Shortcuts in the Oceanic Nitrogen Cycle: Fluxes and Microbial Pathways of Nitrogen Remineralization in the Ocean's Twilight Zone

Lead Research Organisation: University of Southampton
Department Name: School of Ocean and Earth Science

Abstract

The Ocean's twilight (mesopelagic, 100-1000m) zone lies beneath the sunlit surface ocean, with too little light for photosynthesis but above the pitch-black deep ocean, where large animals can no longer see their prey. Of all organic matter that sinks out from the surface, >90% is degraded in the mesopelagic with only a small fraction escaping into the deep ocean (>1000 m). The mesopelagic thus represents an important barrier: most material falling into it is prevented from sinking further by remineralisation - the degradation process that breaks down organic matter and releases CO2 and inorganic nutrients to solution. Eventually, physical mixing or ocean circulation deliver the nutrients back to the surface to fuel phytoplankton growth. Hence, remineralisation in the mesopelagic is critical to controlling the oceanic biological pump, and can affect the ocean's ability to sequester atmospheric CO2. Nitrogen is often the limiting nutrient for biological production in global oceans, its remineralisation would thus be key to biological pump efficiency. However, the mechanisms of N-remineralisation are poorly characterized, and there are no rate measurements of this process in the mesopelagic.

The remineralisation of nitrogen (N) in the oceans encompasses ammonification: the degradation of organic N to ammonium (NH4+), and subsequently nitrification: the oxidation of NH4+ to nitrite and then nitrate. However, a recent study suggests that some nitrifiers (microorganisms conducting nitrification) can utilise organic N directly, thus presenting a possible shortcut in the N-cycle. Because the respective organisms have different feeding styles regarding carbon (CO2- fixing or producing), the relative abundance and activities of these functional groups of organisms will have different direct impacts on the CO2 balance, and the existence of the potential shortcut will likely cause a shift towards autotrophy (CO2 fixation).

This project aims to determine exactly how and how much nitrogen is remineralised in the twilight ocean, using a combination of state-of-the-art geochemical rate measurements and molecular biological analyses. In particular, we will determine whether the above-mentioned shortcut exists in the remineralisation of organic nitrogen (N) to nitrate (NO3-), and quantitatively assess its potential significance to the oceanic N-cycle relative to the conventional ammonification-nitrification pathway. Together, these planned analyses will give the most complete dataset of directly measured N-remineralisation fluxes ever attempted in the oceans.
State-of-the-art 15N-stable-isotope-labeling experiments will be conducted to measure rates of concurrent N-conversions for a more accurate assessment of upper ocean N-budget: ammonification, nitrification, assimilation (incorporation of N into biomass) and release of dissolved organic N. We will do this by tracing 15N (the heavy stable isotope of N that is rare in nature, as opposed to the common 14N) from various amended organic substrates into different N-pools at the same time, to determine whether organic-N is directly channeled to nitrification or via ammonification. In parallel, major remineralisation pathways will be identified by elucidating the expression of biomarker enzymes key to these N-conversions at both gene transcript and protein levels, as quantifiable activity indicators for the respective processes.

Sampling is planned along the Atlantic Meridional Transect (AMT) from the north (UK) to south Atlantic (Falklands/Chile) to examine N-remineralisation in diverse nutrient regimes, while temporal variability is explored via seasonal sampling at the Bermuda Atlantic Time Series (BATS) site. Such spatiotemporal coverage and complementary, interdisciplinary dataset would yield a highly representative depiction of mesopelagic N-remineralisation in the oceans, and the most comprehensive assessment to date on the significance of the twilight zone in oceanic N-cycle.

Planned Impact

Increasing human activities and pressures on our planet have resulted in, in addition to the greater emissions of greenhouse gases, higher inputs of anthropogenic nitrogen into oceans. In particular, out of ~67 Tg N/y of atmospheric nitrogen deposition into the ocean, ~80% is anthropogenic, an increase from 29% in pre-industrial times (Duce et al., 2008, Science 320:893-897). This enhanced nutrient level to the surface ocean, whether from the atmosphere, rivers or land, has significantly stimulated surface production and so CO2 drawdown. How long this enhanced export production may last, however, depends on a combination of feedbacks and factors, including the efficiency of the biological pump mediated by the remineralisation of organic matter in both of the euphotic zone and the mesopelagic.

Since this proposed study plans to address the efficiency of remineralisation in both the lower euphotic and the mesopelagic, while comparing different nutrient regimes and seasons, data from this study will provide invaluable information on how such enhanced N inputs may impact the export of organic matter into the deep, the degree of remineralisation within the mesopelagic and how the increase in nitrogen may alter such balance, and if there exist other possible feedbacks via the little known resident microbial communities. These data can be further coupled to e.g. global circulation models for more comprehensive assessment, and to yield better understanding on how increased surface production may impact subsurface processes and their feedbacks to surface production. These model outputs may provide the necessary basis and guidance for governments and policy makers, such as DEFRA and the Environmental Agency in the UK and other countries, to the design of appropriate policies to control the amounts and forms of anthropogenic nitrogen released into the environment from different channels. Therefore, this proposed study would have direct and indirect impacts on government policy makers, and would also be beneficial to the well being of wider general public.

The mesopelagic zone harbours microbial communities distinct from the surface ocean. As a result, there is a good chance of encountering new organisms, new proteins and new enzymes from the metaproteomics analyses. Of particular interests are the detection of enzymes and proteins involved in the degradation of organic matter, which may find applications in bioprocessing and bioremediation and thus interest industrial sectors like biotechnology or wastewater treatment.

Publications

10 25 50
 
Description The objectives of this project were: 1) to determine whether a shortcut exists in the remineralisation of organic nitrogen to nitrate, thus bypassing the need for external
ammonium sources; and 2) to quantify the significance of this shortcut to oceanic nitrogen cycle relative to the conventional ammonification-nitrification
pathway. [Nitrification = oxidation of ammonium to nitrite and then nitrate; Ammonification = remineralisation of organics to release ammonium.] We used a combination of omics analyses and incubation experiments with various 15N-labeled inorganic and organic substrates to distinguish the direct vs indirect pathways, and to quantitatively compare the significance of the two in different seasons and ocean provinces (regions of the oceans marked by distinct levels of nutrients and subsequently biological productivity structured by various oceanographic factors).
After 4 repeated sampling at the Bermuda Atlantic Time-Series site (BATS) over 3 seasons, and along the Atlantic Meridional Transect (AMT) that traversed across nine ocean provinces, we found that nitrifying organisms can indeed directly use small organic nitrogen compounds, including urea, amino acids and amino sugars, and convert them to nitrite/nitrate without relying on another organism for the ammonification step. However, ammonium remain the predominant substrate used for nitrification in ocean basin wide scale. In absolute terms, the use of organics in nitrification was higher in more productive seasons and waters; yet the relative importance of organics in nitrification appears to increase in the less productive depths and times, such as in the lower twilight ocean and in the middle of summer when/where ammonium is virtually depleted.
Exploitation Route We are in the process of finalising our data analyses for publication in peer-reviewed journals, aiming at least 2 in higher impact journals (e.g. Nature Communications, PNAS, Science) that have wider readership to maximise our impacts in the research community. We have so far presented our results in four international research conferences, and more will be anticipated in the coming couple of years. Findings from this project would also be included in the teaching materials used in various undergraduate modules the PI/Co-I coordinate at both undergraduate and graduate levels (e.g. Introduction to Biological Oceanography/ Ocean Biogeochemistry, Marine Molecular Biology, and Biogoechemical Cycling in Earth System). We have also organised outreach activities focussing on the roles of ocean microbes in our regional Science and Engineering Festival, and will refine this for a new exhibit/ interactive activities in the coming year, as our ongoing efforts to transfer knowledge and broaden public interests in biogeochemical functions of microorganisms in Earth system, accompanied by a regularly updated website and blog on our research and outreach activities. Environmental microbiology, especially marine and aquatic microbiology, are in a broad sense lagging behind counterparts in e.g. continental Europe and North America. Our research outcomes from this project would thus help in closing this undesirable gap, and serve as a basis to further develop leading programmes in microbiological research in the twilight and deep dark ocean.
Sectors Education,Environment,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Masters thesis research training
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Work from this grant resulted in thesis research training opportunities for 3 MSc/MRes candidates - successful completion of MSc degree in 2017 by both Rachel Rayne and Matthew Crossley, who both moved on to become PhD candidates in related environmental research fields. Rachel Rayne 2017 - CHARACTERISING DISSOLVED ORGANIC MATTER USING FOURIER-TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY Matthew Crossley 2017 - A METATRANSCRIPTOMIC INVESTIGATION OF GENE EXPRESSION AND ACTIVE METABOLIC PATHWAYS WITHIN THE SARGASSO SEA OVER SPATIAL AND TEMPORAL SCALES Ongoing MRes thesis research by Emily Cooper in academic year 2018/19.
 
Title Optimisation of conversion method for ammonification and nitrification rate measurements using stable isotope tracers 
Description The use of stable-isotope-labeled organic nitrogen compounds in incubation experiments to determine nitrification and ammonification rates are often challenged by contamination of the stable isotopes in various inorganic nitrogen pools. Method refinement and optimisation has now been made to establish a working protocol that allows accurate measurements of these nitrogen conversions using gas chromatography-isotope ratio mass spectrometry. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The working protocol will be published later with our research findings. 
 
Title Tracing production of dissolved organic nitrogen (DON) compounds in mesopelagic ocean 
Description Combining incubation experiments with 15N-labeled tracers with Fourier Transform - Ion Cyclotron Resonance Mass Spectrometry (FT-ICRMS) analyses to characterise DON compounds produced from specific inorganic and organic nitrogen compounds. This involved a series of optimisation and innovative methods in data analyses to pinpoint the compounds specifically produced from various nitrogen-containing substrates in the ocean's twilight zone 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact DON composition have been treated as a big black box of organic compounds, but recent developments of the FTICRMS have enabled us to identify the various components of DON in seawater. However, the production pathways of the diverse marine DON compounds remain unexplore. Here, we combined the 15N-tracer experiments with FT-ICRMS to examine which DON compounds would be respecitvely produced by the common inorganic nutrient nitrogen (ammonium and nitrate) and various simple organic nitrogen compounds (e.g. urea, amino acids, amino sugars). To our knowledge, this is the first investigation to dissect the production mechansim of the complex DON pool in seawater. 
 
Title DOM characterisation from BATS 
Description FT-ICRMS analyses have been performed on dissolved organic matter samples collected from the 4 BATS cruises in 2016/17. Novel DOM characterisation was performed on experimental samples incubated with various 15N-labeled tracers to decipher the transformations of organic nitrogen compounds. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact These data and analyses have led to the completion of a Master thesis - Rachel Rayne (MSc 2017, University of Southampton). Further analyses are ongoing and aim to be published in peer-reviewed journal by end of the year/ early next year. Dataset would then be made publically available via BODC after publication. 
 
Title Molecular biological analyses for samples from BATS and AMT 
Description Metatranscriptomics analyses have been performed on samples collected from the 4 research cruises to the Bermuda Atlantic Time Series (BATS) site on April, July, November 2016 and April 2017, while they are ongoing for Atlantic Meridional Transect (AMT) in 2017 Catalysed Reporter Deposition-Fluorescence in situ Hybridisation (CARD-FISH) on selected samples to enumerate specific microbial groups. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact Sample collection and subsequent data analyses have led to the completion of a Master thesis - Matthew Crossley (MSc 2017, University of Southampton). Further advanced analyses are taking place and anticipate of publication of data in 2019. Sequence data would then be made publically available via GenBank (NCBI). AMT sample and data analyses ongoing. CARD-FISH cell counts would be made available upon publication of relevant paper. 
 
Title Nitrogen cycling rate measurements 
Description Rate measurements resulted from incubation experiments with 15N-labeled tracers conducted on the 5 participated research cruises, and subsequently measured in shorebased laboratories (QMUL and MPI-MM). Thus far, data include rates of nitrification and assimilation for 4 BATS cruises, and partially nitrification rates for AMT cruise. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? No  
Impact Measurements started for the first cruises in 2016, but due to necessary method optimisation, measurements are ongoing and full dataset will not be available until later this year. We anticipate dissemination of some of our results through presentation in conferences this summer - e.g. Gordon Research Conference on Marine Microbes and International Society of Microbial Ecology Conference. 
 
Description Atlantic Meridional Transect (AMT) programme 
Organisation Plymouth Marine Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution The Atlantic Meridional Transect (AMT) is a multidisciplinary programme which undertakes biological, chemical and physical oceanographic research during an annual voyage between the UK and destinations in the South Atlantic. Our team joined the AMT research expedition in September-November 2017, to conduct sampling and experiments as laid out in our proposal, while also adding measurements of ammonium and urea to the AMT core measurements. Our nitrogen-remineralisation and cycling rate measurements are highly complementary to the ongoing investigations on the distribution and cycling of nitrous oxide, general nitrogen cycling and primary productivity along the transect, while our molecular data (metatranscriptomics and metaproteomics) could give additional insights into differentiating the microbial processes occurring in these regions.
Collaborator Contribution Over the past 2 decades, the AMT programme has conducted 26 annual cruises from the UK to either Falklands or Cape Town in the South Atlantic, traversing multiple biogeochemical provinces of contrasting productivity and nutrient regimes. It provides an invaluable dataset of core parameters in physical oceanography, nutrient distribution, primary production and respiration, as well as optical data on particle distribution in the water column, and now also greenhouse gases methane and nitrous oxide. Hence, not only the AMT programme provides us a sampling platform and framework, the AMT background data are essential to the interpretation to our experimental results.
Impact Two master theses (both at Ocean and Earth Science, University of Southampton) - Jack Harrington (MSci 2014), Alexander Moody (MSci 2014)
Start Year 2013
 
Description Bermuda Atlantic Time Series Study (BATS) 
Organisation Bermuda Institute of Ocean Sciences
Country Bermuda 
Sector Academic/University 
PI Contribution Bermuda Atlantic Time Series (BATS) study is an ongoing programme that has been undertaking monthly biogeochemical oceanographic measurements at an open ocean site 82km south-east to Bermuda since 1988. We joined the BATS team on 3 sampling cruises in 2016 (April, July, November) and 1 more in April 2017, in order to conduct our sampling and experiments as planned in our project proposal - to assess the fluxes and pathways mesopelagic nitrogen cycling, and to identify the active microorganisms mediating these processes. These process studies on marine nitrogen cycling are highly relevant and complementary to the carbon cycling and microbial ecology investigations that are ongoing with the BATS team. We will exchange necessary data and work closely together in subsequent data analyses and interpretation.
Collaborator Contribution Our collaborators at BATS (Prof. Nicholas Bates and Dr. Rodney Johnson) have provided us with the necessary platform and logistical support for sampling onboard the research vessel Atlantic Explorer during their scehduled BATS cruises. At the same time, they provide us with the physical and chemical oceanographic data during the same research cruises (e.g. CTD data, nutrients and primary production data) that enable more comprehensive data interpretation.
Impact Basic BATS data such as CTD and nutrients are made public soon after research according to their funding agency's data policy (More details on the BATS project website - http://bats.bios.edu/). From our side, samples are still being analysed and data being processed for various biogeochemical and molecular biological parameter.
Start Year 2016
 
Description FT-ICR MS analyses of marine DOM 
Organisation Carl von Ossietzky University of Oldenburg
Country Germany 
Sector Academic/University 
PI Contribution We conducted experiments while at sea and subsequently provided samples to examine the production pathways of dissolved organic matter in the mesopelagic ocean, with emphasis on nitrogen containing compounds.
Collaborator Contribution Prof. T. Dittmar from ICBM Oldenburg provides the expertise on DOM cycling and instrumentation (Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, FT-ICRMS) to analyse the composition of DOM.
Impact Samples are currently being processed
Start Year 2016
 
Description GC-IRMS analyses 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution 15N-labelling experiments to decipher complex nitrogen cycling processes
Collaborator Contribution Expertise and access to GC-IRMS measurements
Impact 15N-rates data.
Start Year 2017
 
Description Blog post - A Typical Atypical Day at Sea 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact It is a blog post describing a 'typical' day at sea on a research vessel, whilst I was on a research expedition sailing from North to South Atlantic. The blog post was written for the Mass Open Online Course - Exploring our Oceans hosted at University of Southampton, though the blog was open to the public, and not just for registered participants. The blog usually get 100-200 hits per day. Audience ranged from school pupils at age 6 to retirees, from the general public with interests in the Oceans.
Year(s) Of Engagement Activity 2017
URL http://moocs.southampton.ac.uk/oceans/2017/11/15/typical-atypical-day-sea/
 
Description Project Introduction Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact We gave a talk to introduce our project to a wider audience in our department in the second month after the commencement of our project.
Year(s) Of Engagement Activity 2016
 
Description Science and Engineering Festival - stand on "Ocean of Microbes" - open to all ages at the University of Southampton 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We are responsible for one of the activities "Oceans of Microbes" (Ecosystem in a Bottle) at the annual Science and Engineering Festival that is to be held on 18th March, 2017, at the Boldrewood Innovation Campus, University of Southampton. This annual science and engineering festival is aimed to inspire and educate the general public of all ages, and to showcase the research-led activities relevant to the general public in this open event. This science and engineering festival is publicised and brocasted via various channel on social media. (Full details can be found at http://www.sotsef.co.uk/science-and-engineering-day/).
Year(s) Of Engagement Activity 2017
URL http://www.sotsef.co.uk/science-and-engineering-day/