Identification of Metolachlor Mineralizing Bacteria in Aerobic and Anaerobic Soils Using DNA-Stable Isotope Probing.

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of a popular soil applied herbicide like metolachlor is unknown. To address this knowledge gap, we utilized DNA based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor, ¹³C-metolachlor or ¹⁴C-metolachlor. The extracted DNA from unlabeled (¹²C-metolachlor) and labeled (¹³C-metolachlor) herbicide treated soil samples were subjected to ultracentrifugation, followed by fractionation of the centrifuged samples. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts after 28 days of incubation, facilitated the identification of different characteristic fragments of target populations in aerobic and anaerobic incubations.  T-RFLP profiles from the ultracentrifugation fractions illustrated that these characteristic fragments experienced an increase in relative abundance at higher buoyant density (BD) in the labeled fractions, when compared to the unlabeled herbicide amendment fractions. The shift in BD of characteristic T-RFLP fragments in the density resolved fractions suggested the incorporation of ¹³C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the ¹³C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soils.