Article pubs.acs.org/est
Characterization of Dissolved Organic Matter in Full Scale Continuous Stirred Tank Biogas Reactors Using Ultrahigh Resolution Mass Spectrometry: A Qualitative Overview Sepehr Shakeri Yekta,*,† Michael Gonsior,‡ Philippe Schmitt-Kopplin,§,∥ and Bo H. Svensson† †
Department of Thematic Studies - Water and Environment, Linköping University, SE-581 83 Linköping, Sweden Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, Maryland, USA § German Research Center for Environmental Health, Helmholtz Zentrum Munich, Neuherberg, Germany ∥ Chair of Analytical Food Chemistry, Technische Universität München, Germany ‡
S Supporting Information *
ABSTRACT: Dissolved organic matter (DOM) was characterized in eight full scale continuous stirred tank biogas reactors (CSTBR) using solid-phase extraction and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS). An overview of the DOM molecular complexity in the samples from biogas reactors with conventional operational conditions and various substrate profiles is provided by assignments of unambiguous exact molecular formulas for each measured mass peak. Analysis of triplicate samples for each reactor demonstrated the reproducibility of the solid-phase extraction procedure and ESI-FT-ICR-MS which allowed precise evaluation of the DOM molecular differences among the different reactors. Cluster analysis on mass spectrometric data set showed that the biogas reactors treating sewage sludge had distinctly different DOM characteristics compared to the codigesters treating a combination of organic wastes. Furthermore, the samples from thermophilic and mesophilic codigesters had different DOM composition in terms of identified masses and corresponding intensities. Despite the differences, the results demonstrated that compositionally linked organic compounds comprising 28−59% of the total number of assigned formulas for the samples were shared in all the reactors. This suggested that the shared assigned formulas in studied CSTBRs might be related to common biochemical transformation in anaerobic digestion process and therefore, performance of the CSTBRs. amender.7 The bioavailability and mobility of organic compounds in soil depend upon their degree of reactivity and degradability, which in turn regulate their transport and residence.8,9 Accordingly, unveiling the detailed molecular properties of DOM in biogas reactors will facilitate our understanding of the nature of organic compounds and related biochemical reactions in anaerobic digestion with potential implications in biogas production and waste management. We applied electrospray ionization (ESI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for the characterization of DOM in samples from eight full scale continuous stirred tank biogas reactors (CSTBR), a commonly used reactor type in Sweden.10 The method allows characterization of DOM at the molecular level and assignment of unambiguous exact molecular formulas for each measured mass peak in samples with various matrices.9,11−15 The main objective pursued via DOM characterization using ESI-FT-ICR-MS was to provide a qualitative
1. INTRODUCTION The anaerobic digestion process is widely applied for production of methane as a renewable biofuel source from various types of organic wastes. A network of microbially mediated reactions leads to the production of methane and carbon dioxide (i.e., biogas). These reactions include hydrolysis of complex organic compounds such as polysaccharides, proteins, and lipids followed by acidogenesis of monosaccharides, amino and long chain fatty acids, and further acetogenesis and methanogenesis as the final steps in anaerobic digestion.1−3 Dissolved organic matter (DOM) is a key component in the reaction network of anaerobic digestion. DOM composition comprises the dissolved fraction of influent organic matter, which is a part of available nutrient sources, and the intermediates of microbial processes during degradation of composite organic compounds.4 Furthermore, DOM interacts with micronutrients (i.e., trace metals) and influences their bioavailability, 5 which is critical for sustaining the stability and the efficiency of the anaerobic digestion process.6 In addition to the operational considerations, the characteristics of DOM in the effluents from biogas reactors influence the fate of the residual organic matter and the associated trace metals in the soil when applied in agricultural practices as fertilizer and soil © 2012 American Chemical Society
Received: Revised: Accepted: Published: 12711
June 18, 2012 October 30, 2012 October 31, 2012 October 31, 2012 dx.doi.org/10.1021/es3024447 | Environ. Sci. Technol. 2012, 46, 12711−12719
Environmental Science & Technology
Article
solid-phase extraction was performed on three replicates for each reactor to ensure the reproducibility of the extraction procedure and mass spectrometric analysis. Prior to solid-phase extraction, the filtered liquid fractions were acidified to pH 2 to allow protonation of organic acids, which enables them to be adsorbed on the functionalized styrene-divinyl-benzene polymer resin (PPL). This resin was designed to extract also relatively polar compounds such as phenols. Small highly polar organic compounds escape the applied extraction procedure, but low molecular weight compounds could not be detected using FT-ICR-MS because of the mass cutoff at about 150 Da of the ion cyclotron detector. As a result of these limitations, low molecular weight organic compounds (mass 10% of base peak. Three clustered mesophilic CD4, CD5, and CD7 reactors (i.e., group D, cf. Figure 2) had 7, 5, and 16 assigned formulas with the relative intensities >10% of base peak and 4 of them were shared between these reactors. Furthermore, the SS1 reactor had 12 assigned formulas with relative intensities >10% of base peak, and 8 of these compounds also appeared with high intensities (i.e., >10% of base peak) in the mass spectra of SS4 samples. These observations imply the presence of shared highly abundant peaks in the samples from the reactors with similar operational conditions and substrate profiles (cf. Figure 2) suggesting that the high intensity DOM formulas might be indicative of certain operational conditions and substrate types of the biogas reactors. The details about the relationships between reactor classes and high intensity mass peaks are not discernible based on the present observations and their elucidation requires further research. 3.3.3. H/C and O/C versus Mass. The H/C and O/C versus mass diagrams are plotted together with van Krevelen diagrams (Figure 3 and Figure S2) indicating the trend of saturation and oxidation of DOM with mass. An overall higher O/C values
under the anoxic conditions and subsequent reaction of sulfide with organic compounds.11,39 The CHOS formulas with H/C between ca. 2 and 2.3 and O/C between ca. 0.2 and 0.6 can be attributed to the presence of aliphatic sulfur-containing organic compounds (e.g., aliphatic sulfonic acids and sulfates) and their relatively high intensities (as illustrated by size of the dots in van Krevelen diagrams) is possibly related to their high ionization efficiency during ESI.15 These compounds might be related to the presence of surfactants as was previously shown by Gonsior et al.15 in the samples from the effluent of wastewater treatment plants. Differences were observed in terms of DOM diversity for the formulas with H/C and O/C ratios of ca. 0.6−1.2 and ca. 0.6−1, respectively (e.g., tannin-like substances).25 The number of assigned molecular formulas in these H/C and O/C ranges was larger in SS1, SS4, CD5, and CD7 compared to the other reactors including mainly the CHO group (Figure 3 and Figure S2). Similar to the lignin-like compounds, the tannin-like compounds have presumably aromatic nature limiting their degradability in the CSTBRs. 38 3.3.2. High Intensity Mass Peaks. Despite the fact that parameters other than DOM concentrations might influence the DOM intensities (e.g., solid-phase extraction and ionization efficiencies), the recorded intensities might be used as additional information to indicate the relative significance of the compounds in the samples.21 The intensity data are represented by the size of the dots for the individual molecular formulas in the van Krevelen diagrams (Figure 3 and Figure S2) as based on the average intensities of the DOM formulas identified in the triplicate samples. The presence of shared high intensity DOM mass peaks for the triplicate samples confirmed the significance of these assigned formulas in each reactor. 12716
dx.doi.org/10.1021/es3024447 | Environ. Sci. Technol. 2012, 46, 12711−12719
Environmental Science & Technology
Article
symmetric pattern was observed when the shared formulas in all the biogas reactors are plotted in KMD/z* versus mass diagrams (Figure 4 and Figure S4). The shared CHO compounds in the mass range of 210 −250 are depicted in Figure S4 to visually clarify the plane of symmetry and the relationship between molecular composition of the compounds. The symmetric pattern indicates that the formulas might be related to each other via chemical transformation e.g. methylation/demethylation and alkyl chain elongation (difference in CH2), oxidation/reduction (difference in exchange of 1C, 4H, and 1O), and hydrogenation/dehydrogenation (difference in H2). Therefore, the observed symmetric pattern suggest that the shared DOM formulas might include the end products of the biochemical reactions and/or residues of degradation processes. The shared and compositionally linked molecular formulas occurred at lower masses (mass 400 Da (Figure 4). This implies that the differences in molecular compositions among the reactors are more pronounced at high molecular masses (mass >400 Da) presumably due to the contribution of different substrate sources and the effects of specific operational conditions on biodegradation of organic substrates. Thus, it might be perceived that the high molecular weight compounds (i.e., mass >400 Da) could reflect the characteristics of DOM related to substrate sources and/or certain operational conditions and the low molecular weight compounds (i.e., mass
