Isolation and identification of polybrominated diphenyl ethers (PBDEs) degrading microalgae from wastewater and their degradation mechanism
Student thesis: Doctoral Thesis
Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants which cause adverse effects to human health and environments. Wastewater treatment plants (WWTPs) receive PBDEs from various discharges but also release them back to the environment via treated effluent and sludge, as the conventional treatment processes are not designed to remove PBDEs. It is essential to develop effective remediation technologies for PBDE contamination. Microalgae have been demonstrated to be effective in the removal of nutrients, heavy metals and toxic organic pollutants other than PBDEs from domestic and industrial wastewaters. Microalgal species with resistance to toxic pollutants have been isolated from wastewater. It is possible that the sewage from WWTPs with PBDE contamination is a potential source of PBDE-tolerant microalgal species, and the tolerant isolates may be employed for PBDE removal. The present study aims to investigate the physiological responses and the ability of microalgae, especially the microalgal strains isolated from influents of Hong Kong WWTPs, to degrade and transform PBDEs. The contamination of PBDEs in four WWTPs in Hong Kong was investigated by analyzing samples of primary influent, final effluent and dewatered sludge collected from October 2011 to January 2013. Results showed that the concentrations and composition profiles of eight PBDE congeners (BDE-28, -47, -99, -100, -153, -154, -183 and -209) differed among WWTPs and fluctuated during the study period. Higher concentrations of PBDEs were detected in the influent and dewatered sludge from the two WWTPs receiving both domestic and industrial wastewaters than the two serve mainly residential and commercial districts. The concentrations of Σ8PBDEs (total of eight congeners) in the influent of all WWTPs ranged from 1 to 254 ng L-1, decreased to 12-27 ng L-1 in effluent, with removal efficiency ranged from 20 to 53% but high concentrations of PBDEs, ranging from 9 to 307 ng g-1 dry weights, were detected in dewatered sludge. Every day, 0.66-73 g PBDEs entered the four WWTPs, while 0.38-38 g and 0.17-17 g PBDEs were discharged to the surrounding waters via effluent and disposed to landfill sites in sludge form, respectively. These indicated that WWTPs in Hong Kong were not designed for effectively removal of PBDEs and both outputs (effluent and sludge) became substantial contamination sources. Nine PBDE-tolerant microalgal strains were isolated from influents of four WWTPs spiked with a mixture of DE-71 and BDE-209 (at a ratio of 5:1) at 6 μg L-1. Based on the morphological features and phylogenetic analysis of 18S rDNA sequences, they belonged to five genera, namely, Chlorella (STCh and SICh), Parachlorella (STPa1 and STPa2), Scenedesmus (STSc, TPSc1 and TPSc2), Nitzchia palea (YLBa) and Mychonastes (TPMy). Toxicity assessments revealed that four isolates, SICh, STCh, STPa1 and TPSc1, were PBDE-tolerant strains as their growths, in terms of cell numbers and chlorophyll a content, were not affected after exposed to mixtures of DE-71 and BDE-209 (at a ratio of 5:1) at low (6 μg L-1), medium (60 μg L-1) and even high (600 μg L-1) levels for seven days. On the contrary, the growth of YLBa was significantly inhibited even at the low level and the inhibition to the other three species TPSc2, STPa2, and TPMy was only found at the high level of mixed PBDEs. The removal of PBDEs by the isolate SICh, one of the tolerant isolates, was the highest, with 85% Σ8PBDEs removed after 7-day exposure to the high level of PBDE mixture. The results implied that the removal of PBDEs by microalgae was not related to their tolerance. Bioaccumulation and biotransformation were important processes for PBDE removal. The removal mechanism of the tolerant and efficient Chlorella isolate (SICh) was further investigated by BDE-47 exposure experiments. The results showed that the dominant removal process was adsorption, with efficiencies ranged from 83.6% to 99.7% after exposed to 1.6-320 μg L-1 BDE-47 for one hour. In the 7-day long-term study, the adsorbed BDE-47 was gradually taken up into cell, which accounted for 17% of the spiked BDE-47 (10 μg L-1) at the end of the exposure. During the 7-day exposure, hydroxylated products, 6-OH- and 5-OH-BDE-47, were most important metabolites and accounted for 0.4-3% and 3-15% of the spiked BDE-47, respectively, while the other two metabolites, BDE-28 and 6-MeO-BDE-47 accounted less than 0.3% of the spiked BDE-47. These results demonstrated that the removal mechanism involved a rapid physic-chemical adsorption, followed by long-term absorption and metabolism. 6-day semi-static exposure and cytochemical experiments were conducted to study the physiological response of SICh isolate to BDE-47 stress. The production of hydrogen peroxide (H2O2) in SICh treated with 10 μg L-1 BDE-47 increased significantly in the first day, and such increase was found in cell wall, plasma membrane and chloroplast, but the production decreased in the following five days. The H2O2 induced by BDE-47 in almost all organelles was due to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, while the contribution of H2O2-producing peroxidase (POD) to H2O2 generation was mainly in cell wall, cytosol, peroxisome and vacuole. The activity of H2O2-consuming POD and the content of glutathione (GSH) also increased significantly after BDE-47 treatment, and the most pronounced increase was found after two days of exposure, suggesting their roles in detoxifying the oxidative stress. In vivo metabolism study suggested that BDE-47 first transformed to 5-OH and 6-OH-BDE-47 by hydroxylation, and the hydroxylated products were further methylated to 6-MeO-BDE-47. BDE-47 also debrominated to BDE-28 and -17, which further debrominated to BDE-7, -8 and -15. In vitro study also demonstrated that BDE-47 could be oxidized by H2O2, produced metabolites such as 3-OH, 5-OH and 6-OH-BDE-47. All these studies revealed the oxidative metabolism of BDE-47 by microalgae. In conclusion, this is the first comprehensive study on the spatial and temporal variations of PBDEs in influent, effluent and dewatered sludge in four WWTPs in Hong Kong. It is also the first time isolated and identified PBDE-tolerant microalgae from influent. A Chlorella strain, SICh, with a high tolerance to BDE-47 and high removal ability was obtained. Although this isolate was tolerant to BDE-47 without any significant changes in its growth, BDE-47 still induced oxidative stress in the cell organelles, which was regulated by NADPH oxidase and peroxidase. Nevertheless, this oxidative stress could be detoxified by H2O2-consuming POD and GSH. The removal of BDE-47 by Chlorella sp. involved three major processes, that is, adsorption, uptake and metabolism via debromination, hydroxylation and methoxylation.
- Biological treatment, Polybrominated diphenyl ethers, Biotechnology, Biodegradation, Purification, Microalgae, Sewage