Changes in the phytochelatin levels in the freshwater green alga Stigeoclonium tenue exposed to heavy metal mixture at different pHs, bicarbonate and suspended matter content

Barbara Pawlik-Skowrońska, Institute of Ecology, P.A.S., Experimental Station, Niecala 18, 20-080 Lublin, Poland; e-mail: pawlik@golem.umcs.lublin.pl

Abstract

Production of phytochelatins (PCn) in Stigeoclonium tenue in response to heavy metal mixture under various environmental conditions was studied. S. tenue exposed to the mining water containing different heavy metals (17 μM), mainly Zn (15 μM), produced phytochelatins (PC2 - PC3). The total PC level was about 600 nmol SH/ g dry wt. Acidification of the mining water to pH 6.8 caused a significant increase of the concentration of the labile Zn forms in the water, the total PC level increase and the appearance of the longer chain PC₄ in the alga. In a non-complexing solution of pH 8.2, the heavy metal mixture (similar to those in mining water) induced in S. tenue higher PC production than at pH 6.8. Increased concentrations of bicarbonate in the heavy metal solution resulted in the increased PC level, while the addition of particulate matter resulted in the decrease of the total PC level and PC₄ disappearance in the heavy metal exposed alga.

Introduction

The occurrence of the filamentous green algae belonging to the genus Stigeoclonium in freshwaters polluted with a number of heavy metals have been reported (Say and Whitton, 1981; Takamura et al.,1990; Pawlik-Skowronska et al., 1999). Stigeoclonium species or ecotypes which accumulate heavy metals from the polluted water and survive under chronic metal exposure seem to be metal-tolerant. As reported for some green algae and diatoms (Takamura et al.,1989) the alga population isolated from the heavy metal polluted water reveals higher metal-tolerance than the population from unpolluted environment. It has been assumed that phytochelatins ( sulfur-rich oligopeptides of the general structure γ (Glu-Cys)n-Gly, n = 2-11 (Zenk, 1996), synthesized in response to the excess of heavy metals, are involved in the heavy metal homeostasis and detoxification in higher plants, some fungi and eukaryotic algae. In polluted freshwaters usually not one but a number of heavy metals of different concentrations can occur. The aim of this paper was to study the phytochelatin production in the alga Stigeoclonium tenue exposed to the mixture of heavy metals under different environmental conditions like pH, bicarbonate and suspended particulate matter contents .

Methodology

Stigeoclonium tenue Kűtz. isolated from an unpolluted lake water and kindly donated by Dr. J. Simons form the Free University of Amsterdam, was cultivated in the liquid nutrient medium Wood's Hole.

The ability of S. tenue to cope with 15 μM Zn was studied in the liquid nutrient medium (pH 7.5) by the determination of the biomass and chlorophyll content during 8-day experiment. To determine the phytochelatin production, the alga was exposed for 17 h to the mining water (collected from a mine drainage stream in Poland) containing heavy metals (17 μM) or the similar heavy metal mixture (17 μM ) prepared in the non-complexing buffer solution (5 mM Hepes/NaOH) of different pH values (5 - 8.2), in the light, at the temperature 23° C. The mining water originated from the mine drainage stream in southern Poland and contained 15 μM Zn, 0.85 μM Cu, 0.25 μM Pb, 0.14 μM Cd.

In the carried out experiments NaHCO₃ (p.a.) and the particulate matter collected from the mining water were used.

Algal material were collected by filtration and extracted by homogenizing in the ice-cold 5% SSA + DTPA. The fresh extracts were immediately assayed by HPLC. Thiol-containing peptides were determined using post-column derivatization with DTNB and absorbance measurements at 412 nm. Identification of individual PCs was based on the comparison of their retention times with those of the standard PC samples from Silene cucubalus (kindly donated by Prof. M. Zenk).

The total concentrations of metals was determined using AAS and the labile forms of heavy metals by anodic stripping voltammetry.

Results

The studied strain of S. tenue does not seem to be Zn-tolerant because the 8-day exposure to 15 μM Zn under laboratory conditions caused the chlorophyll content decrease by 45% compared to the control. S. tenue is able to produce phytochelatins in response to heavy metals contained in the mining water (Table 1). The total level of phytochelatins as well as the composition and the concentrations of phytochelatins changed significantly with the pH lowering of the mining water. In the water of pH 8.2 the alga produced only PC₂ and PC₃ ( the total PC level was about 600 nmol SH/ g dry wt), while in the acidified water of pH 6.8 the total level of phytochelatins increased 2.8-fold and the longer chain oligopeptide PC₄ also appeared. The acidification of the water caused an essential increase of the labile forms of Zn from 3.4 μM at pH 8.2 to 14 μM at pH 6.8, and no change of the Pb speciation (Table 1). No labile forms of Cd and Cu were detected in the water. However, in the alga exposed to the similar heavy metal concentrations in the non-complexing buffer, the phytochelatin level increased significantly with the external pH increase (Table 2). When the heavy metal solution (pH 8.2) was enriched with bicarbonate to the levels equal to the bicarbonate concentrations in acidified and alkaline mining water (2.5 mM and 5 mM, respectively), the PC level in the exposed alga increased significantly as compared to the non-enriched solution (Table 2). The addition of particulate matter to the heavy metal solution resulted in the decreased level of PCs in S. tenue (Table 2).

Discussion

Some populations of S. tenue are able to grow at higher heavy metal (especially Zn) concentrations, accumulate Zn and other heavy metals, and seem to be Zn-resistant (Silverberg, 1975; Kelly and Whitton, 1989). The mechanism of metal-resistance in algae is not clear, although there are some explanations concerning some higher plants and water macrophytes i.e. intravacuolar binding of zinc by organic acids or histidine (Hermens, 1993). It is assumed that phytochelatins are heavy metal complexing agents involved in the heavy metal detoxification (Zenk, 1996) They can traverse vacuolar membrane, therefore the role of thiol peptides in the complex resistance phenomenon should not be excluded. As observed under laboratory conditions (nutrient medium), the studied strain of S. tenue, isolated from unpolluted water, was rather sensitive to 15 μM Zn (the total zinc concentration in the studied mining water). The studied alga respond to the increased heavy metal bioavailability in the mining water ( a result of the pH lowering of the water) by fast, increasing synthesis of different oligomers of phytochelatins (PC₂ - PC₄). The pH change of the water was responsible for the significant change in the zinc speciation, leading to the increase of the more available, labile metal forms. It seems that the observed higher production of PCs in the alga exposed to the mining water at the lower pH reflected mainly the increased zinc bioavailability to the alga. Also the appearance of the longer chain oligopeptide PC₄ (of the higher metal complexing capacity) suggested that at the lower pH more metal entered the alga cells. The observation of Harding and Whitton (1977) concerning the decreased Zn toxicity to the population of S. tenue with a rise in pH from 6.1 to 7.6 is in agreement with the presented results suggesting the lower zinc availability in the rich natural water of alkaline pH. However, the observed increased production of PCs with the pH increase of the metal solution in the non-complexing buffer can suggest that the algae growing in the alkaline (but non-complexing environment) can take up more metal than at acidic conditions and/or that the PC production is more efficient . There are reports (Skowroński, 1986, Harrison et al., 1986) showing the higher heavy metal uptake at the neutral or slightly alkaline pH than at the acidic one. However, in the alkaline water the concentration of bicarbonate can also essentially increase compared to pH 6.5 (Rybova et al., 1991). In the presented study the bicarbonate content in the mining water of pH 8.2 was twice as high (5 mM) as that of pH 6.8 (2.5 mM). The observed increase of the PC production in the alga exposed to heavy metal at the higher bicarbonate concentration suggests that S. tenue is able to produce phytochelatins very efficiently under conditions optimal for its growth , i.e. at the alkaline pH and high bicarbonate concentration in water. The availability of heavy metals in aquatic environment depends also on the inorganic and organic ligands as well as on the content of suspended particulate matter (Santschi, 1988). The observed lower PC production in S. tenue in the presence of suspended matter in comparison with the solution free of suspension seems to reflect the lower metal availability to the alga cells. The obtained results suggest that the low PC level observed in S. tenue exposed to heavy metals contained in the complex, hard, alkaline mining water was a consequence of the limited metal availability under such conditions. However, the alkaline pH and high bicarbonate content can simultaneously enhance the PC synthesis and hence the defence process in the heavy metal exposed S. tenue.

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Table 1. The changes in the phytochelatin content in S. tenue and the labile forms of metals in the mining water, depending on the water pH.

pH of the mining water	PC content (nmol SH/g dry wt)	labile Zn (μM)	labile Pb (μM)	labile Cd (μM)
8.2	604 ± 49	3.4	0.22	nd
6.8	1682 ± 110	14.0	0.22	nd

Table 2. Phytochelatin levels in S. tenue exposed to the heavy metal mixture (17 μM) in non-complexing solution at different pHs, bicarbonate and suspended matter content.

exposure conditions		PC level (nmol SH/g dry wt.)
pH	6.8	524 ± 38
	8.2	1544 ± 84
bicarbonate content in solution (mM)	0.008	595 ± 48
	5.0	1623 ± 98
suspended matter in solution (mg/l)	0.0	1507 ± 101
	6.0	748 ± 32