Nitrogen Source Governs Community Carbon Metabolism in a Model Hypersaline Benthic Phototrophic Biofilm

Abstract

Increasing anthropogenic inputs of fixed nitrogen are leading to greater eutrophication of aquatic environments, but it is unclear how this impacts the flux and fate of carbon in lacustrine and riverine systems. Here, we present evidence that the form of nitrogen governs the partitioning of carbon among members in a genome-sequenced, model phototrophic biofilm of 20 members. Consumption of NO₃ ^- as the sole nitrogen source unexpectedly resulted in more rapid transfer of carbon to heterotrophs than when NH₄ ⁺ was also provided, suggesting alterations in the form of carbon exchanged. The form of nitrogen dramatically impacted net community nitrogen, but not carbon, uptake rates. Furthermore, this alteration in nitrogen form caused very large but focused alterations to community structure, strongly impacting the abundance of only two species within the biofilm and modestly impacting a third member species. Our data suggest that nitrogen metabolism may coordinate coupled carbon-nitrogen biogeochemical cycling in benthic biofilms and, potentially, in phototroph-heterotroph consortia more broadly. It further indicates that the form of nitrogen inputs may significantly impact the contribution of these communities to carbon partitioning across the terrestrial-aquatic interface.IMPORTANCE Anthropogenic inputs of nitrogen into aquatic ecosystems, and especially those of agricultural origin, involve a mix of chemical species. Although it is well-known in general that nitrogen eutrophication markedly influences the metabolism of aquatic phototrophic communities, relatively little is known regarding whether the specific chemical form of nitrogen inputs matter. Our data suggest that the nitrogen form alters the rate of nitrogen uptake significantly, whereas corresponding alterations in carbon uptake were minor. However, differences imposed by uptake of divergent nitrogen forms may result in alterations among phototroph-heterotroph interactions that rewire community metabolism. Furthermore, our data hint that availability of other nutrients (i.e., iron) might mediate the linkage between carbon and nitrogen cycling in these communities. Taken together, our data suggest that different nitrogen forms should be examined for divergent impacts on phototrophic communities in fluvial systems and that these anthropogenic nitrogen inputs may significantly differ in their ultimate biogeochemical impacts.

Keywords: carbon cycling; cyanobacteria; mass spectrometry; nitrogen cycling; stable isotopes.

FIG 1

The available nitrogen source affects bulk nitrogen incorporation, but not bulk carbon incorporation into the model phototrophic biofilm. Unicyanobacterial consortium UCC-O was grown in media that contained either NO₃⁻ with NH₄⁺ or NO₃⁻ without NH₄⁺, and both were supplemented with HCO₃⁻. The bulk isotope uptake of ¹⁵N and ¹³C was measured in a 7-day-old biofilm, where the unicyanobacterial consortium was fed H¹³CO₃⁻ with either ¹⁵NO₃⁻ only or with NO₃⁻ supplemented with ¹⁵NH₄⁺ and incubated for 8 h.

FIG 2

The available nitrogen source affects community dynamics within the model phototrophic biofilm. Unicyanobacterial consortium UCC-O was grown in media that contained either NO₃⁻ with NH₄⁺ or NO₃⁻ without NH₄⁺, and both media were supplemented with HCO₃⁻. (A) Comparison of the relative abundance differences in the top eight most-abundant community members as a function of NH₄⁺ supplementation (the 12 other members account for 1% of the total population combined). The qPCR primer and probe sequence for each of these members can be found in Table S1 in the supplemental material. Significant shifts in HL-49, HL-109, and bin04 population occurred depending on whether the consortium was supplied a reduced form of N (Welch’s independent t test, P < 0.01; indicated by an asterisk). (B) Bulk proteomic data illustrating the relative number of expressed proteins (by peptide count) per each community member. The total number of proteins being expressed by each member is proportional to the community member’s biomass. str., strain.

FIG 3

High-lateral-resolution isotopic imaging (256 pixels by 256 pixels, 40 μm by 40 μm) measurements of the biofilms using nanoscale secondary ion mass spectrometry (NanoSIMS) after an 8-h incubation with isotope media. Under the unicyanobacterial consortium growth conditions noted in Fig. 1 and 2, we observed differences in the NanoSIMS images of these biofilms based on ¹³C enrichment (left panels) and ¹⁵N enrichment (right panels). Using the spatial segmentation methodology developed in our lab for analyzing these images (20), we determined the enrichment of both ¹³C and ¹⁵N across the entire heterotroph population imaged (histograms in bottom panels). These results quantify the trends observed in the NanoSIMS images themselves. The segmentation images that correspond to this data are in Fig. S6.

FIG 4

A deeper look into the proteomic data for Phormidium sp. strain OSCR. These data suggest changes in C and N metabolism when NH₄⁺ is added to the media (red), as opposed to when only NO₃⁻ is available (blue). With respect to nitrogen metabolism, we detected differences in nitrate assimilation via nitrite/nitrate transport transporter (Nrt) and nitrate reductase (EC 1.7.7.2) and in ammonium cycling through glutamine synthetase type 1 (EC 6.3.1.2), glutamate synthase (EC 1.4.13), and glutamate dehydrogenase (EC 1.4.1.4). Proteins in pyruvate metabolism were differentially expressed, and these proteins included pyruvate:ferredoxin oxidoreductase (1.2.7.1), l-lactate dehydrogenase (EC 1.1.1.27), and pyruvate formate-lyase (EC 2.3.1.54). Peptides related to iron acquisition, transport, and use in electron carriers were differentially expressed between treatments.