Effects of Blue-Light Laser Irradiation on the Enzymatic Activities and Sporulation of Trichoderma atroviride Grown on Rice Husks

Round 1

Reviewer 1 Report

The manuscript submitted by Ioana-Alexandra Bala et al. entitled "Effects of blue-light laser irradiation on the enzymatic activities and sporulation of Trichoderma atroviride grown on rice husks" is well written, well organized, the discussion of the results is acceptable, providing a good reading experience. The submitted manuscript is perfectly suitable for the Applied Sciences journal's scope.

However, I have some suggestions and comments that the authors should address, such as:

a) The optical apparatus used to irradiate the samples is made very straightforwardly, perfect for the reader's understanding and reproduction. Although, the authors must include the average optical power used in the experiments in the revised version of the manuscript. Please, also indicate which type of optical power meter was used to make this measurement.

b) Did the authors check the laser emission stability? If yes, please report the uncertainty associated with the laser emission over time (mainly covering the time range used in the experiment).

c)  The last but not least important laser parameter is the transversal spatial profile of the laser. Did the authors check which is the profile of the laser emission?

d) In line 95, the authors wrote, "... compared to the laser light which is very concentrated." Please, re-write this sentence once the term concentrated is unsuitable for the laser field of context.

e) The optical intensity of the laser beam (line 150), must be calculated in terms of the ratio between optical average power and the area of the laser beam (collimated in the sample). Therefore, in line 150, the authors must modify the "laser intensity of 1.5 A (0.271μmoles/s)" by W/cm^2, for example. If they wish to present the irradiated dose, they can do it once they have the time exposure (or irradiation time).

f) It needs to be clarified if the authors used the transmittance of reflectance mode to record the Absorbance in Figure 4a). It needs to be clarified which equipment (model) they used to record the Absorbance.

1) Please indicate the model of Ocean Insight (former Ocean Optics) optical spectrometer used;

2) If transmittance mode is used, please specify the type of cuvette as well as its optical path;

3) The authors should also report the concentration of the samples (if transmittance mode is used);

This is highly relevant because I need to understand better the true nature of the high energy absorbance peaks (220 to 250 nm) in Figure 4a) once the authors claim such peaks in the discussion of their results.

g) Regardless of the method to acquire the absorbance spectra, the physical quantity absorbance is unitless! It is, unfortunately, a common mistake to use units in the physical quantity absorbance. Please, remove the arbitrary units from Absorbance once this quantity is unitless! Absorbance (A) = -log(I/I₀);

I will decide on the manuscript's acceptance only after the authors address my comments (well addressed).

 The English language is almost fine; a few minor editing of the English language is required.

Author Response

The manuscript submitted by Ioana-Alexandra Bala et al. entitled "Effects of blue-light laser irradiation on the enzymatic activities and sporulation of Trichoderma atroviride grown on rice husks" is well written, well organized, the discussion of the results is acceptable, providing a good reading experience. The submitted manuscript is perfectly suitable for the Applied Sciences journal's scope.

However, I have some suggestions and comments that the authors should address, such as:

a) The optical apparatus used to irradiate the samples is made very straightforwardly, perfect for the reader's understanding and reproduction. Although, the authors must include the average optical power used in the experiments in the revised version of the manuscript. Please, also indicate which type of optical power meter was used to make this measurement.

Response: Thank you for the positive appreciations and suggestions to improve the manuscript. We have added the required Information, please check 2.3 (lines 150-166), including Table 1 with the power determined for several intensities of the laser, from the minimum to the maximum. The values used in this study were underlined in Table 1. The optical power meter used was Coherent PM2 (Coherent, Santa Clara, CA, USA).

b) Did the authors check the laser emission stability? If yes, please report the uncertainty associated with the laser emission over time (mainly covering the time range used in the experiment).

Response: The laser manufacturer declares the RMS stability of 2%, measured over a 4 hours period after the initial warm-up phase. We added this Information (lines 152-153)

c)  The last but not least important laser parameter is the transversal spatial profile of the laser. Did the authors check which is the profile of the laser emission?

Response: The laser manufacturer declares that the transversal spatial profile of the laser is near TEM₀₀. We have added this Information (lines 153-154)

d) In line 95, the authors wrote, "... compared to the laser light which is very concentrated." Please, re-write this sentence once the term concentrated is unsuitable for the laser field of context.

Response: We have re-written the sentence, changing to: “compared to the laser light which is focused and highly localized”. We hope this is more appropriate.

e) The optical intensity of the laser beam (line 150), must be calculated in terms of the ratio between optical average power and the area of the laser beam (collimated in the sample). Therefore, in line 150, the authors must modify the "laser intensity of 1.5 A (0.271μmoles/s)" by W/cm^2, for example. If they wish to present the irradiated dose, they can do it once they have the time exposure (or irradiation time).

Response: We have added the equivalent requested expression and the calculated doses for each time period of irradiation, 60 s and 300 s. (lines 169-173). We also added Appendix A with a short description and theory of parameter transformations, for the non-specialist reader to understand easier (lines 482-501).

f) It needs to be clarified if the authors used the transmittance of reflectance mode to record the Absorbance in Figure 4a). It needs to be clarified which equipment (model) they used to record the Absorbance.

1) Please indicate the model of Ocean Insight (former Ocean Optics) optical spectrometer used;

Response. We added this information - UV-VIS-NIR USB 2000+(Ocean Optics (now Ocean Insight), Orlando, FL, USA),

2) If transmittance mode is used, please specify the type of cuvette as well as its optical path;

Response: a quartz cuvette (Quartz SUPRASIL, Hellma Analytics, Müllheim, Germany), light path = 10 mm.

3) The authors should also report the concentration of the samples (if transmittance mode is used);

This is highly relevant because I need to understand better the true nature of the high energy absorbance peaks (220 to 250 nm) in Figure 4a) once the authors claim such peaks in the discussion of their results.

Response: The transmittance mode was used to record the absorbance. We added this Information and the requested Information at points 1 and 2 in subchapter 2.6 (lines 229-234). We have determined the refractive index and dry mass of the samples, as this was the only way to determine the concentration. We have presented the analysis details in subchapter 2.7 (lines 235 – 239) and the results were presented at lines 376-384.

g) Regardless of the method to acquire the absorbance spectra, the physical quantity absorbance is unitless! It is, unfortunately, a common mistake to use units in the physical quantity absorbance. Please, remove the arbitrary units from Absorbance once this quantity is unitless! Absorbance (A) = -log(I/I₀);

Response: Thank you for the correct observation. We removed arbitrary units from Figure 4.

I will decide on the manuscript's acceptance only after the authors address my comments (well addressed).

We hope we have addressed all comments as expected. If not the case, we welcome any additional observation that would help improve our manuscript. Thank you again for all the suggestions.

Reviewer 2 Report

The authors showed that blue laser light irradiation had an inducing effect on cellulase and protease activities and on conidia dimensions of Trichoderma. The authors show very interesting research results in this study.

 The authors need to make some revisions to the content of this paper.

 The authors should make the title of 2.3 more specific. For example (method of laser light irradiation), etc.

 The authors need to make the data in Figure 8 more comprehensible. This data should be supplemented with data from non-irradiated samples and short irradiations.

Author Response

The authors showed that blue laser light irradiation had an inducing effect on cellulase and protease activities and on conidia dimensions of Trichoderma. The authors show very interesting research results in this study.

 The authors need to make some revisions to the content of this paper.

Point 1: The authors should make the title of 2.3 more specific. For example (method of laser light irradiation), etc.

Response 1: Thank you for your positive appreciations. We improved the title of 2.3 and we hope it is more specific now:

Experimental design of Trichoderma irradiation with blue-violet-light laser

Point 2: The authors need to make the data in Figure 8 more comprehensible. This data should be supplemented with data from non-irradiated samples and short irradiations.

Response 2: Thank you for your valuable comment. We have added supplementary figures for non-irradiated and short-irradiated samples and modified the legend accordingly. We also highlighted in the figures of 3x300s with arrows and geometrical forms how the conidia migrate and where they cluster. We hope the data in Figure 8 are more comprehensible now.

Round 2

Reviewer 1 Report

The revised manuscript submitted by Ioana-Alexandra Bala et al. addressed most of this reviewer's comments. Still, a couple of issues must be corrected for the manuscript to have my acceptance to be published.

1) The authors decided to include Table 1 in the revised version of the manuscript. However, modifications must be made to Table 1:

- The authors correctly use the quantity Average Power (mW). However, because they are using Electrical Intensity (pumping source of the diode laser), not Optical Intensity, they must replace Average Power with Optical Average Power (mW). If they wish to keep the Electrical Intensity they must change it by Pump Electrical Current (mA).  

- I suggest that they remove the "Intensity (mA). Moreover, the Intensity of the driving current to pump the diode laser is not helpful for the reader once the essential parameters are the Optical Average Power, Optical Intensity, Beam Size, and Irradiation Time. All these parameters are needed to calculate the Irradiation Dose, or simply, Dose.

- Regarding the importance of the Dose, it is clear that the authors and this reviewer are in explicit agreement. Still, they need to calculate the Dose correctly!

- As an example, the authors could check Table 1 from a recent work published in MDPI journals by Zaidem, Antonia, et al. "New Biocompatible Technique Based on the Use of a Laser to Control the Whitefly Bemisia tabaci." Photonics. Vol. 10. No. 6. MDPI, 2023. Moreover, the second paragraph above the Table from the mentioned paper briefly discusses the physical quantity Dose. I suggest the authors follow such a discussion.

2) The discussion of lines 169-174 must be deleted for two reasons:

i) In the photonics field, using moles as a unit for counting individual photons is not shared. Moreover, the approach provided by the authors could mislead the reader! Counting photons is typically done on an individual basis rather than in moles. As the authors well know, moles are used as a unit for counting a large number of particles, such as atoms, molecules, or ions, based on Avogadro's number. 

ii) Having an optical power meter available to quantify the optical average power, knowing the beam spot size* and the irradiation time, is enough to estimate the Dose, which is the crucial parameter of this work.

* If the authors do not have access to any equipment to measure the laser beam size precisely, they can do it roughly since they provide a suitable uncertainty to this measure, which will be carried out to the irradiation dose.

Finally, the authors must understand that if one decides to use this technique will only be able to achieve the same results if the optical parameters are well described and reported!

Minor editing of English language required

Author Response

The revised manuscript submitted by Ioana-Alexandra Bala et al. addressed most of this reviewer's comments. Still, a couple of issues must be corrected for the manuscript to have my acceptance to be published.

 We want to thank the reviewer for the very useful and well-informed comments. We think we have corrected the issues described by the reviewer.

1) The authors decided to include Table 1 in the revised version of the manuscript. However, modifications must be made to Table 1:

- The authors correctly use the quantity Average Power (mW). However, because they are using Electrical Intensity (pumping source of the diode laser), not Optical Intensity, they must replace Average Power with Optical Average Power (mW). If they wish to keep the Electrical Intensity they must change it by Pump Electrical Current (mA).  

We have made the suggested modifications. We thank the reviewer for the informative suggestions. We have also moved the Table to Appendix A.

- I suggest that they remove the "Intensity (mA). Moreover, the Intensity of the driving current to pump the diode laser is not helpful for the reader once the essential parameters are the Optical Average Power, Optical Intensity, Beam Size, and Irradiation Time. All these parameters are needed to calculate the Irradiation Dose, or simply, Dose.

We replaced the Intensity (mA) Pump Electrical Current (mA). We have payed close attention to use the essential parameters described by the reviewer with the required names.

- Regarding the importance of the Dose, it is clear that the authors and this reviewer are in explicit agreement. Still, they need to calculate the Dose correctly!

We completely agree. We have reviewed our calculations carefully and have made sure that we have calculated the dose correctly.

- As an example, the authors could check Table 1 from a recent work published in MDPI journals by Zaidem, Antonia, et al. "New Biocompatible Technique Based on the Use of a Laser to Control the Whitefly Bemisia tabaci." Photonics. Vol. 10. No. 6. MDPI, 2023. Moreover, the second paragraph above the Table from the mentioned paper briefly discusses the physical quantity Dose. I suggest the authors follow such a discussion.

We have checked the suggested article and have employed the relevant information into our article whilst also citing the article when discussion how the dose was calculated and what assumptions were made. This was done in an added paragraph in section 2.3. (lines 164 to 173).

 2) The discussion of lines 169-174 must be deleted for two reasons:

1. i)In the photonics field, using moles as a unit for counting individual photons is not shared. Moreover, the approach provided by the authors could mislead the reader! Counting photons is typically done on an individual basis rather than in moles. As the authors well know, moles are used as a unit for counting a large number of particles, such as atoms, molecules, or ions, based on Avogadro's number. 
2. ii)Having an optical power meter available to quantify the optical average power, knowing the beam spot size*and the irradiation time, is enough to estimate the Dose, which is the crucial parameter of this work.

We have removed the discussion of lines 169-174 and agree with the reasons suggested by the reviewer. We have also added a table which presents the relative experimental error concerning beam size, beam surface, average optical power and optical intensity and the associated error values calculated through propagation of errors.

* If the authors do not have access to any equipment to measure the laser beam size precisely, they can do it roughly since they provide a suitable uncertainty to this measure, which will be carried out to the irradiation dose.

This suggestion is very useful and we have added a paragraph (lines 176 - 183) explaining how the uncertainty was estimated. We have also estimated the uncertainty of the irradiation doses as required by the reviewer.

Finally, the authors must understand that if one decides to use this technique will only be able to achieve the same results if the optical parameters are well described and reported!

We completely agree with the reviewer and we are very thankful for the well-informed comments. We hope that the optical parameters are now well described, and that this technique can be reproduced

Round 3

Reviewer 1 Report

The manuscript can be accepted for publication once the authors have corrected the optical parameters used in their work.

Minor editing of English language required