Diagenetic Transformation of Dissolved Organic Nitrogen Compounds

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Diagenetic Transformation of Dissolved Organic Nitrogen Compounds under Contrasting Sedimentary Redox Conditions in the Black Sea Frauke Schmidt,†,* Boris P. Koch,‡,§ Marcus Elvert,† Gunnar Schmidt,^ Matthias Witt,|| and Kai-Uwe Hinrichs† †

MARUM
Center for Marine Environmental Sciences, Leobener Strasse, D-28359 Bremen, Germany Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany § University of Applied Sciences, An der Karlstadt 8, D-27568 Bremerhaven, Germany ^ Wilhelm-Schickard-Institute, Sand 14, D-72076 T€ubingen, Germany Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany

)

‡

bS Supporting Information ABSTRACT: Remineralization of organic matter in reactive marine sediments releases nutrients and dissolved organic matter (DOM) into the ocean. Here we focused on the molecular-level characterization of DOM by high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in sediment pore waters and bottom waters from contrasting redox regimes in the northern Black Sea with particular emphasis on nitrogen-bearing compounds to derive an improved understanding of the molecular transformations involved in nitrogen release. The number of nitrogen-bearing molecules is generally higher in pore waters than in bottom waters. This suggests intensified degradation of nitrogen-bearing precursor molecules such as proteins in anoxic sediments: No significant difference was observed between sediments deposited under oxic vs anoxic conditions (average O/C ratios of 0.55) suggesting that the different organic matter quality induced by contrasting redox conditions does not impact protein diagenesis in the subseafloor. Compounds in the pore waters were on average larger, less oxygenated, and had a higher number of unsaturations. Applying a mathematical model, we could show that the assemblages of nitrogen-bearing molecular formulas are potential products of proteinaceous material that was transformed by the following reactions: (a) hydrolysis and deamination, both reducing the molecular size and nitrogen content of the products and intermediates; (b) oxidation and hydration of the intermediates; and (c) methylation and dehydration.

1. INTRODUCTION Dissolved organic matter (DOM) in marine sediment pore waters is a heterogeneous mixture of organic compounds with wide spectra of both reactivities and molecular sizes.1 It is released during the degradation of particulate organic matter (POM) through reactions such as hydrolysis, oxidative cleavage, leaching, cell autolysis, and remineralization by sedimentary organisms.2 The resulting DOM either accumulates in the sediment pore water or is released to the ocean by diffusion possibly providing an important nutrient pool for benthic organisms.3 Up to now, a large fraction of the pore water DOM remains uncharacterized at the molecular level. It could, however, contain important information for a comprehensive understanding of the factors controlling preservation and remineralization of organic matter in marine sediments. Similarly, knowledge about the composition and structures of dissolved organic nitrogen (DON) compounds may help to understand the role of DON as source of N-bearing nutrients that are returned to the oceans. The abundance of N in labile r 2011 American Chemical Society

biomolecules such as proteins, amino acids, RNA, DNA, and so forth implies fast degradation and utilization of DON compounds during the early diagenetic transformations of organic matter.4,5 However, intact proteins have been identified in seawater6 and the predominance of amide-derived DON in seawater7,8 points to proteinaceous sources for these compounds and their preservation in modified form.9 An important factor controlling the preservation or remineralization of organic matter is the prevailing redox regime.10,11 The redox processes controlling the fate of N-nutrients in the ocean are currently the subject of intense research but the impact of contrasting redox processes on the fate of N in organic matter is still poorly constrained. A thorough understanding of the mechanisms involved in N-remineralization will become even more important Received: January 29, 2011 Accepted: April 29, 2011 Revised: April 17, 2011 Published: May 13, 2011 5223

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Environmental Science & Technology in the future ocean with projected increased bottom water anoxia and extension of oxygen minimum zones in response to climate change.12 To obtain insights into early diagenetic processes on a molecular level, we analyzed DOM in bottom and pore waters from the Black Sea by high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). FT-ICRMS resolves complex organic mixtures by providing exact masses of single molecules. Although tentative structure assignments were only accomplished for isolated molecules,13 FT-ICR-MS provides a substantial amount of new information on the molecular composition of DOM.14
16 In a previous study, we have for the first time applied FT-ICR-MS to marine sedimentary pore water and identified sources and early diagenetic processes in the polymeric low molecular weight-DOM fraction (200
600 Da) in surface sediments.17 Now, we aim at constraining the initial steps of organic matter remineralization and remobilization of nutrient N during early diagenesis in a set of DOM samples from the Black Sea. The modern Black Sea is permanently stratified with anoxic conditions below 150 to 200 m water depth18 and is therefore well suited for systematic comparison of organic matter remineralization under contrasting redox conditions. The two sampling sites are located on the northern continental shelf where the effect of different redox conditions on the DOM pool in bottom water and sediment pore water was investigated with a focus on N-bearing compounds.

2. MATERIAL AND METHODS 2.1. Sampling and Sample Preparation. As a reference for the marine DOM pool, 2 L of the anoxic water column of the northern Black Sea were sampled from 150 m water depth (44330 01 N, 36200 00 E) using a Pump-CTD during a cruise with RV Meteor in May 2007. Bottom waters and sediment pore waters were taken from multicorers (MUC) at two sampling sites on the shelf, one in the oxic zone in 71 m water depth (44420 98 N, 36240 86 E; 16-MUC) and one in the anoxic zone in 268 m water depth (44330 01 N, 36200 00 E; 14-MUC). 50 mL of bottom water was sampled from the sediment water interface of each MUC and filtered through GF/F filters (Whatman, 0.7 μm pore size). Sediment pore water (50 mL) was obtained by rhizon sampling (Eijkelkamp, pore size 0.1 μm) from diagenetically stable sediment in 15
17 cm sediment depth. All samples were acidified to pH 2 with hydrochloric acid (HCl, ultrapure, Merck) and stored in sealed, precombusted vials at þ4 C in the dark until further preparation. In addition, the associated sediment (0
1 cm and 15
17 cm) was sampled for analysis of total organic carbon (TOC), total nitrogen (TN), and its stable carbon isotopic composition (δ13CTOC). Sediment samples were stored in precombusted glass vials at
20 C in the dark until analysis. Bottom and pore water samples (50 mL) were concentrated by a factor of 50, and the water column sample was concentrated by a factor of 2000 with solid phase extraction on precleaned SPE cartridges. Extraction efficiency ranged between 55% for the water column, 38% and 55% for the pore waters from the oxic and the anoxic site, respectively (Table S1 of the Supporting Information). After DOM adsorption onto PPL cartridges, salt was removed from the samples by rinsing with 6 mL 0.1 M HCl-solution and DOM was eluted with 1 mL methanol (LiChrosolv, Merck). DOC concentrations were analyzed in aliquots of the SPE extract by high-temperature catalytic oxidation using a Shimadzu TOC/TN analyzer equipped with an

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infrared and a chemiluminescence detector (gas flow oxygen: 0.6 L min
1) as described previously.17 The detection limit (5σ of the blank) was 7 μM C with an accuracy of (2 μM C and reported DOC concentrations were average values of triplicate measurements. 2.2. Total Hydrolyzable Dissolved Amino Acids (THDAA). Stereoisomeres of THDAA were analyzed using a modified method after Fitznar et al.19 Briefly, aliquots of the DOM methanol extract were hydrolyzed with 32% HCl in glass ampules, which were flushed with nitrogen, sealed, and kept for 24 h at 110 C. The solution was neutralized with 1.78 mL borate buffer (pH 8.5), and the pH was adjusted to 8.5 with 32% sodium hydroxide. After precolumn derivatization with orthophthaldialdehyde and N-isobutyryl-L/D-cysteine, samples were analyzed from an injection volume of 20 μL on a high-performance liquid chromatography (HPLC) system equipped with a C18 column (Phenomenex Hyperclone 5 μ, BDS C18; 250  4 mm; pre column, 2  4 mm) and a fluorescence detector using a binary gradient of sodium acetate buffer and acetonitril (LiChroSolv, Merck) at a flow rate of 0.85 mL min
1 (for more details see Table S2 of the Supporting Information). THDAA include D- and L-stereoisomers of alanine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, methionine, serine, threonine, tryptophan, tyrosine, valine, and additionally glycine, L-isoleucine, L-leucine, D-phenylalanine, L- and D-Raminobutyric acid, and γ-aminobutyric acid. The relative standard deviation determined from four replicate measurements of amino acid standards was below 2.7%. 2.3. FT-ICR-MS Analyses. DOM extracts were analyzed in methanol/water 50:50 with electrospray ionization (ESI, Apollo II electrospray source) in negative ion mode (capillary voltage:
4 kV) at an infusion flow rate of 2 μL min
1 on an Apex Qe mass spectrometer (Bruker Daltonics Inc. Billerica, USA) equipped with a 9.4 T superconducting magnet (Bruker Biospin, Wissembourg, France). Prior to analyses, blanks were measured to check for contaminations. Spectra were calibrated with arginine clusters and 300 scans were added to one spectrum. Internal calibration with compounds, which were repeatedly identified in marine DOM yielded a mass accuracy below 0.3 ppm. Molecular formulas were calculated in a mass range of 200
600 m/z and were restricted to a molecular element ratio of O/C e 1.2 and to integer double bond equivalent (DBE) values. A formula tolerance of (0.5 ppm was considered as valid formula and for the final data set we focused on ions with a relative abundance I g 4% (the most abundant sample compound in each spectrum was set to 100%), using the following restrictions with respect to the molecular composition: 1H0
120, 12 C0
50, 16O0
35, 14N0
2. For more details on the FT-ICR-MS formula assignments, see the Supporting Information. Because of the lack of standard substances and possible variations in the ionization efficiency of different substance classes during ESI ionization, FT-ICR-MS is not a quantitative method for DOM. However, the variability in peak heights for ions in replicate FTICR-MS analyses are low20 and the relative peak magnitude approach allows for semiquantitative statements for samples analyzed under similar conditions. Weighted average values were calculated for several parameters from the magnitude of the assigned peaks in the spectrum. 2.4. Mathematical Model. A simple mathematical model was used for the calculation of transformation reactions. Compounds were defined as a tuple C of two vectors C = (E, F), where E = [C, H, O, N] describes the elemental composition and F = [(OH þ NH), 5224

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Table 1. Sediment and Organic Matter Properties of the Sampling Locations in the Northern Black Sea and Their Interpretation TOC

TN

TOC/

δ13CTOC

sampling site

sediment

(%)

(%)

TN

(%)

16-MUC 0
2 cm

green to gray shelly mud

1.19

0.21

6.46


22.9

organic matter derived from marine sources; increase in TOC/TN and decrease in δ13CTOC indicate selective

(oxic) 16-MUC 15
17 cm

interpretation

gray mud

0.89

0.13

8.04


23.5

dark-gray to black mud,

2.60

0.31

9.92


24.1

1.68

0.20

10.01


24.3

remineralization in deeper sediment

71 m bsla 14-MUC 0
2 cm (anoxic) 14-MUC 15
2 cm 268 m bsl a

fluffy layer on top laminated dark-gray to black mud

preferential remineralization of labile marine organic matter under anoxic conditions; accumulation of recalcitrant possibly terrestrial organic matter in surface and deep sediment

bsl
below sea level.

CH2] the reaction potentials, that is, reaction centers involved in specific reactions. A reaction R was also a tuple of two vectors R = (G, L), where G = [C, H, O, N] describes the change in the elemental composition and L = [(OH þ NH), CH2] the change in reaction centers. To apply a reaction to a compound, a vector addition was used (Supporting Information). After the combination of amino acids to glycosylated and nonglycosylated di- and tripeptides, reactions were tested in various combinations until no further reaction occurred. Reaction products were compared to the molecular formulas of the FT-ICR-MS data set. The model was implemented in the PYTHON programming language. 2.5. TOC, TN, and δ13CTOC. The TOC content in the freezedried homogenized sediment was analyzed using a Leco CS 200. δ13C was measured on an Heraeus Elemental analyzer connected to a Finnigan MAT Delta Plus. Prior to analysis, samples were treated with 12.5% HCl to remove carbonates. Standard deviations for isotope analyses were below 0.1%, determined from routine and replicate measurements of a reference sample. Values are quoted in the δ13C notation in per mil relative to the Vienna-Pee Dee Belemnite (V-PDB) standard. For TN measurements, 25 mg of the freeze-dried homogenized sediment were weighted into tin boats and analyses were carried out using a Vario EL III Elemental Analyzer.

3. RESULTS AND DISCUSSION 3.1. Characterization of Sedimentary and Dissolved Organic Matter. The two sampling sites differed considerably

regarding the sediment type, TOC content (Table 1), and bulk composition of the DOM pool (Table 2). Whereas TOC decreased with sediment depth, the pore water DOC concentration was a factor of 2.5 to 4 higher than in the bottom water reflecting remineralization of POM as well as accumulation of degradation products and intermediates in the pore water. Consistent with the DOC increase, THDAA concentrations were around five times higher in the pore waters. Likewise the numbers of total identified N-bearing molecules (nIon) in the FTICR-MS data set (Table 3) were higher in the pore waters. Glycine (G) concentrations in the pore waters from the oxic (16MUC PW) and the anoxic (14-MUC PW) shelf were elevated compared to the bottom water from the same site indicating increased diagenesis of the THDAA pool. The surface sediment and the bottom water at the anoxic site consisted both of rather recalcitrant organic matter such as bacterial peptidoglycan, indicated by the elevated percentage of D-amino acids of the total concentrations of the enantiomers of alanine, glutamic acid, and serine

Table 2. DOC, THDAA, and Amino Acid Compositional Data Analyzed from the Solid Phase Extract of Water Column, Bottom Water (BW), and Pore Water (PW) DOMa DOCSPE

THDAA

%DAES

G

sampling site

(μM)

(nM)

(%)

(%THDAA)

16-MUC BW

151

298

22.8

13.5

16-MUC PW

380

1541

18.7

14.7

14-MUC BW

70

156

34.8

15.9

14-MUC PW

278

794

21.1

16.6

water column 150 m

68

98

24.1

19.0

a

Elevated %DAES indicate higher contributions from bacterial remains,27 whereas higher relative glycine (G) concentrations reflect increased diagenesis.

(%DAES),21 and possibly a higher fraction of terrestrial organic matter compared to the oxic site (lower δ13C value in Table 1), whereas under oxic bottom water conditions degradation is generally less selective22,23 and bacterial contributions are lower (Table 2). The reference sample from the water column in 150 m depth represents refractory DOM24 and had by far the lowest THDAA and DOC contents as well as nIon suggesting high consumption and degradation of labile and semilabile DOM in the surface waters and the subsequent relative enrichment of N-depleted DOM with high (CHO/CHNO)wa in the water layers below. The sediments on the other hand provide an extensive POM pool for the early diagenetic formation of DOM, as it is also reflected in the increased number of total identified N-bearing molecules (nIon, Table 3). 3.2. Variations of N-Bearing Compounds in DOM and the Effect of Early Diagenesis on Molecule Size and Saturation. We identified up to 416 molecular formulas with one and two N atoms in the FT-ICR-MS data set (Table 3); compounds with three or more N atoms were not detected. The N-bearing compounds were distributed over a mass range from 250 to 550 Da and had relative peak magnitudes of up to 24%. Larger and more unsaturated molecules tended to accumulate in the pore water compared to the bottom water (Figure 1, Table 3). Generally, the molecular formulas showed a relatively high similarity between samples, especially among the molecules with high relative peak magnitudes (I = 10
24%, maximum peak magnitude for C15H19N1O9). Compositional differences between individual samples are evident among molecules with relative peak magnitudes below 10%. Figure 2 shows the elemental composition of the DOM samples in van Krevelen diagrams, where molecular formulas 5225

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are arranged according to their O/C and H/C ratios. Pore and bottom water samples share a high number of molecular formulas in the range of O/C = 0.40
0.90 and H/C = 0.39
1.65, whereas the water column DOM was restricted to smaller ranges of O/C = 0.44
0.69 and H/C = 1.06
1.53 of compounds, shared with the bottom and pore water samples (data not shown). The Table 3. Characteristic Parameters Derived from FT-ICRMS Analysis of Water Column, BW and PW DOMa sampling site

(CHO/ nion (m/z)wa DBEwa (O/C)wa (H/C)wa CHNO)wa

16-MUC BW 16-MUC PW 14-MUC BW 14-MUC PW water column 150 m

292 358 260 416 60

374.93 377.09 368.56 379.77 370.65

7.12 7.21 6.93 7.58 7.33

0.58 0.55 0.56 0.55 0.55

1.31 1.32 1.33 1.28 1.28

6.20 5.14 6.70 4.70 26.97

a

Displayed are numbers of identified N-bearing molecular formulas (nIon), the peak magnitude weighted average values of the molecular weight ((m/z)wa), double bond equivalents (DBEwa), molar oxygen to carbon ((O/C)wa) and hydrogen to carbon ratios ((H/C)wa), and the ratio of the total peak magnitude of CHO formulas versus CHNO formulas ((CHO/CHNO)wa).

Figure 1. Molecular mass distribution of N-bearing compounds in all samples. Black bar indicates compounds with N = 1, gray bar refers to compounds with N = 2. Arrows indicate maximum molecule sizes of (glycosylated = glyc.) di- and tripeptides.

oxic bottom water contained a set of unique compounds with molecular formulas at higher O/C ratios (O/C = 0.40
0.90), whereas a set of exclusive molecular formulas with lower O/C ratios (O/C = 0.15
0.75) was present in the anoxic bottom water. The pore water-specific compounds at both sites are again in the range of lower O/C ratios (O/C = 0.35
0.64) with various H/C ratios and mostly relative high DBE values (average DBE of 8.87). Thus, the contrasting redox conditions are to a certain degree directly reflected in the molecular pattern of dissolved organic compounds in the bottom water, and accordingly the sedimentary pore waters were anoxic at both sites. 3.3. N-Bearing Compounds As Products of Protein Degradation. Because most N in biomass is bound in proteins and only a minor fraction is attributed to amino sugars (e.g., in chitin), nucleic acids in DNA and RNA and tetrapyrroles in pigments,25 it is most likely that a larger fraction of DON was derived from proteinaceous material. The high O content in the molecular formulas of our sample set (up to 13 O atoms in one molecule) suggests extensive oxidation or that carbohydrate-like structural elements are involved in the molecules. Proteins in organisms are mainly glycoproteins, that is, sugar groups are attached to polypeptides via an O- or N-glycosidic linkage, and both glycoproteins and possible degradation products were detected in marine DOM before.6,9 Recent studies showed that aminosugars become enriched relative to THDAA during the decomposition of proteinaceous material.21,26 The mass range of molecular formulas in the samples from the Black Sea includes potential protein-derived glycosylated and nonglycosylated di- and tripeptides (Figure 1). Although the contribution of amide-N to the DOM pool in sediment pore waters has not yet been verified, there are clear indications that a fraction of the DOM derives from protein degradation.27 Protein degradation starts rapidly after cell death, initially, and most importantly by hydrolysis of the peptide bonds, which successively breaks down the macromolecular protein structure into smaller oligopeptides, dipeptides, and amino acids.28 In addition to abiotic mechanisms, protein hydrolysis proceeds via enzymatic reactions, performed intracellularly by macroorganisms or extracellularly by bacteria that are not able to transport molecules larger than 600 Da across their cell membranes.29 Molecular formulas in the range of O/C ≈ 0.15
0.5 and H/C ≈ 1.5
2.0 were previously detected by FT-ICR-MS in DOM from protozoa cultures30 and in continental slope sediment pore waters;17 both studies suggested proteins as sources for such compounds. Assuming that the N-bearing

Figure 2. Sample comparison of nitrogen-bearing compounds in van Krevelen diagrams for a) oxic bottom water (16-MUC BW) and anoxic bottom water (14-MUC BW), b) bottom water (16-MUC BW) and pore water from the oxic site (16-MUC PW), and c) bottom water (14-MUC BW) and pore water from the anoxic site (14-MUC PW). Gray dashes indicate compounds common in both samples. Symbols refer to sample-specific compounds with one or two nitrogen. 5226

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Figure 3. Possible precursor peptides and deamination reactions for exemplary sum formulas from the FT-ICR-MS data set: a) oxidative deamination of a peptide built from glutamic acid (E) and lysine (L) results in the substitution of two ammonium groups by oxygen and in the sum formula C11H15NO7 which was identified in all samples, b) reductive deamination substitutes the ammonium group by hydrogen in the peptide built from tyrosine (Y) and threonine (T), and c) in the Stickland reaction of the peptide build from Y, glutamine (Q) and T. Y is deaminated reductively, whereas Q is deaminated oxidatively. Sum formulas corresponding to the hypothetical products from reaction b) and c) were only detected in the anoxic bottom water and the pore water samples.

compounds detected by FT-ICR-MS are protein degradation products, we conclude that processes in addition to hydrolysis alter the structural and elemental composition of proteins. Additional processes related to protein, peptide, and amino acid degradation involve deamination and decarboxylation.31 For example, several molecular formulas in our samples could derive from oxidative peptide deamination, which is a microbially catalyzed process. During this reaction, the terminal amino group is transformed to a carbonyl group (part a of Figure 3). Such a reaction shifts molecular formulas to higher O/C and lower H/C ratios and several N-bearing compounds from our data set could be indeed formed by this pathway. Because oxidative deamination is not restricted to oxic environments,32 we observe high similarities of N-bearing compounds and high relative peak magnitudes from oxic and anoxic sites. Furthermore, deamination can also proceed in the reductive form,33 which also lowers the H/C ratios but does not change the O/C ratios of the altered peptides (part b of Figure 3) due to the loss of two hydrogen atoms. Another possible deamination reaction performed by anaerobic bacteria (e. g., Clostridia) is a coupled oxidation and reduction of amino acids in the form of a Stickland reaction,34 which results in an increase in O/C and decrease of H/C values (part c of Figure 3). We computed the formulas of the deamination products for a range of di- and tripeptides and compared it with our sample set using a dedicated program (Supporting Information). Accordingly, 26 to 34% of the formulas with one N atom are potentially reaction products of deamination (Figure 4). The number of matches is even higher for the formulas with two N atoms with a maximum of 84% in the pore water from the oxic site. Most formulas were formed via the oxidative way and there is no

Figure 4. a) van Krevelen diagram of the DOM sample from the oxic bottom water displaying molecular formulas with N = 1 as a result of degradation reactions (blue
deamination, red
deamination plus oxidation and/or hydration, black
deamination plus oxidation and/or hydration and/or methylation and/or dehydration); gray dots represent all glycosylated and nonglycosylated dipeptides, which were considered as source compounds in the computer program (Table S7 of the Supporting Information); b) shows the relative proportion of the reactions in each sample according to color code.

obvious relationship between the redox conditions at the sampling sites and the nature of deamination reaction. Because amino acids and peptides are highly reactive, additional transformations may occur in the DOM. To address this, we additionally considered the following transformation reactions in the data analysis program, which are depicted in part a of Figure 4: 1 Oxidation of hydroxyl groups; this results in a shift to higher O/C ratios and increases m/z by 13.98352 Da. 5227

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Environmental Science & Technology 2 Hydration of molecules increases m/z (by 17.00734 Da) as well as O/C and H/C. 3 Methylation of amino- and hydroxyl-groups reduces O/C, whereas H/C and m/z increase. The latter increases by 14.02658 Da for each newly introduced methyl group. 4 Dehydration of a hydroxyl or carboxyl group results in a decrease of the molecule size by 18.01528 Da and increases the DBE. By adding oxidizing reactions 1 and 2 to hydrolysis and deamination, up to 61% of the formulas with one N atom and almost all formulas with two N atoms can be explained (for examples, Table S6 of the Supporting Information). Generally, oxidation of DOM compounds was higher in the bottom water at the oxic site (part b of Figure 4). Methylation and dehydration transformations, however, are the dominant reactions under anoxic conditions and especially in the pore waters. Two thirds of the pore water-specific molecular formulas and all of the molecular formulas from the water column could be formed via deamination, methylation, and dehydration. The latter reaction increases the DBE as observed for the pore water samples (Table 3) and may lead to ring formation and aromatization, which is expected in the course of diagenesis.35,36 This process also transforms the molecules from the aliphatic structure of peptides to the carboxyl-rich alicyclic molecules (CRAM) commonly observed in water column DOM14,37 and fulvic acids.13 3.4. Other Sources for N-Bearing Compounds in DOM. Alternative formation pathways for the production of N-bearing molecules in the samples from the Black Sea could be reactions of ammonium with reactive functional groups of N-free DOM in the pore water. Such reactions were recently observed for esters in organic-rich anoxic sediments38 and could represent a sink for ammonia released by deamination. The exclusive set of N-bearing compounds with low O/C ratios in the sediment pore waters (parts b and c of Figure 2) could be intermediates of such rearrangement reactions, which shift the molecular composition to higher H/C and lower O/C ratios. These reactions are restricted to anoxic environments and therefore they can only explain a small subset of N-bearing molecules because the oxic and anoxic sites share a high number of N-bearing molecules (Figure 2, Table S6 of the Supporting Information). The conclusive results from our calculations of peptide degradation reactions suggest that a large part of the molecular formulas represent degradation products and intermediates of proteinaceous biomolecules with the latter being rapidly formed during bacterial protein degradation in general.5,39 Our study sheds new light on molecular-scale mechanisms related to the remineralization of N-bearing organic compounds in sedimentary environments. The detected N-bearing compounds are presumed intermediates and/or products in geochemical reactions returning nutrient-N to the ocean. Our data set suggests that the redox regime in the water column has no significant impact on the inferred mechanisms of protein degradation in the sediment, despite the large differences in the sedimentary POM quality. Whereas our data set does not provide evidence for a strong influence of the oceanic redox regime on the molecular mechanisms involved in recycling of nutrient-N, we cannot decipher potential differences in the kinetics of N release. More research is required to fully assess the impact of expanding oxygen minimum zones12 on nutrient-N recycling.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed description of the THDAA and FT-ICR-MS analyses as well as the computer

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program. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected], phone: þ49-42121865705, fax: þ49-421-21865715.

’ ACKNOWLEDGMENT We thank the crew, Christian Borowski, and the scientific shipboard party of the RV Meteor cruise M72-5. We are grateful to Brit Kockisch for TOC measurements, Hella Buschoff for TN and TC analysis, Monika Segl for δ13CTOC, and Kai-Uwe Ludwichowski for amino acid measurements. Furthermore, we thank Xavier Prieto and Kevin Becker for support in the lab, and Travis Meador for comments on an earlier Version. Funding was provided by the “Deutsche Forschungsgemeinschaft” through DFG-Research Center/Excellence Cluster “The Ocean in the Earth System” and the Bremen International Graduate School for Marine Sciences (GLOMAR). ’ REFERENCES (1) Arnosti, C.; Holmer, M. Carbon cycling in a continental margin sediment: contrasts between organic matter characteristics and remineralization rates and pathways. Estuar. Coast. Shelf S. 2003, 58 (1), 197–208. (2) Henrichs, S. M. Early diagenesis of organic matter in marine sediments: progress and perplexity. Mar. Chem. 1992, 39 (1
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