Year

2009

Degree Name

Doctor of Philosophy

Department

School of Chemistry, Faculty of Science

Abstract

Microalgae are sensitive indicators of environmental change and, as the basis of most freshwater and marine ecosystems, are widely used in the assessment of risk and the development of environmental regulations for metals. However, inter-species differences in sensitivity to metals are not well understood and laboratory-based toxicity tests with planktonic microalgae are often criticised for their lack of environmental relevance. One such criticism is that algae do not exist in isolation in the field, interacting with bacteria and other algal species, as well as higher order organisms, both in the plankton and when attached to substrates in the form of biofilms. The environmental relevance of toxicity tests could be improved by having a better fundamental understanding of the interactions between metals and algae and how this relationship is affected by the presence of bacteria and biofilms. Copper was the metal of choice for this research because it is both essential and toxic to microalgae, and because it is a contaminant of concern in aquatic environments, with toxicity occurring at low µg/L concentrations. The relationship between metal-algal cell binding and copper sensitivity of marine microalgae was investigated using a series of 72-h growth rate inhibition bioassays and short-term (1-h) uptake studies (Chapter 3). A range of marine algae from different taxonomic groups were screened to determine whether copper adsorption to the cell membrane was influenced by biotic factors, such as the ultrastructure of cell walls and cell size. Minutocellus polymorphus was the most sensitive species to copper and Dunaliella tertiolecta the least sensitive, with 72-h IC50 values (concentration to inhibit growth rate by 50%) of 0.6 and 530 µg Cu/L, respectively. Copper solution-cell partition coefficients (Kd) were calculated for six species of algae on a per cell and surface area basis. The largest and smallest cells had the lowest and highest Kd values, respectively (on a surface area basis), with a general (non-linear) trend of decreasing Kd with increasing cell surface area (p = 0.026). However, no relationship was found between Kd and copper sensitivity. Interspecies differences in copper sensitivity were not related to cell size, cell wall type, taxonomic group or Kd values. Variation in sensitivity may be due to differences in uptake rates across the plasma membrane, differences in internal binding processes and/or in detoxification mechanisms between the species. A more in-depth study investigated copper adsorption, internalisation, exudate production and changes in cell ultrastructure in three algal species with differing sensitivities to copper (Chapter 4). The diatom Phaeodactylum tricornutum was particularly sensitive to copper, with a 72-h IC50 of 8.0 µg Cu/L, compared to the green algae Tetraselmis sp. (72-h IC50 47 µg Cu/L) and Dunaliella tertiolecta (72-h IC50 530 µg Cu/L). At these IC50 values, Tetraselmis sp. had much higher intracellular copper concentrations (1.97 ± 0.01  10-13 g Cu/cell) than P. tricornutum (0.23 ± 0.19  10-13 g Cu/cell) and D. tertiolecta (0.59 ± 0.05  10-13 g Cu/cell), suggesting that Tetraselmis sp. effectively detoxifies copper within the cell. By contrast, at the same external copper concentration (50 µg/L), D. tertiolecta appears to better exclude copper than Tetraselmis sp., having a slower copper internalisation rate and lower internal copper concentrations at equivalent extracellular concentrations. These results suggest that the use of internal copper concentrations and net uptake rates alone cannot explain differences in algal species sensitivity. The cell ultrastructure of the three algal species was examined in control cells and cells exposed to copper concentrations similar to each species’ IC50 value, i.e. 15 µg Cu/L (P. tricornutum), 50 µg Cu/L (Tetraselmis sp.) and 500 µg Cu/L (D. tertiolecta) using transmission electron microscopy (TEM) (Chapter 4). Swelling and clumping of P. tricornutum cells occurred, potentially related to changes in the glutathione ratio in the cell resulting in inhibition of mitosis. Size changes were not evident in the other species. The main changes observed in these cells were an increase in the size and number of vacuoles. At higher magnification, disruption to plasma or thykaloid membranes was not observed. The production of exudates as a potential detoxification mechanism for copper in algal cells was investigated (Chapter 4). Following exposure to 50 µg Cu/L for 72-h, anodic stripping voltammetry (ASV)-labile copper concentrations were 92 ± 9%, 94 ± 9% and 114 ± 3% of the dissolved copper concentrations for P. tricornutum, Tetraselmis sp. and D. tertiolecta, respectively. This suggests that major production of exudates in response to 50 µg Cu/L was unlikely, and analysis of control samples also suggested limited exudate production in the absence of copper. However, examination of spent test media containing D. tertiolecta after 72-h exposure to 500 µg Cu/L suggested that carbohydrate and protein concentrations at the cell surface of this alga increased in response to copper. An increase in organic matter (as protein or carbohydrate) in solution was not detected however, suggesting that any additional ligands were not excreted into the test medium. The role of exudates in ameliorating copper toxicity for these marine species requires further research. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter the sensitivity of algal species to contaminants, which is not currently taken into account in laboratory toxicity tests. Chapter 5 describes an investigation of the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth rate inhibition bioassays. Four species of microalgae were used, with two isolates of each species tested; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72 h) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46 µg Cu/L, while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208 µg Cu/L. The bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rDNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE). It was found that bacteria were present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The “axenic” cultures contained less bacterial phylotypes than an equivalent non-axenic culture, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment where bacteria are from the same environment as the algal cell and bacterial numbers are low. Cell-cell or cell-exudate interactions in biofilms or periphyton may help alleviate metal stress exerted on microalgae. In Chapter 6, 72-h growth rate inhibition bioassays were used to assess the sensitivity of the test species Tetraselmis sp. (Prasinophyceae) to copper in the absence and presence of field-collected marine biofilms. Harvested biofilm homogenate (initial cell density 1.5-15  104 fluorescent cells/mL) and biofilms attached to slides were added to bioassays. Using flow cytometry, Tetraselmis sp. cells were easily distinguished from the biofilm for the 72-h test duration. The addition of field-collected marine biofilms to toxicity tests improved the control growth rate of the test species (Tetraselmis sp.), probably due to the provision of extra nutrients not otherwise available in the minimal nutrient test medium. The addition of biofilm material at cell densities  6  104 fluorescent cells/mL effectively ameliorated the toxicity of copper to Tetraselmis sp. in some tests, with the 72-h IC50 significantly increasing from 43 to 67 µg Cu/L upon the addition of biofilm (initial biofilm cell density, 6  104 fluorescent cells/mL). However, results using biofilm added as a homogenate were variable and with the copper-sensitivity of Tetraselmis sp. varying only by a factor of two for controls (no biofilm) and tests with biofilms (1.5-15  104 fluorescent cells/mL), this may well be within the natural variation in sensitivity for Tetraselmis sp. A similar decrease in sensitivity was observed regardless of the season the biofilm was collected (winter or spring). Tests in which biofilm material was added to bioassays while still attached to slides appeared to ameliorate toxicity in a more consistent fashion, and this type of bioassay is likely to be more appropriate. Biofilm cell densities did not increase under laboratory toxicity test conditions unless a critical initial cell density of 15  104 fluorescent cells/mL was used, in which case a centric diatom grew rapidly. Biofilms collected in spring were visibly thicker upon collection, had higher cell counts, higher chlorophyll a content, higher concentrations of proteins and carbohydrates in the extracellular polymeric substances (EPS) and a greater diversity and abundance of bacteria when compared to biofilms collected in winter (using denaturing gradient gel electrophoresis). Field-collected biofilm material used in toxicity tests should be used within one or two weeks of collection, as the biofilm can change upon storage. This work has clearly shown that biotic factors such as cell size and cell wall structure are not linked to algal species-sensitivity to copper. Adsorption of copper to the cell surface, although integral to further uptake, cannot be used as a factor to define species-sensitivity, and is rapid and non-discriminatory of cell wall structures. While uptake rates and copper cell loadings are also important factors in defining species-sensitivity, quantifying these characteristics for three species of varying sensitivity did not easily elucidate why some species are more tolerant than others, with the species of moderate sensitivity exhibiting the highest uptake rates and highest internal copper loadings of the three species tested. Further knowledge of specific detoxification mechanisms in these species is required to fully understand why some species are so much more sensitive to copper. Some work into detoxification mechanisms showed that effects on cell ultrastructural features like plasma or thykaloid membranes, or effects on exudate production are only likely to be observed upon high copper exposures. Examination of membrane potential changes or membrane damage using cell staining and flow cytometric techniques, rather than imaging techniques, may be more successful in assessing cell damage. Biofilms were successfully incorporated into toxicity testing regimes, but further research is required to improve the relevance of these tests. If future work can accomplish this task it would be beneficial to understanding the importance of biofilms in the dynamics of trace metals in aquatic systems, and could potentially improve the regulation of metals in aquatic environments.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.