Micro- and Nano-Plastics Induced Release of Protein-Enriched Microbial Exopolymeric Substances (EPSs) in Marine Environments
, Peter H. Santschi 2,*, Antonietta Quigg 3, Chen Xu 2, Peng Lin 4 and Manoj Kamalanathan 5
Round 1
Reviewer 1 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsDear Authors,
I have some major comments for the MS that are listed in the attachment.
Comments for author File: 
Comments.pdf
Author Response
Answers to Comments for Environments-3002550
Reviewer 1
The manuscript titled “Micro- and nano-plastics induced release of protein-enriched microbial exopolymeric
substances (EPS)” was revised, however, it still need to be re-organized and improved. I have some major comments as below:
- It would be better to shorten the previous paragraphs and extend the last paragraph from line 76 to
82 to focus on the main topic and purpose of the study. Moreover, it should be add more reference in
this paragraph as providing proofs.
Answer: We feel that the previous paragraph addresses the lack of studies on nanoplastics, due to the fact that the majority of separation and analytical techniques in aquatic sciences are not directly applicable for nanoplastic investigations. The next paragraph has been rewritten as follows:
“Various aquatic microbes can reduce plastic stress by producing EPS with distinctive characteristics that promote aggregation to MPS, reduce disaggregation, and thus allow sedimentation. This review will focus on the molecular mechanisms activated by microbes in response to plastic pollution, for example by the release of protein-rich EPS, which facilitate their efficient removal from the water column. Most importantly, this review highlights the areas of uncertainty that should be taken into account in future research efforts designed that promote the self-cleaning capacity of aquatic ecosystems.
- Please remove line 107 to after Fig.1.
Answer: We understand this comment as moving this line below Fig. 1, which we carried out.
- Please point out in detail of all processes in 1 to make it clear and understandable for readers. For
example: In the aggregation…….,….In the disaggregation……, and in subsequent settling
(scavenging)……….
Answer: We revised the figure caption as follows: “Figure 1. Model of hypothetical pathways and processes for micro- and nano-plastics in the environment. This includes plastic particles, when contacting microbes, induce the generation of radical oxygen species (ROS), which themselves induce genetic changes to produce more protein-enriched EPS with higher protein to carbohydrate (P/C) ratios that renders EPS more hydrophobic and sticky. Such EPS is more sticky and tends to promote aggregation with other particles, including denser mineral particles (scavenging), producing aggregates (marine plastic snow, MPS) that allow the lighter plastic particles in the MPS aggregate to sink to sediments without disaggregation.”
- It is better to collect and list some typical/general secreted EPS algal and bacterial species separately in
Answer: Since this appears to be a general reaction of both bacterial and algal species when exposed to pollutants such as plastic particles. Therefore, we did not include such a table as we feel that such a table would be excessively large.
Author Response File: 
Author Response.docx
Reviewer 2 Report (New Reviewer)
Comments and Suggestions for AuthorsThis review refers on the effects of microbial exposure to micro- and nano-plastics focusing on the production of exopolymeric substances (EPS) by microbial cells (phytoplankton and bacteria).
In my opinion the mechanisms involved in EPS release and formation of aggregates with plastic particles (marine plastic snow) are well described. My comments (detailed below) are mostly aiming at shortening the sentences (sometimes too long) and minor changes. Attention must be paid at revising the section References (which do not seem to be reported according to the Authors' instructions, abbreviated Journal name, not in full, DOI indication and not PubMed PMID). To improve reference list, making this review as much exhaustive as possible, other articles should be added, regarding the effects of plastic particles on the production of EPS, as reported below:
Hua Wang, Cheng Qiu, Shaochen Bian, Lei Zheng, Yongmin Chen, Yali Song, Chengran Fang,
The effects of microplastics and nanoplastics on nitrogen removal, extracellular polymeric substances and microbial community in sequencing batch reactor, Bioresource Technology (2023) , 379,129001, https://doi.org/10.1016/j.biortech.2023.129001.
Yaxin Wang, Xiuhong Liu, Runyu Liu, Weipeng Han, Qing Yang, Mechanisms of interaction between polystyrene nanoplastics and extracellular polymeric substances in the activated sludge cultivated by different carbon sources,
Chemosphere (2023) 314, 137656, https://doi.org/10.1016/j.chemosphere.2022.137656.
Tianran Ye, Ao Yang, Yulai Wang, Na Song, Ping Wang, Huacheng Xu,
Changes of the physicochemical properties of extracellular polymeric substances (EPS) from Microcystis aeruginosa in response to microplastics,
Environmental Pollution (2022) 315, 120354, https://doi.org/10.1016/j.envpol.2022.120354.
Irem Demir-Yilmaz, Nadiia Yakovenko, Clément Roux, Pascal Guiraud, Fabrice Collin, Christophe Coudret, Alexandra ter Halle, Cécile Formosa-Dague,
The role of microplastics in microalgae cells aggregation: A study at the molecular scale using atomic force microscopy, Science of The Total Environment (2022) 832, 155036, https://doi.org/10.1016/j.scitotenv.2022.155036.
Other comments
Abstract, line 13, particle through physical/mechanical mechanisms
line 15 RPS cause oxidative damage to microalgae and bacteria; this induces....
line 19, and afterwards: identify the knowledge gaps .....in future research. In particular, the microbial communities reply to plastic pollution reducing the plastic burden to ecosystems through the production of EPS that may induce reduce the plastic impacts via marine plastic snow (MPS) formation, allowing particles to settle into sediments and facilitating their removal from the water column.
Introduction
Line 42, micro- and nano-plastics are dispersed rapidly and can be transferred along the food webs,.....ranging from.... in the aquatic medium.
Line 42, for polar environments, a recent review should be included in the Reference list (Caruso, Bergami, Singh, Corsi, 2022. Plastic occurrence, sources, and impacts in Antarctic environment and biota, Water Biol. Security 1 (2) 100034, https://doi.org/10.1016/j.watbs.2022.100034.
Line 51, to uptake instead of to consume
line 53, food chains of aquatic environments
line 59, physical effects (instead of outcomes)
line 61, to play a potential role as vectors of harmful algae species....
line 74, applications to deepen our understanding
line 75 reduce plastic-derived stress
line 80, from the water column
line 78 and afterwards, on the molecular mechanisms activated by microbes in response to plastic pollution, for example by the release of protein-rich EPS...
line 81, efforts designed to favour the self-cleaning capacity of aquatic ecosystems.
Section 2. Plastics incorporation into biological aggregates (instead of aggregates and plastics)
line 88, marine snow was reported (not published)
line 89, remove Remains of
line 92, total microbial community
line 99, rapidly colonized by microbial biofilms (to this I suggest to add another reference, Caruso, G. Microbial Colonization in Marine Environments: Overview of Current Knowledge and Emerging Research Topics. J. Mar. Sci. Eng. 2020, 8, 78. https://doi.org/10.3390/jmse8020078
line 102, interactions of plastic particles with natural colloids
line 105. consequent settling processes are needed (Fig. 1). Further studies to get new insights......are needed [47, 48].
Figure 1, line 109, micro- and nano-plastics interactions with aquatic microbes
line 110, remove This includes
line 114, mineral particles, producing aggregates ( marine plastic snow, MPS) that finally sink into the sediment.
line 117, 40% of microplastics that were....MPS) and this material might potentially settle
line 123, major vector
line 126, biological properties (instead of makeup)
line 127 significance of this research topic,
line 127, has been conducted to understand the interactions
line 130, drive (instead of govern)
line 131, the major variables that may act on....
line 141, heterogeneous variety of organic material
line 142, remove Which function as particles
line 156, A similar EPS secretion process has been....
lines 161 and 163, nanoplastics; line 233, microplastics (without hyphen)
line 177, Ultraviolet ray exposure (UVA)....
line 178, remove pathways including
line 185, DOM concentration
line 198, can be activated to reduce the negative impacts of ROS
line 203, ROS were produced
line 205, the greater was the protein to carbohydrate......, the greater was the.....of the formed microgel aggregates
line 209 en enzyme involved in ...
line 211, EPS from phytoplankton cells.
line 216, In order to draw a final conclusion
line 239 has largely been focused
line 243, in the context of oxidative stress consequent to microbial exposure to micro- and nanoplastics and in the formation of MPS
line 250, to carbon, is used as a proxy
line 251; this ratio has been related to their interactions....
line 260, nanoplastics. Conversely, in another study, the P/C ratios
line 265, [113], reducing the entrance of toxins into microbial cells
line 269, in relation to their P/C ratios of EPS extracted (remove repetition P/C ratios)
line 289, The lack of a right understanding of these processes prevents the determination of the fate of pollutants such as microplastics and the set up of measures for their removal...
line 299, increase (instead of ameliorate)
line 317, remove In order to test this question. Experimental tests.....
Figure 3: PCA (Principal Component Analysis), NMDS (Not MultiDimensional Scaling) should be reported in the caption
line 334, BET techniques, please provide in full (instead of acronym)
line 369, needs (instead of warrants)
line 390, remove to oil
line 406, should be investigated in future
line 415, unique features that undergo aggregation, formation......(remove that are ameliorating the impacts via)
line 419, identify the knowledge gaps
line 425, MPS compared to marine snow
line 427, processes and the molecular mechanisms
Comments on the Quality of English LanguageOnly minor changes to improve reading are suggested.
Author Response
Answers to Comments for Environments-3002550
Reviewer 2
This review refers on the effects of microbial exposure to micro- and nano-plastics focusing on the production of exopolymeric substances (EPS) by microbial cells (phytoplankton and bacteria).
We included references suggested by Reviewer 2 and incorporated all the revision suggestions, except one suggestion, “line 390, remove to oil”. “Marine oil snow” is a particular type of marine snow that was reported after Deepwater Horizon oil spill containing oils. We decided to keep the term “marine oil snow” as we intended.
In my opinion the mechanisms involved in EPS release and formation of aggregates with plastic particles (marine plastic snow) are well described. My comments (detailed below) are mostly aiming at shortening the sentences (sometimes too long) and minor changes. Attention must be paid at revising the section References (which do not seem to be reported according to the Authors' instructions, abbreviated Journal name, not in full, DOI indication and not PubMed PMID). To improve reference list, making this review as much exhaustive as possible, other articles should be added, regarding the effects of plastic particles on the production of EPS, as reported below:
Hua Wang, Cheng Qiu, Shaochen Bian, Lei Zheng, Yongmin Chen, Yali Song, Chengran Fang,
The effects of microplastics and nanoplastics on nitrogen removal, extracellular polymeric substances and microbial community in sequencing batch reactor, Bioresource Technology (2023) , 379,129001, https://doi.org/10.1016/j.biortech.2023.129001.
Yaxin Wang, Xiuhong Liu, Runyu Liu, Weipeng Han, Qing Yang, Mechanisms of interaction between polystyrene nanoplastics and extracellular polymeric substances in the activated sludge cultivated by different carbon sources,
Chemosphere (2023) 314, 137656, https://doi.org/10.1016/j.chemosphere.2022.137656.
Tianran Ye, Ao Yang, Yulai Wang, Na Song, Ping Wang, Huacheng Xu,
Changes of the physicochemical properties of extracellular polymeric substances (EPS) from Microcystis aeruginosa in response to microplastics, Environmental Pollution (2022) 315, 120354, https://doi.org/10.1016/j.envpol.2022.120354.
Irem Demir-Yilmaz, Nadiia Yakovenko, Clément Roux, Pascal Guiraud, Fabrice Collin, Christophe Coudret, Alexandra ter Halle, Cécile Formosa-Dague,
The role of microplastics in microalgae cells aggregation: A study at the molecular scale using atomic force microscopy, Science of The Total Environment (2022) 832, 155036, https://doi.org/10.1016/j.scitotenv.2022.155036.
Answer: Added
Answers to the following comments: accepted and carried out, shown as strike through of comment
Other comments
Abstract, line 13, particle through physical/mechanical mechanisms
line 15 RPS cause oxidative damage to microalgae and bacteria; this induces....
line 19, and afterwards: identify the knowledge gaps .....in future research. In particular, the microbial communities reply to plastic pollution reducing the plastic burden to ecosystems through the production of EPS that may induce reduce the plastic impacts via marine plastic snow (MPS) formation, allowing particles to settle into sediments and facilitating their removal from the water column.
Introduction
Line 42, micro- and nano-plastics are dispersed rapidly and can be transferred along the food webs,.....ranging from.... in the aquatic medium.
Line 42, for polar environments, a recent review should be included in the Reference list (Caruso, Bergami, Singh, Corsi, 2022. Plastic occurrence, sources, and impacts in Antarctic environment and biota, Water Biol. Security 1 (2) 100034, https://doi.org/10.1016/j.watbs.2022.100034.
Line 51, to uptake instead of to consume
line 53, food chains of aquatic environments
line 59, physical effects (instead of outcomes)
line 61, to play a potential role as vectors of harmful algae species....
line 74, applications to deepen our understanding
line 75 reduce plastic-derived stress
line 80, from the water column
line 78 and afterwards, on the molecular mechanisms activated by microbes in response to plastic pollution, for example by the release of protein-rich EPS...
line 81, efforts designed to favour the self-cleaning capacity of aquatic ecosystems.
Section 2. Plastics incorporation into biological aggregates (instead of aggregates and plastics)
line 88, marine snow was reported (not published)
line 89, remove Remains of
line 92, total microbial community
line 99, rapidly colonized by microbial biofilms (to this I suggest to add another reference, Caruso, G. Microbial Colonization in Marine Environments: Overview of Current Knowledge and Emerging Research Topics. J. Mar. Sci. Eng. 2020, 8, 78. https://doi.org/10.3390/jmse8020078
line 102, interactions of plastic particles with natural colloids
line 105. consequent settling processes are needed (Fig. 1). Further studies to get new insights......are needed [47, 48].
Figure 1, line 109, micro- and nano-plastics interactions with aquatic microbes
line 110, remove This includes
line 114, mineral particles, producing aggregates ( marine plastic snow, MPS) that finally sink into the sediment.
line 117, 40% of microplastics that were....MPS) and this material might potentially settle
line 123, major vector
line 126, biological properties (instead of makeup)
line 127 significance of this research topic,
line 127, has been conducted to understand the interactions
line 130, drive (instead of govern)
line 131, the major variables that may act on....
line 141, heterogeneous variety of organic material
line 142, remove Which function as particles
line 156, A similar EPS secretion process has been....
lines 161 and 163, nanoplastics; line 233, microplastics (without hyphen)
line 177, Ultraviolet ray exposure (UVA)....
line 178, remove pathways including
line 185, DOM concentration
line 198, can be activated to reduce the negative impacts of ROS
line 203, ROS were produced
line 205, the greater was the protein to carbohydrate......, the greater was the.....of the formed microgel aggregates
line 209 en enzyme involved in ...
line 211, EPS from phytoplankton cells.
line 216, In order to draw a final conclusion
line 239 has largely been focused
line 243, in the context of oxidative stress consequent to microbial exposure to micro- and nanoplastics and in the formation of MPS
line 250, to carbon, is used as a proxy
line 251; this ratio has been related to their interactions....
line 260, nanoplastics. Conversely, in another study, the P/C ratios
line 265, [113], reducing the entrance of toxins into microbial cells
line 269, in relation to their P/C ratios of EPS extracted (remove repetition P/C ratios)
line 289, The lack of a right understanding of these processes prevents the determination of the fate of pollutants such as microplastics and the set up of measures for their removal...
line 299, increase (instead of ameliorate)
line 317, remove In order to test this question. Experimental tests.....
Figure 3: PCA (Principal Component Analysis), NMDS (Non-metric multidimensional scaling) should be reported in the caption
line 334, BET techniques, please provide in full (instead of acronym)
line 369, needs (instead of warrants)
line 390, remove to oil (The reviewer is not correct here, marine oil snow is a correct term)
line 406, should be investigated in future
line 415, unique features that undergo aggregation, formation......(remove that are ameliorating the impacts via)
line 419, identify the knowledge gaps
line 425, MPS compared to marine snow
line 427, processes and the molecular mechanisms
Author Response File: Author Response.docx
Reviewer 3 Report (New Reviewer)
Comments and Suggestions for Authors
This topic, which connects emerging contaminants and the dynamics of natural organic matter in the environment, is very interesting. References are up to the date. The authors have been working on related topics and research fields. This timely review is a welcome addition to the literature.
Specific comments.
1. EPS are produced from both phytoplankton and microbes in aquatic environments (see Fig. 1 and examples in Table 1). In the title, the authors used the term, “microbial EPS”, and similarly, section 3, Line-293 states “EPS is produced by microbes”. Regardless of whether EPS is also produced by phytoplankton, it is important to be consistent about the source of EPS throughout the manuscript.
2. Second, EPS contain mostly polysaccharides (carbohydrates), but the P/C ratio has been shown to be crucial in regulating the fate and transport of contaminants and many biogeochemical processes (section 2.3.3). It is commendable that the authors linked and extend section 2.3.3 into future research to better understand detailed molecular mechanisms in the production of protein-enriched EPS.
3. Lastly, the authors did not specify “aquatic environments” in the title although their hypothetical pathways and processes depicted in Figure 1 and examples in Table 1 are mostly for marine or oceanic environments. I suggest that the authors either add “marine environments” to the title or include a small sub-section related to freshwater environments either in section 2 or section 3.
Author Response
- Both phytoplankton and bacteria produce and release EPS. Phytoplankton, a photosynthetic autotrophic microbial component of aquatic systems, are considered microbes. We use “microbes’” and “microbial” to include both phytoplankton and bacteria.
- We appreciate Reviewer 3’s support.
3. As suggested by Reviewer 3, we change the title to “Micro- and nano-plastics induced release of protein-enriched microbial exopolymeric substances (EPS) in marine environments”
Author Response File: Author Response.docx
Reviewer 4 Report (New Reviewer)
Comments and Suggestions for AuthorsThe paper is not strictly speaking a research paper as it does not provide data. It is a bibliographical work. However, I consider it very interesting and that it should be published as it helps other researchers within the framework in which it is framed.
The work can serve as a reference for other researchers in the field of microplastics and help them understand the behavior of this pollutant in the environment.
Are the conclusions consistent with the objectives intended to be achieved at the start of the work, with all the intended objectives having been addressed.
The number of references cited is very high and coherent with the line of research in which the work is framed.
The tables and figures are of adequate quality, except for figure 2 which should be improved.
Author Response
As Academic Editor pointed out in his/her review, “most of the requests of Reviewer 1 have been accepted or reasonably denied”, in most cases, we agree with your suggestions and have tried to make changes that we hope will serve to alleviate most, if not all, of Reviewer 1’s concerns. We appreciate all Reviewer 1’s time and effort, which we believe has served to significantly improve the work. We believe we have made a good-faith effort to address the concerns in our response and revised the manuscript based on the suggestions from Reviewer 1.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsDear Authors,
This manuscript has been revised partially. However, it is still not enough to improve the quality. Three main points that need to be revised I mentioned in the previous revision. However, you did not tend to make an effort on it. You tried to revise the first point but it is not enough to focus on the purpose of research. Moreover, the two remaining points were not dealt with significantly. You have the right to protect your opinions if you do not want to revise more following my comments. I respect that. To me, it was not revised and improved enough to be accepted in the current version.
Author Response
As Academic Editor pointed out in his/her review, “most of the requests of Reviewer 1 have been accepted or reasonably denied”, in most cases, we agree with your suggestions and have tried to make changes that we hope will serve to alleviate most, if not all, of Reviewer 1’s concerns. We appreciate all Reviewer 1’s time and effort, which we believe has served to significantly improve the work. We believe we have made a good-faith effort to address the concerns in our response and revised the manuscript based on the suggestions from Reviewer
Author Response File: Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe review on “Micro- and nano-plastics induced release of protein-enriched exopolymeric substances (EPS) from phytoplankton and bacteria” is interesting. The authors describe various features of EPS deposited on the micro- and/or nano-plastic particles. There is some confusion in this review. The EPS is deposited on the surface of the particles by the algae or microbes as a cushion or creating a familiar surface for further growth or colonization. In fact, the EPS act as a barrier or adhesive for the microbes or algae present on the substrate. The authors have taken this into a different context and argue that the microplastics present outside and separated by EPS induce ROS production or gene expression inside the microbes or algae. What is missing is the connection between how the microplastics or nanoplastic particles or substrates sitting outside the cells of these organism induces such biochemical changes inside the cells. The composition changes in EPS such as protein-to-carbohydrate (P/C) ratio, molecular weight, and hydrophobicity changes are usually accompanied by the pollution level (may be caused by leachate from the plastic particles) and chemical nature of the environment where microbes or algae are growing and the type or family of algae or microbes. There are many reports on the impact of EPS on microplastics, but the authors need to provide more details on such issues. In addition, micro- or nanoplastic particles are not all the same in chemical composition or size or surface features etc., most of which will influence the deposition of EPS and growth of the algae or microbes. The authors are strongly encouraged to reorganize the review and resubmit with a focused review. Some minor points…
Fig. 1-4 are not a well thought out one. Need more clarifications and focus on the topic of the review.
On page 4 and other places, the authors have written Ding et al. ([70]) and Ding et al. ([71]). It should be Ding et al. (70, 71).
Comments on the Quality of English LanguageNo major problem with the language.
Author Response
Reviewer #1
In fact, the EPS act as a barrier or adhesive for the microbes or algae present on the substrate. The authors have taken this into a different context and argue that the microplastics present outside and separated by EPS induce ROS production or gene expression inside the microbes or algae.
There are many published reports and reviews that indicate that plastic particles (microplastics (MP) and nanoplastics (NP)) can induce ROS levels, related gene expression variations which subsequently (thus indirectly) cause EPS composition changes in microbes in aquatic systems. The produced ROS could also directly modify the ambient DOM which includes EPS. This idea is also expressed well in Figure 1. We now explicitly mention this text “Creating a buffer zone with secreted EPS between the living cell and the surrounding environment in order to chemically mitigate the effects of ROS is yet another defense tactic that can be implemented ([88]). Many cyanobacteria ([89]), e.g., Nostoc commune ([90]; [91]), Microcoleus vaginatus ([92]; [93]), produce EPS under UV-induced oxidative conditions. Such EPS produced under micro- and nano-plastics induced ROS ‘toxicity’ would be different in amounts and composition as compared to those produced under pristine conditions. In addition to this above mentioned indirect effects, ROS can directly modify ambient DOM, including EPS. Sun et al. ([94]) showed that ROS was produced by irradiation of seawater, and ROS were responsible for chemically crosslinking proteins. This research also found that the greater the protein to carbohydrate ratio of EPS, the greater the increase in size of the microgels (aggregates) that formed ([94]).”.
Some example publications:
- Das, Amlan. "The emerging role of microplastics in systemic toxicity: Involvement of reactive oxygen species (ROS)." Science of The Total Environment (2023): 165076.
- Junaid, Muhammad, and Jun Wang. "Interaction of nanoplastics with extracellular polymeric substances (EPS) in the aquatic environment: A special reference to eco-corona formation and associated impacts." Water research201 (2021): 117319.
- Li, Ze, et al. "Toxicological effects of microplastics and sulfadiazine on the microalgae chlamydomonas reinhardtii." Frontiers in Microbiology 13 (2022): 865768.
- Liu, Xiaomei, et al. "Nano-and microplastics aided by extracellular polymeric substances facilitate the conjugative transfer of antibiotic resistance genes in bacteria." Acs Es&T Water12 (2022): 2528-2537.
- Sun, Xuemei, et al. "Toxicities of polystyrene nano-and microplastics toward marine bacterium Halomonas alkaliphila." Science of the total environment642 (2018): 1378-1385.
- Wang, Rongyu, et al. "Bacterial Interactions with Nanoplastics and the Environmental Effects They Cause." Fermentation11 (2023): 939.
- Ye, Tianran, et al. "Changes of the physicochemical properties of extracellular polymeric substances (EPS) from Microcystis aeruginosa in response to microplastics." Environmental Pollution315 (2022): 120354.
- Yokota, Kiyoko, et al. "Finding the missing piece of the aquatic plastic pollution puzzle: interaction between primary producers and microplastics." Limnology and Oceanography Letters 2.4 (2017): 91-104.
- Zakaria, Basem S., et al. "Responses of syntrophic microbial communities and their interactions with polystyrene nanoplastics in a microbial electrolysis cell." Science of the Total Environment903 (2023): 166082.
Fig. 1-4 are not a well thought out one. Need more clarifications and focus on the topic of the review.
We added more clarifications to improve focus on topic of manuscript, which are on the physical-chemical connections to molecular mechanisms of microbes in response to exposure to micro- and nano-plastic particles. This has been done mainly in the figure captions, but also in the text. We maintain that all figures are essential to this review, as they illustrate the focus of this review on the connections between the physical-chemical processes and relate to molecular mechanisms of microbes in response to exposure to micro- and nano-plastic particles. In particular, the novel focus on the relationship between extent and speed of self-assembly of EPS and its stickiness to the protein to carbohydrate ratio of EPS. This then allows plastic particles to sink to the bottom, where they are found to be most abundant.
On page 4 and other places, the authors have written Ding et al.([70]) and Ding et al. ([71]). It should be Ding et al. (70, 71).
Agreed. We have revised the manuscript accordingly.
Author Response File: Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThe review entitled “Micro- and nano-plastics induced release of protein-enriched exopolymeric substances (EPS) from phytoplankton and bacteria” focused on the effects of micro and nanoplastics on bacteria and phytoplankton inducing the release of EPS.
In this study, I only read data relating to bacteria, as also underlined by the authors who stated in their conclusions that “This review will thus examine what is known about the molecular mechanism’s microbes use to regulate the fate and transport of this material and identify the unknowns which should be considered in future research endeavors”.
So, I suggest fixing the title of the review. The review is interesting and in my opinion, requires minor revisions before publication.
I also noted that:
From line 55 to line 73 the text does not seem integrated with the above, I suggest you improve it to make it smoother and easier to interpret. It also misses the purpose of the review.
Reduce paragraph 2.3.3.
Please explain the paragraph better. Furthermore, I don't understand the need to include Figure 3 and Table 2. Please explain the reasons and importance.
Highlight the importance of this review in the conclusions
Author Response
Reviewer #2
suggest fixing the title of the review.
Agreed. We have revised the manuscript accordingly. The new title is “Micro- and nano-plastics induced release of protein-enriched microbial exopolymeric substances (EPS)”.
From line 55 to line 73 the text does not seem integrated with the above, I suggest you improve it to make it smoother and easier to interpret.
We have revised the manuscript.
Reduce paragraph 2.3.3. Please explain the paragraph better.
We have revised the manuscript Figure 3 was deleted.
I don't understand the need to include Figure 3 and Table 2. Please explain the reasons and importance.
As requested by the reviewer for brevity purpose, we deleted Figure 3 yet kept Table 1 (from Chen et al. 2021). Table 1 provides quantitative evidence to support that microbial EPS with higher P/C ratio have higher stickiness (are stickier).
Highlight the importance of this review in the conclusions
The importance of this review lies in revealing the connections between the physical-chemical processes as they relate to molecular mechanisms of microbes in response to exposure to micro- and nano-plastic particles. Another novel focus is on the relationship between exposure to pollutants, ROS generation, the extent and speed of self-assembly of microgels, stickiness of EPS, its hydrophobicity, and protein to carbohydrate ratio. As suggested by the reviewer, we added this to the manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
The manuscript tilted "Micro- and nano-plastics induced release of protein-enriched exopolymeric substances (EPS) from phytoplankton and bacteria have addressed the significance of microbes' reaction on micro- and nano-plastics. The MS has been presented logically, however, it still need to be revise some points as below:
1. There is no section 2 beofe a subsection of 2.1.
2. It would be better to add more a target/goal of this review briefly to end the introduction.
3. In each subsection, please make a table for listing bacterial/algal strains that contribute in each relevant mechanism. It helps understanding easily for readers.
4. Please provide more proof as the examples of the role of extremophilic microbes in stressful environments.
Thank you.
Author Response
Response to Reviewer #3
There is no section 2 before a subsection of 2.1.
We have revised the manuscript accordingly.
It would be better to add more a target/goal of this review briefly to end the introduction.
We have revised the manuscript accordingly.
In each subsection, please make a table for listing bacterial/algal strains that contribute in each relevant mechanism. It helps understanding easily for readers.
There is no reason to believe that not all microorganisms would respond similarly to plastic particles by ROS and MPS generation. Making a table with a comprehensive list of bacterial and algal strains would be a massive undertaking and beyond the scope the paper. However, we have cited relevant research and review publications in the revised manuscript that should direct the readers towards relevant examples of bacterial and algal strains. In addition, we have included results from our own experiment with relevant species to demonstrate the mechanisms. Lastly, there are plenty of review papers doing this kind of thing already and readers would likely seek out those papers if this is the information they are seeking.
Please provide more proof as the examples of the role of extremophilic microbes in stressful environments.
We appreciate the suggestion. For the role of extremophilic, it is unclear why this suggestion is arising or how it would contribute to the manuscript. The current discussion is more about the bacteria’s potential for plastic degradation (their unique enzymes). Our review is more about the "transport" aspect of plastics, e.g., aggregation, sedimentation etc., and their relationship to EPS composition. Plastic degradation is an important issue, but it is not the focus of our manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThank you for revising the main points.
Please check and revise other minor ones as below:
1. Arrows' position in Fig.4.
2. It seems missing Fig.3?
3. Please check again the format of whole context.
Author Response
We forgot to renumber figures in the text after we deleted fig. 3. As requested, we fixed Fig. 4 (now Fig. 3).
Author Response File: Author Response.docx
