In the study, the authors used art-of-the-state technologies to understand the transformation of pyrogenic dissolved organic matter by soil microbes. The authors reveal that the alternation of pyDOM was uniform and that a large portion of the bio-produced compounds is peptide-like. The results of this study can definitely improve the current understanding of the biogeochemical cycle of pyDOM.

We thank the referee for their review of our manuscript and are pleased that they see its value for improving the current understanding of the biogeochemical cycle of pyDOM.

1 Method. FT-MS.Positive or negative ESI? These two modes are suitable for acidic and basic compounds, respectively. If only one mode was used, only partial results could be obtained.

We apologize for omitting this critically important detail. Samples were analyzed in negative electrospray ionization (ESI) mode. We recognize the limitations of the use of only one mode of ionization. The manuscript will be edited accordingly to specify the ionization mode as well as to include a short discussion section in the supporting information that contextualizes how the choice of ionization mode impacts the results obtained for the present study.

2 Results 3.1 The pyDOM produced at a higher temperature is more recalcitrant than that produced at a lower temperature. The photo-irradiated pyDOM should be more labile than the fresh one. However, it was not the case in this study (as shown in Figure 1). Why? How about the results using TOC loss and CO2 respiration?

The four samples of this study behaved as expected though we avoided quantifying bio-lability in this manuscript due to the non-quantitative character of ESI-FT-ICR-MS. The bio-lability of these samples is quantified in a companion paper using carbon loss as a proxy for lability (Bostick et al., 2021). The quantitative results do follow our expectations (Figure 1 of Bostick et al., 2021): Oak 400 Photo (48% C loss) is the most bio-labile followed by Oak 400 Fresh (45% C loss), Oak 650 Photo (44% C loss), and Oak 650 Fresh (37% C loss). As the referee notes, using the number of formulas lost as a proxy for lability here in our study, Oak 400 Photo (1242 bio-labile formulas) appears less bio-labile than its non-photo-irradiated counterpart (Oak 400 Fresh, 1646). What is also surprising is that Oak 650 Photo (1410) appears to be more bio-labile than Oak 400 Photo (1242) and we expect the opposite based on quantitative carbon loss data.  Clearly, the trends observed by mass spectrometry are different.

The difference is likely due to the difference in the analytical windows of the techniques used to describe lability. From the four samples of this study, Oak 400 Photo is likely the sample with the highest number of low molecular weight (LMW) compounds. Microbes would have preferentially consumed LMW compounds before moving onto consuming larger molecules. LMW compounds are not detectable by the FT-ICR-MS either due to the analytical m/z range of 250 – 900, and/or because such molecules are generally lost during PPL extraction. We only detect acetate, formate, and methanol using proton nuclear magnetic resonance spectroscopy (Bostick et al., 2021) but there are certainly many more LMW that remain undetectable. We recognize that this has not been made clear in the manuscript and this will be corrected in the results section of the revised version of the manuscript.

Regarding CO₂ respiration, nearly all carbon (within 4%) was lost during the incubations as respired CO₂ (based on CO₂ quantification).  

3 Result. As stated by the authors, the photo-degradation of pyDOM is also very interesting; why not to compare the structure change before and after photo-irradiation in the supplement.

The photochemical changes to these pyDOM samples have been a focus of two previously published manuscripts: we report the quantitative changes in Bostick et al. (2020) and the qualitative (structural and molecular) changes are described in detail in Goranov et al. (2020). As the current manuscript and its supplement are already of significant length, we prefer to not further focus on the photochemical changes of these samples. Rather, we will clarify that their photochemical trends have been previously published. This clarification will be made in the revised version of manuscript.

4 Discussion. 4.1.1 It should be very careful to draw a conclusion on the biodegradability of pyDOM with various structures. Only polar compounds can be ionized by the ESI. Therefore, the results obtained by the ESI-FT-MS are biased. Some compounds are with high aromaticity index; they are still polar if the ESI can ionize them. If possible, more ionization modes can be tested using the same samples.

We agree with the referee that the observed trends from negative-mode ESI-FT-ICR-MS are biased by the analytical window of this technique. This will be addressed in the revised version of the manuscript and we will incorporate a higher level of caution when discussing bio-labile (molecules that appear to have degraded) or bio-produced molecules. We will edit the discussion section accordingly and provide an additional section in the supporting information describing the biases of ESI pertaining to the current study. However, the fact that we observe 1000+ formulas of significant spectral magnitude being removed suggests that the sample composition had been significantly altered after the biotic treatment. This conclusion is validated by findings from the quantitative techniques (NMR, absorbance, fluorescence, etc.) employed in this and the companion study (Bostick et al., 2021). Unfortunately, due to sample and funding limitations, we were not able to employ positive-mode ESI or other types of ionization.

References

Bostick, K.W., Zimmerman, A.R., Goranov, A.I., Mitra, S., Hatcher, P.G. and Wozniak, A.S. (2020) Photolability of pyrogenic dissolved organic matter from a thermal series of laboratory-prepared chars. Science of The Total Environment 724, 1-9.

Bostick, K.W., Zimmerman, A.R., Goranov, A.I., Mitra, S., Hatcher, P.G. and Wozniak, A.S. (2021) Biolability of Fresh and Photodegraded Pyrogenic Dissolved Organic Matter From Laboratory-Prepared Chars. Journal of Geophysical Research: Biogeosciences 126, e2020JG005981.

Goranov, A.I., Wozniak, A.S., Bostick, K.W., Zimmerman, A.R., Mitra, S. and Hatcher, P.G. (2020) Photochemistry after fire: Structural transformations of pyrogenic dissolved organic matter elucidated by advanced analytical techniques. Geochimica et Cosmochimica Acta 290, 271-292.

This work investigated the molecular changes of water extracted chars (pyDOM) during microbial degradation using FT-ICR-MS and other methods. The topic is interesting and the manuscript is well written. But I have some major comments about the discussion about the role of ROS in the transformation of pyDOM.

We thank the referee for their review of our manuscript and we are pleased that they find the topic of research interesting.

The author said this is a parallel study of the same samples (Bostick et al., 2020a), and “Over the 96-day incubation, up to 48% of the carbon was respired to CO2 following first-order kinetics,” However, this study only incubated 10 days. The DOC loss or mineralization is very important in the biodegradation of DOM, but I did not see any contens about this in results or discussions in this paper.

The quantitative losses have been described in detail in the companion paper by Bostick et al. (2021). After 10-days of bio-incubation, the following DOC losses were observed: Oak 400 Fresh: 16% loss, Oak 400 Photo: 25% loss, Oak 650 Fresh: 15% loss, Oak 650 Photo: 23% loss. We recognize that these results have not been clearly discussed in our manuscript and thank the referee for reminding us of their importance. These data will be incorporated in the results and discussion sections of the revised manuscript.

My biggest concern: The results and discussions about “Radical oxygenation as a potential source of molecular diversity” contained too many over-interpretations. Only the results of FT-ICR MS cannot support the obtained conclusions. (1) no data about the detection of ROS were present in this study. In addition, the control experiment by addition of ROS inhibitors during incubation was lacking. (2) the conclusions like “the bio-produced formulas could be classified as products of oxygenation reactions, likely driven by ROS species such as the hydroxyl radical (•OH)” obtained by the KMD analysis using oxygen (O) series (eg. Figure 4) are severe over-interpretation of the FT-ICR MS data. There is no evidence to support that C_cH_hO_o+1 is produced from C_cH_hO_o via oxygenation by hydroxyl radical (•OH) attacks. Combined (1) and (2), no evidence support the conclusions about the pathway of radical oxygenation of pyDOM.

 We agree with the referee that our results here are not definitive. During the experimental design of our study, we did not hypothesize that radicals could play an important part in the bio-incubations we were going to perform and thus, we did not prepare any controls to include ROS quenchers to specifically test for radical reactions. As this is the first study to incubate pyDOM with microbes, our overarching study hypothesis had to be broad: pyDOM will be degraded/transformed by microbes (H₁) vs pyDOM will not be degraded/transformed by microbes (H₀). Thus, our control samples were designed to allow for testing this hypothesis.

We strongly disagree that FT-ICR-MS data cannot be used to imply radical oxygenation. Waggoner et al. (2015) performed lab-controlled Fenton reactions (producing hydroxyl radical species) on a lignin concentrate. In a sequential study, the same lignin concentrate was fractionated using HPLC and then exposed to hydroxyl radicals (Waggoner and Hatcher, 2017). Later, singlet oxygen (¹O₂) and superoxide (O₂^-•) were also added to the lignin concentrate in lab-controlled conditions. These three studies showed that C_cH_hO_o+1 are indeed produced from C_cH_hO_o molecules via radical oxygenation. As the same trends can be seen in our data, it is reasonable to speculate that the observed molecular changes to pyDOM potentially result from radical oxygenation. Similar trends have been also observed in fungal incubations (Khatami et al., 2019). We recognize that this evaluation is not definitive and have provided an alternative in which the observed C_cH_hO_o+1 oxygen series are biologically unrelated to C_cH_hO_o and the observed oxygen series are coincidental:

• Lines 696-700: The observed diversity can be explained by a scenario wherein the microbes secreted labile molecules whose identities differed depending on the growth medium and/or food source, yielding high variability among bio-produced formulas after the incubation of pyDOM. Additionally, it is possible that different microbial species (different bacteria, fungi, archaea, etc.) have proliferated in response to the sample-specific pyDOM composition, yielding different microbial populations growing during each different incubation, sequentially producing different bio-produced compounds (Fitch et al., 2018).

Because we have no evidence from DOC loss or other quantitative measurements proving that there were radical oxygenation reactions in these pyDOM systems, the proposed radical oxygenation pathways were labeled as a potential source of molecular diversity. This will be emphasized in the revised version of the manuscript and we will describe in better detail the two explanations of observed molecular diversity and C_cH_hO_o+1 oxygen series. We will also recommend that future studies perform specific experiments to test for radical reactions.

m was converted to square, eg. line 150, line 469

We thank the referee for spotting this. We are unsure why the Greek symbols on lines 150 (μL) and 469 (β-hydrogens) were formatted into squares. These will be corrected in the revised manuscript.

Figure 1: Present bio-resistant formulas in Figure 1?

Unfortunately, when bio-resistant formulas are plotted in Figure 1, the figure becomes cluttered and the trends are very difficult to see. To overcome this, we tried using several different color schemes, marker sizes and shapes. No figure that had the bio-resistant formulas present was visually appealing. Furthermore, the bio-resistant formulas are not important for understanding the major findings in our study. Thus, if bio-resistant formulas are plotted on Figure 1 they will likely distract the readers from the main trends in the data. Therefore, we prefer to keep the bio-resistant formulas shown on Figures S3 and S7.

References

Bostick, K.W., Zimmerman, A.R., Goranov, A.I., Mitra, S., Hatcher, P.G. and Wozniak, A.S. (2021) Biolability of Fresh and Photodegraded Pyrogenic Dissolved Organic Matter From Laboratory-Prepared Chars. Journal of Geophysical Research: Biogeosciences 126, e2020JG005981.

Fitch, A., Orland, C., Willer, D., Emilson, E.J.S. and Tanentzap, A.J. (2018) Feasting on terrestrial organic matter: Dining in a dark lake changes microbial decomposition. Global change biology 24, 5110-5122.

Khatami, S., Deng, Y., Tien, M. and Hatcher, P.G. (2019) Formation of water-soluble organic matter through fungal degradation of lignin. Organic Geochemistry 135, 64-70.

Waggoner, D.C., Chen, H., Willoughby, A.S. and Hatcher, P.G. (2015) Formation of black carbon-like and alicyclic aliphatic compounds by hydroxyl radical initiated degradation of lignin. Organic Geochemistry 82, 69-76.

Waggoner, D.C. and Hatcher, P.G. (2017) Hydroxyl radical alteration of HPLC fractionated lignin: Formation of new compounds from terrestrial organic matter. Organic Geochemistry 113, 315-325.