TEN-YEAR INVESTIGATION OF THE SUBSURFACE MICROBIOME IN A VARIABLY SATURATED CONTAMINANT PATHWAY WITH TWO CARBON-AMENDMENTS

The filterable ultramicrobacterial (UMB) size-fraction is largely understudied, and consists of many unknown bacterial lineages with unique metabolic pathways. Very little work has been done to understand the community of organisms and their response to long-term nutrient presence in the subsurface. This study focused on a shallow gravel back-filled, and fractured saprolite unconfined aquifer with characteristically high hydraulic conductivity, preferential contaminant transport, and where UMB movement can be up to half a meter a day or more. Two Emulsified Vegetable Oil (EVO) injections were conducted in 2009 and 2017 with seasonal and periodic monitoring through 2019. The EVO induced reducing conditions for microbial consortia causing radionuclide, and heavy metal bioimmobilization in 8 monitoring wells which showed quick recovery of the microbial community, geochemistry, ions, organic acids, acridine orange cell counts, transmission electron microscopy, and hydraulic conductivity. The microbial community was collected on 10µm, 0.2µm and 0.1µm filters from which 16S rRNA amplicons were sequenced on an Illumina MiSeq. The area underwent rapid sulfate reduction and acetate production post-injection, but the microbial community recovered to background composition. EVO persistence and microbial community structure were greatly influenced by pH, hydraulic conductivity, and reductive potential with community variability for both 0.2µm and 0.1µm microbial communities related to these parameters. Unweighted UniFrac beta diversity of the 0.2µm community identified an order of magnitude increase in families of sulfate reducing bacteria including Geobacteraceae, Desulfobacteraceae,and Desulfovibrionaceaein both injections with the 0.2µm community showing a similar community response to the earlier EVO injection indicating a memory response. UMB taxa community response suggests these organisms play a role in the biostimulation of the 0.2µm consortia and indicate a potential physical relationship between these groups. The site continues to show higher levels of total organic carbon, and organic acids, and findings for this site have further informed the development of in-situ devices with proper resolution to investigate the unculturable UMB size-fraction.