{'fraction_non_alphanumeric': 0.04339883753113756, 'fraction_numerical': 0.0204262385828951, 'mean_word_length': 4.946675444371297, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 11, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Linkage Between Dissolved Organic Matter Transformation, Bacterial Carbon Production, and Diversity in a Shallow Oligotrophic Aquifer: Results From Flow-Through Sediment Microcosm Experiments

Aquifers are important reservoirs for organic carbon. A fundamental understanding of the role of groundwater ecosystems in carbon cycling, however, is still missing. Using sediment flow-through microcosms, long-term (171d) experiments were conducted to test two scenarios. First, aquifer sediment microbial communities received dissolved organic matter (DOM) at low concentration and typical to groundwater in terms of composition (DOM-1x). Second, sediments received an elevated concentration of DOM originating from soil (DOM-5x). Changes in DOM composition were analyzed via NMR and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Carbon production, physiological adaptations and biodiversity of groundwater, and sediment prokaryotic communities were monitored by total cell counts, substrate use arrays, and deep amplicon sequencing. The experiments showed that groundwater microbial communities do not react very fast to the sudden availability of labile organic carbon from soil in terms of carbon degradation and biomass production. It took days to weeks for incoming DOM being efficiently degraded and pronounced cell production occurred. Once conditioned, the DOM-1x supplied sediments mineralized 294(±230) μgC L−1sed d−1, 10-times less than the DOM-5x fed sediment communities [2.9(±1.1) mgC L−1sed d−1]. However, the overall biomass carbon production was hardly different in the two treatments with 13.7(±4.8) μgC L−1sed d−1 and 14.3(±3.5) μgC L−1sed d−1, respectively, hinting at a significantly lower carbon use efficiency with higher DOM availability. However, the molecularly more diverse DOM from soil fostered a higher bacterial diversity. Taking the irregular inputs of labile DOM into account, shallow aquifers are assumed to have a low resilience. Lacking a highly active and responsive microbial community, oligotrophic aquifers are at high risk of contamination with organic chemicals.


Details on compositional changes of DOM with sediment passage
High-field NMR spectroscopy provides the capability for quantitative and non-destructive de novo determination of atom-specific chemical environments (describes chemical bonding to neighboring atoms) of polydisperse and molecularly heterogeneous DOM Powers et al., 2019). While NMR allows analysis and quantification of carbon-and hydrogen-based aliphatic, oxygenated and aromatic chemical environments of atoms, FT-ICR mass spectrometry enables to assign thousands of CHO, CHOS, CHNO and CHNOS molecular formulas directly out of complex mixtures (Koch et al., 2005;Hertkorn et al., 2008;Kujawinski et al., 2009). FT-ICR mass spectrometry of DOM is however subject to ionization selectivity, which regularly causes mass peak intensities to deviate from actual molecular concentrations (Hertkorn et al., 2008). Moreover, many (several thousands and more) isomeric molecules may project on any individual mass peaks, further obstructing direct relationships between mass spectra and actual defined individual molecules. Because NMR and FT-ICR mass spectra each provide valuable but rather complementary information about structure and molecular composition of DOM , both technologies were applied. 1 H NMR spectra served to constrain compositional changes of DOC in selected samples collected from non-amended groundwater and the DOM-5x approach. The 1 H NMR spectra of groundwater DOM and the feeding solution DOM-5x were largely dissimilar as expected ( Figure 4 in the manuscript). Groundwater DOM featured the highest content of pure aliphatics (CCCH units; Table 1) and the lowest content of oxygenated aliphatic groups and protons bound to unsaturated carbon (OCH and Csp2H units; Table 1), with olefinic protons particularly depleted. In contrast, feeding solution DOM-5x was much more oxidized than the groundwater DOM sample and contained large fractions of potentially lignin-derived aromatic and phenolic units but only minor proportions of olefins ( Figure S1). The group of oxygenated aliphatics included a large complement of carbohydrates, aromatic and aliphatic methyl esters and methyl ethers, peptides and carboxyl-rich alicyclic matter (CRAM) (Figure 4 in the manuscript).
The small compositional changes between the pure groundwater infiltrated into the microcosm before any feeding solution was supplied and the outflow at t0 ( Table 1 in the manuscript) most probably reflected slight attenuation of DOM due to adsorption to the sediment as well as some production of limited amounts of microbial metabolites in a nearly equilibrated system. These showed sharp NMR resonances in all sections of the 1 H NMR spectrum ( Figure S1-C & S1-D), including CarH, OCHn and CCCH derived aliphatic metabolites, likely including derivatives of short-chain fatty acids (δH ~ 1.4-0.9 ppm).
The DOM-5x feeding solution at the inflow and the DOM in the outflow at t7 were overall comparable in their structural characteristics (Figure S1-C & S1-D), underlining a delayed onset of microbial transformation of soil leachate DOM added to groundwater. However, the DOM from the microcosm outlet at t7, already showed a selective depletion in aromatic methylesters and methyl ethers by ~35% ( Figure S1-D), in line with a known preferential sorption of aromatic compounds. Besides sorption being involved in DOM attenuation, indication of microbial processing of DOM came from the presence of different sharp NMR resonances in eluates from t7 and t171, which probably originated from a few but rather abundant microbial metabolites. Compared with groundwater inflow, pure and functionalized aliphatics were found relatively enriched at t7. In particular, aliphatics became likely more branched upon processing of DOM as shown by the overall broadened NMR resonances at δC ~ 0.8-1.7 ppm ( Figure S1-B). A higher abundance of CRAM molecules (carboxyl-rich alicyclic molecules; Hertkorn 2006), pointed at a higher overall extent of carboxylation, and depletion of certain oxygenated molecules (δH > 4 ppm) at t7 (Figure S1-A & S1-B) implied early preferential processing of DOM molecules with higher proportions of oxygen-containing functional groups. After 171 days of experiment, DOM samples at the outflow of the DOM-5x fed sediment zones were found very similar in composition to the DOM in the natural groundwater. The long-term supply of DOM-5x thus led to an altered dynamic equilibrium, with increased proportions of aromatic and olefinic molecules as well as aliphatic methyl esters, compared with the DOM from t0 ( Figure S1-A & S1-B). In fact, initial sorption of these compounds later dropped out and these compounds quantitatively passed the sediments.
The high sensitivity, excellent mass resolution and mass accuracy of FT-ICR mass spectrometry allowed profiling of the temporal changes of organic matter for both the DOM-1x and the DOM-5x approach. Several thousands of mass peaks were assigned to CHO, CHNO, CHOS and CHNOS molecular formulas ( Figure 5 in the manuscript; Table S1; Table S2). FT-ICR-MS spectra of groundwater as well as all samples collected at the microcosm outlets showed the skewed, near Gaussian distribution of mass peaks typical of common organic matter, with mass differences dominated by the most abundant ±DBE (∆m = 2.1057 Da) and ±CH2 (∆m = 14.0156 Da) groups ( Figure 5 in the manuscript). Feeding solution DOM-5x showed a higher mass range and a more complex pattern than the other SPE-DOM samples, in line with the prominent signature of oxygenated aliphatic and aromatic functional groups observed already in NMR spectra ( Figure S1). In accordance with NMR results for the DOM-5x experiment, hierarchical cluster analysis (HCA) and principal component analysis (PCA) grouped the feeding solution DOM-5x and its eluate from t7 together, well separated from the other three DOM samples ( Figure 4-A & 4-B in the manuscript). In the DOM-1x experiment, the PCA-based distinction of all eluates was less pronounced than with the DOM-5x approach that showed a more distinct temporal evolution ( Figure 5-A in the manuscript; Figure S1-S3).
FT-ICR mass spectrometry revealed a different temporal evolution of DOM processing for CHO, CHNO, CHOS and CHNOS molecular series in DOM-1x and DOM-5x treatments ( Figure S4, Figure S5). Figures S4 and S5 display differences between mass spectra from samples at time points t0 [t0 minus [DOM-1(5)x + ground water)] , t7 and t171, and therefore refer to molecular compositions affected by degradation and processing of organic matter. The molecular diversity of organic molecules affected by degradation was, broadly spoken, higher in DOM-5x samples than in DOM-1x samples, because their van Krevelen and mass-edited H/C ratio diagrams occupied larger areas. The effects of feeding solution on the composition of outflow waters were more distinct in case of the DOM-5x experiment ( Figure S5). While the DOM-1x experiments showed lesser variations between t0 and t7 samples and even smaller ones between t171 and natural groundwater DOM ( Figure S4), the respective changes in the DOM-5x experiment showed a distinct processing of DOM.
General FT-ICR-MS-derived bulk characteristics, like averaged elemental ratios and relative unsaturation as expressed by DBE/C varied only slightly across all samples (Table S1; Table   S2) reflecting the overall gross similarity of eluates and effects of massive projection of structural isomers onto common molecular compositions  and ionization selectivity out of the very complex mixture of DOM molecules (Hertkorn et al., 2008). The temporal evolution of the average mass in DOM-5x experiments was consistent with a continual degradation from larger to smaller molecules (Table S2). Comparative analysis of CHO, CHNO, CHOS and CHNOS molecular series of individual samples in comparison with groundwater inflow revealed significant information about the temporal evolution and selective processing of DOC molecules ( Figure S4; Figure S5). Overall, the molecular selectivity of processing of CHO and CHNO compounds appeared similar, whereas processed less numerous CHOS compounds were in general more depleted in oxygen and of lower mass. Processing of the very numerous CHNOS compounds was clearly distinct as described below, Turnover of CHO compounds between t0 and t7 moved towards smaller molecules and slightly more deoxygenation at otherwise similar H/C ratios (H/C ratio ~ 0.8-1.5). At t171, the group of oxygenated, lignin-like compounds of relatively high mass (m/z > 450 Da) was depleted ( Figure S4-F, Figure S5-F). Furthermore, only highly oxygenated CHO (with m/z < 450) and CHNO compounds (O/C ratio > 0.4) disappeared. Selectively processed CHOS molecules were lipid-like and of weak to considerable unsaturation.
Overall, the FT-ICR-MS derived molecular selectivity of processing of CHO and CHNO compounds appeared grossly similar, however, processed CHNO compounds were less numerous than CHO molecules and also chemically less diverse as seen by the smaller space occupied in the van Krevelen diagrams ( Figure S4, Figure S5). The temporal evolution of the less numerous CHOS compounds primarily involved aliphatic compounds with a wide range of oxygenation, comprising common DOM molecules as well as sulfolipid-like compounds.
At t7, the proportions of processed DOM molecules had increased in comparison, whereas lipid-like compounds and less oxidized CHOS compounds were transformed only later (t171), indicating a slow progressive defunctionalization toward more saturated and less oxygenated compounds ( Figure S4, Figure S5). CHOS compounds were in general more depleted in oxygen and of lower mass for both DOM-5x and DOM-1x treatments. This is remarkable, because common sulfur functional groups in DOM will most probably carry several oxygen atoms (e.g. -OSO3H and -OSO2H groups) under oxic conditions. Hence, the parent molecules of selectively reacted CHOS compounds were rather oxygen-depleted and of considerably lower mass compared with their CHO counterparts ( Figure S4-E, Figure S5 When passing through the sediments, CHO and CHNO molecules of average H/C and relatively high O/C ratios in the groundwater have been selectively attenuated at t0 (previous to DOM supply) in experiment DOM-5x, suggesting initial degradation or selective adsorption of highly functionalized DOM molecules ( Figure S5-A). At t7 ( Figure S5-B), the profile of processed CHO and CHNO molecules had shifted toward highly oxidized lignin-(O/C ratio > 0.5) and tannin-like (O/C ratio > 0.7) compounds of high mass, in line with the appearance of forest-soil derived compounds in the 1 H NMR spectra ( Figure S1). Preferential processing of CHOS compounds shifted from common DOM molecules and marginally unsaturated sulfolipid-like compounds at t0 to a much more restricted set of less oxidized molecules at t7, whereas at t171 more unsaturated CHOS compounds of substantial overall chemical diversity have been selectively transformed ( Figure S5-A-C). At t0, a very few CHNOS compounds of average H/C and O/C ratios and a large set of oxygen-depleted (O/C ratio < 0.4) molecules with a wide-ranging diversity of relative unsaturation from aromatic to near fully saturated (H/C ratio ~ 0.8-1.9) molecules with m/z < 500 Da have been selectively transformed (Table   S2, Figure S5-D). A few aliphatic and oxidized CHNOS compounds had also been depleted.
At t7 and t171, two very distinctive major sets of CHNOS compounds, one compact but molecularly widely diverse set of minimal to considerable oxygenation (O/C ratio ~ 0.05-0.5) 8 and aromatic to nearly fully saturated (H/C ratio ~ 0.6-1.85), and a similarly diverse but less numerous assembly of oxygenated (O/C ratio ~ 0.4-0.9) and rather aliphatic (H/C ratio ~ 1.1-2.2) CHNOS compounds of relatively smaller mass (m/z < 650 Da) have been substantially depleted ( Figure S5-E & Figure S5-F).
The temporal evolution of DOM-1x was less distinct than that in DOM-5x experiments ( Figure   S4). In particular, turnover of CHO compounds between t0 and t7 moved towards smaller molecules and slightly more deoxygenation at otherwise similar H/C ratios (H/C ratio ~ 0.8-1.5); at t171 a limited set of oxygenated, lignin-like compounds of relatively high mass (m/z > 450 Da) was depleted. At t171, only highly oxygenated CHO (with m/z < 450) and CHNO compounds (O/C ratio > 0.4) had been depleted, reflecting a greater relative stability of these highly oxidized compounds when compared with common DOM/CRAM molecules of more average H/C and O/C ratios ( Figure S4-C & Figure S4-F). Selectively processed CHOS molecules were lipid-like and of weak to considerable unsaturation. Area normalized spectra computed solely within the zone of C sp2 H units (δ H : 5-10 ppm) more instructively visualize structural variations resulting from microbial processing, and the difference between the two latter spectra is highlighted in light brown (outflow day 7 minus forest soil leachate) and light green (forest soil leachate minus outflow day 7) for improved contrast. Forest soil leachate displays higher proportions of polycarboxylated benzene derivatives and polyaromatic compounds (δ H >7.6 ppm) whereas outflow at day 7 exhibits relatively higher proportions of olefins, likely indicative of natural products, lignins and oxygenated aromatic molecules (δ H ~ 5.1-6.7 ppm). Figure S2. 2D NMR spectra of forest soil leachate, section of C sp2 H units (panel A): 1 H, 1 H JRES NMR spectrum shows near absence of trans-double bonds and abundant ortho-and meta-protonated aromatic molecules; (panel B) 1 H, 1 H TOCSY NMR spectrum shows dominance of 3/4 J-couplings between carboxylated aromatic molecules (COR), indicating multiple carboxylation; couplings between COR (electron withdrawing substituents; δ H > 7.3 ppm), R (neutral substituents H, R alkyl ; green shade, δ H : 7.0 -7.3 ppm), and OR (electron-donating substituents; ; δ H : 6.5 -7.0 ppm) substituents also occur, testifying to a substantial diversity of chemical environments. Section a: minor set of polarized double bonds (-CH=CH-C=O units) and (panel C) 1 H, 13 C HSQC NMR spectrum indicates major contributions of lignins (annotation equals that in ref. Kim and Ralph, 2010); the green section indicates the chemical shift range accessible by single aromatic rings; the numerous cross peaks at δ H > 7.3 ppm indicate abundance of (poly)carboxylated molecules (SPARIA: substitution patterns in aromatic rings by increment analysis; Perdue et al., 2007); section b: polarized double bonds, with other HSQC cross peaks originating from double bonds in vicinity. Figure S3. 2D NMR spectra of forest soil leachate (panel A): 1 H, 1 H JRES NMR spectrum; (panel B) 1 H, 13 C HSQC NMR spectrum: section a: C-CH 3 cross peaks; section b: C=C-CH 3 and -SCH3 cross peaks; section c: -CHn-CH n -COOH cross peaks (n: 1, 2); section d: -C-CHn-COOH cross peaks (n: 1, 2); section e: -CONH-CH2glycine in peptides; section f: CONH-CHn-CHα in peptides (amino acids other than glycine); section g: methoxy (OCH3) cross peaks: aliphatic and aromatic methyl esters H3CO-C(=O)-C-; section h: aromatic and aliphatic methyl ethers H3CO-C-C; section i: oxomethylene (OCH2) cross peaks, likely from carbohydrates; section j: OC 2 CH cross peaks, carbohxydrates, ethers, esters, oxidation products of carotenoids; section k: O 2 CH anomeric cross peaks from carbohydrates; dotted box cf. Figure S2 (Csp2H units).  The plotted data reflect difference spectra, i.e. t0 minus [DOM-1(5)x + ground water at ti], which were computed to emphasize DOM processing of groundwater inflow SPE-DOM and show CHO, CHOS, CHNO and CHNOS compounds, which were declining throughout the experiment (cf. text). Bubble areas represent relative mass peak amplitudes. The purple triangle indicates presence of aromatic molecules with aromaticity index AI > 0.5 (Koch and Dittmar, 2006).