Abstract
Previous studies have suggested that protozoa prey on Fe(III)- and sulfate-reducing bacteria that are enriched when acetate is added to uranium contaminated subsurface sediments to stimulate U(VI) reduction. In order to determine whether protozoa continue to impact subsurface biogeochemistry after these acetate amendments have stopped, 18S rRNA and ß-tubulin sequences from this phase of an in situ uranium bioremediation field experiment were analyzed. Sequences most similar to Metopus species predominated, with the majority of sequences most closely related to M. palaeformis, a cilitated protozoan known to harbor methanogenic symbionts. Quantification of mcrA mRNA transcripts in the groundwater suggested that methanogens closely related to Metopus endosymbionts were metabolically active at this time. There was a strong correlation between the number of mcrA transcripts from the putative endosymbiotic methanogen and Metopus ß-tubulin mRNA transcripts during the course of the field experiment, suggesting that the activity of the methanogens was dependent upon the activity of the Metopus species. Addition of the eukaryotic inhibitors cyclohexamide and colchicine to laboratory incubations of acetate-amended subsurface sediments significantly inhibited methane production and there was a direct correlation between methane concentration and Metopus ß-tubulin and putative symbiont mcrA gene copies. These results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
Highlights
Methanogenic microbial communities exemplify the importance of interspecies interactions
METHANE PRODUCTION FOLLOWING INJECTION OF ACETATE INTO THE SUBSURFACE Acetate was pumped into the subsurface for 68 days to promote in situ uranium bioremediation
PROTOZOA KNOWN TO HARBOR METHANOGENIC ENDOSYMBIONTS DETECTED IN GROUNDWATER As previously reported (Holmes et al, 2013), the Fe(III) reduction phase of the bioremediation process was associated with the growth of Geobacter species and a specific enrichment of protozoa from the genus Breviata
Summary
Methanogenic microbial communities exemplify the importance of interspecies interactions. These include the various forms of interspecies electron transfer between bacteria and methanogens (Stams and Plugge, 2009; Malvankar and Lovley, 2014; Rotaru et al, 2014), and the symbiotic association of protozoa and endosymbiotic methanogens (van Hoek et al, 2000; Fenchel and Finlay, 2010). The majority of methanogenic endosymbionts are associated with ciliated protozoa, but methanogens have been found in the cytoplasm of anaerobic amoebae and flagellates (Vogels et al, 1980; Vanbruggen et al, 1983; van Hoek et al, 2006; Nowack and Melkonian, 2010; Hackstein, 2011). The acetate formed by this fermentation reaction is used by the ciliate as an energy and carbon source, while the symbiont can utilize the H2 and CO2 for methanogenesis
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