Geochemistry of Dissolved Organic Matter in a Spatially Highly

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Geochemistry of Dissolved Organic Matter in a Spatially Highly Resolved Groundwater Petroleum Hydrocarbon Plume Cross-Section Sabine Eva-Maria Dvorski, Michael Gonsior, Norbert Hertkorn, Jenny Uhl, Hubert Müller, Christian Griebler, and Philippe Schmitt-Kopplin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00849 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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Environmental Science & Technology

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Geochemistry of Dissolved Organic Matter in a Spatially Highly

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Resolved Groundwater Petroleum Hydrocarbon Plume Cross-

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Section

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Sabine E.-M. Dvorski a, Michael Gonsior b, Norbert Hertkorn a, Jenny Uhl a, Hubert Müller c, Christian Griebler c, Philippe Schmitt-Kopplin* a, d

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a

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Analytical BioGeoChemistry, D-85764 Neuherberg, Germany

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b

Helmholtz Zentrum München - German Research Center for Environmental Health, Research Unit

University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland 20688, USA

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c

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Groundwater Ecology, D-85764 Neuherberg, Germany

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d

Helmholtz Zentrum München - German Research Center for Environmental Health, Institute of

Technische Universität München, Chair of Analytical Food Chemistry, D-85354 FreisingWeihenstephan, Germany

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* Corresponding author: Philippe Schmitt-Kopplin, Helmholtz Zentrum München, - German Research

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Center for Environmental Health, Research Unit Analytical BioGeoChemistry, Ingolstädter

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Landstraße 1, 85764 Neuherberg, Germany, phone: (+49) 089 3187 3246, fax: (+49) 089 3187 3358,

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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At numerous groundwater sites worldwide, natural dissolved organic matter (DOM) is quantitatively

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complemented with petroleum hydrocarbons. To date, research has been focused almost exclusively

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on the contaminants, but detailed insights of the interaction of contaminant biodegradation, dominant

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redox processes, and interactions with natural DOM are missing. This study linked on-site high

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resolution spatial sampling of groundwater with high resolution molecular characterization of DOM

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and its relation to groundwater geochemistry across a petroleum hydrocarbon plume cross-section.

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Electrospray- and atmospheric pressure photoionization (ESI, APPI) ultra-high resolution mass

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spectrometry (FT-ICR-MS) revealed a strong interaction between DOM and reactive sulfur species

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linked to microbial sulfate reduction, i.e. the key redox process involved in contaminant

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biodegradation. Excitation emission matrix (EEM) fluorescence spectroscopy in combination with

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Parallel Factor Analysis (PARAFAC) modeling attributed DOM samples to specific contamination

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traits. Nuclear magnetic resonance (NMR) spectroscopy evaluated the aromatic compounds and their

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degradation products in samples influenced by the petroleum contamination and its biodegradation.

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Our orthogonal high resolution analytical approach enabled a comprehensive molecular level

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understanding of the DOM with respect to in situ petroleum hydrocarbon biodegradation and

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microbial sulfate reduction. The role of natural DOM as potential co-substrate and detoxification

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reactant may improve future bioremediation strategies.

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Introduction

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Natural dissolved organic matter (DOM) is an essential energy source for heterotrophic organisms in

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aquatic ecosystems.1 Its concentration and composition are pivotal parameters related to water

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quality.2 Groundwater ecosystems are typically poor in organic carbon and energy and, consequently,

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systems of low productivity.3 DOM concentrations in pristine oxic groundwater are as little as 0.1

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mg/L to a few mg/L.4 However, numerous groundwater sites worldwide are contaminated with

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organic chemicals and exhibit significantly higher DOM concentrations.5,

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groundwater contaminants are petroleum hydrocarbons with benzene, toluene, ethylbenzene and

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xylenes (BTEX) and polycyclic aromatic hydrocarbons (PAHs) being the most common.7,

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contaminated systems, natural DOM may act as a reactant, chelator, and sorbent for hydrophobic

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organic contaminants influencing their bioavailabilty, mobility, and fate.1, 9 Thus far, detailed insight

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into the role of natural DOM for petroleum hydrocarbon contaminated groundwater ecosystems is

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missing on a molecular level.

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From the point source, pollutants partly dissolve in groundwater and are transported along with the

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water flow, thereby forming a contaminant plume.5 The fate and transformation of organic

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contaminants in a mature plume are related to several abiotic processes such as dilution, diffusion, and

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microbial degradation.10 Current research underlined that contaminant plumes in porous aquifers are

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characterized by steep and small-scale gradients in the range of centimeters to decimeters with high

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biodegradation activities occurring at the fringes of the plume.11, 12 This “plume fringe concept” is

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founded on the depletion of dissolved electron acceptors in the plume core.5, 13 Biodegradation with

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oxygen, nitrate, or sulfate reduction can then only take place at the fringes of the plume where

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electron acceptors are replenished from surrounding groundwater by dispersion and diffusion. In the

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plume core, biodegradation may alternatively be driven by reduction of solid electron acceptors such

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as Fe(III)- or Mn(IV), and also by methanogenesis.14

6

Prominent organic

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In

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Natural DOM interacts with not only organic pollutants but also inorganic reactive species that may

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directly be related to the dominant terminal redox processes coupled to contaminant biodegradation.

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Reactive sulfur species such as sulfide are a prominent example. In fact, sulfate reduction is the

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quantitatively most important redox process in petroleum hydrocarbon contaminated aquifers.10 An

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improved fundamental understanding of contaminant biodegradation bottlenecks and development of

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successful remediation strategies need detailed investigations of the mutual influence, the dominating

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redox reactions and products, and characteristics and distribution of contamination as well as its

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interaction with natural DOM. As natural DOM is a complex organic mixture, often consisting of

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thousands of organic compounds, high resolution non-targeted analytical characterization by means of

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Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), excitation emission

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matrix (EEM) fluorescence spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy are

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techniques of choice for an adequate DOM assessment.1, 15-19

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The diverse molecular characteristics of complex DOM can be unraveled using FT-ICR-MS as shown

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in previous studies.1, 20 Here, the application of negative mode electrospray ionization ((-)ESI) FT-

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ICR-MS led to a significantly improved mechanistic understanding of natural biotic and abiotic as

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well as anthropogenic processes that characterize DOM in aquatic ecosystems.21-26 Recently, the FT-

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ICR-MS analysis of DOM alteration in wastewater treatment and drinking water plants improved the

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assessment of water quality.27-29 While (-)ESI FT-ICR-MS is selective towards DOM molecules

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containing carboxylic acids and other polar functional groups, positive mode atmospheric pressure

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ionization (+)APPI FT-ICR-MS favors the detection of aromatic and less polar compounds.30-33

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(+)APPI FT-ICR-MS has also been used to characterize nonpolar complex mixtures such as shale oils,

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asphaltenes, and PAHs and is targeted towards molecules with π-electron systems.34-37

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Complementary to FT-ICR-MS, EEM fluorescence spectroscopy in conjunction with Parallel Factor

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Analysis (PARAFAC) is a powerful tool used to study fluorescent dissolved organic matter

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(FDOM).38, 39 PARAFAC modeling has been utilized to study water treatment and recycling schemes Page 4 of 28


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as well as processes in natural aquatic systems.15,

40-44

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molecules in mineral oils and their degradation products, EEM fluorescence spectroscopy is useful to

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assess distribution, influence, and transformation of petroleum products in the environment.45-48

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Additionally, 1D and 2D NMR spectroscopy offers quantitative in-depth information of proton and

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carbon chemical environments from which extended substructures can be assembled.1,

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combined use of NMR and FT-ICR-MS produced an informative assessment of DOM structural

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details according to ecosystem characteristics.1,

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employed for the analysis of oils from various sources and to study the interaction of natural organic

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matter and organic pollutants.50-53

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The aim of this study was to combine on-site high resolution spatial sampling across a well-defined

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petroleum hydrocarbon plume with high resolution molecular characterization of DOM in an effort to

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study the interaction of the contaminants in addition to redox processes and their reaction products

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with natural DOM. We performed comprehensive analyses of solid phase extracted DOM (SPE-

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DOM) from groundwater of the petroleum hydrocarbon contaminated sandy aquifer in Düsseldorf

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Flingern by means of high-field (-)ESI and (+)APPI FT-ICR-MS, NMR, and EEM fluorescence

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spectroscopy.

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Methods

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Site description and sampling

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Groundwater was sampled in September 2013 at the former gasworks site in Düsseldorf Flingern,

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Germany (51°13’19.83” N; 6°49’05.44” E). The aquifer is predominantly composed of quaternary

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sand and gravel. The disposal of tar oil in the shallow subsurface for many decades (i.e. during

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operation from 1893 – 1967) caused a contaminant plume mainly consisting of BTEX and PAHs. A

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detailed description of the test site is provided elsewhere.11, 12

18, 19, 49

Owing to the high abundance of aromatic

17, 18

The

Furthermore, NMR spectroscopy has been

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Groundwater samples were collected from a high resolution multilevel well (HR-MLW), which

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allowed a spatial vertical resolution of 3 to 10 cm. Thirty-two small volume samples were taken

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simultaneously to prevent mixing of water from different depths. A list of sampling depths is provided

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in the Supporting Information (SI). The sampling scheme using this HR-MLW is described in detail

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elsewhere.11, 12

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Groundwater geochemistry and occurrence of (parent) contaminants

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Methods and detailed protocols for the analysis of aromatic hydrocarbons, i.e., BTEX and selected

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PAHs, are given by Anneser et al.11 Basic physical-chemical parameters such as pH and redox

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potential (EH) were determined in freshly collected groundwater samples with field sensors (WTW,

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Weilheim, Germany). Major ions, e.g., sulfate, were analyzed within 2 days by ion chromatography

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(Dionex DC-100, Idstein, Germany) from samples stored in the dark without headspace and cooled.

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Fe(II) concentrations were measured by the ferrozine assay.54 For sulfide determination, 200 µL of

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sample were fixed in duplicates in 1 mL of a 2% (w/v) zinc acetate solution and analyzed on-site

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following a protocol, which was adapted to smaller volumes, from Cline.55

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Solid phase extraction of DOM

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DOM from the 0.2 µm filtered groundwater samples was isolated by an established solid phase

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extraction (SPE) method described by Dittmar et al.56 A detailed description is given in the SI

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Methods.

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Ultra-high resolution mass spectrometry

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Ultra-high resolution spectra were acquired with a Bruker (Bremen, Germany) SolariX Qe FT-ICR-

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MS equipped with a 12 Tesla superconducting magnet. Reproducibility of the mass spectra was

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assured by measuring SPE-DOM from 250 mL and 1 L samples collected at the same depths, also

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indicating no extraction volume effect. Additionally, FT-ICR mass spectra of samples taken in close

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vicinity to each other (enabled by the high spatial resolution sampling in the centimeter range)

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resemble in principle composition, indicating indirectly reproducibility of FT-ICR mass spectra. Page 6 of 28


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Further details on the mass spectra acquisition and formula assignment from mass spectra are

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described in the SI. Hierarchical cluster analyses of assigned FT-ICR-MS peaks were performed with

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Hierarchical Clustering Explorer Version 3.0 and R.

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FT-ICR-MS is a semi-quantitative method owing to the lack of calibration standards in non-targeted

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analysis and differential ionization efficiency in complex and chemically diverse mixtures. The signal

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intensities of mass peaks are not necessarily directly proportional to their respective concentration;

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however increase and decrease of mass peak intensities follow the changes in relative concentrations.

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To address the limited detection of some compounds due to low ionization efficiency and suppression,

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we performed two orthogonal ionization modes. Additionally, ionization and mass independent

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quantitative 1H NMR and optical EEM fluorescence spectroscopy was utilized.

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EEM Fluorescence Spectroscopy and PARAFAC Modeling

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Excitation emission matrix fluorescence spectroscopy of SPE-DOM was measured using a Jobin

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Horiba Instruments (Kyoto, Japan) Aqualog spectrofluorometer. Details on the measurements and

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PARAFAC modeling, which were performed by the drEEM MATLAB toolbox, are given in the SI

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(Figures S1, S2).38 Principal component analysis (PCA) of Fmax values was carried out with SIMCA-

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P 9.0 to evaluate the influence of the components on the individual samples.

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NMR

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1D and 2D 1H and 13C NMR spectra were acquired from re-dissolved SPE-DOM in CD3OD at 283 K

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with a Bruker (Karlsruhe, Germany) AV III 800 spectrometer operating at B0 = 18.7 Tesla with

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Bruker standard pulse sequences and cryogenic detection. A detailed description of the NMR

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acquisition conditions is described in the SI.

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Results and Discussion

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Biogeochemical gradients and distribution patterns of major contaminants and signature metabolites

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provide critical information on the redox processes involved in transformation of contaminants and Page 7 of 28



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active biodegradation pathways.5 Typically, biodegradation in organic contaminant plumes in porous

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aquifers is most pronounced at the plume fringes as repeatedly confirmed by (1) steep and opposing

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gradients of contaminants and electron acceptors such as sulfate, (2) peaking of the metabolic end

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products sulfide and Fe(II) from sulfate and iron reduction, respectively, and (3) bacterial cell counts

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and contaminant specific stable carbon isotope values at the plume fringes.5, 11, 13

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Samples collected from the petroleum hydrocarbon contaminated aquifer in Düsseldorf Flingern

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showed steep small-scale gradients for BTEX and naphthalene as the major contaminants (Figure

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1A). The contaminant plume, at the time of sampling, was located at the groundwater table extending

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to 0.8 m beneath (6.51–7.31 m below surface (bls)). According to the scheme of Anneser et al., the

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contaminant plume was subdivided into three compartments (Figure 1).57 The first, the plume core,

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was defined as the zone exhibiting BTEX concentrations >50% of the observed maximum of 616

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µmol/L (6.61–7.01 m bls). The second compartment, the upper plume fringe (6.51–6.61 m bls), was

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bound at the upper side by the groundwater table including the capillary fringe, which was located

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right above the groundwater table and only partially filled with water. The lower plume fringe, which

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was the third compartment and located below the plume core, extended from 7.01–7.31 m bls.

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PAHs, in particular naphthalene, exhibited a high abundance (maximum of 89.5 µmol/L at 6.78 m

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bls) in the plume core (Figures 1A, B). The three-ring PAHs exhibited the highest abundances ~0.3 m

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deeper (Figures 1A, B). Acenaphthene was present at approximate concentrations of 7 µmol/L all

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along the sampled vertical profile (Figure 1B). Previous studies at this site had shown a comparable

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distribution of the major contaminants, however, with slightly different concentrations of the

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individual compounds.11, 57

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Redox potentials between +89 mV at the groundwater table and −149 mV in the plume core indicated

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strictly anoxic conditions except for the capillary fringe (Figure 1D). The pH profile showed elevated

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values of up to pH 8.05 in the plume core (Figure 1D). Sulfate as the predominant electron acceptor

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was almost absent in the plume core and showed a steep counter gradient to the major contaminants at Page 8 of 28


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the lower plume fringe. Outside of the contaminant plume, sulfate exhibited a rather constant high

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concentration of approximately 1.5 mmol/L (Figure 1C). Thus, hydrocarbon degradation was sulfate-

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limited in the plume core. Sulfide peaked at concentrations of up to 0.17 mmol/L in the plume core

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and lower plume fringe, indicating a hot spot of sulfate reduction between 6.75 and 7.21 m bls. The

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discrepancy between the amount of sulfate reduced within the contaminated area and the free sulfide

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detected in the water is explained by precipitation of reduced sulfur with iron as well as incorporation

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of reduced sulfur into DOM (see below). As already shown in previous studies11,

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reduction was the key redox process involved in biodegradation of organic contaminants in the

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Düsseldorf Flingern aquifer. However, in contrast to earlier surveys, sulfate, sulfide, and BTEX

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profiles indicated biodegradation by sulfate reduction taking place not only at the lower plume fringe

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but also in parts of the plume core. These changing patterns may be related to temporal dynamics of

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contaminant concentrations and biodegradation activities as evaluated by contaminant-specific stable

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isotope analysis (CSIA) and microbial community analysis (unpublished data). Extraordinary high

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Fe(II) concentrations of up to 0.38 mmol/L were found close to the groundwater table, thereby

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hinting, in accordance with previous studies11, at iron reduction to be a relevant redox process at the

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upper plume fringe.

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(-)ESI FT-ICR-MS of DOM

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DOM in pristine groundwater is generally composed of organic molecules from natural carbon

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sources, such as plants, microbes and fungi, whereas DOM at contaminated sites considerably derives

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from the anthropogenic carbon input.2 (-)ESI FT-ICR-MS spectra from SPE-DOM showed distinct

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compositional variation along the aquifer (Figure S3B). In general, sample similarity followed spatial

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proximity, and hierarchical cluster analysis revealed three distinct subclusters, 6.51–6.64 m bls, 6.67–

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7.06 m bls, and 7.08–10.20 m bls (Figure 2A). The extensive chemical dissimilarity between the

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subclusters is depicted in Figure 2B. The van Krevelen diagrams and ring charts display the most

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representative molecular formulas (correlation coefficient r > 0.9) for each subcluster. More details on

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the selection of characteristic molecular formulas are given in the SI.

12, 57, 58

, sulfate

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The differences between (-)ESI FT-ICR mass spectra of the SPE-DOM isolates corresponded to the

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respective prevailing and clearly distinct zone-dependent redox chemistry (Figures 1, S3B). The

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specific DOM chemistry in the upper part of the aquifer, including the upper fringe, plume core, and 5

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cm of the lower plume fringe (cf. blue and red subcluster, Figure 2), was dominated by sulfur organic

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molecules. This is in agreement with sulfate reduction being the major redox process in the plume and

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its fringes.12 The samples from 6.67–7.06 m bls (red subcluster, Figure 2) were dominated by CHOS

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compounds with a contribution to the total intensity of assigned molecular formulas of up to 52%

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(Figure S4A). This was most likely a result of high sulfide concentrations in these depths generated by

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sulfate reduction (Figure 1C). The relative proportion of CHO compounds from 6.67–7.06 m bls

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decreased (Figure S4A), thus, indicating that CHOS compounds were formed from CHO compounds.

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The formation of sulfur organic compounds at elevated sulfide concentrations is a common

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biogeochemical process

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DOM and model organic compounds.63-66 Sulfide reacted with organic matter functionalities such as

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single and activated double bonds (Michael systems, Quinones) at the boundary of the contamination

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plume.63-66 Therefore, we anticipate direct reaction of highly nucleophilic sulfide with DOM

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functional entities to result in CHOS molecules. CHOS compounds might have been also formed

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directly during sulfate reduction. Sulfide concentrations in the upper plume fringe were low because

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of the supposed dynamic re-oxidation of sulfide to sulfate close to the capillary fringe.67 Furthermore,

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the Fe(II) concentration was elevated in the upper fringe (Figure 1), which likely led to precipitation

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of sulfide with Fe(II) and implied a dominance of iron over sulfate reduction in this zone.12 Thus,

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DOM chemistry in the upper fringe and the topmost centimeters of the plume core was affected

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differently compared to the area ~20 cm below. Einsiedl et al. found elevated sulfite concentrations

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close to the capillary fringe; thus, characteristic CHOS compounds from 6.51–6.64 m bls (blue

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subcluster, Figure 2) potentially resulted from sulfonation reactions.67 With respect to contaminants,

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the influence of ionizable aromatic molecules (H/C < 1) was higher in the top centimeters close to the

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groundwater table, thereby indicating fewer degraded contaminants present inside the upper plume

59-62

that has also been confirmed in several laboratory experiments with

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fringe, which was likely due to the limited access to electron acceptors (Figures 1D, 2).11 A

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substantial portion of characteristic CHNO and especially CHNOS compounds suggested significant

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nitrogen DOM chemistry in the upper plume fringe where dissolved sulfide was almost absent.

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The characteristic mass peaks of SPE-DOM from the deep zone below 7.08 m bls showed mainly

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CHO and CHNO compounds in the polyphenolic region of the van Krevelen diagram (1.5 > H/C >

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0.7 and 0.7 > O/C > 0.1) (Figure 2B). Hence, DOM characteristics of less contaminated deep zone

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groundwater showed similarities to previously published (-)ESI FT-ICR-MS results of groundwater

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DOM.68, 69 The comparably low number of characteristic mass peaks for deep zone SPE-DOM arose

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from the fact that these samples represented general DOM patterns of the sampling site, which was

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only marginally impacted by petroleum contaminants, degradation products, and intense sulfur redox

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chemistry (Figure S3A).

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(+)APPI FT-ICR-MS of DOM

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To address limited sensitivity of some compounds due to low ionization efficiency and suppression in

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(-)ESI FT-ICR-MS we performed (+)APPI FT-ICR-MS as a nearly orthogonal ionization mode

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(Figure S3C).30-32 (+)APPI is the method of choice for the detection of molecules with extended,

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conjugated π-electron systems such as petroleum contaminants and their degradation products.30 As

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an effect of the sample preparation, which was not optimized for the extraction of nonpolar

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contaminants but for DOM extraction, a mere 6% of the annotated molecular formulas of the whole

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data set were oxygen-free molecules. Therefore, only the results of the oxygen containing CHNOS

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molecular formulas are presented and discussed.

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Hierarchical cluster analysis of (+)APPI FT-ICR mass peaks and the associated van Krevelen

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diagrams of characteristic molecular formulas of the subclusters showed that all samples above 7.06

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m bls were dominated by aromatic and even condensed aromatic molecules (low H/C and O/C ratios)

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(Figures 3B, 3C). These molecules are typically derived from petroleum, thus the segmentation of the

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cluster was clearly driven by the elevated contamination input in the top ~50 cm beneath the Page 11 of 28



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groundwater table (6.54 m bls). Analogous to the (-)ESI FT-ICR-MS results, the proportion of

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characteristic CHNO and CHNOS compounds in the contamination plume and fringes was greater in

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the samples taken close to the groundwater table further corroborating the hypothesis that organic

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nitrogen chemistry played a role in this depth region (Figure 3C). Furthermore, (+)APPI FT-ICR-MS

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showed that a large proportion of characteristic formulas were sulfur organic compounds, which was

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in accordance with the (-)ESI FT-ICR-MS results and the intensive bacterial sulfate reduction

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(Figures 3B, 3C, S5).12,

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formulas did not show any clear trend in the highly sulfidic region (Figure S4B). Thus, CHOS

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compounds formed as a result of bacterial sulfate reduction were proposed to derive from highly

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functionalized DOM molecules (i.e. carboxylic acids, carbonyl derivatives, alcohols,…), which have

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a greater (-)ESI ionization efficiency.30

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On the contrary, SPE-DOM isolates from the deeper zone of the aquifer were characterized by a large

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combined abundance (greater 80% of the overserved characteristic mass peaks describing the overall

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diversity) of CHO and CHNO compounds with 1.5 > H/C > 1 and 0.6 > O/C > 0.2 (Figure 3C, note

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that due to limitations in the visualization, CHO compounds are partially hidden by the CHNO

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compounds); therefore, corresponding to APPI FT-ICR mass spectra of DOM from previous

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studies.32, 31 This implied that DOM from the lower fringe and below was presumably not heavily

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affected by the contamination and its degradation products at the time of sampling.

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Field and culture degradation studies of BTEX, PAHs and heteroatom PAHs at the same or similar

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conditions were able to identify typical degradation products.7,

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molecular formula annotation combined with the profound literature knowledge, the respective mass

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peaks most probably derive from these known degradation products. Figure 3D shows the relative

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mass peak intensities for the four highest abundant putative degradation products originating from

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BTEX, PAHs and heteroatom PAHs as well as their methylated relatives methylnaphthyl-2-

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methylsuccinic acid (MNMS, C16H16O4), naphthyl-2-methyl-succinic acid (NMS, C15H14O4),

67

However, the intensity-weighted contribution of assigned molecular

71-75

Due to the high accuracy in

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methylnaphthoic acid (MNA, C12H10O2), and acenaphthene-5-carboxylic acid (AC, C13H10O2).7, 70-74

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The depth profile was in accordance with the distribution of plume marker compounds,

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hydrochemical parameters, and redox chemistry markers (Figures 1, 3D). Moreover, MNMS, NMS,

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AC, and an unknown compound (C15H12O2) represented the highly abundant characteristic masses of

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the 6.75 to 7.03 m bls subcluster (Figure 3C). Hence, the initial steps of biodegradation, which have

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been identified in pervious degradation studies

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plume core where bacterial sulfate reduction was highly active.

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EEM fluorescence spectroscopy of DOM

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EEM-PARAFAC modeling was validated for a three component model and the relative contribution

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of each component was identified for the SPE-DOM samples taken along the aquifer (Figures 4, S1,

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S2). The first component (C1) showed an excitation/emission (Ex/Em) maximum at 237(288)/360

299

nm, which was similar to the PARAFAC components modeled in Deepwater Horizon oil spill studies

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resembling oil and its degradation products as well as BTEX- and PAH-like fluorescence (Table

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S2).46, 47, 48 The second component (C2) identified at an Ex/Em maximum of 255(315)/420 nm, which

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was in accordance with humic-like fluorescence, has been found in many PARAFAC studies on

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organic matter.75, 44, 76 Also, PARAFAC studies on the Deepwater Horizon oil spill showed similar

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components, which are described as humic-like therein (Table S2).45-48 The third component (C3)

305

exhibited its Ex/Em maximum at 255/333 nm matching previously published components that were

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reported as oil, BTEX, and PAH related (Table S2).46, 48 High bacterial activity was expected to show

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protein-like fluorescence, however this signal was absent from zones of highly active sulfate reducing

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bacteria. In fact, the typical protein-like fluorescence has never been observed under anoxic

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conditions from sulfate reducing bacteria but has often been associated with aerobic heterotrophic

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bacteria.77 At this point, it is not clear why anaerobic conditions would prevent the release of water

311

soluble proteins that contain tyrosine and/or tryptophan, however this was not the aim of this study.

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Furthermore, Wang et al. 2015 showed significant quenching of tyrosine and tryptophan fluorescence

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as well as shift of the peak position in the presence of humic substances.78

7, 71-75

, were mainly located in the lower part of the

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To distinguish fluorescence spectra of SPE-DOM dominated by contamination-related versus typical

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DOM fluorescence, PCA of Fmax values of the components was performed (Figure 4). PCA revealed

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that SPE-DOM from 6.59 to 6.96 m bls were mainly driven by C1, which was annotated to oil, BTEX

317

and, PAH, in addition to its degradation products. The samples from 7.01 to 7.21 m bls showed a

318

strong correlation with C3, which was also described as oil, BTEX, PAH, and its degradation

319

products. Therefore, C3 fluorescence most likely derived from further degraded products and three-

320

ring PAHs that were more abundant in this region (Figure 1B). Samples in close vicinity of the

321

groundwater table and the ones from the deep zone (highlighted in green, Figure 4B) were

322

characterized by C2, showing that their fluorescence was humic-like and not strongly affected by the

323

contamination. The ratios of C1/C2 and C3/C2, which indicated oil/humic contributions to SPE-DOM

324

composition, were elevated for the samples ranging from 6.61–7.08 m bls. Therefore, EEM

325

fluorescence spectroscopy also successfully differentiated SPE-DOM influenced by the contamination

326

plume.

327

NMR spectroscopy of DOM

328

In addition to FT-ICR-MS spectrometry, with its already discussed susceptibility to selective

329

ionization and suppression effects, and EEM fluorescence spectroscopy, which is only able to detect

330

fluorophores, NMR spectroscopy was utilized. NMR does not suffer from any of these potential

331

distortions and provides quantitative depiction of relevant structural features in complex organic

332

mixtures.18 But 1H and

333

EEM fluorescence spectroscopy and is rather limited in its capability to provide in depth information

334

about nitrogen and sulfur containing compounds compared to FT-ICR mass spectrometry.18 1H NMR

335

spectroscopy of selected SPE-DOM sampling positions that were indicative of changing geochemical

336

parameters and contamination levels provided detailed structural information and substantial variance

337

according to zonation (Figures 1, 5). These variations at first indicated variable contributions of

338

common bulk DOM with broad NMR resonances and of petroleum degradation products showing

339

sharp NMR resonances with well resolved splittings from J-couplings (Figure 5) (cf. Tables S3, S4 for

13

C NMR spectroscopy is less sensitive compared to both FT-ICR-MS and

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340

1

341

units of observed DOM and petroleum degradation products is listed in Table S5.

342

In general, the relative contribution of aromatic structures (δ(1H) = 9.5–7.0 ppm) ranged up to 30% in

343

the plume core and fringes, while it was below 15% for SPE-DOM close to the groundwater table

344

(6.64 m bls) and the deeper zone (10.20 m bls) (Tables S3, S4). Figure S6 depicts overlaid 1H NMR

345

spectra adjusted to common amplitude of the aliphatic bulk DOM signature (δ(1H) = 0.8–2.0 ppm;

346

Figure S6A) and alternatively area-normalized 1H NMR spectra across the entire range of chemical

347

shifts (δ(1H) = 0.5–9.5 ppm; Figure S6B). 1H NMR spectra clearly showed a nearly constant but

348

somewhat variable background bulk DOM signature present in all SPE-DOM (Figure S6A) and

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highly variable proportions of PAH and its degradation products. In particular, not only the ratios of

350

aromatic protons (δ(1H) > 7 ppm) and those of HOOC-CαH-protons (δ(1H) ~ 2.1–2.7 ppm) differed

351

quite substantially, but the line shapes within these sections of chemical shifts also greatly differed.

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This demonstrated a huge variance of PAH-degradation products present at each individual depth of

353

sampling and again testified to the unique capability of 1H NMR spectroscopy in the elucidation of

354

complex environmental unknowns.16-19

355

As the petroleum contamination arose mainly from BTEX and naphthalene, only a very small

356

percentage of the NMR total integral derived from multi (>3) ring condensed aromatics with δ(1H) >

357

8.3 ppm. Most of the aromatic structures were found in the chemical shift region δ(1H) = 8.3–7.3

358

ppm, which was indicative of single and two-ring aromatics, six membered N-heterocycles, and

359

especially oxidized aromatics (i.e., mainly polycarboxylated). These types of structural features were

360

in general matching the contaminants and their degradation products.7, 70 The two SPE-DOM from the

361

top and bottom of the aquifer, while assumed representative of common groundwater DOM,

362

nevertheless showed elevated proportions of petroleum derivatives compared to pristine DOM

363

levels.69 This background concentration was drastically increased in the plume core, and the co-

364

occurrence of contaminants and their degradation products was confirmed by co-occurring sharp

H NMR section integrals according to DOM substructures). A detailed description of key structural

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NMR resonances at δ(1H) > 7 ppm (aromatics) and δ(1H) ~ 2.1–2.7 ppm (aliphatic carboxylic acids).

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Additionally, J-resolved (JRES) and total correlation (TOCSY) spectroscopy indicated the presence of

367

multiring polycyclic aromatic compounds. Most of them showed two rings (Figure S7), in accordance

368

with common composition of groundwater contamination by petroleum products.11, 12 Furthermore,

369

aliphatic cross peaks in TOCSY and methylene-edited 1H, 13C distortion less enhanced by polarization

370

transfer heteronuclear single quantum coherence (DEPT HSQC) NMR spectra showed typical DOM

371

signatures as well as succinic acid side chains attached to aromatics (Figure S8) that are typical for

372

PAH degradation products such as NMS, which were also detected in the (+)APPI FT-ICR mass

373

spectra (Figure 3D).7, 70, 72, 73 The proportion of non-functionalized monoaromatic structures (δ(1H) =

374

7.3–7.0 ppm) was lower but might be underestimated because of losses during the sample preparation

375

as a consequence of their high volatility.

376

The region δ(1H) = 7.0–6.5 ppm of Csp2H likely originated from OH and OR substituted aromatics of

377

common SPE-DOM and partially indicated aromatics with fused alicyclic units, which are

378

constituents of partly biodegraded mineral oil.52 Otherwise, conjugated (δ(1H) = 6.5–6.0 ppm) and

379

isolated double bonds (δ(1H) = 6.0–5.1 ppm) likely represented natural product derivatives present in

380

groundwater SPE-DOM.

381

The slight depletion of carbohydrates (δ(1H) = 4.9–3.1 ppm) in SPE-DOM from depths with high

382

contamination levels might be an artifact of the sample preparation as PPL cartridges discriminate

383

against carbohydrates.19 Also, the region of carboxyl-rich alicyclic molecules (CRAM) from δ(1H) =

384

3.1–1.9 ppm showed no clear trend because of the interference of CRAM and contamination

385

degradation products.

386

Contaminant abundance and that of “original” SPE-DOM signatures were inversely related across the

387

aquifer (Figure S6B, radar diagram). However, the region of non-functionalized aliphatics (CCCH;

388

δ(1H) = 1.9–0.5 ppm), a major contributor of common SPE-DOM, maintained its core integrity with

389

limited but clearly recognizable variance (Figure 5A) across the entire range of sampling.19 Page 16 of 28


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DOM characteristics and contamination biodegradation

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Non-targeted high resolution analytics in combination with spatially highly resolved sampling

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revealed DOM signatures from petroleum contaminants and specific degradation products (Figures 3–

393

5, Tables S2–S5, Figures S6–S8). Additionally, our approach enabled a comprehensive molecular

394

level understanding of natural DOM and its role in petroleum hydrocarbon bioremediation. Previous

395

studies at the investigated site identified bacterial sulfate reduction as the major redox process

396

involved in the contaminant biodegradation by isotope ratio analysis.11,

397

demonstrates that the DOM characteristics were highly affected by bacterial sulfate reduction. Both

398

(+)APPI FT-ICR-MS, which favors the detection of petroleum derived compounds, and (-)ESI FT-

399

ICR-MS, which is preferably applied for the characterization of natural DOM, revealed characteristic

400

CHOS compounds at the depths where sulfate reduction is active. Organosulfur compounds were

401

found by far more abundant in the (-)ESI FT-ICR mass spectra (Fig. S4). Thus, reduced sulfur mainly

402

reacts with natural DOM. CHOS compounds represented >50% of the total mass peak intensity of the

403

corresponding assigned molecular formulas derived from (-)ESI FT-ICR mass spectra in the zone

404

with maximum sulfide concentrations (Figures 1, 2, S4A). Especially in zones where Fe(II) and other

405

complexation partners were limited, sulfide remained in solution. Contaminant biodegradation is

406

limited by the availability of sulfate as electron acceptor at the investigated site. Elevated sulfide

407

concentrations may even further decelerate petroleum biodegradation at low sulfate concentrations;

408

indeed, high concentrations of sulfide are toxic to many microbes.79 In this context, natural DOM may

409

act as a beneficial key player in the mediation of sulfide resulting from bacterial sulfate reduction. In

410

consequence, natural DOM may be applied in bioremediation. Future work will thus address the

411

linkage of contaminant biodegradation, redox processes, DOM, and the microbial communities.

412

Acknowledgements

413

We thank C. Kellermann and K. Hörmann for their contribution in the field. We are grateful to J.

414

Luek for support with the EEM fluorescence spectroscopy measurements. The authors S.E.-M. D. and

12

The current study

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H.M. thank the Helmholtz WasserZentrum München for funding. This is contribution (xxxx, to be

416

filled in after acceptance of manuscript) of the University of Maryland Center for Environmental

417

Science.

418

Supporting Information Available

419

The SI contains extended information on methods (Table S1 and Figures S1, S2) and results and

420

discussion (Tables S2–S5 and Figures S3–S8). This material is available free of charge via the

421

Internet at http://pubs.acs.org.

422

TOC/Abstract Art

423

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425 426 427 428 429 430 431 432

Figure 1. Vertical concentration profiles of main contaminants (A) BTEX and (B) PAHs, (C) sulfide, sulfate and Fe(II) as well as (D) the hydrochemical parameters pH and redox potential (depth in m below surface (m bls)). The black dashed line depicts the groundwater table. Values represent single or means of duplicate and triplicate measurements (error bars depict standard deviations), respectively. The assumed contamination plume is highlighted in gray and the plume core defined by >50% of the maximal BTEX concentration is depicted by the solid gray line. For further information, see text.

433

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Figure 2. (A) Hierarchical cluster analysis of the assigned (-)ESI FT-ICR-MS derived molecular formulas observed in SPE-DOM along the aquifer with samples named according to their sampling depth (bls = below surface). (B) Van Krevelen diagrams depict the most representative characteristic molecular formulas (correlation coefficient > 0.9; cf. SI) for the three main subclusters, representing the specific DOM chemistry. Elemental composition of the molecular formulas are represented as CHO (blue), CHNO (orange), CHOS (green), and CHNOS (red), and the bubble area depicts the mean mass peak intensity within the respective subcluster.

442 443

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Figure 3. (A) Hierarchical cluster analysis of the assigned (+)APPI FT-ICR-MS derived molecular formulas observed in SPE-DOM along the aquifer with samples named according to their sampling depth. (B) Van Krevelen diagram of shared characteristic molecular formulas of the two upper subclusters, which were highly affected by the contamination plume and (C) van Krevelen diagrams of the characteristic molecular formulas for the three main subclusters representing the specific SPEDOM chemistry. Elemental composition of the molecular formulas are represented as CHO (blue), CHNO (orange), CHOS (green), and CHNOS (red), and the bubble area depicts the mean mass peak intensity within the respective subcluster. Note that the size of the three and four most abundant CHO (blue) bubbles of the purple (B) and red subcluster (C) had to be down-sized because of their very high mass peak intensity. (D) (+)APPI FT-ICR-MS derived abundance profile of main petroleum contamination degradation products.

456

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Figure 4. (A) EEM fluorescence spectra of three PARAFAC-derived components C1–C3. (B) PCA of SPE-DOM along the aquifer based on the Fmax values of the PARAFAC components. Samples are labeled according to their sampling depth in m bls. H corresponds to 250 mL samples, and L corresponds to 1 L samples.

462

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Figure 5. (A) 1H NMR spectra (800 MHz, CD3OD) of selected SPE-DOM with depth provided and color coded according to similarity of contamination patterns and geochemical parameters. (B) Bar charts indicate relative 1H NMR section integrals according to substructures shown in Tables S3 and S4 and the main structural sections are depicted with dotted lines.

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Geochemistry of Dissolved Organic Matter in a Spatially Highly

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