the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 03 Feb 2025
Anaerobic biodegradation of Miocene lignites from an opencast mine by autochthonous microorganisms stimulated under laboratory conditions
Abstract. The supplementation and provision of appropriate nutrients to microorganisms, which are often lacking in the natural environment are essential and critical for microbial growth. One such element is nitrogen, most of which is found in the Earth's atmosphere. In this study, we present evidence of nitrogen processing and anaerobic N2-fixation by microorganisms naturally present in sedimentary organic matter. Miocene detritic lignite from the opencast mine was incubated under anaerobic conditions in the dark (headspace atmosphere 85 % N2, 10 % CO2, 5 % H2) for three years. The natural microbial community of these coal materials was stimulated for growth through the addition of trace elements, vitamins, and carbon-bearing compounds such as yeast extract, nutrient broth, methanol, and sodium acetate. A visual indicator of microbial activity was observed as the color of the fermentation solutions changed over time: from colorless to light yellow (after 3 months), dark brown (after 6 months), and finally black (after more than 1 year). This progression suggests the dissolution of fulvic and humic acids. At the end of the cultivation period, the total nitrogen (TN) and total inorganic nitrogen (TIN) contents in the solutions were significantly reduced whereas in incubations with sodium acetate, total organic nitrogen (TON) content significantly increased compared to the initial levels. In most cases, total carbon (TC) content significantly increased due to biodegradation, except for the incubations where methanol was added. A GC-MS analysis of the total extracts from lignite revealed that the main macromolecule decomposed by microorganisms was lignin, along with its diagenetic derivatives. The biogas released during the process contained CO2 and trace amounts of CH4 (up to 50 ppm). Isotopic data indicated the occurrence of anaerobic CH4 oxidation. Notably, 16S rRNA gene sequencing identified the presence of N2-fixing microorganisms in all investigated samples, members of the order Rhizobiales (families Beijerinckiaceae, Rhizobiaceae). Our findings demonstrate that N2-fixation may play a pivotal role in coal decomposition under anaerobic conditions.
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RC1: 'Comment on egusphere-2025-236', Anonymous Referee #1, 20 Mar 2025
The manuscript by Bucha et al. described lignite degradation in a microcosm supplemented by nutrients. The authors set 12 conditions of incubations (each two replicates) that varied in nutrient additions and compared the difference in gaseous components and in-liquid carbon and nitrogen species in the incubations. The key point in this study was the comparison among different incubation conditions to find factors affecting the anaerobic degradation of lignite. Thus, the statistically validated comparisons of incubations are critically necessary. Unfortunately, the presented results lack the statistical significance necessary to draw valid conclusions from the experiments. The only "standard deviation" I could find in this manuscript was in line 142, which seemed to be standard deviations of analytical standard measurements. The authors described placing "around 10g" of lignite samples in their vials, which indicated an uncertainty in the precision of replicates in preparation for the incubation. In addition, the natural heterogeneity of the used lignite should add another layer of the experimental error. Even with this situation, the authors did not show any measurements on any of the replications. I do not understand why the authors prepared only two replicates, which cannot give a good standard deviation and, thus, statistical significance. Therefore, I was not entirely convinced by the authors' claim that the data variation was caused by the incubation conditions. Clear statistical validity is needed to exclude the possibility of an error in the experimental setup as a cause of the variation. As written above, this is critical to assess the validity of the experimental data shown and needs to be carefully addressed.
Specific comments;
- 142: What standard deviation did the authors mention here? Is it the standard deviation of repeated analysis of the same standard? Or analysis of replicates of the standard? Clarification is necessary.
- 187: Have the authors checked the cell extraction recovery? What gravity force the 120 rpm centrifugation, and why was 250xg centrifugation added to it?
- 263-266: Methane concentration of M9 only condition was 37.2, and 47.2 was for MG.
- 266-267: Is there any possibility of just a release of coal-bearing methane?
- 288 and Table 3: Is T1 3 years time point? What does "END" mean?
- 341: What denote the "1" for each condition?
Citation: https://doi.org/10.5194/egusphere-2025-236-RC1 -
AC1: 'Reply on RC1', Michal Bucha, 27 Jun 2025
We sincerely thank you for your review of our manuscript. We greatly appreciate your critical insights, which help us clarify the scope and limitations of our study.
We agree that statistical analysis for our research could significantly improve the quality of data discussion and interpretation.
However, our study was focused on finding similar tendencies rather than differences among cultivations with different carbon sources. Such carbon sources as yeast extract, nutrient broth methanol and sodium acetate are often used for stimulation of microorganisms from lignite and other raw coal materials. Our intention was to study the common products of lignite biodegradation. Specific products typical for lignite incubation with addition of different carbon sources are another topic, which was not the aim of this study.
In the revised version of the manuscript, we will underline the major goals more precisely and adjust the discussion accordingly to reflect this scope. As one of the main conclusions, we identified groups of organic compounds (Table 5, mean content for all tested samples) that are typical for coal decomposition processes regardless of the applied carbon-bearing additive and trace solution. While microbiological data is difficult to obtain, we see that further research should take a closer look at the nitrogen cycle in coal-rich sediments.
We appreciate your comment regarding the natural heterogeneity of lignite and the potential impact on experimental variability. We would like to clarify that no mechanical processing (e.g., grinding or homogenization) was applied to the lignite samples. This decision was intentional: any physical manipulation of the lignite could have negatively affected the indigenous microbial communities, which were central to our investigation. Moreover, such processing could introduce external contamination or alter the native microenvironment, compromising the biological relevance of the microcosms. Therefore, to preserve the integrity of the microbial assemblages, we used the lignite in its natural, unprocessed form. This is a difficult approach and is closer to natural processes, but at the same time more difficult for interpretation.
The phrase “approximately 10 g” used in the methods section was intended to reflect the minor deviations due to the natural moisture present in lignite sample. Unfortunately, we could not dry the lignite material before the experiment because it would affect the microorganisms living inside the samples. In practice, all samples were carefully weighed, and deviations did not exceed ±0.2 g. We will clarify this point in the revised manuscript to avoid any misunderstanding regarding sample preparation precision.
Once again, we are grateful for your valuable feedback. Your comments have helped us identify key areas for clarification and improvement in our manuscript. We hope that the planned revisions will address your concerns and contribute to a clearer presentation of our exploratory findings.
Specific comments
1. 142: What standard deviation did the authors mention here? Is it the standard deviation of repeated analysis of the same standard? Or analysis of replicates of the standard? Clarification is necessary.
Thank you for this comment. The presented SD are replicates of the standards (5 replicates for each).
2. 187: Have the authors checked the cell extraction recovery? What gravity force the 120 rpm centrifugation, and why was 250xg centrifugation added to it?
Thank you for raising interesting issues. The cell recovery has not been checked as this would be extremely difficult and unreliable or impossible at all. Initially the DNA isolation from lignite directly was attempted but did not succeed. It can be assumed that the reasons for that were 1) relatively low abundance of cells in the lignite, and 2) high content of humic substances that were released during the isolation which would interfere with the process and downstream analysis. Therefore attempt similar to this from other environments with expected low relative abundance of the microorganisms has been applied (e.g. Schulz-Makuch, et al. 2018). Extraction of cells allowed to process large (relatively) amount of initial material and by that increase the cell amount per volume of the sample used for DNA isolation – making it possible to isolate enough material for downstream analysis while reducing the amount of humic substances and other potential inhibitors. Checking the cell recovery rate would be tricky as culture-based approach would not be reliable. Majority of environmental microorganisms cannot be cultivated in laboratory conditions. And reviewing the whole material, 5 g per repeat in this case, would not be feasible at all. Furthermore, there is no method that would allow to do it with certainty – no microscopic methods and flow cytometry/molecular methods which would require some type of cell extraction again. Therefore, we cannot be certain that all the cells were extracted, although this is a type of uncertainty that is connected with most environmental studies based on nucleic acids extraction, along e.g. cell lysis efficiency, binding of DNA to external substrate material after lysis etc. As the downstream analysis is based on the relative abundance of the microorganism taxa, even not complete extraction of all cells should not interfere with obtained data. Additionally, during bioinformatic analysis rarefaction curves are generated which are sampling if with increased depth of sequencing (number of reads) the number of detected ASVs increases. The curves reached plateau, which means the further increase will not result in detection of new unique sequences. That together with relative character of data analysis make any cells not extracted from lignite neglectable for overall results.
The speed of 120 rpm has been used for rocking of the tubes on the rotatory shaker and mixing material with cell extraction buffer to increase the extraction efficiency. In this case the tube was attached in parallel position to the shaking table surface and rotation of the table was applied, as this was not a centrifugation, the multiplication of gravitational force did not take place.
The centrifugation at 250xg was applied after shaking to remove larger chunks of lignite while leaving the cells in suspension, which allowed to separate one from another. The process was repeated three times (once with Ringer’s solution supplemented with 0.5% Tween and twice with cell extraction buffer) to maximize efficiency of cell extraction from the lignite. It is important to notice that in case of lignite samples, still large number of finer particles were present in solution, which if cell extraction from the solid material was not completely efficient should further contribute to the increase in the cell amount in samples used for downstream processing.3. 263-266: Methane concentration of M9 only condition was 37.2, and 47.2 was for MG.
Thank you for this comment. The highest concentration 47.2 ppm of CH4 was for MG (which contained M9 and trace solution). This mistake will be corrected in the revised manuscript.4. 266-267: Is there any possibility of just a release of coal-bearing methane?
Thank you for this comment. Of course, we always allow for such a possibility, but previous studies in which we used lignite collected from an opencast mine from layers near the surface never showed enrichment in methane, even in trace amounts. In the working conditions in the Konin mine, where the samples come from, gas appeared in the past only in places of water outflow (most often it was hydrogen sulphide, which is an inhibitor of methanogenesis via the CO2 reduction path) or in piezometers collecting groundwater. In near-surface conditions, where lignite deposits have been in contact with atmospheric air and rainwater for years, the original methane would certainly have been consumed, especially since these sediments are of Middle Miocene age, i.e. between 15-11 million years old.5. 288 and Table 3: Is T1 3 years time point? What does "END" mean?
Thank you for the comment. This is end of incubation – 3rd year. We will add this information in the revised manuscript.6. 341: What denote the "1" for each condition?
Thank you for this comment. The correct names are MG, MGC, MGD and MGE. The denote “1” will be deleted in the revised manuscript.Literature:
Bucha, M., Detman, A., Pleśniak, Ł., Drzewicki, W., Kufka, D., Chojnacka, A.,Mielecki, D., Krajniak, J., Jędrysek, M.O., Sikora, A., Marynowski, L., 2020: Microbial methane formation from different lithotypes of Miocene lignites from the Konin Basin, Poland: Geochemistry of the gases and composition of the microbial communities. International Journal of Coal Geology; 229, 103558.Bucha, M., Jędrysek, M.O., Kufka, D., Pleśniak, Ł., Marynowski, L., Kubiak, K., Błaszczyk, M., 2018: Methanogenic fermentation of lignite with carbon-bearing additives , inferred from stable carbon and hydrogen isotopes. International Journal of Coal Geology; 186:65–79.
Detman, A., Bucha, M., Simoneit, B.R.T., Mielecki, D., Piwowarczyk, C., Chojnacka, A., Błaszczyk, M.K., Jędrysek, M.O., Marynowski, L., Sikora, A., 2018: Lignite biodegradation under conditions of acidic molasses fermentation. International Journal of Coal Geology; 196:274-287.
Schulze-Makuch, D., Crawford, I.A., 2018: Was there an early habitability window for Earth's Moon? Astrobiology; 18(8):985-988.
Citation: https://doi.org/10.5194/egusphere-2025-236-AC1
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RC2: 'Comment on egusphere-2025-236', Anonymous Referee #2, 03 May 2025
In this study, Michał Bucha et al. demonstrated the nitrogen fixation capacity of autologous microorganisms in lignite by long-term anaerobic culture in a reactor and explored the nitrogen fixation mechanism by visual analysis to determine whether the microorganisms degrade organic matter, biogas and isotope analysis of the gas changes at the beginning and end of the reactor, fermentation broth analysis of different nitrogen forms in the liquid in the reactor, and GC-MS determination of the molecular composition changes of detrisulous lignite organic matter (OM). There are a few things to be aware of before publication.
- 1. Lines 80-85, unify paragraph spacing and improve the clarity of charts.
- 2. Lines135, recommend the process and timing of the addition of complementary isotopes to materials and methods.
- 3. Lines105-110, recommend supplementing the reasons for setting up experiments with different groups and adding trace mineral solutions.
- Lines250 color change as visual evidence of microbial activity, which needs to exclude the interference of abiotic factors such as lignite self-oxidation or chemical dissolution, can supplement data such as enzyme activity to prove that color change is directly related to microbial metabolism.
- Lines445, why may TON in fermentation broth contain 27.7 to 91.5mg/L lignin-degrading organic compounds?
- The discussion focused on the nitrogen fixation of Rhizobiales, and did not deeply analyze the potential contribution of other coexisting microorganisms (e.g., Burkholderiales, Pseudomonadaceae) to the nitrogen cycle, nor did it explore the possible influence of carbon sources (e.g., methanol, sodium acetate) on metabolic pathway selection.
- Lines 580, adjusts the format of references, adds their DO numbers or page numbers, and Lines700 pays attention to the whitespace of words.
Citation: https://doi.org/10.5194/egusphere-2025-236-RC2 -
AC2: 'Reply on RC2', Michal Bucha, 27 Jun 2025
Thank you very much for your positive evaluation of our study and all the comments which help us to further improve this work. We have addressed all the critical comments and missed information.
1. Lines 80-85, unify paragraph spacing and improve the clarity of charts.
The text will be proofread, and graphics (e.g. Figure 2A and Figure 2B) will be corrected in the revised manuscript.
2. Lines 135, recommend the process and timing of the addition of complementary isotopes to materials and methods.
Thank you for the comment. We did not add complementary isotopically labeled tracers (e.g., ¹³C or ¹⁵N). Instead, we measured natural abundance δ¹³C values of CH₄ and CO₂ from headspace gases at the end of incubation - to study microbial activity. Biogas samples (5 ml) were taken from bacterial cultures using a syringe and transferred to a 2 L Tedlar bag filled with nitrogen. This was to dilute the concentrations of the gases being analyzed (particularly CO2 to concentrations in the near atmospheric range). The information regarding specific conditions of measurements will be added in the revised manuscript.3. Lines 105-110, recommend supplementing the reasons for setting up experiments with different groups and adding trace mineral solutions.
Additives were used to stimulate the growth of microorganisms, which is not so obvious in the case of lignite cultivation without prior preparation of bacterial inoculum. The additives used are a source of carbon easily metabolized by microorganisms. Lignite is a raw material poor in nutrients compared to, for example, modern plant material. Therefore, the addition of a trace mineral solution was to provide access to vitamins and elements for microorganisms. We will include this information in the revised manuscript.
4. Lines 250 color change as visual evidence of microbial activity, which needs to exclude the interference of abiotic factors such as lignite self-oxidation or chemical dissolution, can supplement data such as enzyme activity to prove that color change is directly related to microbial metabolism.
From our previous works and experiments we know that dissolution of organic compounds from lignite concerns fulvic acids and occurs without the participation of microorganisms. The change in the color of the solution is then from colorless to light yellow at neutral pH. It is also important to note that lignite is specific, and difficult raw material for microbial studies. Abiotic, negative control of lignite incubation is basically impossible to perform, because, for example, autoclaving the lignite material will cause its chemical decomposition (extraction in an aqueous solution) - such autoclaving leads to the dissolution of even more organic acids. We will add this information in the revised manuscript.
5. Lines 445, why may TON in fermentation broth contain 27.7 to 91.5mg/L lignin-degrading organic compounds?
Thank you for this comment. This is a mistake. Assuming maximal amount of gaseous N2 in the liquid which equaled 20 mg/L, the TON values from lignin degradation products are in the range from 3.0 to 71.5 mg/L (mean value 30.8 mg/L). These values will be corrected in the revised manuscript.
6. The discussion focused on the nitrogen fixation of Rhizobiales, and did not deeply analyze the potential contribution of other coexisting microorganisms (e.g., Burkholderiales, Pseudomonadaceae) to the nitrogen cycle, nor did it explore the possible influence of carbon sources (e.g., methanol, sodium acetate) on metabolic pathway selection.
We acknowledge this limitation. Although other taxa (e.g., Burkholderiales, Pseudomonadaceae) was detected, our focus was on Rhizobiales due to their consistent presence and N₂-fixation role. A more detailed microbial pathway analysis, particularly focused on carbon source influence can be a futuristic approach to our study. A full answer to this question would require repeating the experiment and measuring the enzymatic activity periodically, in planned and narrow time intervals. Measurement after a year or two might not capture the activity of some enzymes, e.g. bacterial laccase. Stimulation by adding various carbon-containing additives may in principle lead to the preferential growth of specific microorganisms, but in our work, we focus on finding common features rather than discussing the influence of these ingredients on the microbial community.7. Lines 580, adjusts the format of references, adds their DO numbers or page numbers, and Lines 700 pays attention to the whitespace of words.
Thank you for the comment. The reference list was prepared using Mendeley software and manually corrected when necessary. All missed mistakes will be corrected in the revised manuscript.Citation: https://doi.org/10.5194/egusphere-2025-236-AC2
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