PROJECT SUMMARY

Quantifying the Photochemical Reactivity of Deep Ocean Water

Intellectual Merit: The dissolved organic carbon (DOC) pool in the ocean contains a mass of carbon about equal to that in the earth’s atmosphere, with over 70% of the marine DOC pool residing in the deep ocean (Hansell et al., 2009). Any quantitative description of the role of DOC in the global carbon cycle demands comprehensive data on the oceanic distribution, sources, and sinks of DOC in the deep sea. Carbon dynamics in the surface ocean are relatively well studied, but the extremely slow organic carbon conversion rates, along with the size and inaccessibility of the deep sea pool, has prevented an equivalent level of observation. Data is sparse, often contradictory and the sinks for DOC in the deep ocean remain largely unknown.

One improbable, yet particularly interesting removal pathway for deep refractory DOC is thought to be photochemistry, a view based largely on limited measurements of photochemical products in deep Atlantic water exposed to sunlight (Mopper et al., 1991). These experiment-based estimates of photochemical half life for marine DOC of about 500 to 2,100 yrs are much shorter than the accepted mean age of DOC in the deep ocean (4000-6000 yrs; Bauer et al., 1992). This proposal is to revisit this long neglected issue by directly measuring the photochemical reactivity of abyssal, mesopelagic, and surface ocean waters in the north Pacific, where the abyssal DOC concentrations are the lowest in the ocean, presumably reflecting the presence of the most aged and refractory reduced carbon pool in the sea. Our project will combine field measurements and laboratory irradiations to quantify the photochemical rates controlling (1) direct loss of DOC and photoproduction of CO, a significant product resulting from DOC oxidation, (2) common optical tracers of organic carbon (CDOM & FDOM fading), and (3) two reactive oxygen species (HOOH & O2−) that reflect the role of O2 in DOC photochemistry. In doing so, we will quantitatively reevaluate this basic question that now constrains global DOC models: Does photochemistry have a significant role in the removal of the massive amount of refractory DOC that is pooled in the deep sea? This study will contribute directly to the identification of fundamental factors driving carbon fluxes within the earth system and estimating their sensitivity to climate change which has been identified as a priority for most US Climate/Carbon Programs (e.g. workshop & strategic reports compiled at the OCB webpage; http://www.us-ocb.org). Results will inform new global carbon models with data on the impact of photochemistry on deep ocean DOC reservoirs.

Broader Impacts: This project will provide support for 1 doctoral and 3 undergraduate students that will be encouraged to take on an aspect of the research for a senior thesis project for credit. Participants will receive training in field and lab research, optics, data analysis, modeling photochemical impact, presentations at meetings and manuscript preparation. Data collected will be used by Miller as specific examples in undergraduate and graduate classes related to carbon cycles and biogeochemical processes. The project will use UGA’s Summer Undergraduate Research Program (SURP) which recruits undergraduate students from historically underrepresented groups in the STEM (Science, Technology, Engineering and Math) areas. Links with K-12 area teachers (specified in proposal) will be used to share “Deep Ocean” science in the form of classroom visits by Miller and his students before and after the expedition and live blogs from the Pacific cruise. UGA’s Office of Vice President for Research will promote public information exchange and arrange other outreach venues throughout the project.

TABLE OF CONTENTSFor font size and page formatting specifications, see GPG section II.B.2.

Total No. of Page No.*Pages (Optional)*

Cover Sheet for Proposal to the National Science Foundation

Project Summary (not to exceed 1 page)

Table of Contents

Project Description (Including Results from Prior

NSF Support) (not to exceed 15 pages) (Exceed only if allowed by aspecific program announcement/solicitation or if approved inadvance by the appropriate NSF Assistant Director or designee)

References Cited

Biographical Sketches (Not to exceed 2 pages each)

Budget (Plus up to 3 pages of budget justification)

Current and Pending Support

Facilities, Equipment and Other Resources

Special Information/Supplementary Documents(Data Management Plan, Mentoring Plan and Other Supplementary Documents)

Appendix (List below. )

(Include only if allowed by a specific program announcement/solicitation or if approved in advance by the appropriate NSFAssistant Director or designee)

Appendix Items:

*Proposers may select any numbering mechanism for the proposal. The entire proposal however, must be paginated.Complete both columns only if the proposal is numbered consecutively.

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PROJECT DESCRIPTION

Quantifying the Photochemical Reactivity of Deep Ocean Water

1. INTRODUCTION

To fully understand the global carbon cycle, quantitative data on the distribution, sources, and sinks of the oceanic dissolved organic carbon (DOC) pool is essential. This reservoir represents the largest mass of reduced carbon in the ocean (~662 PgC), containing over 200 times more organic carbon than contained in marine particulate organic carbon (POC) (Hansell et al., 2009) and roughly the same amount of carbon as the global atmospheric CO2 reservoir. Consequently, the relative rates of DOC production and oxidation in the ocean, and the turnover time for the oceanic DOC pool, are critical elements in the overall global carbon balance between the ocean and atmosphere (Najjar et al., 2007). Evaluation of carbon dynamics in past and future climate scenarios requires a quantitative knowledge of the processes that keep these systems in balance and the rates at which carbon can be transferred among reservoirs.

Considering a global picture, the main sources for DOC in the sea are terrestrial runoff and in situ marine production with the subsequent removal of this reduced carbon pool dominated by biological oxidation (Carlson, 2002). Diverse organic source material and distributed sinks for these molecules in the sea, together with oceanic mixing patterns (Beaupré & Aluwihare, 2010), create a global distribution of DOC that is by no means homogenous. Quantifying the rates at which DOC is removed from the ocean by both biotic and abiotic processes remains challenging, but it is particularly difficult when considering DOC in the deep ocean. The pool is huge (> 70% the oceanic total; Hansell et al., 2009), removal rates are extremely slow, data is sparse and often contradictory.

One ostensibly improbable, yet particularly interesting challenge in estimating global removal rates for deep refractory DOC revolves around the role of photochemistry. With a mean age of 4000-6000 years (Bauer et al., 1992), this refractory DOC pool (RDOC) must be extremely resistant to removal, apparently surviving multiple cycles through both the deep and surface ocean; passing repeatedly through the photon rich, clear surface waters of the open ocean unscathed. While this argues for resistance to loss by photochemical oxidation, other evidence seems incompatible with this idea. Deep ocean water is generally capable of absorbing more UV radiation than surface water (CDOM and FDOM are both higher in deep, aged water; Jorgensen et al., 2011; Yamashita et al., 2010; Nelson et al., 2010) and this quality is normally correlated with increased photochemical reactivity. Additionally, the best and most influential papers examining this issue with direct measurements (Mopper and Zhou, 1990; Mopper et al., 1991) showed Sargasso Sea water from 1000-4000m to be much more photochemically reactive that surface water from the same location, leading to half-life estimates for DOC as short as ~500 yr. While some photochemical studies of “deep” water down to 1000m have been published since (e.g. Benner and Biddanda, 1998), the additional direct measurements of irradiated water from deeper than 1000m and from other locations that is needed to fully explore this potentially critical element in the marine DOC cycle are surprisingly missing.

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Possibly funding has not been forthcoming for studies of “photochemistry in the dark,” or maybe the issue did not seem pressing until now, but regardless of the reason, global ocean models developed to evaluate the sensitivity of deep ocean DOC reservoirs to climate change (e.g. Najjar et al., 2007), now find themselves limited by an extremely small, and spatially limited data set for the photoreactivity of DOC polls in all but the surface ocean. New details of DOC distributions in the deep ocean is leading to better models for organic carbon circulation and exchange (e.g. Hansell et al., 2012) and it has become absolutely essential that we better define the role of photochemistry in removing old, “refractory” DOC from the oceanic pool.

With this proposal, we intend to revisit this long neglected issue by directly measuring the photochemical reactivity of abyssal, mesopelagic, and surface ocean waters in order to more clearly define the role of photochemistry in the ocean carbon cycle. This effort is not meant to be a comprehensive examination of the myriad of photochemical reactions found in the ocean, nor is it a definitive measure of total global carbon conversion rates due to photochemistry. It is, rather, a first survey to reevaluate the very basic question that limits advancement of global DOC models: Does photochemistry have a significant role in the removal of the massive amount of refractory DOC that is pooled in the deep sea? Our area of concentration will be the north Pacific, where the deep ocean DOC concentration is the lowest anywhere, and presumably reflects the presence of the most aged and refractory reduced carbon pool in the sea. Layered in the top 1000m above this deep DOC is a concentration gradient that will allow comparison of waters with different DOC concentrations, ages and apparent refractivity. Our project will combine field measurements and laboratory irradiations to quantify the photochemical rates controlling (1) direct loss of DOC and consequent photoproduction of CO, a significant product resulting from DOC oxidation, (2) common optical tracers of organic carbon (CDOM & FDOM fading), and (3) production of two reactive oxygen species (hydrogen peroxide & superoxide) that reflect the role of O2 in DOC photochemistry. Results will inform new global carbon models by beginning the quantitative evaluation of t h e t o t a l i m p a c t o f photochemical processes on deep DOC reservoirs in the ocean.

2. BACKGROUND

Global DOC Distribution and Reactivity: Over the past decade, improved analytical methods and e x t e n s i v e d a t a s e t s g e n e r a t e d b y g l o b a l hydrographic surveys have forced oceanographers to

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Fig. 1. Distribution of DOC (μmol/kg) in the central Atlantic, central Pacific, and the eastern Indian oceans from CLIVAR lines A16, P16, and I8/I9 respectively (from Hansell et al., 2009).

contemplate a new, highly detailed picture of DOC distributions in the ocean such at the one found in Figure 1 (Hansell et al., 2009). One useful approach to examine the global marine DOC reservoir is to define the major fractions based on their turnover times in the ocean (Bauer, 2002; Hansell et al., 2012). In general, both supply and removal rates are rapid in the surface ocean, with most DOC contributed by marine phytoplankton (~ 30 PgC/yr) being quickly respired to CO2 by marine bacteria. The most biologically labile pool (LDOC) is short-lived (minutes to days) and almost all of this surface pool is remineralized each year in the surface ocean, allowing only about 2 PgC/yr to escape as a semi-labile DOC pool (SLDOC) with turnover times on the order of months to years. SLDOC penetrates to depth but is largely constrained to the upper 500 to 1000 meters (Fig 2). These turnover times for LDOC and SLDOC are consistent with the extensive CLIVAR DOC data collected over the last decade and can reasonably constrain the inventory of dissolved organic carbon in the upper 1000 meters to about 185 PgC (Hansell et al., 2009). By difference, this indicates that a DOC inventory ~2.5 times larger than LDOC and SLDOC combined (477 PgC) must exist in the deep sea .

Carlson (2002) provides a remarkably complete review of the production and removal processes for the DOM pool in the ocean, providing a summary of the general characteristics found in the LDOC, SLDOC, and RDOC pools based on a large number of direct obse rva t i ons and DOC distribution data (references within). Jiao et al. (2010) provide a synthesis of the major connected processes operating on the oceanic DOC cycle in their paper on the “microbial pump.” (Fig 3). On ave ra rge , LDOC i s composed of a mix of high and low molecular weight (HMW/LMW) compounds with labile, nM level monomers that support most of the bacterial production in the surface

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Fig. 2. DOC, Δ14C, and depth along CLIVAR line P16 in the Pacific (from Hansell et al., 2009).

Fig 3. Linked rapid and slow cycles demonstrating the microbial pump from labile to refractory DOC for export to the deep sea (from Jiao et al., 2010)

ocean. SLDOC is also composed of a HMW and LMW mixture with a C:N ratio from 6 to above 20, is dominated by carbohydrates that decrease with depth as microbial activity continually modifies DOC to create biosynthetically altered molecules (Gogou & Repeta, 2010; Jiao et al., 2010); altogether increasing the chance that SLDOC is exported from its point of origin to the deep ocean. RDOC is a more consistent carbon pool with a concentration of about 34μM, a dominance of LMW compounds, a C:N ratio constrained to between 14 and 20, with a high resistance to biological oxidation. This last characteristic is consistent with a general continuum of bioreactivity for organic matter found in the ocean of POM HMW DOM LMW DOM, and matches well the DOC vs. Age distributions in the Pacific seen in Figure 2. Bauer (2002), using 14C data and a two-pool model, estimated that 45% and 65% of the DOC in the surface Pacific and Atlantic respectively is made up of this old, refractory pool. This large presence of RDOC is also estimated by Carlson (2002) for the surface waters of both the Mediterranean and the Ross Sea.

Changing Deep Sea DOC and AOU Relationships: Using the P16 line data shown in Figure 2, it appears that once below ~1000 m, the DOC concentration in the deep Pacific decreases slowly by ~ 10-15 μM over the whole trip. Hansell et al. (2009) show that by following constant density lines along this same P16 line at ~ 4000m, (i.e. Lower Circumpolar Deep Water moving from the south to north) DOC is almost conservative with only ~ 4-6 μM DOC loss from its origin in the southern ocean (~ 42 μM) to its global low between 30ºN and 58ºN. This is an oversimplification of the deep ocean circulation when the Atlantic, Indian, Arctic, and Southern Ocean are all considered, but it does show that with limited mixing in the deep Pacific, the loss of DOC is extremely slow. This is confirmed by AOU vs DOC relationships that suggest DOC flux can only support about 10-20% of measured deep sea microbial respiration (Aristegui et al., 2002) with the remaining carbon fuel for bacterial production coming from sinking particles.

Related to DOC changes in the deep ocean, Figures 4 & 5 show results from recent publications reporting extensive study of deep transect data that demonstrate strong positive relationships between AOU and fluorescent and chromophoric dissolved organic matter (FDOM & CDOM) in the Pacific Ocean (Yamashita & Tanoue, 2008; Nelson et al., 2007). Jørgensen et a l . (2011) have fur ther investigated this relationship between FDOM and AOU using their extensive transect data from the north and south Atlantic, the Indian and Southern Oceans, and the eastern south Pacific. Using exc i ta t i on -emiss ion mat r i x spec t roscopy (EEMS) and

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Fig. 4. Contour of AOU and FDOM in a N-S Pacific trans-ect at 160ºW and 170ºW (from Yamashita & Tanoue, 2008).

PARAFAC statistical analysis (Stedmon & Bro, 2008), they identified 7 distinct fluorescent components in their global data set. The same strong positive correlation with AOU was seen for 2 specific EEMS components (C1 & C4) that represent “humic-like” fluorescence in marine samples. Since FDOM is a subset of CDOM, and both are components within the DOM pool (albeit admittedly small parts), these optical tracers clearly show the in situ addition of optical compounds to the deep sea DOC pool associated with microbial consumption of either new carbon from particles, or the conversion of older uncolored DOM to new fluorescent/absorbing dissolved compounds by heterotrophic bacteria.

This process could be similar to results reported by Nelson et al. (1998) that have shown the main source of new CDOM in the upper 1000 m at the BATS station to be produced near the bottom of this layer and mixed back up into the surface ocean. If this is the case for abyssal waters, then this pool of FDOM/CDOM will be well preserved since both are much more likely to be produced by bacterial activity than to be consumed by it, and the main global sink for both is photochemical fading (i.e. conversion to uncolored dissolved organic matter (UDOM)). While likely comprising only a small component of the overall deep DOC pool, Yamashita & Tanoue (2008) point out that annual FDOM production in the deep Pacific is of the same magnitude as the global input of FDOM from rivers. This said, it should also be noted that neither FDOM nor CDOM may qualify as RDOC if it represents a small, “new” component in the deep ocean reservoir from particle dissolution, and may then be expected to have distinctly different photochemical reactivity than RDOC. However, not ALL colored or fluorescent characteristics of the deep ocean are contributed in situ, as can be inferred by the small but positive intercepts when plotted against AOU in Figure 5.

With a mean age of 4000-6000 years (Bauer et al., 1992; Carlson, 2002), the underlying RDOC pool, on which the new CDOM/FDOM signal is laid down, must be resistant to removal processes of any kind and consequently is well-mixed throughout

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Fig. 5. Correlation of AOU with (1) FDOM in the deep waters of the Pacific (left panel; Yamashita & Tanoue, 2008) and (2) CDOM (right panel; Nelson et al., 2007). Note both FDOM and CDOM have small positive intercepts vs AOU, indicating source water values.

the ocean. RDOC apparently survives multiple cycles through both the deep and surface ocean; repeatedly mixed with LDOC as it passes through the photon rich clear surface waters of the open ocean. On the face of it, this apparent old age of RDOC seems to be at odds with what we have come to learn about the impact of marine photochemistry on DOC.

Marine Photochemistry Related to DOC: Over the last several decades, it has been clearly shown that the sum result of photochemical reactions in the surface ocean, driven mainly by solar ultraviolet radiation (UVR), is to remove a significant amount of dissolved organic carbon (DOC) from the oceanic carbon pool (Zepp et al., 2011). The comprehensive review by Mopper & Kieber (2002) of independent photochemical studies related to DOC, supports the significance of photochemical removal of reduced organic carbon by both direct and indirect photochemical conversion. Direct photochemical production of CO2 and CO represent removal from the DOC pool (Miller & Zepp, 1995; White et al., 2010). Incomplete oxidation and molecular transformations of refractory DOC can produce a multitude of biologically labile photoproducts that are rapidly utilized by microorganisms, leading to increased heterotrophic mineralization and a pathway for indirect photooxidation of DOC to CO2 through the microbial loop (Moran & Covert, 2002; Mopper & Kieber, 2002). Together, these photochemical processes have been estimated to remove 12-16 PgC yr (Moran & Zepp, 1997), oxidizing a carbon mass 10 times greater than estimates of global net DOC production (Carlson, 2002). It is clear this estimate needs refinement but it does, however, make the point that photochemistry is potentially a significant loss term for DOC in the ocean.

Not surprisingly, almost every study of marine DOC photochemistry has been done using sunlit water. Most have investigated coastal systems where great quantities of DOC are deposited to the ocean from terrestrial runoff and the absorbance of UVR needed to drive photochemical reactions is highest. This is understandable since the surface ocean is where ALL marine photochemical reactions take place and the combination of high UV absorbance and high DOC creates significant photochemical rates. However, considering the overwhelming mass of the RDOC pool in the ocean, a truly relevant question to any consideration of global DOC cycles is “What happens when the refractory DOC from the oceanʼs depths returns to the sunlit surface waters?” This simply has not been addressed by the chemical oceanography community. Most global models of DOC make reference to removal by photochemistry (and in fact, most require sinks for RDOC that have not yet been identified) with very little supporting data except by extrapolating results from the well-studied surface mixed pool (LDOC+SLDOC+RDOC) (Hansell et al., 2012; Anderson & Williams, 1999).

Are results from surface water irradiations relevant to the much larger RDOC or SLDOC pools or do they simply measure the rapidly cycling LDOC pool? Are the predominately short-term experiments done thus far even useful in determining long-term oceanic rates of photochemical DOC turnover? These critical questions remain virtually neglected.

A few good studies have looked at photochemical reactions in “deep” waters from the top 1000m. Benner & Bidanda (1998) used water collected at depth from the Gulf of Mexico and subsequently exposed to sunlight and saw clear increases in its ability to

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stimulate bacterial production relative to unirradiated controls while “re-irradiation” of surface samples showed a marked decrease in its ability to support microbial production (Fig 6). This suggests that “ d e e p ” w a t e r i s m o r e photochemically reactive in supply ing new organic substrate for bacteria. The question remains, however, as to what DOC fraction is supplying these substrates when exposed to sunlight. Cherrier et al. (1999), using 14C analysis of surface living bacteria, show that some of the very old RDOC pool can end up in bacteria in the surface ocean. This later finding does not necessarily require photochemistry, but preserves the notion that it may be involved.

An obvious way to examine the photochemical reactivity of the enormous, well mixed RDOC pool apart from less refractory DOC, is to simply irradiate the oldest water in the ocean, yet these experiments are totally lacking. The only direct photochemical measurements of irradiated abyssal ocean water are from the early 1990ʻs (Mopper et al., 1991; Mopper & Zhou, 1990). Both studies irradiated “younger” deep ocean water (relative to the N. Pacific) collected to 4000m in the Sargasso Sea. While the 1990 paper focused on OH• radical production and inferred a role in carbon cycles, the Mopper et al. (1991) paper seemingly stands alone as the only published record of direct measurements of photochemically generated carbon compounds (form-aldehyde, acetaldehyde, gly-oxal, glyoxylate, pyruvate, & CO) from seawater collected below 1000m. Figure 7 shows the average profile results for formaldehyde production on two

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Fig. 6. (A) DOC in the Gulf of Mexico and (B) its ability to enhance bacterial production following exposure to the sun relative to dark controls (from Benner & Biddanda, 1998).

Fig. 7. Profiles from the western Sargasso Sea showing the photochemical formation of formaldehyde (a & b) and FDOM (c) (from Mopper et al., 1991), showing increased photochemical rates correlated to DOM fluorescence in deep water samples. Depths are collection points, subsequently exposed on deck to natural sunlight.

cruises and FDOM from the surface to 4000m. This correlation of increased FDOM and higher photochemical CH2O production is typical of many photoproducts in the ocean.

Based on their measured photochemical production rates for LMW carbonyl compounds plus CO (12.9-26.7nM-C hr-1) pooled for discrete depth intervals, and using a 1-box model, they calculated the half-life for DOC in the ocean to be in the range of 1000 to 4,200 yrs. Recognizing that < 50% of the known carbon products were measured for this study, they suggested that true half-lives for DOC may be as much as 50% lower. This would mean that photochemistry could remove about half the DOC in the ocean on the same time frame as a single oceanic mixing cycle, a result that is not compatible with the 14C ages for DOC in the deep ocean.

As the first, and till now apparently the only estimate of photochemical oceanic DOC turnover times based on direct field observations, Mopper et al. (1991) were required to make a number of assumptions. Estimating the average global DOC as 50μM and the UV penetration as 0.13% of the depth of the ocean, they then used reasonable global solar irradiance values and assumed that their measured short-term rates were sustained over the long-term exposure of DOC in the surface ocean. As in all half-life calculations using one reservoir and a single rate, the assumption is that the entire DOC pool is subject to photo-oxidation. With the dearth of knowledge about deep sea DOC distributions and almost no marine photochemical context in the early 1990ʼs, this approach was very reasonable, representing one of the first notable insights into the possibility that photochemistry could be a significant process in the global DOC cycle.

Over the past two decades, a wealth of new knowledge about marine photochemistry and DOC turnover times in the deep ocean has been acquired. We now realize that the latter two assumptions in the Mopper et al. (1991) half-life calculations are probably not valid. Photo-fading of CDOM and FDOM dominate in the surface ocean (Nelson et al., 2010; Omori et al., 2011) and as less UVR is absorbed, photochemical reactions slow down. Miller & Zepp (1995) measured DIC/CO2 photoproduction rates (a photochemical carbon product shown to be 15-20 times > CO in coastal waters) and compared this “loss” of DOC to the CDOM fading rate. They showed that only ~15% of the DOC could be oxidized to DIC/CO2 before CDOM was near zero. DOM with no ability to absorb UVR translates to a photochemical rate of zero. This argues that the entire DOC pool is not susceptible to photo-oxidation. Photochemical fading (CDOM & FDOM) is far faster than direct removal of carbon from the DOC pool. Omori et al. (2011) showed that ~36% of the FDOM signal from a deep sample was lost in just 0.5 days of irradiation while most of the faded UDOM remained. This demonstrates strong photochemical reactivity in the short term but argues that these reactions do not always remove carbon from the DOC pool and may not be sustained over longer irradiations. One can argue that photochemical sources are replenished as part of the day-night cycling of photochemical reactions and biological resupply, thus sustaining daily rates. While this is possible, and even likely for the LDOC component of the global cycle, by definition the RDOC pool cannot be replenished on this time scale. If sustained long-term rates rely on replenished supply of photoreactive compounds, this does not apply to RDOC. With much slower, longterm DOC loss rates, the half-life calculation for DOC removal by photochemistry would be much longer, and possibly more compatible with 14C ages.

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It remains remarkable that, given the strong start made by Mopper et al. into the investigation of photochemistryʼs role in removing deep, RDOC from the sea, virtually nothing has been done since. Conflicting and dubious extrapolations from coastal photochemical results to the open ocean have raised many questions. Models for DOC in the whole ocean await answers as to the susceptibility of the RDOC pool to removal by photochemical reactions in the surface ocean. Given the importance of oceanic DOC in the global carbon cycle and evaluations of reservoir transfer rates of carbon in future climate scenarios, new data on potential removal processes for the RDOC pool of carbon are desperately needed.

3. OBJECTIVES AND HYPOTHESES

The primary objectives of the proposed research are to provide direct measurements of the photochemical reactivity of the deep marine DOC reservoirs obtained from the north Pacific for (1) evaluation of photochemistry as a RDOC removal mechanism and (2) comparison of waters collected at abyssal (RDOC), mesopelagic (SDOC+RDOC) and shallow (LDOC+SDOC+RDOC) depths. Specific objectives include experimental measurements of the samples mentioned above to establish (1) depth profiles and photochemical fading dynamics for CDOM, FDOM, & EEMS as optical tracers and (2) the photochemical reactivity of DOC pools as quantified by [DOC] loss together with the photoproduction rates and quantum efficiencies for CO, HOOH, and O2−. Based on measurements of these quantities in photochemical experiments, we pose the following two main null hypotheses:

Hypothesis 1: The photochemical ʻreactivity,ʼ as quantified by changes in optical properties (EEMS, FDOM, & CDOM fading), production of CO, HOOH, and O2−, and direct loss of DOC during controlled laboratory irradiations, will be the same for abyssal water (RDOC) from the north Pacific as it is for surface and mesopelagic waters from the same location.

Hypothesis 2: Extended exposure of RDOC water (alone and in mixtures with surface water) to solar irradiance will show no change in photochemical efficiency and/or photochemical ʻreactivityʼ with time and consequently, results from short term exposure can be used to estimate long term exposure (i.e. cumulative photon dose does not alter the photochemical ʻreactivityʼ of RDOC).

4. PROPOSED RESEARCH

Water Collection

Cruise Plans: We will proceed on two fronts to examine the photochemical reactivity of Pacific DOC pools. First, Dr. Lisa Miller at the Canadian Institute for Ocean Science in Sidney, BC, has agreed to have water collected for us from the August 2012 Line P cruise (and additional cruises if warranted). For each deep cast below 1000m (usually 4-5), samples from the surface, mid-depth, and deepest casts will be 0.2um filtered into 2L ashed amber glass bottles, refrigerated, and shipped cold back to Athens. The results of our first photochemical experiments with these samples will inform our analytical methods and refine our protocols to maximize results for the extensive fieldwork planned for the summer of 2013 (below).

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Secondly, we propose an extensive sampling strategy and cruise track for 2013 (Fig. 8) that was conceived by Dr. Dennis Hansell, University of Miami, for a separate project that has now been funded by NSF-OCE. His survey of the deep north Pacific for DOC distributions along density gradients to track and locate areas of loss for refractory DOC in the deep Pacific is a perfect field companion for our proposal. Lengthy conversations with Dr. Hansell have led to this proposal of a coordinated effort that we both feel is greatly compatible. Consequently, we propose Dr. Hansellʼs cruise track (described below) as our first option for this project. A letter of support from Dr. Hansell is included as a supplementary document with this proposal. If this proves impossible, any similar east-west transect in the Pacific north of 30ºN with a component east of 180ºW would allow access to the lowest deep DOC water.

The proposed cruise would sample areas where the deep western boundary current enters the region of Pacific Deep Water (PDW) formation as well as within the Gulf of Alaska, providing good coverage of deep ocean DOC variations. Leaving from Dutch Harbor, AK, sampling would begin at the shelf break (52.5ºN, 170ºW) at 1º intervals

south to 47ºN, turn to sample an eastward track along 47ºN at 2º intervals that would reoccupy a s e c t i o n o f t h e 2 0 0 7 Japanese CLIVAR P01 line p r o v i d i n g c o n t e x t u a l hydrograph ic da ta fo r reference, intersect the CLIVAR P16N line at 152ºW for more contextual data, turn NE to sample the deep Gulf of Alaska in route to Station Papa (50ºN 145ºW) to intersect Line P (where we will have water from the

previous summer) and continue sampling to the shelf break at 59ºN 141ºW where we conclude sampling and steam to Sitka, AK where we disembark.

Field Sampling: We will collect water samples with a 24 bottle CTD/rosette system (at ~ 40 stations during the 2013 cruise). Samples for optical and photochemical work will be collected at multiple depths at each station and filtered through a 0.2 μm nylon cartridge filter (Whatman Polycap AS75). At sea, we will first characterize EEMS, FDOM, and CDOM spectra on fresh samples to be used in photochemical exposures which will be initiated as soon as possible to minimize any aging effects on our results (methods below). We will focus photochemical exposures on representative samples from deep, mid-depth, and surface water. The rest of the profile samples will be analyzed for optical data at sea with those that cannot be processed immediately, refrigerated (not frozen) for later use or shipment to Millerʼs lab (as was the case for the 2012 samples from Line-P). Our experience shows this treatment will preserve optical characteristics and photochemical reactivity for CO and HOOH in ocean waters.

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Fig. 8. Proposed east to west cruise track for summer 2013 to collect deep, low DOC, refractory N. Pacific water along 47N, to

Experimental and Analytical Methods

Choice of Measurements to Define Photochemical ʻReactivityʼ: The specific measurements in this study (detailed below) were chosen to represent some of the more practical, useful, and comparable photochemical products and proxies available. We will measure TOC/DOC loss directly in our longer-term solar simulations for obvious constraints on the potential for photochemical carbon reactions that remove DOC. HOOH and CO have the most extensive published AQY and production databases for any of the multitude of marine photochemical products, allowing our work to be comparable to other studies (e.g. Yocis et al., 2000; Zafiriou et al., 2003). HOOH and CO are confidently measured (LOD = 5nm & 5ppm respectively) with accepted methods at the very low photoproduction rates found in blue ocean water.

Reactive oxygen species (ROS) originating from photochemical redox reactions involving DOC and molecular oxygen are implicated in secondary oxidation of organic compounds in natural waters (Goldstone et al., 2002; Mopper & Zhou,1990; White et al., 2003) and generally correlate with carbon-driven photochemistry in natural waters (Burns et al., 2012). Altered concentrations of molecular oxygen, and presumably ROS originating from its reaction with irradiated DOC, alters common photochemical rates such as CDOM fading, and DIC and CO formation (Gao & Zepp, 1998). Superoxide, O2−, is a ubiquitous ROS, a proxy for DOC photoreactivity, and the primary photoproduct leading to formation of HOOH, an important component in redox chemistry in surface waters, and can be measured simultaneously with HOOH by chemiluminescence.

CO was the most significant carbon photoproduct analyzed in the work by Mopper et al. (1991) and remains the central element in many subsequent arguments for the large role of photochemical oxidation in the oceanic carbon cycle. While newer work does not suggest a large role for CO in direct loss of DOC, showing that CO photoproduction rates are ~15 to 20 times less than those observed for DIC/CO2 (Reader & Miller, 2012), the extreme analytical challenges to quantify DIC production in blue water has led to the suggestion of using a defined CO2/CO ratio with CO production for large scale estimates of total DOC oxidation by photochemistry (Mopper & Kieber, 2002).

Extensive work has been done recently on FDOM & CDOM distributions (e.g. Jørgensen et al., 2011; Nelson et al., 2010), profiling to all depth of the oceans with observations informing knowledge of vertical transport processes to the deep sea. New direct knowledge about photoinduced changes in these two optical tracers when they return to sunlit waters will add to the confidence with which they can be applied.

Optical Measurements and DOC: Absorption spectra (250–800 nm) on 0.2um filtered water samples will be measured using a UltraPath liquid waveguide cell (World Precision Instruments; Miller et al., 2002) with fiber optic attachment to a Perkin Elmer Lamda 40 spectrophotomer. Blanks and corrections for refractive index variations due to salinity changes will be performed as in Nelson et al. (2010; & references within). A SPEX Fluoromax-4C will be used to determine FDOM and Excitation-Emission Matrix Spectra (EEMS) of discrete, filtered samples and its “Quanta Phi” integrating sphere attachment will be used to determine fluorescent quantum yield directly. EEMS will be obtained at sea using established methods for instrument settings and corrections for Raman scattering and using quinine sulfate standards (Zepp et al., 2004; Stedmon &

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Bro, 2008; Jørgensen et al., 2011 & references therein). Distinct excitation/emission spectra located within the complete EEMS will be quantified with parallel factor analysis (PARAFAC) using the published MATLAB toolbox, DOMFluor (Stedmon & Bro, 2008). EEMs has been used effectively for surface-to-bottom profiling in the major ocean basins (Jørgensen et al., 2011; Yamashita et al., 2010). Loss and alteration of CDOM and FDOM optical properties by photochemistry will be examined by quantifying spectral changes in UV absorbance and EEMS using PARAFAC analysis. We will also calculate the specific UV absorbance (SUVA = Absnm / [DOC]) of DOC at 254 nm and 325 nm for comparison with previous studies (ex. Nelson et al., 2007) and for potential insight on the presence of dissolved aromatic carbon (Weishaar et al., 2003).

Samples at sea for TOC/DOC will be collected filtered through ashed GF/F filters and frozen in 60 ml HDPE bottles for post-cruise analysis by standard seawater methods using a Shimadzu TOC-V system with auto injection, daily KHP standard curves, and regular analysis of Consensus Reference Materials (CRMs) obtained from the University of Miami (Hansell, 2005; Sharp et al., 2002). In the coordinated cruise with Hansellʼs group (as described above), his group will be focused on TOC/DOC measurements and cruise samples will be trusted to his expertise. Samples for long-term solar simulator experiments will be analyzed before and after exposure to quantify the loss of DOC (0.2um filtered) relative to measured changes in FDOM, CDOM and photochemical reactivity.

Photochemical Irradiations for Apparent Quantum Yield (AQY): We will irradiate deep, mid-depth, shallow, and mixed samples to evaluate photochemical reactivity relative to dark controls. With a starting assumption based on the three-component global DOC model of Hansell et al. (2012), deep samples contain almost exclusively RDOC, mid-depth samples will have mixed RDOC+SLDOC, and shallow samples will have mixed RDOC+SLDOC+LDOC. Using deep RDOC carbon concentrations as a base, we will develop a matrix of mixed samples (e.g. 50:50, 25:75, etc. mixtures of deep RDOC source water with shallow and mid-depth water) for photochemical exposures. By quantifying each original sample and mixture for TOC/DOC, optical qualities and their photochemical reactivity, we will develop a matrix of results that better simulates real ocean conditions with RDOC irradiated in the presence of possible organic and inorganic photosensitizers added to deep water as it mixes to the surface. We will optimize mixing matrices and irradiation times using the IOS supplied Line P samples from 2012 prior to the 2013 expedition. As the suite of standard chemical measurements are finalized for the cruise, we will work to ensure each sample and mixture processed undergoes as many auxiliary analyses as possible by collaborators and/or onboard data systems (i.e. nutrients, carbon components, salinity, metals, etc.).

Two DSET Suntest CPS Solar Simulators (1500W Xe lamp) are available for photochemical experiments at sea. While “bleed” from UV radiation below 290nm has been reported for similar DSETs used for solar simulation (Kromer et al., 2004), secondary Schott glass filters eliminate this problem in our system as confirmed by careful measurement of UV radiation in our system with our Optronic spectroradiometer (see below). We will undertake shipboard full spectral solar simulations on fresh samples, primarily to evaluate changes in our photochemical “indicators” from full sunlight for model scaling of results, and to allow long-term, and/or repeated exposures

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needed to address our second hypothesis regarding the effect of total photon dose on observed reactivity. With the capability of producing a continuous photon flux of ~3X the intensity of mid-latitude summer noon sun, we can simulate months of surface layer exposure in days, after which we will reevaluate photochemical reactivity and direct changes in DOC concentrations. It is well-documented that O2 is consumed via ROS formation and subsequent reactions during photochemical irradiations (Andrews et al., 2000) and that depletion of O2 alters reaction rates and pathways. Consequently, our long-term irradiations will only involve those measurements that donʼt require gas-tight containers (i.e. all but CO) and care will be taken to re-equilibrate with clean air at regular intervals. Samples for study of short-term photochemical changes under full sunlight in FDOM (EEMS) will be exposed directly in quartz fluorometer cells to minimize contamination and allow time course measurements on individual samples.

One of the DSET Suntest CPS units will be configured for apparent quantum yield (AQY) determinations of HOOH and CO in short-term irradiations using the multispectral, statistical methods established and described in detail for AQY studies by Ziolkowski & Miller (2007) and by Reader & Miller (2011). Generally, fifteen 10 cm quartz spectrophotometer cells filled with no headspace are precisely oriented vertically in a water-cooled Al block beneath a range of Schott long-pass optical filters (280, 295, 305, 320, 335, 345, 380, 400, & 480 nm cutoff wavelengths) and irradiated from above. This setup allows each sample cell to receive a controlled, varied, and known spectral irradiation. A 2-inch integrating sphere attached with a 60 cm fiber optical cable to an Optronic Laboratories OL Series 756 portable UV-VIS spectroradiometer, calibrated on site with an OL 752-10 plug-in spectral irradiance NIST lamp, is used to quantify the spectral downwelling irradiance entering each cell, E0(λ) (mol(photons) m-2 s-1 nm-1). The photons absorbed over the course of an experiment is calculated using the following equation from Hu et al. (2002) to avoid overestimating the absorption of photons due to inner filter effects (i.e. self shading)

Qa (λ) = E(0)λ (aCDOM(λ ) / at(λ) ) S [1− exp(−at(λ) L)]

where Qa is the mol photons absorbed sec-1 by the sample, E(0) is the irradiance entering the top of the cell (mols photons/m2/sec), S is the irradiated surface area of the spectrophotometric cell (m2), aCDOM and at are the absorption coefficients for CDOM and for the total solution respectively (m-1), and L is the pathlength of the spectrophotometric cell (m). The experimental photochemical production under each cutoff filter is calculated according to the equation

dP/dt = AQY × Qa

where the dP/dt is the production rate of the species in question (CO or HOOH) per experiment. The intentional variation of Qa using multiple irradiation spectra gives production results that allow use of an in-house MATLAB® routine for iterative, non-linear fitting to solve for the single AQY spectrum that best describes the production measured under all of the cutoff filters for a single experiment (White et al., 2010; Xie et al., 2009; Ziolkowski & Miller, 2007). This general multispectral approach can also be used for photochemically induced changes in optical spectra (FDOM & CDOM fading, changes in EEMS, FDOM efficiency, etc.) by defining dP/dt as a rate of change in

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specific optical characteristics, allow quantification of differences in photochemical fading susceptibility.

Analysis of CO, HOOH and O2− in Photochemical Experiments: CO is measured as detailed previously (Xie et al., 2002; Ziolkowski & Miller, 2007), using an SRI gas chromatograph equipped with a reduced gas analyzer (mercuric oxide reactor and UV detector). Post-irradiation, the sealed sample cells are injected with a known amount of CO-free air (Schutze Reagent) via a septa to form a headspace while simultaneously removing an equivalent volume of sample to equalize pressure. With a known sample volume and temperature, CO measured in the equilibrated headspace allows calculation of total CO photoproduction in the sample. The volume removed from the quartz cell to make room for the headspace equilibration will be pumped directly through teflon tubing for chemiluminescent analysis of hydrogen peroxide and superoxide, thus allowing all three photochemical products to be measured from the same sample cell.

HOOH and O2− will be determined by flow injection analysis chemiluminescence (FIA-CL). An acridinium ester-based CL reagent (AE-CL), 10-methyl-9-(p-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate, is used for the analysis of hydrogen peroxide in seawater (King et al., 2007) and 2-Methyl-6-(4-methoxyphenyl)-3,7- dihydroimidazo[1,2-a]pyrazin-3(7H)-one (MCLA) chemiluminescence is used for superoxide measurements (Rose et al., 2008; Hansard et al., 2011). Standards for HOOH and O2− are prepared as described in references above. Our Fe-Lume instrument (Waterville Analytical) is configured with duel channels (two PMTs (Hamamatsu HC135 photon counter) and two, 10-port valves) that allow simultaneous detection of AE-CL and MCLA-CE from a single input stream. This pumped system is relatively fast, measuring both HOOH and O2− in real time with a reported limit of detection for HOOH and O2− of 350 pM and 10 pM for open ocean seawater respectively (King et al. 2007; Hansard et al., 2011).

5. PROJECT MANAGEMENT AND SCHEDULE OF ACTIVITIES

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Schedule of Activities 2012 2013 2014

J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D

Exp. method setup, cruise prep. ---------- ---------

Field Sampling (2013 w/shipboard anal.)

-- ----

Sample analysis & lab followup(FDOM, CDOM, DOC, AQY, SolSim)

------- --------- ---------

Data Analysis & Synthesis ------ -------- -------- ---------------

Presentations at meetings & paper submission

----- ---------------------

Miller will direct the proposed research activities and supervise all students involved to ensure quality and productivity. A PhD student, Leanne Powers, will be involved in all aspects of the project, participating with Miller on the cruise to support her dissertation.

6. BROADER IMPACTS

Results will inform new global carbon models with data on the impact of photochemical processes on deep ocean DOC reservoirs. This project will provide support for 1 doctoral and 3 undergraduate students that will be encouraged to take on an aspect of the research for a senior thesis project for credit. All participants will receive training in field and lab research, optics, data analysis, modeling photochemical impact, presentations at meetings and manuscript preparation. Data collected from this project will be used by Miller as specific examples in undergraduate and graduate classes covering the carbon cycle and biogeochemical processes. The project will acquire students from UGAʼs Summer Undergraduate Research Program (SURP) which recruits undergraduate students from historically underrepresented groups in the STEM (Science, Technology, Engineering and Math) areas. Established links from previous visits with local K-12 area teachers (Ms.Soutar, Oconee High; Ms.Bennett, Social Circle Middle School; Ms.Brown, J.J. Harris Elementary) will be used to share “Deep Ocean” science from the N. Pacific cruise in the form of classroom visits by Miller and his students before and after the expedition, an “ask the oceanographer” website, and a live ship blog to support related classroom instruction. UGA public relations and the Office of the Vice President for Research (T. Hastings) will be kept informed of progress and involved in public information and outreach as opportunities arise.

7. RESULTS FROM PRIOR NSF SUPPORT

W. Miller: NSF Collaborative: Photodegradation of Dissolved Organic Matter and its Contribution to Surface Water CO2 fluxes and the Carbon Cycle in a River Dominated Ocean Margin (OCE0850677) $188,413, 2/1/2009 - 2/30/2012 (no-cost extension to 2013). The primary objective of this project is to provide a quantitative regional-scale estimate of the influence of DOM photodegradation on inorganic carbon fluxes in surface waters of the northern Gulf of Mexico margin. Specific objectives include the determination of apparent quantum yield spectra for photoproduction of CO2 and CO, the photochemical removal of DOC and the coupled photochemical-microbial removal of DOC. We are specifically investigating the photodegradation of terrigenous DOM and its fate in northern Gulf of Mexico surface waters. The research provides an integrative approach that combines field measurements, modeling, and satellite imagery to provide a regional scale assessment of photochemically-mediated carbon remineralization. We have completed 5 cruises in the northern Gulf of Mexico where we collected water samples, made in situ optical measurements, and are completing photochemical experiments in support of these objectives. A graduate student is supported in this effort and is making good progress toward a thesis, presenting her first paper, “Depth resolved rates of photochemical carbon monoxide production in the Gulf of Mexico, a river-dominated coastal margin” at the Ocean Optics 2010 in Anchorage, AK and another at the Ocean Science Meeting in February, 2012. discussions with R. Benner for joint publications are underway. This is the sole NSF project awarded to Dr. Miller since returning from Canada to the US in 2004.

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