Investigating the role of bacterial raw milk community members in chlorate reduction
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Chlorine-based detergents, used in the dairy industry for cleaning, often degrade into chlorate, contaminating milk and dairy products. Consumption of chlorate has been linked to thyroid dysfunction in adults and impaired neurological development in infants. Despite the ban on chlorine-based detergents in Ireland since 2021, chlorate contamination remains a problem in the dairy supply chain. A recent study found chlorate-reducing bacteria naturally present in raw milk, highlighting their potential for chlorate mitigation. In this study, shotgun metagenomic sequencing was applied to determine the effects of chlorate concentration and incubation conditions on the raw milk microbiome, specifically focusing on chlorate-reducing bacteria within the community. Chlorate-spiked milk samples from different farms showed reductions in chlorate levels over time, from day 10 onwards when stored at 4°C and after 24 hours when incubated at 25°C. Pseudomonas and Lactococcus were observed as the most dominant taxa in raw milk samples stored at 4°C and 25°C, respectively. High abundances of ydeP and narG genes were observed for 4°C samples and were attributed to Pseudomonas and various low abundance genera, respectively. High abundances of the napA gene were noted in 25°C samples and were attributed to the Lactococcus genus. Overall, this study highlights the presence of naturally occurring chlorate-reducing bacteria as part of the raw milk microbiome and identifies multiple genes linked to various pathways potentially involved in chlorate reduction. Furthermore, incomplete pathways potentially involved in chlorate reduction were found, suggesting metabolic cross-feeding and underscoring the community roles bacteria play in chlorate reduction in raw milk. Additionally, a few previously uncharacterised genes, such as ydeP, belonging to the dimethyl sulfoxide (DMSO) reductase gene family were identified at high abundances in samples that showed chlorate reduction, emphasising the need for further biochemical characterisation of these genes to better understand the pathways involved in chlorate reduction in milk.
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The paper entitled "Investigating the role of bacterial raw milk community members in chlorate reduction" presents novel research that is of interest to the wider researcher community. Previous work in this area introduced well and allows for those not familiar with the field understand the rationale for the work reported. The methodology is sounds and results were well presented and discussed. I recommend this paper for publication in Access Microbiology after a few minor comments I have on the manuscript have been addressed (see below). Line 298 - Which data was imported into R and how was it imported before generating diversity measures? Lines 337 - 338: should be moved to discussion (if not already mentioned). Lines 535 - 539: Same as last comment Could a paragraph on limitations of the work be …
Comments to Author
The paper entitled "Investigating the role of bacterial raw milk community members in chlorate reduction" presents novel research that is of interest to the wider researcher community. Previous work in this area introduced well and allows for those not familiar with the field understand the rationale for the work reported. The methodology is sounds and results were well presented and discussed. I recommend this paper for publication in Access Microbiology after a few minor comments I have on the manuscript have been addressed (see below). Line 298 - Which data was imported into R and how was it imported before generating diversity measures? Lines 337 - 338: should be moved to discussion (if not already mentioned). Lines 535 - 539: Same as last comment Could a paragraph on limitations of the work be added to the discussion? I think some have been briefly mentioned but would be good to have a stand-alone paragraph on any potential limitations of the study design and results Supplementary figure 2: Can the resolution be increased? It is currently hard to read the key and axis labels.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
This study highlights the risks associated with chlorate contamination in milk and provides a novel insight into the role of the milk microbiome at different storage temperatures in mitigating this risk. Overall the topic is introduced well, and the methodological approach is robust. However, the methods section is lacking in detail, which currently hinders the repeatability of this research. It could also benefit from being written in a more concise tone, as currently much of the information is difficult to follow. The discussion section would benefit from a more critical analysis of the limitations of this study, while also delving more into the reasons of this research, why it is so important, and what the next steps would be for this to have a real-world impact. 1. Methodological rigour, …
Comments to Author
This study highlights the risks associated with chlorate contamination in milk and provides a novel insight into the role of the milk microbiome at different storage temperatures in mitigating this risk. Overall the topic is introduced well, and the methodological approach is robust. However, the methods section is lacking in detail, which currently hinders the repeatability of this research. It could also benefit from being written in a more concise tone, as currently much of the information is difficult to follow. The discussion section would benefit from a more critical analysis of the limitations of this study, while also delving more into the reasons of this research, why it is so important, and what the next steps would be for this to have a real-world impact. 1. Methodological rigour, reproducibility and availability of underlying data The methodological approach itself is robust and demonstrates a successful analysis of the milk microbiome. However many details are missing in the methodology section which should be provided prior to publication: Line 151-152: Was the chlorate concentration initially measured in the raw milk samples before spiking? Is it true to say that the control was 0 ppb, given that the introduction suggests raw milk to naturally contain chlorite? Line 179: Why were triplicates pooled here and throughout the methodology? Would it not be better to analyse them individually to see the spread of the data? Lines 180-184: The information regarding UHPLC and MS has been cited, however it might be useful to have some additional information here regarding the parameters and the columns used. Line 193: PBS manufacturer information is needed Line 202: What was the DNA eluted into? Lines 203-204: How were these quantifications performed? Lines 231-233: As so many samples were found to have low yields, were any additional analyses conducted to try and fix this? Could a greater number of PCR cycles have been justified, or could you have increased the original input of dairy product before DNA extraction? Lines 248-259: This reads more as a discussion. 2. Presentation of results Overall the results are of a good quality and presented well. Figure legends need to be amended throughout, and some other adjustments could be made to make this section more polished: Lines 333-334: 'an average microbial load ranging from 103 to 105 CFU/mL'. Here I would either specify the specific value of each condition, or I would remove this sentence and simply state the level of increase. This is a big range to display as an average and isn't consistent with the later specification of increases for each condition. Line 349. Figure 2. These figures should be split up, have one figure for bacterial growth and another for chlorate residue analysis. The figure legend should also have more detail, which will be easier to do with these separated. For all figure legends, you should have a short title. You also must include your number of repeats (n numbers) in all figure legends, in addition to any statistical tests used and the p number thresholds. All figure legends in this paper need to be edited to get them to a publication standard. Line 387: Full species names need to be given every time an organism is first mentioned. Line 469. Figure 3. Again these should be separated, and figure legends should be more detailed for each specific graph. Line 524: Is there an increase abundance of the Ydep gene if there is no statistical significance? Line 524. Figure 4. Same comments as previously, n numbers should be stated? 3. How the style and organization of the paper communicates and represents key findings The paper is organised well throughout, and the key findings are communicated clearly. 4. Literature analysis or discussion The literature review provides key insight into the literature and prepares the reader well for the following chapters. The discussion is well-written but is currently missing some key points and overall doesn't provide much of an insight into the reason for this research project. More should be added here to justify the methodological approach, assess the study limitations and demonstrate the applicability of this study to the real-world. More detailed comments are provided below: Lines 556 and 557: These points need to be referenced. Line 596: These genes have been found to be present, but do we know if they are being expressed? Lines 617-620: Can you explain why a higher concentration would make chlorate reduction more challenging? If this is the case, why were higher concentrations used in this study? The association between bacteria and chlorate reduction currently is quite hypothetical, especially with many of the genes only having assumed roles in chlorate reduction. Is there anything else that could be causing chlorate reduction in these samples. For example other microbiome components, such as fungi which are unaccounted for. Also, does the temperature itself impact chlorate reduction? The discussion would benefit from exploring these questions, as it currently seems there is little evidence to suggest the bacteria themselves are driving chlorate reduction. The discussion and conclusion could benefit from an explanation as to the value of this research. What value do the findings contribute, why does this study matter and does this study have any application to the real-world?
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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