Bioprospection of Bacterial Strains from Chromite Process Industry Residues from Mexico for Potential Remediation

Round 1

Reviewer 1 Report

The paper entitled "Bioprospection of bacterial strains from chromite process industry residues (Mexico) for potential remediation potentials" presents an interesting investigative study regarding chromium bioremediation.  However, to be considered for publication some revisions are necessary. Please, see below.

- What is new about this work in relation to others already published? The novelty character.

- "After separation of liquid and biomass fractions, we verified that nearly 75, 67 and 94% of the total chromium were adsorbed on the biomass (for the cultures 50, 75 and 100 ݉݃Cr(VI) respectively), suggesting it as a good option for further studies with focus in an application for chromate remediation." In other words, this means that the main mechanism involved is not the reduction of Cr(VI) into Cr(III). It is necessary to include in the introduction an approach to the adsorption of CrVI in biomass. Also include clear objectives, challenges, hypotheses...

- "... but maybe also for other metals." Why do the authors suggest this?

Moderate editing of English language required.

Author Response

Answers in red after each reviewer comment, additions in the text in blue

The paper entitled "Bioprospection of bacterial strains from chromite process industry residues (Mexico) for potential remediation potentials" presents an interesting investigative study regarding chromium bioremediation.  However, to be considered for publication some revisions are necessary. Please, see below.

Thank you for the general positive  comment

• What is new about this work in relation to others already published? The novelty character.

The novelty of the work is in the completeness of the bioprospecting process with a view of bioremediation. We comprehensively carry out all the steps of the investigation process: Sampling, characterization of the samples, bacterial biodiversity, screening stages of aerobic bacteria, evaluation of the consortia obtained in the presence of  chromium, isolation and phylogenetic identification analyses of the strains and selection of the most resistant strains.

We think this is clearly stated in the last paragrph of the introduction

• "After separation of liquid and biomass fractions, we verified that nearly 75, 67 and 94% of the total chromium were adsorbed on the biomass (for the cultures 50, 75 and 100 ݉݃Cr(VI) respectively), suggesting it as a good option for further studies with focus in an application for chromate remediation." In other words, this means that the main mechanism involved is not the reduction of Cr(VI) into Cr(III). It is necessary to include in the introduction an approach to the adsorption of CrVI in biomass.
•  
• yes , as we did not detect either Cr(VI) or total Cr in the medium at the end of the experiments, it is likely that a Cr(VI) adsorption process is occurring, but unfortunately it was not possible to analyze the chromium residual on the biomass.
•  
• the following sentence has been added in the introduction (lines 64 to 69)
  

The chromium bacterial resistance includes a combination of several mechanisms, such as, biosorption, bioaccumulation, biotransformation, efflux, enzymatic and non-enzymatic reduction, precipitation, cytosolic biding. In the case of adsorbtion, the adsorbed Cr (VI) may be reduced to Cr (III) since most of chromium resistant bacteria exhibits chromium reductase activity whether membrane-bound or intracellular.

• Also include clear objectives, challenges, hypotheses...

added at the end of introduction (lines 98 to 105)

The main  objective of this work was to isolate aerobic bacterial strains resistant to Cr (VI) with ability to diminish this ion from liquid media. Abandoned waste from industries and mines represents a serious and frequently socio-environmental problem. Environmental mitigation bioprocess can be an economic option for underdeveloped countries. However, obtaining extremophilic microorganisms in axenic culture is a major challenge for microbial ecology, since under stress conditions, the microorganisms live and maintain synergistic relationships with themselves, which makes their isolation and maintenance in laboratory conditions difficult to carry out.

The hypothesis is described  on lines 95-98: Due to the chemical characteristics and bacterial biodiversity of these residues, it has been assumed that they may be a source of microorganisms physiologically adapted to both alkaline and saline conditions, resistant to chromate ions and eventually able to biologically deplete chromate.

• "... but maybe also for other metals." Why do the authors suggest this?

Generally, bacterial strains that live under stress conditions caused by different metals and/or contaminants shows resistance to multiple metals/contaminants [Mukherjee et al., 2017]. Ours strains were isolated from an industrial residue of cromite mine whose previous analysis (Brito et al, 2013) detected other toxic metals like  Copper or Zinc in addition to Cr. It's possible that these are also resistant to other metals like Cu and Zn but we agree that it remains an hypothesis

Mukherjee, P., Roy, M., & Mitra, A. (2017). Isolation and characterization of a multiple heavy metal-resistant, halo-rhizobacterium Oceanobacillus sp. strain Exo5 from Avicennia marina of Indian Sundarbans. Journal of Science, Engineering, Health and Management

Reviewer 2 Report

1. What is the specific mechanism of action in Cr(VI) removal? Is it oxidation-reduction or biosorption?

2. There are many hazardous substances in industrial residues, should the influence of other hazardous substances be considered in the application of the strain?

3. What is the innovation of this microbial community compared to previous microorganisms for Cr(VI) removal?

4. What is the maximum concentration tolerated? Are there any advantages at this concentration compared to chemical treatment?

good

Author Response

Answers in red after each reviewer comment, additions in the text in blue

thank you for the good general evaluation

1. What is the specific mechanism of action in Cr(VI) removal? Is it oxidation-reduction or biosorption?

The chromium bacterial resistance includes a combination of several mechanisms, such as, biosorption, bioaccumulation, biotransformation, efflux, enzymatic and non-enzimatic reduction, precipitation, cytosolic biding, biofilm formation etc [see Purhlkar et al, 2021]. The interaction Cr-cell membrane may be carried out by its molecules (anionic lipopolysaccharides, phospholipids and proteins) or by interaction to a specific functional group (such as, carboxyl, amines, hydroxyl, sulphonate, etc) by electrostatic interactions, that is influenced by the surround pH. Since there is differences of membrane structure among  Gram negative and Gram positive bacteria, the interaction with the Cr on these also will be distinct. Gram negative bacteria can reduce more efficiently Cr (VI) extracellular as compared to Gram positive, while the last have cell surface ligands (SO₃^2-, RNH₂, COO^-, etc) increasing the affinity to transition metal cations, as Cr(VI). The adsorbed Cr (VI) may be reduced to Cr (III) and then to be adsorbed on the surface of bacterial cell, or it may to enter to the cell by membrane transporters (e.g., by sulphate transporters). The Cr (VI) reduction mechanism may be by enzymatic or non-enzymatic process and the most of chromium resistant bacteria exhibits chromium reductase activity whether membrane-bound or intracellular. Sometimes, the last is also associated to a chromium efflux mechanism. 

As we did not detect either Cr(VI) or total Cr in the medium at the end of the experiments, it is likely that a Cr(VI) adsorption process is occurring, but unfortunately was not possible to analyze the chromium residual on the biomass.

in accordance whith Reviewer 1 we add a sentence in introduction about adsorption (lines 64 to 69) , make some changes in the discussion (lines 3971-392 and 548-550)

2. There are many hazardous substances in industrial residues, should the influence of other hazardous substances be considered in the application of the strain?

we can expect no influence of the other coupounds  (Cu, Zn and Fe)

Generally, bacterial strains that live under stress conditions caused by different exposures to metals and/or contaminants shows resistance to multiple metals/contaminants [ej., Mukherjee et al., 2017]. Our strains were isolated from an industrial residue of cromite mine whose previous analysis (Brito et al, 2013) detected other toxic metals (like  20 mg/kg of Cu and 430 mg/Kg of Zn) in addition to Cr (18 g/kg). Its possible that these are also resistant to other metals like Cu and Zn. 

Mukherjee, P., Roy, M., & Mitra, A. (2017). Isolation and characterization of a multiple heavy metal-resistant, halo-rhizobacterium Oceanobacillus sp. strain Exo5 from Avicennia marina of Indian Sundarbans. Journal of Science, Engineering, Health and Management (JSEHM)

3. What is the innovation of this microbial community compared to previous microorganisms for Cr(VI) removal?

As described in the discussion, "practically all" species like ours isolates were already been studied in relation to some xenobiont or some biotechnological potential. But the highlight of the strains obtained in this work is they are extremophiles microorganisms (halo-alkaline) enabling their use in the search for a bioprocess for transforming Cr (VI) using mining effluents with high pH.

we added this sentence as conclusion

Constructing consortia on the basis of these results will be the next challenge. This challenge will be easier to develop because the isolated strains are extremophiles microorganisms (halo-alkaline) enabling their use in the search for a bioprocess for transforming Cr (VI) using mining effluents with high pH.

4. What is the maximum concentration tolerated? Are there any advantages at this concentration compared to chemical treatment?

400 mg/kg (see figure4)

Chemical treatment is more efficient than biological treatment. But, chemical treatment is very expensive, not applied, and produces a lot of waste (explained in introduction lines 59 to 61). On the other hand, environmentally friendly approaches have received a lot of attention recently, Application of technological innovation to solve environmental problems is a bet for the future of the planet.

Reviewer 3 Report

The manuscript is of particular interest to specialists. It is good that the authors understand the difference between the results obtained in the laboratory on nutrient media and in natural soil conditions. In general, there are no critical comments.

From the recommendations: To determine the concentration of Cr(VI), it would be better to use a more sensitive method, for example, atomic absorption or inductively coupled plasma method. In order to enhance the practical significance, it would be nice, based on the basic parameters of the tested media (pH, nutrient content), to propose the types of soils on which, according to the authors, the best ecological effect would result from the introduction of strains of microorganisms cultivated in the laboratory.

Author Response

Answers in red following the reviewer'd text, additions in blue

"The manuscript is of particular interest to specialists. It is good that the authors understand the difference between the results obtained in the laboratory on nutrient media and in natural soil conditions. In general, there are no critical comments."

we would like to thank Reviewer for these positive comments

From the recommendations: To determine the concentration of Cr(VI), it would be better to use a more sensitive method, for example, atomic absorption or inductively coupled plasma method.

Yes we are aware that determining the speciation of chromium in both the liquid phase and the biomass would be valuable for future studies, specially if we will find some chromium reductases in the isolated strains

A sentence related to that has benn added (lines 548-550)

In order to enhance the practical significance, it would be nice, based on the basic parameters of the tested media (pH, nutrient content), to propose the types of soils on which, according to the authors, the best ecological effect would result from the introduction of strains of microorganisms cultivated in the laboratory.

According to the nutrient content of the medium it is rather possible to use these strains (mixture of several strains) in bioprocesses rather than use them directly in the tailing wastes.

it has been stated in the last sentence 

It will be rather possible to use a mixture of several of these strains in bioprocesses /bioreactors rather than use them directly in the tailing wastes.

Round 2

Reviewer 1 Report

The authors responded and/or corrected all the comments mentioned.