Great Lakes Cladophora harbors phylogenetically diverse nitrogen-fixing microorganisms

Nitrogen-fixing microorganisms are among the epiphytic communities in Cladophora, potentially benefitting the algae in nutrient-deficient waters, but their abundance and diversity remain unexplored. In this study, we determined the abundance and taxonomic composition of these nitrogen-fixing microorganisms in Cladophora growing on rocks, breakwall structures, or submerged dreissenid mussel beds around southern Lake Michigan (N = 33) during the summer 2015, using two complementary genomic techniques: quantitative PCR (qPCR) and shotgun metagenomic sequencing. Genomic DNA was extracted from processed algal pellets, and the nitrogen-fixing microbes were quantified by qPCR by targeting the nifH gene. Mean nifH concentrations (log₁₀ copy numbers/gram algae fresh weight ± SE) were 5.54 ± 0.09, ranging from 4.31 to 6.57. Mean nifH concentrations in water samples (log₁₀ copy numbers/milliliter of water ± SE) were: 3.25 ± 0.06, ranging from 2.41 to 3.90. Shotgun sequencing of a subset of algal samples representing the four sampling locations (N = 10) revealed as many as 267 nifH reads from among the sequences of the 10 shotgun metagenomes (averaging 27 reads per metagenome), ranging from 5 to 91 reads from Jeorse Park (September) and North Beach (September) locations. Taxonomic assignment of nifH sequences identified members from bacteria and archaea domains showing a clear separation of reads at domain and lower taxonomic levels. Bacteria were relatively more abundant than archaea. Anabaena, Bradyrhizobium, Geobacter, Methylocystis, Oscillatoria sp., and Skermanella (all bacteria), and Methanoregula, Methanothrix, and Methanosarcina (archaea) were among the nitrogen-fixing genera identified by the MEGAN Community Edition program. Collectively, these findings show that phylogenetically diverse nitrogen-fixing microbial communities are part of the Cladophora microbiome, likely contributing to the algal nitrogen needs.