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Sampling campaigns are performed periodically by KEPLER at a site contaminated with heat transfer fluid (HTF), a thermal oil used in solar thermal plants. The results obtained showed that when the concentration of HTF decreased in piezometers nitrites (NO2-) production was detected in groundwater. This fact suggested a possible removal of HTF via biological denitrification of the pollutant where nitrate is used as an electron acceptor under anoxic conditions. The denitrification metabolic pathway consists of the reduction of nitrates (NO3-) to nitrogen gas. This process occurs in successive stages, catalyzed by different enzymatic systems, with nitrites (NO2-), nitric oxide (NO) and nitrous oxide (N2O) appearing as intermediate products. Thus, if HTF could be being used as a source of organic carbon during a denitrification process, an active natural attenuation process could be demonstrated in the aquifer.
To study this hypothesis, KEPLER carried out a metagenomics study in which the genomic region of the 16S ribosomal gene was analyzed to identify the autochthonous population of the aquifer and thus verify if there were denitrifying microorganisms. From the 16S sequences identified, a functional analysis was also carried out to infer the metabolic functions that could take place in the aquifer and check if denitrifying enzymes were present. After the metagenomics analysis, a laboratory treatability study was also carried out to evaluate a possible removal of HTF in the aquifer groundwater by this biological denitrification pathway. For that, a continuous test was performed in which a reactor under anoxic conditions was fed with HTF and nitrate for 6 months to evaluate the potential of the natural attenuation process.
Metagenomics study demonstrated that among the most abundant genera in the autochthonous microbial community were some that participate in denitrification processes using nitrate as an electron acceptor under anoxic conditions, such as the genus Thauera spp. The ability of this genus to degrade aromatic compounds, as are HTF and its by-products, was also observed in denitrification reactors. Other microorganisms that play an important role in the nitrogen cycle were also identified, such as the genera Nitrosomonas spp. and Nitrospira spp. and, within the archaea group, the genus Nitrosarchaeum spp. Among the potential metabolic functions, enzymes described as denitrifying agents were identified such as nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase. The presence of these microorganisms and these enzymes indicates that denitrification may occur in the aquifer and, furthermore, reinforces the hypothesis that HTF can be degraded by denitrification in an anoxic environment.
Treatability study in a continuous reactor confirmed the presence of a denitrification metabolic pathway. In this test it was observed that when HTF or NO3- were added separately, the concentrations of these two compounds did not decrease, however, when adding them simultaneously, HTF consumption and transformation of NO3- into NO2-, confirming that HTF can be removed from groundwater via biological denitrification.
In conclusion, this study provides the first evidence of the effectiveness of denitrification to eliminate HTF during groundwater treatment and highlights the potential of using genetic tools in the evaluation of soil and groundwater natural attenuation. In this case study, the demonstration of natural biodegradation of HTF to local authorities made it possible the application of a groundwater control and monitoring program and discard an active remediation strategy in the aquifer.
Key words: denitrification, HTF-removal, natural attenuation, metagenomics