Removal and biodegradation of polycyclic aromatic hydrocarbons by immobilized microalgal beads

Transcription

1 Wste Mngement nd the Environment V 391 Removl nd iodegrdtion of polyyli romti hydrorons y immoilized mirolgl eds N. F. Y. Tm 1, M. N. Chn 1 & Y. S. Wong 2 1 Deprtment of Biology nd Chemistry, City University of Hong Kong, Chin 2 Vie-President for Administrtion nd Business, Hong Kong University of Siene nd Tehnology, Chin Astrt Polyyli romti hydrorons (PAHs) re lss of toxi, rinogeni nd/or mutgeni orgni ompounds, whih must e removed from ontminted environments suh s wstewter. The present study ims to explore the potentil of fresh wter green mirolg, Selenstrum priornutum, immoilized in lginte eds to remove mixed PAHs nd determine the optiml density for n effetive removl. Results demonstrted tht the immoilized mirolgl eds were effetive in removing nd iodegrding mixed PAHs t their dissolved onentrtions, nmely phennthrene (PHE, 1 mg l -1 ), fluornthene (FLA, 0.25 mg l -1 ) nd pyrene (PYR, 0.1 mg l -1 ). These ilities were signifintly ffeted y ell density, rnging from 2.5x10 5 to 5x10 6 ells ed -1. At the optiml ell density of 2.5x10 6 ells ed -1, 92% PHE nd ll FLA were removed in 168 hours, nd it only took 24 hours to ompletely remove PYR. In 168-hour tretment, 73% PHE ws iodegrded while FLA nd PYR were ompletely degrded. The removl nd iodegrdtion of PAHs were further enhned y inresing the numer of eds used nd the optiml ed density ws 20 eds ml -1 wstewter. These findings suggested tht PAHs were quikly dsored onto ells nd the lginte mtrix, sored within ells nd further degrded y live ells immoilized in lginte eds. Keywords: lge, lginte, persistent orgni pollutnt, PAHs, Selenstrum, wstewter tretment. WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line) doi: / WM351

2 392 Wste Mngement nd the Environment V 1 Introdution Polyyli romti hydrorons (PAHs), lrge lss of persistent, rinogeni nd/or mutgeni orgni ompounds onsisting of two or more fused romti rings in liner, ngulr or luster struture, re generted from oth nturl nd nthropogeni soures. With inresing humn tivities, PAHs hve een widely ontminting our environment nd hve ttrted inresing onern. Bioremedition y teri nd fungi hs een suggested s n lterntive mens to remove PAHs [1]. Reltively little work hs een done on PAH removl y mirolge, lthough this miroil group hs een reported to e effetive in removing toxi metls nd other orgni pollutnts, suh s triutyltin [2-4]. One of the mjor prolems of employing miroorgnisms, inluding mirolge, to remove pollutnts from wstewter is the diffiulty in seprting them fter tretment. Immoiliztion tehniques y emedding miroil ells into mtrix to mke ig prtiles ould solve the hrvesting prolem. Mtries, suh s lginte, k-rrgeenn, grose, hitosn, polyrylmide, et., hve een suggested [5-7]. Among these, lginte hs een very populr nd is ommonly used mtrix euse of its reliility, stility nd simpliity of preprtion, nd reltively non-toxi to miroorgnisms. Growth of C. vulgris immoilized in lginte ws not ffeted nd its effiieny to remove nitrogen nd phosphorus ws even higher thn the free ell ounterprt [6]. Trvieso et l. [7] reported tht the originl shpe nd struture of lginte pellets of C. vulgris, C. kessleri nd Senedesmus qudriud were still mintined, ut the k-rrgeenn pellets of S. qudriud disintegrted with ell leking during the ten-dy nutrient removl experiment. The lginte immoilized C. vulgris ould remove nd regenerte nikel for t lest ten yles without losing ny removl pity, inditing the superior stility of lginte [8]. The performne of immoilized mirolge in removing ontminnts is ffeted y the ell density emedded within the mtrix, nd the numer of eds employed for tretment, tht is, ed density. Prdhn nd Ri [9] reported tht the removl perentges of opper(ii) y ynoterium, Miroystis sp., immoilized in lginte inresed when iomss inresed from to g ut further inrese of the iomss did not enhne the removl perentges. Similr to free ells, higher iomss would provide more ell surfe, volume nd enzymes for dsorption, sorption nd degrdtion of the ontminnt. However, ompetition of resoures, suh s nutrients nd light, self-shding nd limittion in the mss trnsfer of ontminnts, will our when the ell density is too high [10]. Tm nd Wong [11] found tht the optiml ed density for the removl of mmonium nd phosphte from wstewter y lginte immoilized C. vulgris ws 12 eds ml -1. Although lginte immoilized mirolge hve een optimized for the removl of nutrients nd hevy metls from wstewter [5-8, 11, 12], knowledge on treting PAH-ontminted wstewter using immoilized mirolge is very limited. The present study ims to determine the optiml initil ell density inside eh ed, s well s the ed density, on the removl nd degrdtion of mixture of phennthrene (PHE), fluornthene WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

3 Wste Mngement nd the Environment V 393 (FLA) nd pyrene (PYR) y lginte-immoilized lgl eds. Selenstrum priornutum ws hosen s model mirolgl speies euse of its uiquitous ourrene nd ese of ultivtion. The free ells of S. priornutum were lso found to hve higher ility to degrde PAHs thn other green mirolge, suh s Chlorell [13, 14]. 2 Mterils nd methods 2.1 Preprtion of immoilized eds The fresh wter uniellulr mirolg, Selenstrum priornutum, ws purhsed from Crolin Biologil Supply Compny (USA). Cells ultured in modified SE medium under xeni ondition were hrvested fter dys [15]. The lgl suspension ws mixed with het-dissolved sodium lginte to form 3% lgl lginte mixture, dropped into 2.5% lium hloride solution y peristlti pump (Cole-Prmer Instrumentl Compny Msterflex L/S model ) to produe immoilized lgl eds, eh with dimeter of 3 mm. Blnk eds were prepred in the sme wy with sterilized de-ionized wter insted of ell suspension. 2.2 Effets of initil ell densities per ed on PAH removl A totl of 90 sterilized onil flsks (250 ml), eh with stopper onsisting of otton wool plug wrpped y luminium foil ontining ml SE medium dissolved with 1 mg L -1 PHE, 0.25 mg L -1 FLA nd 0.1 mg L -1 PYR, were prepred. Mirolgl eds t initil ell densities of 0 (without ells, lnk ed ontrol), 2.5x10 5, 1.25x10 6, 2.5x10 6 nd 5x10 6 ells ed -1 were exmined. 0 lginte-immoilized mirolgl eds were inoulted to eh of the flsks (equivlent to the ed density of 4 eds ml -1 ). The flsks were shken t 150 rpm nd were illuminted y ool fluoresent light (50 E s -1 ) with 16/8 hour light/drk yle. At 1, 24, 48, 72, 96, nd 168 hours, triplite flsks from eh tretment were retrieved. The eds, fter seprted from the medium, were dissolved y ml sodium itrte (0.2 M). The dissolved lginte mtrix ws seprted from the ells y entrifugtion t 2,0 g for 15 min. The PAH onentrtions in the medium, in lginte mtrix nd tken up y ells were extrted y ethyl ette following the stndrd method [15], nd determined y Hewlett Pkrd 5890 gs hromtogrphy (GC) equipped with flme ioniztion detetor (FID). The fused pillry olumn (J & W Sientifi HP- 5MS) ws 30 m long with 0.25 mm internl dimeter nd ws oted with methyl silione of 0.25 µm film thikness. The temperture progrm ws mintined t C for 1 min, inresed to 214 C t rte of 2.5 C min -1, then to 222 C t rte of 1 C min -1 nd mintined t 222 C for 1 min. The injetor nd detetor tempertures were 2 C (splitless for 1.5 min) nd 300 C, respetively. The rrier gs ws helium. The PAH removl perentge ws lulted y: (Amount of PAH dded Amount remined in the medium) / Amount of PAH dded x %. The PAH WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

4 394 Wste Mngement nd the Environment V degrdtion perentge ws lulted: (Amount of PAH dded Amount of PAH remined in medium Amount of PAH dsored on lginte mtrix Amount of PAH tken y ells) / Amount of PAH dded x %. 2.3 Effet of different numer of mirolgl eds on PAH removl The sme experimentl set-up ws used to determine the effet of ed density. The numer of eds pplied to eh flsk ws 12, 20 nd eds ml -1 with ed-to-medium volume rtios of 0.9:5, 1.5:5 nd 3:5, respetively. Only one initil ell density, 2.5x10 6 ells ed -1, ws used. Triplite flsks from eh tretment were retrieved t regulr time intervl to determine the removl nd degrdtion of PAHs s desried ove. 3 Results 3.1 Effets of ell density per mirolgl ed on PAH removl The removl of ll three PAHs inresed signifintly from lnk eds (no mirolge) to mirolgl eds of higher initil ell densities (Figure 1). In the first hour of tretment, the density of 2.5x10 6 ells ed -1 hd the highest PHE removl (61%) when ompred to the other densities. From 24 hours onwrds, the removl perentges t the two highest densities (2.5x10 6 nd 5x10 6 ells ed -1 ) were omprle, inresed from % in 24-hour to 92% PHE removl in 168-hour tretment. At the lowest density, 2.5x10 5 ells ed -1, the removl perentges inresed from 42% in 1-hour to 56% in 24-hour, nd only % PHE ws removed t the end of the experiment (164-hour). Similr removl ptterns were oserved for FLA nd PYR ut with different mplitudes. Sme s PHE removl, the initil density of 2.5x10 6 ells ed -1 hd the highest FLA removl (70%) in the first hour, the removl then inresed to 90% in 24-hour nd there ws no signifint differene etween this density nd the highest one (5x10 6 ells ed -1 ) from there onwrds. At the lowest density, 2.5x10 5 ells ed -1, only 76% FLA ws removed in 24 hours. After 168 hours, FLA ws ompletely removed y eds t the two highest densities while 95 nd 83% FLA removl were hieved y eds t 1.25x10 6 nd 2.5x10 5 ells ed -1, respetively. For the removl of PYR, 2.5x10 6 ells ed -1 tretment hd 73% removl in the first hour while its initil removl t the other ell densities ws lower, rnging from 50 to 55%. All PYR ws removed y immoilized mirolge t the three highest densities in 24 hours. At the lowest density (2.5x10 5 ells ed -1 ), PYR removl ws 78% in 24 hours nd inresed to 85% in 168 hours (Figure 1). 3.2 Effets of ell density per mirolgl ed on degrdtion of PAHs The effets of ell density on PAH degrdtion y immoilized S. priornutum (Figure 2) ws very different from tht of PAH removl (Figure 1). The perentges of PHE, FLA nd PYR degrded y lnk eds WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

5 Wste Mngement nd the Environment V 395 % Removl Blnk eds 2.5 x x x x 10 6 d d (A) PHE d d % Removl (B) FLA d d % Removl (C) PYR Time (hour) Figure 1: The removl of (A) PHE, (B) FLA nd (C) PYR y immoilized Selenstrum priornutum t vrious initil ell densities (lnk eds, 2.5x10 5, 1.25x10 6, 2.5x10 6 nd 5x10 6 ells ed -1 ) during 168-hour tretment (the ed density for ll the tretments ws 4 eds ml -1 ; men nd stndrd devition of three replites re shown; signifint differenes etween ell densities t eh tretment time re shown y different lowerse letters ording to one-wy ANOVA test t p 0.05). WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

6 396 Wste Mngement nd the Environment V (without ells) were 18, 11 nd 12% t ll tretment times, respetively (Figure 2), inditing tht degrdtion without lge ws miniml. For the mirolgl eds, the perentges of PAH degrdtion inresed signifintly with tretment time nd the effets of ell density were PAH-dependent. At the end of 168 hours, PHE degrdtion inresed with ell densities, from 14% t the lowest density to 55% t the seond lowest density, nd the two highest densities hd omprly high degrdtion perentges (>65%). Similrly, the lowest density (2.5x10 5 ells ed -1 ) only hieved 1% FLA degrdtion, while FLA ws ompletely degrded t 2.5x10 6 ells ed -1 in 168 hours. All PYR ws lso degrded y immoilized eds t the two highest densities (2.5x10 6 nd 5x10 6 ells ed -1 ) in 168 hours, while only 12% PYR ws degrded t the lowest ell density. The smll degrdtion found t the lowest initil ell density suggested tht ertin numer of ells were needed for n effiient degrdtion of PAHs. It is ler tht the seond highest density (2.5x10 6 ells ed -1 ) not only hd the est removl, it lso hieved the est degrdtion perentges for PHE, FLA nd PYR, nd ws the optiml ell density for the removl nd degrdtion of mixed PAHs. 3.3 Effets of ed density on the removl nd iodegrdtion of PAHs When ompred to the PAH removl y the immoilized S. priornutum t ed density of 4 eds ml -1 (Figure 1), initil removl of PAHs, s well s the removl effiieny, were signifintly enhned t high ed densities (Figure 3). The removl lso inresed grdully with time nd rehed the mximum removl within 6 to 12 hours, depending on the ed density nd the types of PAH. For PHE removl, the ed density t 12 eds ml -1 ould remove 79% PHE in the first hour while 84% PHE ws removed t the two highest ed densities, 20 nd eds ml -1. In 96-hour tretment, PHE ws ompletely removed t 20 eds ml -1 while the removl t 12 nd eds ml -1 ws 96 nd 99%, respetively. These PHE removl perentges were muh higher thn tht t 4 eds ml -1 (Figure 1). Similrly, lmost 168 hours were needed for omplete removl of FLA when the ed density ws only 4 eds ml -1 (Figure 1), ut FLA ws ompletely removed in 24 hours t ed density of 12 eds ml -1 nd the time redued to 6 hours t higher ed density (20 nd eds ml -1 ). In the first hour, PYR ws ompletely removed t 20 eds ml -1, while it took 6 hours to remove ll PYR t ed densities of 12 nd eds ml -1 (Figure 3). PAH degrdtion y immoilized S. priornutum t high ed densities inresed signifintly when ompred to tht t the low ed density of 4 eds ml -1 (Figures 1 nd 4). The degrdtion of PAHs y immoilized S. priornutum inresed with time (Figure 4). For PHE degrdtion, the 12 eds ml -1 tretment ws higher thn tht t 20 eds ml -1. However, the reverse ws found for FLA nd PYR with more degrdtion t ed density of 20 eds ml -1. In 96-hour tretment, FLA ws ompletely degrded t 20 eds ml -1 while only 88% FLA degrdtion ws reorded in the 12 eds ml -1 tretment. All PYR ws degrded in 48 hours t 20 eds ml -1 ut it took 96 hours when the ed density deresed to 12 eds ml -1 (Figure 4) nd lmost 168 hours t the WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

7 Wste Mngement nd the Environment V 397 % Degrded 20 0 Blnk eds 2.5 x x x x 10 6 d (A) PHE d d d -20 % Degrded 20 0 d d (B) FLA d -20 % Degrded 20 0 d d d (C) PYR d d Time (hour) Figure 2: Degrdtion of (A) PHE, (B) FLA nd (C) PYR y immoilized Selenstrum priornutum t vrious initil ell densities (lnk eds, 2.5x10 5, 1.25x10 6, 2.5x10 6 nd 5x10 6 ells ed -1 ) during 168-hour tretment (the ed density for ll the tretments ws 4 eds ml -1 ; men nd stndrd devition of three replites re shown; signifint differenes etween ell densities t eh tretment time re shown y different lowerse letters ording to one-wy ANOVA test t p 0.05). WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

8 398 Wste Mngement nd the Environment V % Removl eds ml eds ml -1 eds ml -1 (A) PHE 70 % Removl (B) FLA 70 % Removl (C) PYR 70 Figure 3: Time (hour) Removl of (A) PHE, (B) FLA nd (C) PYR y immoilized Selenstrum priornutum t vrious ed densities (12, 20 nd eds ml -1 ) during 96-hour tretment (men nd stndrd devition of three replites re shown; signifint differenes etween ed densities t eh tretment time re shown y different lowerse letters ording to one-wy ANOVA test t p 0.05). WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

9 Wste Mngement nd the Environment V 399 low ed density (4 eds ml -1 ) s shown in Figure 2. The ed density of 20 ells ed -1 hd the est removl rte of PYR nd the est degrdtion performne on the more toxi nd persistent PAHs, FLA nd PYR, nd ws the optiml ed density for PAH removl nd degrdtion. 4 Disussion A mixture of PAHs, inluding PHE, FLA nd PYR, were suessfully removed nd iodegrded y lginte-immoilized S. priornutum in the present study. PAHs in the medium were quikly dsored onto lginte mtrix nd ell surfes, pssed through ell wlls nd ell memrnes, umulted inside ells nd grdully degrded y live ells immoilized in lginte eds. Lee et l. [16] found tht oth lginte-immoilized nd free ells of mixture of teri ould effiiently iodegrde phenol t low onentrtions ut only immoilized ells ould mintin their degrdtion effiieny t phenol onentrtion of 300 mg l -1. The nikel(ii) iosorption pity of C. sorokinin ws inresed y 25.3% fter loof sponge immoiliztion [17]. However, the effets of immoiliztion on the removl nd degrdtion of pollutnts y miroorgnisms re not lwys positive. A mirolg, Protothe zopfii, ws found to e less effiient in degrding n-lknes (C 14 to C 16 ) fter polyurethne fom immoiliztion nd omplete degrdtion of hydrorons y the mirolge ws delyed from 210 to 290 hours fter immoiliztion [18]. The removl effiieny of immoilized eds, sme s free ells, ws relted to the mounts of iomss used. Mfie et l. [19] reported tht higher ell density or iomss provided more surfe re nd ell volume for dsorption nd sorption of pollutnts leding to etter removl t shorter period of time. Prdhn nd Ri [9] lso showed tht the removl of opper(ii) y lginte-immoilized Miroystis inresed with inresing iomss up to g ut further inrese from to g did not enhne removl perentges. The removl nd degrdtion of PAHs y immoilized S. priornutum in this study ws found to inrese with initil ell density per ed, from 2.5x10 5 to 2.5x10 6 ells ed -1, ut further enhnement effet ws not oserved t the highest initil ell density (5x10 6 ells ed -1 ). The ells entrpped in immoiliztion mtrix would ompete for spe, nutrients, ron dioxide nd light supply, similr to or even more severely thn tht in free ells, espeilly when the ell density eme very high [10]. The mss trnsfer of nutrients to the ells inside the immoilized ed ws lso limited. Another possile men to enhne the removl nd degrdtion of ontminnts y immoilized miroorgnisms is to inrese ed density (numer of eds used). The removl of mmonium y lginte-immoilized C. vulgris t 8 nd 12 eds ml -1 were signifintly higher thn tht t other densities, 4, 16 nd 20 eds ml -1, while the phosphte removl ws not ffeted y ed density [11]. Dense mirolgl eds would limit the light penetrtion nd enhne the self-shding prolem mong eds whih in turn redued ell growth nd metoli tivities [20]. Self-shding might lso ffet the mss WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

10 0 Wste Mngement nd the Environment V (A) PHE % Degrded 12 eds ml eds ml % Degrded (B) FLA 20 (C) PYR % Degrded Time (hour) Figure 4: Degrdtion of (A) PHE, (B) FLA nd (C) PYR y immoilized Selenstrum priornutum t ed densities of 12 nd 20 eds ml -1 during 96-hour tretment (men nd stndrd devition of three replites re shown; signifint differenes etween ed densities t eh tretment time re shown y different lowerse letters ording to t-test t p 0.05). WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

11 Wste Mngement nd the Environment V 1 trnsfer of the ontminnts to the mirolgl eds. This explined why the removl nd degrdtion performne of PAHs in the present study inresed signifintly from 4 to 20 eds ml -1 ut further enhnement effet ws not ovious when the ed density ws inresed to ells ed -1. Aknowledgement The work desried in this mnusript ws supported y the Are of Exellene Sheme estlished under the University Grnts Committee of the Hong Kong SAR (Projet No. AoE/P-04/2004). Referenes [1] Juhsz, A.L. & Nidu, R. Bioremedition of high moleulr weight polyyli romti hydrorons: A review of the miroil degrdtion of Benzo[]pyrene. Interntionl Biodeteriortion nd Biodegrdtion, 45, pp , [2] Semple, K.T., Cin, R.B. & Shmidt, S. Biodegrdtion of romti ompounds y mirolge. FEMS Miroiology Letters, 170, pp , [3] Chong, A.M.Y., Wong, Y.S. & Tm, N.F.Y. Performne of different mirolgl speies in removing nikel nd zin from industril wstewter. Chemosphere, 41, pp , [4] Lun, T.G., Jin, J., Chn, S.M.N., Wong, Y.S. & Tm, N.F.Y. Biosorption nd iodegrdtion of triutyltin (TBT) y lginte immoilized Chlorell vulgris eds in severl tretment yles. Proess Biohemistry, 41, pp , [5] Aksu, Z., Egretli, G. & Kutsl, T. A omprtive study of opper(ii) iosorption on C-lginte, grose nd immoilized C. vulgris in pked-ed olumn. Proess Biohemistry, 4, pp , [6] Lu, P.S., Tm, N.F.Y. & Wong, Y.S. Wstewter nutrients (N nd P) removl y rrgeenn nd lginte immoilized Chlorell vulgris. Environmentl Tehnology, 18, pp , [7] Trvieso, L., Benitez, F., Weilnd, P., Snhez, E., Dupeyron, R. & Dominguez, A.R. Experiments on immoiliztion of mirolge for nutrient removl in wstewter tretments. Bioresoure Tehnology, 55, pp , [8] Lu, A., Wong, Y.S., Tong, Z. & Tm, N.F.Y. Metl removl studied y lortory sle immoilized mirolgl retor. Journl of Environmentl Sienes, 10, pp , [9] Prdhn, S. & Ri, L.C. Optimiztion of flow rte, initil metl ion onentrtion nd iomss density for mximum removl of Cu 2+ y immoilized Miroystis. World Journl of Miroiology nd Biotehnology, 16(6), pp , WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)

12 2 Wste Mngement nd the Environment V [10] Aksu, Z. & Kutsl, T. A omprtive-study for iosorption hrteristis of hevy-metl ions with C. vulgris. Environmentl Tehnology, 11(10), pp , [11] Tm, N.F.Y. & Wong, Y.S. Effet of immoilized mirolgl ed onentrtions on wstewter nutrient removl. Environmentl Pollution, 107, pp , [12] Mllik, N. & Ri, L.C. Influene of ulture density, ph, orgni ids nd divlent tions on the removl nutrients nd metls y immoilized Anen doliolium nd Chlorell vulgris. World Journl of Miroiology nd Biotehnology, 9, pp , [13] Lei, A.P., Wong, Y.S. & Tm, N.F.Y. Removl of pyrene y different mirolgl speies. Wter Siene nd Tehnology, 46, pp , [14] Lei, A.P., Hu, Z.L., Wong, Y.S. & Tm, N.F.Y. Removl of fluornthene nd pyrene y different mirolgl speies. Bioresoure Tehnology, 98, pp , [15] Chn, S.M.N., Lun, T.G., Wong, M.H. & Tm, N.F.Y. Removl nd iodegrdtion of polyyli romti hydrorons y Selenstrum priornutum. Environmentl Toxiology nd Chemistry, 25, pp , [16] Lee, C.M., Lu, C.J. & Chung, M.S. Effets of immoilized ells on the iodegrdtion of hlorinted phenols. Wter Siene nd Tehnology, 30(9), pp , [17] Akhtr, N., Iql, J. & Iql, M. Removl nd reovery of nikel(ii) from queous solution y loof sponge-immoilized iomss of Chlorell sorokinin: hrteriztion studies. Journl of Hzrdous Mterils, 108(1-2), pp , [18] Ymguhi, T., Ishid, M. & Suzuki, T. An immoilized ell system in polyurethne fom for the lipophili miro-lg Protothe zopfi. Proess Biohemistry, 34, pp , [19] Mfie, S.M., Trmohmed, Y. & Welourn, P.M. Effets of dmium, olt, opper nd nikel on growth of the green lg Chlmydomons reinhrdtii: the influenes of the ell wll nd ph. Arhives of Environmentl Contmintion nd Toxiology, 27, pp , [20] Gryo, I., Leon, R., Vigr, J. & Vilhez, C. Inhiition of nitrte onsumption y nitrite in entrpped Chlmyodomons reinhrdtii ells. Bioresoure Tehnology, 81, pp , WIT Trnstions on Eology nd the Environment, Vol 1, ISSN (on-line)