Environ. Sci. Technol. 1994, 28, 1577-1581
Monitoring the Cellular Response of Stichococcus bacillaris to Exposure of Several Different Metals Using in Vivo 31P NMR and Other Spectroscopic Techniques Weixlng Zhang and Vahid Majidi' Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506
The cellular response of a green algae, Stichococcus bacillaris, to sudden exposure of Zn2+,Mn2+,Cd2+,and Cu2+was investigated. The algae adsorbed these metal cations on the cell wall initially, and the adsorption equilibrium was established within 3 min. Later, some of the adsorbed metals were either transported inside the algae or released into the solution. The transport of metals inside the algae was demonstrated by their interaction with intracellular polyphosphate. The release of the adsorbed metals into solution is probably the result of detoxification actions. The interaction of copper with algae was found to be unique in many ways. For instance, Cu2+was adsorbed more efficiently than the other metals investigated. The algae detoxification process for copper involved the intervention by both polyphosphate and protein from the algae. At high copper/algae ratio, the algae cells were lysed and unable to excrete the toxic metal. Introduction
The toxicity of heavy metals to algae has been investigated by numerous researchers (1-4). It is generally believed that the toxic metals initially attack the algae cell wall by binding to it, and then they are transported inside the cell. These metals exert their toxic effects by competing with essential metals for active enzymes or biologically active groups. Thus, an understanding of the toxicological responses of algae to heavy metals is paramount in order to predict their influence on aquatic systems. The identity of the functional groups that are responsible for metal uptake on the cell wall is unclear in most cases. Gardea-Torresdey and collaborators (5) used chemical modification of carboxyl groups on the cell wall of nonliving microorganisms to investigate the binding of metals to five strains of algae. It was demonstrated that the binding capacity for Cu2+and AP+was reduced after esterification of the carboxyl groups on the cell wall of algae. Thus, they concluded that carboxyl groups on the cell wall of algae are responsible for a great portion of copper and aluminum binding. Majidi and co-workers (6,7) examined the binding of cadmium to Stichococcus bacillaris with nuclear magnetic resonance (NMR) technique and concluded that the binding sites for cadmium were carboxylate groups based on the chemical shift of bound cadmium in l13Cd NMR spectra. Other researchers have examined the algae cells with electron microscopy and X-ray microanalysis after exposure of the cells to toxic metals (8-14). Inorganic polyphosphates are long-chained linear polymers of orthophosphate found in a wide variety of microorganisms, such as bacteria, fungi, yeast, and algae
* Author to whom correspondence should be addressed; e-mail address:
[email protected]. 0013-936X/94/0928-1577$04.50/0
0 1994 American Chemical Society
(15-18). Polyphosphates may function as an energy reservoir since the phosphoanhydride bond in polyphosphate has a free energy close to that of ATP (17). Polyphosphates are generally associated with K+, Na+, Mgz+, and Ca2+,which are considered osmotically inert sinks for metallic cations required for cellular metabolism (10, 19). 31PNMR spectroscopy is an invaluable tool for probing metabolic processes in living organisms. Measurement of pH and metabolite concentrations (20-24), evaluation of the polyphosphate concentrations inside the algae (25, 261, study of polyphosphate metabolism (27-291, and determination of polyphosphate chain length (30,31) are a few examples highlighting the utility of 31PNMR. The purpose of this study is to examine the dynamic response of a green algae, S. bacillaris, to the exposure of several different metals, including both toxic and essential elements in relatively large quantities. This is accomplished by monitoring the 31PNMR signals inside the algae cells along with the metal and metabolite concentrations outside the cells. Experimental Section
Cell Culture. The unialgal species of S. bacillaris (UTEX 419) was obtained from Department of Botany, The University of Texas a t Austin. Algae cells were cultured in a Bristol's medium modified from Wong and Beaver (32). The culture was stirred with filtered air and subjected to 16 h of light followed by 8 h of dark cycles. The organism was harvested by centrifugation (1OOOg for 5 min) and washed twice with deionized water. NMR Measurements. In vivo 31PNMR experiments were carried out in the dark at 20 OC using a VXR-400s spectrometer (Varian Associate Inc., Palo Alto, CA) operating at 161.9 MHz. Field-frequency lock was employed by placing a sealed capillary containing DzO into the 5-mm NMR tubes which were spun a t 20 rps. A rf pulse width of 40" at a repetition time of 0.5 s was applied. In order to increase the signal-to-noise (S/N) ratio, a 10Hz line broadening function was employed during the transformation of the free induction decays (FIDs). Phosphoric acid (85 % ) was used as an external reference. Positive chemical shift values denote higher frequencies or lower fields. Phosphorus and Metal Analysis. Phosphorus concentration (from inorganic orthophosphate) in solution was determined by the molybdenum blue method modified from Murphy and Riley (33). In this method, a daily prepared solution mixture containing 2.5 M HzS04,3.0 X M ammonium molybdate, 3.0 X M ascorbic acid, and 3.0 X lo4 M potassium antimonyl tartrate is used as a colorimetric reagent. Spectrophotometric measurements were made approximately 10min after 1.0 mL of the above solution was mixed with 4.0 mL of a sample solution. The absorbance was determined at 882 nm using an AVIV 14DS Environ. Sci. Technol., Vol. 28, No. 9, 1994
1577
UV-Vis-IR spectrophotometer (Lakewood, NJ). Total phosphates (orthophosphate and polyphosphate) were determined using the same method followingthe hydrolysis of polyphosphate in 0.5 M H2SO4 at 100 "C for 4 h. Polyphosphate does not react with the colorimetric reagent, thus the amount of polyphosphate in solution was calculated by taking the difference between the total phosphate and the orthophosphate concentrations. For the analysis of polyphosphate and inorganic orthophosphate inside the algae, the freeze-dried algae cells were suspended in 1.0 M sulfuric acid and heated at 100 "C for 8 h, and the released orthophosphate in solution was subsequently analyzed by spectrophotometry. Metal concentrations were determined by graphite furnace atomic absorption spectroscopy (GFAAS) using a Perkin Elmer 3100 atomic absorption spectrometer (Norwalk, CT). Protein Determination. To evaluate the protein content in solutions, 1.0 mL of the solutions was freezedried and hydrolyzed in 0.20 mL of 6.0 M HC1 for 24 h at 110 "C. The remaining HC1 was removed under vacuum, and amino compounds were then analyzed using the ninhydrin colorimetric method, modified from Magne and Larher (34). Glutamic acid was used as the standard amino compound.
Results and Discussion Binding of Metals to Intracellular Polyphosphate. In order to evaluate the response of 5'. bacillaris to selected metal cations, 40 mg (dry weight) of algae was suspended in 1.0 mL of phosphorus-deficient culture medium for phosphorus NMR studies. The resulting 31PNMR spectra are presented in Figure 1. The resonances observed at 2.2,1.6,0.7,and -22.2ppm are from cytoplasmic phosphate, vacuolar phosphate, phosphodiester, and interior polyphosphate, respectively (26). In line A, the resonance intensity from the interior polyphosphate was very strong, indicating that the algae cells had accumulated a large amount of polyphosphate. The absence of resonances from terminal phosphate groups (around -6 ppm) indicates that the polyphosphate chains are very long. Based on the fact that the signal to noise ratio of the interior polyphosphate resonance is about 100,the average chain length of the polyphosphate was estimated to be at least 200 phosphate units (30). It is also possible that the lack of terminal phosphate signals is due to the binding of these phosphate groups to the cell wall or the cytoplasmic membrane (26). After the addition of 5.0 X M Cu2+ to the algae suspension for 5 min, the intensity of polyphosphate resonance was reduced (line B), and the resonance signal disappeared after 2 h (line C) due to the binding of paramagnetic copper cations to the polyphosphate. After 7 h (line D) the resonances from vacuolar phosphates were broadened, indicating the sequestering of copper by these phosphorus-containing bodies. The influence of copper at lower concentrations and other paramagnetic metals (Fe2+,Mn2+,and Co2+)as well as diamagnetic zinc on polyphosphate resonance was also investigated with 31P NMR. In order to minimize the osmotic pressure and the effect of paramagnetic metals (Fe2+,Mn2+,and Co2+etc.) in the original culture medium, the algae were suspended in a salt solution containing M KN03, 3.0 X lo4 M MgSO4, and 2.0 X only 3.0 X 10-3 M NaC1. 3lP NMR spectra of algae suspensions (40 1578
Environ. Scl. Technol., Vol.
28, No. 9, 1994
4
C
4
2
0
-2
-4
-6
-8
-12
-16
-20
ppm
Figure 1. 31P NMR spectra of S. baclllaris. Line A was obtained immediately after 40 mg (dry weight) of algae was suspended in 1.O mL of phosphorus-deficient culture medium. Lines B-D were obtained 5 min, 2 h, and 7 h after the addition of 5.0 X M CuCI,, respectively. The number of transients accumulated was 1024. The assignments of resonance peaks are (1) cytoplasmic phosphate, (2) vacuolar phosphate, (3) phosphodiester, and (4) interior polyphosphate.
Table 1. Influence of Selected Metal Cations on Peak Height (PH) and Ratio of Peak Height to Peak Area (PH/ PA) of Interior Polyphosphate Resonance in 3lP NMR Spectra as a Function of Time after Addition of Specified Metals
3 control
time (min) 80 180 420
PH 100 101 PH/PA 1.00 1.01 PH 92.7 93.0 2.0 X lo3 M ZnSO4 PH/PA 1.01 1.01 PH 94.2 91.9 2.0 X loa M CoClz 1.01 1.01 PH/PA PH 99.2 88.2 2.0 X 10" M FeS04 PH/PA 1.03 0.94 PH 96.9 92.1 2.0 X lo4 M MnCl2 PH/PA 1.03 0.95 2.0 x 104 M cuciz PH 83.3 45.2 0.96 0.67 PH/PA 93.6 96.4 2.0 X 10-3 M ZnSO4 + PH 2.0 X 10" M CoClz PH/PA 1.02 1.03 2.0 X 10-3 M ZnSO4 + PH 95.3 92.9 0.98 2.0 X 104MFeS04 PH/PA 1.02 2.0 X M ZnSO4 + P H 92.5 89.0 2.0 X 1 0 4 M MnCl2 PH/PA 0.99 0.98 2.0 X 10-3 M ZnSOd + PH 85.1 38.5 0.65 2.0 X 10" M CuClz PH/PA 0.93
96.9 1.04 97.2 1.05 86.5 0.98 84.6 0.90 90.9 0.95 52.9 0.78 96.5 1.07 92.2 1.00 92.1 1.00 30.6 0.60
93.5 1.03 98.5 1.18 74.0 0.93 78.2 0.94 83.9 1.01 71.0 1.03 89.3 1.13 94.0 1.11
91.5 1.11
29.0 0.65
mg of dry algae/mL) were recorded a t different times. The peak height (PH) and the ratio of peak height to peak area (PA)of polyphosphate resonance in the NMR spectra were measured and are listed in Table 1. The values of PH/PA are nearly the same for the control sample in all M ZnS04, measurements. In the presence of 2.0 X PH/PA increased gradually in a 7-h period, while in the
presence of 2.0 X lo4 M Co2+,PH/PAdecreased gradually. In the case of Fe2+, Mn2+,and Cu2+,the PH/PA ratio decreased during the first several hours and reached a minimum, after which time this ratio recovered and returned to its initial value for Mn2+and Cu2+. After the addition of zinc into the algae suspension along with the paramagnetic metals, PH/PA increased for Co2+,Fe2+, and Mn2+and decreased for Cu2+. We know that Zn2+ is a diamagnetic cation. The narrowing of the polyphosphate signal was ascribed to the transport of Zn2+ into the algae cell, where Zn2+replaced some of the paramagnetic metals that were originally bound to polyphosphates. After the paramagnetic metals were removed, the polyphosphate signal became narrower. This result indicates that some paramagnetic metals from the nutrient solution were associated in polyphosphate bodies. Cobalt is a paramagnetic cation, its interaction with polyphosphate will cause broadening of the polyphosphate resonance and decrease in PH/PA. When Co2+ was added into the algae suspension,the peak height (PHI as well as the ratio of peak height to peak area (PH/PA) from polyphosphate decreased slowly. Both phenomena indicated the transport and the interaction of the paramagnetic cobalt with polyphosphate. In the case of other paramagnetic metal cations (e.g., Fe2+,Mn2+,and Cu2+), some of these cations were slowly transported inside the algae where they interacted with polyphosphate during the early stages, as demonstrated by the decrease in PH/ PA ratio. Later, following a minimum, the PH/PA ratio from polyphosphate resonances began to increase. The increase in PH/PA was most likely due to the release of the paramagnetic cations from the polyphosphate bodies. The addition of zinc prevented the access of Co2+,Fe2+, and Mn2+ to the polyphosphate bodies, but zinc did not inhibit the interaction between copper and polyphosphate. Furthermore, the copper cations that were bound by polyphosphate were not easily released in presence of zinc. Adsorption, Transport, and Desorption of Metals by Algae. The interaction between algae cells and metal cations was further studied by monitoring the metal and metabolite concentrations outside the cells. The algae M KN03, was suspended in a solution containing 3.0 X 3.0 X 10-4 M MgSO, and 2.0 X M NaCl at a density of 4.0 g of algae/L, and the suspension was illuminated. After the addition of the specified metals, a portion of the algae suspension was removed and centrifuged at different time intervals. The metal and metabolite concentrations in the supernatant were analyzed and the results are presented in Figures 2-5. When 4.0 X M Zn2+ was added to the algae suspension for 3 min, about 82 % of the Zn2+cations were adsorbed on the algae as demonstrated by the drastic decrease in the free zinc concentration (Figure 2). Then, the concentration of zinc in solution further decreased during the first 2 h. This phenomenon has been observed by other researchers and is attributed to the transport of zinc inside the algae (35,36). Later, the zinc concentration in solution began to increase. After 20 h, only 75 % of the initially adsorbed zinc remained on the algae. When either copper or cadmium was added along with zinc, the amount of zinc adsorbed decreased due to competitive binding, and the transport of zinc into the algae was inhibited. After the algae cells were mixed with a solution containing 4.0 X lo4 M Mn2+for 3 min, 68% of the metal cations were taken up by the algae (Figure 3). Then, the
53
0.22
P
ti
Y
8 P a
0.18
3
8
e;
.Ci
a
0.14
W
0
8 4
z8
0.10
8
U
0.06
1
10
100
1000
10000
Time (mid Flgure 2. Concentration of zinc in solution as a function of time. The initial metal concentrations were (0)4.0 X M Zn2+, (A)4.0 X M Zn2+and 1.0 X M Cu2+, and (0) 4.0 X M Zn2+and 1.0 X M Cd2+.
E 0.24
-
P
8 .* Y
a
3
8
e; .*
3 W
0
8 4
z8
8
U
Time ( m i d Flgure 3. Concentration of manganese in solution as a function of time. The initial metal concentrations were (0)4.0 X M Mn2+and (0) 4.0 X lo-' M Mn2+ and 1.0 X M Cd2+.
concentration of manganese in solution decreased gradually and reached a minimum after 2 h. The decrease in manganese concentration was, again, due to the transport of metal cations into the algae. The addition of cadmium reduced the amount of bound manganese due to competitive binding on the cell wall, but the transport of Mn2+ was not affected. The algae took up 94 % of the cadmium after 1.0 X lo4 M Cd2+was present in the solution for 3 min (Figure 4). When zinc or manganese was present along with the cadmium, the amount of bound cadmium was reduced due to competitive binding. The competition effect was stronger for zinc than that for manganese. In the case of copper, 99% of the metal cations were adsorbed by the algae after 1.0 X lo4 M Cu2+ was mixed with the algae for 3 min (Figure 5). It is interesting to note that some phosphate and polyphosphate were also released by the algae cells as some of the adsorbed copper was released into solution. This observation indicates that polyphosphate was probably involved in the release of copper. Skowronski (35,361 studied the uptake and transport of cadmium by S. bacillaris based on the measurement of Environ. Sci. Technol., Vol. 28, No. 9, 1994
1579
Efi ."83
0.05
-
0
0.04
-
0.03
-
v
.L
3
.5
rn
u
W
0
."8 e
0.02 -
Y
1
e 8 e
0.01
-
0,
I
10
1
100
1000
10000
Time (min) Flgure 4. Concentration of cadmium in solution as a function of time. The initial metal concentrations were (0) 1.0 X M Cd2+, (A)1.0 X M Cd2+ and 4.0 X M Mn2+, and (0) 1.0 X M Cd2+ M Zn2+. and 4.0 X
1
10
1000
100
10000
Time ( m i d Flgure 5. Concentrations of copper (0),inorganic orthophosphate (O), and polyphosphate expressed as mM of orthophosphate units (A)
in solution as a function of tlme. The lnltial copper concentration was 1.0 x 10-4 M.
radioactivity from l15Cd. He demonstrated that S. bacillaris took up cadmium by means of both adsorption and energy-dependent transport, but the energy-dependent transport of cadmium was not clearly observed in our study. Since our studies focused mainly on metal toxicity at low concentrations, most of the cadmium was initially adsorbed on the algae, and the cadmium concentration remaining in the solution was toolow for an active transport process to initiate. We did, however, noticed the active transport of zinc and manganese (Figures 2 and 3). The metal adsorption and transport process can be described by the following scheme:
-
-
slow
fast
M"+ (solution)
M"' (cell wall)
M"' (inside algae)
The metal cations (M"+)in solution were rapidly adsorbed on the cell wall of algae, and this process reached equilibrium within several minutes. Some of the adsorbed metals were slowly transported inside the algae cells, freeing the binding sites on the cell wall of algae. Then more free metal cations from the solution were bound to the cell wall. In this process, the amount of metal cations 1580
Envlron. Sci. Technol., Vol. 28, No. 9, 1994
transported inside the algae is equal to the net loss of free metals from the solution. The average rate of zinc transport (expressed as the decrease in zinc concentration in the supernatant of an algae suspension with a density of 4.0 g of algae/L) was 5 X lo4 M h-l, whereas that for manganese is 1.0 X M h-l in the 2-h period following the initial adsorption. Cadmium inhibited zinc transport but not manganese. Thus, the sequence of transport inhibition is that Mn2+inhibited Cd2+transport (36)and Cd2+inhibited Zn2+transport. The crystal ionic radii of Mn2+,Cd2+,and Zn2+are 0.67,0.78,and 0.60A, respectively (37). Therefore, the metal transport does not depend on the size of the metal cations. Algae Detoxification. A unique phenomenon observed in our study was the release of the initially adsorbed metals on the algae after a period of time (Figures 2-5). Release of the bound metals led to the increase in free metal concentrations in solution. One explanation for this phenomenon is the decrease in external pH. It has been reported that the limitation of oxygen and light supply may cause the accumulation of COZ or lactate in the cytoplasm of algae and a subsequent decrease in cytoplasmic pH (22,23).Since the cytoplasm was metabolically controlled, the excess COZaccumulated in cytoplasm was released into solution and, thus, resulted in a decrease in external pH. The decrease in external pH was confirmed by monitoring the 31PNMR signal of inorganic orthophosphate outside the algae. For example, when 40 mg of algae was suspended in a 1.0 mL solution, sealed in an NMR tube, and placed in the dark for 1day, the external pH decreased by 0.3 pH unit. In this study, we used dense algae suspension (4.0 g of dry algae/L). The oxygen supply and light intensity were not sufficient for this suspension, which may have caused a decrease in cytoplasmic pH and external pH, thus facilitating the release of bound metals. However, the pH change for this experimetal condition is negligible, because the algae suspension for metal analysis was 10 times less dense than that for NMR measurement and sufficient light was supplied. The amount of metals released due to this small pH change would be insignificant. Thus, it is more likely that the release of the bound metals back into the solution is due to a detoxification mechanism. This conclusion is also supported by the data in Table 1. Another evidence for detoxification is the release of the adsorbed metals in presence of sufficient oxygen and light (Figure 6). For an algae suspension of 1.5 g of algae/L when both oxygen and light were normally supplied, the algae took up 92 % of the copper initially presented in the solution (1.0 x 10-4M). Then, some of the adsorbed copper was slowly released by the algae. When the initial copper M, a similar result concentration was reduced to 1.0 X was obtained. It is most likely that the detoxification action originated from the conformational changes of the cell wall, which reduced its affinity for the adsorbed metal cations. With the amount of toxic metal on the algae cell wall reduced, its transport into the cell is hindered, and hence, its toxic action is inhibited. For an algae suspension of 0.60 g of algae/L in 1.0 x lo4 M copper solution, the algae cells took up 71% of the copper initially, but the amount of adsorbed copper increased with time as demonstrated by the gradual decrease in relative copper concentration in the supernatant. This is probably due to the death of algae caused by copper toxicity. The dead cells took up more copper from the solution instead of releasing the adsorbed metals. Subsequently, the free
"
1
-
7
0
I
20
.
I
40
,
I
60
.
I
80
'
Takamura, N.; Kasai, F.; Watanabe, M. M. J.Appl. Phycol. 1989, 1, 39-52. Visviki, I.; Rachlin, J. W. Arch. Environ. Contam. Toxicol. 1991,20, 271-275. Twiss, M. R.; Nalewajko, C. J.Phycol. 1992,28, 291-298. Gardea-Torresdey, J. L.; Becker-Hapak, M. K.; Hosea, J. M.; Darnall, D. W. Environ. Sei. Technol. 1990,24, 13721378. Majidi, V.; Laude, D. A.; Holcombe, J. A. Environ. Sei. Technol. 1990,24, 1309-1312. Zhang, W.; Majidi, V. Appl. Spectrosc. 1993,47,2151-2156. Baxter, M.; Jensen, T. E. Protoplasma 1980, 104, 81-89. Jensen, T. E.; Baxter, M.; Rachlin, J. W.; Jani, V. Environ. Pollut., Ser. A 1982, 27, 119-127. Rachlin, J. W.; Jensen, T. E.; Warkentine, B. Arch. Environ. Contam. Toxicol. 1984, 13,143-151. Pettersson, A.; Kunst, L.; Bergman, B.; Roomans, G. M. J. Gen. Microbiol. 1985, 131, 2545-2548. Sicko-Goad,L.; Ladewski, B. G.; Lazinsky, D. Arch.Environ. Contam. Toxicol. 1986,15, 291-300. Sicko-Goad, L.; Lazinsky, D. Arch. Environ. Contam. Toxicol. 1986, 15, 617-627. Rai, L. C.; Jensen, T. E.; Rachlin, J. W. Arch. Environ. Contam. Toxicol. 1990, 19, 479-487. Harold, F. M. Bacteriol. Rev. 1986, 30, 772-794. Jensen,T. E.;Sicko,L. M. Can.J.Microbiol. 1974,20,12351239. Kulaev, I. S.; Vagabov, V. M. Adv. Microb. Physiol. 1983, 24, 83-171. Roberts, M. F. In PhosphorusNMR in Biology;Burt, C. T., Ed.; CRC Press, Inc.: Boca Raton, FL, 1987; pp 85-94. Peverly, J. H.; Adamec, J. Plant Physiol. 1978,62,120-126. Roberts, J. K. M.; Jardetzky, 0.Annu. Rev. Plant Physiol. 1984, 35, 375-386. Wray, V.; Schiel, 0.; Berlin, J.; Witte, L. Arch. Biochem. Biophys. 1985,236, 731-740. Wray, S.; Tofts, P. S. Biochim. Biophys. Acta 1986, 886, 399-405. Sianoudis, J.; Kusel, A. C.; Mayer, A.; Grimme, L. H.; Leibfritz, D. Arch. Microbiol. 1987, 147, 25-29. Kusel, A. C.; Sianoudis, J.; Leibfritz, D.; Grimme, L. H.; Mayer, A. Arch. Microbiol. 1990, 153, 254-258. Oh-hama, T.; Siebelt, F.; Furihata, K.; Seto, H.; Miyachi, S. J.Phycol. 1986, 22, 485-490. Sianoudis, J.; Kusel, A. C.; Mayer, A.; Grimme, L. H.; Leibfritz, D. Arch. Microbiol. 1986, 144, 48-54. Lundberg, P.; Weich, R. G.; Jensen, P.; Vogel, H. J. Plant Physiol. 1989, 89, 1380-1387. Kuesel, A. C.; Sianoudis, J.; Leibfritz, D.; Grimme, L. H.; Mayer, A. Arch. Microbiol. 1989, 152, 167-171. Bental, M.; Pick, U.; Avron, M.; Degani, H. Biochim. Biophys. Acta 1991, 1092, 21-28. Jacobson, L.; Halmann, M.; Yariv, J. Biochem. J. 1982,201, 473-479. Rao, N. N.; Roberts, M. F.; Torriani, A. J.Bacteriol. 1985, 162,242-247. Wong, S. L.; Beaver, J. L. Hydrobiologia 1980,74,199-208. Murphy, J.; Riley, J. P. Anal. Chim. Acta 1962,27, 31-36. Magne, C.; Larher, F. Anal. Biochem. 1992,200, 115-118. Skowronski, T. Chemosphere 1984,13,1379-1384. Skowronski, T. Appl. Microbiol.Biotechnol. 1986,24,423425. Lide, D. R., Ed. In CRC Handbook of Chemistryand Physics, 74th ed.; CRC Press, Inc.: Boca Raton, FL, 1993/1994; pp 8-9. Wood, H. G.; Clark, J. E. Annu. Rev. Biochem. 1988,57, 235-60.
I
100
Time (hours) Flgure 6. Amount of free copper in solution (percentage of total cu) as a function of time for different initial copper concentrations and different amounts of algae. The algae suspensions were stirred with water-saturatedair and illuminated at a 16-h light18-h dark cycle: (0) 1.0 X lo-' M Cu2+ and 1.5 g of algae/L, (0)1.0 X M Cu2+ and 1.5 g of algae/L, and (A) 1.O X M Cu2+ and 0.60 g of algae/L.
copper concentration in solution decreased. In this case, the detoxification process was inhibited due to the high Cu/algae ratio. Some of the cells were lysed as observed under a light microscope, while the algae cells from other experiments remained complete. The response of S. bacillaris to copper is unique in this study. As the copper cations were released, inorganic orthophosphate and polyphosphate were also released into the solution by the algae (Figure 5 ) . When 1.0 X lo4 M Cu2+was initially present in the algae suspension (4.0 g of algae/L),the amount of phosphates released in 20 h was 30% of the total inorganic phosphate and polyphosphate originally present in the algae cells. Some amino compounds were also found in the hydrolysates of the supernatant collected 20 h following the addition of copper into the algae suspension. These amino compounds were from protein since no such compounds were detected beforehydrolyzing the sample in 6.0 M HC1 for 24 h. The release of phosphate and protein is most likely related to algae detoxification. Two types of enzymes involved in polyphosphate degradation have been identified (15,17, 38). One type is polyphosphatase (also called exopolyphosphatase), which catalyzes hydrolysis at the end of an open polyphosphate chain. The other is polyphosphate depolymerase (also called endopolyphosphatase), which catalyzes the cleavage within the polyphosphate chain. The presence of both inorganic orthophosphate and polyphosphate in solution suggests that both enzymes are present in the algae cell and that they were activated by the Cu2+cation. The degraded polyphosphate along with some protein transported copper out of the cell and, thus, reduced its toxic action to photosynthesis of the green algae. Acknowledgments
The technical assistance of Claude Dungan is gratefully acknowledged. Literature Cited (1) Skowronski, T.;Pawlik, B.; Jakubowski, M. Bull. Environ. Contam. Toxicol. 1988, 41, 915-920.
Received for review December 7, 1993. Revised manuscript received May 16, 1994. Accepted May 20, 1994.' @
Abstract published in Advance ACS Abstracts, July 1, 1994. Envlron. Sci. Technol., Vol. 28,
No. 9,1994 1581
