The sensitivity of two Monoraphidium species to zinc: their possible future role in bioremediation

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1 Int. J. Environ. Si. Tehnol. () : 66 DOI.7/s ORIGINAL PAPER The sensitivity of two Monorphiium speies to zin: their possile future role in ioremeition I. Bási Z. Novák M. Jánószky V. B-Béres I. Grigorszky S. A. Ngy Reeive: Jnury / Revise: 8 April / Aepte: 8 July / Pulishe online: July Islmi Az University (IAU) Astrt Effets of zin on growth, ell morphology, oxitive stress responses n zin removl tivity of two ommon phytoplnkton speies, Monorphiium pusillum (Printz) Komárková-Legnerová n Monorphiium griffithii (Berkeley) Komárková-Legnerová were investigte t onentrtion rnge of. 6 mg l - zin. Cell ensities n hlorophyll ontent erese ompre with ontrols in ultures of oth speies, effetive onentrtions using % growth inhiition within 7 h on the sis of ell numers were.69 n.6 mg l - zin for M. pusillum n M. griffithii, respetively. Chnges in ell morphology n elevte lipi peroxition levels ppere in zin-trete ultures of oth speies, ut only t higher ([ mg l - ) zin onentrtions. The most effetive zin removl ppere t n mg l - zin onentrtion for M. pusillum n M. griffithii, respetively. Remove zin is minly oun on the ell surfe in the se of oth speies. This stuy provies new t for the zin tolerne n zin removl ility of the green lge M. pusillum n M. griffithii n shows tht green lgl speies ommon in surfe wters oul hve zin tolerne n zin-ining ilities, whih mkes them fesile in tretment of wters ontminte with mg l - zin. I. Bási (&) Z. Novák I. Grigorszky S. A. Ngy Deprtment of Hyroiology, University of Dereen, Egyetem tér, Dereen, Hungry e-mil: istvn.si@gmil.om M. Jánószky V. B-Béres Environmentl Protetion n Nture Conservtion Authority, Trns-Tisznin Region, Htvn u. 6, Dereen, Hungry Keywors Common green lgl speies Zin Growth inhiitions Morphologil hnges Lipi peroxition Removl Introution It is well known tht the presene of hevy metls in quti environments uses serious mge to quti life. These metls n reue the effetiveness of purifition proesses uring iologil tretment of wstewter y mging miroorgnisms (e.g., teri, protozo). Lots of metl slts re solule in wter n onsequently nnot e seprte y physil seprtion (Hussein et l. ). Physio-hemil methos, suh s evportive reovery, hemil oxition or reution, hemil preipittion n filtrtion, eletrohemil tretment, ion exhnge, or memrne tehnologies hve een wiely use to remove hevy metl ions from wstewter. The min prolem of these proesses is tht they my e ineffetive n/or rther expensive, when the onentrtion of issolve metl ions is lower thn mg l - (Volesky 99, ). Biologil methos for the removl of metl ions my provie n ttrtive n ost-effetive lterntive to physio-hemil methos (Kpoor n Virrghvn 99), sine mny miroorgnisms re le to remove metl ions from quti solutions y metolilly tive (ioumultion) n/or pssive (iosorption) proesses (Chojnk ). Alge re in the fous of sientifi interest euse of their mss proutions (looms) n possile toxi properties (Perovi et l. ; Vss et l. ; Zhng et l. ). Alge ply more n more importnt roles in wie rnge of eonomil n environmentl issues in the lst few yers, oviously they re lso involve in wstewter tretment n sequestering of toxi, essentil or preious

2 6 Int. J. Environ. Si. Tehnol. () : 66 metls (Munoz n Guieysse 6; e Bshn n Bshn ; Ds ). Algl ell wll hs key role in metl removl, espeilly in iosorption proesses, whih re lso importnt in metl ining of living iomss. Cell wll of green lge (Chlorophyt) is ompose minly from ellulose, n high perentge of the ell wll is struture from glyoproteins, proteins oun to polyshries (Romer et l. 6). Over the metl-ining pility of ell wll, mirolge n some fungi re le to proue pepties ple to in hevy metls. These moleules, s orgnometlli omplexes, re further prtitione insie vuoles. They filitte the ontrol of the ytoplsmi onentrtion of hevy metl ions, thus preventing or neutrlizing the potentil toxi effets (Coett n Golsrough ). It is iffiult to eie whether lgl iomss is etter or not thn others (teril or fungl). The results of vrious stuies re not esy to ompre iretly ue to the ifferent t presenttion of vrious uthors (Wng n Chen 9). Nevertheless, stuy of photosyntheti orgnisms is justifile, euse there re generlly epte proeures in wstewter tretment tehnology, in whih the presene of these orgnisms n e onsiere, or esy to implement (Perles-Vel et l. 6). Over the well-stuie spets of toxi hevy metls (Cr; C; P), effets of tre elements (Fe; Cu; Zn) on the growth of phytoplnkton speies still hve een in the fous of interest for long time. Zin is n essentil tre element, neessry for living orgnisms (Sugrmn 98; Broley et l. 7; Prs8). Zin is foun in gret numer of speifi enzymes (US NRC ), serves s struturl ion in trnsription ftors n is store n trnsferre in metllothioneins (Cotton et l. 999). Zin is known s the seon most unnt trnsition metl fter iron in orgnisms, n it is the only metl tht ppers in ll enzyme lsses (Broley et l. 7). Although zin is essentil, exess of this element n e toxi. Exessive sorption of zin suppresses opper n iron sorption (Fosmire 99). Free zin ion in solution nehighlytoxi(eisler99),nmnytinthe literture show tht just miromolr mounts of the free ion use the eth of some orgnisms (Muyssen et l. 6). Zin resistne of mirolge vries within very wie rnge (from severl mg l - to severl hunres of mg l - (Trvieso et l. 999; Nishikw n Toming ; Lietl.6; Monteiro et l. 9,, ). In generl, it n e si tht mny mirolge n in signifint mounts of zin (Brinz et l. 7). The ove-mentione ell wll properties n phytoheltin proution ilities re ehin this phenomenon. Similrly to other metls, elevte zin levels my use morphologil hnges or oxitive stress in miroorgnisms, inluing lge (Gol et l. ; Morinn Coste 6; Tripthi n Gur 6; Li et l. 6). Effetive onentrtions epen on the tolerne/sensitivity of lgl speies. Lortory stuies showe tht zin pretretment oul enhne the tolerne of lge towr toxi metls others thn zin (e.g. C, Hg, Cu, P; Lehmnn et l. 998; Tsuji et l. ), so zin-tolernt speies oul e goo sujets of iosorent, metllothionein or phytoheltin reserh (Anre et l. ; Perles-Vel et l. 6). To tke these fts into onsiertion, it is resonle to stuy the metl tolerne n removl ility of inln lgl speies, whih oul hve relevnt potentil in wter tretment. Monorphiium speies re pplie for severl iotehnologil events (Fujii et l. 9; Gttullo et l. ; Yng et l. ; Yu et l. ). There re few t out metl tolerne n metl removl ility of Monorphiium speies (Hornstrom et l. 99; Plmieri et l. ; Tkmi et l. ; e Queiroz et l. ), ut oring to our knowlege, there re no t out their zin tolerne. Although the verge zin onentrtion rnges from to lg l - in Hungrin surfe wters, zin is one of the most unnt metl ontminnt in wstewters in Hungry. Zin onentrtion n e inrese in onsequene of mining or inustry (eletroplting plnts, phrmeutil ompnies, pint ftories), moreover househol n trffi tivities (Horváth n Gruiz 996; Óor et l. 998; Bir et l. ). The im of this stuy ws to investigte the effets of zin on growth, ell morphology, oxitive stress responses, n zin removl tivity of phytoplnkton speies, whih re ommon in Hungrin surfe wters. Green lge Monorphiium pusillum n Monorphiium griffithii were trete with zin in wie onentrtion rnge (. 6 mg l - )to get etile t of the sensitivity n zin removl ility, herey the possile future role of these wiespre lge of surfe wters in ioremeition. Experiments were rrie out in, on the Deprtment of Hyroiology, University of Dereen, Dereen, Hungry. Mterils n methos Strins, ulturing onitions, n experimentl esign Monorphiium pusillum (Printz) Komárková-Legnerová (ACCDH-UD9) n M. griffithii (Berkeley) Komárková-Legnerová (ACCDH-UD8) re ulture in the Algl Culture Colletion of Deprtment of Hyroiology, University of Dereen (ACCDH-UD). Cultures re mintine in Jworski s meium (ph: 7 7.), in -ml Erlenmeyer flsks ( ml ulture per flsk), ule with

3 Int. J. Environ. Si. Tehnol. () : 66 7 sterile ir, t C, uner ontinuous irrition. Tretments were hieve within the sme onitions, in -ml Erlenmeyer flsks ( ml ulture per flsk). Control ultures ontine no e zin, n ZnSO (Renl, Hungry) ws e to the trete ultures to reh.,.,.,.,.,,.,,,, 8, n 6 mg l - zin onentrtions. Negtive ontrols (meium? zin, without lge) were pplie for ll zin onentrtions to hek the mintenne of the metl onentrtion e to the ulture meium uring the timefrme of the experiments (7 ys). All experiments were one in triplites. Growth of the ontrol n trete ultures ws followe y ounting the ell numer n mesuring the hlorophyll ontent n ry mss of the ells. Cell numers were ounte oring to the Europen Stnr EN (6) using n Olympus BX fluoresent mirosope t 9 mgnifition. Chnges in ell morphology were followe y the sme mirosope t,9 mgnifition, on ys, n 7 of the experiments. For hlorophyll ontent n ry mss mesurements, 6 ml smples were ollete ily, entrifuge ( min,, rpm, Miro-entrifuge Type-), n fter removl of superntnts, pellets were lyophilize (Christ Alph LD plus) n weighe (Ohus Aventurer TM Pro). Chlorophyll ontents were mesure from the weighe smples pplying the methnoli extrtion metho of Felföly (987). Spetroqunt Phro spetrophotometer ws use for the spetrophotometri mesurements. Mesurement of hnges in lipi peroxition Lipi peroxition levels were mesure in signifintly ffete, ut still onsiere s living ultures n were expresse s the mount of thiorituri i retive sustnes (TBARS), following the metho of Verm n Duey (). Smples of 6 ml were ollete t ys n fter 7 h, smples were entrifuge ( min,, rpm, Miro-entrifuge Type-) n fter removl of superntnts, pellets were use for the retions. Briefly: pellets were resuspene in ml of. % (w/v) -thiorituri i (TBA) in % (w/v) trihloroeti i (TCA). Mixtures were inute t 9 C for min, n then, they were quikly oole in ie th n entrifuge ( min,, rpm, Miro-entrifuge Type- ). The sorne of superntnts ws mesure t n 6 nm (for orretion of unspeifi turiity). TBA solution in TCA serve s lnk. Amounts of TBARS were lulte to unit of ell numer using the extintion oeffiient of.6 9 M - m -. Results re given in perentge, n % is the vlue tht ws mesure in ontrol ulture t the eginning of the tretments. Mesurement of the totl mount of remove zin Zin ontents of the ell-free superntnts were mesure oring to the Hungrin Stnr MSZ 8-:6. y Atom Asorption Spetrometry (AAS, UNICAM 969 with GF9 plus oven trnsformer n FS9 plus oven smple try unit), in the lortory of Environmentl Protetion, Nture Conservtion n Wter Authority, Trns-Tisznin Region. Smples of 6 ml from signifintly ffete, ut still onsiere s living ultures, were ollete t ys,, n 7 of the experiments n entrifuge ( min,, rpm, Miro-entrifuge Type-) n zin ontents of superntnts were mesure. Ten-fol ilute smples of ll were mesure, n ll mesurements were one in triplite. For lirtion, MERCK ICP IV multielement stnr ws use. Zin preipittion in negtive ontrols (meium? zin, without lge) were mesure n lulte y the sme wy. Amounts of zin remove y the ells were lulte se on eresing zin ontents of the superntnts, orrete with the mount of preipitte zin. Results were given to unit of ry mss. Mesurement of intrellulr level of zin Intrellulr zin ontent ws mesure oring to the metho esrie y Tripthi n Gur (6). To efine intrellulr zin ontent, 6 ml smples from signifintly ffete, ut still onsiere s living ultures, were ollete t ys,, n 7 of the experiments. The lgl suspensions were entrifuge ( min, rpm, Miroentrifuge Type-), n fter removl of superntnts, pellets were wshe in ml of mm EDTA for min to remove ll of the surfe-oun zin without ny effet on the internl zin ontent. After entrifugtion, pellets were trnsferre to ml of igestion mixture ontining HNO (7 %), H O ( %), n eionise wter t :: rtio (Btes et l. 98). Digestion ws performe on hot plte t 8 C until the solutions eme olorless. The resiues were issolve in % (v/v) nitri i n were juste to finl volume of ml. The smples were nlyze for zin ontent y AAS. Sttistil nlyses One-wy ANCOVA ws use to etermine the signifines mong the teneny ifferenes of growth urves of ontrol n trete ultures (Zr 996; Hmmer et l. ). For sttistis of morphology hnges, oxitive stress n zin removl t were sujete to nlysis of vrine (two-wy ANOVA). Tukey s test s multiple omprison proeure ws use to show the signifint ifferenes etween mens t the % level.

4 8 Int. J. Environ. Si. Tehnol. () : 66 () Cell numer ( 6 ml - ) () Cell numer ( 6 ml - ) M. pusillum Zn (Control). Zn. Zn. Zn Zn (Control). Zn. Zn Zn. Zn 6 8 () Cell numer ( 6 ml - ) () Cell numer ( 6 ml - ) Zn (Control).. Zn. Zn.6. Zn 6 8 M. griffithii Zn (Control).. Zn. Zn.6 Zn. Zn 6 8 (e) Cell numer ( 6 ml - ) Zn (Control) Zn Zn Zn Zn (f) Cell numer ( 6 ml - ) Zn (Control) Zn Zn Zn Zn (g) Cell numer ( 6 ml - ) Zn (Control) 8 Zn Zn 6 Zn (h) Cell numer ( 6 ml - ) Zn (Control) 8 Zn Zn 6 Zn 6 8 Time (ys) 6 8 Time (ys)

5 Int. J. Environ. Si. Tehnol. () : 66 9 Fig. Chnges of ell numers in ontrol ultures n in ultures with ifferent initil zin onentrtions of M. pusillum n M. griffithii green lge. Men vlues (n = ) n stnr evitions re plotte, n ifferent lowerse letters inite signifint ifferenes (P \.) Results n isussion Growth of the ultures Both in M. pusillum n M. griffithii ultures,.,., n. mg l - zin use slight, ut not signifint inrese in ell numer ompre with ontrol ultures (Fig., ). Zin onentrtions from. to mg l - i not use signifint ltertions in ell numers (Fig., ). Trete ultures iffere signifintly from ontrol ultures only from n mg l - zin in the se of M. pusillum n M. griffithii, respetively (P \.; Fig. e, f), so the sensitivity of the two speies ws ifferent on the sis of ell numer. There were no signifint ifferenes mong the effets of zin in onentrtions higher thn mg l - (8, n 6 mg l -, Fig. g, h). Growth of the ultures ws strongly inhiite, ut it hs to e note tht M. pusillum showe slight inrese in ell numers even t the presene of 6 mg l - zin. As it ws oserve in the se of ell numer hnges, M. pusillum ws less sensitive to zin thn M. griffithii lso on the sis of hlorophyll ontent. Lower zin onentrtions (. mg l - ) i not use signifint ltertions in hlorophyll ontent of M. pusillum ultures, ut signifintly lower hlorophyll level ws mesure in M. griffithii ultures trete with. mg l - n higher zin onentrtions (Fig. ). While there ws etetle inreses in ell numers t high zin onentrtions, hlorophyll ontent remine t low level or erese t higher zin onentrtions (from n mg l - in the se of M. pusillum n M. griffithii, respetively, Fig. e h). Tle summrizes the tul zin onentrtions n the lulte mount of preipitte zin in negtive ontrols (meium? zin, without lge) for signifintly ffete, ut still onsiere s living ultures. Growth urves n EC vlues showe tht inhiition of hlorophyll synthesis ourre t lower zin onentrtion thn the inhiition of ell ivision (Figs., ; Tle ). As fr s we know, these re the first t out zin tolerne of these Monorphiium speies, whih n e hrterize s zin-tolernt ones. Comprison of literry t with eh other n with our results my not e entirely urte euse of ifferent mesure growth prmeters n exposition times, yet it seems tht the zin tolerne of the stuie Monorphiium speies is in the meium rnge in omprison with the zin tolerne of some other green lge. Growth rtes of extremely zin-tolernt speies, suh s Chlorell vulgris n Seneesmus utus strin, i not iffer from ontrols in the presene of n mg l - zin, respetively (Trvieso et l. 999). EC ws mg l - zin in the se of Chlmyomons iophil strin (Nishikw n Toming ). On the other hn, there re speies muh more sensitive to zin thn the ones in our stuy. Tripthi n Gur (6) oserve tht.6 mg l - zin inhiite the growth of Seneesmus strin within 8 h. Zin onentrtion of 6. mg l - use growth inhiition n oxitive stress in Pvlov viriis strin (Li et l. 6). Chnges in ell morphology Morphologil hnges were followe in signifintly ffete, ut onsiere s living ultures (from to mg l - tretments). Zin tretments use hnges in ell morphology of oth stuie speies, ut the hnges were ifferent. The hloroplsts n lter the whole ells of M. pusillum eme frgmente, n the ells preipitte n sunk to the ottom of the flsks. Liner orreltion ws oserve etween these phenomen n the inresing onentrtion of zin. In the se of M. pusillum, the numer of eforme ells in ontrol n in trete ultures iffere signifintly from eh other in every se (P \., Fig. ). Cell morphology hnges of M. griffithii were rely etetle: M. griffithii ells eme slightly more swollen thn in ontrol ultures, floultion lso ourre. Signifint ifferenes (P \.) were mong numers of eforme ells in ontrol n trete ultures of M. griffithii, ut numers of eforme ells i not iffere signifintly from eh other, only in n mg l - tretments (Fig. ). Morphology hnges of lgl tx s response to zin ontmintion strongly epen on eotype of the tx (Pwlik-Skowronsk ), on the onentrtion of zin n on the time of exposure (Gol et l. ; Morin n Coste 6). In sensitive speies (e.g., filmentous green lge s Stigeolonium tenue or ertin itoms), these morphologil hnges pper t low zin onentrtions (Pwlik-Skowronsk ; Gol et l. ; Morin n Coste 6). It is known tht metls le to inhiit norml ell ivision ue to ining to sulfhyryl groups, whih re importnt in regulting ell ivision (Visviki n Rhlin 99). In the se of the stuie Monorphiium speies, ell morphology hnges ppere only from reltively high zin onentrtion ( mg l - ). There ws etetle inrese in ell numer in M. pusillum ultures trete with mg l - zin, this oul e the reson of highest unne of norml ells. Inresing mitoti normlities use y zin oul e one of the resons of this phenomenon. Morphologil hnges seeme not to e highly

6 6 Int. J. Environ. Si. Tehnol. () : 66 () Chlorophyll- ontent (ug ml - ) () Chlorophyll- ontent (ug ml - ) 6... Zn (Control). Zn.. Zn. Zn M. pusillum... Zn (Control). Zn.. Zn Zn.. Zn 6 8 () Chlorophyll- ontent (ug ml - ) () Chlorophyll- ontent (ug ml - ).... Zn (Control). Zn. Zn. Zn M. griffithii Zn (Control). Zn. Zn Zn.. Zn 6 8 (e) Chlorophyll- ontent (ug ml - ) 6.. Zn (Control) Zn Zn. Zn Zn.. e 6 8 (f) Chlorophyll- ontent (ug ml - ). Zn (Control) Zn Zn Zn Zn (g) Chlorophyll- ontent (ug ml - ) 6... Zn (Control) 8 Zn Zn. 6 Zn. 6 8 Time (ys) (h) Chlorophyll- ontent (ug ml - ). Zn (Control) 8 Zn Zn 6 Zn Time (ys)

7 Int. J. Environ. Si. Tehnol. () : 66 6 Fig. Chnges of hlorophyll ontents in ontrol ultures n in ultures with ifferent initil zin onentrtions of M. pusillum n M. griffithii green lge. Men vlues (n = ) n stnr evitions re plotte, n ifferent lowerse letters inite signifint ifferenes (P \.) speifi or hrteristi to zin tretment in the stuie green lge, ontminnts ffeting ell ivision other thn zin, my use similr morphologil hnges. Chnges of lipi peroxition levels Oxitive stress responses were followe in signifintly ffete, ut onsiere s living ultures (from to mg l - tretments). The mount of TBARS inrese in proportion to the inresing zin onentrtion in M. pusillum ultures. There were no signifint ifferenes etween ontrol n mg l - tretment. The other tretments iffere signifintly from ontrol, ut not from eh other (Fig. ). TBARS levels i not iffere signifintly in ontrol n in mg l - zin-trete M. griffithii ultures, ut signifint ifferenes were oserve etween ontrol n mong the other tretments (P \.). The highest TBARS onentrtion ws mesure t mg l - zin tretment (Fig. ). n mg l - initilly e zin use signifintly higher inrese in TBARS levels in M. griffithii ultures thn in M. pusillum ultures (P \.). M. griffithii seeme to e more sensitive from enzymologil point of view: mg l - initilly e zin use % inrese in the mount of TBARS within 7 h ompring to ontrol. This vlue is..-fol higher thn tht of M. pusillum. Alge show gret iversity in oxitive stress responses to zin, s it is inite y our results n literry t. There were etetle inresing mount of TBARS in Seneesmus sp. lrey from lm zin (*. mg l -, Tripthi n Gur ). Zin ws oserve to use n inrese in glutthione pools, in prtiulr of glutthione isulfie (GSSG), using erese of its reox rtio, i.e., to use oxitive stress in Enteromorph speies lrey from. to. mg l - (Mle et l. 6). On the other hn, lipi peroxition levels in Chlorell pyreonios inrese only from lm zin (* mgl - ; Vvilin et l. 998), or from 6. mg l - in P. viriis (Li et l. 6). Tle EC vlues lulte to ell numer n to hlorophyll ontent fter n 7 ys of exposition EC mg l - Zn Green lge use in our stuy n e regre s not sensitive to zin from oxitive stress spets, n mount of TBARS reveling enhne lipi peroxition inrese only from reltively high (C mg l - ) zin onentrtion. Removl of zin Bse on ell numer, ys (7 h) Bse on ell numer, 7 ys (68 h) Bse on Chl ontent, ys (7 h) Bse on Chl ontent, 7 ys (68 h) M. pusillum M. griffithii Zin removl ws followe in signifintly ffete, ut onsiere s living ultures (from to mg l - tretments). Both speies were le to remove notle mount of zin y the en of the experiments (Fig. ; Tle ). In the se of M. pusillum, zin removl ility iffere signifintly in the ifferent tretments n t ifferent exposition times (P \.). The highest removl tivity ppere in the mg l - zin-trete ultures on the 7th y, 9. mg g - zin ws remove (Fig. ), whih ment.8 % of the initil zin ontent (Tle ). A signifint mount of zin oun quikly t the eginning of the experiments in, n mg l - zin-trete ultures. There ws no signifint orreltion etween the zin onentrtion, exposition time, n the mount of remove zin (Fig. ). Removl ility of M. griffithii ells ws ifferent: lthough signifint ltertions ourre in zinining tivity of M. griffithii in ifferent trete ultures (P \.), the most mount of zin per unit of ry mss ws oun in the mg l - trete ultures, on the r y (9.7 mg g -, Fig. ). Highest removl pity s perentge of initil zin ontent ourre in the sme ultures on the 7th y (8 %, Tle ). As it ws oserve in M. pusillum ultures, signifint mount of zin oun quikly t the eginning of the experiments in, n mg l - zin-trete M. griffithii ultures. The results show tht only smll prt of totl mount of remove zin Tle Atul zin onentrtions n the lulte mount of preipitte zin in negtive ontrols (meium? zin, without lge) fter n 7 ys of exposition Conentrtion of e Zn (mg l - ) Atul onentrtion of Zn (mg l - ) on the r y Preipitte Zn (mg l - ) on the r y Atul onentrtion of Zn (mg l - ) on the 7th y Preipitte Zn (mg l - ) on the 7th y

8 6 Int. J. Environ. Si. Tehnol. () : 66 is intrellulr in the se of oth speies (Fig., ). There were no signifint effets of zin onentrtion n exposition time on intrellulr zin ontent (Fig., ). The results show tht removl pity of M. pusillum ws the highest t mg l - zin onentrtion on the 7th y, while in the se of M. griffithii mximl zin removl ppere on the r y in mg l - trete ulture. This phenomenon is just the opposite of the oservtions of Tripthi n Gur () or Monteiro et l. (, ), they esrie tht the highest zin mss removl ourre in Seneesmus ultures expose to the highest metl onentrtion. On the other hn, Omr () lso oserve erese in the mount of oun zin t higher onentrtions. It seems tht there ws strong primry ining of zin (t mg l - ), followe y slower seonry ining. This oul explin why the mount of remove zin inrese signifintly to the r n 7th ys in mg l - zin-trete Monorphiium ultures. The signifint erese in zin iosorption in mg l - trete ultures my inite tht there is some () Rtio of morphologilly hnge ells (%) M. pusillum e i h k j f g (Control) () Rtio of morphologilly hnge ells (%) M. griffithii g e f (Control) Conentrtion of initilly e Zn (mg l - ) Conentrtion of initilly e Zn (mg l - ) y r y 7th y y r y 7th y Fig. Rtio of norml n eforme ells in ontrol n in zin-trete M. pusillum () n M. griffithii () ultures on y,, n 7 of ultivtion. Men vlues (n = ) n stnr evitions re plotte, n ifferent lowerse letters inite signifint ifferenes (P \.) () M. pusillum () M. griffithii TBARS (%) *, *, TBARS (%) (Control) (Control) Conentrtion of initilly e Zn (mg l - ) Conentrtion of initilly e Zn (mg l - ) hours 7 hours hours 7 hours Fig. Lipi peroxition levels expresse s the mount of TBA retive sustnes (%) in ontrol n in zin-trete M. pusillum () n M. griffithii () ultures on y n fter 7 h of ultivtion. Men vlues (n = ) n stnr evitions re plotte, n ifferent lowerse letters inite signifint ifferenes (P \.)

9 Int. J. Environ. Si. Tehnol. () : 66 6 () M. pusillum () M. griffithii Remove Zn (totl, mg g - ) e f g i Remove Zn (totl, mg g - ) e f g h h i () Remove Zn (intrellulr, mg g - ) M. pusillum () Remove Zn (intrellulr, mg g - ) M. griffithii Conentrtion of initilly e Zn (mg l - ) y r y 7th y Conentrtion of initilly e Zn (mg l - ) y r y 7th y Fig. Totl (, ) n intrellulr (, ) mount of remove zin (mg g - ) in zin-trete M. pusillum n M. griffithii ultures, on y,, n 7 of ultivtion. Men vlues (n = ) n stnr evitions re plotte, n ifferent lowerse letters inite signifint ifferenes (P \.) mehnism involve in zin removl, whih moify the ining sites of the ell wll of vitl ells, similrly to heting or rying proesses (Monteiro et l. ). A prtil isruption of struturl omponents in the ell wlls owing to high zin onentrtion is presumle, whih les to protein enturtion n is thus responsile for the erese in numer of the funtionl sites ville to intert with zin ions (Monteiro et l. ). Proof of this hypothesis requires further experiments. The results show tht lrger proportion of remove zin is oun y sorption onto the ell surfe in the se of oth stuie strins. Only smll inreses in intrellulr zin were oserve with inresing mounts of zin in the ulturing mei, whih is in orne with the t of other stuies (Hssler et l. ; Monteiro et l., ). In ontrst, in some ses umulte quntity of metls oul e higher thn sore, s for ertin C. vulgris strins or other ioumultors (Nor et l. 7). As in the se of growth, omprison of the literture t with eh other n with our results my not e entirely urte euse of ifferent mesure growth prmeters, exposition times, n ifferent extrpoltion of mesure t. It seems tht zin-ining ility of the stuie speies is in the meium rnge in omprison with tht of some other green lge, similrly to their zin tolerne. There re more ple isoltes: Shmitt et l. () lime mximum speifi sorption pities of 7.6 mg g - for Seneesmus suspitus (expose to. mg l - zin for ys), Ahuj et l. () lso reporte higher zin removl ility for the ynoterium Osilltori

10 6 Int. J. Environ. Si. Tehnol. () : 66 Tle Amount of remove zin interprete s per unit of ry mss n s perentge of initil zin ontent M. pusillum M. griffithii Mx. remove Zn (mg g - ) 9. (7th y, mg l - ) 9.7 (r y, mg l - ) Mx. remove Zn, % of initil Zn onentrtion.8 (7th y, mg l - ) 8 (7th y, mg l - ) Exposition times n e zin onentrtions elonging to the mximl remove vlue re lso inite ngustissim (.7 n 8. mg g - ; expose to n mg l - zin, respetively for fter ys). High zin sorption ws reporte for n other O. ngustissim strin n for Miroystis sp. (6. n 999. mg g -, respetively; Ahuj et l. 999; Prhn et l. 998). Otherwise, the pplition of these ynoteri s iosorents is questionle, euse of their possile toxi properties (Vss et l., ). Some speies of the fmily Seneesmee n e onsiere s outstning zin iners, 6 n mg g - oun zin were reporte for Desmoesmus pleimorphus isolte n lortory strin, respetively, (Monteiro et l. 9) n 86, mg g - ws reporte for Seneesmus oliuus strin (Monteiro et l. ). On the other hn, there re strins, whih n e hrterize more omprle or even weker zin-ining ility, thn the ones in our stuy: Romer et l. (7) reporte tht the filmentous green lg Spirogyr insignis remove mximum of. mg g - of zin, Pwlik- Skowrońsk () reporte 8. mg g - for n other filmentous green lg, Stigeolonium tenue (exposure to mgl - zin for weeks). A strin of Seneesmus quriu remove. mg g - zin fter h inution from solutions rnging from. to mg l - (Omr ), similr mount ( mg g - ) ws reporte for Euglen grilis (Fukmi 988). The most ommon rrngements use for the removl of inorgni ontminnts re high-rte lgl pons (HRAP) n the ptente lgl turf sruer (ATS), whih employs suspene iomss of ommon green lge, ynoteri or onsorti of oth (Perles-Vel et l. 6). Algl speies, whih re ommon in the environment of the plnne lolities of these kins of pons, oul e more suitle to inoulte n mintin these systems thn speies from n entirely ifferent environment. On the sis of the results presente here, it n e onlue tht there re ommon green lgl speies in surfe wters, whose tolerne to zin n zin-ining ility mkes them fesile in tretment of wstewters with mg l - zin ontmintion. Conlusion Bse on these results, it n e onlue tht the stuie green lgl speies re zin tolernt; they re le to survive reltively high zin onentrtions. This pper provies new t for the zin tolerne n zin removl ility of the green lge M. pusillum n M. griffithii. Both speies re le to remove signifint mount of zin, n the lrger proportion of remove zin is oun y sorption onto the ell surfe. Living iomss of the stuie speies oul e pplile for iosorption, 7 ys exposition up to mg l - zin onentrtion t ph It n e onlue tht living iomss of the stuie strins ommon lgl speies in Hungrin surfe wters s newly introue metl tolernt speies oul e goo lterntives for pplition n/or ses of future reserh. Aknowlegments The reserh ws supporte y the EU n ofinne y the Europen Soil Fun uner the projet ENVIKUT (TÁMOP-...A-//KONV--), y the Internl Reserh Projet of the University of Dereen n y the János Bolyi Reserh Sholrship of the Hungrin Aemy of Sienes. Referenes Ahuj P, Gupt R, Sxen RK (999) Zn? iosorption y Osilltori ngustissim. Pro Biohem :77 8 Ahuj P, Mohptr H, Sxen RK, Gupt R () Reue uptke s mehnism of zin tolerne in Osilltori ngustissim. Curr Miroiol : Anre LR, Frin M, Amo-Filho GM () Effets of opper on Enteromorph flexuos (Chlorophyt) in vitro. Eotoxiol Environ Sf 8:7 Btes SS, Tessier A, Cmpell PGC, Buffle J (98) Zin sorption n trnsport y Chlmyomons vriilis n Seneesmus suspitus (Chlorophyee) grown in semiontinuous ulture. J Phyol 8: 9 Bir G, Brewer PA, Mklin MG, Bltenu D, Drig B, Sern M, Zhri S () The soli stte prtitioning of ontminnt metls n As in river hnnel seiments of the mining ffete Tis ringe sin, northwestern Romni n estern Hungry. Appl Geohem 8:8 9 Brinz L, Dring MJ, Gvrilesu M (7) Mrine miro- n mrolgl speies s iosorents for hevy metls. Environ Eng Mng J 6:7 Broley MR, White PJ, Hmmon JP, Zelko I, Lux A (7) Zin in plnts. New Phytol 7:677 7 Chojnk K () Biosorption n ioumultion the prospets for prtil pplitions. Environ Int 6:99 7 Coett C, Golsrough P () Phytoheltin n metllothioneins: roles in hevy metl etoxifition n homeostsis. Annu Rev Plnt Biol :9 8 Cotton FA, Wilkinson G, Murillo CA, Bohmnn M (999) Avne inorgni hemistry, 6th en. John Wiley & Sons In., New York Ds N () Reovery of preious metls through iosorption review. Hyrometllurgy :8 89

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