Enhancing the Extraction Process Efficiency of Thyme Essential Oil by Combined Ultrasound and Microwave Techniques

Round 1

Reviewer 1 Report

The paper was easy to read and the results were well presented.

Abstract.

In line 15: Authors should mention the yields obtained and then make the comparison

When the ultrasound pre-treatment (using the ultrasonic processor with 14 an amplitude of 70%) was applied, the EO content was 23% higher compared to the extraction without pre-treatment.

Line 24: an average of yield in essential oil??

Line 61-64: At least some yields should be mention, authors only mention in general the effects of ultrasound in extraction, but if they were trying to compare as pre-treatment yield of reported studies should be mention.

Line 135,136 add standard deviation in yields

Line 148: authors should present more information about bio-148 conversion of γ-terpinene in thymol, possible reactions, etc.

Results:

Authors mention that “All measurements were carried out in triplicate and the data were expressed as mean 116 value ± SD (standard deviation) for triplicate of samples”, table 1. Mean values in composition and standard deviation should be added.

Besides, there is not discussion and contrast with another research, the results obtained in this work it´s better than reported by other authors, the 1.73 g EO/100 g DM is and average yield? Is lower? And carvacrol? There was not carvacrol in the EO?

In conclusions:

Authors should make a deep analysis more than repeat what was showed in results section.  

Author Response

We very much appreciate your constructive and critical suggestions and comments, which have been very helpful in improving the quality of the manuscript. It has been revised in detail according to the comments, and all the comments were incorporated into the revised manuscript. The corrections have been marked up in the revised manuscript in red color. Our responses (in RED color) to all your comments are stated below:

The paper was easy to read and the results were well presented.

Abstract.

In line 15: Authors should mention the yields obtained and then make the comparison “When the ultrasound pre-treatment (using the ultrasonic processor with 14 an amplitude of 70%) was applied, the EO content was 23% higher compared to the extraction without pre-treatment.”

Response: Thank you for your useful suggestion on the manuscript, which are very helpful to our article. The sentence “When the ultrasound pre-treatment (using the ultrasonic processor with an amplitude of 70%) was applied, the EO content was 23% higher compared to the extraction without pre-treatment.” was modified as follow: “When the ultrasound pre-treatment (using the ultrasonic processor with an amplitude of 70%) was applied, the EO content was 23% higher compared to the extraction without pre-treatment (2.67±0.06 g EO/100 g DM for the MWHD with ultrasound pre-treatment compared with 2.18±0.07 g EO/100 g DM for the MWHD without pre-treatment).” Please, see page 1, lines 16 and 17. 

Line 24: an average of yield in essential oil??

Response: Thank you for the suggestion. An information about the yield in essential oil of thyme leaves was added. Please, see page 1, line 26. 

“Thyme is rich in essential oil (EO) which is part of the secondary metabolites of the plants (up to 2.5% EO for the dried herb [2]).”

Line 61-64: At least some yields should be mention, authors only mention in general the effects of ultrasound in extraction, but if they were trying to compare as pre-treatment yield of reported studies should be mention.

Response: Thank you for the valuable suggestion. For a better comparison with other studies, the following sentence was inserted into the manuscript:

“In this study, a maximum yield of 2.4% (v/w) was achieved, meaning approximatively 2.15 g EO/100 g of dried plant material [29].” Please, see page 2, lines 64 and 65.

Line 135,136 add standard deviation in yields

Response: Thank you for the comment. At your suggestion, we added the standard deviation. Please, see page 3, lines 142 and 143.

 

Line 148: authors should present more information about bio-148 conversion of γ-terpinene in thymol, possible reactions, etc.

Response: Thank you for the suggestion. The following paragraph regarding the bioconversion of thymol was inserted into the manuscript (please, see page 4, lines 158-163):

“Krause et al. proposed a biosynthetic pathway of thymol, starting from the cyclization of geranyl diphosphate forming γ-terpinene. The latter is oxidized by the P450 monooxygenases enzyme to cyclohexadienol intermediates. These products are unstable being dehydrogenated by a dehydrogenase/reductase enzyme to allylic ketone intermediates. Further, through keto-enol tautomerisms thymol is formed [33].”

Results:

Authors mention that “All measurements were carried out in triplicate and the data were expressed as mean 116 value ± SD (standard deviation) for triplicate of samples”, table 1. Mean values in composition and standard deviation should be added.

Response: Thank you for your useful suggestion. The standard deviation of the values shown in table 1 were added. Please, see page 4, table 1.

Besides, there is not discussion and contrast with another research, the results obtained in this work it´s better than reported by other authors, the 1.73 g EO/100 g DM is and average yield? Is lower?

Response: Thank you for the comment. A discussion regarding other studies was introduced into the manuscript (please, see page 8, lines 283-287):

“These strategies (the ultrasound pre-treatment using an ultrasonic probe and milling the plant material to a particle size under 0.1 cm) lead to a higher extraction yield compared with other studies. For example, Kowalski et al., after applying ultra-sonic pre-treatment using an ultrasound bath, followed by conventional steam distillation method, achieved an extraction yield of 2.15 g EO/100 g DM.”

And carvacrol? There was not carvacrol in the EO?

The components of the EO higher than 0.2% were selected to be presented in the manuscript. Carvacrol was less than 0.2% and, for this reason, it was not considered a major component. 

In conclusions:

Authors should make a deep analysis more than repeat what was showed in results section. 

Response: Thank you for the valuable suggestion. The Conclusion section was modified as follow (please see page 12-13, lines 402-422):

“The purpose of this study was to investigate the influence of ultrasound pre-treatment of thyme leaves, before MWHD, on the thymol content and on the EO extraction yield. Comparative extractions, without pre-treatment, by both MWHD and CHD were also performed. The influence of several parameters (solvent to plant ratio, particle size of the vegetal material, ultrasound equipment to pre-treat the extraction mixture, and amplitude of the ultrasonic processor) on the extraction of thyme EO by MWHD was investigated. The composition of the EO resulted from all methods was analyzed by GS-MS, the major constituents being thymol, γ-terpinene, p-cymene, α-terpinene, and β-pinene. The results showed that the ultrasound pre-treatment of the extraction mixture enhance the EO content which can be extracted from thyme leaves. Moreover, combining the strategies: ultrasound pre-treatment using an ultrasonic probe and milling the plant material to a particle size under 0.1 cm, a maximum yield of 2.67±0.06 g EO/100 g DM was achieved. These strategies were also favorable for the targeted constituent, thymol, leading to a maximum content of 1764±52.68 mg/100 g DM. However, the ultrasound pre-treatment requires additional energy to the MWHD. The energy consideration study showed that the ultrasound pre-treatment using an ultrasonic probe leads to a lower specific energy (442.8 kJ/g of EO) compared with the extraction without pre-treatment (508.8 kJ/g of EO). On the contrary, the energy consumption of the ultrasonic bath was high, leading to a higher specific energy compared with the extraction without pre-treatment, although the EO content was higher. This means that the use of an ultrasonic probe is a better and greener choice to pre-treat vegetal materials.”

Reviewer 2 Report

Development of novel methods for the extraction of bioactive components and/or increase their efficiency can provide useful information not just for the science but also for the practice. Thyme can be found in many areas and contains essential oil with strong antioxidant, antimicrobial and anticancer properties. There are known several conventional processes for the extraction of essential oils from thyme, but they have long process time demand and high solvent ratio (which makes difficulties for further processing, such as concentration/separation steps). Microwave and ultrasound assisted extraction process are known as fast, good controllable and efficient methods in the literature. Authors focused on the applicability and efficiency of sonication as pre-treatment followed by microwave process.  Therefore, the topic of the manuscript can be considered as relevant and can provide interesting information for the readers. The manuscript is generally well structured. Introduction section summarizes well the background and relevance of the study, and, furthermore, the specific research motivations. Applied methods are adequate and described clearly. Results are presented well and discussed with relevant references. The manuscript contains interesting results obtained from lab-scale study, but need revision to make it clear and complete.

 

Comments, suggestions:

Authors mentioned the role of the temperature in the extraction efficiency in line 130-132, but temperature data are not provided.

How affect frequency the extraction efficiency? Is it possible to compare the two sonication methods with different frequencies?

Please give the frequency of microwave processor (2450 or 915 MHz, for instance).

It is not clear how was the amplitude range of sonication selected/determined? (90% why not used?, for example,)

Please discuss briefly the scaling-up possibilities of the combined US+MW method for industry scale, as well.

Authors conclude in line 331-335 ’ using the ultrasonic probe to pre-treat the thyme leaves leads to a specific energy with 14 and 16% lower for an amplitude of 50 and 70%, respectively. This difference is lower for a thyme leaves particle size under 0.1 cm (a specific energy with 6 and 13% lower for an amplitude of 50 and 70%, respectively as compared with the extraction without pre-treatment)’. In my opinion, particle size decreasing/chopping etc. need energy, as well. At industry scale it should be added to the total energy demand of the process.

Author Response

We very much appreciate your constructive and critical suggestions and comments, which have been very helpful in improving the quality of the manuscript. It has been revised in detail according to the comments, and all the comments were incorporated into the revised manuscript. The corrections have been marked up in the revised manuscript in red color. Our responses (in RED color) to all your comments are stated below:

Development of novel methods for the extraction of bioactive components and/or increase their efficiency can provide useful information not just for the science but also for the practice. Thyme can be found in many areas and contains essential oil with strong antioxidant, antimicrobial and anticancer properties. There are known several conventional processes for the extraction of essential oils from thyme, but they have long process time demand and high solvent ratio (which makes difficulties for further processing, such as concentration/separation steps). Microwave and ultrasound assisted extraction process are known as fast, good controllable and efficient methods in the literature. Authors focused on the applicability and efficiency of sonication as pre-treatment followed by microwave process.  Therefore, the topic of the manuscript can be considered as relevant and can provide interesting information for the readers. The manuscript is generally well structured. Introduction section summarizes well the background and relevance of the study, and, furthermore, the specific research motivations. Applied methods are adequate and described clearly. Results are presented well and discussed with relevant references. The manuscript contains interesting results obtained from lab-scale study, but need revision to make it clear and complete.

Comments, suggestions:

Authors mentioned the role of the temperature in the extraction efficiency in line 130-132, but temperature data are not provided.

Response: Thank you for your useful comment. For a better understanding regarding the temperature, the sentence was modified as follow:

“As shown in Figure 1, the time required to achieve the reflux temperature (the time after the EO starts to be collected into the Clevenger apparatus) for MWHD (10 min) is lower compared with CHD (60 min).” Please, see page 3, lines 136-138.

How affect frequency the extraction efficiency? Is it possible to compare the two sonication methods with different frequencies?

Response: Thank you for the comment. According to the manufacturer of Vibracell and ultrasound bath apparatuses the frequency is 20 kHz and 30 kHz, respectively. Neither of these apparatuses allow modifying the frequency. Thus, using the equipment described in this work, it is not possible to perform experiments at different frequencies.    

Please give the frequency of microwave processor (2450 or 915 MHz, for instance).

Response: Thank you for your valuable suggestion. The frequency of microwave equipment is 2450 MHz. it was also introduced into the manuscript. Please, see page 2, line 85.

It is not clear how was the amplitude range of sonication selected/determined? (90% why not used?, for example,)

Response: Thank you for the comment. One of the major issue associated with the extraction of EOs using ultrasounds is oxidation at high temperatures. We chose the 70% as maximum amplitude to avoid decomposition of valuable compounds, because at higher ultrasonic amplitudes the mixture is auto-heated.

Please discuss briefly the scaling-up possibilities of the combined US+MW method for industry scale, as well.

Response: Thank you for the useful suggestion. The following two paragraphs regarding the scaling-up possibilities were introduced into the manuscript.

“For scaling up the microwave and ultrasound technologies an important parameter to evaluate is the specific energy.” Please, see pages 11, lines 369-370.

“The aim of current study was to develop a procedure appropriate to small scale with scale-up possibilities. The reduced cost of combined ultrasound and microwave extraction is evidently beneficial for the proposed methods related to energy and time. The specific energy necessary to perform these extraction methods are 638.2 kJ/g of EO for MWHD and 534.5 kJ/g of EO for MWHD + ultrasonic horn pre-treatment. The extraction time is reduced compared with the conventional extraction. To scale-up these methods, Sairem has already commercialized different industrial microwave-assisted equipment [39] and also the ultrasound pre-treatment can be applied at industrial scale. Relating to the environmental effect, the amount of CO₂ fingerprint was calculated based on the supposition that 800 g CO₂ will be released for each 1 kWh obtained by combustion of fossil fuel [40]. It was observed that for the conventional extraction the CO₂ emission (553 g CO₂/g of EO) is higher compared with MWHD (142 g CO₂/g of EO) and MWHD + ultrasonic horn pre-treatment at an amplitude of 70% (119 g CO₂/g of EO). According to these calculations the combined ultrasound pre-treatment with MWHD is an environmentally friendly method.” Please, see pages 12, lines 387-400.

Authors conclude in line 331-335 ’ using the ultrasonic probe to pre-treat the thyme leaves leads to a specific energy with 14 and 16% lower for an amplitude of 50 and 70%, respectively. This difference is lower for a thyme leaves particle size under 0.1 cm (a specific energy with 6 and 13% lower for an amplitude of 50 and 70%, respectively as compared with the extraction without pre-treatment)’. In my opinion, particle size decreasing/chopping etc. need energy, as well. At industry scale it should be added to the total energy demand of the process.

Response: Thank you for the valuable suggestion. The energy for the grinding step was calculate and the following paragraph was introduced into the manuscript:

“The plant material grinding step implies energy consumption. In order to achieve a particle size under 0.1 cm (starting from 1-2 cm size) 20 s of milling were required. Thus, the grinding step will add 3 kJ to the energy of the overall process of EO extraction. However, this quantity of energy is insignificant, the specific energy for the experiments performed with a thyme leaves particle size under 0.1 cm increasing with only 0.25%.” Please, see pages 11, lines 380-384.

Reviewer 3 Report

Review of the paper

"Enhancing the extraction process efficiency of thyme essential oil by combined ultrasound and microwave techniques"authors Adina Ionuta Gavrila , Ciprian Gabriel Chisega-Negrila, Laura Maholea, Mircea Leontin Gavrila, Oana Cristina Pârvulescu, Ioana Popa

            The present paper aims to study the extraction of essential oil (EO) from thyme. The intensification methods of this process, based on ultrasonic and microwave treatment, as well as their combinations, have been studied experimentally in this work. The Introduction clearly identified the paper's place in its field of study. The differences of the present paper from closely related articles were described. The work is well structured and the material is presented in easy to understand manner. The presented experimental results clearly demonstrate the influence of various factors on the extraction efficiency.

            The choice of the journal "Agronomy" raises questions, as the article rather belongs to the following fields: chemical engineering, separation and purification processes, food technology, green technology. In this regard, it appears that journals such as: AppliedChem, ChemEngineering, Clean Technologies are much more relevant to the field of paper.

            The main drawback of the paper is that the experimental results were not presented numerically. The most of the experimental results were presented only graphically. The numerical values of the amount of extracted essential oil (g EO/100 g DM) and the concentration of the components are not given in the paper. This makes it impossible to verify the statistical analysis presented in the paper and makes it difficult to use the results of this paper. In my opinion, all the results presented in Figures 1-5 should also be presented in the form of tables.

            In the discussion of the results (3. Results), in cases where there is a decrease in the concentration of a component with increasing EO yield, the authors explain this solely to chemical transformations (bioconversion, degraded). However, the explanation for a decrease in the concentration of one component may be an increase in the yield of another component. For example, in the case of increasing ultrasound amplitude (Fig. 4.), the concentration of all components except thymol decrease. This may be due to the fact that these components were fully extracted even at an amplitude of 50%, and increasing the amplitude resulted in an increase in yield for thymol only. This can be checked by comparing the absolute amounts of extracted components (component g/100 g DM) instead of concentrations in % of total.

Another explanation for the decrease in component concentration with increasing total EO yield, could be a difference in the kinetics of the extraction process. Different treatments have a different influence on the permeability of the cell membrane.

Specific comments

2.1. Materials

What type of oven was used for leaf drying (model, characteristics)?

Why was a temperature of 60^oC chosen?

How exactly was dried vegetal material chopped to 1-2 cm pieces?

How was the water content of the dried leaves determined?

In what units is the water content represented (mass or mole %)?

            2.2.1. Essential Oil Extraction Procedure

Is the multimode microwave oven (Plazmatronika, Wroclaw, Poland) in which the process of EO extraction from thyme was carried out the same as the one presented in [10]? If yes, this should be explicitly mentioned in the article. If not, provide a scheme and description of the equipment in the paper.

The authors wrote: «the mixture was subjected to extraction until no EO was obtained (80 min)». Meaning that the extraction was carried out before extracting all the essential oil from the vegetal material? If yes, how was this determined?

            3.1. Microwave-assisted Hydro-distillation (MWHD) vs. Conventional Hydro-distillation (CHD)

The authors wrote: «As shown in Figure 1, 130 the heating time required for MWHD (10 min) is lower compared with CHD (60 min)». However, Figure 1 does not present any heating or temperature information at all. In the paper there is no data at what temperatures the described processes were carried out. In my opinion this is important information that needs to be added to the paper.

In what % is Composition in Table 1, mass or mole?

            3.2. Influence of solvent to plant ratio on the extraction of thyme essential oil

In this part, the authors make a choice of the most efficient water/plant ratio of 8/1, 10/1 or 12/1. Their choice is understandable, but the explanation raises questions.

The authors wrote: «The one-way ANOVA analysis demonstrated that by increasing the solvent to plant ratio from 8/1 to 10/1 the EO content increases significantly (p < 0.05). By further increase of solvent to plant ratio, the EO content increases non-significantly (Figure 2a). Regarding the thymol extraction, the one-way ANOVA analysis showed that by increasing the solvent to plant ratio, the thymol concentration increased significantly (p < 0.05, Figure 2b)». From what has been written, it seems that the one-way ANOVA analysis for EO yield and for thymol concentration showed different results, although according to Figure 2 this is not the view. When the ratio is increased from 8/1 to 10/1, both the EO content and thymol concentration increase significantly, while when the ratio is increased from 10/1 to 12/1, both increase not- significantly.

The authors wrote: «Although the amount of EO is similar for both 10/1 and 12/1 ratios, the further experiments were performed for a 12/1 (v/w) ratio of solvent to plant material since the amount of the targeted compound (thymol) is higher and the specific energy (see Table 2) is lower compared with the other ratios used». As noted above, in both cases the value for 12/1 is higher than 10/1, but in both cases according to one-way ANOVA analysis the difference is not significant. Then why is an increase in thymol concentration a reason to choose the 12/1 ratio but an increase in EO yield is not?

Regarding the decrease in specific energy, according to Table 5, the difference between 10/1 and 12/1 is less than 3%. The extent to which this difference can be considered significant is not clear, as the paper does not give the accuracy with which the energy input is measured.

            3.7. Energy considerations

In Table 5, the authors give the total and specific energies in sum. It would be interesting to see separately the energy expended for ultrasound and microwaves.

Typos

Figure 2 does not have the letter «c» but the caption does: "Different letters (a-c) within graph show the significant difference between groups"

In line 182 should probably refer to Table 5, not Table 2.

There is no definition of the abbreviation DM (g/100 g DM) in the text of the paper.

Comments for author File: Comments.pdf

Author Response

We very much appreciate your constructive and critical suggestions and comments, which have been very helpful in improving the quality of the manuscript. It has been revised in detail according to the comments, and all the comments were incorporated into the revised manuscript. The corrections have been marked up in the revised manuscript in red color. Our responses (in RED color) to all your comments are stated below:

"Enhancing the extraction process efficiency of thyme essential oil by combined ultrasound and microwave techniques"authors Adina Ionuta Gavrila , Ciprian Gabriel Chisega-Negrila, Laura Maholea, Mircea Leontin Gavrila, Oana Cristina Pârvulescu, Ioana Popa

The present paper aims to study the extraction of essential oil (EO) from thyme. The intensification methods of this process, based on ultrasonic and microwave treatment, as well as their combinations, have been studied experimentally in this work. The Introduction clearly identified the paper's place in its field of study. The differences of the present paper from closely related articles were described. The work is well structured and the material is presented in easy to understand manner. The presented experimental results clearly demonstrate the influence of various factors on the extraction efficiency.

The choice of the journal "Agronomy" raises questions, as the article rather belongs to the following fields: chemical engineering, separation and purification processes, food technology, green technology. In this regard, it appears that journals such as: AppliedChem, ChemEngineering, Clean Technologies are much more relevant to the field of paper.

Response: Thank you for the comments. The Agronomy journal was chosen due to its special issue “It Runs in the Family: The Importance of the Lamiaceae Family Species”. The manuscript was summited to this special issue.

The main drawback of the paper is that the experimental results were not presented numerically. The most of the experimental results were presented only graphically. The numerical values of the amount of extracted essential oil (g EO/100 g DM) and the concentration of the components are not given in the paper. This makes it impossible to verify the statistical analysis presented in the paper and makes it difficult to use the results of this paper. In my opinion, all the results presented in Figures 1-5 should also be presented in the form of tables.

Response: Thank you for your useful comments and suggestions on the manuscript. Unfortunately, the authors’ guide says that the results must be presented either as a graph or as a table. So, we can not add tables for the results presented as graphs. However, for a better understanding of the obtained values, we inserted within text numerical values of the results for both EO content and components amount. They are highlighted within manuscript in red color.

In the discussion of the results (3. Results), in cases where there is a decrease in the concentration of a component with increasing EO yield, the authors explain this solely to chemical transformations (bioconversion, degraded). However, the explanation for a decrease in the concentration of one component may be an increase in the yield of another component. For example, in the case of increasing ultrasound amplitude (Fig. 4.), the concentration of all components except thymol decrease. This may be due to the fact that these components were fully extracted even at an amplitude of 50%, and increasing the amplitude resulted in an increase in yield for thymol only. This can be checked by comparing the absolute amounts of extracted components (component g/100 g DM) instead of concentrations in % of total.

Another explanation for the decrease in component concentration with increasing total EO yield, could be a difference in the kinetics of the extraction process. Different treatments have a different influence on the permeability of the cell membrane.

Response: Thank you for the valuable comments. At your useful suggestion we expressed the content of the components in absolute amounts (mg of component/100 g DM). As shown in figs 2-5, the results are clearer now. Also, for a better understanding of the component’s behavior, we introduced into the manuscript the following paragraph (please, see pages 8-9, lines 302-315):

“As shown in Figure 4b, this behavior of α-terpinene can be also observed for the ex-traction with thyme leaves of a 1-2 cm particle size (51±4.5 and 46±4.02 mg/100 g DM for an amplitude of 50 and 70%, respectively). Indeed, according to ANOVA analysis, the decrease at an amplitude of 70% is insignificant (see Figure 4b), but the higher decrease when smaller particles are used can be explained by a more considerable exposure of ultrasound energy. Moreover, the p-cymene content is higher for an amplitude of 70% compared with 50% when the plant is milled (Figure 5b - 100±9.9 and 170±14.06 mg/100 g DM for an amplitude of 50 and 70%, respectively), while for the extraction performed with leaves of 1-2 cm particle size an opposite behavior is observed (Figure 4b - 90±7.28 and 63±5.99 mg/100 g DM for an amplitude of 50 and 70%, respectively). This could be due to a possible oxidation of some components (such as α-terpinene and γ-terpinene) yielding p-cymene [38]. However, further research is required for a possi-ble oxidation under combined ultrasound and microwave treatments.”     

Specific comments

2.1. Materials

What type of oven was used for leaf drying (model, characteristics)?

Response: Thank you for the comment. The oven used to dry the vegetal material was the Memmert UNE 500 Universal Oven. The oven used to dry the plant material was also introduced into the manuscript. Please, see page 2, line 73.

Why was a temperature of 60^oC chosen?

Response: Thank you for the comment. The industries which use plants as raw material employ a temperature of 40-60 °C for the drying step. Based on this, the drying temperature of thyme leaves and stems used in this study was chosen to be 60 °C.

How exactly was dried vegetal material chopped to 1-2 cm pieces?

Response: Thank you for your comment. There was an error in the chopping of plant material description. The sentence was modified as follow (please, see page 2, lines 72-75):

“The fresh leaves and stems were chopped into pieces of 1-2 cm, then dried in an air flow-heating oven (Memmert UNE 500 Universal Oven, Germany) at 60 °C to a constant weight. Part of the dried vegetal material was used as such, and another part was ground using an electric grinder and screened to a particle size under 0.1 cm.”

How was the water content of the dried leaves determined?

Response: Thank you for the comment. The water content of dried leaves was determined using the moisture analyzer PMB 202, Adam Equipment Co Ltd, United Kingdom. The equipment used for the water content determination was also introduced into the Materials section (please, see page 2, lines 77-79).

In what units is the water content represented (mass or mole %)?

Response: Thank you for your comment. The water content is expressed by weight. We also introduced this information in the Materials section (please, see page 2, lines 77).

2.2.1. Essential Oil Extraction Procedure

Is the multimode microwave oven (Plazmatronika, Wroclaw, Poland) in which the process of EO extraction from thyme was carried out the same as the one presented in [10]? If yes, this should be explicitly mentioned in the article. If not, provide a scheme and description of the equipment in the paper.

Response: Thank you for the useful suggestion. The multimode microwave oven Plazmatronika used for the EO extraction is the same as the one presented in our previous work. Thus, the following sentence was inserted into the manuscript (please, see page 2, line 86):

“This apparatus is described in our previous work Calinescu et al. [17].”

The authors wrote: «the mixture was subjected to extraction until no EO was obtained (80 min)». Meaning that the extraction was carried out before extracting all the essential oil from the vegetal material? If yes, how was this determined?

Response: Thank you for the comment. There were preliminary tests (part of the research project PN-III-P2-PED-2019-2118, project: Technologies for obtaining of natural products with immunostimulatory properties - “IMUNOSTIM” financed by contract: PED, no. 381PED/2020) for which the extraction of EO from thyme leaves was conducted for 120 min. However, these tests revealed that in the 80-120 min interval, the amount of EO extracted remained the same. Thus, the extraction time for MWHD in the present study was chosen to be 80 min.

3.1. Microwave-assisted Hydro-distillation (MWHD) vs. Conventional Hydro-distillation (CHD)

The authors wrote: «As shown in Figure 1, 130 the heating time required for MWHD (10 min) is lower compared with CHD (60 min)». However, Figure 1 does not present any heating or temperature information at all. In the paper there is no data at what temperatures the described processes were carried out. In my opinion this is important information that needs to be added to the paper.

Response: Thank you for the valuable suggestion. For a better understanding regarding the temperature, the sentence was modified as follow:

“As shown in Figure 1, the time required to achieve the reflux temperature (the time after the EO starts to be collected into the Clevenger apparatus) for MWHD (10 min) is lower compared with CHD (60 min).” Please, see page 3, lines 136 - 138.

In what % is Composition in Table 1, mass or mole?

Response: Thank you for the comment. At your useful suggestion, we presented the composition of thyme EO in absolute amounts (mg EO/100 g DM) for table 1 too.

3.2. Influence of solvent to plant ratio on the extraction of thyme essential oil

In this part, the authors make a choice of the most efficient water/plant ratio of 8/1, 10/1 or 12/1. Their choice is understandable, but the explanation raises questions.

The authors wrote: «The one-way ANOVA analysis demonstrated that by increasing the solvent to plant ratio from 8/1 to 10/1 the EO content increases significantly (p < 0.05). By further increase of solvent to plant ratio, the EO content increases non-significantly (Figure 2a). Regarding the thymol extraction, the one-way ANOVA analysis showed that by increasing the solvent to plant ratio, the thymol concentration increased significantly (p < 0.05, Figure 2b)». From what has been written, it seems that the one-way ANOVA analysis for EO yield and for thymol concentration showed different results, although according to Figure 2 this is not the view. When the ratio is increased from 8/1 to 10/1, both the EO content and thymol concentration increase significantly, while when the ratio is increased from 10/1 to 12/1, both increase not- significantly.

The authors wrote: «Although the amount of EO is similar for both 10/1 and 12/1 ratios, the further experiments were performed for a 12/1 (v/w) ratio of solvent to plant material since the amount of the targeted compound (thymol) is higher and the specific energy (see Table 2) is lower compared with the other ratios used». As noted above, in both cases the value for 12/1 is higher than 10/1, but in both cases according to one-way ANOVA analysis the difference is not significant. Then why is an increase in thymol concentration a reason to choose the 12/1 ratio but an increase in EO yield is not?

Response: Thank you for the useful comments. On the new graphical representation of components’ content, it can be noticed that for thymol, the one-way ANOVA analysis showed that by increasing the solvent to plant ratio, the thymol concentration increased significantly (p < 0.05, Figure 2b).

Regarding the decrease in specific energy, according to Table 5, the difference between 10/1 and 12/1 is less than 3%. The extent to which this difference can be considered significant is not clear, as the paper does not give the accuracy with which the energy input is measured.

Response: Thank you for the comment. The equipment used for the experiments (microwave and ultrasound equipment) show, on the display, the power introduced into the system during the experiment. These powers were used to calculate the energy and then to calculate the specific energy using the equation given into the manuscript. The aim of the energy determination was to establish if the process is feasible given the fact that a supplementary step which consume energy (the pre-treatment) is added to the microwave extraction of the EO. For example, although the EO amount is higher for the pre-treatment using the ultrasonic bath compared with the MWHD without pre-treatment, the energy consumption of the ultrasound bath is too high to obtain a lower specific energy. Thus, this process (which use the ultrasonic bath) is not more efficient than the one without pre-treatment.   

For the conventional extraction the power input was measured at the heater power supply using a wattmeter. The following sentence was also introduced into the manuscript (please, see page 11, lines 355 and 356):

“The power input for the electrical heater was measured at the heater power supply using a Wattmeter.”

3.7. Energy considerations

In Table 5, the authors give the total and specific energies in sum. It would be interesting to see separately the energy expended for ultrasound and microwaves.

Response: Thank you for the valuable suggestion. For a better understanding, the following paragraph was introduced into the manuscript (please, see page 11, lines 362-368):

“The energy consumption for MWHD is 1104 kJ. The ultrasound pre-treatment step in-creases this quantity of energy concordantly with table 5. For example, the highest in-crease in energy is given by the ultrasonic bath, the overall process energy increasing from 1104 kJ (for the extraction without pre-treatment) to 1896 kJ (for the extraction with pre-treatment using the ultrasound bath). Regarding the pre-treatment performed using the ultrasonic horn, the energy increase is much lower, meaning between 47 and 82 kJ, as shown in table 5.”   

Typos

Figure 2 does not have the letter «c» but the caption does: "Different letters (a-c) within graph show the significant difference between groups"

Response: Thank you for noticing. In accordance with your suggestion the composition of thyme EO was expressed as absolute amount, thus all the graphs were modified.

In line 182 should probably refer to Table 5, not Table 2.

Response: Thank you very much for noticing. The table number was modified. Please, see page 5, line 193.

There is no definition of the abbreviation DM (g/100 g DM) in the text of the paper.

Response: Thank you for noticing. The “DM” abbreviation was defined in the Methods section. Please, see page 3, line 97-98.