MS-172 Journal Home page: wuveb.con * Email; eitor eb coin Review Article Journal of Environmental Biology JEB/ ASSN 02548704 PISSN. 23840379 Dol: 10.22438 CODEN: JEBIOP Perspective of nitrate assimila and bioremediation in Spirulina platensis (a_ non-nitrogen \fixing cyanobacterium): An overview ~ @ Q. Feriduddin*, P. Varshney' Cyanobacterium, Spiruine/plaiensi, has been used for many centuries as a food product and ica ‘also has important applcatons in industry end environmental remediation Apart om being avery good source ofessenialnutrens such as proviains, minerals, polvunsalurtedfaty acids, S.platenssis also ‘Plant Physiology and Biochemistry _characterzed by it high pros conn, The nate assimlaton genes of S.platensis is organised in a Section, Department of Botany, systemic operon with the structure: ni (firite reductase}-permease gene(s}-nar6 (nitrate reductase ‘Agath Musi University, ‘Genomic loealsaton nS. plteai erent rom the operons found in other cyanobacteria duet the ‘Nigarh-202 002, India presence ofboth types of irate anspeiters(ntP and ABC types). Both nitrate uptake and transcription of “Depavent fe Sciences, ‘heniateassimiatry gnesin Spleens areregdated. However, the mechanismofreguatinofitate Uanestyat ities: assimiatory genes erent tom abe on-itogen fing cyanobacterial species. nthe last decade, isabel 400 036, ce has aso been exploited fais obey fo deconlamiate water ed enviranmental poston caused by hazardous mates. Role of Spetonsis in removal of toxic metal ons from the polutedeffuent have taken moreimpotanceinshisaregbecauseof thelr small size have ahigh surface areao-clumeratioand therefore provide-8lrge céntct are for metal binding. The biosorpton processes have been studied extensively using S plotnsis microbial biomass as basorbent for heavy metal ions removal because of ‘adherent advantage*of mass culation. Al of these desirable physioogical characterises and ‘applications have made S pltensis as amodel organism toundestandnivogen metabolism. Ths review eal with recent advances in the characterization of nitate assimilation and bioremediation poental ofS. pltensis: “Sercponding har Bally : cat. fanedyaoo con ¢ Oo co Inrodution Key words_ me aS Me Ria posession tng, ritrate-uiising fiamentous cyanobacteria best Gitoperon i= its high protein content (70% by dry weight) and has remarkable neutraceutical, Rita assinlation ‘sttvironmental and biotechnological signcance (Vonshak, 1997; Gumbo and Nesamvuri, Kploraee “Sexzoi7). There ae fw studies onthe inducion, actly, sabity and themotolerance of N- assimilatory enzymes in S. platensis (Jha ef al, 2007; Ali etal, 2008; Lochab eta, 2010 and +2014) butitis not understandable how the organism contrlits nitrogen metabolism to produce so much protein, The N-metabolc pathway and its regulation responsible for its high protein Publication Info contentremainto be well characterized atthe gene o protein level P 4: 07.10.2015, : fee tie Nitrate assimilation is the major process of nitrogen acquisition in non-N-fixing Revevised received: 19052017 cyanobacteria Guerrero etal, 1961). Accordingly the N metabolism andits regulation has been Accepiod ; 18.08.2017 E> specs _ 7 a Sen Email Fit :Locaaenl iat sian gers and gee css ina Cannes (BE, ch v0 de} Be pres represanthe pale ransrion unin ferent cyanobeceia. The locations of exra genes i he cstr have been shown by an inverse thang above te pnlagos (gene namenotshownber) T3].and 504 aminoacids. The deduced amino acd sequence of NrPy protein showed high sequence identity with that of Tachodesmium erjthraeum IMS1O1 (84%). The highest uence identities were found with Lyngbya sp. PCC 8106 and Tchodesmium erjttraeum IMS101 (77%). Characterization of genomic cones of otber nate assimiltory genes of S. PCC 7942 (Espinosa et a, 2006) by enhancing the 2-0G- )peplaensisreveaed separate ORFs for i186), gna (1422 dependent nicA-mediated uprequlatonofnicA operon. Due tothe ( lack of information regarding the reguators in S. platens their flinreqdangheablsnrenans tobe deseced nea Molecular characterization of nitrate assinilatny, pathway remains an explored area ofresearchin the non xing fiamentous cyanobacterium S. patensis, despite il nce in nutiional and environmental biotechnologyesThe “fist comprehensive characterization of the genes of ritate uptake, assimilation and regulation in S. platensis PCC T345.has been done, The uHlengh genes of nt, nar, nirkginAand gt and the regulator nicA have been doned, sequenced and their prylogeny, genomic organization and fanscriptional reguation by nitrate andits downstream metabolites have been determined (Lochab et al, 2014). The genes navB and nrP were found ina ‘single genomic done of 5.2 kb wih separate reading frames of 2.211 and 1,513 nucleotides, respectively, ving for proteins of 'bp) and gS (4607 bp), indicating the absence of operon \eetarhileture. The ntcA gene fom S-platnsis PCC 7345 showed ‘a high degree of simlarty with previously reported nicA gene ‘sequences from ther cyanobacteria, such as S.plafensis NES- 39 (69%), Lyngoya sp. PCC 8106 (96%), Leptelyngbya sp. PCC 7316 (90°) and Tchodesmium ertraeum IMS101 (89%). The hylogenetic. analyses. were done using deduced protein ‘sequences of al the ve N-assimiatory genes as wel as forthe regulator nc showed very high degre of aminoacid sequence similaty ofthe various genes of N uptake and assimaon ‘among diferent cyenobacteria adapted to diferent habitats (Fujsawaetal, 2010) Localization of the genes encoding NRT, NR and NiR in S. platensis: The genes ofriate uptake and assimilation areoten Cuslered as operons in cyanobacteria. The most common arrangement ofthe nitrate asimiaton genesis the operon nid- ‘Tablet: Genes related tonal assimlaton and its equation wth completely sequenced genome i S.psensis 'S.platenss POCTAS fresh water strain of feeyanobacteria) Sein Taapaaee NON, (wtABCOY nt) Engen NR nar) and NR (ic) Sensor! Regulators NO}, (ne), Zexoghsarate (2-06), nk inki ze Q.Feriduadin tat. ‘™tABCO-narB (ered the nr operon), The niroperon i esh water cyenabactera ke Synechococcus elongatus string PCC8901 and Anabaena sp. is comprised ofthe genes nik. ‘\MABCO-narB (Ohashi tal, 2011) (Fig 1A), whereas marine ‘cyanobacteria like Tichodesmium sp, have ntP in place of ‘tABCDin te operon 18) (Wang aa 2000; Ohashiet 2011) asthe NRT encoting gene. hough many cyanobacleral Genomes have the genes encoding NR NRT and NR ina single ‘operon there are other arrangements ao, The most common among these is separation ofthe gene enceing NR fom the coer genes. The ir gene is separated from nrtABCD-nar8 in ‘Synechocystis, PCCE03 (Fig 1) androm natP-nrBin the atne fcyarobacterum Syrechoroceus sp. PCCTOO2 (Fi. 10), Recent progress in genome sequencing projeds has ‘revealed mor complex arangements ofthe nite asiniaion genes. Lochab ta. (20'4) showed that S.platenss PCCT34S ‘ con me pene hasthe ntABCD gene custerin adationto the tanscion units é sonny pany So Canying nik and rr (Fg. 1). The anP and na awe Groaned together while ntABCD, nic, nA and gtSexstsin Ff Repulton tate asiaonins platens feparte denon leans, indicating thal 8. patensis desrot__ The rtteenaion ene plese cen be ented by wo ‘clowtve opsonarciecare typical ofohercynobacea Ts Steet nadanans Ammotacroe testy cheesey i in spt ofthe fact that belongs fo the same order ainzmesolniastcmisin trugherepcesontnen are Oscilatoricles as Trichodesmium, itis inleresting and significant iserthenceofnrate the Ove core nitrate asimiatory genes (ntP, thal S.plotensis PCC 7245 has both ypesoftranspartes, nas» M8214") areupoquatd well 28 ABCD, which are otherwise known fo be specic 0 Imatne ond freshwater organisms, respecvely, baring 2 few (Luque and Forchammer, 2008; Ohashi et al, 2011). This é 7 7 folect the evcubonryadapaion of. stonce at =D Rolé"of transcription factor-NteA : Characterisation of nfcA gene and is binding elements inthe promoter regions of nt P, cane eee an esae ‘FA, Gna, gS ofS. patensis pave the way fr the more detailed i senes ni, verb, Studies on the mechanisms of N regulation inthis organism The te ascanforniate assimilation genesinSpatensis Om 8 itogencontlmachansmmediatody nck etarceptony the basis of accumulating S. platensis genomic infgrmation, the <2ativaor belonging to CRP (cAMP receptor protein family Being ‘ealolndftaesssmictonissunmarodinfig2y, ttl fre achaton of many genes sated eae : and assimilation ofivoge, NcAs defined as global ntogen seeratontn ge abolts on the regulation ofitate equator Genero of a, 2001) 8 nee: genome algo assinilationinS. platensis v caniains 14 genes encoding cNMP-bindng domains, One ot Etfect of ammonium: ammoniuah iiition of Bika ma te OF Pea aay ‘Genes : Ammoniumashowedlear inhibitory Conserved gene sequence for a transcriptional regulator felons nciond gana Fotoage (FHS2WG oll 2010) The pronitisachatedby teks oe as" . a) peaciviiesot NRignd NRin S. platensis the consensus Sequence GTANGTAC-NaarTANGT, in which (Lochab et al, 2014). In non-N fining cyanobacteria, expression GTAN,TAC represents the NcA‘ining ma and equates ty ofthe nivale assinilaton genesis repressed bythe addon of ‘genes ‘of multiple metabolc and: devwlomn eeesestne ccroporleeratumant Raed sma dexeesson, aca ta MtcAactates the exprestonotin ggtmorl of arrnium rm he medium oes and ener, 2s well s of ther genes involved in ioe mate Herero eta, 2001). In Ndepleted cultures, the fal nthe ote tiles of he above enzymes i fntaraceniuted bythe 2 (Luque ef af on Te es at cae CN addition of ammonium tons. Therefore, in acdllion te cyancbacteriaby the clin oct, arated in caeboratng he wel known ammonium pression ofthe above 20), Maries of 2.06 WuoPas gereslenzymes inher cyoncbatera was aso nocd (Mur. Pastor et al, 2001), They may be explained in par by the ack of Operonarchitecture, Lochab et al (2014) have reported that cA induction of irate upleke and assimilation operates at tha highest levelLLochab eta. (2014) showed forthe frst time that tate ‘upregulates the gene expression of narB, nirA and nrfP in S. blatonsis. Their transcript levels dropped drastically when the Cultures were harvested and resuspended in Nfree media and recovered when irate was reintroduced, Similarly, nitrate induction was also found atthe enzyme activity level for NR and NNR. Eafier studies showed that nitrate causes induction of NR activity based ona different strain ofS. patensis (Jha eal, 2007) "not only confims ita the transcriptional leveln the type strain PCC7345, butalso provides the frst evidence foritrateindueton ‘of mutiple genes ofitate assimiatory pathwayin S. platensisor in any other filamentous, non-N-fixing, nitrateuilizing {yanobacterium. Another group has also shown recent thatthe activity of NR, NiR, GS and GOGAT increased with increasing Concentrations of sodium nitrate (Esen and Urek2044), In a Similar study they have shown that highest growthgpigmeritand ‘metaboite levels and enzyme activites of nitrate assimilating enzymes in Spiruina platensis were achieved’ by,supplying ‘ammonium nitrate and ron Esen and Urek, 20*8)athe presence ‘of carbon in the medium is one ofthe mosLimportant factors for the optimum growth and enzyme a InSthis organism (Chauhan etal, 2013), /, Effects of downstream N metabolites, nitrite, ammonium and glutamine using N-depleted cuitiresis the presence or sbsence ofnitrate have also been studied byLochab etal (2014). ‘Two pattems of response to nitrite emerged, depending on the ‘genes involved. Inthe case ofnPand nirA, tite enhanced ther {ene expression to levels comparable to that ofnitate alone, ora ‘Combination of ntite and nitrate, Incase ofnaB, nite alone had no inductive effect and instead inhibited nitrate induction of gene expression when provided in combination. Glutamine had no efiect at the RNA evel while ammonium had significant inhibitory «effect on the expression of these genes, either on its own or on 5 nitrate response, However, atthe level of enzyme activity allthree ‘downstream N metabolites had inhibitory effects on NR and NiR activites, ‘The only nitrite regulation known so far ‘operates through nicB, a lysR-type transcriptional activator that upregulates the :nirA operon in S. elongatus strain PCC 7942 (Aichi et al, 2004). It also revealed that nite induces nrtPand nr gene expression in ‘amanner similar to Whrate, butinhibits induction of narB by nitrate, Upregulation of operate i known in Symechecoras ‘Sp. strain PCC 7942 and:Plectonema boryanum (Kikuchi ef al., 1996). Nitrite inhibition of nitrate induction is ‘not known in non-N,- {xing flamentousgyanobactria, because nitrate induction Iself was not known earlier. However, this is a ‘well-known Phenomenon} ‘blants lke maize (Raghuram and Sopory, 1999) and tice (Ali eta 2007). tet factor sitolated withthe regulation of nite assimilating genes: on-N, metabolites in S, platensis : The studies*are, in Progress to understand the mechanism of regulation ofgene exxression by nicA and other rgulatrs, cB, PlLand LysR (ntcBIK489170, Pik. 3X431864, lysRUX431863) Which have been cloned recently from S. Platensis (Lochab etal, 2044), The ole of i andppX equator inenhancing the. OG, apendent ntcA-mediated ‘upregulation of nirA operon has been showmin unicellarnonfsing cyanobacterium § elongotg 7942 (Espinosa ef a, 2006). However, none of hese wegulators have been studied at ‘any level in S. platensis, and erelore, her rol in regulating Neetabolsm remain tee 00d, Salient features of nitrate assimilatory enzymes of S, platonsis: higher specific activities and thermostability : Purified nitrate assimilating enzymes are used for environmental testing (Campbell etal, 2006), nitrate decontamination (Mellor et al, 1992), biosensors (Chen etal, 2008) and other applications (Angeby et al, 2002), while whole organisms are useful for bioremediation (Hu eta, 2000), However, the poor stability and {hermosenstvty of these enzymes represents a major imitation in their widespread use. S. platensis, whose N-metaboic Pathwayis similar to higher plants, produces tenfold more protein in comparison torice (O12 sata) ndicating a higher capacity fornitrogen use efficiency and nitrate utizaionkemoval ‘Ate al (2008) showed that the N-assimilating enzymes, nivale reductase (NR), nite reductase (NR) and glutamine synthetase (GS) ofS. platensishave higher specific actives and Sabilties atroom temperature than those of rice. They carried out ‘in vitr enalysis in crude extracts revealing that the higher specific activities (¢-6 fold) and halves (12-44 fold) ofthe NR, NiR and Sin S. patensis which ely contibute to the far higher nate ulizing capacity of Spruina as compared to that of oe leaves, ‘Thisis futher enhanced by the higher stabilities of NR and GS in SS platensis at room temperature, assuming that the in vitro6 stabilities of these enzymes in crude extracts are a reflection of theirlongerhal-ves in vivo, thas been also shown that S. platensis could be an attractive natural source of NR for environmental, diagnostic and other applications (Chuntapa etal, 2003), considering that thas (ver 3fold higher specific activity and almost6 fldhigher stability than that ofrce, with ahal-ife of over 22 hrs atroom temperature in viro. The specific activity of NR in S. platensis isnot only sh- fold higher than that of rice, but the fold dference between NiR ‘and NRis far higher nS. platensis (8 fold) than in ice (5 fold) (Al et al, 2008). This may reflect the abiliy of S. platensis to effectively prevent the accumulation ofnitt, whichis toxic tothe cell. Lochab et a. (2009) have assessed the thermotolerance ‘OfNR, NiR, and GS of S. platensis compared with those of rice in crude extract in vito, since higher plants are tratonal sources for these enzymes. When the extracts were pre-exposed to 80°C {orth the tree S.platensis enzymes retained higher activity by 3.4, 1.7 and 3.7 fold, respectively than corresponding enzymes in ‘ice. These results revealed that S. platensis enzymes have relatively higher thermotolerance which renders S. platensis an atracive natural source of sturdy enzymes. for varous applications, Bioremediation : Environmental contamination by toxic metals isa serious problem worldwide. Toxicmetals ae often discharged by a number of industial processes and this in tum to the Contamination of sol, freshwater and marine envronmentn Due to their non-biodegradabiliy and persistence these mitals Gan accumulate inthe environment elements such as foodchaingand may thus pose a signiicent danger to human Reali, Decontamination of heavy metalsin the soll and waterhas been & challenge for along time. Number of efcent mettads such as evaporation, on exchange, electroplating, membrane prosesses ‘or precipitation etc. has been developed forthe removal ofheavy ‘metals particularly from liquid wastes ¢ Microorganisms have evolved varioug proc88es such as transport across the cell membrane, biasortion ja cell walls and entrapment in extracellular capsules, precpitalion, omplexation, and oxidation reduction reactions taprotea themselves rom the harmful effects of heavy metals pfesent in environment. thas been proved that they are capable oFadsabing heavy metals from aqueous solutions, especially for the metal concentration below 50 maf’ (Lu and Wikins, 1995). The metal-inding capacities of several biological materials have been identified to be very high, inducing marine algae, fungi and yeasts Microorganisms can also accumulate a wide range of metal species. Metal ons can bind tothe biomass by ether non ving {Ciosorption) olin (bioaccumulation) technique, Biosorpon i ‘mostly useful fr the waste wate treatment and bioaccumuaton Q Faridualin etal finds the application of microalgae. Selection of biomass (microalgae), pre-reatment, immobilzation etc are some of the procedure involved in the metal recovery by biosorption, The biological materials can accumulate heavy metals from waste Water either metabolically or by physicochemical pathway. The provess of biosorption ullizes the above mentioned capacity of ‘accumulating heavy metals (Fourest and Roux, 1992). Several studies have beef dane to look for the more efficient and economical | osorbents from the microbial sources (Chang et alyig97"Ring eta, 2007). Mcroalgal species have become matéfia of @hoice because oftheir greater ability to bind metals by vitdeoftheresence of some functional groups in the cel wall cnttugns (Yuet al, 1999; Schiewer and Volesky 2000), Thé Second technique, bioaccumulation, can be defined a thetransfeFtexganic or norgaricpoltans into the interior of livingcels (Barron, 1985). Asarsul ofthis transfer there can be anffecurlation of toxic elements inthe organism. Tis process can be USadfo remove the nuents from the treated effuents (nitrates, phosphates, sulfates, organic and inorganic carbon ompounds). Ehrich (1986) hes reported the use of artificial ond COntaning photosynthetic microorganisms to reat mining Ieahates. Bioaccumulation occurs when an organism takes up a Yoxicsubstance rom the environment at rte higher han hat of the|J685 of the substance. This may pose a greater risk of wl esi on neh ys 19 Bioremediation of heavy metal by S. platensis: The industial development and urbanization have caused polution of water resources. One ofthe major pollutant in the water bodies isthe Presence of heavy metals. The presence, accumulation and magnification of toxic metas lke Cu, Zn, Cd, Cr, Pb and Ni eto. has imposed a greater threat to the ecology by causing imbalances in aquatic ecosystems (Pergent and Pergent-Marini, 1989). Biological materials have been proved to be capable of adsorbing heavy metals from aqueous solutions, especialy for themetal concentration below { mgi, S.platensis is curently being investigated as a potential Bioremediation agent forthe removal ofheavy metals from waste water. miningandoterindustal eluents (Chen and Pan, 2008), When the toxic effects of metals were investigated, Spiruina was {ound to havea threshold level of about 30 uM for Cu, Zn and Pb Gund Zn appeared to havea direct effect onthe photosynthetic Bathway, thereby causing a rapid decne in cell growth, Lead on [he hher hand seemed to afc surface properties and hence binding meta ions, as well Metals. S. platensis does £28 2 preciptation agent asi is able to maina Surrounding medium, possibly through the