Abstract
The eastern oyster (Crassostrea virginica) is a foundation species providing significant ecosystem services. However, the roles of oyster microbiomes have not been integrated into any of the services, particularly nitrogen removal through denitrification. We investigated the composition and denitrification potential of oyster microbiomes with an approach that combined 16S rRNA gene analysis, metabolic inference, qPCR of the nitrous oxide reductase gene (nosZ), and N2 flux measurements. Microbiomes of the oyster digestive gland, the oyster shell, and sediments adjacent to the oyster reef were examined based on next generation sequencing (NGS) of 16S rRNA gene amplicons. Denitrification potentials of the microbiomes were determined by metabolic inferences using a customized denitrification gene and genome database with the paprica (PAthway PRediction by phylogenetIC plAcement) bioinformatics pipeline. Denitrification genes examined included nitrite reductase (nirS and nirK) and nitrous oxide reductase (nosZ), which was further subdivided by genotype into clade I (nosZI) or clade II (nosZII). Continuous flow through experiments measuring N2 fluxes were conducted with the oysters, shells, and sediments to compare denitrification activities. Paprica properly classified the composition of microbiomes, showing similar classification results from Silva, Greengenes and RDP databases. Microbiomes of the oyster digestive glands and shells were quite different from each other and from the sediments. The relative abundance of denitrifying bacteria inferred by paprica was higher in oysters and shells than in sediments suggesting that oysters act as hotspots for denitrification in the marine environment. Similarly, the inferred nosZI gene abundances were also higher in the oyster and shell microbiomes than in the sediment microbiome. Gene abundances for nosZI were verified with qPCR of nosZI genes, which showed a significant positive correlation (F1,7 = 14.7, p = 6.0x10-3, R2 = 0.68). N2 flux rates were significantly higher in the oyster (364.4 ± 23.5 μmol N-N2 m-2 h-1) and oyster shell (355.3 ± 6.4 μmol N-N2 m-2 h-1) compared to the sediment (270.5 ± 20.1 μmol N-N2 m-2 h-1). Thus, bacteria carrying nosZI genes were found to be an important denitrifier, facilitating nitrogen removal in oyster reefs. In addition, this is the first study to validate the use of 16S gene based metabolic inference as a method for determining microbiome function, such as denitrification, by comparing inference results with qPCR gene quantification and rate measurements.
Highlights
Chesapeake Bay, the largest estuary in the United States, is one of many systems that has experienced the detrimental effects of excess nitrogen (N) and cultural eutrophication, including bottom water hypoxia, reduced fisheries harvests, and loss of submerged aquatic vegetation [1,2]
Of the oyster-related microbiomes, the sediment microbiome showed the greatest number of families (n = 12.5 ± 1.7) and the lowest percent of sequences identified (47.7 ± 6.7%), the oyster digestive gland microbiome showed the lowest number of families (n = 1.3 ± 0.5) and the highest number of sequences identified (73.1 ± 24.5%), and the oyster shell microbiome fell somewhere in the middle (n = 8.8 ± 2.5; 59.7 ± 7.7%) (Fig 1)
Our study found Alphaproteobacteria to be the dominant class in the oyster shell microbiome, while Gammaproteobacteria were more dominant in the sediment microbiome
Summary
Chesapeake Bay, the largest estuary in the United States, is one of many systems that has experienced the detrimental effects of excess nitrogen (N) and cultural eutrophication, including bottom water hypoxia, reduced fisheries harvests, and loss of submerged aquatic vegetation [1,2]. Oysters may stimulate denitrification by supplying organic carbon (C) and N in the form of biodeposits to denitrifying communities in sediments [4,10,11]. Oyster shells were found to have denitrification activity even though the rates were much lower than those measured in live oysters [19]. The shell microbiome is exposed to increased C and N from biodeposits and excretions, which may enhance denitrification. Both the gut and shell microbiomes are likely important contributors to oyster denitrification, no previous studies have identified denitrifying taxa or genes in the oyster microbiome
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