The Upper Mississippi River (UMR) basin contributes over 50,000 metric tons of nitrogen (N) to the Gulf of Mexico each year, resulting in a “dead zone” inhospitable to aquatic life. Land-applied N (fertilizer) in the corn-belt is attributed with a majority of the N-load reaching the Gulf and is difficult to treat as run-off is considered a non-point source of pollution (i.e. not from a pipe). One solution to this “grand challenge” of intercepting N pollution is utilizing filter-feeding organisms native to the UMR. Freshwater mussel (order Unionidae) assemblages collectively filter over 14 billion gallons of water, remove tons of biomass from overlying water, and sequester tons of N each day. Our previous research showed mussel excretions increased the sediment porewater concentrations of ammonium by 160%, and indirectly increased nitrate and nitrite by 40%, presumably from microbial degradation of ammonium. In response, the goal of this research was to characterize how mussels influenced microbial communities (microbiome) to determine the fate of N in UMR sediment.
First, we used qPCR and non-targeted amplicon sequencing within sediment layers to identify the N-cycling microbiome and characterized microbial community changes attributable to freshwater mussels. qPCR identified that anaerobic ammonium oxidizing (anammox) bacteria were increased by a factor of 2.2 at 3 cm below the water-sediment interface when mussels were present. Amplicon sequencing of sediment at depths relevant to mussel burrowing (3 and 5 cm) showed that mussel presence reduced microbial species richness and diversity and indicated that sediment below mussels harbored distinct microbial communities. Furthermore, mussels increased the abundance of ammonia oxidizing bacteria (family Nitrosomonadaceae), nitrite oxidizing bacteria (genus Nitrospira), but decreased the abundance of ammonia oxidizing archaea (genus Candidatus Nitrososphaera), and microorganisms which couple denitrification with methane oxidation. These findings suggested that mussels may enhance microbial niches at the interface of oxic and anoxic conditions, presumably through excretion of N and burrowing activity.
In response, we performed metagenomic shotgun sequencing to identify which genes of the microbiome were most impacted by mussels. We hypothesized that genes responsible for ammonia and nitrite oxidation would be greater in the sediment with mussel assemblages. We found the largest abundance of N-cycling genes were responsible for nitrate reduction and nitrite oxidation, which is corroborated by the high concentration of nitrates in UMR water. Linear discriminant analysis statistical analyses showed nitrification genes were most impacted by mussels, and this presented an opposing effect on genes responsible for producing nitrous oxide, a potent greenhouse gas. Further investigation showed an increased abundance of a novel organism capable of completely oxidizing ammonia to nitrate (Candidatus Nitrospira inopinata) and coexisted with metabolically flexible Nitrospira (sp. moscoviensis), likely enhancing both carbon and N-cycling.
We demonstrated that native mussels harbor a unique niche for N-cycling microorganisms with large metabolic potentials to degrade mussel excretion products. Our findings suggest the ecosystem services of mussels extend beyond water filtration, and includes enhanced biogeochemical cycling of carbon, N, and reduces the potential for a potent microbially-produced greenhouse gas. Ultimately, this research could be used to advocate for mussel habitat restoration in the UMR to lessen the impacts of non-point pollution.