Rhamnolipids Mediate the Effects of a Gastropod Grazer in Regards to Carbon–Nitrogen Stoichiometry of Intertidal Microbial Biofilms

Round 1

Reviewer 1 Report

1. Novelty of this work must be stated in the Introduction and a strong justification why this work is important.

2. Why additional surfactants were not tested?

3. Why just rhamnolipids? How it is released in to marine environment? The details has to be included for a clear understanding on the importance of using rhamnolipids.

4. Measurement of macromolecular compositions of the biofilm could give additional details on the study conducted.   

5. In figure 3, it is not clear why the carbon and nitrogen content of biofilm is ~3% and 0.2%. If we assume that biofilm comprises both cells and EPS, then the biofilm should have atleast 50% of carbon, and atleast 10-20% nitrogen which is not obtained in this case?

6. A conclusion section may be included.

 

Author Response

Main comments:

1. Novelty of this work must be stated in the Introduction and a strong justification why this work is important.

Further justification has been added into the introduction. The introductory paragraphs containing the justification are added (Lines 73-92):

2. Why additional surfactants were not tested?

Rhamnolipids are one of the most commonly studied and used biological surfactant groups as alternatives to synthetic surfactants for biofilm control. Due to this we made the decision to focus our funds and allocated mesocosm space to higher replicate numbers for rhamnolipids alone. We also had local expertise and history working with rhamnolipids, which made it the obvious choice for us. The following sentence was added into our methodology (lines 112-114)

3. Why just rhamnolipids? How it is released in to marine environment? The details has to be included for a clear understanding on the importance of using rhamnolipids.

We previously had the following sentence within our methodology explaining why rhamnolipids were chosen (lines 114-115):

“Rhamnolipids were chosen as our biosurfactant due to their potential application as an alternative biofilm control agent to chemical surfactants.”

We also added in the following sentence to the introduction to explain how surfactants reach the marine environment (lines 78-79):

“Surfactants specifically are being introduced in great diversity and quantity to the marine environment due to their heavy use in cargo ship and oil cleanups, and ocean drilling [32].”

4. Measurement of macromolecular compositions of the biofilm could give additional details on the study conducted.   

While we do agree that additional measurements are useful to any study, however at this stage we have no more samples to measure and this would be a useful parameter to consider in future work. A sentence to this effect has been added please see line 288-290

5. In figure 3, it is not clear why the carbon and nitrogen content of biofilm is ~3% and 0.2%. If we assume that biofilm comprises both cells and EPS, then the biofilm should have at least 50% of carbon, and at least 10-20% nitrogen which is not obtained in this case?

 

The experimental site’s water source has an intake near the bed of an Irish sea lough. After passing through a sand trap the water still contains small amounts of inorganic silt particles. During the flow through our system a low level of deposition was therefore inevitable and led to the observed relatively low carbon and nitrogen contents in percent of biofilm dry mass. A parallel arrangement within the same mesocosm table ensured that all replicates had equal exposure to suspended solids and other quality parameters of the water source. Therefore, the observed differences between C/N ratios of treatment and control were due to surfactant exposure.

The following sentence was added into the discussion (lines 269-271):

“Relatively low C and N contents in percent of biofilm dry mass were attributable to the inevitable low-level deposition of inorganic solids from the water flowing through the system.”

6. A conclusion section may be included.

We previously had a concluding paragraph at the end of the discussion (lines 317-336) and feel that it reads best as part of the discussion, and not as a separate section.

 

Reviewer 2 Report

This article discusses how rhamnolipids and a grazer can interact on a phototrophic marine biofilm, below are my main comments:   Introduction: Add a little information regarding published preview studies on biofilm and the relationship with holobiont, and whether the resistance to biosurfactants can affect biofilm development.  

Line 72-74: How does the presence of chemicals within marine environments reduce the exposure of marine biota to the action of rhamnolipids?

 Materials and methods: Where the biofilm was collected? What is the composition of the biofilm considered? Otherwise the experimental plan involves the analysis of C/N ratio but if this index decreased or increased with the increase of biofilm stress is not specified.   Conclusion: in the introduction and materials and method authors justify the use of rhamnolipids as their increase in several applications. Furthermore in the introduction and conclusion it isn't mentioned what these applications are. Improve this aspect and if possible compare results obtained with ones of other studies.   Minor comments: line 109 = missing a space between 2 and cm (elsewhere there is) lines 129-130 = is it reproducible? line 138 = the term ''sterilized'' is excessive for a 70% alcohol solution; it should have a bacteriostatic effect. line 139 = missing space between 2 and mL line 140 = wrong degree sign between 20 and C line 148 = ml misspelled, L should be capitalized

Author Response

This article discusses how rhamnolipids and a grazer can interact on a phototrophic marine biofilm, below are my main comments:  

1. Introduction: Add a little information regarding published preview studies on biofilm and the relationship with holobiont, and whether the resistance to biosurfactants can affect biofilm development.  

Some adjustments were made within the introduction concerning biofilm holobiont relationships ( Please see lines 53-59):

“However, they also have holobiontic (where a symbiotic relationship forms between hosts and the surrounding microbiota creating a unique ecosystem [22]) and mutualistic relationships with other marine organisms. Holobiontic relationships have been noted among grazers where snails [23] and limpets [24] removed unproductive canopy from the biofilm. This encouraged new biofilm growth and greater productivity, leading to benefits to both the grazer, which also acts as the host for microorganisms, and the surrounding biofilm itself.”

Regarding the general impact of biosurfactants on biofilm, the introduction had already stated that biosurfactants can affect biofilm development by controlling EPS matrix size and structure. This is precisely why biofilm dwelling bacteria such as Pseudomonas aeruginosa produce rhamnolipids, to control the EPS matrix around the individual cells. Biofilm dwelling microorganisms have, therefore, co-evolved with biosurfactants such as rhamnolipid. If a biofilm were resistant to the biosurfactant it would resist developmental changes in structure. This would require investigation at both the microscopic scale to understand how it may influence the composition of the biofilm and its physical structure. However, we are not clear what influence this would have on a short term study such as ours.

 

2. Line 72-74: How does the presence of chemicals within marine environments reduce the exposure of marine biota to the action of rhamnolipids?

The referenced section does not discuss the presumably many ways how the presence of anthropogenic chemicals in the marine environment could affect the rhamnolipid mode of action on biofilm. We agree with the reviewer that questions concerning the combined impacts of chemicals on both bacterial response to rhamnolipid exposure and rhamnolipid production itself are important topics for future studies. However, these topics were not the focus of our particular experiment presented in this paper.

 

3. Materials and methods: Where the biofilm was collected? What is the composition of the biofilm considered? Otherwise the experimental plan involves the analysis of C/N ratio but if this index decreased or increased with the increase of biofilm stress is not specified. 

Biofilms were grown within the mesocosms during the experiment. The following sentence was added in the methods (lines 130-131):

“Tiles were used as substrata for marine biofilm growth during the experiment.”

We assume the reviewer’s comment is referring to the bacterial composition of the biofilm. Bacterial composition was not analysed due to funding limitations, which is why biofilm compositional changes in regards to stress were not included in the discussion. The changes in C and N content have been discussed in the paper with specific reference to observed changes in C:N ratios. For example (lines 279-284):

“Although not significant, loss of C within our biofilms in the rhamnolipid treated systems led to a decreased C:N ratio. Rhamnolipids can encourage EPS material disintegration [52]. It is likely that as the EPS matrix of a biofilm is heavily made up of lipids, polysaccharides, proteins, and extracellular DNA [17], and therefore heavy in C content, rhamnolipids affected the EPS matrix and decreased overall C within the biofilm.”

 

4. Conclusion: in the introduction and materials and method authors justify the use of rhamnolipids as their increase in several applications. Furthermore in the introduction and conclusion it isn't mentioned what these applications are. Improve this aspect and if possible compare results obtained with ones of other studies.  

Additional information has been added concerning sources of surfactants in the marine environment as requested by Reviewers 1 and 2. The following referenced statement was included (lines 78-79):

“Surfactants specifically are being introduced in great diversity and quantity to the marine environment due to their heavy use in cargo ship and oil cleanups, and ocean drilling [32].”

Due to the novelty of this work, extensive comparisons are not possible; however, our discussion did contain comparisons where relevant references were available. For example:

Lines 240-251:

“Prior research has shown that biofilm exposure to synthetic surfactants can decrease respiratory activity temporarily, with full recovery occurring after 12 hours [40]. Conversely, antibiotic exposure has temporarily increased respiration for several hours for affected single species biofilms [41]. Biomass has similarly been negatively affected by surfactant exposure over a short time period. Rhamnolipid exposure reduced single species biofilms by 74-98% over a 4 day exposure of a 4 day old biofilm [42]. Grazing on biofilms from known biofilm feeders has also been seen previously to significantly decrease biofilm biomass after a period of 3-4 days [43]. Our study took place over a longer time period than the previous studies, and exposure started before initial growth occurred. Therefore, it is likely that the lack of significant differences in GPP, CR, and biomass occurred due to the longer experimental timeline allowing biofilm time to recover and adapt its growth to the presence of a surfactant stressor.”

Lines 260-274:

“The formation of a beneficial relationship between biofilms and grazers in our study are supported by previous studies where similar results occurred, although we obtained our evidence over a much smaller timeframe. Limpet grazing of epilithic bio-films over the summer when growth and grazing rates peak led to biofilm biomass growth increases of 20% later on in the season [47]. Nutrients from grazer excretion can also specifically increase microphytobenthic growth, which would then affect local biofilm productivity [48]. Nutrients obtained from grazer excretion are typically P [49] and N [50], which would explain why biofilms exposed to only grazers within our mesocosms had altered stoichiometry with decreased C:N ratios and increased N. Therefore, in line with previous research our results show an indication that marine biofilms, even after 14 days of grazing, may be able to develop beneficial relationships with grazers and demonstrate high tolerance for grazing disturbances.”

 

Minor comments:

5. line 109 = missing a space between 2 and cm (elsewhere there is)

 

Space has been added there and elswhere.

 

6. lines 129-130 = is it reproducible?

Within this section we described as detailed as possible how we obtained primary productivity and community respiration methods. For this reason, the section highlighted where we used plastic bags to simulate a nighttime environment as a reproducible and low cost method to obtain these results. Specifically, lines 129-130 (now lines 144-145) are in reference to pressing the bags against the water line, which is a very reproducible action. Particularly, the low standard deviations observed for GPP measurements are a testament for good reproducibility.  

 

7. line 138 = the term ''sterilized'' is excessive for a 70% alcohol solution; it should have a bacteriostatic effect.

 

“Sterilized” has been replaced by “cleaned…for bacteriostatic effect”. (Line 154)

8. line 139 = missing space between 2 and mL

Space added.

 

9. line 140 = wrong degree sign between 20 and C

The degree sign has been changed.

 

10. line 148 = ml misspelled, L should be capitalized

“L” has been capitalized. 

Round 2

Reviewer 1 Report

The authors have tried to address all queries and they have succeeded. However, the response for comment no. 5, is still not clear. As per the response, the biofilm has more inorganic contents due to deposition happened over time and reduced organic contents. If that's the case, then in figure 3, the y-axis title may be changed accordingly, so that readers can have a clear idea. The same details should be incorporated in the manuscript.

Author Response

Kindly note we have incorporated details into the Figure 3 caption to highlight the low-level deposition of inorganic solids within each system. We included this information into the figure caption rather than the Y-axis title so as not to exceed the space available for the axis title. As all of our samples were taken at the same time, and all systems used the same water source, we are confident that all replicates were affected in the same way and that any results obtained were solely due to treatment effects.

Author Response File: Author Response.pdf