Uptake and metabolism of arsenate by anexic cultures of the microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum
Introduction
Microalgae (phytoplankton) are key contributors to arsenic cycling in the marine environment primarily as a food source for higher organisms (Sanders et al., 1989, Edmonds et al., 1997). The formation of arsenite (As(III)), methylarsonate (MA) and dimethylarsinate (DMA) are positively correlated with primary productivity (Andreae, 1978, Andreae, 1979) in oceans and phytoplankton are considered to be responsible for the greater proportion of these arsenic species present in marine waters (Edmonds and Francesconi, 1998). Microalgae have been examined in food chain studies, primarily to elucidate the formation of arsenic species such as arsenobetaine (AB) in higher organisms (Sanders et al., 1989, Lindsay and Sanders, 1990, Edmonds et al., 1997).
Arsenic is thought to be taken up by microalgae from seawater in the form of arsenate (As(V)) via the phosphate transport systems located in cell membranes and converted to As(III) as As(V) is known to interfere with metabolic processes associated with phosphorylation (Slater, 1963). Arsenic uptake in microalgae is often examined under arsenic concentrations far exceeding environmental concentrations found in uncontaminated environments (Planas and Healey, 1978, Sanders and Windom, 1980, Cullen et al., 1994), which can influence total arsenic concentrations and the proportions of inorganic and organic arsenic species. Moreover, the presence of phosphate in microalgae culture studies has been shown to have varying effects on the uptake of arsenate either by reduced arsenic uptake in phosphate replete conditions (Sanders and Windom, 1980) or concomitant uptake of arsenic and phosphate (Andreae and Klumpp, 1979). It is evident that the ratio of As/P influences arsenic concentrations in marine microalgae and the response is not consistent across all phytoplankton species.
Marine microalgae have been shown to metabolise inorganic arsenic by forming methylated arsenic species, MA and DMA (Andreae, Andreae and Klumpp, 1979, Sanders and Windom, 1980, Sanders, 1979, Wrench and Addison, 1981, Cullen et al., 1994, Edmonds et al., 1997, Yamaoka et al., 1999). Previous studies on the uptake of arsenic by microalgae have reported that arsenic species are distributed between chloroform–methanol-soluble, water-soluble and insoluble (residue) cellular components (Lunde, 1973, Andreae and Klumpp, 1979; Wrench et al., 1981). Earlier studies have reported on the proportion of inorganic/organic arsenic present in microalgae (Sanders and Windom, 1980) on the assumption that the organic arsenic species comprised simple methylated compounds MA and DMA. However, base digestion of aqueous extracts of arsenic species in marine algae has resulted in the increase of MA and DMA indicating the presence of more complex arsenic species (Andreae and Klumpp, 1979). The formation of arsenoribosides has subsequently been detected in the diatom Chaetoceros concavicornis (Edmonds et al., 1997) and freshwater algae Chlorella vulgaris (Murray et al., 2003), Chlorella sp. (Levy et al., 2005) and Monoraphidium arcuatum (Levy et al., 2005), yet the formation of arsenoribosides across all classes of marine microalgae as a major mechanism of metabolising arsenic has not been established. Further identification of the organoarsenic compounds has not occurred either due to concentrations being below detection limits or as a limitation of the analytical techniques used. It has been speculated that microalgae sequester arsenic species into the lipids of cells (Lunde, 1968, Irgolic et al., 1977, Wrench and Addison, 1981). The lack of suitable techniques for arsenic lipid determination has hampered research in identifying arsenolipids in marine algae with detection and identification of arsenic lipids occurring mostly in marine macroalgae and marine animals or animal products (Morita and Shibata, 1988, Hanaoka et al., 1999, Hanaoka et al., 2001, Devalla and Feldmann, 2003, Kohlmeyer et al., 2005, Schmeisser et al., 2005). Residue arsenic, the portion of arsenic not accounted for in lipid and water-soluble components of algae, is thought to be bound to insoluble constituents of cells (Kuenhelt et al., 2001). Additionally, the induction of phytochelatins (PC) in microalgae as a response to As(V) exposure supports the binding of arsenic to thio-groups such as glutathione and PCs (Pawlik-Skowrońska et al., 2004, Morelli et al., 2005).
To gain a more comprehensive understanding of arsenic uptake and metabolism in marine microalgae across all biochemical fractions of the cell, axenic cultures of the microalgae species, Dunaliella tertiolecta (green microphyte) and Phaeodactylum tricornutum (diatom) were grown under different phosphorus concentrations commonly used in laboratory cultures with arsenic concentrations that are typically found in uncontaminated marine environments. These two species were chosen as green microphytes and diatoms are two major classes of oceanic phytoplankton. A secondary objective was to determine whether phosphate concentrations found in uncontaminated marine environments influence uptake and metabolism of arsenic in these species of marine microalgae.
Section snippets
Stock culture maintenance
Axenic cultures of D. tertiolecta and P. tricornutum obtained from the Centre for Analytical Chemistry (CSIRO, Lucas Heights Science and Technology Centre, NSW, Australia), were transferred into separate acid washed (2% HNO3) Erlenmeyer flasks containing 100 mL of fresh, sterilised f/2 medium (Guillard and Ryther, 1962) for D. tertiolecta and 50 mL of f/2 medium for P. tricornutum. Cultures were maintained at 21 °C in an incubator at approx 4000 lux on a 12:12 h light:dark cycle. To maintain
Growth of D. tertiolecta and P. tricornutum in batch cultures
Lower growth for D. tertiolecta and P. tricornutum was found for the medium containing phosphorus at f/10 compared to the nutrient media at f/2 and f/5. Cell numbers indicated exponential growth until day 5 and that cultures were entering stationary phase by day 7 (Fig. 1). Lower growth of f/10 phosphate cultures was more pronounced in D. tertiolecta than in P. tricornutum.
Arsenic uptake by microalgae
A significant difference was seen in total arsenic concentrations between phytoplankton species (Table 2) with higher
Uptake of arsenic by microalgae
Total arsenic concentrations found in D. tertiolecta are similar to arsenic concentrations found in this species by Sanders et al. (1989) grown under ambient seawater arsenic concentrations (∼ 1.0 μg L− 1) and phosphorus concentrations equivalent to f/5 and f/10 used in this study (Lindsay and Sanders, 1990). Arsenic concentrations of P. tricornutum were significantly lower (1.62–2.08 μg g− 1 As) compared to D. tertiolecta (13.3–14.5 μg g− 1 As) but are similar to arsenic concentrations found for
Acknowledgments
We thank Frank Krikowa for assistance with chemical analyses. Merrin Adams and Jenny Stauber for provision of microalgae cultures. D. Thomson was supported by an Australian Postgraduate Award. S. Foster was supported by a University of Canberra Vice Chancellors award.
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