Native Bacteria Isolated from Phosphate Deposits Reveal Efficient Metal Biosorption and Adhesion to Ore Particles

Round 1

Reviewer 1 Report

The article has scientific and practical interest. It is shown the perspectives of using the bacterial strains isolated from Djebel Onk phosphate ore (Algeria). The introduction gives a general idea of the problem, from which the aim of this study follows logically. The technique is described in sufficient detail. This section shows that the study was carried out using a wide range of methods for the analysis of both microorganisms and substances. The article has a clear structure. Data is statistically confirmed. The obtained results are compared with the data of other authors and show the effectiveness of the new bacterial strains for biosorption processes and removing of various metals over a wide pH range. The most interesting result was obtained for bacteria of the genus Bacillus, which demonstrated the highest adhesive ability at neutral pH. Tables and figures illustrate the results well. Data is statistically confirmed.

There are several comments and questions.

It would be good to clarify the principle by which samples of phosphate ore were taken for setting up experiments and isolation of microorganisms.

It is also not very clear on the what basis 4 strains of bacteria were selected for experiments from 160 species of isolated microorganisms, emphasising on the degree of study of these bacteria in this direction and providing additional references to such works.

On fig. 5 there is no designation "b" in the upper right figure of the presented insert.

In the discussion, the authors describe their previous work but there is no clear connection between previous data and new results because in the current study quite different species of bacteria were used.

The mechanism of biosorption cadmium ions by bacteria of the genus Bacillus, which are capable of producing extra-polymeric substance (EPS) and forming biofilms, has not been described. How these features of Bacillus sp. might affect metal adhesion. It would also be useful to look at possible approaches to using the obtained results for practical applications for bioremediation in natural ecosystems.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

I receive many papers for review where authors claim that bio processes such as bioleaching or biosorption are "enironmentally friendly and low cost technologies" which are predicted to become solution for many enivironmental or economic issues. Although, these claims sound nice, the reality is different. Authors of this stduy also claim that biosorption is a "low cost and environmentally friendly solution" for removal of Cd from phosphate ores. If that claims are true, why  are there no commercial aplications of biosorption after decades of research and thousands of published papers? Maybe because it is not a low cost option at all, and environmental frinedliness is often comparable to other more efficient chemical processes. Bioleaching technology has been around for decades, but now only 1 % of global Cu comes from biloeaching. From my point of view, research papers like this have little or no value without even basic techno-economic analysis. There were tons of smilar research papers and finding novelty in this research is difficult. If authors could provide some proof of economic, environmental and technologicial feasibility of this process on industrial scale, than this reseach would have certain value for scientific community.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

This article reports a study investigating selected bacteria strains to adsorb Mg and Cd. The article was prepared generally well with concrete data and discussion. However, some issues/concerns have been found. Please see comments below.

 

General comments:

 

In-depth discussion is missing but needed. The authors described the results well, while did not discuss the reasons underlying the results and the associated mechanisms. Some more investigations can be carried out for verification of the mechanisms.

 

The study is primarily a case study with testing a specific scenario. The authors found a native stain, called HK4, performed better than other tested strains. However, this conclusion seems limited and hard to be extrapolated to other situations. Can the authors say that HK4 will be always better or that native strains will be always better? I think neither can be drawn unless more scenarios with a wide range of sources of samples are investigated.

 

The background of biosorption was not shown clearly. Were the bacteria used to adsorb metals from water or from phosphate ores in the solid phase? If they are for solid phase adsorption, how can the adsorption process be set up? Do you need to first elute the ores to let metals dissolve into water, then use bacteria adsorb metals? Such general introduction to the biosorption process is not shown in the article.

 

Specific comments:

 

Lines 18 to 19: What disadvantages do conventional processes have? Please specify them.

 

Lines 22 to 24: Show quantitative results. Is HK4 always the best strain? How about other scenarios? Based on this study, do you propose HK4 to be the best or native strains to be the best? If you recommend native strains to be the best, you need to test multiple scenarios. In this case, since you only test one specific scenario, such a conclusion seems hard to be drawn.

 

Line 26: Specify the metals and the pH range.

 

Line 41: Coherence between first and second paragraphs is poor. You first mentioned mining then jumped to the cadmium issue without a smooth connection.

 

Line 49: This is not clear. Do you mean these countries cannot sell cadmium rich ores to EU?

 

Lines 56 to 58, “Microorganisms might also be successfully used to treat post-flotation wastes…”: Why do you think microorganisms might be a good choice? Please give a review of previous studies and show references.

 

Lines 70 to 71: After pollutants are removed through biosorption, do they change to other substances? How to treat the microorganisms that have adsorbed the pollutants?

 

Line 93: What criteria did you use to select these four strains?

 

Line 95: Why did you investigate Mg biosorption? I do not think Mg is toxic. You need to explain the reason for analyzing Mg.

 

Line 110: Did the ore samples appear naturally as powder? What colour is it? It would be good to describe its physical appearance.

 

Lines 117 to 118: Did you invent this procedure or follow previous studies? Please show references if applicable.

 

Line 129: Was anything not dissolved as precipitates?

 

Line 150: This is not clear. How little is the difference?

 

Line 162: Figure 1 should be improved. Some lines do not have an arrow. What do the plus and minus signs mean?

 

Line 177: Indicate the concentration of NaCl.

 

Lines 191 to 193: This is confusing. Please improve the writing and check the data. The logic seems not right. In addition, it would be good to create a table to summarize the sequences, if this information is important.

 

Lines 211 to 213, “Next, the solution containing microorganisms was…”: The expression is not clear. Did you test the microorganism performance to remove Cd from water? What water did you test?

 

Lines 252 to 253: What criteria did you use to judge an efficient accumulation?

 

Lines 255 to 257: Please discuss why pH at 10 was associated with the lowest performance and why HK3 and HK4 worked best.

 

Line 260, “HK4 and 259 the reference strain Bacillus subtilis USK1 were independent of pH”: Why? Explain the reason.

 

Line 266, “HK4 had the highest variance in Cd accumulation at pH 4 compared to other strains”: This expression is not clear. If you say "highest variance", it sounds that the standard deviation of the replicates is higher. It seems not your meaning.

 

Lines 267 to 269, “Contrary, Cd biosorption by the other studied strains…”: Why? Explain the reason. You said the values at pH 10 were highest. Is it correct? I see the values are lowest in Figure 3.

 

Figure 3: It could be better to show the lower limits of the error bars. Please indicate experimental conditions in the caption for better reference.

 

Figure 4: It could be better to show the lower levels of the error bars. Please indicate experimental conditions in the caption for better reference.

 

Line 286: How about HK4? It had highest efficiency at pH 7, as shown in Figure 4.

 

Line 287: Is it correct? HK1 at pH 10 seems not good, as shown in Figure 4.

 

Lines 294 to 296, “Post-hoc testing revealed the highest variance…”: Please improve the discussion to make it clearer.

 

Line 302: Please discuss the mechanisms why HK4 worked the best.

 

Lines 315 to 316: Was HK4 better to adhere to those ore particles than USK1? Did you test USK1 to adhere to the ore that was used to test HK4?

 

Line 342 to 343, “The most effective strain, Bacillus sp. HK4, isolated from raw ore…”: Discuss why this strain works best.

 

Lines 347 to 348, “Also, during phosphate ore processing…”: This sentence is not complete.

 

Lines 362 to 363: Indicate why the cells in the absence of Cd reached higher accumulation.

 

Lines 367 to 369: Can you discuss the reason for such phenomena.

 

Line 370: It would be good to explore the mechanisms in this study. Here, you only cite previous studies.

 

Lines 383 to 384, “The importance of phosphoryl sites was…”: Improve the writing of this sentence.

 

Line 397: The discussion in this paragraph is very confusing. Please streamline this paragraph and possibly divide it. Some discussion on Mg should be moved to Introduction to justify why you tested it.

 

Lines 415 to 416: The discussion here and above is quite confusing. Why does the biofilm develop easier?

 

Line 417: Please indicate the constituents of apatite and dolomite.

 

Line 424: Discuss why HK4 has a higher adhesion capacity.

 

Lines 436 to 437: You tested four native strains in a specific case. How about other scenarios? Can you suggest native strains are always promising?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

From my ponit of view, after improvements, the paper is suitable for publishing.