PhD Candidate, Duke University Visiting Researcher, University of Oslo Investigating interactions between the major biogeochemical cycles through understanding ecosystem-scale organic matter dynamics I’d like to take today as an opportunity to talk more about some of the projects and research that I have going on outside of the work we’re doing here in Norway. My dissertation research is focused on understanding interactions between the C and N cycles through investigating organic matter dynamics in, and in particular organic matter losses, from forested watersheds. Brian Lutz PhD Candidate, Duke University Visiting Researcher, University of Oslo

Outline Research Background and Justification Ch 1: Preliminary Test of Hypotheses Synoptic Surveys of Stream Chemistry Ch 2: Ecosystem Scale Manipulations Stream Nutrient Spiraling Ch 3: Geochemical Controls on DON Losses Soil Sorption Assays Ch 4: DOM Characterization Fluorescence and PARAFAC Modeling Eutropia Project I’ve structured this talk to be very general. I will spend the first third or half of the talk discussing the background and context for my dissertation research, and then will spend the balance of the time only briefly describing some of the projects I have ongoing. While I won’t be able to go into much detail for each of the specific projects, my hope is that this will give many of you who I haven’t had much chance to talk with more insight into my interests and, perhaps, lead to opportunities for more detailed discussions throughout the weekend.

Human Domination of the Global N Cycle It is well known that humans have had a major impact on the global nitrogen cycle – doubling the amount of reactive N supplied to the biosphere on an annual basis. Credits: Netherlands Environmental Assessment Agency, www.pbl.nl

Nitrogen Saturation: Environmental Consequences It is also well known that this increase in N pollution has environmental consequences, perhaps most noticeably is the increase in frequency of dead zones in coastal marine ecosystems due to increased N runoff from the landscape. Credits: Diaz and Rosenberg, Science, 2008

Nitrogen Saturation: Symptoms and Diagnosis Much of our understanding of N losses from the landscape is based solely on inorganic N dynamics. Models of ecosystem N saturation, such as the one here by Aber and others, focus almost exclusively on nitrate. Because the nitrate anion is highly mobile in most soils and because nitrate is often highly biologically available, nitrate is a good indicator species for assessing ecosystem N demand – when biotic N demand is saturated, nitrate losses are high. But this framework has led to an overemphasis on inorganic N losses from ecosystems. Credits: Aber, BioScience, 1998

What about organic nitrogen? But what about organic nitrogen? Soil organic N is the single largest pool of fixed N on the planet. In most ecosystems, almost all of the N is contained in organic matter at any given time—as shown here in the nitrogen budget for one of the reference watersheds at Hubbard Brook where more than 99% of the total ecosystem N is contained in organic matter. Credits: Bormann et al., Science, 1977

What about organic nitrogen? In addition, a large portion of the organic N in a given ecosystem cycles dynamically. It is the turnover of organic N that fuels the majority of primary production globally. Only in the most polluted ecosystems does the rate of anthropogenic supply of inorganic N exceed the rate of organic N turnover. And because so much organic N is turning over – entering soil solution, being broken down by microbes, and both taken up and produced by plants – some amount of organic N is subject to loss hydrologically. And while organically bound N is often not nearly as mobile in most soils as nitrate, if only a few percent of the organic N in an ecosystem is lost this amount could easily rival or surpass the amount of inorganic N losses. Organic N turnover supports the majority of ecosystem production in most ecosystems Credits: Bormann et al., Science, 1977

DON Losses from the landscape are important And this is often the case. Currently, despite humans having doubled the amount of inorganic N supplied to the biosphere annually, organic N losses continue to dominate the flux of N from terrestrial to aquatic ecosystems globally. Even in the United States, where fertilizer application and N deposition rates are high, N losses from many watersheds are dominated by organic N. Moreover, even in the Midwest and Northeast regions where pollution is high, organic N still often accounts for 20-40% of the total N runoff. Credits: Scott et al., Global BGC Cycles, 2007

Nitrogen Saturation: Symptoms and Diagnosis DON?? And although we have a good understanding of how inorganic N losses change under N saturation, we know very little about how organic N losses respond to increased N avaialbility. We do know that inorganic N often dominates runoff when N pollution is high. But this does not mean that there are also not important increases in organic N losses as well. But there has been extremely little work done on this. Credits: Aber, BioScience, 1998

‘Breaks in the Cycle’: Increased organic N losses? - Neff et al., Frontiers in Ecology, 2003 Saturated Plant Demand Anthropogenic N Additions Neff and others proposed a conceptual model for the potential response of organic N losses under N saturation. They suggest that inorganic N loading might ultimately saturated both plant and microbial demand for N and, therefore, any organic N that would have otherwise been consumed to satisfy N demand may be lost from the ecosystem. INCREASED ORGANIC N LOSSES

DON Release Hypothesis: Schematic This may be better represented using this schematic, where we show DOM as being a heterogenous pool of molecules varying in their N content. When N is limiting, the dark circles representing N-rich molecules are consumed to satisfy ecosystem N demand. Another portion of the DOM pool is consumed to satisfy heterotrophic C demand, and a small N-poor portion of the DOM pool is subject to hydrologic loss. Under the Neff model, when systems reach N saturation the N-rich molecules that were previously being consumed are instead exported.

DON Release Hypothesis: Predictions This could be illustrated in the Aber model with a response curve that looks like this. And the prediction would be that some positive relationship exists where sites with high inorganic N losses should also have high organic N losses. [DIN]

Outline Research Background and Justification Ch 1: Preliminary Test of Hypotheses Synoptic Surveys of Stream Chemistry Ch 2: Ecosystem Scale Manipulations Stream Nutrient Spiraling Ch 3: Geochemical Controls on DON Losses Soil Sorption Assays Ch 4: DOM Characterization Fluorescence and PARAFAC Modeling Eutropia Project The first project I’ll introduce is a preliminary test of this hypothesis.

DON Release Hypothesis: Preliminary Test We conducted multiple synoptic surveys in about 30 forested watersheds spanning on of the largest N deposition gradients in North America, located in the Great Smoky Mountains National Park in the Southern Appalachians.

DON Release Hypothesis: Preliminary Test During our first survey, conducted in September 2004 just before litterfall, we found a strong positive relationship between DON and nitrate concentrations. We also saw a significant decrease in the DOM C:N ratio – suggesting the N-rich DOM molecules may have been lost from systems with higher N loading. These observations are consistent with the predictions from Neff and others that DON losses increase with N saturation.

DON Release Hypothesis: Preliminary Test However, when we repeated the survey the following spring immediately prior to leaf out, we found these trends had reversed. There was a significant negative correlation between DON and nitrate concentrations and the DOM C:N ratio decreased with increasing nitrate. We were uncertain these relationships changed, but we speculated that it might have been due to seasonal differences – perhaps associated with instream processing due to the open canopy. So we repeated the survey again in May 2007 just after leaf out in the early growing season.

DON Release Hypothesis: Preliminary Test While we expected this result may have been more similar to the initial survey, it was nearly identical to the second survey – indicating that this may not have been due to canopy effects. But we repeated the survey again, consistent with the timing of the first survey – just before leaf fall – in October of 2008.

DON Release Hypothesis: Preliminary Test We again found very similar results to the previous survey, but we could not replicate the finding from the first survey. The first survey was conducted about 10 days following a major storm – which dropped approximately 35cm of rain in a single event. While we waited until streams were back to baseflow before surveying, we speculate that overland flow resulted in litter deposits in the stream channels and that litter leachates dominated streamwater chemistry – not groundwater inflow. Because of this, we think the the first survey is atypical and misrepresentative of normal conditions. What is interested, however, is the consistent negative relationship between DON and nitrate concentrations in the last three surveys. This is opposite what is predicted and requires an alternative explanation. To explain why N containing organic matter decreased when there should have been decreasing demand for N, we hypothesize that increasing demand for C when N limitation is alleviated could result in an indirect demand for DON. To test this, for the last two surveys we brought back extra sample volume to conduct bioavailability assays.

DON Release Hypothesis: Preliminary Test Bioavailability Assays In these bioavailability assays, we manipulated C and N availability in a full-factorial design by either adding dextrose or potassium nitrate. We found that when we added N, DON consumption increased relative to the control. And when we added labile C, either alone or in conjunction with N, DON consumption was halted. Moreover, we found that when N was abundant and DON consumption with high, the N-rich DOM molecules were being preferentially consumed as sources of C over the bulk DOC pool. These experimental results are consistent with our survey patterns – providing support for the negative correlation between DON and nitrate, and the positive corellation between the DOM C:N ratios and nitrate across these watersheds. +C +N +CN +DI

Indirect Carbon Control Hypothesis Given our findings, we’ve proposed an alternative hypothesis for predicting DON losses from the landscape with takes into account both C and N demands on the DOM pool, where the N-rich DOM molecules that would have otherwise been consumed as an N source when N is limiting, may instead be the most bioavailable sources of C when N is saturating.

Outline Research Background and Justification Ch 1: Preliminary Test of Hypotheses Synoptic Surveys of Stream Chemistry Ch 2: Ecosystem Scale C/N Manipulations Stream Nutrient Spiraling Ch 3: Geochemical Controls on DON Losses Soil Sorption Assays Ch 4: DOM Characterization Fluorescence and PARAFAC Modeling Eutropia Project As an extension of the bioavailability assays we conducted, we conducted similar whole-system manipulations of C and N availability in a stream at one of our long-term research sites.

Ecosystem Scale Experimental Manipulations NUTRIENT SPIRALING THEORY We use Nutrient Spiraling Theory to quantify demand for DON. Nutrient Spiraling Theory takes advantage of the advective movement of water within stream channels to describe the relative demand, or uptake length, of a given element or compound. For an element in high demand, the distance traveled downstream before removal from the water column should be short, and the retention time relative to transport time should be long, as in the top panel. Longer spiral lengths, as depicted in the bottom panel, indicate less demand.

Ecosystem Scale Experimental Manipulations NUTRIENT SPIRALING THEORY Pump Metering in C/N We can slowly add either C or N to the system using a pump and change the spiral length of DON. If DON is functioning mainly as an N source, adding NO3 should reduce the demand for DON and increase its spiral length. If DON is functioning primarily as a source of C, adding labile C to the stream should increase the spiral length of DON.

Ecosystem Scale Experimental Manipulations Day 1: N Enrichment Day 2: C Enrichment We conducted these types of additions on back-to-back days under similar environmental conditions. We also conducted the enrichments at multiple levels. The left panel shows the possible predictions for the N addition. If DON is functioning mainly as an N source, each progressive enrichment level should result in an increase in DON concentrations in the stream channel. If DON is functioning mainly as a source of C, increasing N may lead to a reduction in DON concentrations. The converse is true for the labile C additions. We have these experiments completed, and we were able to measure significant changes in DON in response to the additions—but I’ve yet to work up the data fully.

Outline Research Background and Justification Ch 1: Preliminary Test of Hypotheses Synoptic Surveys of Stream Chemistry Ch 2: Ecosystem Scale Manipulations Stream Nutrient Spiraling Ch 3: Geochemical Controls on DON Losses Soil Sorption Assays Ch 4: DOM Characterization Fluorescence and PARAFAC Modeling Eutropia Project While biological influences on DON losses can be important, geochemical controls also deserve much attention.

The Importance of Geochemical Controls We have also been conducting synoptic surveys throughout the Oak Ridge National Lab Research Park in Tennessee. This is where the atomic bomb was developed. The area has banded parent geologies of alternating shale and dolomite mineralogy. By chance, many of the watersheds we monitored were almost entirely of one or the other geology.

Large differences in DOM stoichiometry with different parent geology We found the streamwater DOM C:N ratios between these two geologies to differ significantly. Yet these sites are all located in a very small geography—experiencing identical climates—with nearly identical vegetation dynamics. Because of this, these differences are likely driven almost exclusively by geologic factors alone. Given the stark differences in DOM elemental composition we are interested in: (1) what are the geochemical mechanisms responsible for producing such different streamwater DOM compositions?, and (2) what is the ecological significance (such as bioavailability and reactivity) of the DOM lost from each of these different types of watersheds?

Batch incubation soil sorption assays Unprocessed Soil Removal of Fe/Al Removal of Native Organic Matter We’ve collected and processed soils from each horizon and from each of the different watersheds with a variety of treatments – intact soils, Fe And Al removal, and Native OM removal. When I return in the fall, we will be using these processed soils in batch incubations where we will apply a constructed DOM solution of varying concentration to construct sorption isotherms. Artificial Leachates

Batch incubation soil sorption assays We will construct these isotherms for both DOC and DON so that we can identify which treatments and which soil horizons are responsible for the largest influences on DOM elemental composition. We will also be characterizing the equilibrium DOM solutions using a variety of methods to gain insight into their bioavailability and ecological significance. Credits: Qualls, Forest Ecol. & Managmnt, 2001

Outline Research Background and Justification Ch 1: Preliminary Test of Hypotheses Synoptic Surveys of Stream Chemistry Ch 2: Ecosystem Scale Manipulations Stream Nutrient Spiraling Ch 3: Geochemical Controls on DON Losses Soil Sorption Assays Ch 4: DOM Characterization Fluorescence and PARAFAC Modeling Eutropia Project Everything we’re doing can benefit from more advanced methods for organic matter characterization. One of the most promising methods being developed in recent years is DOM fluorescence and PARAFAC modeling.

DOM Characterization: Fluorescence and PARAFAC This simply involves collecting an excitation-emission matrix for each sample, compiling hundreds of scans, and using a Parallel Factor analysis to identify common ‘components’ or unique fluorescence signatures common to certain classes of organic compounds. This approach has large limitations—including limited ability to be quantitative, as well as limited qualitative information… for example, it identifies distinct fluorescent components we may have no idea what these components are chemically. But for our purposes, where we are interested in answer questions such as: (1) are different fractions of the organic matter pool being consumed as C vs. N sources when we manipulate C and N availability? Or (2) Are geochemical reactions removing specific compoenents from solution and how do those components relate to the solution’s elemental composition? For these types of questions, the fluorescnce signature may be helpful – and the data are inexpensive and easy to collect.

Outline Research Background and Justification Ch 1: Preliminary Test of Hypotheses Synoptic Surveys of Stream Chemistry Ch 2: Ecosystem Scale Manipulations Stream Nutrient Spiraling Ch 3: Geochemical Controls on DON Losses Soil Sorption Assays Ch 4: DOM Characterization Fluorescence and PARAFAC Modeling Eutropia Project And a brief word about the work we’re doing here in Norway related to the Eutropia project…

What am I doing in Norway? Working with Eutropia Project Flux of Organic N and P from catchment soils to stream Event (storm) export of OM from catchments End Member Mixing Models (EMMA) X Y Our goal is to increase our understanding of how storm events influence the export of organic N and P from a forested catchment to its stream. To do this, we have a forested watershed with 3 hillslope plots, each instrumented with lysimeters in the dominant soil horizons. Through characterizing soil solution chemistries and monitoring streamwater compositions across storm events, we hope to construct End Member Mixing Models that will increase our understanding of storm losses of DON and P from forests to receiving aquatic ecosystems may vary, and how the composition (and potentially the ecological significance) of storm versus baseflow organic matter exports may also vary. Z

Acknowledgments Host: Funding: Rolf Vogt UiO Chemistry Department Research Council of Norway National Science Foundation

Implications for Ecosystem N Losses The importance of organic N forms in the N cycle has received increasing attention in the last several years. In a paper in Ecology in 2004, Schimel and Bennett argue that incorporating organic N dyamics into our current conceptual understanding of the N cycle requires a change in paradigm. Their focus, however, was on how explicit consideration of organic N forms can change the relationships between key players involved in the cycling of N within ecosystems – how plants may be able access much N without obligate dependence on microbial mineralization. But it is no less important to consider the implications this revision has on our understanding of ecosystem N losses. Credits: Schimel and Bennett, Ecology, 2004