Light and depth dependency of nitrogen fixation by the non‐photosynthetic, symbiotic cyanobacterium UCYN‐A

The symbiotic cyanobacterium UCYN-A is one of the most globally abundant marine dinitrogen (N2)-fixers, but cultures have not been available and its biology and ecology are poorly understood. We used cultivation-independent approaches to investigate how UCYN-A single-cell N2 fixation rates (NFRs) and nifH gene expression vary as a function of depth and photoperiod. Twelve-hour day/night incubations showed that UCYN-A only fixed N2 during the day. Experiments conducted using in situ arrays showed a light-dependence of NFRs by the UCYN-A symbiosis, with the highest rates in surface waters (5–45 m) and lower rates at depth (≥ 75 m). Analysis of NFRs versus in situ light intensity yielded a light saturation parameter (Ik) for UCYN-A of 44 μmol quanta m−2 s−1. This is low compared with other marine diazotrophs, suggesting an ecological advantage for the UCYN-A symbiosis under low-light conditions. In contrast to cell-specific NFRs, nifH gene-specific expression levels did not vary with depth, indicating that light regulates N2 fixation by UCYN-A through processes other than transcription, likely including host–symbiont interactions. These results offer new insights into the physiology of the UCYN-A symbiosis in the subtropical North Pacific Ocean and provide clues to the environmental drivers of its global distributions.En prens

A Critical Review of the \u3csup\u3e15\u3c/sup\u3eN\u3csub\u3e2\u3c/sub\u3e Tracer Method to Measure Diazotrophic Production in Pelagic Ecosystems

Dinitrogen (N2) fixation is an important source of biologically reactive nitrogen (N) to the global ocean. The magnitude of this flux, however, remains uncertain, in part because N2 fixation rates have been estimated following divergent protocols and because associated levels of uncertainty are seldom reported—confounding comparison and extrapolation of rate measurements. A growing number of reports of relatively low but potentially significant rates of N2 fixation in regions such as oxygen minimum zones, the mesopelagic water column of the tropical and subtropical oceans, and polar waters further highlights the need for standardized methodological protocols for measurements of N2 fixation rates and for calculations of detection limits and propagated error terms. To this end, we examine current protocols of the 15N2 tracer method used for estimating diazotrophic rates, present results of experiments testing the validity of specific practices, and describe established metrics for reporting detection limits. We put forth a set of recommendations for best practices to estimate N2 fixation rates using 15N2 tracer, with the goal of fostering transparency in reporting sources of uncertainty in estimates, and to render N2 fixation rate estimates intercomparable among studies

Growth of <i>C. watsonii</i> WH8501 batch cultures over a 6-day period under three CO₂ treatments.

<p>Shown are concentrations of PN (a), PC (b), and cells (c), molar C:N ratios (d), and <i>p</i>CO₂ in <i>µ</i>atm (e) within each treatment. For (a–c), the concentrations for each time point (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110660#pone.0110660.s001" target="_blank">Table S1</a>) were first normalized to the concentration at the day 1 L6 time point, then ln-transformed. The derived slopes between day 1 L6 and day 3 L6 time points correspond to the exponential growth rates (<i>µ</i>) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110660#pone-0110660-t002" target="_blank">Table 2</a>. The lines in (a) represent linear regressions through the day 1, day 2, and day 3 L6 time points for high-CO₂ (dashed line) and low-CO₂ (dotted line) treatments. The regression lines have been extended to the full time period (day 0–5) for visualization of exponential growth (day 0–3 L6 time points) transitioning to early stationary growth (L6 time points after day 3). The dotted line in (d) represents the 6.6 C:N ratio expected from Redfield stoichiometry. Shaded areas represent the dark periods. Error bars represent standard deviations from three replicates.</p

Carbon-normalized PN (a) and PC production rates (b) of <i>Crocosphaera</i> WH8501 cultures grown under three CO₂ treatments during periods of exponential (day 1–day 3) and early stationary (day 3–day 5) growth phases.

<p>Production rates are calculated as increases in PC and PN concentrations (data provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110660#pone-0110660-t001" target="_blank">Table 1</a>) per time normalized to initial PC concentrations within the time interval. Error bars represent standard deviations from three replicates.</p

Time series biomass measurements for cultures of <i>Crocosphaera watsonii</i> WH8501 grown under three CO₂ treatments.

<p>Time series biomass measurements for cultures of <i>Crocosphaera watsonii</i> WH8501 grown under three CO₂ treatments.</p

Biomass-specific growth rates of <i>Crocosphaera watsonii</i> WH8501 cultures grown under three CO₂ treatments.

<p>Biomass-specific growth rates of <i>Crocosphaera watsonii</i> WH8501 cultures grown under three CO₂ treatments.</p

Physiological Response of <i>Crocosphaera watsonii</i> to Enhanced and Fluctuating Carbon Dioxide Conditions

<div><p>We investigated the effects of elevated <i>p</i>CO₂ on cultures of the unicellular N₂-fixing cyanobacterium <i>Crocosphaera watsonii</i> WH8501. Using CO₂-enriched air, cultures grown in batch mode under high light intensity were exposed to initial conditions approximating current atmospheric CO₂ concentrations (∼400 ppm) as well as CO₂ levels corresponding to low- and high-end predictions for the year 2100 (∼750 and 1000 ppm). Following acclimation to CO₂ levels, the concentrations of particulate carbon (PC), particulate nitrogen (PN), and cells were measured over the diurnal cycle for a six-day period spanning exponential and early stationary growth phases. High rates of photosynthesis and respiration resulted in biologically induced <i>p</i>CO₂ fluctuations in all treatments. Despite this observed <i>p</i>CO₂ variability, and consistent with previous experiments conducted under stable <i>p</i>CO₂ conditions, we observed that elevated mean <i>p</i>CO₂ enhanced rates of PC production, PN production, and growth. During exponential growth phase, rates of PC and PN production increased by ∼1.2- and ∼1.5-fold in the mid- and high-CO₂ treatments, respectively, when compared to the low-CO₂ treatment. Elevated <i>p</i>CO₂ also enhanced PC and PN production rates during early stationary growth phase. In all treatments, PC and PN cellular content displayed a strong diurnal rhythm, with particulate C:N molar ratios reaching a high of 22∶1 in the light and a low of 5.5∶1 in the dark. The <i>p</i>CO₂ enhancement of metabolic rates persisted despite <i>p</i>CO₂ variability, suggesting a consistent positive response of <i>Crocosphaera</i> to elevated and fluctuating <i>p</i>CO₂ conditions.</p></div