Interactions of Metals and Protons with Algae Ray H. Crlst, Karl Oberholser, Dwight Schwartz, James Marzoff, and Darryl Ryder Messiah College, Grantham, Pennsylvania 17027
DeLanson R. Crlst * Department of Chemistry, Georgetown University, Washington, D.C. 20057
H Proton uptake by intact algal cells was found to consist of two processes: (1)a fast (95% pure. Elemental analyses by Desert Analytics gave the following C, H, N, and S percentages: for Vuucheria, 40.91,5.51,3.06,0.00; for Spirogyra, 46.10,6.10, 5.77,O.w for Oedononium, 34.09, 3.92, 1.88, 0.00. Algal stocks were stored as a compact wet mass at 0 OC. Several types of observations indicate the suitability of stored material for adsorption studies: (1)Just prior to use, samples were washed with distilled water and in-
0 1988 American Chemical Society
Environ. Sci. Technol., Voi. 22, No. 7, 1988 755
spected microscopically to verify that cells were intact; (2) A fresh sample of Vaucheria was tested for Cu adsorption after storage for 3 months; values of K for fresh and stored material (2110 f 150 and 1711 f 120 M-l) and y , (302 and 298 pmollg) were reasonably similar; (3) When fresh stock was collected, it was used in parallel experiments with stored material; at no time was any significant difference noted that could be attributed to storage; (4) A sample of Vaucheria, which has been stored for 3 months, was placed in spring water and exposed to light under conditions similar to its native habitat; over a 3-week test it maintained its usual green color and showed no cell deterioration; (5) A 10-mL wet mass of Spirogyra after 6 months of storage was placed in spring water in sunlight; after 1 h it became bright green and bloomed to the surface by bubbles of photosynthetic oxygen. It would appear that our storage conditions simulate winter months in Pennsylvania, since Spirogyra settles to the bottom of a pond in winter (dark, near freezing temperature) and blooms under a March sun. Weight Basis for Calculations Involving Cells and Cell Components. All experiments were conducted on algal samples that had been partially dried by pressing with adsorbent paper and hereafter referred to as moist samples. All results, even for cell components, are expressed per gram (dry weight) of intact alga. The following procedure was used to determine dry weight for proton studies and obtain cell components. A moist algal sample was split and part was dried to constant weight at 70 O C to give the gram of dry weight per gram of moist weight. With this ratio, results on a certain weight of another moist portion could be expressed per gram of dry weight. As an example, a 0.50-g moist sample of Vaucheria corresponded to 0.17 g dry weight. Cell components were obtained from another moist portion that was mixed with 30 mL of water and subjected to grinding by a Fisher tissue grinder. After 70 mL of water was added and the mixture allowed to settle for 15-30 min, 50 mL of the supernatant liquid, referred to as cytoplasm, could be corrected for dilution to correspond to a dry weight of alga. The bottom portion containing settled material was washed 3 times by decantation, filtered, pressed dry, and used for proton uptake experiments. This material, ca. 0.09 g of a 0.17-g (dry weight) Vaucheria sample, is referred to as cell walls and consisted of wall fragments, membranes, and any other dense matter. Microscopic observation showed transparent pieces with perhaps 2% of pigmented material. Further separation of these components was impractical considering the amounts required for analysis. However, the walls and cytoplasm were reasonably well separated as demonstrated by the absence of the characteristic slow proton uptake of the walls and unseparated suspension. In general, the weights of cell components were not measured, since they were not relevant to the point of the experiments: for 1 g of dry alga with a certain slow proton uptake, how much was due to absorption into walls and how much to reaction with cytoplasm. To answer this question, one only needed to separate the components and measure their different uptake values. Proton Reactions. A technique resembling a pH titration was developed to measure rapid surface pkoton uptake alone. Five separate suspensions at pH 7.0 were prepared, each with about 0.05 g (dry weight) of alga in 20 mL of water. Each suspension was injected with either 0.2, 0.4, 0.6, 0.8, or 1.0 mL of 0.02 M HC1. The peak of the pH recorder tracing for each suspension (see Figure 1A) then gave the solution pH before substantial proton 756
Environ. Sci. Technol., Vol. 22, No. 7, 1988
I Rapid
i
'
1
601
Time,sec
I
B
4.0L ~
I
'
Protons in
-
__
HCL Added Time
-
Protons out
NaOH Added
Flgure 1. Reactions of protons with algae. (A) A rapid proton uptake followed by slow diffuslon of protons into cells when 2.0 mL of 0.02 M HCI is added to 0.12 g (dry weight) of Vaucheria in 20 mL of water. Top curve (HCI added to distilled water) shows the response time of the electrode. (B) Reversibility of proton transport into cells. Successive 0.20-mL amounts of 0.02 M HCI were added to the above sample at about 9-s intervals. Although the solution becomes increasingly acidic, there is the slow diffusion into ceiis as indicated. When similar injections of base are added, the reverse occurs, and there is a slow diffusion of protons out of cells.
"I 40
Flgure 2. pH titration measuring surface sites of Vaucheria and Spirogyra. Each point is a separate determination In which the mi-
cromole of HCI indicated for that point was injected into an algal suspension of about 0.050 g (dry weight) in 20 mL, initially at pH 7. The pH for this point was taken from the peak reading on the recorder tracing (see Figure 1A) and represents only the rapid surface reaction.
transport into the cell. These data were used to make a pH titration curve, shown for Vaucheria and Spirogyra in Figure 2. Slopes of these curves gave the micromoles of H+ (rapid uptake) per gram of alga per pH unit.
Table I. Proton Reactions with Vaucheria and Oedogoniuma (A) First-Order Rate Constants for Slow Uptake Process
Vaucheria, k PH
cells
3.5 4.0 5.0
18.1 f 3.3 6.8 f 0.9 1.0 f 0.2
X
Oedogonium, k
lo4 s-l walls
X
cells
lo4 s-l walls
13.6 f 1.3 6.7 0.3 1.40 f 0.05
*
7.5 f 0.8
10.6 f 0.7
(B) Total Uptake at pH 4.0
Vaucheria, pmollg
fast slow total
Oedogonium, pmollg
components cytoplasm
cells
walls
374 f 10 953 f 65 1327
209 f 12 479 f 28
328 f 25 1016
components cytoplasm
cells
walls
532 f 30 5613 f 335 6145
369 f 8 4174 f 58
936 f 226 5475
total (av) 1171 f 156 5810 f 335 OAn algal suspension was taken quickly (
