Samuel T. Wilson

Comparative Assessment of Nitrogen Fixation Methodologies, Conducted in the Oligotrophic North Pacific Ocean

ABSTRACT Resolution of the nitrogen (N) cycle in the marine environment requires an accurate assessment of dinitrogen (N2) fixation. We present here an update on progress in conducting field measurements of acetylene reduction (AR) and 15N2 tracer assimilation in the oligotrophic North Pacific Subtropical Gyre (NPSG). The AR assay was conducted on discrete seawater samples using a headspace analysis system, followed by quantification of ethylene (C2H4) with a reducing compound photodetector. The rates of C2H4 production were measurable for nonconcentrated seawater samples after an incubation period of 3 to 4 h. The 15N2 tracer measurements compared the addition of 15N2 as a gas bubble and dissolved as 15N2 enriched seawater. On all sampling occasions and at all depths, a 2- to 6-fold increase in the rate of 15N2 assimilation was measured when 15N2-enriched seawater was added to the seawater sample compared to the addition of 15N2 as a gas bubble. In addition, we show that the 15N2-enriched seawater can be prepared prior to its use with no detectable loss (<1.7%) of dissolved 15N2 during 4 weeks of storage, facilitating its use in the field. The ratio of C2H4 production to 15N2 assimilation varied from 7 to 27 when measured simultaneously in surface seawater samples. Collectively, the modifications to the AR assay and the 15N2 assimilation technique present opportunities for more accurate and high frequency measurements (e.g., diel scale) of N2 fixation, providing further insight into the contribution of different groups of diazotrophs to the input of N in the global oceans.

Ecogenomic sensor reveals controls on N2-fixing microorganisms in the North Pacific Ocean.

Nitrogen-fixing microorganisms (diazotrophs) are keystone species that reduce atmospheric dinitrogen (N2) gas to fixed nitrogen (N), thereby accounting for much of N-based new production annually in the oligotrophic North Pacific. However, current approaches to study N2 fixation provide relatively limited spatiotemporal sampling resolution; hence, little is known about the ecological controls on these microorganisms or the scales over which they change. In the present study, we used a drifting robotic gene sensor to obtain high-resolution data on the distributions and abundances of N2-fixing populations over small spatiotemporal scales. The resulting measurements demonstrate that concentrations of N2 fixers can be highly variable, changing in abundance by nearly three orders of magnitude in less than 2 days and 30 km. Concurrent shipboard measurements and long-term time-series sampling uncovered a striking and previously unrecognized correlation between phosphate, which is undergoing long-term change in the region, and N2-fixing cyanobacterial abundances. These results underscore the value of high-resolution sampling and its applications for modeling the effects of global change.

Particulate dimethylsulphoxide and dimethylsulphoniopropionate in phytoplankton cultures and Scottish coastal waters

Abstract.Dimethylsulphoniopropionate (DMSP), an algal compatible solute, has for many years been considered to play a key role in dimethylsulphide (DMS) production, influencing the concentrations of DMS in sea water available to be transferred to the atmosphere. However, in recent years it has been shown that dimethylsulphoxide (DMSO) can also be produced directly within the cells of marine phytoplankton. The exact role DMSO plays in cells is still subject to debate, but it is thought that it may act as an antioxidant or cryoprotectant. Whatever the reason, it has been suggested that release through algal mortality and permeative loss of DMSO from cells may contribute to dissolved DMSO concentrations and as such this pathway must also be considered an important component of DMS biogeochemistry. Experiments were conducted to investigate the intracellular concentrations of DMSO and the ratio of DMSP:DMSO in a range of phytoplankton species and in natural samples. Results indicate that prymnesiophytes and dinoflagellates are the main producers, generating relatively higher concentrations of particulate DMSO than diatoms. Results from both laboratory and field experiments show that there is a strong relationship between DMSOp and DMSPp, with DMSO generally representing between 10 and 20% of the intracellular sulphur pool. Field data also indicates that dissolved DMSO concentrations in surface waters were not significantly correlated with those for particulate DMSO, but were significantly correlated with DMS concentrations.

Functional group-specific traits drive phytoplankton dynamics in the oligotrophic ocean

Significance Blooms of phytoplankton can shift the ecosystem state of low-nutrient ocean regions between net heterotrophic and autotrophic conditions, increasing carbon sequestration and driving carbon export to the deep sea. Little is known about the traits that govern the timing and magnitude of these bloom events. We used metatranscriptomics to assess phytoplankton functional group-specific metabolic shifts experimentally during simulated blooms in the North Pacific Subtropical Gyre. The results indicate blooms form when phytoplankton are released from limitation by resources (nutrients, vitamins, and trace metals) and that the mechanistic basis for the success of one functional group over another may be driven by how efficiently the transcriptome is modulated following a nutrient pulse. A diverse microbial assemblage in the ocean is responsible for nearly half of global primary production. It has been hypothesized and experimentally demonstrated that nutrient loading can stimulate blooms of large eukaryotic phytoplankton in oligotrophic systems. Although central to balancing biogeochemical models, knowledge of the metabolic traits that govern the dynamics of these bloom-forming phytoplankton is limited. We used eukaryotic metatranscriptomic techniques to identify the metabolic basis of functional group-specific traits that may drive the shift between net heterotrophy and autotrophy in the oligotrophic ocean. Replicated blooms were simulated by deep seawater (DSW) addition to mimic nutrient loading in the North Pacific Subtropical Gyre, and the transcriptional responses of phytoplankton functional groups were assayed. Responses of the diatom, haptophyte, and dinoflagellate functional groups in simulated blooms were unique, with diatoms and haptophytes significantly (95% confidence) shifting their quantitative metabolic fingerprint from the in situ condition, whereas dinoflagellates showed little response. Significantly differentially abundant genes identified the importance of colimitation by nutrients, metals, and vitamins in eukaryotic phytoplankton metabolism and bloom formation in this system. The variable transcript allocation ratio, used to quantify transcript reallocation following DSW amendment, differed for diatoms and haptophytes, reflecting the long-standing paradigm of phytoplankton r- and K-type growth strategies. Although the underlying metabolic potential of the large eukaryotic phytoplankton was consistently present, the lack of a bloom during the study period suggests a crucial dependence on physical and biogeochemical forcing, which are susceptible to alteration with changing climate.

Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria

Aerobic degradation of methylphosphonate (MPn) by marine bacterioplankton has been hypothesized to contribute significantly to the oceans methane supersaturation, yet little is known about MPn utilization by marine microbes. To identify the microbial taxa and metabolic functions associated with MPn-driven methane production we performed parallel metagenomic, metatranscriptomic, and functional screening of microcosm perturbation experiments using surface water collected in the North Pacific Subtropical Gyre. In nutrient amended microcosms containing MPn, a substrate-driven microbial succession occurred. Initially, the addition of glucose and nitrate resulted in a bloom of Vibrionales and a transcriptional profile dominated by glucose-specific PTS transport and polyhydroxyalkanoate biosynthesis. Transcripts associated with phosphorus (P) acquisition were also overrepresented and suggested that the addition of glucose and nitrate had driven the community to P depletion. At this point, a second community shift occurred characterized by the increase in C-P lyase containing microbes of the Vibrionales and Rhodobacterales orders. Transcripts associated with C-P lyase components were among the most highly expressed at the community level, and only C-P lyase clusters were recovered in a functional screen for MPn utilization, consistent with this pathway being responsible for the majority, if not all, of the methane accumulation we observed. Our results identify specific bacterioplankton taxa that can utilize MPn aerobically under conditions of P limitation using the C-P lyase pathway, and thereby elicit a significant increase in the dissolved methane concentration.

Metabolic balance in the mixed layer of the oligotrophic North Pacific Ocean from diel changes in O2/Ar saturation ratios

In situ measurements were made to determine oxygen (O2) metabolic balance in the upper oligotrophic ocean from diel changes in O2 to argon (Ar) ratios. The study took place during 13–24 March 2014, at the Hawaii Ocean Time-series Station ALOHA (A Long-term Oligotrophic Habitat Assessment), in the North Pacific Subtropical Gyre. Microbial community respiration and gross O2 production, estimated from in situ diel changes in O2/Ar saturation, agreed well with those calculated using other independent methods. Net oxygen production (NOP), estimated from in situ diel changes in O2/Ar saturation, showed large day-to-day variability. However, when averaged over the entire observational period, mean diel NOP was in relatively good agreement with the estimated mean steady state NOP (9.2 ± 9.3 mmol O2 m−2 d−1 compared to 11.7 ± 1.1 mmol O2 m−2 d−1, respectively).

Bacterial Dimethylsulfoniopropionate Degradation Genes in the Oligotrophic North Pacific Subtropical Gyre

ABSTRACT Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound that is rapidly metabolized by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methiolpropionate. The abundance and diversity of genes encoding bacterial DMS production (dddP) and demethylation (dmdA) were measured in the North Pacific subtropical gyre (NPSG) between May 2008 and February 2009 at Station ALOHA (22°45′N, 158°00′W) at two depths: 25 m and the deep chlorophyll maximum (DCM; ∼100 m). The highest abundance of dmdA genes was in May 2008 at 25 m, with ∼16.5% of cells harboring a gene in one of the eight subclades surveyed, while the highest abundance of dddP genes was in July 2008 at 25 m, with ∼2% of cells harboring a gene. The dmdA gene pool was consistently dominated by homologs from SAR11 subclades, which was supported by findings in metagenomic data sets derived from Station ALOHA. Expression of the SAR11 dmdA genes was low, with typical transcript:gene ratios between 1:350 and 1:1,400. The abundance of DMSP genes was statistically different between 25 m and the DCM and correlated with a number of environmental variables, including primary production, photosynthetically active radiation, particulate DMSP, and DMS concentrations. At 25 m, dddP abundance was positively correlated with pigments that are diagnostic of diatoms; at the DCM, dmdA abundance was positively correlated with temperature. Based on gene abundance, we hypothesize that SAR11 bacterioplankton dominate DMSP cycling in the oligotrophic NPSG, with lesser but consistent involvement of other members of the bacterioplankton community.

Hydrogen Cycling by the Unicellular Marine Diazotroph Crocosphaera watsonii Strain WH8501

ABSTRACT The hydrogen (H2) cycle associated with the dinitrogen (N2) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H2 production and acetylene (C2H2) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H2 production was coincident with the maximum rates of C2H2 reduction. Theoretical stoichiometry for N2 fixation predicts an equimolar ratio of H2 produced to N2 fixed. However, the maximum rate of net H2 production observed was 0.09 nmol H2 μg chlorophyll a (chl a)−1 h−1 compared to the N2 fixation rate of 5.5 nmol N2 μg chl a−1 h−1, with an H2 production/N2 fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H2 production was hypothesized to be a result of H2 reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H2 production rates ∼40-fold to a maximum rate of 3.5 nmol H2 μg chl a−1 h−1. We conclude that the reassimilation of H2 by C. watsonii is highly efficient (>98%) and hypothesize that the tight coupling between H2 production and consumption is a consequence of fixing N2 at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H2 cycle provides unique insight into N2 fixation and associated metabolic processes in C. watsonii.

Quantifying subtropical North Pacific gyre mixed layer primary productivity from Seaglider observations of diel oxygen cycles

Using autonomous underwater gliders, we quantified diurnal periodicity in dissolved oxygen, chlorophyll, and temperature in the subtropical North Pacific near the Hawaii Ocean Time-series (HOT) Station ALOHA during summer 2012. Oxygen optodes provided sufficient stability and precision to quantify diel cycles of average amplitude of 0.6 µmol kg−1. A theoretical diel curve was fit to daily observations to infer an average mixed layer gross primary productivity (GPP) of 1.8 mmol O2 m−3 d−1. Cumulative net community production (NCP) over 110 days was 500 mmol O2 m−2 for the mixed layer, which averaged 57 m in depth. Both GPP and NCP estimates indicated a significant period of below-average productivity at Station ALOHA in 2012, an observation confirmed by 14C productivity incubations and O2/Ar ratios. Given our success in an oligotrophic gyre where biological signals are small, our diel GPP approach holds promise for remote characterization of productivity across the spectrum of marine environments.

Coordinated regulation of growth, activity and transcription in natural populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera

The temporal dynamics of phytoplankton growth and activity have large impacts on fluxes of matter and energy, yet obtaining in situ metabolic measurements of sufficient resolution for even dominant microorganisms remains a considerable challenge. We performed Lagrangian diel sampling with synoptic measurements of population abundances, dinitrogen (N2) fixation, mortality, productivity, export and transcription in a bloom of Crocosphaera over eight days in the North Pacific Subtropical Gyre (NPSG). Quantitative transcriptomic analyses revealed clear diel oscillations in transcript abundances for 34% of Crocosphaera genes identified, reflecting a systematic progression of gene expression in diverse metabolic pathways. Significant time-lagged correspondence was evident between nifH transcript abundance and maximal N2 fixation, as well as sepF transcript abundance and cell division, demonstrating the utility of transcriptomics to predict the occurrence and timing of physiological and biogeochemical processes in natural populations. Indirect estimates of carbon fixation by Crocosphaera were equivalent to 11% of net community production, suggesting that under bloom conditions this diazotroph has a considerable impact on the wider carbon cycle. Our cross-scale synthesis of molecular, population and community-wide data underscores the tightly coordinated in situ metabolism of the keystone N2-fixing cyanobacterium Crocosphaera, as well as the broader ecosystem-wide implications of its activities.