The effect of droplet size on syntrophic dynamics in droplet-enabled microbial co-cultivation

James Y. Tan; Tatyana E. Saleski; Xiaoxia Nina Lin

doi:10.1371/journal.pone.0266282

Peer Review History

Original SubmissionSeptember 27, 2021
3 Nov 2021 Decision Letter - Mehmet A Orman, Editor PONE-D-21-31201The effect of droplet size on syntrophic dynamics in droplet-enabled microbial co-cultivationPLOS ONE Dear Dr. Lin, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec. 18, 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors describe an interesting observation that droplet size impacts the growth dynamics of a two member cross-feeding auxotrophic E coli system coloaded at low cell density into droplets. The experiment is very well laid out and the findings clearly demonstrate a difference in growth dynamics vs droplet size -- a result which I think is surprising and I wouldn’t have necessarily expected. The interpretation of the results that growth dynamics vs droplet size is caused by diffusion I think is plausible, but the authors need to describe this interpretation in more detail. What are the diffusion rates and timescales for the relevant metabolites? Also, how does this compare to the fact that the E coli are highly mobile? As the E coli are swimming around, don’t they leave a wake of metabolites? Numerical order of magnitude estimations are needed here which describe all relevant scales -- diffusion, E coli mobility, mass transport. Growing microbes in droplets is a fast moving field -- which makes the authors publication very relevant -- but the authors also need to update their references to cover more recent droplet investigations of bacteria growth. A starting list is Villa, Max M., et al. "Interindividual variation in dietary carbohydrate metabolism by gut Bacteria revealed with droplet microfluidic culture." Msystems 5.3 (2020): e00864-19. Mahler, Lisa, et al. "Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities." Elife 10 (2021): e64774. Watterson, William J., et al. "Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes." Elife 9 (2020): e56998. Reviewer #2: The authors present a very interesting and coherent dataset of E. coli cultivation and co-cultivation experiments in microfluidic droplets of different sizes, demonstrating strong culture and metabolite interaction dependence on droplet size in various relevant growth parameters. The study is timely and relevant as the throughput of microfluidic droplet studies is desired for microbial co-culture and microbiome community studies. The here-studied droplet size parameter may be relevant for the design of future droplet based studies to choose a suitable size, as well as to include size as a parameter to look at different interaction dynamics. I recommend the study for publication in general, but also note a few inaccuracies, omissions and questions that I recommend to address first: (important point) Line 56-57: The authors claim that droplets are increasingly used to study interactions. I think this is true and that what has been published so far is only the early beginning of many such methods to be developed and used. But the references 1-3 do not involve droplets, and the following references provide an incomplete overview mainly based on older publications, not reflecting the increment. I suggest a few relevant and incomplete recent examples from third party research groups that I think help show this trend and its relevance: DOIs: 10.1073/pnas.1811250115; 10.1039/D0LC01204A; 10.1016/j.copbio.2019.09.001; 10.7554/elife.64774 (just individual strains) Line 85: "In bulk culture, convective mass transfer is ...seldom a limiting factor...". This sounds a lot like gut microbiome to me but I don't think it generally holds for other habitats such as soil, tissues, or even ocean zones. Please correct. Line 111: A wide range of droplet sizes can be made with the same device, especially when switching between jetting and dripping by changing flow ratios and speeds. The current sentence sounds like this is not possible and I recommend an adjustment. (important) Line 415 and after: The results section contains large amounts of interpretation, which I would normally expect in the discussion section. In particular, in my view, the claim that diffusion of metabolites is the underlying mechanism of the observed variable changes in different droplet sizes is not supported by the data. It is a reasonable hypothesis to explain the trends, but as the authors point out in other parts of the manuscript, other parameters such as quorum sensing also influence the growth behaviour. I could not follow the conclusion that the diffusion mechanism is the sole reason, for this I would expect to see more molecular-level evidence. However, in my view this makes the study no less interesting. It still shows the droplet size dependence of an example microbial co-culture, with all its complex dependencies. Line 490: The droplet-to-droplet variation is an interesting side story of the article that I would ideally like to see explored in more detail as a reader (beyond the short discussion from line 549 onwards), as it is equally relevant for interaction study design in droplets. To what degree is this variation explained by the stochasticity of the poisson distribution at lambda = 5? Also, it would be great to see more justification or explanation around this value "5" as a choice for almost all experiments presented. How much can be assigned to secretion, and how much to fluorescent protein generation? Are there other systematic studies to give partial answers? And in particular for interactions: What is the relevance of the stochastic co-encapsulation? For example if one partner is encapsulated at higher cell numbers as the other by chance (or systematically by varying the lambda value for one, but not the other strain). Related to this, please clarify the sentence in line 520: Does the variability only depend on the limited growth capacity in smaller droplets? How did you reach that conclusion? Line 528 onwards: Very nice summary of recommendations based on the presented data. On data availability: All the used code seems to have been made available in detail, including scripts to generate the figures, but I did not see the raw data. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes:** Tobias Wenzel [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0266282.r001
Revision 1
10 Feb 2022 Author Response Reviewer #1 Comment: The interpretation of the results that growth dynamics vs droplet size is caused by diffusion I think is plausible, but the authors need to describe this interpretation in more detail. What are the diffusion rates and timescales for the relevant metabolites? Also, how does this compare to the fact that the E coli are highly mobile? As the E coli are swimming around, don’t they leave a wake of metabolites? Numerical order of magnitude estimations are needed here which describe all relevant scales -- diffusion, E coli mobility, mass transport. Response: We appreciate this very thoughtful comment. As suggested, we carried out an order of magnitude estimation for the time scale of diffusion in microdroplets, and the results were very helpful in allowing us to re-evaluate our interpretation of experimental observations. Specifically, we estimated the average time required for a representative amino acid molecule to travel the longest distance in a droplet based on statistical mechanics and recognized that the time scale involved in molecular diffusion is much shorter than that associated with cell growth. Hence, diffusion is not likely a major contributing factor underlying our experimental results. We have included these calculations and discussions throughout the revised manuscript (in Introduction, Results and Discussion). Regarding cell mobility, E. coli is indeed motile and can move on the scale of microns/sec. If E. coli is moving while secreting, this will also contribute to the secreted metabolites being spread in the droplet and then quickly diffused. However, as discussed above, we now speculate that transport of the secreted metabolites within the droplet is most likely not the limiting factor. Comment: Growing microbes in droplets is a fast moving field -- which makes the authors publication very relevant -- but the authors also need to update their references to cover more recent droplet investigations of bacteria growth. A starting list is: • Villa, Max M., et al. "Interindividual variation in dietary carbohydrate metabolism by gut Bacteria revealed with droplet microfluidic culture." Msystems 5.3 (2020): e00864-19. • Mahler, Lisa, et al. "Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities." Elife 10 (2021): e64774. • Watterson, William J., et al. "Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes." Elife 9 (2020): e56998. Response: The field truly does move fast and we appreciate the reviewer’s thoughtful suggestion! These more recent references, along with several others, have been added in the introduction. Reviewer #2 Comment: (important point) Line 56-57: The authors claim that droplets are increasingly used to study interactions. I think this is true and that what has been published so far is only the early beginning of many such methods to be developed and used. But the references 1-3 do not involve droplets, and the following references provide an incomplete overview mainly based onolder publications, not reflecting the increment. I suggest a few relevant and incomplete recent examples from third partyresearch groups that I think help show this trend and its relevance: DOIs: 10.1073/pnas.1811250115;10.1039/D0LC01204A; 10.1016/j.copbio.2019.09.001; 10.7554/elife.64774 (just individual strains) Response: We thank the reviewer for looking very carefully at the reference list in our original submission and providing these excellent suggestions. We have removed the wrong references and added the suggested references, along with several others, in the first paragraph. Comment: Line 85: “In bulk culture, convective mass transfer is ...seldom a limiting factor...". This sounds a lot like gut microbiome, but I don't think it generally holds for other habitats such as soil, tissues, or even ocean zones. Please correct. Response: The original intention was to describe laboratory flask cultures which are typically well-mixed, but the reviewer brings up a good point to put this in perspective with natural systems where there is a continuum of homogeneity and heterogeneity. We have revised this part as suggested and expanded our discussion. Comment: Line 111: A wide range of droplet sizes can be made with the same device, especially when switching between jetting and dripping by changing flow ratios and speeds. The current sentence sounds like this is not possible and I recommend an adjustment. Response: We agree. This study itself uses two droplet devices to generate 5 different sizes in the range of 55 to 150 μm. This sentence has been removed. Comment: (important) Line 415 and after: The results section contains large amounts of interpretation, which I would normally expect in the discussion section. In particular, in my view, the claim that diffusion of metabolites is the underlying mechanism of the observed variable changes in different droplet sizes is not supported by the data. It is a reasonable hypothesis to explain the trends, but as the authors point out in other parts of the manuscript, other parameters such as quorum sensing also influence the growth behaviour. I could not follow the conclusion that the diffusion mechanism is the sole reason, for this I would expect to see more molecular-level evidence. However, in my view this makes the study no less interesting. It still shows the droplet size dependence of an example microbial co-culture, with all its complex dependencies. Response: This is an excellent comment. This feedback and a similar one from another reviewer have pushed us to re-think about our previous interpretation. We have also conducted new investigations in an attempt to dissect more thoroughly the mechanisms underlying our experimental observations. In particular, using an ODE model we have previously developed, we examined how the growth dynamics change when key parameter values are varied and have been able to recapitulate part of the complex trends observed experimentally. These new findings suggest that in addition to or even instead of diffusion, inherent properties related to intercellular interactions in complex ecosystems give rise to specific trends of growth dynamics. Please see details in the new first paragraph in the “Discussion” section and a new figure in the supplement (Fig. S10). Comment: Line 490: The droplet-to-droplet variation is an interesting side story of the article that I would ideally like to see explored in more detail as a reader (beyond the short discussion from line 549 onwards), as it is equally relevant for interaction study design in droplets. To what degree is this variation explained by the stochasticity of the poisson distribution at lambda = 5? Also, it would be great to see more justification or explanation around this value "5" as a choice for almost all experiments presented. How much can be assigned to secretion, and how much to fluorescent protein generation? Are there other systematic studies to give partial answers? And in particular for interactions: What is the relevance of the stochastic co-encapsulation? For example if one partner is encapsulated at higher cell numbers as the other by chance (or systematically by varying the lambda value for one, but not the other strain). Related to this, please clarify the sentence in line 520: Does the variability only depend on the limited growth capacity in smaller droplets? How did you reach that conclusion? Response: These are all very insightful questions. We have carried out an additional experiment to determine if an increase in the initial lambda value would decrease the amount of droplet-to-droplet variability. We investigated this hypothesis in a simple monoculture experiment and added two new figures (Fig. 2 and Fig. S5 in the revised manuscript) to demonstrate that variability can be decreased by increasing the initial lambda. We have clarified the choice of lambda = 5 in the text, that it was to keep the initial cell number per droplet low while trying to ensure that almost every droplet had at least one cell. It is difficult to fully assess how much the variability is assigned to each of these components (Poisson distribution, cell viability, growth/secretion, and fluorescent protein generation). The added experiment with higher initial lambda demonstrates that even with variation introduced from the Poisson distribution, the variability is decreased with higher lambda. This result suggests that the cell-to-cell variability is what is contributing the most to the droplet-to-droplet variability observed at low lambda values. It would be interesting to try systematically varying the lambda value of one strain and keeping the other constant. Extrapolating from our experiment where we increased the lambda, we would expect the characteristics of the one strain at a higher lambda to be more consistent across droplets, whereas the lower lambda strain would be expected to exhibit a higher degree of droplet-to-droplet variability. This could be investigated in future studies. The statement about variable community composition due to limited growth capacity was confusingly worded. It was a statement on the change of community composition across different droplet sizes for the same condition, not on droplet-to-droplet variability. The statement has been removed. Comment: On data availability: All the used code seems to have been made available in detail, including scripts to generate the figures, but I did not see the raw data. Response: We appreciate the reviewer’s attention to detail. The raw data was not included in our original submission, but the Github repository has the raw data and description of how the raw data was converted to normalized data required for the MATLAB scripts. Attachments Attachment Submitted filename: Tan_etal_Rebuttal_PLOSONE_v3.docx https://doi.org/10.1371/journal.pone.0266282.r002
18 Mar 2022 Decision Letter - Konstantinos Papadimitriou, Editor The effect of droplet size on syntrophic dynamics in droplet-enabled microbial co-cultivation PONE-D-21-31201R1 Dear Dr. Lin, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The authors addressed all reviewer comments thoroughly. They have added new simulations and experiments that provide valuable additional insights into the topic of the manuscript, clarifying all previously speculative aspects including optional questions of interest. Well done! ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes:** Tobias Wenzel https://doi.org/10.1371/journal.pone.0266282.r003
Formally Accepted
23 Mar 2022 Acceptance Letter - Konstantinos Papadimitriou, Editor PONE-D-21-31201R1 The effect of droplet size on syntrophic dynamics in droplet-enabled microbial co-cultivation Dear Dr. Lin: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Konstantinos Papadimitriou Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0266282.r004

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