Laccase Production from Local Biomass Using Solid State Fermentation
, Zahid Anwar 1, Waseem Khalid 2, Fareed Afzal 2, Muddassar Zafar 1, Usman Ali 1, Mohammed Y. Refai 3, Mohamed Afifi 3,4,5, Ammar AL-Farga 3 and Moneera O. Aljobair 6,*
Round 1
Reviewer 1 Report (New Reviewer)
Materials and Methods
The pure culture of A.niger IBP2013 available in Biochemistry Lab, Department of Biochemistry and Biotechnology, University of Gujrat Pakistan.
Add the reference of this strain or Accession no In Gen Bank.
The media medium needs to.
Medium is singular and media for plural. Please check in the whole text
produced prepared PD medium,
at 30°Cat 120rpm. Add space at 30°C at 120 rpm
Agitation improves dissolved oxygen ------- cell structures, varying fungal growth, and altering yield formation, it can affect the morphology of the fungus [28].
This is discussion not in the M&M part
All selected substrates were inoculated by adding 3mL of spore suspension of A. niger. Then adjusting temperature at 35±1 °C for 4-5 days.
Is this temp. the most suitable for Fungi a and laccase production. Add ref.
Laccase activity as -- was assayed
guaiacol : Mention the source of this chemical and all the chemical used in the manuscript
spectrophotometer Mention the description of this instrument
According to Kalra et al., 2013. Follow the journal format style
The supernatant of the cultured fermented culture.
Comments for author File: 
Comments.pdf
Author Response
Comments and Suggestions for Authors
Materials and Methods
The pure culture of A.niger IBP2013 available in Biochemistry Lab, Department of Biochemistry and Biotechnology, University of Gujrat Pakistan.
Add the reference of this strain or Accession no In Gen Bank.
Reference no 26 added.
The media medium needs to.
Medium is singular and media for plural. Please check in the whole text
produced prepared PD medium,
Corrected in whole text.
at 30°Cat 120rpm. Add space at 30°C at 120 rpm
Corrected
Agitation improves dissolved oxygen ------- cell structures, varying fungal growth, and altering yield formation, it can affect the morphology of the fungus [28].
This is discussion not in the M&M part
Remove from M&M part and added in discussion part.
All selected substrates were inoculated by adding 3mL of spore suspension of A. niger. Then adjusting temperature at 35±1 °C for 4-5 days.
Is this temp. the most suitable for Fungi a and laccase production. Add ref.
Reference no 18 added
Laccase activity as -- was assayed
Corrected
guaiacol : Mention the source of this chemical and all the chemical used in the manuscript
spectrophotometer Mention the description of this instrument
Source and description added.
According to Kalraet al., 2013. Follow the journal format style
Corrected
The supernatant of the cultured fermented culture.
Corrected
Comments and Suggestions for Authors
The publication is interesting and provides new insights into the use of renewable raw materials for the production of laccases. Laccase from Aspergillus niger is commercially available, its applications are increasing especially for the modification of fibrous materials and for the treatment of dyes in water.
The abstract is complete.
The introduction as well as literature and basic principles are presented and described in great detail, some of the sources are older and too broad. Please check the sources, there are duplications, please format more carefully. The introduction does not focus on the essentials with respect to the experiments.
Duplications have been removed. Introductory part has modified with respect to experiments.
The experimental design for the many parameters is correct and commendable, there is a lack of information on the replicates (n=?) of the experiments of the experimental design. The use of 5.0 g of material for a solid-state fermentation is very small in a vessel volume of 250 ml.
In section 2.8 numbers of replicates have been added. In section 3.2, experimental design (CCD) has been discussed.
The 5g material of substrate was used basically to check the concentration of substrates on the enzyme production. And due to availability of 250ml vessel, it was used for experiments.
The methods lack information on the equipment and chemicals used (suppliers, manufacturers, material and equipment type designations). Information on the experiments, such as residence times and the number of replicates, is missing in some cases. A protein determination, e.g. according to Bradford or Lowry, for the detection of active/inactive enzymes and possible foreign proteins is missing. The evaluation of the statistical experimental design should be tabulated and the individual results should be presented more clearly. Results of chromatographic separation/elution are missing in one figure.
The information about chemicals and description with working principle of instruments has added. Protein determination assay was not performed due to lack of chemicals required for this assay anyhow other purification techniques (ammonium sulfate precipitation, dialysis and chromatography) have applied for obtaining pure enzyme.
Error calculations and bars are missing in the diagrams for pH and temperature screening.
Line chart was used for the characterization of temperature and pH. So there was no information about error.
The text should be significantly shortened overall and concentrated on the essentials.
Text modification done.
What is with reference 21 and 29, they are the same.
Reference 29 has removed and written as 21.
I have not marked up all points of criticism
Author Response File: 
Author Response.docx
Reviewer 2 Report (New Reviewer)
The publication is interesting and provides new insights into the use of renewable raw materials for the production of laccases. Laccase from Aspergillus niger is commercially available, its applications are increasing especially for the modification of fibrous materials and for the treatment of dyes in water.
The abstract is complete.
The introduction as well as literature and basic principles are presented and described in great detail, some of the sources are older and too broad. Please check the sources, there are duplications, please format more carefully. The introduction does not focus on the essentials with respect to the experiments.
The experimental design for the many parameters is correct and commendable, there is a lack of information on the replicates (n=?) of the experiments of the experimental design. The use of 5.0 g of material for a solid-state fermentation is very small in a vessel volume of 250 ml.
The methods lack information on the equipment and chemicals used (suppliers, manufacturers, material and equipment type designations). Information on the experiments, such as residence times and the number of replicates, is missing in some cases. A protein determination, e.g. according to Bradford or Lowry, for the detection of active/inactive enzymes and possible foreign proteins is missing. The evaluation of the statistical experimental design should be tabulated and the individual results should be presented more clearly. Results of chromatographic separation/elution are missing in one figure.
Error calculations and bars are missing in the diagrams for pH and temperature screening.
The text should be significantly shortened overall and concentrated on the essentials.
What is with reference 21 and 29, they are the same.
I have not marked up all points of criticism
Comments for author File: Comments.pdf
Author Response
Comments and Suggestions for Authors
The publication is interesting and provides new insights into the use of renewable raw materials for the production of laccases. Laccase from Aspergillus niger is commercially available, its applications are increasing especially for the modification of fibrous materials and for the treatment of dyes in water.
The abstract is complete.
The introduction as well as literature and basic principles are presented and described in great detail, some of the sources are older and too broad. Please check the sources, there are duplications, please format more carefully. The introduction does not focus on the essentials with respect to the experiments.
Duplications have been removed. Introductory part has modified with respect to experiments.
The experimental design for the many parameters is correct and commendable, there is a lack of information on the replicates (n=?) of the experiments of the experimental design. The use of 5.0 g of material for a solid-state fermentation is very small in a vessel volume of 250 ml.
In section 2.8 numbers of replicates have been added. In section 3.2, experimental design (CCD) has been discussed.
The 5g material of substrate was used basically to check the concentration of substrates on the enzyme production. And due to availability of 250ml vessel, it was used for experiments.
The methods lack information on the equipment and chemicals used (suppliers, manufacturers, material and equipment type designations). Information on the experiments, such as residence times and the number of replicates, is missing in some cases. A protein determination, e.g. according to Bradford or Lowry, for the detection of active/inactive enzymes and possible foreign proteins is missing. The evaluation of the statistical experimental design should be tabulated and the individual results should be presented more clearly. Results of chromatographic separation/elution are missing in one figure.
The information about chemicals and description with working principle of instruments has added. Protein determination assay was not performed due to lack of chemicals required for this assay anyhow other purification techniques (ammonium sulfate precipitation, dialysis and chromatography) have applied for obtaining pure enzyme.
Error calculations and bars are missing in the diagrams for pH and temperature screening.
Line chart was used for the characterization of temperature and pH. So there was no information about error.
The text should be significantly shortened overall and concentrated on the essentials.
Text modification done.
What is with reference 21 and 29, they are the same.
Reference 29 has removed and written as 21.
I have not marked up all points of criticism
Author Response File: Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The authors report on an optimization procedure of laccase production by solid state fermentation using a native Aspergillus niger and locally available substrates. In addition, the enzymatic activity was subjected to different purification steps and the resulting extract was then partially characterized.
It is clear the technological importance of the research done. However, the article has some issues that should be addressed, as outlined below.
1) The novelty of the results should be highlighted.
2) Abstract, line 27:
Please revise the expression “inoculum size”
3) Line 87
Please revise the inclusion of reference 13.
4) Line 143
A reference should be added
5) Line 168
Please revise de section “inoculum preparation”
-Was a suspension of conidia or a mycelium used as inoculum?
-What do the authors mean with the following sentence?
“Shaking incubator delays the process of mycelia formation so the completely grown suspension of fungal spore was stored at 4°C”
6) Line 273
The purity of the enzymatic preparation should be tested in some way.
7) Line 278
What do the authors mean with “crude enzyme”?
8) Line 229
Please revise if there is a significant difference between wheat straw and guava leaves.
9) Line 310
Please revise here and throughout the manuscript the use of the expressions enzyme activity or enzyme production.
10) Line 320
As the experimental design studies all the factors at the same time, the authors should present the data taking this aspect into account. Thus, in my opinion, the subsections 3.1.1 to 3.1.4 are not necessary. The statistical analysis should also be included in this section. In addition, the physiological/technological meaning of factors interactions and quadratic terms should be discussed more deeply.
Author Response
Comments and Suggestions for Authors
The authors report on an optimization procedure of laccase production by solid state fermentation using a native Aspergillus niger and locally available substrates. In addition, the enzymatic activity was subjected to different purification steps and the resulting extract was then partially characterized.
It is clear the technological importance of the research done. However, the article has some issues that should be addressed, as outlined below.
1) The novelty of the results should be highlighted.
The goal of the study was to develop laccase from A. niger while also choosing low-cost, readily accessible high-yielding substrates.
The commercial development of laccases is being pursued in a cost-effective manner and through the screening of low-cost substrates. The use of agro-industrial waste as substrates adds value to the sector and raises knowledge of recycling and energy usage.
The current study focuses on producing, screening, optimizing, and purifying laccase from the local strain of A. nigerthat is readily available substrates in our vicinity.
2) Abstract, line 27:
Please revise the expression “inoculum size”
At 45°C, pH 5.0 and 2mL inoculum size, the isolated enzyme produced its highest level of activity.
3) Line 87
Please revise the inclusion of reference 13.
Correction done.
4) Line 143
A reference should be added
Reference no. 26 added.
5) Line 168
Please revise de section “inoculum preparation”
-Was a suspension of conidia or a mycelium used as inoculum?
Mycelium via spore germination
-What do the authors mean with the following sentence?
“Shaking incubator delays the process of mycelia formation so the completely grown suspension of fungal spore was stored at 4°C”
We need stock culture for further experiments. By continuously putting the inoculum into shaking incubator will slow down the mycelia formation due to components of media consumption by A. niger. So we need to preserve it for further culturing process.
6) Line 273
The purity of the enzymatic preparation should be tested in some way.
The purity of enzyme has been checked in 3 ways including Ammonium Sulfate precipitation, Dialysis and Colum Chromatography.
The particular substrate Guaiacol has also been used to ensure the production of Laccase enzyme.
7) Line 278
What do the authors mean with “crude enzyme”?
Crude enzyme means the 1st extraction of enzyme from sample. (without purified form)
8) Line 229
Please revise if there is a significant difference between wheat straw and guava leaves.
As the biomass screening graph (figure 2) clearly shown the value of enzyme activity of Guava leaves (2.21 U/mL). has less than wheat straw (2.551 U/mL). So our aim was that picking the highest activity gaining substrate.
9) Line 310
Please revise here and throughout the manuscript the use of the expressions enzyme activity or enzyme production.
Our aim is production of enzyme laccase and its production rate is checked by its activity. So we check the enzyme activity because enzyme activity relates to the number of substrates transformed to outputs every unit time.
Enzyme activity refers to the general catalytic properties of an enzyme, and enzyme assays are standardized procedures for measuring the amounts of specific enzymes in a sample.
10) Line 320
As the experimental design studies all the factors at the same time, the authors should present the data taking this aspect into account. Thus, in my opinion, the subsections 3.1.1 to 3.1.4 are not necessary. The statistical analysis should also be included in this section. In addition, the physiological/technological meaning of factors interactions and quadratic terms should be discussed more deeply.
In section 3.2 basically these all factors have been interrelated with previous studies to make it unique from other types of strains as well as substrates. In section 3.4 statically analysis has been done among different combinations of factors to check the interaction lf these factors for the production of enzyme.
Author Response File: Author Response.pdf
Reviewer 2 Report
This study tried to study the laccase production through solid state fermentation using various agricultural wastes. This manuscript needs a revision as listed below.
Introduction
Introduction should be more focused. Most part of the introduction is explaining only the laccase enzyme. Other aspects like solid state fermentation (SSF), the importance of optimization, what is the present need of SSF for laccase production should be included.
Line# 83: It can easily grow on pH from 1.4-9.8 and temperature 83 (6-47oC) while optimum temperature is 35-37oC – include the reference
No need to mention the full genus name Aspergillus after first mentioning. A. niger is enough- Correct it.
Materials and Methods
Line# 140: In 2.1 - AspergilllusnigerIBP2013 –correct it
Table 1: PDA is a well-known medium, no need to include the composition as a table. If authors willing to keep this information, then it should be in the text in a few lines. The composition should be g/L, not as g/200 mL
Remove the table 2
Line# 188: before being autoclaved for two hours to sterilize them – why the substrate was sterilized for 2 h?
Table 3 – correct the Ph
Line# 245: After that, the pellet was thrown away, and 1 mL of the supernatant was added to test tube together with 3 mL of sodium acetate 246 buffer pH 5 and 1 mL of guaiacol for the enzyme assay – ammonium sulfate precipitates the enzyme. Now the laccase is in the precipitate not in the supernatant. How did the enzyme assay conducted using the supernatant?
Results and Discussion
3.1. Biomass Screening – change it as Screening of biomass for laccase production. Similarly, the caption for Figure 2.
3.2 Repeatedly using Response Surface Methodology (RSM) – correct this (use only the abbreviation)
Include the type of design used in RSM
Line #327: 5 days when maximum laccase was produced – Include the activity of laccase
Table 4 –Include the predicted value by RSM. Similarly, the R2 , predicted R2, and the F-value of the
Design to show the significance of the model.
How about the validation experiment for the model? Nothing mentioned about running the experiment
under optimal conditions. Include the details.
Both contour and 3D plots are given for RSM analysis. No need to duplicate the presentation for the same data. It is unnecessarily increasing the figs. Keep only one type and delete the other.
I wonder to see the R sq. 1. Hence, include the values predicted by the design in the RSM table (4)
Finally, the authors’ decision on selecting the optimal conditions using RSM is not correct. They simply took the run number 6 and claimed that it is the optimal condition. Instead, they should analyze solutions using the RSM runs and then run a separate validation experiment under the optimum conditions in the solution given by the software.
Author Response
Comments and Suggestions for Authors
This study tried to study the laccase production through solid state fermentation using various agricultural wastes. This manuscript needs a revision as listed below.
Introduction
Introduction should be more focused. Most part of the introduction is explaining only the laccase enzyme. Other aspects like solid state fermentation (SSF), the importance of optimization, what is the present need of SSF for laccase production should be included.
Line # 70-91 added on need of SSF with references.
Line# 83: It can easily grow on pH from 1.4-9.8 and temperature 83 (6-47oC) while optimum temperature is 35-37oC – include the reference
Reference no. 18 added
No need to mention the full genus name Aspergillus after first mentioning. A. niger is enough- Correct it.
Corrected.
Materials and Methods
Line# 140: In 2.1 - AspergilllusnigerIBP2013 –correct it
Corrected
Table 1: PDA is a well-known medium, no need to include the composition as a table. If authors willing to keep this information, then it should be in the text in a few lines. The composition should be g/L, not as g/200 mL
Remove the table 2
Table 1 and Table 2 have been removed.
Line# 188: before being autoclaved for two hours to sterilize them – why the substrate was sterilized for 2 h?
Substrate without sterilization will most probably cause undesired microbial contamination to negatively affect the fermentation process. So we need to sterilize it for the better results of desired fermentation process.
Table 3 – correct the Ph
Corrected
Line# 245: After that, the pellet was thrown away, and 1 mL of the supernatant was added to test tube together with 3 mL of sodium acetate 246 buffer pH 5 and 1 mL of guaiacol for the enzyme assay – ammonium sulfate precipitates the enzyme. Now the laccase is in the precipitate not in the supernatant. How did the enzyme assay conducted using the supernatant?
Correction has been done. The pellet was taken for the enzyme assay.
Results and Discussion
3.1. Biomass Screening – change it as Screening of biomass for laccase production. Similarly, the caption for Figure 2.
Corrected
3.2 Repeatedly using Response Surface Methodology (RSM) – correct this (use only the abbreviation)
Corrected
Include the type of design used in RSM
Central composite design (CCD) added
Line #327: 5 days when maximum laccase was produced – Include the activity of laccase
3.206 U/mL added
Table 4 –Include the predicted value by RSM. Similarly, the R2 , predicted R2, and the F-value of the
Design to show the significance of the model.
Analysis of Variance
Source DF Adj SS Adj MS F-Value P-Value
Model 19 7.79432 0.410228 * *
Linear 6 0.40646 0.067744 * *
Substrate Size (g) 1 0.05847 0.058473 * *
pH 1 0.01027 0.010266 * *
Temperature 1 0.09015 0.090151 * *
Inoculum size (ml) 1 0.03451 0.034515 * *
Moisture Level (%) 1 0.00796 0.007958 * *
Incubation Days 1 0.29379 0.293788 * *
Error 0 * *
Total 19 7.79432
Model Summary
S R-sq R-sq(adj) R-sq(pred)
* 100.00% * *
That above was the design for the significance of model.
How about the validation experiment for the model? Nothing mentioned about running the experiment
under optimal conditions. Include the details.
The sub-sections of 3.2 explain all the optimal conditions of this experiment and shown optimal conditions of temperature, pH, inoculum size, incubation time and substrate size for gaining highest enzyme activity.
Both contour and 3D plots are given for RSM analysis. No need to duplicate the presentation for the same data. It is unnecessarily increasing the figs. Keep only one type and delete the other.
In a contour plot, the response surface is viewed as a two-dimensional plane where all points that have the same response are connected to produce contour lines of constant responses. A contour plot allows you to visualize three-dimensional data in a two-dimensional plot.
A surface plot generally displays a three-dimensional view that may provide a clearer picture of the response.
So both plots will be helpful to understand the results of different variables by 2D angle as well as 3D angle.
I wonder to see the R sq. 1. Hence, include the values predicted by the design in the RSM table (4)
Finally, the authors’ decision on selecting the optimal conditions using RSM is not correct. They simply took the run number 6 and claimed that it is the optimal condition. Instead, they should analyze solutions using the RSM runs and then run a separate validation experiment under the optimum conditions in the solution given by the software.
The run numbers basically were the trials to pick the right number under optimum conditions. So we need to run RSM to show the actual results and validate them with actual run number (Factors) picked by highest activity gained by run number 6.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors report on an optimization procedure of laccase production by solid state fermentation using a native Aspergillus niger and locally available substrates. In addition, the enzymatic activity was subjected to different purification steps and the resulting extract was then partially characterized.
It is clear the technological importance of the research done. However, the article has some issues that should be addressed, as outlined below.
1) The novelty of the results should be highlighted.
The goal of the study was to develop laccase from A. niger while also choosing low-cost, readily accessible high-yielding substrates.
The commercial development of laccases is being pursued in a cost-effective manner and through the screening of low-cost substrates. The use of agro-industrial waste as substrates adds value to the sector and raises knowledge of recycling and energy usage.
The current study focuses on producing, screening, optimizing, and purifying laccase from the local strain of A. niger that is readily available substrates in our vicinity.
Concerning the importance of research, I totally agree with the authors. However, the novelty of results should be highlighted. For laccase production, the use of agro-industrial residues such as wheat straw in solid state fermentation is not novel.
2) Abstract, line 27:
Please revise the expression “inoculum size”
At 45°C, pH 5.0 and 2mL inoculum size, the isolated enzyme produced its highest level of activity.
The authors write (line26): “Additionally, column chromatography was used to further purify laccase. The next step was enzyme characterization to evaluate how temperature, pH, and inoculum size affected enzyme activity. At 45°C, and pH 5.0 and 2mL inoculum size, the isolated enzyme produced its highest level of activity.”
The parameters temperature and pH are appropriate to characterize an enzyme activity, while the inoculum size is not. Instead, the inoculum size is an appropriate parameter to optimize a microbial enzyme production.
3) Line 87
Please revise the inclusion of reference 13.
Correction done.
4) Line 143
A reference should be added
Reference no. 26 added.
5) Line 168
Please revise de section “inoculum preparation”
-Was a suspension of conidia or a mycelium used as inoculum?
Mycelium via spore germination
In the abstract section, the authors write:
“The RSM results demonstrated that after five days of incubation, the enzyme activity was at its highest at 45 °C, pH 5.5, and 30% moisture level inoculated with 2 mL spore suspension.”
A conidia suspension is not the same as a pre-grown mycelium. Please revise.
-What do the authors mean with the following sentence?
“Shaking incubator delays the process of mycelia formation so the completely grown suspension of fungal spore was stored at 4°C”
We need stock culture for further experiments. By continuously putting the inoculum into shaking incubator will slow down the mycelia formation due to components of media consumption by A. niger. So we need to preserve it for further culturing process.
The authors should include a reference for the procedure. Alternatively, the impact of the mycelium physiological state used as inoculum on the bioprocess should be checked.
6) Line 273
The purity of the enzymatic preparation should be tested in some way.
The purity of enzyme has been checked in 3 ways including Ammonium Sulfate precipitation, Dialysis and Colum Chromatography.
The particular substrate Guaiacol has also been used to ensure the production of Laccase enzyme.
Ammonium sulfate precipitation, dialysis and column chromatography are methods or procedures applying in protein purification protocols. Concerning the purity of an enzymatic preparation, it can be tested in several ways. The most common method is by SDS-PAGE analysis.
7) Line 278
What do the authors mean with “crude enzyme”?
Crude enzyme means the 1st extraction of enzyme from sample. (without purified form)
In the Section 2.13. Characterization, the authors write: “The purified laccase was subjected to characterize…”. Thus, subsections 2.13.1 and 2.13.2 should be described for the purified laccase. The subsection 2.13.3 doesn't make sense. Please revise.
8) Line 229
Please revise if there is a significant difference between wheat straw and guava leaves.
As the biomass screening graph (figure 2) clearly shown the value of enzyme activity of Guava leaves (2.21 U/mL). has less than wheat straw (2.551 U/mL). So our aim was that picking the highest activity gaining substrate.
In my opinion, Student's t-test should be done.
9) Line 310
Please revise here and throughout the manuscript the use of the expressions enzyme activity or enzyme production.
Our aim is production of enzyme laccase and its production rate is checked by its activity. So we check the enzyme activity because enzyme activity relates to the number of substrates transformed to outputs every unit time.
Enzyme activity refers to the general catalytic properties of an enzyme, and enzyme assays are standardized procedures for measuring the amounts of specific enzymes in a sample.
The expressions “enzyme activity” and “enzyme production” are not synonyms. For example (line 335):
The authors write: “The enzyme activity is also influenced by other factors, including as nutrient content, temperature, pH, culture state, and media composition.”
The authors should write: “The enzyme production is also influenced by other factors, including …”
10) Line 320
As the experimental design studies all the factors at the same time, the authors should present the data taking this aspect into account. Thus, in my opinion, the subsections 3.1.1 to 3.1.4 are not necessary. The statistical analysis should also be included in this section. In addition, the physiological/technological meaning of factors interactions and quadratic terms should be discussed more deeply.
In section 3.2 basically these all factors have been interrelated with previous studies to make it unique from other types of strains as well as substrates. In section 3.4 statically analysis has been done among different combinations of factors to check the interaction lf these factors for the production of enzyme.
The response surface methodology allows the analysis of process variables as well as their interactions.
Author Response
Comments and Suggestions for Authors
The authors report on an optimization procedure of laccase production by solid state fermentation using a native Aspergillus niger and locally available substrates. In addition, the enzymatic activity was subjected to different purification steps and the resulting extract was then partially characterized.
It is clear the technological importance of the research done. However, the article has some issues that should be addressed, as outlined below.
1) The novelty of the results should be highlighted.
The goal of the study was to develop laccase from A. niger while also choosing low-cost, readily accessible high-yielding substrates.
The commercial development of laccases is being pursued in a cost-effective manner and through the screening of low-cost substrates. The use of agro-industrial waste as substrates adds value to the sector and raises knowledge of recycling and energy usage.
The current study focuses on producing, screening, optimizing, and purifying laccase from the local strain of A. niger that is readily available substrates in our vicinity.
Concerning the importance of research, I totally agree with the authors. However, the novelty of results should be highlighted. For laccase production, the use of agro-industrial residues such as wheat straw in solid state fermentation is not novel.
The novelty in this research is production of laccase enzyme from A.niger. As A niger is classified as Generally Recognized as Safe (GRAS) by US Food and Drug Administration for use in food production. And A.niger is easily available. Other points of novelty are the parameters i.e Temperature 45oC and pH 5.0 that are suitable conditions for the production of laccase. In previous research the parameters conditions were different so we have work on these present condition to make it novel.
2) Abstract, line 27:
Please revise the expression “inoculum size”
At 45°C, pH 5.0 and 2mL inoculum size, the isolated enzyme produced its highest level of activity.
The authors write (line26): “Additionally, column chromatography was used to further purify laccase. The next step was enzyme characterization to evaluate how temperature, pH, and inoculum size affected enzyme activity. At 45°C, and pH 5.0 and 2mL inoculum size, the isolated enzyme produced its highest level of activity.”
The parameters temperature and pH are appropriate to characterize an enzyme activity, while the inoculum size is not. Instead, the inoculum size is an appropriate parameter to optimize a microbial enzyme production.
Corrected. Inoculum size has excluded from this section.
3) Line 87
Please revise the inclusion of reference 13.
Correction done.
4) Line 143
A reference should be added
Reference no. 26 added.
5) Line 168
Please revise de section “inoculum preparation”
-Was a suspension of conidia or a mycelium used as inoculum?
Mycelium via spore germination
In the abstract section, the authors write:
“The RSM results demonstrated that after five days of incubation, the enzyme activity was at its highest at 45 °C, pH 5.5, and 30% moisture level inoculated with 2 mL spore suspension.”
A conidia suspension is not the same as a pre-grown mycelium. Please revise.
Corrected, as mycelium were used as inoculum.
-What do the authors mean with the following sentence?
“Shaking incubator delays the process of mycelia formation so the completely grown suspension of fungal spore was stored at 4°C”
We need stock culture for further experiments. By continuously putting the inoculum into shaking incubator will slow down the mycelia formation due to components of media consumption by A. niger. So we need to preserve it for further culturing process.
The authors should include a reference for the procedure. Alternatively, the impact of the mycelium physiological state used as inoculum on the bioprocess should be checked.
Correction added with Reference no. 27, 28 have been added.
6) Line 273
The purity of the enzymatic preparation should be tested in some way.
The purity of enzyme has been checked in 3 ways including Ammonium Sulfate precipitation, Dialysis and Colum Chromatography.
The particular substrate Guaiacol has also been used to ensure the production of Laccase enzyme.
Ammonium sulfate precipitation, dialysis and column chromatography are methods or procedures applying in protein purification protocols. Concerning the purity of an enzymatic preparation, it can be tested in several ways. The most common method is by SDS-PAGE analysis.
Yes you are right. But we have lack the instrument of SDS-PAGE so we can’t run this analysis. Anyhow the above mentioned techniques are also enough for the purification of enzyme.
7) Line 278
What do the authors mean with “crude enzyme”?
Crude enzyme means the 1st extraction of enzyme from sample. (without purified form)
In the Section 2.13. Characterization, the authors write: “The purified laccase was subjected to characterize…”. Thus, subsections 2.13.1 and 2.13.2 should be described for the purified laccase. The subsection 2.13.3 doesn't make sense. Please revise.
The subsection 2.13.3 has been removed. The detail about characterization has been written in 3.13 section.
8) Line 229
Please revise if there is a significant difference between wheat straw and guava leaves.
As the biomass screening graph (figure 2) clearly shown the value of enzyme activity of Guava leaves (2.21 U/mL). has less than wheat straw (2.551 U/mL). So our aim was that picking the highest activity gaining substrate.
In my opinion, Student's t-test should be done.
As we have not bulk amount of guava leaves as compared to wheat straw and we have also highest activity from wheat straw so we move on this substrate because it’s available in bulk amount.
9) Line 310
Please revise here and throughout the manuscript the use of the expressions enzyme activity or enzyme production.
Our aim is production of enzyme laccase and its production rate is checked by its activity. So we check the enzyme activity because enzyme activity relates to the number of substrates transformed to outputs every unit time.
Enzyme activity refers to the general catalytic properties of an enzyme, and enzyme assays are standardized procedures for measuring the amounts of specific enzymes in a sample.
The expressions “enzyme activity” and “enzyme production” are not synonyms. For example (line 335):
The authors write: “The enzyme activity is also influenced by other factors, including as nutrient content, temperature, pH, culture state, and media composition.”
The authors should write: “The enzyme production is also influenced by other factors, including …”
Corrected.
10) Line 320
As the experimental design studies all the factors at the same time, the authors should present the data taking this aspect into account. Thus, in my opinion, the subsections 3.1.1 to 3.1.4 are not necessary. The statistical analysis should also be included in this section. In addition, the physiological/technological meaning of factors interactions and quadratic terms should be discussed more deeply.
In section 3.2 basically these all factors have been interrelated with previous studies to make it unique from other types of strains as well as substrates. In section 3.4 statically analysis has been done among different combinations of factors to check the interaction lf these factors for the production of enzyme.
The response surface methodology allows the analysis of process variables as well as their interactions.
Author Response File: Author Response.pdf
Reviewer 2 Report
· · Line# 188: before being autoclaved for two hours to sterilize them – why the substrate was sterilized for 2 h?
Substrate without sterilization will most probably cause undesired microbial contamination to negatively affect the fermentation process. So we need to sterilize it for the better results of desired fermentation process.
My question is, "Did you sterilize it for 2 hours?" But it is not answered.
· Both contour and 3D plots are given for RSM analysis. No need to duplicate the presentation for the same data. It is unnecessarily increasing the figs. Keep only one type and delete the other.
The given explanation is not acceptable
· I wonder to see the R sq. 1. Hence, include the values predicted by the design in the RSM table (4)
Not answered
· Finally, the authors’ decision on selecting the optimal conditions using RSM is not correct. They simply took the run number 6 and claimed that it is the optimal condition. Instead, they should analyze solutions using the RSM runs and then run a separate validation experiment under the optimum conditions in the solution given by the software.
It looks like the authors don’t have a good idea of how to use RSM and find the optimal conditions. They didn’t understand my question and once again explained what they had done.
Author Response
Comments and Suggestions for Authors
Line# 188: before being autoclaved for two hours to sterilize them – why the substrate was sterilized for 2 h?
Substrate without sterilization will most probably cause undesired microbial contamination to negatively affect the fermentation process. So we need to sterilize it for the better results of desired fermentation process.
My question is, "Did you sterilize it for 2 hours?" But it is not answered.
Basically the whole process (washing, crushing, drying, and packaging) was taking 2 hours, the sterilization process was done only for 15 minutes. The correction has been done.
Both contour and 3D plots are given for RSM analysis. No need to duplicate the presentation for the same data. It is unnecessarily increasing the figs. Keep only one type and delete the other.
The given explanation is not acceptable
Contour plots have been excluded.
I wonder to see the R sq. 1. Hence, include the values predicted by the design in the RSM table (4)
Not answered
Predicted values have been added.
Finally, the authors’ decision on selecting the optimal conditions using RSM is not correct. They simply took the run number 6 and claimed that it is the optimal condition. Instead, they should analyze solutions using the RSM runs and then run a separate validation experiment under the optimum conditions in the solution given by the software.
It looks like the authors don’t have a good idea of how to use RSM and find the optimal conditions. They didn’t understand my question and once again explained what they had done.
Response Surface Methodology, RSM, is a collection of mathematical and statistical techniques that are useful for the analysis of problem in which a response of interest is influenced by several variables and the objective is to maximize this response.
Your statement is right but our interest of response was run number 6 because it gives highest enzyme activity then the other optimal conditions were obtained by checking the interaction of different parameters to get a high response from this run number.
Author Response File: Author Response.pdf
