the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 06 Jan 2026
Suspended sediment transport modulated by microbial activities in estuarine waters: Insights from molecular and structural perspectives
Abstract. Suspended sediment transport in coastal estuaries is profoundly shaped by microbial activities, yet the underlying molecular mechanisms remain poorly constrained during their flocculation. Here, we demonstrate that the estuarine bacterium Stutzerimonas decontaminans acts as a key mediator of sediment flocculation. Compared to purely physical aggregation, microbially-induced flocculation developed more slowly but yielded flocs fourfold larger, with looser fractal structures and higher organic carbon content, indicating strong microbial-mineral coupling. Bacteria modulated flocculation both physically via flagella-driven adhesion and biochemically through extracellular polymeric substances, which enhanced particulate organic carbon accumulation. Transcriptomic analyses revealed an early upregulation of flagellar genes initiating particle adhesion, followed by the activation of polysaccharide biosynthesis pathways to stabilize aggregates. This sequential regulation highlights a genetic trade-off between motility and biofilm-like stickiness in controlling floc growth. Our findings provide direct molecular and structural evidence that microbial activities fundamentally reshape sediment aggregation dynamics, thereby regulating suspended sediment transport and carbon cycling in coastal systems.
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RC1: 'Comment on egusphere-2025-6516', Anonymous Referee #1, 24 Feb 2026
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AC1: 'Reply on RC1', Leiping Ye, 04 Mar 2026
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Thanks a lot for the valuable and very helpful comments and we do really appreciate.
We totally agree with the reviewer that most living microorganisms can participate in estuarine flocculation processes through the secretion of TEP or EPS which needs to be added and combined in the comparison and discussions. Here in our this work, we originally and primarily focused on microbial EPS which used to be more difficult to quantified and an extension from the living microorganisms function on flucculation. We very much appreciate your suggestion to broaden the perspective. Following your advice, we have now carefully reviewed the relevant literature and have cited the following works to support this point in the References section (Page 17, 18, 19, 21, 23, 25 in the revision):
Deng, Z., He, Q., Chassagne, C., & Wang, Z. B. (2021). Seasonal variation of floc population influenced by the presence of algae in the changjiang (yangtze river) estuary. Marine Geology, 440, 106600. https://doi.org/10.1016/j.margeo.2021.106600
Deng, Z., He, Q., Safar, Z., & Chassagne, C. (2019). The role of algae in fine sediment flocculation: In-situ and laboratory measurements. Marine Geology, 413, 71～84. https://doi.org/10.1016/j.margeo.2019.02.003
Bar-Zeev, E., & Rahav, E. (2015). Microbial metabolism of transparent exopolymer particles during the summer months along a eutrophic estuary system. Frontiers in Microbiology, 6, 403. https://doi.org/10.3389/fmicb.2015.00403
Lin, Y., Ye, L., Li, C., Cui, Y., & Wu, J. (2024). Morphology and distribution of suspended particles during typhoon-induced algal bloom in the pearl river estuary. (11), 1499002. https://doi.org/10.3389/fmars.2024.1499002
Salehizadeh, H., & Yan, N. (2014). Recent advances in extracellular biopolymer flocculants. Biotechnology Advances, 32(8), 1506-1522. https://doi.org/10.1016/j.biotechadv.2014.10.004
Sheng, G.-P., Yu, H.-Q., & Li, X.-Y. (2010). Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnology Advances, 28(6), 882-894. https://doi.org/10.1016/j.biotechadv.2010.08.001
Ye, F., Ye, Y., & Li, Y. (2011). Effect of C/N ratio on extracellular polymeric substances (EPS) and physicochemical properties of activated sludge flocs. Journal of Hazardous Materials, 188(1-3), 37-43. https://doi.org/10.1016/j.jhazmat.2011.01.043
Zhang, Y., Ren, Jie, & Zhang, Wenyan. (2020). Flocculation under the control of shear, concentration and stratification during tidal cycles. (586), 124908. https://doi.org/10.1016/j.jhydrol.2020.124908
Also, in the manuscript, we have added in a specific explanation for this issue in the 2nd paragraph of the Discussion section (Page 13, Line 374), i.e. ‘Microbial flocculation is strongly modulated by bacterial growth phase, hydrodynamic shear, and nutrient availability (Sheng et al., 2010; Ye et al., 2011; Salehizadeh & Yan, 2014; Tang & Maggi, 2016). For instance, Maalej et al. (2017) reported that in Pseudomonas stutzeri (now reclassified as Stutzerimonas stutzeri), EPS production and viscosity increased concurrently during exponential growth, followed by a decline upon starch depletion’.
We also have incorporated the relevant studies from both field in situ and laboratory research to support this point in the 3rd paragraph the Discussion section (Page 13, Line 386). The current logic of the Discussion section is as following: "However, the extremely swift (a few minutes) biologically-induced flocculation in estuarine waters as a result of Transparent Exopolymer Particles (mucus) secreted not just by microbes, but also bacteria and plankton and other biota (Droppo, 2001; Passow, 2002; Labille et al., 2005; Morelle et al., 2017; Mari et al., 2017). In eutrophic estuarine environments, high phytoplankton biomass drives TEP production, which fuels microbial growth and accelerates the transformation of TEP into biologically-induced aggregates (Bar-Zeev & Rahav, 2015). Eukaryotic microorganisms, especially diatoms, often bloom in estuarine waters and flocculate rapidly (within minutes), forming visible macroaggregates (Deng et al., 2019, 2021, 2023; Ho et al., 2022). Our previous work in the Pearl River Estuary showed that tidally modulated floc sizes typically range from 20 to 200 μm (Zhang et al., 2020). In contrast, typhoon-induced algal blooms enhanced bioflocculation by a factor of 1 to 8 compared to this study, yielding flocs as large as 118–920 μm (Lin et al., 2024). In addition, the composition and quantity of EPS secreted by microorganisms differ among species (Flemming & Wingender, 2010), which in turn modulate flocculation efficiency."
Specifically, we acknowledge that our controlled laboratory conditions cannot fully replicate the complexity of natural estuarine environments. We also explicitly recommend that future research should integrate field observations with laboratory experiments to more comprehensively evaluate the real-world implications of bio-flocculation (Page 16, Line 474). The current logic of the Discussion section is as following: "Given that the bioflocculation observed in laboratory settings may not fully capture those occurring in natural environments, future studies should aim to bridge this gap through in situ validation experiments or by incorporating more complex, multi-species communities into mesocosm designs. Specifically, combining controlled experiments with field measurements of TEP/EPS concentrations, floc size distributions, and settling velocities across estuarine gradients would help validate the conceptual framework proposed here."
We hope the revision and improvement will strengthen the manuscript and satisfactory. A full revision will be formally uploaded after all the other potential reviewers' comments been addressed. Thank you again for your constructive feedback.
Citation: https://doi.org/10.5194/egusphere-2025-6516-AC1 -
AC2: 'Reply on RC1', Leiping Ye, 04 Mar 2026
reply
Thanks a lot for the valuable and very helpful comments and we do really appreciate.
We totally agree with the reviewer that most living microorganisms can participate in estuarine flocculation processes through the secretion of TEP or EPS which needs to be added and combined in the comparison and discussions. Here in our this work, we originally and primarily focused on microbial EPS which used to be more difficult to quantified and an extension from the living microorganisms function on flucculation. We very much appreciate your suggestion to broaden the perspective. Following your advice, we have now carefully reviewed the relevant literature and have cited the following works to support this point in the References section (Page 17, 18, 19, 21, 23, 25 in the revision):
Deng, Z., He, Q., Chassagne, C., & Wang, Z. B. (2021). Seasonal variation of floc population influenced by the presence of algae in the changjiang (yangtze river) estuary. Marine Geology, 440, 106600. https://doi.org/10.1016/j.margeo.2021.106600
Deng, Z., He, Q., Safar, Z., & Chassagne, C. (2019). The role of algae in fine sediment flocculation: In-situ and laboratory measurements. Marine Geology, 413, 71～84. https://doi.org/10.1016/j.margeo.2019.02.003
Bar-Zeev, E., & Rahav, E. (2015). Microbial metabolism of transparent exopolymer particles during the summer months along a eutrophic estuary system. Frontiers in Microbiology, 6, 403. https://doi.org/10.3389/fmicb.2015.00403
Lin, Y., Ye, L., Li, C., Cui, Y., & Wu, J. (2024). Morphology and distribution of suspended particles during typhoon-induced algal bloom in the pearl river estuary. (11), 1499002. https://doi.org/10.3389/fmars.2024.1499002
Salehizadeh, H., & Yan, N. (2014). Recent advances in extracellular biopolymer flocculants. Biotechnology Advances, 32(8), 1506-1522. https://doi.org/10.1016/j.biotechadv.2014.10.004
Sheng, G.-P., Yu, H.-Q., & Li, X.-Y. (2010). Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnology Advances, 28(6), 882-894. https://doi.org/10.1016/j.biotechadv.2010.08.001
Ye, F., Ye, Y., & Li, Y. (2011). Effect of C/N ratio on extracellular polymeric substances (EPS) and physicochemical properties of activated sludge flocs. Journal of Hazardous Materials, 188(1-3), 37-43. https://doi.org/10.1016/j.jhazmat.2011.01.043
Zhang, Y., Ren, Jie, & Zhang, Wenyan. (2020). Flocculation under the control of shear, concentration and stratification during tidal cycles. (586), 124908. https://doi.org/10.1016/j.jhydrol.2020.124908
Also, in the manuscript, we have added in a specific explanation for this issue in the 2nd paragraph of the Discussion section (Page 13, Line 374), i.e. ‘Microbial flocculation is strongly modulated by bacterial growth phase, hydrodynamic shear, and nutrient availability (Sheng et al., 2010; Ye et al., 2011; Salehizadeh & Yan, 2014; Tang & Maggi, 2016). For instance, Maalej et al. (2017) reported that in Pseudomonas stutzeri (now reclassified as Stutzerimonas stutzeri), EPS production and viscosity increased concurrently during exponential growth, followed by a decline upon starch depletion’.
We also have incorporated the relevant studies from both field in situ and laboratory research to support this point in the 3rd paragraph the Discussion section (Page 13, Line 386). The current logic of the Discussion section is as following: "However, the extremely swift (a few minutes) biologically-induced flocculation in estuarine waters as a result of Transparent Exopolymer Particles (mucus) secreted not just by microbes, but also bacteria and plankton and other biota (Droppo, 2001; Passow, 2002; Labille et al., 2005; Morelle et al., 2017; Mari et al., 2017). In eutrophic estuarine environments, high phytoplankton biomass drives TEP production, which fuels microbial growth and accelerates the transformation of TEP into biologically-induced aggregates (Bar-Zeev & Rahav, 2015). Eukaryotic microorganisms, especially diatoms, often bloom in estuarine waters and flocculate rapidly (within minutes), forming visible macroaggregates (Deng et al., 2019, 2021, 2023; Ho et al., 2022). Our previous work in the Pearl River Estuary showed that tidally modulated floc sizes typically range from 20 to 200 μm (Zhang et al., 2020). In contrast, typhoon-induced algal blooms enhanced bioflocculation by a factor of 1 to 8 compared to this study, yielding flocs as large as 118–920 μm (Lin et al., 2024). In addition, the composition and quantity of EPS secreted by microorganisms differ among species (Flemming & Wingender, 2010), which in turn modulate flocculation efficiency."
Specifically, we acknowledge that our controlled laboratory conditions cannot fully replicate the complexity of natural estuarine environments. We also explicitly recommend that future research should integrate field observations with laboratory experiments to more comprehensively evaluate the real-world implications of bio-flocculation (Page 16, Line 474). The current logic of the Discussion section is as following: "Given that the bioflocculation observed in laboratory settings may not fully capture those occurring in natural environments, future studies should aim to bridge this gap through in situ validation experiments or by incorporating more complex, multi-species communities into mesocosm designs. Specifically, combining controlled experiments with field measurements of TEP/EPS concentrations, floc size distributions, and settling velocities across estuarine gradients would help validate the conceptual framework proposed here."
We hope the revision and improvement will strengthen the manuscript and satisfactory. A full revision will be formally uploaded after all the other potential reviewers' comments been addressed. Thank you again for your constructive feedback.
Citation: https://doi.org/10.5194/egusphere-2025-6516-AC2
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AC1: 'Reply on RC1', Leiping Ye, 04 Mar 2026
reply
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RC2: 'Comment on egusphere-2025-6516', Anonymous Referee #2, 19 Apr 2026
reply
This manuscript presents an investigation into the effects of microbial activities on sediment flocculation in estuarine environments using a single bacterial strain and microstructural characterization. While the dataset is extensive and the interdisciplinary effort is evident, several key issues, including severely limited environmental relevance, critical logical flaws, methodological inadequacies, and overstatement of conclusions, render the current version unsuitable for publication. Thus, I recommend rejecting the manuscript.
Major comments:
- The experimental design uses only one bacterial strain (Stutzerimonas decontaminans) and a fixed shear rate of 65 s⁻¹ without justification or variation, yet the study extrapolates to complex natural estuarine processes involving diverse microbial communities and highly variable hydrodynamic regimes. Moreover, the maximum experimental duration of 72 h is insufficient for biofilm development, which typically requires weeks to months, and this temporal mismatch is not adequately acknowledged.
- The causal interpretation that upregulation of flagellar genes drives initial attachment is not supported, as transcriptomic data only show temporal sequences and both processes could be independently triggered by unknown signals such as quorum sensing. Additionally, the lack of direct comparison between the mixed system and the pure SD at identical time points, together with unreported initial bacterial size and floc size for the SD group, prevents isolating the effect of mineral-induced flocculation from intrinsic growth dynamics.
- The study contains internal inconsistencies (total EPS, polysaccharide, and protein contents did not change significantly), yet transcriptomic data show late activation of polysaccharide biosynthesis pathways and a significant increase in POC, and this contradiction is not resolved. The mineral control group was run for only 160 min while the biomineral group ran for 48 h, and the potential impact of long-term shear on mineral fragmentation cannot be ruled out despite claims of early equilibrium.
- The microscopic characterization using air-dried SEM and AFM samples is inappropriate for highly hydrated EPS-rich flocs, as air-drying causes polymer network collapse and alters morphology and roughness, meaning the observed structures do not represent in-situ conditions. Alternative approaches such as cryogenic preparation or ESEM should have been used.
- The discussion is too brief and largely descriptive, failing to adequately explain biological mechanisms, and the manuscript over-extrapolates from an idealized laboratory system to complex estuarine processes without a dedicated “Limitations” section, while several interpretative statements appear in the Results section rather than the Discussion.
Minor comments:
In Section 3.1 (line 242), “As shown in Fig. 1e” should be revised to “As shown in Fig. 3e”.
In Figures 3(a) and 3(c), the y‑axis scale should be refined with finer increments in the range of 30 μm to 100 μm to better reflect the size distribution of D_m and D_50 after 15 hours, and in Figures 3 and 7 the meanings of labels such as “a”, “ab”, “abc” are unclear and should be explained.
Abbreviations and notations are inconsistent (e.g., “fig. 9” vs. “Figure 10”) and should be unified throughout the manuscript.
The title emphasizes estuarine environment and hydrodynamics, but the manuscript focuses extensively on biological changes with minimal discussion of hydrodynamics. Thus, either the title or the content should be revised to resolve this discrepancy.
Citation: https://doi.org/10.5194/egusphere-2025-6516-RC2
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There are publications in the literature that show the extremely swift (a few minutes) biologically-induced flocculation in estuarine waters as a result of Transparent Exopolymer Particles (mucus) secreted not just by microbes, but also bacteria and plankton and other biota. None of them are cited. There are also other publications that show that experimental studies in the laboratory with selected microbes to start the biological flocculation underestimate by an order of magnitude the biologically-induced flocculation. These are not cited.
This manuscript fails to address these pitfalls.