Using root soil & pore water microcosm incubations coupled with metagenomic and qPCR techniques to probe primary Hg-methylating guilds in the paddy rhizosphere

• Main Authors: Yi-Ting Liao, 廖怡婷
• Other Authors: Chu-Ching Lin
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/gscb7n

碩士 === 國立中央大學 === 環境工程研究所 === 105 === Recent studies have shown that in addition to intake of piscivorous fish, rice consumption is another critical route of human expose to methylmercury (MeHg), the most toxic form of mercury (Hg) in the environment. Nonetheless, there is still a paucity of data on the biogeochemical mechanisms that control the formation (in the rhizosphere), uptake (by root), and eventual accumulation of MeHg (in rice grain) in the paddy ecosystem. To gain an in-depth understanding of this undesirable environmental process, in 2014 we began our investigation and initiated fieldwork at rice paddies that were proximal to the coal-fired power station in Taichung. Preliminary results of microcosm incubations (of root soil samples) suggested that sulfate-reducing bacteria (SRB) might be the primary Hg methylators at our study sites. However, because the incubation tests were conducted with synthetic media instead of pore water, there was a potential fraud in our methodology that might have resulted in a bias in our observations. Further, a detail look at the microbial community structure has not yet carried out. In light of this, here we aimed at rectifying our former protocols of microcosm incubations to confirm the role of SRB as the principal Hg methylating guild. More importantly, this study incorporated certain advanced molecular biology techniques including real-time polymerase chain reactions (qPCR), metagenomics, as well as the next-generation sequencing (NGS) into this inquiry, hoping to obtain a complementary interpretation of methylation results at the cellular level. Results from root soil/pore water incubations assayed with Hg methylation & demethylation confirmed that SRB indeed were the major Hg-methylating guild in the rhizosphere of our study sites. Relative quantification of the hgcA level by qPCR also indicated that Deltaproteobacteria was the principal Hg-methylators at the class level, consistent with the aforementioned role of SRB. In addition, our data suggested that iron-reducers and methylotrophic- and hydrogentrophic-methanogens, while not prominent, might as well play a certain role in MeHg production in paddies. However, metagenomic analysis of 16S rRNA genes showed that Geobacter was the most abundant genus in all samples, suggesting that there are a significant amount of unknown Hg-methylating microbes inhabiting in paddies that await to be identified. On the basis of all these results, time-course experiments focusing on RT-PCR and RT-qPCR of mRNA transcribed from the hgcAB gene cluster are warranted for the future study to pinpoint Hg-methylators at the species level. Ultimately, information gain from this type of investigations may entail us to devise more efficient and sounder remediation strategies to deal with Hg contamination issues in farmland.