Toxicity, uptake and biotransformation of estrogens by different microalgae
Student thesis: Doctoral Thesis
Estrogens, a group of steroid hormones, are serious environmental pollutants due to their persistence, ubiquity and endocrine-disrupting toxicity. Natural and synthetic estrogens, especially the more potent ones, such as estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), have been found in the influent and effluent of municipal wastewater treatment plants (WWTPs). Alternate technologies are needed to eliminate these compounds from the environment. The removal of estrogen compounds by bacteria, and even fungi, has been reported widely, but the role of microalgae has seldom been reported. The microbial species suitable for the removal of the estrogens present in wastewater must be able to tolerate the toxicity of estrogens and some studies proposed that a native species, such as those isolated from contaminated sites had higher tolerance than the commercially available species. However, there is still considerable debate as to whether native species perform better than commercially available species. The present study therefore compared the tolerance of six microalgal species, including three local isolates, Chlamydomonas sp. (WW), Chlorella sp. (2f5aia) and Chlorella sp. (1uoai), and three commercially available species, Scenedesmus quadricauda (SQ), Selenastrum capricornutum (SC) and Chlorella vulgaris (CV) to the toxicity of mixed E2 and EE2 (1:1) at concentrations ranging from 0 to 20 mg L-1. The research also investigated the antioxidant responses, uptake, biotransformation and biodegradation of mixed estrogens of different microalgae. Results showed that the sensitivity of the microalgae to estrogen toxicity was highly dose-, incubation time-, and species-dependent. The increase in cell numbers of SQ at the end of the 7-day incubation reduced significantly with increasing E2 and EE2 concentrations. Two-way multivariate analysis of variance (MANOVA) results showed that the changes in the cell numbers of SQ were significant with time. Accompanied with the changes in cell number, other growth responses, including increases in cell volume and decreases in cell division, were observed with increasing E2 and EE2 concentrations. The other five species presented different degrees of adaptations to E2 and EE2. For species of 2f5aia and CV, E2 and EE2 had an adverse effect on the increase in cell number, but such response correlated poorly with concentrations. For species of SC and WW, E2 and EE2 had no (1 mg L-1) or even stimulating (4-20 mg L-1) effect on cell numbers. No significant differences were found in the cell numbers of 1uoai when exposed to different E2 and EE2 concentrations (Two-way MANOVA, P=0.349). In addition to cell numbers, other growth parameters, including reduction in chlorophyll a fluorescence, in the five species also showed significant changes in response to estrogen toxicity. The percentages of inhibition to E2 and EE2 at 96 hours of exposure demonstrated that the sensitivity of the six microalgal species followed the order of SQ>CV=WW=2f5aia≥1uoai=SC. The biotransformation and biodegradation of single E2 and EE2, each at 200 μg L-1, as well as mixed E2 and EE2 at 100 μg L-1 each, by all six microalgae were measured in terms of the residual amount and uptake in cells at different time intervals, up to seven days. The biological removal of estrogen was incubation time- and species-dependent, but there were no differences in the removal percentages of E2 and EE2 between commercial and local species. Among the three Chlorella species, 1uoai and 2f5aia showed the same removal efficiency, both reached the highest removal efficiency of E2, either in mixed or single treatment, at Day 2. The biological removal of E2 and EE2 by SC increased with incubation time and achieved the highest removal percentage at the end of incubation. After seven days of incubation, the removal percentage of mixed EE2 by SC was much higher than that of single EE2 (one-way ANOVA, P<0.001). In all microalgal species, the percentages of E2 or EE2 taken up in cells were very low when compared to the respective biological removal percentages. E2 easily transformed into estrone (E1) and some unknown products by all microalgal species, while the products for EE2 transformation have not yet to be identified, due to the limited availability of the standards for metabolites. It is clear that SC and 2f5aia (a Chlorella isolate) had the highest removal ability. The antioxidant responses, including reactive oxygen species (ROS), esterase activity, superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH) and malonaldehyde (MDA) content, of different microalgae to single and mixed E2 and EE2 in artificial wastewater at a concentration of 200 μg L-1 were explored. The temporal changes of the esterase activity and ROS level in these two species were very different, with a continuous increase with time in Chlorella but peaking at Day 2 in SC. The esterase activity of SC was not affected by estrogen treatments; however, the activity in estrogen treated Chlorella decreased significantly from Day 2 onwards. The single EE2 and mixed E2 and EE2 treatments induced more ROS of SC than the single E2 treatment and control at Day 4, but no other difference was found between treatments and control of Chlorella at the same day. The other oxidative damage responses of SC were not affected by estrogen treatments, except MDA increased in EE2 treatment. Interestingly, POD and GSH of Chlorella increased in both single and mixed estrogen treatments at Day 4. These results suggested that microalgal species were tolerant to E2 and EE2, but some species-specific changes occurred to combat the oxidative stress posed by estrogens. The study further examined whether SC could grow and remove a mixture of E2 and EE2, each at a contamination level of 100 μg L-1, from commercial medium and municipal sewages (primary settled sewage and final effluent) collected from Yuen Long WWTP in four repeated exposure cycles, each lasted for seven days. Estrogen in commercial media did not have any significant effect on the growth of SC during four repeated cycles. In all cycles, the mixed estrogen (E2 and EE2) in primary settled sewage stimulated the growth of SC when compared to the control without estrogen. Sterilization of wastewater also affected the tolerance of cells to estrogens. Cell growth in non-sterilized, estrogen-contaminated sewage was higher than that in sterilized sewage, suggesting that the presence of other microorganisms (such as indigenous bacteria and microalgae) in the sewage had a synergistic effect on the growth of SC under estrogen exposure. At the end of the four repeated exposure cycles, more than 80% of the cells in commercial media or sterilized wastewater were still alive, with significant removal rates of the mixed estrogens from contaminated water. The mean removal percentages of E2 in the control of non-sterilized sewage, that is, without the inoculation of SC but with the presence of other indigenous microorganisms, were more than 90% at the first day of each cycle. This suggests that E2 could be removed easily by the indigenous bacteria and microalgal species in the sewage, thus the role of SC was insignificant. Conversely, the control only removed a small amount of synthetic EE2, and the inoculation of SC significantly enhanced its removal in both non-sterilized primary settled sewage and final effluent, with mean removal percentages at 53.1% and 62.9%, even at the end of the fourth cycle, respectively. The removal percentages of E2 and EE2 in sewage positively correlated with the cell numbers of SC. These results indicated that SC, the most resistant species to continuous exposure of E2 and EE2 among the six microalgal species investigated in the present study, could be repeatedly used to remove synthetic EE2 from wastewater and may be a suitable microbial candidate for treating estrogen-contaminated environments.
- Microalgae, Toxicology, Estrogen, Microbiology