A simple guideline to apply excitation-emission matrix spectroscopy (EEMs) for the characterization of dissolved organic matter (DOM) in anoxic marine sediments
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Abstract: Marine sediments represent a major carbon reservoir on Earth. Dissolved organic matter (DOM) in pore waters accumulates products and intermediates of carbon cycling in sediments. The application of excitation-emission matrix spectroscopy (EEMs) in the analysis of subseafloor DOM samples is largely unexplored due to the redox-sensitive matrix of anoxic pore water. Therefore, this study aims to investigate the interference caused by the matrix on EEMs and propose a guideline to prepare pore water samples from anoxic marine sediments. The parameters determined by fluorescence spectra include 3D-index derived from EEMs after parallel factor analysis (PARAFAC), fluorescence index (FI) (contribution of terrigenous DOM), biological index (BIX) and humification index (HIX) derived from 2D emission spectra. First, we investigated the impacts of extensively-presented ions as typical electron acceptors, which are utilized by anaerobic microbes and stratified in marine sediments: Fe(II), Fe(III), Mn(II) and sulfide in anoxic pore water resulted in biases of fluorescent signals. We proposed threshold concentrations of these ions when the interference on EEMs occurred. Effective removal of sulfide from sulfide-rich samples could be achieved by flushing with N2 for 2 min. Second, the tests based on DOM standard were further verified using pristine samples from marine sediments. There was a significant change in the fluorescence spectra of DOM in anoxic sediments from the Rhône Delta. This study demonstrated that the change was caused by oxidation of the matrix rather than the intrinsic alteration of DOM. It was confirmed by extracted DOM via both EEMs analysis and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Slight oxidation of sulfur-containing compounds (e.g., sulfhydryl) and polyphenol-like compounds occurred. Finally, a sample preparation sequence is proposed for pore water from anoxic sediments. This method enables measurement with small volumes of the sample (e.g., 50 µL in this study) and ensures reliable data without the interference of the redox-sensitive matrix. This study provides access to the rapid analysis of DOM composition in marine sediments and can potentially open a window into examining the carbon cycling of the marine deep biosphere.
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Key words:
- marine subsurface sediment /
- EEMs /
- PARAFAC /
- FT-ICR-MS /
- anaerobic pore water
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Figure 1. Excitation-emission matrix spectroscopy (EEMs) components identified by parallel factor analysis (PARAFAC). Components 1, 2, 3, 4 and 5 represent Peaks A(C), M, C, T and B, respectively. More information is shown in Table S1. The em and ex represent emission and excitation wavelength, respectively.
Figure 2. Composition of Suwanee River Fulvic Acid Standard (SRFA), natural samples and yeast extract (YE) after parallel factor analysis.
Figure 3. Effect of redox-sensitive ions on humic-like peaks. a. Excitation-emission matrix spectroscopy (EEMs) spectra of original Suwanee River Fulvic Acid Standard (SRFA) samples; b–d. EEMs spectra of SRFA samples with addition of Fe(II), Mn(II), S2– at concentrations of 0.06 mmol/L, 0.06 mmol/L, 0.3 mmol/L, respectively; e–g. EEMs of SRFA samples with addition of Fe(II), Mn(II), S2– at concentrations of 0.6 mmol/L, 0.6 mmol/L, 1 mmol/L, respectively. The em and ex represent emission and excitation wavelength, respectively.
Figure 4. Effect of redox-sensitive ions on protein-like peaks. a. Original excitation-emission matrix spectroscopy (EEMs) of yeast extract (YE); b–d. EEMs of YE samples with addition of Fe(III), Mn(II), S2– at concentrations of 0.2 mmol/L, 0.6 mmol/L, 1 mmol/L, respectively. The em and ex represent emission and excitation wavelength, respectively.
Figure 5. Effect of two-month O2 exposure on pore water dissolved organic matter (DOM) characterized by FT-ICR-MS. Change of DOM sample from the North Sea (a) and Rhône Delta (b) in van Krevelen diagram; change of O/C ratio in DOM sample from the North Sea (c) and Rhône Delta (d). Type 1: aliphatic compounds, AI<0; Type 2: highly unsaturated compounds, 0.5≥AI≥0; Type 3: aromatic compounds (including condense aromatic compounds), AI>0.5. The peak magnitude shown in the figure is relative intensity normalized to the sum of all peaks, i.e., rIntn= IPeak/∑IallPeaks. The color represents the difference of rIntn after air exposure (
$\Delta r_{\rm{Intn}} $ ), i.e., rIntn-end−rIntn-start. rIntn>0: relative abundance of formulae increases after air exposure. The y-axis in c and d shows the difference of rIntn at a narrower range. The definition of AI refers to Eq. (3).
Figure 6. Effect of O2 exposure on Excitation-Emission Matrix Spectroscopy (EEMs) of pristine sample from Rhône Delta. EEMs of fresh sample without exposure to O2 (a), exposure to O2 for 2 h (b) and without precipitation (c). The em and ex represent emission and excitation wavelength, respectively.
Table 1. Summary of main subseries experiments and controls
Experiment Sample, matrix or treatment Purpose of tests Incubation of sediments North Sea sample (20℃) sulfide-rich samples North Sea sample (85℃) samples enriched in protein-like compounds and humic-like compounds Rhône Delta sample (20℃) metal-ion-rich samples Matrix effects S2– impacts of anions involved in anoxic mineralization Mn(II), Fe(II), Fe(III) impacts of metal ions involved in anoxic mineralization matrix-removed DOM method to avoid matrix effects Storage effects DOM extracts, O2 exposure oxidation of DOM pristine samples, Rhône impacts of matrix oxidation during storage under O2 and method to avoid storage effects pristine samples, North Sea impacts of matrix oxidation during storage under O2 and method to avoid storage effects Note: The pristine sample refers to original pore water without solid phase extraction (SPE); the dissolved organic matter (DOM) extracts refer to purified sample without inorganic matrix after SPE. 
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Table 2. Change of fluorescent signal along with metal ions, sulfide and O2 exposure. Acceptable ranges of concentrations are listed
Acceptable range Index Fe(III)
0–0.007 mmol/LFe(II)
0–0.06 mmol/LMn(II)
0–0.06 mmol/LNa2S
(N2-flushed)Extracted DOM after O2 exposure
2 monthsBias of 2D index FI ↑ NS NS ↑ ↓ HIX ↓ ↓ ↓ ↓ ↑ BIX ↑ NS NS ↑ NS Bias of 3D index protein-like peaks ↓ ↓ ↓ ↑ / humic-like peaks ↓ ↓ ↓ NS / AC/M ↓ ↓ ↓ ↑ NS Note: The relative changes below 5% were defined as NS, i.e., no significant impact. For 2D index with variation coefficient ranging from 5% to 15%, P<0.05 in ANOVA were defined as NS. Results of ANOVA are presented in Table S3. ↑ and ↓ represent increase and decrease of the parameters with higher concentrations of added ions or O2 exposure. FI: fluorescence index; BIX: biological index; HIX: humification index. AC/M: peak height ratio of Peak AC to Peak M.
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Table 3. Comparisons of dissolved organic matter (DOM) fluorescence spectra before and after solid phase extraction (SPE) by pre-cleaned Bond Elut-PPL cartridge
Sample P/H AC/M FI BIX HIX Before SPE-pristine sample 0.5 1.2 1.5 0.9 4.3 After SPE-DOM extract 0.2 1.5 1.6 0.6 8.1 After SPE-residue liquid 1.5 0.6 1.8 1.2 0.5 Standard deviation-pristine sample 0.01 0.01 0.02 0.1 0.2 Note: A 20-mL liquid sample was used for SPE. In-house-prepared samples from the North Sea sediments after incubation were tested since the pristine sample contains both substantial protein-like and humic-like compounds. FI: fluorescence index; BIX: biological index; HIX: humification index. AC/M: peak height ratio of Peaks A and C to Peak M; P/H: peak height ratio of Peak P to Peak H.
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