The Ecology of Nitrogen Cycling Microorganisms in the Subtropical North Pacific Ocean

Thank you… Daniela Böttjer, Brenner Wai, Donn Viviani, Sara Thomas, Christina Johnson Dave Karl, Jon Zehr, Ed DeLong NSF

A Dedicated HOT Team NSF NSF

N cycle stories from the open sea How do seasonal to episodic changes in the upper ocean habitat influence distributions, abundances, and activities of N cycling microorganisms? N2 fixing cyanobacteria Ammonia oxidizing Crenarchaea Focus on epipelagic N- cycle dynamics: Concentrations of inorganic N low Rapid N turnover Intense vertical gradients in microbial habitat structure (light, nutrients, temperature, etc.) Sensitive to physical perturbations (seasonal to episodic)

Time, water, and change The 24 years of Hawaii Ocean Time-series (HOT) measurements provide a rich time-history for assessing change to a persistently oligotrophic ocean ecosystem. The resulting data provides insight into the mean ecosystem state, and variability around the mean state. ALOHA Photo: Paul Lethaby

The upper ocean habitat 14C-PP PAR Chl a N+N

These processes are time variable Physical and biological controls on nitrogen availability to the upper ocean N2 fixation Physical: Mixing Upwelling, downwelling Biological: N2 fixation Remineralization (e.g. ammonification, nitrification) Organic matter NH4+ NO2- NO3- These processes are time variable Organic matter NO3- NO2- NH4+

Seasonal variations in upper ocean temperature, light, and nutrients

Seasonal climatology of primary production and particle export Productivity and export both elevated in summer when upper ocean nutrient concentrations are lowest. What processes supply nitrogen to support new production in this ecosystem?

Biological N supply to the ocean: N2 fixation At ALOHA, N2 fixation fuels ~50% of new production (and hence carbon export) Several groups of N2 fixing microorganisms identified Regular occurrence of large blooms of N2 fixing microorganisms; Trichodesmium spp. and/or diatoms with endo/epi-symbionts Changes in elemental stoichiometry of particulate and dissolved nutrient pools (decreased P availability) attributed to N2 fixation.

Motivating Questions How variable in time are N2 fixing microbes and rates of N2 fixation? What processes control variability in diazotroph population structure and rates of N2 fixation?

nifH → Nitrogenase (N2 fixers) Approach Combined rate measurements of N2 fixation together with analyses of time/space variability in distributions and activities of N cycle microbes in the sea. nifH → Nitrogenase (N2 fixers)

N2 fixing cyanobacteria at Station ALOHA based on nifH gene transcripts Richelia-1 Richelia-2 Richelia-1 20-50 mm Heterocystous cyanobacteria Richelia-1 >10 mm Richelia-2 Calothrix 2-10 mm Group A Unicellular cyanobacteria Calothrix Crocosphaera Trichodesmium / Katagynemene 20-50 mm Trichodesmium spp. Katagynemene spp. Images courtesy of Angel White, Rachel Foster, Grieg Steward 20-100 mm 2-2000 mm

Several cultivated strains (in culture since 1985) Uncultivated Crocosphaera “Group A” Several cultivated strains (in culture since 1985) Uncultivated Fixes N2 at night Likely fixes N2 during day Photosystem I & II Photosystem I Functional TCA cycle No TCA cycle Photoautotroph Photoheterotroph “Free” living Symbiont? Church et al. (2005)

0-125 m Abundances of unicellular N2 fixing cyanobacteria vary seasonally and interannually

Temporal variability in N2 fixation Major fraction (~70%) of annual N2 fixation associated with microorganisms <10 mm. Rates of N2 fixation tend to increase in the summer, driven by microorganisms > 10 mm.

Unicellular N2 fixers are dominate diazotroph abundances and activities most of the year Filamentous diazotrophic cyanobacteria increase episodically through the summer and fall 0-125 m Modified from Church et al. (2009)

Spatiotemporal history of a January 2005 Spatiotemporal history of a downwelling eddy April 2005 Fong et al. 2008 July 2005 July 2005 “typical profile”

Time series measurements of near-surface ocean N2 fixation at Station ALOHA Episodic increases in N2 fixation by diazotrophs >10 mm appear associated with mesoscale physical forcing Modified from Church et al. (2009, GBC) nmol N L-1 d-1

OPEREX: July 2009 CMORE cruise in the subtropical North Pacific Ocean Meso- and submesoscale physical processes may drive enhanced carbon export in the open sea OPEREX: July 2009 CMORE cruise in the subtropical North Pacific Ocean Chlorophyll a N2 fixing microorganism biomass accumulation SSHa 2 white transects Cruise track across eddies Guidi et al. 2012 Large particle concentration

What is the time history associated with these processes? Property (0-200 m) SSHa - + Primary production  Carbon export NO3-   NO3- : PO43- Chl a Prochlorococcus Diatoms Pelagophytes Haptophytes N2 fixation What is the time history associated with these processes? How does such mesoscale variability influence plankton ecology?

Variability in diazotrophs and N2 fixation Unicellular N2 fixing microorganisms are important contributors to the upper ocean N cycle Different groups of N2 fixing microorganisms have very different physiologies and population dynamics Larger, filamentous N2 fixing microorganisms (Trichodesmium, heterocystous N-fixers) increase seasonally, but episodically Mesoscale physical processes appear important drivers of episodic N2 fixation supported plankton blooms

Nutrient recycling: Dynamics of ammonia oxidizing Thaumarchaea N2 fixation amoA gene as a molecular marker to examine the time/space variability in Thaumarchaeal distributions and transcriptional activities Organic matter NH4+ NO2- NO3- amoA Organic matter NO3- NO2- NH4+

Nitrification and ammonia oxidation Nitrification is the two-step process that converts ammonia to nitrate. Ammonia oxidation: NH3 + O2 → NO2− + 3H+ + 2e- Nitrite oxidation: NO2− + H2O → NO3− + 2H+ + 2e- Different steps are mediated by different groups of prokaryotic microorganisms Ammonia oxidation historically thought to be catalyzed by ammonia oxidizing bacteria (b and g-Proteobacteria) Now thought to be predominately driven by members of the Thaumarchaeota (previously classified as mesophilic Crenarchaeota).

Karner et al. (2001) Könneke et al. (2005) Archaea first identified in seawater in 1992 Found to dominate plankton biomass in the meso- and bathypelagic waters at Station ALOHA Sequencing efforts in Sargasso Sea discovered gene encoding ammonia monooxygenase from planktonic Thaumarchaea (Venter et al. 2004) Isolation of marine Thaumarchaea (Nitrosopumilus maritimus) from Seattle Aquarium found to grow using ammonia as sole source of energy, CO2 as carbon source Könneke et al. (2005)

Only cultivated representative of marine Thaumarchaea “Upper ocean” Thaumarchaea Only cultivated representative of marine Thaumarchaea “Deep ocean” Thaumarchaea Comparison of Thaumarchaea amoA genes indicates at least two vertically partitioned groups (upper and deep ocean) – Mincer et al. (2007), Beman et al. (2008). At Station ALOHA there appear to be “sub-clades” of these vertically partitioned major groups.

Brenner Wai, Christina Johnson Vertical distributions of these major groups are distinct, with the transition region between the euphotic zone and the upper mesopelagic comprising a dynamic transition point. 1% PAR 0.1% PAR Brenner Wai, Christina Johnson

Distributions of Thaumarchaea amoA genes appear sensitive to upper ocean mixing 108 107 50 106 100 amoA genes L-1 105 Depth (m) 104 150 103 200 102 2007 2008 2009 2010 Year What processes control the vertical distributions of Thaumarchaea? Physics (mixing, upwelling, downwelling)? Light? Competition for substrates? Top down (predators, viruses)? Brenner Wai et al.

Thaumarchaea amoA expression and rates of ammonia oxidation at Stn Thaumarchaea amoA expression and rates of ammonia oxidation at Stn. ALOHA Transcription of the amoA gene often peaks in the transition zone between the euphotic zone and the upper mesopelagic Ammonia oxidation rates courtesy of Mike Beman

Gene abundances increase by ~1000-fold between the near-surface ocean and the lower euphotic zone (200 m). Gene transcripts increase ~10- fold toward the base of the euphotic zone. Why are the upper ocean populations apparently so active, yet not abundant?

Conclusions Time-varying changes in ocean habitat structure influence the population dynamics of N cycling microorganisms. Ecology matters: Changes in population structure of N cycling microbes are directly influence ocean biogeochemistry. Interactions between biogeochemistry and ecology occur across a range of scales, from decadal to seasonal to episodic.