Effect of Chromium(VI) on growth, element and photosynthetic pigment composition of Chlorella pyrenoidosa

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1 Volume 5(-2):9-23, 26 At Biologi Szegediensis ARTICLE Effet of Chromium(VI) on growth, element nd photosyntheti pigment omposition of Chlorell pyrenoidos Zsolt Hörsik, Viktor Oláh 2, Árpád Blogh, Ilon Mészáros 2, László Simon 3, Gyul Lktos 2 Institut of Biology, College of Nyíregyház, Nyíregyház, Hungry, 2 Deprtment of Botny, Debreen University, Debreen, Hungry, 3 Deprtment of Lnd nd Environmentl Mngement, College of Nyíregyház, Nyíregyház, Hungry ABSTRACT The effets of Cr(VI) were investigted on the growth rte, element, photosyntheti pigment nd mino id omposition of Chlorell pyrenoidos. Cr(VI) is toxi to Chlorell pyrenoidos. The influene of hromium on ell density nd ell number followed very similr trends, inditing tht these growth responses might be orrelted. The EC 5 vlue for Cr(VI) were 2. mg dm -3, the lethl onentrtion of hromium ppers to be pproximtely 2 mg dm -3 for Chlorell pyrenoidos. We hve investigted the onentrtions of hromium, lium, mgnesium nd iron. The ells were frtionted into three frtions: ell wll frtion, membrne frtion, soluble frtion fter the ells were disrupted. The mount of metls in whole ells nd in eh ell frtion ws determined. Chromium uptke t eh onentrtion ws high within 3 dys. Chlorell pyrenoidos n umulte hromium minly (pproximtely 7%) in the ell wll. The onentrtions of hromium nd lium show prllel hnges with eh other. A higher lium onentrtion n be observed long with n inresing hromium onentrtion, both in the ell wll system nd in the whole ells. Iron nd mgnesium onentrtion show deresing tendeny. Cr(VI) used hnges both in free mino ids nd proline ontent. Both free mino ids nd proline ontent inrese with the inresing onentrtion of hromium. Chlorophyll nd b ontent show deresing, while OH-hlorophylls show inresing tendeny. Rte of rotene β nd α hnge grows with the inresing hromium onentrtion. The toxi properties of Cr(VI) n rise from the possibly free diffusion ross ell membrnes nd strong oxidtive potentil. The toxiologil impt of Cr(VI) origintes from the tion of this form itself s n oxidizing gent, s well s from the formtion of free rdils during the redution of Cr(VI) to Cr(III) ouring inside the ell. At Biol Szeged 5(-2):9-23 (26) KEY WORDS Chlorell pyrenoidos hromium element omposition pigment pttern Chlorell pyrenoidos is n uniellulr green lg, whih is found in both fresh nd mrine wters. Its physiology, biohemistry nd photosyntheti pprtus re similr to higher plnts but its growth is very quik. For these resons Chlorell is often studied in vrious metboli nd stress investigtions (Rhlin nd Grosso 993; Lustigmn et l. 995). When the onentrtion of metl ions in the environment rises bove speifi threshold hevy metl ions inhibit vriety of metboli tivities nd prove toxi to most orgnisms. Interest in hromium origintes from widespred use of this metl in vrious industries, suh s metllurgil (steel, ferro nd nonferrous lloys) nd hemil (pigments, eletroplting, tnning, others). Due to industril run-off, proess, lrge quntities of Cr ompounds re dishrged in liquid, solid nd gseous wstes into the environment, resulting in signifint dverse biologil nd eologil effets (Kbt- Pendis nd Pendis 2). Chromium n exist in severl Aepted My 5, 26 *Corresponding uthor. E-mil: horsik@nyf.hu hemil forms, displying oxidtion numbers from to VI. Only trivlent nd hexvlent hromium, re stble enough to our in the environment. Cr(IV) nd (V) form unstble intermedites in retions of trivlent nd hexvlent oxidtion sttes with oxidizing nd reduing gents respetively (Bll nd Nordstrom 998; Shriver et l. 2). Cr(III) is the best known form displying stbility t neutrl ph, if the omplextion n be negleted. Under redox nd ph onditions normlly found in nturl systems, hromium is removed from the solution s Cr(OH) 3, or in the presene of Fe(III) in the form of (Cr x Fe -x )(OH) 3 where the x is the mole frtion of hromium (Sss nd Ri 987). Cr(III) generlly hs lower toxiity thn Cr(VI) ompounds. The Cr(III) is known to be essentil for men nd other mmmls through its importnt funtion in gluose nd lipid metbolism (Mertz 975; Anderson 989). Cr(VI) forms severl oxygen ssoited speies, the reltive proportions of whih depend on both ph nd totl hromium (VI) onentrtion. Within the norml ph rnge in nturl wters CrO 4 2-, 9

2 Hörsik et l. 3 2,5 2. mg dm- 3 Cr ( V I) C o n tr o l mg dm- 3 Cr ( V I) 3,5 7 3, mg d m- 3 Cr( V I) Co n tr o l mg dm- 3 Cr( V I) 5 mg dm- 3 Cr( V I) OD 75,5 5 mg dm- 3 Cr ( V I) mg d m - 3 Cr ( V I) 2 mg d m - 3 Cr ( V I) Cell number 2,5 6 2,5 5 mg d m - 3 Cr ( V I) 2 mg d m - 3 Cr ( V I),5, H o u r s Ho u r s Figure. Effets of Cr(VI) on ell density of Chlorell pyrenoidos in syntheti medium. Figure 2. Effets of Cr(VI) on ell number of Chlorell pyrenoidos in syntheti medium. HCrO 4 - nd Cr 2 O 7 2- ions re the forms generlly found nd onstitute mny of the hromium (VI) ompounds. They re quite soluble nd mobile in the environment (Nieboer nd Jusys 988). Severl lbortory studies hve delt with toxi effets of hromium in higher plnts nd lge (Wong nd Chng 99; Bishnoi et l. 993). The effet of Cr(VI) on the growth of Chlorell pyrenoidos t different onentrtions ws studied under lbortory onditions. We lso studied the element nd photosyntheti pigment omposition of lge, intrellulr distribution of hromium nd the levels of free mino ids nd proline. Mterils nd Methods The lge used in this study were Chlorell pyrenoidos (strin IAM-C28) obtined from the olletion of the Institute of Applied Mirobiology, University of Tokyo (Jpn). Chemils were purhsed from Sigm Chem. Co. (St. Louis, USA), nd Serv Fine Biohem. Gmbh. (Heidelberg, Germny). Alge were mintined on gr. Cells were grown in sterile tubes ontining syntheti medi modified C-3. Cultures were erted by filtered ir bubbled with 5% CO 2, whih served s rbon soure nd stbilized the lgl suspension homogeneity nd ph t 7.2. Cells were permnently illuminted with white fluoresent light (8 Wm -2 ) nd were kept t 25 C during the growing period. The ultures were grown for 3 in some se 4 dys. When ultures rehed pproximtely x 5 ells/ml in the nutrient medium, lge were treted with. - 5 mg dm -3 of hromium (VI). We used K 2 Cr 2 O 7 s hexvlent hromium. For eh experiment ontrol ws lso prepred of untreted Chlorell pyrenoidos ells kept t the sme onditions. Alge were utotrophilly propgted for 72 hours fter hromium tretments. The growth rte of lge ultures ws followed by indiret turbidometri ssy, nd diret ount using hemoytometer. Cells were olleted by entrifuging (5g x min), nd were wshed 2 times with deionized wter. For lgl ell frtiontion ells were hrvested by entrifugtion t 5g x min, were wshed twie with deionized wter nd entrifuged, nd the weight of the ells ws determined. This type of smple ws onsidered s whole ells (smple ). These prepred ells were disrupted using Frenh press t 5 kgf/m 2 three times. Frenh press is the most useful tool for disrupting Chlorell (Tked nd Hirokw 984). The homogente ws entrifuged t 3g x 2 min nd the pellet ws llowed to stnd in ml of,5% sodium n-dodeyl sulphte (SDS) for 3 min. The homogente with SDS ws entrifuged t g x 2 min to remove the soluble omponent, nd the pellet ws boiled in 8% ethnol for 2 min. The ell wll frtion ws prepred by entrifuging the ethnol boiled pellet t g x 2 min (smple 2). The misellneous frtion ws prepred s the mixed superntnt fter the tretment with SDS nd ethnol (smple 3). The homogente ws entrifuged 5g x 45 min fter disruption of ells. The pellet ws the membrne frtion (smple 4) nd the superntnt ws the soluble (ytoplsmi; smple 5; Okmur nd Aoym 994). The wter of the superntnts ws evported before digestion. Smples were digested for dy in onentrted HNO 3 nd 3% H 2 O 2 mixture (6: v/v rte), nd dried. The minerlized residue ws redissolved in 5 ml of 2N HNO 3 solution. The element omposition ws mesured by Spetroflme-type indutively oupled plsm tomi emission spetrophotometer (ICP-AES; Spetro Gmbh Kleve, Germny) with the following prmeters: plsm gs.6 dm 3 min -, nebulizer gs.6 dm 3 min -, oolnt gs 5 dm 3 min -, exittion 27 MHz,.5 kw ross flow nebulizer. When individul photosyntheti pigments were seprted by HPLC tehnique lge pigments were extrted with mixture of hloroform etone isopropyl lohol (2:: v/v) t 4 C, the nlytil proedure ws detiled formerly (Simon et l. 989). To determine free mino id nd proline ontent 2 mg fresh lge were shken in 2 ml of 7% trihloroeti id for two hours, then they were filtered through pper filter nd membrne filter (.45 μm). The nlysis ws rried out using Biotronik LC 3 mino id nlyzer 2

3 Effet of hromium on Chlorell Tble. Element omposition of lgl ell nd ell frtions grown without nd in the presene of, 5 or mg dm -3 of Cr(VI) (the vlues re given in mg dm -3 ). Control mg dm -3 Cr(VI) Cr C Mg Fe Cr C Mg Fe Whole lg ell,2 38± 423±54 48±4 7±.8 64±5 6±82 2±4.8 Cell wll frtion,2 45±.4 52±5 4±7.5 4±2.6 6± ±58 4±7 Membrne frtion Nd 4.8±.4 ±4 32± ±.8.4±.6 98±2 26±3.6 Soluble frtion Nd 3.4±.4 2±7 4±4.9 2.±.7.7±.4 68±9.5±2.8 5 mg dm -3 Cr(VI) mg dm -3 Cr(VI) Cr C Mg Fe Cr C Mg Fe Whole lg ell 28±3.6 74±4.2 98±4 78±8 24± ±4.2 98±8 62±5. Cell wll frtion 28.8±.5 77± ±7 82±3.8 3± ±3.7 72±88 79±4.2 Membrne frtion 4.±.9 6.4±. 44±22 ±2. 2.±.9 7.5±. 94±7.6.2±.4 Soluble frtion 3.4±.2 2.8± 49± 2.5±.9 2.8±.4 3.2±.2 5± ±.3 Nd: not detetble. Vlues represent the men ± SD of one experiment in triplite. (Glib et l. 992). The results re the mens of 3-5 replitions for eh tretment. Three independent repetitions were performed for eh experiment. The dt were sttistilly evluted lulting the stndrd devition, nd by sttistil nlysis using Tukey s b-test. Results The effets of hromium (VI) on Chlorell pyrenoidos ws investigted using inresing onentrtion of hromium from to 5 mg dm -3. As seen in Figures nd 2, exept for slight inrese in growth of the lgl ells t. mg dm -3 in omprison to the ontrol, n inrese in hromium onentrtion used signifint derese in the ell density nd ell number. The influene of hromium on ell density nd ell number followed very similr trends, inditing tht these growth responses might be orrelted. The EC 5 vlue for hromium (VI) bsed on inhibition of ell growth ws 2. mg dm -3. The ell ultures treted with Cr(VI) from 2-5 mg dm -3 showed hloroti symptoms, nd the ells did not grown when we trnsferred them to fresh medium without hromium. This suggests, tht the lethl onentrtion of Cr(VI) ppers to be pproximtely 2 mg dm -3 for Chlorell pyrenoidos. The onentrtions of 2 elements (Al, B, B, C, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, N, Ni, P, Pb, S, Sr, Ti, V, Zn) were determined in lge ells, nd the onentrtions of hromium, lium, mgnesium, iron re presented in Tble. The ells were frtionted fter disruption to ell wll frtion, membrne frtion, nd soluble frtion. The onentrtions of metls in whole ells nd in eh ell frtions were determined. Within 3 dys hromium uptke t eh onentrtion ws high inditing tht lgl ells re ble to bind nd umulte hromium. Generlly 7% of the hromium is lolized in the ell wll region, while the mount of umulted hromium ws lmost the sme in the membrne nd the soluble frtion. Comprison the dt of the ontrol nd treted ells demonstrtes the high rte of umultion of hromium in the ells (Tble ). The hromium ontent of ontrol ells is.2 mg dm -3, while the vlues of treted ells re hundred times higher. Intrellulr distribution of hromium is heterogenous in different frtions. The onentrtions of hromium nd lium show similr hnges. An inresing hromium onentrtion uses higher lium onentrtion, both in the ell wll system nd in the whole ells. The inresed C onentrtion is surprising, beuse n ion exhnge would be expeted. Both iron nd mgnesium onentrtions show de resed onentrtions with inresed hromium onentrtions. All onentrtions of hromium produed hnges in both free mino ids nd in proline onentrtions s seen Figure 3 nd 4. Inresing the onentrtion of hromium to mg dm -3 inresed the onentrtion of free mino ids nd proline. At higher onentrtion of hromium the mount of mino id onentrtion deresed. Proline onentrtion is known to inrese in different type of stresses suh s slinity, drought, low nd high temperture, nd hevy metls (Ibrr-Cbllero et l. 988; Kvi Kishor et l. 995). The toxi properties of Cr(VI) my rise from free diffusion ross ell membrnes nd strong oxidtive potentil. The toxiologil impt of Cr(VI) origintes from its tion s n oxidizing gent, s well s from the formtion of free rdils during the redution of Cr(VI) to Cr(III) ouring inside the ell (Nieboer nd Jusys 988). Deresing levels of free mino ids nd proline t higher onentrtion of hromium mybe due to the degrdtion of the ells. 2

4 Hörsik et l. Tble 2. Photosyntheti pigments onentrtion of lgl ells growth with nd without Cr(VI). 2 8 dd d Cr(VI) Control mg dm -3 5 mg dm -3 mg dm -3 2 mg dm -3 hlorophylls hlorophyllid b,28,24,24,2,8 hlorophyllid,35,25,256,284,26 hlorophyllid,22,86,74,78,82 OH-hlorophyll b-,52,7,64,232,258 OH-hlorophyll b-2,46,82,44,28,22 hlorophyll b,842,872,878,352,734 hlorophyll b,246,272,22,248,82 OH-hlorophyll -,6,58,238,268,276 OH-hlorophyll -2,84,22,38,36,34 hlorophyll 4,338 4,574 4,52 3,88 3,44 hlorophyll,66,756,5,522,58 pheophytin,36,9,38,7,228 rotenoids violxnthin,6,92,76,22,38 nteroxnthin,49,54,458,584,42 lutein,324,372,39,83,96 xntophylls *,22,248,344,42,34 α-riptoxnthin,98,36,3,88,36 β-riptoxnthin,48,48,34,8,6 α-rotene,98,54,58,5,76 β-rotene,344,274,348,368,462 xntophylls * : unidentified xntophylls µg/g b The effets of hromium on onentrtion of photosyntheti pigments nd hlorophyll derivtives re seen in the Tble 2. The mounts of both hlorophyll- nd hlorophyll-b derese with inresing hromium onentrtion, lthough the derese in hlorophyll- is lrger thn in hlorophyllb. Conversely, the mounts of both OH-hlorophyll- nd OH-hlorophyll b (oxidtive produts of the hlorophylls) inresed with the inresing onentrtion of hromium. When Chlorell green lge were grown under similr onditions to this experiment, ddition of metl ions (nmely titnium, gllium nd zironium) to growth medium lso used hnges in photosyntheti pigment onentrtion nd omposition. Apperne of new hlorophyll derivtives ws observed in titnium, gllium nd zironium treted Chlorell ultures (Simon et l. 988; Simon et l. 989; Simon et l. 2). Deomposition of hlorophylls nd forming of hlorophyll derivtives ould be ttributed to in vivo tion of enzymes like peroxidse (Kto nd Shimizu 985; Simon et l. 989). Sine peroxidse is loted minly in the vuoles, nd hlorophyll is omprtmentized in the hloroplst, presumbly peroxidse deomposes hlorophyll in vivo in the proesses where membrnes re dis-integrted (Kto nd Shimizu 985). We suppose tht higher onentrtion of Cr(VI) ts indiretly to hlorophyll metbolism (i.e. by stimultion the dis-integrtion of membrnes, nd enhning the tivity of peroxidse), nd uses pperne of hlorophyll derivtives s OH-hlorophyll- nd OH-hlorophyll-b. There were lso hnges in rotenoid levels. In the ontrol ells the rtio of β - α rotene is bout.5, but the rte beme higher with the inresing hromium onentrtion. This phenomenon ould be proteting proess ginst hevy metl indued oxidtive stress (Slguero et l. 23). Disussion b b Cr(VI) is toxi to Chlorell pyrenoidos. Chlorell green lge n umulte hromium minly in the ell wll. Both free mino ids nd proline ontent inresed with the inresing onentrtion of hromium in the growth medium. Chlorophyll nd b deresed, while OH-hlorophylls inresed in ells. Rtio of rotene β - α inresed with the, Cr(VI) mg dm -3 Figure 3. Totl mino id ontent in Chlorell pyrenoidos grown in syntheti medium treted with Cr(VI). Sttistil nlysis ws done by Tukey s b-test. Dt re mens of 3 replitions. Brs of mens signed by the sme letter re not sttistilly signifint t P=.5. µg/g b b, Cr(VI) mg dm -3 Figure 4. Proline ontent in Chlorell pyrenoidos grown in syntheti medium treted with Cr(VI). Sttistil nlysis ws done by Tukey s b-test. Dt re mens of 3 replitions. Brs of mens signed by the sme letter re not sttistilly signifint t P=.5. 22

5 Effet of hromium on Chlorell inresing hromium onentrtion. These dt suggest tht the toxi effets of hromium on green lge origintes from the tion of Cr(VI) s n oxidizing gent s well s from the formtion of free rdils during the redution of Cr(VI) to Cr(III) ourring inside the ell. The toxi properties of Cr(VI) my rise from the free diffusion ross the ell membrnes nd strong oxidtive potentil. Referenes Anderson RA (989) Essentility of hromium in humns. Si Tot Environ 86:75-8. Bll JW, Nordstrom DK (998) Critil evlution nd seletion of stndrd stte thermodynmi properties for hromium metl nd its queous ions, hydrolysis speies, oxides, nd hydroxides. J Chem Eng Dt 43: Bishnoi NR, Chugh LK, Sowhney SK (993) Effet of hromium on photosynthesis, respirtion nd nitrogen fixtion in pe (Pisum stivum L.) seedlings. J Plnt Physiol 42:25-3. Glib G, Simon-Srkdi L, Kosy G, Slgo A, Sutk J (992) Possible hromosoml lotion of genes determining the osmoregultion of whet. Theor Appl Genet 85: Ibrr-Cbllero J, Villnuev-Verduzo J, Molin-Glm J, Jimenez SD (988) Proline umultion s symptom of drought stress in mize: tissue differentition requirement. J Exp Bot 39: Kbt-Pendis A, Pendis H (2) Tre elements in soils nd plnts (3rd edition). CRC Press LLC, Bo Rton, London, New York, Wshington, D.C. pp Kvi Kishor PB, Hong Z, Mio GH, Hu CA, Verm DPS (995) Overexpression of Δ -pyrroline-5-rboxylte synthse inreses proline prodution nd onfers osmotolerne in trnsgeni plnts. Plnt Physiol 8: Kto M, Shimizu S (985) Chlorophyll metbolism in higher plnts. VI. Involvement of peroxidse in hlorophyll degrdtion. Plnt Cell Physiol 26:29-3. Lustigmn B, Lee LH, Khlil A (995) Effets of nikel nd ph on the growth of Chlorell vulgris. Bull Environ Contm Toxiol 55: Mertz W (975) Effets nd metbolism of gluose tolerne ftor. Nutr Rev 33: Nieboer E, Jusys AA (988) Biologil hemistry of hromium In Nrigu JO, Nieboer E eds., Chromium in nturl nd humn environments. Wiley Intersiene: New York, pp Okmur H, Aoym I (994) Intertive toxi effet nd distribution of hevy metls in phytoplnkton. Environ Toxiol nd Wter Qul 9:7-5. Rhlin JW, Grosso A (993) The growth response of the green lg Chlorell vulgris to ombined tion exposure. Arh Environ Contm Toxiol 24:6-2. Slguero A, de l Moren B, Vigr J (23) Crotenoids s protetive response ginst oxidtive dmge in Dunliell brdwil. Biomol Eng 2(4-6): Sss BM, Ri D (987) Solubility of morphous hromium(iii)-iron(iii) hydroxide solid solutions. Inorg Chem 26: Shriver DF, Atkins PW, Lngford CH (2) Inorgni Chemistry 2nd ed., (App.2., B4) Oxford University Press, Oxford. Simon L, Hjdu F, Blogh A, Pis I (988) Effet of titnium on growth nd photosyntheti pigment omposition of Chlorell pyrenoidos. Prt II. Effet of titnium sorbte on pigment ontent nd hlorophyll metbolism of Chlorell. In Proeedings of the 3rd Interntionl Tre Element Symposium; Budpest, Hungry, pp Simon L, Kiss F, Bkó A, Hjdu F, Hörsik TZS, Blogh Á, Pis I (989) Effet of gllium on photosyntheti pigments nd peroxidse tivity of Chlorell pyrenoidos. J Plnt Nutr 2:23-4. Simon L, Fodor M, Pis I (2) Effets of zironium on the growth nd photosyntheti pigment omposition of Chlorell pyrenoidos green lge. J Plnt Nutr 47(): Tked H, Hirokw T (984) Studies on the ell wll of Chlorell. Comprison of the ell wll hemil ompositions in the strins of Chlorell ellipsoide. Plnt Cell Physiol 25: Wong PK, Chng L (99) Effets of opper, hromium nd nikel on growth, photosynthesis nd hlorophyll synthesis of Chlorell pyrenoidos 25. Env Poll 72: