Performance Evaluation of Selenite (SeO32−) Reduction by Enterococcus spp.
Round 1
Reviewer 1 Report
Paper:
Performance Evaluation of Selenite (SeO32-) Reduction by a mixed culture of Enterococci
General comments:
the present work dealt with the evaluation of the ability of enterococci to reduce selenite, a toxic inorganic form of selenium. The topic of the present study would be of importance if the author provide a speculation of data. Which is the field of application? This should be straight reported in the title. The novelty of this work is not that high, since it is not true that LAB have not been characterized for their capacity to reduce selenite. Please check work of Prof. Madrid-Albarran and Dr. Mozzi in the period 2018 – 2021. However, the focus on Enterococcus is of interest.
I cannot judge the English style because I am not mother tongue. The topic is of interest for the target readers.
Regarding the methodology, it is really poor, no information about the bacterial strains used and all figures do not make any sense. The authors should show which Enterococcus spp. were tested and the results distinguished per strain. As is, the work does not provide any useful information. What is the point of testing a group of strains? Maybe only one is effective, not the pool. The work should be reorganized showing data per strains, so that the authors can conclude on which strains are useful in reducing selenite and can propose some of these strains as starters.
As is, the work is inacceptable unless the microbiological aspects are addressed.
Specific comments:
- Enterococci is a group not genus. So “enterococci” no capital letter and not in italics or “Enterococcus”. Please modify this denomination throughout the text because it is wrong.
- “not much work has been done on LAB”. Please replace “LAB” with “enterococci”.
- All information about Enterococcus used in the study are missing. Which Enterococcus species were tested? How many strains? Which was the source of isolation? The source of isolation is important for future applications. How were they purified and identified? The authors reported a PCR toold in methodology, but there is no sign of identification. How many strains were pooled.
- Figure 1. The effect of increasing selenite concentration on bacterial growth. How can this figure be useful? Which are the bacteria tested? This is a very crucial point of the work.
- Table 1 reports data per three defined strains (Pseudomonas aeruginosa JS-11, Comamonas testosteroni S44, and Stenotrophomonas bentonitica), the last without strain number, then for ENTEROCOCCI. It simply does not make any scientific sense. The authors should show data per all Enterococcus spp.. E.G. Enterococcus faecium XXX, Enterococcus faecalis YYY, Enterococcus durans ZZZ….and so on. Only in this case the work would make sense and data be in a useful form.
- As is, data cannot be of any help for the scientific community.
- The same for all other figures and tables. Data per group are of no use, they should be reported per strain.
- Conclusions not supported by data.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
One of the key issues prevalent in biological treatment of selenium in the mining environment is the competitive inhibition from nitrate, which often exceeds selenium concentrations by factors of 100 to 1000. The study does not take into consideration any inhibitors for LAB. Would it be possible to see if the LAB would grow on real contaminated samples?
At experimental part you mentioned - Culture identification, but no further information is provided: what species of Enterococcus have you identified? This is interesting, since they are published articles with Enterococcus species (E. faecalis, E. faecium, E. durans) used for selenite bioremoval (https://doi.org/10.1016/j.micres.2010.03.005, https://doi.org/10.1016/j.jtemb.2016.09.003, https://doi.org/10.1016/j.jtemb.2016.12.003).
Please explain the novelty of your study in the introduction, mentioning the results previously obtained with Enterococcus species for selenite bioremediation.
Where the samples sterilized ? where you able to confirm only the presence of Enterococcus sp. in your flasks?
The glucose utilisation correlated with the selenite concentration to be reduced. row 179 - please rephrase
Glucose was likely used for fulfilling metabolic needs of the bacteria other than for growth in order to acclimatise better to the high selenite concentrations. row 180 - please explain - on what is based this affirmation? Since glucose was the only source of carbon and energy provided for the cells, what have the microorganisms used for growth, if not glucose ?
The delay in the production of elemental selenium with respect to SeO3
2- reduction was attributed to the formation of metabolic intermediates be-
fore elemental selenium was formed [31], row 164 - the reaction is very slow, in one hour (aprox) there is no SeO3 in the medium, but the production of Se occurs for another 2.5 hours. Have you tried to find these intermediates in the concversion of anorganic to organic selenium? It is important since for bacteria 2.5 hours is a long period in the exponential phase and their number would be very different after this period.
Please make Fig 8 as Protein concentration vs time (using a calibration curve), rather then absorbance.
Have you determined the selenium content in biomass?
How is temperature affecting the bioremediation process?
Author Response
Please see the attachment
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The article can be published.
Author Response
- The issue of strain identification, which was outstanding as per the recent review comments has been addressed. A phylogenetic tree has now been added to the supplementary section and a new section on; “Microbial Characterisation” (from row 114) was also added to the manuscript.
