Nickel uptake and release in nickel-resistant and - sensitive strains of Scenedesmus Acutus F. Alternans (Chlorophyceae)

Environmental and Experimental Botany, Vo|. 36, No. 4, pp. 401 41 l, 1996 Copyright © 1996 Elsevier Science B.V. Printed in Great Britain. All rights reserved 0098- 8472/96 $15.00

S0098--8472(96)01030-1

N I C K E L U P T A K E AND RELEASE IN N I C K E L - R E S I S T A N T AND SENSITIVE STRAINS OF SCENEDESMUS ACUTUS F. ALTER)VANS (CHLOROPHYCEAE) XIAOLEIJIN*, DONNJ. KUSHNER*~ and CZESLAWANALEWAJKO~§ *Department of Botany, University of Toronto, Toronto, Ont. M5S 3B2, Canada; ]'Department of Microbiology, University of Toronto, Toronto, Ont. M5S 1AS, Canada; ++LifeScience Division, Scarborough College, University of Toronto, 1265 Military Trail, Scarborough, Ont, M 1C 1A4, Canada

(Received29 December 1995; acceptedin revisedform 6 June 1996) Jin, X., Kushner, DJ. and Nalewajko, C. Nickel uptake and releasein nickel-resistant and -sensitive strains of Scenedesmus acutus f. alternans (Chlorophyceae).Environmental and Experimental Botany 36, 401 411, 1996.--Intracellular uptake and extracellular adsorption of Ni 2+ were compared in three Niresistant (B4, Cu-Tol, and Ni-Tol) and one Ni-sensitive (UTEX 72) strains of Scenedesmus acutusf alternans, to assess the role of these processes in Ni-resistance. Intracellular uptake was highest in the most sensitive strain (UTEX 72) during a 24 h exposure to 50 fmol Ni 2+ per cell (equivalent to 50 pM). However, B4 could grow longer and accumulated substantially more Ni 2+ than UTEX 72 after a 3 days exposure period, suggesting that B4 is a Ni-accumulator. Ni 2+ transport rate in Cu-Tol and Ni-Tol was only about 1/3 that in UTEX 72, indicating that Ni-Tol and Cu-Tol exclude Ni 2+. A positive linear relationship was found between intracellular uptake and extracellular adsorption and Ni ~÷ dosage in all four strains in the range of 2-500 fmol Ni 2÷ per cell. Although extracellular adsorption as percentage of total uptake was lowest in UTEX 72 in most cases studied, all four strains bound substantial amounts of Ni 2+ to their cell surface, suggesting that extracellular binding contributes only slightly to Ni-resistance in these strains. That low temperature and darkness strongly inhibited but did not abolish Ni 2+ uptake in all four strains suggests that active transport was the major means of Ni 2+ uptake, but that passive Ni 2+ transport also existed. All four strains released more Ni2÷ in dark than under light after 28 h of resuspension in Ni-free medium, suggesting that Ni 2+ release is a passive process which is counteracted by active Ni 2÷uptake in the light. Ni-resistance in these strains can not be attributed to active Ni ~+ release.

Key words: Nickel uptake, nickel release, nickel resistance, Scenedesmus.

INTRODUCTION Elevated Ni concentrations up to 100 /~M have been found in many aquatic systems exposed to industrial pollutionJ 36/ Although Ni is an essential

element in many organisms including algae, at elevated concentrations it is toxic to m a n y physiological processes. (15'27)Singh et aL 13°)demonstrated that Ni 2+ could inactivate PSII ( H 2 0 ~ P B Q ) in Nostoc muscorum by causing alteration and destruction o f p h o -

§Corresponding author. Tel: 416-287-7425; fax: 416-287-7642; e-mail: [email protected] 401

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tosynthetic membranes. Organisms living in a naturally or anthropogenically Ni-enriched or Nicontaminated environment can develop resistance to Ni. Different mechanisms of resistance are involved. Some yeasts and higher plants can accumulate Ni in vacuoles or cytoplasm as organic metal-complexes.)(4'13'16'18-22) Other plants, fungi, yeasts and bacteria exclude Ni by reduced uptake (6'8'17'23'44)or specific efflux systems. (2a'291The mechanisms of resistance involved, especially whether they are due to accumulation or exclusion, can have significant effects on the availability of metals to other forms of life, including their entry into the food chain. There is much less information on Ni 2+ uptake and mechanisms of Ni-resistance in algae than in other organisms. Stokes(37)compared C u 2+ and Ni 2+ uptake in strains ofScenedesmus acutusf alternans. For a sensitive 'laboratory strain', the Cu 2+ and Ni 2+ concentrations that inhibited growth in cell number by 50% were 0.39 and 0.85 ktM respectively; for a resistant strain, B4, isolated from a Ni (42/IM) and Cu (1.69/tM) polluted lake, respective values were 1.57 and 5.1 #M. At Ni 2+ concentrations which permitted 50% growth of the sensitive cells, these bound more metal than the resistant cells. However, at comparable levels of growth inhibition, the resistant cells bound substantially more of both metals than the sensitive cells. These studies measured total metal uptake without distinguishing internal uptake from extracellular binding. We have been concerned with mechanisms of Ni-resistance in algal strains isolated from metalpolluted environments, as well as strains whose metal resistance developed in laboratory cultures. In a previous study, (15/we demonstrated that growth of three resistant strains of Scenede~mus acutusf alternans (henceforth usually designated S. acutus) (B4, Cu-Tol and Ni-Tol) was at least 18 times, and photosynthesis at least 3.5 times, less sensitive to Ni 2+ than in a sensitive strain, U T E X 72. We also found that S. acutus B4 bound more of the cationic dye alcian blue to its cell surface than U T E X 72 (]in unpublished data 1995). Twiss and Nalewajko (41) reported that strain B4 took up more, but strain Cu-Tol took up less, Cu 2+ than strain UTEX 72. Based on this information, we hypothesized that (1) strain Cu-Tol has a Ni exclusion mechanism; (2) strains Ni-Tol and Cu-Tol, derived from S. acutus strain U T E X 72 by exposure to sublethal con-

centrations of metals, have the same resistance mechanism; (3) strain B4 has a Ni accumulation mechanism; and (4) cell surface binding plays a role in Ni-resistance in the resistant strains. We have now measured kinetics and other physiological determinants of uptake of both extracellularly-adsorbed and internally-bound Ni 2+ in these algal strains in order to test these hypotheses. We also looked for evidence of an active Ni 2+ eittux system. The overall focus of this work was to examine the roles of accumulation and exclusion in Niresistance in S. acutusf alternans. MATERIALS AND METHODS

Axenic cultures of S. acutusf alternans, including a Ni-sensitive strain U T E X 72 (UTCC #8) and three Ni-resistant strains: B4 (UTCC # 10) isolated from a nickel- and copper-contaminated lake, (34) Cu-Tol (UTCC # 7) and Ni-Tol (UTCC # 353) derived from U T E X 72 (UTCC # 8), were supplied by the University of Toronto Culture Collection (UTCC). All stock and experimental cultures were grown in autoclaved modified Chu 10 medium pH 7.0 (15) under constant illumination (2.4x 1006) quanta cm 2 s-l) at 24°C with a 12/12 h light/dark cycle, or as specified. In the Ni 2+ uptake and release experiments, exponential-phase cultures were filtered on Millipore cellulose nitrate membrane filters (0.45 /~m pore size) and washed once with fresh medium. Cells were resuspended in fresh medium so that the starting cell densities (106 cells per ml) and other conditions (i.e. nutrients and pH) were the same in all experiments. Twenty ml of the cell suspension were transferred to a capped glass vial which was then spiked with Ni 2+ and 63Ni2+ stock solutions to obtain the required Ni 2+ concentration and total radioactivity of about 30 000 CPM per ml. Ni 2+ stock solution (950 ppm pH 7.0) was prepared from a 1000 ppm Ni 2+ reference standard solution in 25% HNO3 (Fisher Scientific, Nepean, Ont., Canada). The pH was adjusted to 7.0 using l0 N NaOH. 63Ni2+stock solution (pH 6.0) was prepared by a 1000 times dilution of 63Ni2* in 0.5M HC1 (DuPont, NEN Research Products, Boston, MA, USA) in distilled and deionized water (pH 8). For each condition studied, four vials were used. Results are usually presented as mean values _+standard error of four replicates. A number of experiments

NICKEL UPTAKE including time course of Ni 2+ uptake, effect of Ni 2+ dosage, low temperature and darkness on Ni 2+ uptake, and Ni 2+ release in light and dark, were repeated once or twice with similar results to those shown. To measure total Ni 2+ uptake, Ni-exposed cells were collected on a G F / A filter (1.2 #m pore size, from Ahlstrom Filtration Scientific Specialties Group) and washed five times with 20 ml fresh medium (pH 7.0). To distinguish internal uptake from external binding, cells were washed three times with 20 ml of 1 m M E D T A (pH 7.0) after washing with fresh medium. The Ni 2÷ irremovable by E D T A washing was considered to be intracellular, Ni 2+ removable by E D T A washing was considered to be externally bound, i.e. to the cell walls or membranes. Radioactivity of the cell pellet on the filter was measured by scintillation counting on Beckman LS 6000IC scintillation counter (Beckman Instruments, Inc. Fullerton, CA, USA). Abiotic Ni 2+ retention on the filter was measured by filtering an appropriate volume of culture supernatant and was subtracted as a blank for calculation of net Ni 2+ uptake. To determine long term Ni 2+ uptake and its effect on growth, cultures (300 ml, initial cell density 106 cells m l - l) were exposed to 50 #M Ni 2+ (equivalent to 50 fmol per cell) in a 500 ml cotton-plugged Erlenmyer flask. Exposure continued for 3 days under a 12/12 h light/dark cycle, and Ni 2÷ uptake was measured as described above. Control cultures with no Ni 2+, and cultures with the same concentrations of non-radioactive Ni 2÷ as in the radioisotope experiment were grown under the same conditions and used for determination of population density (cell number per ml culture) and dry weight (g per 100 ml culture) after 3 days of growth. Cell density was obtained by microscopic examination using a haemacytometer, and culture dry weight was determined in 100 ml culture filtered on a pre-weighted polycarbonate filter (1 #m pore size, Poretics Corporation, Livermore, CA, USA) and dried at 30°C to a constant weight. T o determine Ni ~+ release in the dark and in the light as a function of duration of exposure, cells were exposed to 50 # M Ni 2+ (equivalent to 50 fmol per cell) under continuous light for 22 h. Cells were then collected on a polycarbonate filter (1 #m pore size) and washed three times with 20 ml modified Chu 10 medium and once with 20 ml of 1 m M

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EDTA. Washed cells were resuspended in 20 ml fresh medium and incubated in the light or in the dark at 24°C for up to 28 h during which 2 ml aliquots of cell suspension were removed at different times and centrifuged at 1625 g on a clinical centrifuge (Model CL, International Equipment Co, Fisher Scientific) for 10 rain. Radioactivity of 1 ml of supernatants was measured by scintillation counting. Student's t-tests were performed on correlation coefficients (r), rates of intracellular Ni 2+ uptake, and percentage of extracellularly hound vs. total Ni 2+ uptake. !33:~ RESULTS

On exposure to 50 p M Ni 2+, all strains ofS. acutus took up Ni 2+ in a roughly linear fashion (r2 > 0.91, t-test P<0.001) for the first 2 h (data not shown), and uptake continued to increase for 24 h (Fig. 1). Although strain B4 had the highest initial (up to 2 h) rate of internal Ni 2+ uptake (2.68 x 10 -3 fmol Ni 2+ per cell per min, t-test P < 0.001 for all the other strains), the overall intracellular uptake rate during 24 h exposure was higher in strain U T E X 72 than in the three resistant strains (P< 0.001) (Fig. 1). The percentage of externally bound Ni 2+ vs. total Ni 2+ uptake fluctuated with time. After 6 h and 24 h exposures, it was lowest in U T E X 72 (for 6 h exposure, t-test P < 0 . 0 1 , for 24 h exposure, P_< 0.005). Intracellular Ni uptake continued in S. acutus strain B4 for at least 3 days, and also increased in strain Cu-Tol, but levelled off or decreased in the other strains after 6 24 h (Fig. 1). The effect of 50 #M Ni 2+ on growth corresponding to the 3 day Ni 2+ uptake was measured both as cell number and dry weight. As found previously,/15~ this Ni 2÷ concentration completely inhibited increase of cell number in strains U T E X 72 and Cu-Tol and almost completely inhibited it in strain Ni-Tol (95%), but not in B4 (70%). However, the dry weight of all cultures were inhibited to a much smaller degree (6% for B4, 35% for U T E X 72, 15% for Cu-Tol and 54% for Ni-Tol), showing that this Ni 2+ concentration had less effect on increase of biomass than on cell division. The 3 day Nitreated cells appeared slightly larger and less green than control ceils; these changes were not measured quantitatively. In 6 h exposure, Ni 2+ uptake per cell in all strains

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Fig. l. Time course of Ni uptake by Scenedesmus acutus strains exposed to 50 fmol Ni per cell or 50/~M). This process was studied from 2 min to 24 h in continuous fight and for 72 h in 12 h light/dark cycles (results for 2-30 rain are omitted). Note the different time scales after 2 h and 24 h. Variation coefficients are usually lower than 20%, about 30% for some points. (Standard error bars are not shown because two sets of data are presented in each bar).

increased linearly (r2 >0.98, P < 0 . 0 1 ) with Ni 2+ dosage (Fig. 2). S. acutus strain U T E X 72 had the highest intracellular uptake for all Ni 2+ concentrations studied (t-test P_<0.01, for all three resistant strains). The proportion of externally bound Ni 2+ increased with Ni 2+ dosage, and was significantly higher (P<0.05, except Ni-Tol exposed to 500 /~M Ni2+: P=-0.405) in the three resistant strains than in strain U T E X 72 at Ni 2+ concentrations > 10 #M. At the highest dose of Ni 2+, extracellular adsorption predominated over intracellular uptake in strain B4. Dark exposure strongly inhibited Ni ~+ uptake in all strains and at all Ni 2+ dosages used (Table 1). Generally, the degree of inhibition increased with Ni 2+ dosage. At the 10 fmol Ni 2+ per cell dosage, total Ni 2+ uptake per cell was most inhibited in strain U T E X 72 (>--90%). The externally bound

Ni 2+ was usually less than 10% of the total and was not greatly affected by dark exposure in all four strains (Table 1). At 50 and 500 fmol Ni 2+ per cell, respectively, the proportion of externally bound Ni 2÷ to total Ni 2+ uptake was reduced significantly by dark exposure in strain B4 (from 31 to 3% and from 50 to 13%), but increased in strain U T E X 72 (from 3 to 28% and from 23 to 42%) (Table 1). Cold exposure was highly inhibitory to Ni 2+ uptake regardless of strains and dosages used (Table 2). In general, the inhibitory effect was more pronounced with the internal than the externally bound Ni 2+. At 10 fmol Ni 2+ per cell dosage, highest inhibition was found in strain B4 (>- 90%). The percentage of externally bound Ni 2+ was increased by cold exposure in all strains, especially in strain Ni-Tol (from 15% to 68%) (Table 2). Inhibitory effects became more pronounced as Ni 2+ dosage

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increased. At 500 fmol Ni 2+ per cell, the degree of inhibition of total, internal and externally bound Ni 2+ was about the same in all strains. At 50 and 500 fmol Ni 2+ per cell, the percentage of externally bound Ni 2+ was increased by cold exposure in U T E X 72 (26-52% and 23-54%), but was increased to a lesser extent or not at all in the other three strains (Table 2). O n resuspension after loading with Ni ~+, all strains released some Ni 2+ rapidly. In the light, this release soon slowed or stopped; in the dark it continued at a substantial rate (Fig. 3). The order of the four strains (dark minus light Ni 2÷ release values as percentage of intracellular Ni 2+ content) during the first 4 h of incubation was U T E X 72 (11.4) > B4(10.7) > Ni-Tol (1.7) Cu-Tol (0.8). DISCUSSION

All four strains of S. acutus f alternans studied continued to take up Ni 2+ over a 24 h treatment

period. This supports our earlier finding that Ni 2+ toxicity to photosynthesis in the four strains increased with duration of exposureJ TM Such continuous uptake has also been demonstrated in other microorganisms, but generally during shorter exposures, and followed by a decrease in uptake rate. (7'3~'38'42'45) The sensitive strain, U T E X 72, had a much higher Ni 2+ transport rate and intracellular Ni 2+ content than the three resistant strains during a 24 h exposure to 50 y M Ni 2+, a concentration that completely inhibited its growth. (15/On longer exposure, strain Cu-Tol took up twice as much Ni 2+ and strain Ni-Tol took up no more Ni 2+ than strain U T E X 72. However, the most resistant strain, B4, which could grow at this Ni z+ concentration, continued to take up Ni 2+ and grew with much higher concentrations of this metal than contained in the other strains (Fig. 1). These results are similar to the finding that a Cu-sensitive strain of the cyanobacterium Anabaena variabilis took up more Cu 2÷ intracellularly than a resistant strain at low Cu 2+

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Fig. 3. Ni release as percentage ofintraceUular Ni content (mean + SE) in the light (Q)) and in the dark (D) by four strains ofScenedesmus acutus f alternans B4, UTEX 72, Cu-Tol and Ni-Tol during a 28 h incubation in Ni-free medium. Cells were exposed to 50 fmol Ni per cell (or 50 #M Ni) for 22 h in the light before being tested for Ni release.

concentrations, but that the resistant strain could grow with much higher intracellular Cu 2+ levelsJ 111 The concentration factor (defined as/~g Ni per g dry weight//2g Ni per ml medium) in strain B4 was 9240, even when population growth was inhibited to 28% of control after 3 days of exposure. An earlier study~35)showed that in the presence of Cu, B4 could accumulate almost this much Ni. Such high Ni concentration factors have been found in only a few algae. ~43/ This confirms that B4 is a 'hyper-accumulator' of Ni. In contrast, the overall Ni 2+ transport rate of strains Ni-Tol and Cu-Tol was only 1/3 of that in strain U T E X 72 during 24 h of exposure, though Cu-Tol had a higher Ni 2+ level after 3 days (Fig. 1). Decreased Ni 2+ transport rate may reduce or prevent Ni 2+ toxicity in Cu-Tol and Ni-Tol as was reported in Anabaena cylindrica, ~5) Saccharomyces cerevisiae,~ 7) and dVeurospora crassaJ 23)We concluded that Cu-Tol and Ni-Tol are Ni

excluders, at least in the early part of their growth cycle. These findings support our hypotheses (See Introduction). All four strains were able to bind substantial amounts of Ni 2+ to their cell surfaces (Fig. 1 and Fig. 2). After 24 h exposure, S. acutus strain B4 had the highest, and U T E X 72 the lowest extracellularly bound Ni 2+, although they took up similar total amounts of Ni 2+. These differences between strains B4 and U T E X 72 suggest major differences in cell surface chemistry. A role of the cell wall in metal tolerance has been postulated in Athyrium yokoscens~ 24) and Agrostis tenuis Sibth. (4°) Although, in many cases, especially at higher Ni 2+ concentrations ( > 10 #M) and longer exposures ( > 2 h), the three resistant strains of S. acutus had significantly higher percentages of extracellularly bound vs. total Ni 2+ uptake, strain U T E X 72 could also bind large amounts of Ni 2+ on its cell

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surface. It is true that strain B4 has a much higher in the dark than under fight even after a short extracellular binding capacity than the other three incubation. However, in strains Ni-Tol and Custrains in the presence of 500 # M Ni 2+ (Fig. 2), but Tol, the difference between fight and dark Ni 2÷ at this concentration its growth would be completely release was only detected after 2 h and 6 h incuinhibited. These findings suggest that extracellular bations, respectively (Fig. 3). This suggests that in B4 binding of Ni 2+ contributes only slightly to Ni-resist- and U T E X 72, Ni 2÷ is released by passive diffusion which is counteracted immediately by a rapid active ance in the three resistant strains. In all strains, Ni 2÷ transport had a strong positive Ni 2+ uptake in the fight, while, in strain Ni-Tol, and especially, in strain Cu-Tol, there is a delay in onset linear correlation with external Ni 2+ concentration or dosage used. The unsaturated Ni 2+ uptake with of active Ni 2+ transport or possibly an enhanced increase in Ni dosage may be due to the relatively Ni 2+ release. Since B4, the most Ni-resistant strain of S. acutus, narrow range of Ni ~+ concentrations used in this study, since saturation of Ni 2+ uptake has been accumulates Ni, our future work will deal with intrashown in strains ofNeurospora crassa at external Ni 2+ cellular binding substances or structures which might cause intracellular detoxification of this heavy concentrations > 0.6 m M after 2 h of exposure./23/ Kinetics of Ni 2+ uptake have not been as well metal. Such agents could include citric, malic and studied in algae as in bacteria./~2/We did not deter- other organic acids which are active in Ni-hypmine the Vm~, of total Ni 2+ uptake and Ni 2+ trans- eraccumulating plants, (4'9'13'21'2~) histidine which port in the four strains. However, the velocities of complexes Ni 2+ in higher plants/2°~ and yeast,/16'~8/ Ni 2+ transport at dosage of 50 fmol Ni 2+ per cell metal-binding peptides and proteins which con(0.175 for strain U T E X 72, 0.097 for B4, 0.057 for tribute to metal tolerance in algae/2'~°/ and other Cu-Tol, and 0.057 for Ni-Tol) were in a similar cells, and polyphosphate granules, which may be range t o Vma x values for Neurospora crassa (0.067)/23/ involved in metal resistance in some algae and and Methanobacterium b~yantii (0.024),/14/but not for cyanobacteria.(11.31,41) Rai et aL 1261found that a Cu Anabaena cylindrica (3.7 x 10-4),/5/ and Methanothrix tolerant strain ofAnabaena doliolum produced a larger concilii (23) (~1 (all figures as/tmol Ni g-1 dry weight lipid fraction even in the absence of the metal, lost less K + and Na + in the presence of the metal, and min-1). The inhibitory effect of low temperature and took up less Cu 2+ than the wild type strain. Their darkness on Ni 2+ transport in all four strains suggests results suggest that a change in permeability of that Ni 2+ transport in all these strains was energy- plasma membrane may be involved in Cu-tolerance in Anabaena doliolum. The fact that strains Cu-Tol dependent, though some energy-independent transport was also present. Energy-dependent Ni 2+ and Ni-Tol are to some extent Ni excluders should transport systems have been identified in a dia- provide interesting systems for studies of differences tom, (~2/ fungi, (23) higher plants (~/ and many in plasma membrane between resistant and senbacteria. (5'6'39'45'46) In contrast, Ni 2+ transport in sitive strains of Scenedesmus acutusf alternans and their brown cells of Mercenaria mercenaria 147) and in the functions in Ni-resistance. bacteria Methanobacterium bryantii, 1141 Azotobacter chroococcum 1251and Methanothrix conciliz~31 appeared to Acknowledgements~This work was supported by a grant be energy-independent. Both dark and cold from the Natural Science and Engineering Research exposure increased the Ni 2+ adsorption relative to Council of Canada to DJ.K. total Ni 2÷ uptake in strain U T E X 72, presumably because adsorption is less energy-dependent than internal transport. REFERENCES Alcaligenes eutrophus has a very efficient plasmid1. Aschmann S.G. and Zasoski RJ. (1987) Nickel and encoded energy-dependent efflux mechanism rubidium uptake by whole oat plants in solution involving a specific Ni2+/H + antiporter. (28'29/There culture. Physiol. Plant. 71~ 191-196. was little evidence for active Ni 2+ release in our 2. Bariaud A. and MestreJ.C. (1984) Heavy metal tolstrains of S. acutus. All released some intracellular erance in a cadmium resistant population of Eug/ena Ni 2+ when placed in Ni-free medium in the dark. gracilis. Bull. Environ. Contam. Toxicol. 32~ 597-601. In strains B4 and U T E X 72, more Ni 2÷ was released 3. Baudet C., Sprott D.G. and Patel G.B. (1988)

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