Labilization and diversification of pyrogenic dissolved organic matter by 1 microbes 2

Abstract

that solar photo-degradation irradiation also significantly affects the composition and reactivity of pyDOM during 23 terrigenousterrestrial-to-marine export, the effects of photochemistry were also evaluated in the context of 24 pyDOM the bio-reactivitydegradability of pyDOM. 25 Ultrahigh resolution mass spectrometry revealed that, after incubation, many different (both aromatic and 26 aliphatic) compounds were bio-degraded. , and nNew labile compounds, 22 -40 % of which were peptide-like, 27 were bio-produced. This These results indicated that a portion of pyDOM has been labilized into microbial 28 biomass during the incubations. Fluorescence excitation-emission matrix spectra revealed that some fraction of 29 these new bio-produced molecules is associated with proteinaceous fluorophores from proteinaceous and/or 30 autochthonous/microbial biomass origin. Two-dimensional 1 H-1 H total correlation NMR spectroscopy identified 31 a peptidoglycan-like backbone within the microbially produced compounds. These results are consistent with 32 previous observations of nitrogen from peptidoglycans within the soil and ocean nitrogen cycles where remnants 33 of bio-degraded pyDOM are expected to be observed. 34 Interestingly, the exact nature of the bio-produced organic matter was found to vary drastically among 35 samples indicating that the used microbial consortium may produce different exudates based on the composition 36 of the initial pyDOM. Another potential explanation for the vast diversity of molecules is that microbes only 37 consume low molecular weight compounds, but they also produce reactive oxygen species (ROS), which initiate Microbial incubations was were performed using a soil-derived microbial consortium as an inoculum. Soil 160 from the Austin Cary Memorial Forest (Gainesville, FL) was chosen, because this area is frequently subjected to 161 prescribed burns (Johns, 2016), and its soil microbes likely interact with pyOM and pyDOM on a regular basis. 162 Taxonomic details of its the used soil microbial characteristics have been published previously (Khodadad et al.,163 2011). The collected soil was treated to remove roots and detritus, and its water-extract was centrifuged to obtain 164 a pellet. The pellet was then dissolved in 10 mL MilliQ laboratory-grade water to obtain an inoculate, 100 μL 165 of which was used to spike 50 mL of each pyDOM substrate. Additionally, microbial nutrients (KH2PO4 and 166 (NH4)2SO4) were provided following Zimmerman (2010) to support a healthy growth medium. Samples were 167 incubated in gas-sealed amber vials on a shaker table at 28 ± 5 O C for 10 days in the dark. Using a double-needle 168 assembly, CO2-free air (Airgas, Zero) was flushed through the samples on days 0, 2, 5, and 10, which oxygenated 169 the samples and removed dissolved inorganic carbon for its measurement (, and is reported by Bostick et al.

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(2020a2021)). A procedural blank and control samples were prepared in the exact same way but were poisoned 171 with HgCl2 immediately following the mixing of the different components (pyDOM, inoculate, nutrients). 172 Additionally, a solution of sucrose (0.5 g C12H22O11 in 40 mL MilliQ laboratory-grade water) was also incubated 173 in the same manner to serve as a positive control. All incubated samples were poisoned with HgCl2 to terminate University (Norfolk, VA). The instrument is externally calibrated daily with a polyethylene glycol standard, and 215 a surrogate laboratory pyDOM standard was analyzed before and after pyDOM analysesthe analytical sequence 216 to verify for the lack of instrumental drift. Additionally, an instrumental blank of methanol was analyzed between 217 samples to verify for the absence of sample carryover. Samples were analyzed in negative ionization mode. ESI 218 spray voltages were optimized for each sample to assure for consistent spray currents among the all samples. For 219 each sample, 300 transients with a 4MWord time domain were collected, co-added, and the resultant free 220 induction decay was zero-filled and sine-bell apodized. After fast Fourier transformation, internal calibration of 221 the resultant mass spectra was performed using naturally abundant fatty acids, dicarboxylic acids, and compounds 222 belonging to the CH2-homologous series as previously described . Then, using an in-house 223 MATLAB script, salt, blank, and isotopologue ( 13 C, 37 Cl) peaks were removed. Molecular formulas within ± 1 224 ppm error were assigned to FT-ICR-MS spectral peaks (S/N ≥ 3) using the Molecular Formula Calculator from 225 the National High Magnetic Field Laboratory (Tallahassee, FL). Formula assignments were restricted to elemental 226 composition of 12 C5-∞, 1 H1-∞, 14 N0-5, 16 O0-30, 32 S0-2, 31 P0-2, and 35 Cl0-4, and were refined using previously established 227 rules (Stubbins et al., 2010). Any ambiguous peak assignments were refined by inclusion within homologous 228 series (CH2, H2, COO, CH2O, O2, H2O, NH3, HCl) following Kujawinski and Behn (2006) and Koch et al. (2007).

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For all samples, at least 80% of the mass spectral peaks were assigned, and they accounted for at least 93% of the 230 mass spectral magnitude.  Kim et al., 2003). Formulas were further categorized using the modified aromaticity index (AIMOD), a proxy 234 for the aromatic character of the associated molecules Dittmar, 2006, 2016), and calculated as shown 235 in Eq.1. 236 237  the summed projections were subtracted from all rows in the spectrum (Klevit, 1985). The same procedure was 278 performed for all columns (F2 dimension). Ultrahigh resolution mass spectrometric analysis of the bio-incubated and corresponding control pyDOM 285 leachates revealed significant changes in molecular composition after the 10-day incubation (Fig. 1). The 286 identified molecular formulas for these samples were classified into one of three groups using a presence-absence 287 approach (Stubbins et al., 2010;Sleighter et al., 2012). This approach identifies any common formulas among the 288 two samples being compared (control and bio-incubated), as well as any formulas that are unique to each sample.

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It is important to note that the electrospray ionization (ESI) source is prone to biases, and the analytical window    resulting in consistent trends between the analyses. Bio-degradability trends derived from FT-ICR-MS molecular 333 data match those from the UV-VIS data from chromophoric pyDOM ( Figure S1) revealing a similar inability of 334 UV-VIS to detect LMW compounds which do not absorb UV-VIS light. In summary, we observe a degradation 335 of a variety of different molecular classes as well as a producion of many molecules that appear to be of high 336 biological lability. However, we caution that there are observed discrepancies among carbon loss and 337 molecular/chromophoric data for the Oak 400 pyDOM systems, an observation that highlights the need to clearly 338 understand methodological analytical windows when interpreting molecular and spectroscopic data.

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Interestingly, for all leachates, the degraded ("bio-labile") molecules were not from a specific area of the 340 vK diagrams but rather represent a broad range of H/C and O/C ratios and compound types (see Fig. S2). This The bio-produced organic compounds can be evaluated in various ways to examine the processes that may have  (Table 1). No significant overlap was 388 found (2 -320 formulas in common, 0 -12%) among the molecules produced in the incubated pyDOM samples. 389 Furthermore, no significant match was found between the bio-produced formulas of incubated pyDOM and those 390 of the sucrose control sample (63 -94 formulas in common, 3%, Table 1). These observations indicate that the substrate. An alternative explanation is that bio-produced formulas or were further altered post-exudation by ROS 393 to result in their molecular diversification.    pPeptide-like microbially-produced formulas comprised 22 -40 % of the bio-produced formulas ( Table S2 in the 406 Supplement). , and tThe results of the comparative analyses described above also imply that these proteinaceous 407 formulas are of highly variable composition. Their molecular diversity is additionally evaluated using one-way  (Table 1). The results from these statistical assessments support the findings by the presence/absence 411 comparisons and Collectively, these findings collectively conclude that the microbial incubations of pyDOM 412 created pools of new, very diverse molecules, a process hereafter referred to as "microbial diversification". As 413 these N-containing molecules is that they were formed by radical processes that coupledcoupling reactions among 416 pyDOM molecules with the NH4 + nutrient that was added to support microbial growth (e.g., via Michael addition but abiotic formation was not tested in the presence of radicals.

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To confirm that these bio-produced formulas were associated with proteinaceous structures and are not 421 just N-containing compounds that coincidentally plotted in the 'peptide region', spectrofluorometric analysis was 422 performed to obtain excitation-emission matrices (EEMs) of the pyDOM samples before and after bio-incubation 423 (Fig. 2). The data for Oak 650 Photo is not reported as the produced EEM spectra were of questionable quality, 424 and as the sample was in very limited amounts, analytical validation replication and quality assessment were not

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Proteinaceous organic matter has a highly characteristic fluorophoric signature due to the distinguishable into question the idea that microbial processes were solely responsible for the high variability of the bio-produced 480 organic matter observed after the microbial incubation of pyDOM.

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In an attempt toTo further elucidate the composition of these bio-produced N-containing substances, we 482 re-evaluated the previously published 1 H NMR data of these samples (Bostick et al., 2020a2021) in greater detail.

483
Additionally, to further elucidate the connectivity between previously observed functional groups, was assessed 484 using two-dimensional 1 H-1 H total correlation NMR spectroscopy (TOCSY) was utilized on a select sample.
485 Figure 3 shows the TOCSY spectra of the bio-incubated Oak 650 Fresh sample.  There are three groups of resonances representing an alicyclic structure, a β-hydrogen to a heteroatom, 525 and a methylene group that were found in all samples, even in the controls (although of small contributions 526 relative to the total spectral signal). These resonances have not been previously observed in the 1 H NMR spectra 30 and τ = 100 ms) in order to evaluate short-range (2 -3 bond) and long-range (4 -6 bond) connectivities. Based 539 on the observed couplings the observed resonances are vicinal to each other (3 bonds away). This indicates that 540 these functional groups are closely bound in the peptidoglycan substances they likely represent.

541
All of these analyses assessments described above of the molecules observed after the biotic incubation 542 of the four pyDOM samples conclude that the observed biochemical processes in these pyDOM systems 543 incubations are complex and difficult to unambiguously interpret. Based on ourthe findings, above it is clear that 544 we summarize that these bio-produced formulas (Figure 1)   The significant degradation of pyDOM and production of these biological compounds indicates that microbes 552 successfully converted the presumably carbon-rich recalcitrant pyrogenic molecules into more labile substances, 553 a process we define as hereafter refer to as "microbial labilization". However, the fact that the observed bio-554 produced labile molecules are not identifiable as simple oligopeptides, and are present in significantly different 555 composition among the four samples, suggests that this molecular diversity may not be caused by predictable  carboxyl groups (Fig. 4).  The mathematics behind the KMD analysis (see Sect. 2.4) convert the mass of the molecular formula (also 591 known as the IUPAC mass) to a "Kendrick" mass placing the formula on a scale that is , whose which mass is on with an OH-group, resulting in the formation of alcohols (C-OH) as shown in Fig. 4c. This is likely the suggested 606 pathway of how the oxygenation products shown in Fig. 4a and 4b have formed. Evidence for such reactions will 607 be found on the KMD plots as evolution of a new molecules within the same KMD series, but with a different 608 number of oxygens. Further radical attacks would produce results in formation of polyols (Fig. 4c). In the case of 609 formation of geminal diols (two alcohol groups on the same carbon atom), they can rearrange to aldehydes or 610 ketones via keto-enol tautomerism (Fig. 4d). Further radical attacks would produce carboxyl groups, which can 611 also be radically cleaved, and pyDOM radicals be formed. PyThese DOM radicals (as well as any other radical  Using KMD analysis, formulas produced that could have been produced by oxygenation were identified 615 and plotted individually (Fig. 5). It is assumed that the smallest molecule in each series is the substrate and any 616 molecules with more increasing number of oxygens are oxygenation products.

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The number of formulas in each of these pools are shown in the legends (along with corresponding percentages).

623
The black lines indicate modified aromaticity index cutoffs (AIMOD; Dittmar, 2006, 2016).  Additional evidence for intense radical processes in these systems is the evolution of bio-produced  Table S4). These correlations suggest that the diversity of bio-degraded (bio-labile) and bio-produced  Table S4 670 of the Supplement).  The inverse relationship between the content of methanol and molecular diversity (Fig. 6) can be 692 interpreted in several ways. Firstly, methanol could be exhibiting toxicity to the microbes that assimilate pyDOM, 693 as has been observed previously (Dyrda et al., 2019). This, however, is unlikely for the pyDOM systems studied    However, if there were abundant radical reactions occurring in the system, as we suggest, it is very possible that 768 these hydrolysates were altered into unrecognizable organic structures that would still be classified as "peptide-769 like" but would have different molecular composition than the predicted linear peptide sequences. It is also 770 possible that instead of peptidoglycan hydrolysis followed by consecutive oxygenation, ROS directly cleaved the 771 peptidoglycans into smaller non-linear substances of peptide-like molecular composition.

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It must be noted that the results of our study were acquired using negative-mode ESI which is only  The observed diversity of bio-produced formulas can be explained by a scenario wherein the microbes 791 secreted labile molecules whose identities differed depending on the growth medium and/or food source, yielding 792 high variability among bio-produced formulas after the incubation of pyDOM. Additionally, it is possible that  Though not directly proven to exist in this study, many of the observed trends in FT-ICR-MS, NMR, and 802 fluorescence data suggest the presence of radicals which diversify the composition of the bio-produced formulas.

803
The observed diversity can be explained by a scenario wherein the microbes secreted labile molecules 804 whose identities differed depending on the growth medium and/or food source, yielding high variability among

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(2015) were of much higher similarity (glucose, glutamic acid, oligosaccharides and oligopeptides). Another 819 possible reason is that due to different the physiology of the soil microbes used here may be producing more 820 diverse molecules biomass than the marine microbes used by Lechtenfeld et al. (2015). It is likely that that aquatic 821 microbes have a much different degradation strategy than soil microbes. As soils are far less rich in labile 822 molecules, it is possible that soil microbes have adapted evolved to produce much higher fluxes of ROS to degrade 823 the more recalcitrant soil organic matter into consumable substrates, which can also explain the larger 824 dissimilarity in bio-produced organic molecules after the incubations of pyDOM. The observed diversity can be 825 explained by a scenario wherein the microbes secreted labile molecules whose identities differed depending on 826 the growth medium and/or food source, yielding high variability among bio-produced formulas after the 827 incubation of pyDOM. Additionally, it is possible that different microbial species (different bacteria, fungi,  An important observation using the H/C versus molecular weightMW plots (Fig. S5) was that the bio-  Clearly, the chemistry behind these microbially induced compositional changes of pyDOM is highly 875 complex, and the observed molecular diversity after these biotic incubations contrasts with previous studies.

876
These discrepancies cannot be interpreted unambiguously using the employed analytical approaches, and future 877 studies need to involve measurements of radicals and their effects, as well as various DNA sequencing and 878 "omics" approaches.

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The present study provides a detailed evaluation of the compounds that microbes degrade and produce in The observed bio-produced labile formulas in our study do not appear to be commonly observed in other 933 environmental samples. This is likely because these labile molecules An alternative idea is that the bio-produced

948
The production of these highly variable and diverse bio-produced molecules, compositionally, is likely a