Unraveling the roles of coastal bacterial consortia in degradation of various lignocellulosic substrates

ABSTRACT Lignocellulose, as the most abundant natural organic carbon on earth, plays a key role in regulating the global carbon cycle, but there have been only few studies in marine ecosystems. Little information is available about the extant lignin-degrading bacteria in coastal wetlands, limiting our understanding of their ecological roles and traits in lignocellulose degradation. We utilized in situ lignocellulose enrichment experiments coupled with 16S rRNA amplicon and shotgun metagenomics sequencing to identify and characterize bacterial consortia attributed to different lignin/lignocellulosic substrates in the southern-east intertidal zone of East China Sea. We found the consortia enriched on woody lignocellulose showed higher diversity than those on herbaceous substrate. This also revealed substrate-dependent taxonomic groups. A time-dissimilarity pattern with increased alpha diversity over time was observed. Additionally, this study identified a comprehensive set of genes associated with lignin degradation potential, containing 23 gene families involved in lignin depolymerization, and 371 gene families involved in aerobic/anaerobic lignin-derived aromatic compound pathways, challenging the traditional view of lignin recalcitrance within marine ecosystems. In contrast to similar cellulase genes among the lignocellulose substrates, significantly different ligninolytic gene groups were observed between consortia under woody and herbaceous substrates. Importantly, we not only observed synergistic degradation of lignin and hemi-/cellulose, but also pinpointed the potential biological actors at the levels of taxa and functional genes, which indicated that the alternation of aerobic and anaerobic catabolism could facilitate lignocellulose degradation. Our study advances the understanding of coastal bacterial community assembly and metabolic potential for lignocellulose substrates. IMPORTANCE It is essential for the global carbon cycle that microorganisms drive lignocellulose transformation, due to its high abundance. Previous studies were primarily constrained to terrestrial ecosystems, with limited information about the role of microbes in marine ecosystems. Through in situ lignocellulose enrichment experiment coupled with high-throughput sequencing, this study demonstrated different impacts that substrates and exposure times had on long-term bacterial community assembly and pinpointed comprehensive, yet versatile, potential decomposers at the levels of taxa and functional genes in response to different lignocellulose substrates. Moreover, the links between ligninolytic functional traits and taxonomic groups of substrate-specific populations were revealed. It showed that the synergistic effect of lignin and hemi-/cellulose degradation could enhance lignocellulose degradation under alternation of aerobic and anaerobic conditions. This study provides valuable taxonomic and genomic insights into coastal bacterial consortia for lignocellulose degradation.

The authors investigated and characterized microbial populations and functional genes attributed to different lignocellulose (hardwood, softwood, and herbaceous) and lignin substrates using in situ lignocellulose enrichment experiments coupled with 16S rRNA amplicon and shotgun metagenomics sequencing methods.The different impacts of substrates and exposure times on long-term bacterial communities and the links of ligninolytic functional traits and taxonomic groups of substrate-specific populations were revealed.Overall, the paper has been well written.However, a few technical concerns need to be addressed for publication.1.Given the relatively complex study design, I suggest authors to add a sub-figure showing how they collected samples including sample size and what techniques have been applied to the samples from different habitats.2. Only the metagenomics method was used in this study, please be aware that DNA from those dead bacterial cells can be also detected.Therefore, although this study provided many DNA-level signals from the bioinformatic analysis, it remains unknown about the gene expression or microbiome activities associated with lignin degradation.On top of this study, more validation work with meta-transcriptome or culturomics would be needed in the future.3. Any particular rationale for selecting the threshold for spearman correlation in the co-occurrence network?I saw you used different thresholds for 16S and whole metagenomics data.4. Typo or grammatical errors can be found sporadically.Please check the language issues thoroughly in the manuscript.5. Line 787, Wilcoxon rank sum test or Wilcoxon signed-rank test?Which method was used should be clearly stated as you can do the mean comparison of certain metrics between time points in this study.6.The reference format needs to be carefully checked.For example, the page number of references needs to be complemented, e.g., lines 990, 965, 855, 859, 868, 918, 938, and 948.
Reviewer #2 (Comments for the Author): Peng et al examined bacterial communities associated with the degradation of lignocellulose substrates via an in-situ enrichment experiments.The bacterial communities were explored by both amplicons and shotgun metagenomic sequencing, and the relevant functional genes associated with lignocellulose and lignin substrates were examined.They included the commercially available lignocellulose of hardwood, softwood and herbaceous.They found the consortia enriched on woody lignocellulose showed higher diversity than those on the herbaceous substrate.A time-dissimilarity pattern with increased alpha diversity over time was observed.Their findings are challenging current opinions of lignin stability in marine ecosystems by revealing a comprehensive set of genes associated with lignin degradation potential, containing 23 gene families involved in lignin depolymerization, and 371 gene families involved in aerobic/anaerobic lignin-derived aromatic compound pathways.Some results may be not surprising but are interesting considering this rarely studied transitional zone.I have only some minor comments for better clarity and hope the authors found them helpful.
More details could be provided in the experimental setups.For instance, the authors included samples from another in-situ experiments (but shorter durations).I would expect more information provided for such inclusions.Are these two experiments started at the same time, and how about the seasons covered for these two experiments?Environmental conditions are not well examined, which is necessary for an in-situ experiments.For instance, how about the importance of temperature and other local environments underlying the community composition, functional compositions, and ecological processes?Better to incorporate important results available in supplementary materials into the main figures?L29.Change to "there are only few studies in"?L43.Traditional view?L46.Better introduction of priming effects here?The readers may not expect the appearance of priming effects at the end of abstract.
L48. Change to indicates.Same for other locations in the following sections.L336.Change to "was constructed with 288 nodes".L360.Rewrite this sentence?I could not understand the "no significantly higher correlation".L392.It is interesting to reveal the network specialization.Would this be linked to other specialization analysis such as Hu et al (Nat Communs 13:3600).
L395.A brief introduction of this synergistic models are needed here.
L402.Too strong to say "should work together"?L430.Any relevant references could be cited for this cross-feeding cooperation as described here?L497.Sharp contrast?Better writing?L578.It is a bit lost here to read something relevant to "Amazon River".Better reorganization of this paragraph is expected.
L605.Rewrite as "zones to link terrestrial and marine ecosystems"?L644-647.I am not sure so much DNA could be yield with 20 mg samples.Better double check.

Comments from editor:
1) Please ensure that you have 10 or fewer supplementary figures.Some will need to be removed to proceed.
Response: Yes.We have revised this section and kept the 10 supplementary figures in the revised version.Please refer to the revised supplementary materials.

Comments from reviewers:
Reviewer comments: Reviewer #1 (Comments for the Author): 2)The authors investigated and characterized microbial populations and functional genes attributed to different lignocellulose (hardwood, softwood, and herbaceous) and lignin substrates using in situ lignocellulose enrichment experiments coupled with 16S rRNA amplicon and shotgun metagenomics sequencing methods.The different impacts of substrates and exposure times on long-term bacterial communities and the links of ligninolytic functional traits and taxonomic groups of substrate-specific populations were revealed.Overall, the paper has been well written.However, a few technical concerns need to be addressed for publication.Response: We very much thank the reviewer for their positive comments!

3) Given the relatively complex study design, I suggest authors to add a sub-figure showing how they collected samples including sample size and what techniques have been applied to the samples from different habitats.
Response: That's an excellent suggestion!We have added the figure, as Figure 1a, in the revised version to present the overview of experimental design.Now it reads (see Figure 1 and its legend).

4)
Only the metagenomics method was used in this study, please be aware that DNA from those dead bacterial cells can be also detected.Therefore, although this study provided many DNA-level signals from the bioinformatic analysis, it remains unknown about the gene expression or microbiome activities associated with lignin degradation.On top of this study, more validation work with meta-transcriptome or culturomics would be needed in the future.Response: We acknowledge this important comment by the reviewer!We also have noticed this point and are performing corresponding experiments (RNA data and culture-omics) to investigate the lignocellulose catabolic processes of coastal bacterial communities.A manuscript is now under preparation specifically for this issue.

5)
Any particular rationale for selecting the threshold for spearman correlation in the co-occurrence network?I saw you used different thresholds for 16S and whole metagenomics data.Response: Based on the literatures and empiric values, we selected the threshold of spearman correlation to maintain appropriate information in the network.We have revised it to ensure accurate communication.The text now reads (see Page 33, Line 798-807): "Only ASVs with relative abundance > 0.01% across all samples and occurring in at least 75% of all samples were selected for the network construction, and the correlations with thresholds value Spearman's | r | > 0.6 and p value < 0.05 were displayed, as previously described (1).
Metagenome samples (n=18) were used to construct functional genes co-occurrence network.Similarly, genes occurring in at ≥ 75% of all samples were chosen, and the correlations with thresholds value Spearman's | r | > 0.7 and p value < 0.05 were displayed (2).Co-occurrence networks were visualized by Cytoscape software (3)."

6)
Typo or grammatical errors can be found sporadically.Please check the language issues thoroughly in the manuscript.Response: Yes.We have thoroughly proof-read and improved English usage (including correcting non-standard expressions and grammatical errors) across the main-text and supplementary materials.

7) Line 787, Wilcoxon rank sum test or Wilcoxon signed-rank test? Which method was used should be clearly stated as you can do the mean comparison of certain metrics between time points in this study.
Response: Wilcoxon rank sum test was used in this study.We have revised it to ensure accurate understanding.The text now reads (see Page 34-35, Line 838-840): "Differences between groups were evaluated using Wilcoxon rank sum test.Those with p values of < 0.05 are considered significant."

8)
The reference format needs to be carefully checked.For example, the page number of references needs to be complemented, e.g., lines 990, 965, 855, 859, 868, 918, 938, and 948.Response: Yes.We have corrected it accordingly.Please refer to Page 37-44, Line 889-1213 in the revised version.
Reviewer #2 (Comments for the Author):

9) Peng et al examined bacterial communities associated with the degradation of lignocellulose substrates via an in-situ enrichment experiments. The bacterial communities were explored by both amplicons and shotgun metagenomic sequencing,
and the relevant functional genes associated with lignocellulose and lignin substrates were examined.They included the commercially available lignocellulose of hardwood, softwood and herbaceous.They found the consortia enriched on woody lignocellulose showed higher diversity than those on the herbaceous substrate.A time-dissimilarity pattern with increased alpha diversity over time was observed.Their findings are challenging current opinions of lignin stability in marine ecosystems by revealing a comprehensive set of genes associated with lignin degradation potential, containing 23 gene families involved in lignin depolymerization, and 371 gene families involved in aerobic/anaerobic lignin-derived aromatic compound pathways.Some results may be not surprising but are interesting considering this rarely studied transitional zone.I have only some minor comments for better clarity and hope the authors found them helpful.Response: We thank the reviewer very much for these positive comments on this work.

10) More details could be provided in the experimental setups. For instance, the authors included samples from another in-situ experiments (but shorter durations). I would expect more information provided for such inclusions. Are these two experiments started at the same time, and how about the seasons covered for these two experiments?
Response: Yes.The two experiments both covered spring and summer.We have added the detailed information to ensure accurate understanding.The text now reads (see Page 28, Line 678-681): "Beginning in April 2019, each treatment with six replicates were enriched in situ for 3-, 6-, 12-and 18-month respectively.The samples were collected in batches, kept at dry ice during transport, and stored at -80°C until further processing."(see Page 29, Line 700-712): "To explore different compositional patterns for functional genes among various lignocellulose enrichments, the DNA samples (n=12) with 6-month exposure were used for metagenomic DNA sequencing on an Illumina PE150 platform at Novogene Co., Ltd., Beijing, China, with 150 bp paired-end sequencing (Fig. 1A).Moreover, to construct functional genes co-occurrence network, another 6 samples, which were enriched in a previous in situ enrichment (6-month) study (4), were also used for metagenomic DNA sequencing.The 6 samples were enriched from April 2018 to October 2018 at the same location, with 3% rice straw powder, corn straw powder and wheat straw powder as the carbon source, respectively (4).As a result, the metagenomic DNA data sets targeting 18 samples were obtained.A total of 1,499,127,964 reads were collected, with an average of 124,927,330 reads per sample and a standard deviation of 13,119,038.All sequencing data have been deposited in the NCBI SRA database under the accession number PRJNA836095."

11) Environmental conditions are not well examined, which is necessary for an in-situ experiments. For instance, how about the importance of temperature and other local environments underlying the community composition, functional compositions, and ecological processes? Better to incorporate important results available in supplementary materials into the main figures?
Response: Yes.We have showed the result in the Figure 2

12) L29. Change to "there are only few studies in"?
Response: Yes.We have revised it accordingly.

14) L46. Better introduction of priming effects here? The readers may not expect the appearance of priming effects at the end of abstract.
Response: Yes.We have revised it to ensure understanding.The text now reads (see Page 2, Line 45-49): "Importantly, we not only observed synergistic degradation of lignin and hemi-/cellulose, but also pinpointed the potential biological actors at the levels of taxa and functional genes, which indicates that the alternation of aerobic and anaerobic catabolism could facilitate lignocellulose degradation."

15) L48. Change to indicates. Same for other locations in the following sections.
Response: Yes.We have revised it at here and across the manuscript.

16) L111. What is DO?
Response: It's dissolved oxygen.We have revised it to ensure understanding.The text now reads (see Page 5, Line 110-112): "Meanwhile, exposure time is the comprehensive factor, including multiple environmental factors (e.g., temperature, pH, salinity and dissolved oxygen (DO))."

17) L178. Wilcoxon text?
Response: Here, we used Wilcoxon rank sum test to compare richness of consortia at each time point.We have revised it to ensure understanding.The text now reads (see Page 8, Line 178-181): "On one hand, richness (the number of unique amplicon sequence variants (ASV)) significantly increased ~3.5-fold from month 3-month to 18-month (Wilcoxon rank sum test, see Fig. S1C at https://doi.org/10.5281/zenodo.7659223)."

18) L188. What is NMDS analysis?
Response: It's non-metric multidimensional scaling analysis for beta diversity.We have revised it to ensure understanding.The text now reads (see Page 9, Line 193-195): "Non-metric multidimensional scaling (NMDS) analysis showed a temporal separation of consortia across all substrates (Fig. 2B)."

19) L225-227. Rewrite this sentence for better understanding.
Response: Yes.We have rewritten it to improve the readability.The text now reads (see Page 10, Line 233-237): "Hence, Mantel test was also used to assess the effects of environmental factors, including pH, temperature, salinity and DO.These factors all significantly associated with the compositional variations of bacterial consortia (p = 0.001, Fig. 2C).Among them, temperature was the most influencing factor associating with the taxonomic composition, followed by pH, salinity and DO, respectively (Fig. 2C)." "To verify this, null model analysis was performed to investigate the relative importance of deterministic and stochastic processes in the assembly of consortia.It showed that homogenous selection (one of deterministic processes) was the most important process, accounting for 53.9%-59.2% of the community variation across all communities, followed by stochastic processes, such as drift ( In addition, relevant references have been cited in the "Methods" section for the description of null mode.Please refer to Page 32-33, Line 774-793 in the revised version.

22) L263. What kind of three tests?
Response: They are permutational multivariate analysis of variance (Adonis), analysis of similarity (ANOSIM) and multiple response permutation procedure (MRPP).We have inserted a detailed explanation to ensure understanding.The text now reads (see Page 12, Line 277-281).
"Interestingly, the compositions of gene families encoding cellulases among the four substrates were similar (p > 0.05), as indicated by the three complementary nonparametric tests, permutational multivariate analysis of variance (Adonis), analysis of similarity (ANOSIM) and multiple response permutation procedure (MRPP) (see Fig. S5

24) L336. Change to "was constructed with 288 nodes".
Response: Yes.We have revised it accordingly.Please refer to our response to Review-Comment No. 23 above.

25) L360
. Rewrite this sentence?I could not understand the "no significantly higher correlation".Response: Yes.We have revised it to improve the readability.The text now reads (see Page 16-17, Line 391-394): "Interestingly, links among aerobic ligninolytic oxidative genes weren't observed, indicating they were likely to independently depolymerize lignin, presenting an aggregation mode for lignin depolymerization."

26) L392. It is interesting to reveal the network specialization. Would this be linked to other specialization analysis such as Hu et al (Nat Communs 13:3600).
Response: That's an excellent suggestion!We have cited this literature and discussed to further reveal such specialization with other complement methods in the next step.The text now reads (see Page 26-27, Line 637-642): "Surprisingly, genes encoding cellulosomes exhibited mostly negative connections with gene encoding anaerobic aromatic compound catabolic enzymes, which is distinct with the positive interactions between genes involved in free hemi-/cellulases and aerobic aromatic compound degradation.Future studies with dissolved organic matter (DOM)-microbe associations would provide insights into such specialization (5)."

27) L395. A brief introduction of this synergistic models are needed here.
Response: Yes.We have revised it to improve the readability.The text now reads (see Page 18, Line 429-431): "Finally, integrating all of the above-stated information, a synergistic model for lignocellulosic degradation was proposed, which identified the potential taxonomic groups and functional genes (Fig. 5)."

28) L402. Too strong to say "should work together"?
Response: Yes.We have revised the statement.The text now reads (see Page 18, Line 436-439): "The interconnection at taxon and gene levels suggest that these potential degraders could work together to efficiently hydrolyze hemi-/cellulose via the aggregation and division of labor strategies (e.g., aerobic and anaerobic hydrolysis)."

29) L430. Any relevant references could be cited for this cross-feeding cooperation as described here?
Response: Yes.We have cited the literatures at here to ensure understanding.The text now reads (see Page 19, Line 465-467): "Such cross-feeding cooperation (6,7) would stimulate aromatic compound metabolism under fluctuating micro-environments."

30) L497. Sharp contrast? Better writing?
Response: Yes.We have revised the statement.The text now reads (see Page 22, Line 534-539): "Lignin substrates are highly crossed-linked aromatic heteropolymers, with complex and varied structures.However, relatively few genes in metagenomes have been predicted to be involved in lignin degradation, as exemplified by 75 gene families from the Amazon River microbiome and 13 gene families from North American forest microbiome participating in lignin degradation (8,9)."31) L578.It is a bit lost here to read something relevant to "Amazon River".Better reorganization of this paragraph is expected.Response: Yes.We have revised the paragraph.The text now reads (see Page 26, Line 620-625): "Cellulose and lignin degradation are mostly decoupled in aquatic ecosystems.For instance, cellulose and lignin degradation occurred in the different sections of Amazon River (9).In contrast, our in situ enrichment experiment not only suggested that lignin and hemi-/cellulose were co-degraded, but also revealed higher lignin degradation rates under lignocellulose substrates than that of purified lignin substrate (Fig. 1; also, see Table S2 at https://doi.org/10.5281/zenodo.7659223)."

32) L605. Rewrite as "zones to link terrestrial and marine ecosystems"?
Response: Yes.We have revised it accordingly."Approximately 20 g of each sample was collected for DNA extraction.DNA was extracted by the CTAB extraction method as previously described (10).As result, ~7-117 μg DNA was generated and preserved at -80°C for subsequent 16S rRNA amplicon sequencing and shotgun metagenomic sequencing."

35) L656-664. More information or reasonings are needed for inclusion of another experiments.
Response: Yes.We have revised this paragraph to improve the readability.Please refer to our response to Review-Comment No. 10 above.

37) L711. Should be richness, evenness.
Response: We have revised the statement.The text now reads (see Page 31, Line 759-761): "For ASV community compositions, several indices of alpha diversity were calculated, including Shannon, Richness and Evenness.Comparisons of the alpha diversity between two groups was performed with Wilcoxon rank sum test."

38) L720. Please check whether Spearman or Pearson analysis were used in the
Results section, and proper results should be reported.Response: Yes.We have revised it to improve the readability.Below are some of the examples.
Response: Yes.We have cited the literatures accordingly.The text now reads (see Page 32-33, Line 774-793):

"Null model
Phylogenetic bin-based null model analysis (iCAMP), with 16S rRNA gene sequencing data, was applied to quantify the contribution of various ecological processes to community assembly (19).It describes assembly processes, e.g., heterogeneous/ homogeneous selection, dispersal limitation, homogenizing dispersal, and drift, based on a quantitative framework (19).Briefly, the observed taxa were first divided into different phylogenetic bins based on their phylogenetic relationships.The process governing each bin was then identified based on null model analysis of phylogenetic diversity using beta Net Relatedness Index (βNRI) and taxonomic β-diversities using modified Raup-Crick metric (RC).For each bin, the fraction of pairwise comparisons with βNRI < −1.96 and > +1.96 were considered as the percentages of homogeneous and heterogeneous selection, respectively (20).Next, RC was used to partition the remaining pairwise comparisons with |βNRI| ≤ 1.96:The fraction of pairwise comparisons with RC < −0.95 and > +0.95 are treated as the percentages of homogenizing dispersal and dispersal limitation (21), and the remaining fraction, with |βNRI| ≤ 1.96 and |RC| ≤ 0.95, represented the percentages of drift (21).The above analysis was repeated for every bin, and then the fractions of individual processes across all bins were further weighted by the relative abundance of each bin and summarized to estimate the relative importance of individual processes at the whole community level (22)."40) L758-817.Please check the results relevant to qPCR and NMR are well reported in the Result section.Response: Yes.We have presented the results in the "Results" section.

41) L768. What kind of filter paper?
Response: We have revised it to ensure understanding.The text now reads (see Page 34, Line 818-819): "Approximately 8 g of each sample was mechanical disrupted, melt agar and then filtered by non-woven fabrics (30 g/m 2 , 30 x 30 cm)." Prof. Lu Lin Shandong University Qingdao China Re: mSystems01283-22R1 (Unraveling the roles of coastal bacterial consortia in degradation of various lignocellulosic substrates) Dear Prof. Lu Lin: Thank you for submitting your manuscript to mSystems.We have completed our review and I am pleased to inform you that, in principle, we expect to accept it for publication in mSystems.However, acceptance will not be final until you have adequately addressed the reviewer comments.
It is my pleasure to accept this manuscript.There are some English grammatical edits that must also be completed, could you please update the grammar and resubmit?Happy to accept once this is complete.
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Figure 1
Figure 1 in situ lignocellulose enrichment experiment.(A) Schematic representation of the experiment design.A: aspen, P: pine, NS: Norway spruce lignin, R: rice straw.(B) Lignocellulosic composition of each substrate.(C-F) Percentage of consumed lignin, cellulose and hemicellulose in each substrate by enriched bacteria consortia over time.3M-18M: different amounts of exposure time, M indicating months.Significant differences between samples are indicated by asterisks (*, p < 0.05, **, p < 0.01).
to sufficiently describe the influence of environmental factors for the community composition and ecological processes.Please refer to Page 10-11, Line 227-253 in the revised version.The Figure2and its legend now read:

Figure 2
Figure 2 The diversity and assembly mechanism of the enriched bacterial communities.(A) Shannon diversity of the bacterial taxonomic community over enrichment time.Significant differences between sampling areas are indicated by asterisks (*, p < 0.05, **, p < 0.01).(B) A non-metric multidimensional scaling (NMDS) profile of the 96 bacterial taxonomic communities based on the Bray-Curtis dissimilarities.Ellipses depict the 95% confidence interval grouping effects of exposure time, represented by different colors.Stress is included in the upper right corner.(C) The association of environmental factors and bacterial taxonomic communities.The compositional variations for taxonomic groups were analyzed.Mantel's r was calculated for association strength.(D) Dynamic changes of stochasticity and determinism during the succession of bacterial taxonomic communities.

20 )
L227.Change to suggest.Response: Yes.We have revised it accordingly.Please refer to Page 10, Line 237-238 in the revised version.21) L228.What is null model analysis?What are stochastic or deterministic processes?Proper citations or better explanations should be provided.Response: Yes.We have revised it to improve the readability.The text now reads (see Page 10-11, Line 238-245): The text now reads (see Page 27, Line 649-650): "In conclusion, coastal intertidal wetlands are important transitional zones to link terrestrial and marine ecosystems."33) L644-647.I am not sure so much DNA could be yield with 20 mg samples.Better double check.Response: We have corrected it.The text now reads (see Page 28, Line 687-690): Please check the results relevant to qPCR and NMR are well reported in the Result section.

15, Line 363-365):
at https://doi.org/10.5281/zenodo.7659223)." Response: It's Spearman's r.We have revised it.The text now reads (see Page Thank you for submitting your paper to mSystems.The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
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