] . A third anatoxin, anatoxin-a(s), is a phosphate ester of a cyclicN-hydroxyguanine, with pharmacological and chemical properties dif-ferent from anatoxin-a, acting as an acetylcholinesterase inhibitor. Thebiosynthetic pathway for anatoxin-a, as recently described based onthe genome sequence of Oscillatoria PCC 6506 [176]. Anatoxin-a is syn-thesized via a PKS pathway. The various types of toxins produced byfreshwater cyanobacteria are summarized in Table 3 .

7.7. UV absorbing pigments

Cyanobacteria have evolved protective mechanisms againstharmful UV radiations, by synthesizing UV absorbing compounds ormycosporine-like amino acids (MAAs) and scytonemin [177] . Schereret al. [178] described a new ultraviolet (UV)-A/B absorbing pigmentwith maxima at 312 and 330 nm from the cosmopolitan terrestrial

cyanobacterium Nostoc commune . According to the Schulz andScherer [179] , cyanobacteria use three different types of strategiesto counteract UV damage: (i) stress avoidance by gliding mecha-nisms, (ii) stress defence by synthesis of UV-absorbing compounds,antioxidants and extracellular polysaccharides, and (iii) repair mech-anisms including DNA repair and resynthesis of UV-sensitive pro-teins. They summarized the effects of and responses to UV-B at thephysiological and molecular levels and the proteome of the terrestrialcyanobacterium N. commune . Matsunaga et al. [180] investigated thephysiological response of marine planktonic cyanobacteria to UV-A(320 390 nm) irradiation and isolated an UV-A absorbing pigmentfrom a UV-A resistant strain of Oscillatoria .

Bohm et al. [181] also reported the secretion of water soluble UV absorbing pigments by the terrestrial cyanobacterium N. commune .

Sinha et al. [182] investigated the presence of ultraviolet-absorbing



12/19

mycosporine-like amino acids (MAAs) and their induction by solarultraviolet-B (UV-B) radiation in three lamentous and heterocystousN2- xing cyanobacteria Anabaena sp., N. commune and Scytonemasp. Torres et al. [183] isolated the mycosporine-like amino acid(MAA-porphyra-334; max =334 nm; =42,300 M

1 cm 1) fromthe aquatic cyanobacterium Aphanizomenon os-aquae (AFA) and veri-

ed its structure by spectroscopic methods. Oren and Gunde-Cinerman[184] in their study observed that mycosporines and mycosporine-like

amino acids (MAAs) are low-molecular-weight water-soluble mole-cules absorbing UV radiation in the wavelength range 310 365 nm.Rastogi and Sinha [185] reviewed various aspects of scytonemin andMAA production, which illustrated the diversity in the structural andfunctional aspects of cyanobacterial response to UV irradiation.

8. Other value added products

8.1. Pigments

Cyanobacterial pigments comprise the most colourful and attractivecomponents, which are extensively utilized in bioindustry. Screeningprograms all over the world have further con rmed the diversity andrich repertoire of pigments, which are revolutionising the industrialuses of colours with their nutraceutical and pharmaceutical value.Cyanobacteriaoptimize theharvesting of light based on available irradi-ance and spectral composition by modulating their antenna pigmentcomposition,through a phenonmenon knownas Complementary Chro-matic adaptation [186] . Among these phycobiliproteins (comprisingphycocyanin, phycoerythrin and allophycocyanin), which account forabout 20% of total dry weight are the most important [187]. Caroten-oids, on the other hand, act as antioxidants, hence, protect cells fromdamage by unstable oxygen molecules. These pigments can boost theimmune system and possibly lower the risk of heart disease, preventonset of cancers and protect against age related diseases such as cata-racts and macular degradation, multiple sclerosis, etc. [188].

Nayak et al. [73] characterized a set of Anabaena strains on thebasis of accumulation of two important pigments carotenoids andphycobiliproteins, in signi cant variability among the Anabaenastrains was observed. More than 60% of the strains accumulated1.01 1.96 g ml 1 of carotenoids. Out of the total cell proteins,phycobiliproteins may constitute up to 50%. The phycobiliproteinssuch as phycocyanin, allophycocyanin and phycoerythrin are foundonly in the cyanobacteria, red algae and cryptophytes. In order to

identify potential strains for pigment production and also understandthe role of different environmental and nutritional conditions on en-hancing pigment accumulation, several studies have been undertaken[189,190] . Tremendous diversity was observed, not only at theintergeneric level [191] , but also at the intra generic and inter/intraspeci c level [192] in the phycobilin composition and relative propor-tion of PC, PE and APC. They can be used as natural food colouringagents (candy, ice creams, dairy products and soft drinks), drug and

cosmetics. The buffer extract of phycocyanin from Spirulina sp. isused in eye shadow, eyeliner and lipsticks, while c-phycocyanin alsoacts as a selective inhibitor of cytochrome oxidase 2, and hashepatoprotective and anti-in ammatory effects [193] .

8.2. Vitamins

Cyanobacteria are the richest source of vitamins. For example,Spirulina ( Arthrospira ) is found to be rich in vitamin B12 (two to sixtimes richer than raw beef liver) and vitamin E, with 20 g of Spirulinaful lling the daily body requirements of Vitamin B12 and 70% of B1(thiamine), 50% of Vitamin B2 (ribo avin) and 12% of Vitamin B3(niacin) [194] . It also contains nutrients, including other B complexvitamins, beta-carotene, vitamin E, manganese, zinc, copper, iron,selenium, and linolenic acid (an essential fatty acid).

8.3. Biopolymers

Cyanobacteria are now included as the potential sources of newpolymers, several species being characterized by the presence of thick capsules surrounding the cells and by the ability to releasepolysaccharide material into culture medium [195] . Carbohydratessuch as glucosyl glycerol, trehalose and sucrose are synthesized bycyanobacteria under different osmoticstresses. Carbohydrateproductionaccounts for 15% to 22% of dry weight [196] , with rhamnose as the mainpolysaccharide in Spirulina . Borowitzka et al. [197] found an increase inosmoregulatory carbohydrates in Synechococcus , when high salinitycon-ditions were provided in growth medium. Niederholtmeyer et al. [198]found that in S. elongatus PCC7942, the transformation of transportergenes encoding invertase, glucose facilitator, lactate dehydrogenaseand lactate transporter stimulates the secretion of glucose, fructose andhigh value hydrophilic products such as lactate.

The diversity of molecules of economic importance produced bythese organisms is immense and prospecting extreme habitats and

Table 3General characteristics of cyanobacterial toxins.

Cyanobacteria Toxins Structure Activity against

Microcystis aeruginosa Microcystin Cyclic peptide Bacterioplankton and zooplanktonCalothrix fusca Calophycins Cyclic undecapeptide and decapeptide Antifungal activityNodularia sp. Nodularin Peptides Antifungal and algicidal activity Anabaena laxa Laxaphycins Decapeptide and cyclic undecapeptide Aspergillus oryzae

Candida albicansPenicillium notatumSaccharomyces cerevisiaeTrichophyton mentagrophy tes

Tolypothrix tjipamasensis Tjipamazoles N-glycosides o f indo le(2 ,3-a)-carbozol Candida albicans Aspergillus avusTrichophyton mentagrophytes

Scytonema hofmanii Scytonemin A Cyclic undecapeptide Colletotrichum gloeosporiodeFusarium oxysporumRhizoctonia solani

Nostoc muscorum Aplysiatoxin Alkylphenols Toxic to mammals (skin)Oscillatoria nigroviridis Aplysiatoxin Alkylphenols Toxic to mammals (skin)Tolypothrix sp. Tolytoxin Polyketide macrolide Bipolaris incurvata

Phytophthora nicotianae Aspergillus oryzae

Schizothrix sp. Schizothrinin A Cyclic undecapeptide Sclerotium rolfsiiLyngbya majuscula Majusculamide-C Cyclic depsi-octapeptide Phytophthora infestans

Plasmopara viticola



13/19

collaboration with industries for scaling up of useful entities can helpto exploit their potential in a more focussed manner.

9. Agricultural implications of cyanobacterial diversity and their role in integrated nutrient and pest management

Cyanobacteria are known to play diverse roles in the environment,as nutrient supplements (inoculants) and soil compaction agents in

agriculture, besides having tremendous ecological signi cance ascarbon sequestering and bio remediating agents [199] . As the primarycolonizers in a diverse range of aquatic and terrestrial habitats, theyare key contributors in the sustenance of fertility and represent thebase of the food pyramids. In the 1970s, algalization or the enrich-ment of soil via inoculation of selected cyanobacterial strains led tothe promotion of these biofertilizers among the farming communityin South East Asia [200,201] . Analyses of the abundance and genera-wise diversity of cyanobacteria isolated from the rice based croppingsystems of North and Eastern India have revealed the dominance of heterocystous forms, with Nostoc and Anabaena comprising 40 90% of the isolates. In general, heterocystous forms were the dominant mem-bers, comprising 70 95% of the isolates. Diversity indices revealed that Jaipur (Orissa), followed closely by Faizabad and Lucknow exhibitedthe greatest diversity. A general pattern of abundance in all locations,with Nostoc > Anabaena > Phormidium , highlighted the omnipresenceand dominance of these genera [202] . Diversity analyses of a set of 70 Anabaena strains (including 67 strains isolated from diverse rice agroecologies of India, and three International Reference/Type strains),and their species wise distribution in different soil types and soil pHrevealed that Anabaena iyengarii was present at pH ranging from 5.5to 8.5 and alluvium soils was the most hospitable, harbouring most of the species of Anabaena , except Anabaena oscillarioides [72].

Studies undertaken to characterize the abundance, genera-wisediversity and metabolic capabilities of cyanobacteria isolated fromthe rice rhizosphere, for the rst time, revealed that the genera Nostoc and Anabaena comprised 80% of the isolates [5]. Such isolates werealso ef cient in enhancing the germination and growth of wheatand rice seeds and exhibited signi cantly high protein accumulationand IAA production, after incubation in light and dark (with 0.5%glucose). Anabaena (40%) and Nostoc (38%) followed by Hapalosiphon ,Calothrix and Westiellopsis represented the dominant genera. Therhizosphere dynamics and plant growth promoting ability of a set of inoculated strains in rice crop revealed the persistence of strains onroots up to harvest stage, entry into roots and enhancement inplant growth and yield attributes. Besides improved soil fertility/microbiological parameters up to harvest stage of crop, observationsclearly demonstrated that cyanobacteria enhanced plant growthparameters (plant height, dry weight, grain yields), besides bringingabout signi cant changes in soil microbial biomass carbon [203] .

Despite reports of cyanobacterial associations with a number of crops [6,203 205] , their role as plant growth promoting inoculantsis sc

Nuevas Luces en La Biodiversidad y Aplicaciones de Cianobacterias Gupta Et Al 2013

Documents

Transcript of Nuevas Luces en La Biodiversidad y Aplicaciones de Cianobacterias Gupta Et Al 2013