Statistical Optimisation and Kinetic Studies of Molybdenum Reduction Using a Psychrotolerant Marine Bacteria Isolated from Antarctica
, Azham Zulkharnain 2, Suriana Sabri 3, Claudio Gomez-Fuentes 4,5, Peter Convey 6, Khalilah Abdul Khalil 7 and Siti Aqlima Ahmad 1,5,8,*Round 1
Reviewer 1 Report
The authors of the article entitled "Statistical Optimisation and Kinetic Studies of Molybdenum-reduction Using a Psychrotolerant Marine Bacteria Isolated 3 from Antarctica” describes the reduction of Mo by a psychrotolerant Antarctic marine bacterium, Marinomonas sp. strain AQ5-A9. Mo reduction was increased optimizing the carbon, nitrogen, and growth conditions by using One-Factor-At-a-Time (OFAT) and Response Surface Methodology (RSM).
The article is well written, and the topic is interesting.
Only a minor revision is suggested for this work:
Line 68 could the authors improve the information about Mo-blue compound
Line 87 Could you provide more information about the sample site where the Marinonomas sp. strain AQ5-A9 has been isolated?
Line 90, please could you clarify why used NB supplemented with 5% of NaCl?
Line 101 when you write “salinity,” you mean NaCl? If yes, you used the maximum salinity of 80 ppt. How much percentage corresponds?
Line 107, in the Materials and methods section, you say that “The sample was incubated for eight days at 150 rpm on an orbital shaker at 10 °C” however, in the result section, the experiments were carried out at 15 °C.
Line 180 Figure 2 error bars were not visible; please increase the quality of the figure.
Line 188-190 The concept of the sentence is not clear. Please could you rephrase this sentence?
Line 310 the optimum salinity “the optimal growth value of salinity “
Line 348 Please could you add more detail “a Mo-reducing Antarctic soil bacterium” which bacterium?
Line 362 Did you suggest the phosphate concentration as another variable to growth/ Mo-reduction of the bacterium?
Line 444 Although the kinetic is not comparable with other studies could you compare the efficiency to reduce molybdenum with other strains?
Author Response
Comment 1
Line 68 could the authors improve the information about Mo-blue compound
Answer: Sentence structure describing the compound was improved. Mo-blue is simply a precipitable compound as product of metal reduction. More information is provided in the supplementary material in the figure on Mo-reduction pathway. Page 2. Line 68. Comment 2 Line 87 Could you provide more information about the sample site where the Marinomonas sp. strain AQ5-A9 has been isolated? Answer: Information on the sample site where the strain was isolated was added. Page 2. Line 90-92. Comment 3 Line 90, please could you clarify why used NB supplemented with 5% of NaCl? Answer: The NB supplemented with 5% of NaCl was used in the preliminary screening i.e., the bacterial isolation stage, as we aim to isolate halotolerant bacterium that is able to grow in a saline condition above the standard salinity percentage of seawater (~3.5%). The 5% was later used to provide saline condition to the bacteria grown in NB before culturing in low phosphate media with various salt concentration for optimisation. Comment 4 Line 101, when you write “salinity,” you mean NaCl? If yes, you used the maximum salinity of 80 ppt. How much percentage corresponds? Answer: Yes, in this study, the term ‘salinity’ means the salt NaCl content. 80 ppt is 8% of NaCl. Page 3. Line 103. Comment 5 Line 107, in the Materials and methods section, you say that “The sample was incubated for eight days at 150 rpm on an orbital shaker at 10 °C” however, in the result section, the experiments were carried out at 15 °C. Answer: As we are doing dependent optimisation, the parameters following the temperature factor (Figure 3-6) applied the value of optimised temperature, 15 °C (Figure 2). The structure of the sentence in Section 2.2 was improved and information on dependent optimisation was added according to the result in 3.1. Page 3. Line 103. Comment 6 Line 180, Figure 2 error bars were not visible; please increase the quality of the figure. Answer: Figure 2 was improved to make the error bars visible. Comment 7 Line 188-190, The concept of the sentence is not clear. Please could you rephrase this sentence? Answer: Sentence rephrased.“Figure 3a exhibited the use of sucrose as carbon source led to the greatest Mo reduction. Tukey’s test (p < 0.01) showed significant difference between sucrose and the second-best carbon source, glucose.” Page 5. Line 193-195. Comment 8 Line 310, the optimum salinity “the optimal growth value of salinity“ Answer: Sentence changed according to the comment. Page 6: Line 334-335. Comment 9 Line 348, Please could you add more detail “a Mo-reducing Antarctic soil bacterium” which bacterium? Answer: The bacterium Pseudomonas sp. strain DRY1 was mentioned. Page 13, 372 -373. Comment 10 Line 362, Did you suggest the phosphate concentration as another variable to growth/ Mo-reduction of the bacterium? Answer: The phosphate concentration was not included as another variable in this study as we referred to previous literature on the optimum concentration of phosphate.(Ahmad, S.A.; Shukor, M.Y.; Shamaan, N.A.; MacCormack, W.P.; Syed, M.A. Molybdate reduction to molybdenum blue by an Antarctic bacterium. BioMedical Res Int 2013, 2013, 871941 https://doi.org/10.1155/2013/871941) Comment 11 Line 444, Although the kinetic is not comparable with other studies could you compare the efficiency to reduce molybdenum with other strains? Answer: We are not able to compare the efficiency to reduce molybdenum with other strains as most papers are reporting on the molybdenum reduction kinetic using enzyme, thus the measuring unit is not using absorbance (OD865nm).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
the Authors investigate the reduction of Mo by a psychrotolerant Antarctic marine bacterium, Marinomonas sp. strain AQ5-A9, and optimize the reduction of Mo reduction using One-Factor-At-a-Time (OFAT) and Response Surface Methodology (RSM).
The rationale of the study is well described and the findings of the study are well described.
Some specific points:
Abstract: English language should be improved.
Line- 54-56: rephrase
Line 119-130: many details included - can be moved to supplementary data with table 1
Line 147- 153: many details included - can be moved to supplementary data
Line 172-174: what does the sentence mean??
The text include many figures that some should move to supplementary data
Line 208: the authors should write the names of nitrogen salts
Line 261-264: more explanation about the results
Line 273-277: the authors repeated some sentences from the material and method part. The authors should improve the description of the results
Line 364: the authors should support some parts of the discussion with further studies.
Author Response
Comment 1
Abstract: English language should be improved.
Answer: A few corrections in the abstract were made.
Comment 2
Line- 54-56: rephrase
Answer: A few changes were made to improve the sentence. Line 54 -55.
Comment 3
Line 119-130: many details included - can be moved to supplementary data with table 1
Answer: In this section, the details are necessary to describe how the experiment was designed and will be analysed. The section was divided into two paragraphs to ensure clarity for readers. Page 3: Line 126-137.
Comment 4
Line 147- 153: many details included - can be moved to supplementary data
Answer: The details are necessary to inform how the experiment will be analysed.
Comment 5
Line 172-174: what does the sentence mean??
Answer: Sentence was paraphrased. “Meanwhile, bacterial growth steadily increased across the temperature range tested. However, there was some indication of a stress response in growth at 20 °C and 25 °C as bacterial cells and Mo-blue were observed to be precipitated at the bottom of the culture flask.” Page 4. Line 178-181.
Comment 6
The text include many figures that some should moved to supplementary data
Answer: All figures is important. Sorry cannot move to supplementary data.
Comment 7
Line 208: the authors should write the names of nitrogen salts.
Answer: The name of the nitrogen salts added. Page 7. Line 215 & 216.
Comment 8
Line 261-264: more explanation about the results
Answer: The explanation of the 3D response surfaces result of was added in section 3.2. Further discussion of the result is in section 4.2.2.
Comment 9
Line 273-277: the authors repeated some sentences from the material and method part. The authors should improve the description of the results.
Answer: Changes in the sentences were made to improve the description of the result. Page 11: Line 294-301.
Author Response File: Author Response.pdf
Reviewer 3 Report
The work entitled “Statistical Optimisation and Kinetic Studies of Molybdenum Reduction Using a Psychrotolerant Marine Bacteria Isolated from Antarctica” by Michal et al has been reviewed. The work is described the interesting data can be considering for publication after the following corrections.
- In the table 1, why the variable D have -1.24 at “-2 experimental value”. in that case how the media is prepared. Why the not displayed the experimental model as separate table?
- The Mo is completely removed or absorbed by the bacterial biomass, proved the mechanism of Mo reduction
- Some of the abbreviations are not explained in the Table or text.
- Why the authors not screened the important factors that influence the reduction of Mo?
- Is it acceptable to use the CCD to optimize more than 4 factors?
- Authors not provided detailed methods to measure the reduction of the Mo by OD value?
- UV- data can only provide the information about the color reduction. How can we confirm that the bacteria removed the Mo or reduced color that formed by dissolving the Mo in water?
- It is better to study the Mo reduction by HPLC rather than UV spectrophotometry.
- Also the RSM methods is not appropriately used in this study that need the critical attention
- Why the temperature is not significantly influenced the reduction of the Mo (table.2.) since the microbe was isolated from the Antarctic region that expected to have the response to temperature. This need the detail explanation or correction.
- Also why not with Molybdate concentration?
- According to the able 2, the significant interaction were found only with combination of AB, AE, BC, BE, BF, CE, DF and EF. But not proved the perturbation graph and SE group as well ?
- The Plackeet Burman is used for screening of factor or CCD?
Author Response
Comment 1
In the table 1, why the variable D have -1.24 at “-2 experimental value”. in that case how the media is prepared. Why the not displayed the experimental model as separate table?
Answer: As variable D is ammonium sulphate concentration, the value -1.24 g/L generated by software is being considered as 0 g/L i.e., no ammonium sulphate is being added in the media. The experimental design is being provided in separate table in the supplementary material of the manuscript.
Comment 2
The Mo is completely removed or absorbed by the bacterial biomass, proved the mechanism of Mo reduction.
Answer: The exact mechanism of Mo reduction in bacteria is still not yet fully understood. However, a study by Ariff et al., 1997 suggests that the enzyme phosphomolybdate reductase was an intercellular enzyme and some may be released out from the cell. High activity of Mo reduction observed in cell extracts, soluble protein and the membrane fraction (after cell disruption) at approximately same levels. (Ariff, A.B.; Rosfarizan, M.; Ghani, B.; Sugio, T.; Karim, M.I.A.Molybdenum reductase in Enterobacter cloacae. World Journal of Microbiology & Biotechnology 1997, 12, 643-647.)
Comment 3
Some of the abbreviations are not explained in the Table or text.
Answer: A few abbreviations in manuscript were edited and/or explained.
Comment 4
Why the authors not screened the important factors that influence the reduction of Mo?
Answer: We have screened the significant factors that influenced the reduction of Mo using Plackett-Burman Design (PBD) before we proceed to Central Composite Design (CCD). However, the data are not included due to the large number of data already included in the manuscript. All six parameters (A-F) screened significantly affect the Mo reduction are being optimised using CCD in Section 3.2.
Comment 5
Is it acceptable to use the CCD to optimize more than 4 factors?
Answer: Yes, it is acceptable as the increase in the number of factors results in the increase of experimental runs in the factorial portion (n = 2k+ 2k +n0, where k is the number of variables and n0 is the number of centre points) to maintain the CCD’s rotatability.
Comment 6
Authors not provided detailed methods to measure the reduction of the Mo by OD value?
Answer: Method to measure Mo reduction was provided in section 2.2.
Comment 7
UV- data can only provide the information about the color reduction. How can we confirm that the bacteria removed the Mo or reduced color that formed by dissolving the Mo in water?
Answer: The culture media containing Mo will not turn blue until the Mo-reducing bacteria is added. This proved that the bacteria and the molybdenum reductase enzyme is responsible for the reduction.
Comment 8
It is better to study the Mo reduction by HPLC rather than UV spectrophotometry.
Answer: Various heterpolymolybdates can be differentiated and seen in the scanning spectra of their reduced form, Mo-blue. The study of Mo reduction using UV spectrophotometry is preferred by previous studies as the product, Mo-blue, can be readily measured at wavelength 865 nm.
Comment 9
Also the RSM methods is not appropriately used in this study that need the critical attention.
Answer: There are two designs being employed in the RSM.
The first design, Plackett-Burman Design (PBD) was used screening the significant factors. All six factors were found to significantly influence the Mo reduction. However, the data is not being included.
The second step was the optimisation of Mo using Central Composite Design (CCD). This design operate around all six significant parameters that were previously screened using PBD. The number of parameters used in CCD are acceptable as the increase in the number of factors results in the increase of experimental runs in the factorial portion. A total of 86 experimental runs were conducted to ensure the optimisation are appropriately done. Comment 10 Why the temperature is not significantly influenced the reduction of the Mo (table.2.) since the microbe was isolated from the Antarctic region that expected to have the response to temperature. This need the detail explanation or correction. Answer: Detailed explanation added in section 4.2.1. “The analysis also confirmed that three factors, B (temperature), E (molybdate concentration) and F (pH) are not significant to the Mo reduction. Although the bacterial strain was isolated from the Antarctic seawater, its metabolism is not largely affected by the temperature due to its ability to thrive in wide range of temperature.” Line 398-401. Comment 11 Also why not with Molybdate concentration? Answer: Detailed explanation added in section 4.2.1. “Molybdate concentration and pH are also not significant to the reduction as the experimental range (Table 1) does not include the inhibitive concentration of the substrate and is within the acidic condition for reduction, respectively.” Line 401-404. Page 14. Comment 12 According to the Table 2, the significant interactions were found only with combination of AB, AE, BC, BE, BF, CE, DF and EF. But not proved with the perturbation graph and SE group as well? Answer: This study included the data from OFAT, RSM and kinetic studies. The addition of the perturbation graph is quite redundant and as the data on the significant interactions have already been provided. Comment 13 The Plackett-Burman is used for screening of factor or CCD? Answer: Plackett-Burman Design (PBD) was used for preliminary screening the significant factors that influenced the reduction of Mo using Plackett-Burman Design (PBD) before we proceed to Central Composite Design (CCD).
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Accept
