Evaluation of Cell Rupture Techniques for the Extraction of Proteins from the Microalgae Tetradesmus obliquus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper compares the efficiencies of three different methods of cell disruption of the green microalgae Tetradesmus obliquus. Cell disruption is determined by microscopic analysis and the recovery of protein. In addition, the paper gives results on the composition of the peptides in the organism under study. 

 

Unfortunately, the results shown in the paper do not exceed or differ from those already described in literature and thus no conclusions can be drawn from these which extend the knowledge on disruption of microalgae cells.

 

In addition I have major concerns about methodology:

1. The authors determined total protein content by Kjeldahl digestion. Using this method organic and inorganic N is determined from which protein was calculated using a factor of 5.89. By doing so the inorganic N which might have been accumulated in the cell was not considered. That this was significant is shown in the discrepancy of the results on total protein content of 40 % and the only 16-18 % of protein recovered during extraction although about 80% of the cells were disrupted according to microscopic analysis.

 

2. The preparation of the biomass included storage at -10 C and freezing at -80 C for freeze drying. It cannot be ruled out that this processing might have already produced cell disruption.

 

3. Mechanical disruption using a ball mill was done on dry biomass. The finding that Ball Milling was the less efficient disruption method might be explained by the use of dry biomass instead of biomass in aqueous suspension.

 

4. The results on the protein analysis do not include the comparison of the disruption methods under study. It thus remains completely unclear for what reason they were obtained.

 

Detailed comments 

·       Numbering of method section is not continuous (there are two chapters of 2.3) 

·       In Line 232 the molecular mass of RUBISCO is given with 52.574 kg/mol. It might be 52.574 kDa.

·       Figure 3 cannot be understood and as mentioned above irrelevant. 

 

 

Author Response

Viçosa, MG, Brazil, November 27^th, 2023.

To the Editor-in-chief

Dear Mr. Louies Liu,

Thank you and the reviews for the comments and suggestions on our manuscript entitled "Evaluation of cell rupture techniques for the extraction of proteins from microalgae Tetradesmus obliquus".

We sincerely appreciate the valuable comments and review suggestions, which helped us improve the manuscript's quality. All their recommendations and suggestions were accepted, and the changes are highlighted in blue within the document.

The manuscript has been formatted according to the Author-Directions of the Phycology. All authors contributed equally to preparing the manuscript, reviewing it, and approving its final version. Moreover, we declare that there are no conflicts of interest.

Best regards,

Jane

On behalf of the authors

Reviewers' comments

REVIEWER 1

Reviewer #1: The paper compares the efficiencies of three different methods of cell disruption of the green microalgae Tetradesmus obliquus. Cell disruption is determined by microscopic analysis and the recovery of protein. In addition, the paper gives results on the composition of the peptides in the organism under study.  Unfortunately, the results shown in the paper do not exceed or differ from those already described in literature and thus no conclusions can be drawn from these which extend the knowledge on disruption of microalgae cells. Response: Thank you for reading our manuscript and for the valuable comments and suggestions, which helped us improve the manuscript's quality. The manuscript was corrected as follows.

Questions and Comments

Question 01. The authors determined total protein content by Kjeldahl digestion. Using this method organic and inorganic N is determined from which protein was calculated using a factor of 5.89. By doing so the inorganic N which might have been accumulated in the cell was not considered. That this was significant is shown in the discrepancy of the results on total protein content of 40 % and the only 16-18 % of protein recovered during extraction although about 80% of the cells were disrupted according to microscopic analysis. Response: Determining total protein in pharmaceutical, biological, food, environmental, and other materials is performed widely by the Kjeldahl method (AOAC, 2007; Persson et al., 2008). The method indirectly quantifies the total protein content from nitrogen measurements. The total nitrogen estimation includes other nitrogenous compounds, such as intracellular inorganic materials. The conversion factor of nitrogen into microalgal proteins varies from species to species and is affected by the processing step. Processing can affect cellular compounds that contain nitrogen, such as nucleotides, glucosamines, inorganic nitrogen, and free amino acids (Silva et al., 2021). For T. obliquus, the percentage of nitrogen was converted into protein percentage using the conversion factor 5.8 established by Afify et al. (2018), based on the amino acid profile of this species. The proximate protein content (w/w) of the T. obliquus biomass found in our study by the Kjeldahl method was 40.29 ± 0.24%. According to Afify et al. (2018), pigments, nucleic acids, glucosamine, and amines could account for approximately 10% of the overall nitrogen content in T. obliquus.

The protein mass yields obtained in the US, HPH, and BM treatments were 20.8%, 17.1%, and 16.1%, respectively. These results were calculated according to the soluble protein extracted from the biomass and the total protein biomass content. These protein contents were determined by the Lowry method. It is important to emphasize that soluble proteins were extracted at pH 10; therefore, insoluble proteins were not recovered from the biomass. The low extraction yields of soluble proteins from microalgae can be explained by their nature and location in cells, as they are found in different parts, such as the cell wall, cytoplasm, chloroplast, and other intracellular organelles (Phong et al., 2018).

In our study, the content of extracted soluble protein was a reliable indirect index to measure the level of cell disruption and compare different mechanical treatments. Suarez Garcia et al. (2018) observed that 72% of proteins are linked to insoluble cellular structures and can only be extracted using surfactants. Other extraction conditions, such as temperature and pH (Gerde et al., 2013) and solvent types (Grossman et al., 2018; Anjos et al., 2022), could improve protein extraction yields. However, it was not the scope of the present manuscript. We added this information to the manuscript.

Now (lines 276-284)

Table 1 shows the results for protein mass yield (%) (g of protein of powder extract/100 g of dried microalgae), % of protein of extracts (g of protein of powder extract/100 g of extract), the visual appearance of protein extracts, and colorimetric parameters of protein extracts. The protein mass yields of the US, HPH, and BM treatments were 20.8%, 17.1%, and 16.1%, respectively. It is important to emphasize that these results describe the extraction of aqueous protein soluble at pH 10. Therefore, insoluble proteins were not extracted under the process conditions evaluated and remained in the residual biomass (pellet) in the centrifugation step. Other extraction conditions could be used to improve protein extraction yield, such as temperature and pH [29], surfactants [30], and solvent type [9,27]. However, it was not within the scope of our research.

Question 02. The preparation of the biomass included storage at -10 C and freezing at -80 C for freeze drying. It cannot be ruled out that this processing might have already produced cell disruption. Response: Before biomass treatment by BM, US, or HPH, the biomass was submitted to freeze drying to avoid biomass deterioration and enable its use for an extended period.

During lyophilization, water in the form of ice under low pressure is removed from a material by sublimation. This process causes some damage to the cell membrane as the intracellular water expands upon freezing, making the cell membrane more porous; however, the cell wall is not ruptured (Guldhe et al., 2014; Lee et al., 2017). Nonetheless, according to Stirk et al. (2020), lyophilization caused some damage to the cell membrane, enabling certain compounds to be extracted. These authors suggest that the damage caused by freeze-drying was sufficient to release the active compounds using water extracts. However, literature regarding the effect of freeze-drying on protein extraction in microalgae is scarce. It is highlighted that all disruption methods were carried out with freeze-dried biomass; thus, the damage caused by freeze-drying in the cell wall was the same in all treatments evaluated. To improve the text, information about the damage to the microalgal cell wall by lyophilization was added.

Now (lines 360-366)

This behavior is probably due to cell disintegration and membrane permeabilization of microalgal species with a rigid cell wall, such as T. obliquus [33], during freeze-drying [39]. The freeze-drying process causes some damage to the cell membrane, making the cell membrane more porous; however, the cell wall is not ruptured [40–42]. It is important to highlight that all disruption methods were carried out with freeze-dried biomass; thus, the eventual damage caused by freeze-drying in the cell wall was the same in all treatments evaluated.

Question 03. Mechanical disruption using a ball mill was done on dry biomass. The finding that Ball Milling was the less efficient disruption method might be explained by the use of dry biomass instead of biomass in aqueous suspension. Response: Thank you for the correction. Grinding in a ball mill (BM) is a unitary operation with high breaking efficiency that is capable of processing a large volume of biomass in batches and easily separating the broken biomass by gravity (D'Hondt et al., 2017). The low extraction yields of soluble proteins by BM can also be explained by using dry biomass instead of wet biomass. The suggestion for using wet biomass in future studies was added to the text. We used dry biomass in the BM rupture because it is a low-cost method that demonstrates its efficiency in disrupting T. obliquus cells (Amorim et al., 2020; Vieira et al., 2021) and avoiding drying biomass after disruption. This information was added to the manuscript.

Now (lines 461-469)

The results observed in Figure 4 show that the amounts of protein extracted from T. obliquus with HPH and BM treatments were lower than that achieved with US treatment. The low extraction yields of soluble proteins with BM can be due to the use of dry biomass instead of wet biomass. The energy for cell rupture is well utilized when the disintegration rate correlates with a specific energy. Schuller et al. [48] used BM to extract lutein and β-carotene from wet and lyophilized biomass of Tetraselmis sp. The break with wet biomass glass beads best responded to the extractive treatments. However, the literature also reported promising results for BM mechanical disruption with dried biomass [13,16].

Question 04. The results on the protein analysis do not include the comparison of the disruption methods under study. It thus remains completely unclear for what reason they were obtained. Response: The protein analysis results were obtained to identify the main protein in the extract. Thus, through the electrophoretic bands, it was possible to determine proteins with molecular masses of 45 kDa, 66.2 kDa, 147.61 kDa, 126.53 kDa, 106.52 kDa, 102.72 kDa, and 83.34 kDa. The bands for molecular masses greater than 200 kDa represent protein aggregates. The diversity in protein composition can be explained by the fact that microalgae do not accumulate distinct proteins as an N source [32]. Furthermore, the large enzyme RuBisCO was identified as the band between 45 and 66.2 kDa with the help of MALDI-TOF MS. The text was changed as follows.

 

Now (lines 332-333):

4.2. Identification of the main protein in the T. obliquus protein extract

The protein analysis results were obtained to identify the main protein in the extract. The electrophoretic bands with higher intensities corresponded to proteins with molecular masses between 45 and 66.2 kDa (Figure 1).

 

Question 05. Numbering of method section is not continuous (there are two chapters of 2.3). Response: The manuscript was corrected according to the reviewer's suggestion.

Question 06. In Line 232 the molecular mass of RUBISCO is given with 52.574 kg/mol. It might be 52.574 kDa. Response: The manuscript was corrected as follows.

 Now (lines 246-251)

The large enzyme ribulose bisphosphate carboxylase (RuBisCO), with a molecular mass of 52.574 kDa, was identified after comparing the masses of peptides arising from protein band digestion with the molecular masses of peptides from protein cleavage available in the SwissProt database using MASCOT software, which indicates a greater homology (score = 80) of the microalgae T. obliquus and the microalgae Acutodesmus obliquus, which belong to the same genus as the organism studied, Tetradesmus sp. [28].

Question 07. Figure 3 cannot be understood and as mentioned above irrelevant.  Response: Figure 3 was removed, as suggested. Its results are described in the text.

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presented by the authors is an interesting addition in understanding how algal biomass can be disrupted to obtain specific metabolites form the cells.

 

Abstract.

lines 31-35. the last part when the results are presented seems redundant, please simplify it .

Please finish the abstract with a remark that highlights the importance of the paper.

 

Introduction

Paragraph 2-4 are loose within the section. those seems to be unrelated idea, please create a single paragraph with those ideas. Unify them please

Please elaborate further the following phrase "to identify the majority of protein in the protein-rich extracts". this is really interesting since most papers focuses only on quantity of protein extract than the "quality" of proteins extracted.

Materials

remove "the" at the beginning of Line 81

how was the biomass concentrated?, floc, centrifuge, filtration?

please open this section with the phrase of lines 83-84 "the biomass was a gift from..."

the first section is quite confusing, please make it clearer and straightforward. 1. who produced the biomass, and how. 1 how was harvested and processed prior to drying. 

Results

Please improve the quality of figure 3, or find a better way to present the results. also improve figure 5.

Discussion

In line 332 Acutodesmus oblíquos is not the correct name, please change it to "Acutodesmus obliquus"

Author Response

REVIEWER 2

Reviewer #2: The paper presented by the authors is an interesting addition in understanding how algal biomass can be disrupted to obtain specific metabolites form the cells. Response: Thank you for reading our manuscript and for the valuable comments and suggestions, which helped us improve the manuscript's quality. The manuscript was corrected as follows.

Questions and Comments

Question 01. Abstract. lines 31-35. the last part when the results are presented seems redundant, please simplify it. Response: The abstract was rewritten.

Now (lines 31-35)

The US treatment promoted the highest yield of total protein extraction (19.95%), while the HPH and BM treatments were 15.68% and 14.11%, respectively. Since the cell break method affects protein extraction from microalgal biomass, protein release can be used as a central indicator reflecting the degree of cell disruption. This study also indicates that T. obliquus can be an alternative and sustainable source of protein, making it attractive for industrial applications in the food industry.

Question 02. Please finish the abstract with a remark that highlights the importance of the paper. Response: The text was modified.

Now (lines 34-37):

Since the cell break method affects protein extraction from microalgal biomass, protein release can be used as a central indicator reflecting the degree of cell disruption. This study also indicates that T. obliquus can be an alternative and sustainable source of protein, making it attractive for industrial applications in the food industry.

Question 02. Introduction: Paragraph 2-4 are loose within the section. those seems to be unrelated idea, please create a single paragraph with those ideas. Unify them please. Response: The Introduction section was rewritten.

 Now (lines 39-78)

Microalgae are a group of organisms with diverse morphological, reproductive, physiological, and ecological characteristics. Microalgae can be used in the cosmetics industry, wastewater treatment, energy production, and human and animal nutrition; have a propensity for growth in any region because their growth is independent of climate conditions; survive in areas unsuitable for growing traditional crops, not competing with food production sites; and are rich in metabolites, such as proteins, lipids, vitamins, enzymes, pigments, mineral salts, antioxidants, and antibiotics [1-2]. Short- and long-term feeding tests have demonstrated that some microalgae are safe for human consumption [3–7]. Thus, in human nutrition, microalgae are an unconventional source of green proteins and bioactive peptides [1]. Some of these proteins have nutritional quality compared to proteins of plant origin [2], exhibiting the composition of essential amino acids in the quantities recommended by the Food and Agriculture Organization/FAO [1]. Depending on the species, microalgal protein content can reach up to 70% [8]. However, approximately 70% of microalgal proteins are intracellularly located and adhere to the cell wall. Thus, the cells must be ruptured to extract or release these components [9].

Cell disruption, as one of the first downstream stages for microalgae biotechnological platforms, promotes the permeabilization or complete breakage of the membrane and wall of cells. Thus, solvents will be able to access intracellular biomolecules [10]. Consequently, selecting a suitable cell-breaking treatment before protein extraction is crucial for downstream processing. Mechanical cell rupture methods, such as high-pressure homogenization, ball milling, and sonication, have been used [11–14] because they provide good cell wall disintegration efficiency and do not use chemical reagents. Since the downstream steps represent a large part of the operational costs to the production chain, cell lysis technologies must be low-cost and energy-efficient, improving product quality and yield [10,15]. Moreover, an effective technique is necessary for monitoring and measuring the degree of microalgal cell disruption.

Question 03. Please elaborate further the following phrase "to identify the majority of protein in the protein-rich extracts". this is really interesting since most papers focuses only on quantity of protein extract than the "quality" of proteins extracted.

Now (lines 71-74):

This study aimed (i) to investigate the effect of three different disruption methods, high-pressure homogenization, ball milling, and ultrasound, on Tetradesmus obliquus cells, (ii) to identify the molecular mass profile for the majority of proteins in protein-rich extracts, and (iii) to evaluate the color parameters of protein-rich extracts.

Question 04. Materials: Remove "the" at the beginning of Line 81. How was the biomass concentrated? floc, centrifuge, filtration? please open this section with the phrase of lines 83-84 "the biomass was a gift from..." the first section is quite confusing, please make it clearer and straightforward. 1. who produced the biomass, and how. 1 how was harvested and processed prior to drying. Response: The paragraph was rewritten.

Now (lines 86-95)

2.1. Tetradesmus obliquus biomass processing

Microalgal biomass was a gentle gift from the Biofuels Laboratory at the Universidade Federal de Viçosa, Brazil. T. obliquus strain was previously isolated from freshwater reservoirs and cultivated in BG11 culture medium [21,22]. The biomass was produced under photoautotrophic growth conditions as described by Vieira et al. [16]. The biomass was collected on the 12^th day of cultivation during the stationary growth phase and concentrated by flocculation (Cationic Polyamine, SNF Floerger, France) and sedimentation, followed by gravity filtration with a 100 μm polyester membrane, resulting in a microalgal slurry with approximately 5% (w v^-1) total solids [16]. The T. obliquus paste was frozen and stored at -10 °C.

Question 05. Results: Please improve the quality of figure 3, or find a better way to present the results. Also improve figure 5. Response: Figure 3 was removed, as suggested by Reviewer 1. Figure 5, now renamed Figure 4, was improved.

Now (line 270)

 

 

Question 06. Discussion: In line 332 Acutodesmus oblíquos is not the correct name, please change it to "Acutodesmus obliquus". Response: The name of the microalgae was corrected to "Acutodesmus obliquus".

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper deals with the comparison of three cell disruption techniques applied to the Chlorophyceae, Tetradesmus obliquus in terms of cells disruption and proteins recovery yields as well as molecular mass and identification of recovered proteins. Thus, high-pressure homogenization, ultrasound and ball milling have been used. The presented work is relevant and topical to the journal. The subject of the presented study is interesting but results are insufficiently discussed and the operating conditions insufficiently justified. The efficiencies of disruption techniques seem to be strongly linked to the operating conditions which had been adopted and unfortunately the results are not discussed facing with these conditions. It would have been better to discuss the reasons that lead to these results, less by comparing with those of literature which are not always well chosen (not the same microalgae and operating conditions) but more on the basis of additional experiments aiming to explain phenomena which take part.

Therefore, I issue an unfavourable opinion on the publication of this paper as a full article, but it could be switched as a short communication following some corrections.

 

Abstract:

Q1. line36: “This study also indicates that T. obliquus can be an alternative and sustainable source of protein, making it attractive for industrial applications in the food industry”: What are the results that lead to this conclusion? To be detailed/explained.

 

Manuscript:

    Introduction:

Q2. line 45: “Their growth is independent of climate conditions”: unclear or even inaccurate

Q3. line 59: “Solvents will be able to access intracellular biomolecules”: To be explained facing with

Q4. line 76-83: Bibliographic references are not judiciously chosen because they concern whole algae whereas authors should try to convince of the added value of extracting proteins from these micro-algae.

 

     Material and methods:

Q5. line 90: “The biomass was produced under photoautotrophic growth conditions as described by Veira et al.”: The conditions should be reminded within this paper for ease of reading. 

Q6. line 95: “washed with deionized water”: The protocol should be added.

Q7. Line 99: “The dry biomass was then sheared through a 59 mm stainless steel sieve”: Better to indicate the size of the holes of the sieve.

Q8. Line 115: “biomass suspensions were prepared with concentrations of 20, 40, and 50 µg of protein”: It is unclear. What is the link between concentrations and µg?

Q9. Line 142-145: Operating conditions of mechanical disruption by ball mill are not indicated in this article nor in this publication of Vieira et al. . Please, indicate them (loading, ball material, ball diameter, etc.).    

Q10. Line 141-160: Operating conditions need to be justified. Why did you choose these conditions? (1.5 %, 5%, 25 passes …). Are these the optimal conditions for each destruction method?

Q11. Line 164: “100 µL of the disrupted cell suspension “: The concentration should be indicated.

Q12. Line 180: “The disrupted biomass in each treatment was suspended in distilled water at a concentration of 10 % w/v”:  This value should be justified.

Q13. 182: “the pH of the microalgal suspension was adjusted to 10”: This value should be justified/explained.  

  

      Results:

Q14. Figure 4, Line 268-274: Letters are not indicated on the figure.

Q15. The different methods of disruption should be compared in view of industrial application (scale-up) since authors talk about “food industry” (for example, Line 312)  

 

Discussion:

Q16. The discussion often resembles a literature review that does not allow the results of this work to be understood and explained. A description of phenomena and mechanisms which take part during each method of disruption should be added since this article deals with the “Evaluation of cell rupture techniques”.

For example, from line 432 to 442: “Furthermore, it is emphasized that acoustic cavitation occurs by increasing the local temperature [50], and this temperature rise might destroy target compounds, especially proteins. As proteins can be used as a techno-functional ingredient in food systems, the protein extraction process must occur without drastic conditions that could make them nonfunctional [9]. González-Fernandez et al. [52] reported temperatures up to 85 °C when 100.7 MJ/kg of energy was supplied to Scenedesmus biomass through US treatment for 15 min. The authors suggested that thermal effects might have accounted for cell disruption of Scenedesmus biomass. US treatment needs to be optimized to avoid thermal overexposure of biomass; once cell disruption is achieved, more energy is absorbed or scattered by the cell debris [53]. Cooling is usually used to prevent overexposure during or after ultrasound operation”. What are the links with the results of this work? The discussion should help to understand the results of this work.

Q17. Line 311-313: “Thus T. obliquus biomass is a strategic ingredient for the food industry, both in nutritional terms and as a raw material for obtaining protein isolates and concentrates”.  To detail the nutritional aspect of the proteins and not the whole microalga since the article deals with the extraction/valorisation of these compounds.   

Q18. Line 319-320: “The observed low lipid content (4.23 ± 0.21%), compared with reported literature for T. obliquus lipid contents [13,16,18], is probably due to the culture media composition and harvest conditions”: The links with the harvest conditions should be explained.

Q19. Line 366-367: “A ball mill (BM) is a simple method for disrupting the cell walls of different microorganisms [43]. However, Figure 3B indicates that the BM treatment did not effectively disrupt the microalgal cells.”: Results must be discussed facing with the operating conditions and scaling-up. Why did you choose these operating conditions because under other conditions, there would have been destruction?. So, it is necessary to discuss the results facing with the operating conditions    

Q20. The discussion focuses on the disruption methods but should integrate a part on the recovery method of proteins. Proteins could be released from the cells but not totally recovered because embedded in aggregates. So, it’s necessary to discuss the couple “disruption method + recovering method” and not just the disruption method.    

Q21. Line 369: “In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with vigorous agitation”: To be placed to the material and methods part and to be more precise. “vigorous agitation”: what does it means?  This applies to the entire document that means it is necessary to set the values of each operating condition.   

Q22. Line 382: “Thus, HPH is especially suitable for emulsification processes”: To be detailed or to be deleted because it does not seem to be the topic of this article.

Q23. Line 384-390: “Dias et al. [12] used HPH, followed by solvent extraction, to simultaneously extract lipids and carotenoids from Rhodosporidium toruloides biomass without any harvesting technique. The combined use of HPH and solvent extraction promoted higher extraction than conventional solvent extraction. Günerken et al. [10] argue some disadvantages of the HPH process, such as nonselective intracellular compound releases, difficulties in breaking rigid cell walls, generation of superfine cell debris, not a mild technique, and inability to isolate fragile functional compounds. However, among the different cell disruption techniques, HPH is easily scalable.”: solvent extraction is not the topic of this article. Moreover, the HPH results of the literature should be discussed facing with those obtained in this work. What about selectivity facing with proteins obtained in the present work, what about cell size debris compared to the present work …   

Q24. Line 416: “Thus, the more intracellular compounds recovered after cell breaking, the greater the efficiency of the disruption method”: Not quite true. What about the efficiency of the recovery method? There are two things, the disruption method but also the recovery method. The disruption could be effective but generate a juice that would be impossible to centrifuge.    

 

  Conclusions:

Q25. “The content of extracted soluble protein was a reliable indirect index to measure the level of cell disruption”: it’s better to discuss the couple disruption plus recovery method than disruption method alone.

Author Response

"Please see also the attachment."

Viçosa, MG, Brazil, December 28^th, 2023.

To the Editor-in-chief

Dear Mr. Louies Liu,

Thank you and the reviewer for the comments and suggestions on our manuscript entitled: "Evaluation of cell rupture techniques on the extraction of proteins from microalgae Tetradesmus obliquus".

We sincerely appreciate the valuable comments and suggestions of the review, which helped us improve the manuscript's quality. All recommendations and suggestions were accepted, and the document highlighted the changes in blue.

The manuscript has been formatted according to the Author-Directions of the Phycology. All authors contributed equally to preparing the manuscript, reviewing it, and approving its final version. Moreover, we declare that there are no conflicts of interest.

 

Best regards,

 

Jane

On behalf of all authors

 

Review comments:

 

REVIEWER 3

Reviewer #3: The paper deals with the comparison of three cell disruption techniques applied to the Chlorophyceae, Tetradesmus obliquus in terms of cells disruption and proteins recovery yields as well as molecular mass and identification of recovered proteins. Thus, high-pressure homogenization, ultrasound and ball milling have been used. The presented work is relevant and topical to the journal. The subject of the presented study is interesting but results are insufficiently discussed and the operating conditions insufficiently justified. The efficiencies of disruption techniques seem to be strongly linked to the operating conditions which had been adopted and unfortunately the results are not discussed facing with these conditions. It would have been better to discuss the reasons that lead to these results, less by comparing with those of literature which are not always well chosen (not the same microalgae and operating conditions) but more on the basis of additional experiments aiming to explain phenomena which take part. Therefore, I issue an unfavourable opinion on the publication of this paper as a full article, but it could be switched as a short communication following some corrections.

Thank you for reading our manuscript and for all the valuable comments and suggestions, which helped us improve the manuscript's quality. As detailed below, the manuscript was corrected in all questions 01 to 25.

 

Abstract

Q1. Line 36: "This study also indicates that T. obliquus can be an alternative and sustainable source of protein, making it attractive for industrial applications in the food industry": What are the results that lead to this conclusion? To be detailed/explained. Response: Thank you for the comment. Our study does not provide the necessary basis to conclude that T. obliquus biomass is an alternative source of proteins. The alternative protein source should have a balanced nutritional profile, providing essential amino acids, vitamins, and minerals comparable to traditional protein sources (Nongonierma and FitzGerald, 2017). Moreover, alternative protein sources must ensure compliance with food safety regulations and obtain necessary approvals from regulatory authorities. Thus, the sources warrant that it can be a viable substitute in various food applications. The T. obliquus protein could be environmentally sustainable, with minimal impact on ecosystems, water usage, and land resources (Soto Sierra et al., 2018). The sentence was removed.

 

Before (lines 34-38): Since the cell break method affects protein extraction from microalgal biomass, protein release can be used as a central indicator reflecting the degree of cell disruption. This study also indicates that T. obliquus can be an alternative and sustainable source of protein, making it attractive for industrial applications in the food industry.

 

Now (lines 34-37): Since the cell breakage method affects protein extraction from microalgal biomass, protein release can be used as a central indicator of the degree of cell disruption.

 

• Nongonierma AB, FitzGerald RJ. (2017). Strategies for the discovery and identification of food protein-derived biologically active peptides. Trends in Food Science & Technology 6, Part B: 289-305, https://doi.org/10.1016/j.tifs.2017.03.003.
• Soto-Sierra et al (2018). Extraction and fractionation of microalgae-based protein products. Algal Research, 36: 175-192, https://doi.org/10.1016/j.algal.2018.10.023.

 

Introduction

Q2. line 45: "Their growth is independent of climate conditions": unclear or even inaccurate. Response: Thank you for the remark. The sentence was removed. In our study, the T. obliquus biomass was produced under photoautotrophic growth conditions, and microalgae grown is influenced by various environmental factors such as temperature, light, nutrient availability, pH, CO₂ concentration, and water availability. Researchers and cultivators often consider and manipulate these environmental parameters to enhance microalgae productivity under controlled conditions.

 

Before (lines 42-48): Microalgae can be used in the cosmetics industry, wastewater treatment, energy production, and human and animal nutrition; have a propensity for growth in any region because their growth is independent of climate conditions; survive in areas unsuitable for growing traditional crops, not competing with food production sites; and are rich in metabolites, such as proteins, lipids, vitamins, enzymes, pigments, mineral salts, antioxidants, and antibiotics [1-2].

 

Now (lines 42-46): Microalgae can be used in the cosmetics industry for wastewater treatment, energy generation, and human and animal nutrition; can survive in areas unsuitable for growing traditional crops; do not compete with food production sites; and are rich in metabolites, such as proteins, lipids, vitamins, enzymes, pigments, mineral salts, antioxidants, and antibiotics [1-2].

 

Q3. line 59: "Solvents will be able to access intracellular biomolecules": To be explained facing with. Response: The text was modified as follows.

 

Before (lines 57-59): Cell disruption, as one of the first downstream stages for microalgae biotechnological platforms, promotes the permeabilization or complete breakage of the membrane and wall of cells. Thus, solvents will be able to access intracellular biomolecules [10].

 

Now (lines 55-63): Cell disruption, as one of the first downstream stages of microalgal biotechnological platform development, promotes the permeabilization or complete breakage of the membrane and wall of cells. Cell wall disruption is necessary for accessing intracellular components since valuable biocomponents, such as lipids and proteins, are located within microalgal cells. Cell breakage enables the release of intracellular contents, which can be further processed, purified, and used in various applications. Rupturing the cell wall also increases the surface area available for extraction processes, enhancing the efficiency of extraction methods through contact between the solvent and the compounds of interest [10].

 

Q4. line 76-83: Bibliographic references are not judiciously chosen because they concern whole algae whereas authors should try to convince of the added value of extracting proteins from these micro-algae. Response: Thank you for the suggestion. Seeking to explore the use of protein concentrates obtained from Tetradesmus spp, we added studies on their application in the food, pharmaceutical, and cosmetic industries as follows.

 

Before (lines 72-84): Microalgae Tetradesmus spp.: are a Brazilian native species abundant in aquatic environments; present a sizeable morphological variation within each species; show a high growth rate; contain 30% to 40% protein on a dry-mass basis [8,13,16]; and can be applied in biofuel production [14,17,18] and the food, pharmaceutical, and cosmetic industries [19,20]. Afify et al. [20] described that T. obliquus contains antioxidative nutraceutical ingredients and is a potential therapeutic agent against Coxsackie B3 virus. Silva et al. [2] identified no toxicity effects of T. obliquus ingestion in Wistar rats. Analyses of the rats' liver, spleen, and kidney organs indicated that the microalga is a potentially safe food, even at high biomass concentrations (23.2%). Furthermore, the consumption of this microalga reduced the triglyceride content (70%), atherogenic index (80%), and serum glucose concentration (42%) in Wistar rats, even on a balanced diet. Thus, T. obliquus may represent a promising sustainable source for preventing and treating diabetes and dyslipidemias.

 

Now (lines 75-90): The microalgae Tetradesmus spp. (i) are native Brazilian species abundant in aquatic environments; (ii) exhibit sizeable morphological variation within each species; (iii) exhibit a high growth rate; and (iv) contain 30% to 40% protein on a dry-mass basis [8,13,16,17]. The literature has also described the great potential of T. obliquus biomass for applications in the food, pharmaceutical, cosmetic, and biofuel industries [13, 17-22]. Silva et al. [21] reported no toxic effects from the ingestion of T. obliquus biomass by Wistar rats. Analyses of the rats' liver, spleen, and kidney indicated that the microalga is a potentially safe food, even at high biomass concentrations (23.2%). Thus, the protein in microalgal biomass is a potential food ingredient. Furthermore, Silva et al. [21] reported that the consumption of T. obliquus reduced the triglyceride content (70%), atherogenic index (80%), and serum glucose concentration (42%) in Wistar rats, even on a balanced diet. Lima et al. [20] reported that lower concentrations (0.5% and 1.0% w/w) of T. obliquus protein concentrate (51.46% w/w) stabilized emulsions after 28 days of storage. Silva et al. [21] observed that the T. obliquus protein concentrate (63.14% w/w) could form stable emulsions that were resistant to pH shifts and tolerated high salt concentrations.

 

Material and methods

Q5. line 90: "The biomass was produced under photoautotrophic growth conditions as described by Veira et al.": The conditions should be reminded within this paper for ease of reading. Response: Thank you for the improvement. The conditions were incorporated into the text as follows .

 

Before (lines 89-90): The biomass was produced under photoautotrophic growth conditions as described by Vieira et al. [16].

 

Now (lines 93-102): Microalgal biomass was a gentle gift from the Biofuels Laboratory at the Universidade Federal de Viçosa, Brazil. The T. obliquus strain was previously isolated from freshwater reservoirs and cultivated in BG11 culture media [23,24]. Biomass production was carried out following the protocol established by Vieira et al. [17]. The biomass was produced under photoautotrophic growth conditions in a concrete raceway pond of 4 m³ with a natural photoperiod and light during the summer (Viçosa, Minas Gerais, Brazil). The average temperature through the growing season was approximately 27°C. The culture media were prepared with macronutrients provided by inorganic fertilizers to increase the protein productivity of T. obliquus through the use of trace elements and without salt stress [25].

 

Q6. line 95: "washed with deionized water": The protocol should be added. Response: The protocol was incorporated into the text as follows.

 

Before (lines 95-97): The frozen biomass was thawed for fractionation, washed with deionized water, filtered in an organza fabric filter medium to remove impurities, packed in containers, and frozen (GE, Brazil) at -80 °C.

 

Now (lines 106-109): The frozen biomass was thawed for fractionation, washed with deionized water three times [21,25,26] to remove salts, filtered in an organza fabric filter medium to remove im-purities, packed in containers, and frozen (GE, Brazil) at -80 °C.

 

Q7. Line 99: "The dry biomass was then sheared through a 59 mm stainless steel sieve": Better to indicate the size of the holes of the sieve. Response: The size of the holes was informed.

 

Before (lines 99-100): The dry biomass was then sheared through a 59 mm stainless steel sieve (Tyler 28).

 

Now (lines 111-112): The dry biomass was then sheared through a stainless-steel sieve (Tyler 28) with a hole of 0.595 mm.

 

Q8. Line 115: "biomass suspensions were prepared with concentrations of 20, 40, and 50 µg of protein": It is unclear. What is the link between concentrations and µg? Response: Thank you. The sentence was corrected as follows.

 

Before (lines 113-115): The microalgal biomass was initially suspended for solubilization in 1 mL of 25 mM Tris-HCl buffer (pH 7.5), and biomass suspensions were prepared with concentrations of 20, 40, and 50 μg of protein.

 

Now (lines 127-129): The microalgal biomass was initially suspended for solubilization in 1 mL of 25 mM Tris-HCl buffer (pH 7.5), and biomass suspensions were prepared with protein concentrations of 20, 40, and 50 μg/mL.

 

Q9. Line 142-145: Operating conditions of mechanical disruption by ball mill are not indicated in this article nor in this publication of Vieira et al. . Please, indicate them (loading, ball material, ball diameter, etc.). Response: The operating conditions for the ball mill were incorporated into the text.

 

Before (lines 142-145): Mechanical disruption by ball mill: The freeze-dried biomass was mechanically disrupted in a ball mill (model MR350, Tecnal Equipamentos Científicos, Brazil) using 10 g of biomass batches for 25 min, following Vieira et al. [16]. The disrupted biomass was kept at 20 °C until use.

 

Now (lines 155-161): Mechanical disruption by ball mill: The freeze-dried biomass (10 g) was mechan-ically disrupted in a cylindrical ball milling constructed using AISI-304 stainless steel with an internal volume of 0.235 L (Tecnal Equipamentos Científicos, model R-TE-350, Brazil), following Vieira et al. [17]. The mill balls (0.635 cm in external diameter) and the biomass swung up and down vertically, approximately 10 times per second and 617 strokes per minute, using batches for 25 min. The dis-rupted biomass was kept at 20 °C until use

 

Q10. Line 141-160: Operating conditions need to be justified. Why did you choose these conditions? (1.5%, 5%, 25 passes …). Are these the optimal conditions for each destruction method? Response: Thank you. The operating conditions in ultrasound followed the optimal conditions of Silva et al. (2021) with adaptations. The aqueous suspension had a biomass concentration of 10%. We corrected the suspension concentration to 10% instead of 5% (as informed previously). Regarding the mechanical disruption caused by high-pressure homogenization, we followed the optimal conditions reported by Shene et al. (2016) with adaptations (suspension with a concentration of 1.5%). Shene et al. (2016) prepared suspensions with 1.25 g:20mL (dry solid content 1.6% w/weight). We used suspensions with a solid content of 1.5% w/volume, similar to the conditions of Shene et al. (2016). In previous tests, we observed that the number of passes equal to  25 promoted greater protein recovery. However, biomass overheating was observed in the tests with a number of passes higher than 25.

 

Before (lines 146-160):  Mechanical disruption by ultrasound: The freeze-dried biomass was mechanically disrupted by ultrasound after resuspension in distilled water (5.0 % w/v), following Silva et al. [19] with adaptations. The suspensions were mechanically stirred (IKA, RW20 digital, Germany) at 25.0 °C overnight and disrupted in a tip sonicator (Sonics, VCX 500, USA) at 20 kHz frequency and 98% amplitude for 6 min of ultrasonication. The cells were disrupted under cooling in an ice bath to avoid overheating the system. After disruption, cell suspensions were frozen, freeze-dried (Terroni, LS 3000, Brazil), and stored at 20 °C until use.

 

Mechanical disruption by high-pressure homogenization: The microalgal biomass was suspended in distilled water (1.5% w/v) and processed in a homogenizer (Alitec, A100, Brazil) at 350 bar, according to Shene et al. [27]. The number of passes of the suspensions in the homogenizer was 25, and the suspension was cooled to avoid compound degradation due to the temperature increase. The homogenized samples were collected, frozen, freeze-dried (Terroni, LS 3000, Brazil), and stored at 20 °C until use.

 

Now (lines 162-170): Mechanical disruption by ultrasound: The freeze-dried biomass was mechanically disrupted by ultrasound after resuspension in distilled water (10.0% w/v) following the optimal conditions determined by Silva et al. [21]. The suspensions were mechanically stirred (IKA, RW20 digital, Germany) at 25.0 °C overnight and disrupted in a tip sonicator (Sonics, VCX 500, USA) at a frequency of 20 kHz and 98% amplitude for 6 min of ultrasonication. The cells were disrupted by cooling in an ice bath to avoid overheating the system. After disruption, the cell suspensions were frozen, freeze-dried (Terroni, LS 3000, Brazil), and stored at 20 °C until use.

 

Now (lines 171-180): •       Mechanical disruption by high-pressure homogenization: The microalgal biomass was suspended in distilled water (1.5% w/v) and processed in a homogenizer (Alitec, A100, Brazil) at 350 bar according to the optimal conditions determined by Shene et al. [30], with modifications. Twenty-five passes of the suspensions in the homogenizer were used, and the suspension was cooled to avoid compound degradation due to the increase in temperature. Based on the results of preliminary tests, we observed that a total of 25 passes promoted greater protein recovery. However, biomass overheating was observed in the tests with more than 25 passes. The homogenized samples were collected, frozen, and freeze-dried (Terroni, LS 3000, Brazil) and stored at 20 °C until use.

 

Q11. Line 164: "100 µL of the disrupted cell suspension ": The concentration should be indicated. Response: The concentration was indicated as follows.

 

Before (lines 163-164): A 100 μL volume of the working solution was added to 100 μL of the disrupted cell suspension to count ruptured biomass cells, according to Gminski et al. [27].

 

Now (lines 183-185): A 100 μL volume of the working solution was added to 100 μL of the disrupted cell suspension in distilled water at a concentration of 0.1% w/v to count the ruptured biomass cells, according to Gminski et al. [31].

 

Q12. Line 180: "The disrupted biomass in each treatment was suspended in distilled water at a concentration of 10 % w/v":  This value should be justified. Response: Our study used the optimal conditions of Silva et al. (2021) for protein extraction, an aqueous suspension of disrupted biomass at a concentration of 10.0% w/v. The sentence was corrected.

 

Before (lines 179-180): The effect of microalgal cell disruption on protein extraction was evaluated using the amount of the total hydrosoluble protein extracted [3].

 

Now (lines 201-205): The effect of microalgal cell disruption on protein extraction was evaluated using the amount of total hydrosoluble protein extracted. The disrupted biomass in each treatment was suspended in distilled water at a concentration of 10.0% w/v and then magnetically stirred for 24 h, following the optimal conditions for protein extraction determined by Silva et al.[21] with modifications.

 

Q13. 182: "the pH of the microalgal suspension was adjusted to 10": This value should be justified/explained.  Response:  The pH 10 was defined because the proteins of the microalga T. obliquus revealed greater solubility in pH value, according to Silva et al. (2021).

 

Before (lines 182-183):  The pH of the microalgal suspension was adjusted to 10, and the suspension was magnetically stirred (MA 502/D, Marconi, Brazil) at 1500 rpm for 2 h at 25 °C.

 

Now (lines 205-209): The pH of the microalgal suspension was adjusted to 10, and the suspension was magnetically stirred (Marconi, MA 502/D, Brazil) at 1500 rpm for 2 h at 25 °C. A pH of 10 was chosen because the proteins of the microalga T. obliquus are more soluble at this pH [21]. According to Afif et al. [18], the further away from the isoelectric point of the T. obliquus proteins (2.5), the greater the solubility of these proteins.

 

Results

Q14. Figure 4, Line 268-274: Letters are not indicated on the figure. Response: Thank you. Figure 4 was improved, and letters were indicated.

 

Before (Lines 268-274):

Now (lines 287-294):

 

Figure 4. Protein amount recovered (Equation (3)) after T. obliquus cell breakage as a percentage of the total protein content. Disruption treatments of biomass included ball mill (BM), high-pressure homogenization (HPH), and ultrasound (US) methods. Different letters indicate a significant difference (p < 0.05). The capital letters indicate differences among the cell disruption treatments in the same extraction step. Lowercase letters indicate the difference between each extraction step and the total extraction for the same cell disruption treatment. The results are expressed as the mean, and the bars indicate the standard deviation (n = 3).

 

Q15. The different methods of disruption should be compared in view of industrial application (scale-up) since authors talk about "food industry" (for example, Line 312). Response: The paragraph was rewritten according to the objectives and results found.

 

Before (Lines 311-313): Thus, T. obliquus biomass is a strategic ingredient for the food industry, both in nutritional terms [3] and as a raw material for obtaining protein isolates and concentrates [18,19].

 

Now (lines 350-353): Thus, T. obliquus biomass is a strategic ingredient for the food industry since it is a potential alternative raw material for obtaining protein isolates and concentrates [20,21] that can be cultivated in large quantities for industrial feed applications in the food industry.

 

 

Discussion

Q16. The discussion often resembles a literature review that does not allow the results of this work to be understood and explained. A description of phenomena and mechanisms which take part during each method of disruption should be added since this article deals with the "Evaluation of cell rupture techniques".

For example, from line 432 to 442: "Furthermore, it is emphasized that acoustic cavitation occurs by increasing the local temperature [50], and this temperature rise might destroy target compounds, especially proteins. As proteins can be used as a techno-functional ingredient in food systems, the protein extraction process must occur without drastic conditions that could make them nonfunctional [9]. González-Fernandez et al. [52] reported temperatures up to 85 °C when 100.7 MJ/kg of energy was supplied to Scenedesmus biomass through US treatment for 15 min. The authors suggested that thermal effects might have accounted for cell disruption of Scenedesmus biomass. US treatment needs to be optimized to avoid thermal overexposure of biomass; once cell disruption is achieved, more energy is absorbed or scattered by the cell debris [53]. Cooling is usually used to prevent overexposure during or after ultrasound operation". What are the links with the results of this work? The discussion should help to understand the results of this work. Response: Thank for the improvement. The discussion section 4.3 was rewritten as follows.

 

Before (Lines 351-496)

4.3. Cell rupture and protein extraction from T. obliquus

• Evaluation of the cell disruption level through cell counting

The optical microscopic examination of cells is a common technique to measure the cell breaking level. Figure 3A shows the control sample used in the disruption studies. It comprises cells of T. obliquus not subjected to any disruption treatment, also named original T. obliquus cells in the present work. The control sample showed an ellipsoidal shape, with a concave or linear fusiform contour and cells grouped in numbers of 4 or 8, the cenobia. Even the control sample showed the presence of cells with a reddish color, suggesting that some of the cells may have been damaged or ruptured. This behavior is probably due to cell disintegration and membrane permeabilization of microalgal species with a rigid cell wall, such as T. obliquus [33], during freeze-drying [39]. The freeze-drying process causes some damage to the cell membrane, making the cell membrane more porous; however, the cell wall is not ruptured [40–42]. It is important to highlight that all disruption methods were carried out with freeze-dried biomass; thus, the eventual damage caused by freeze-drying in the cell wall was the same in all treatments evaluated.

A ball mill (BM) is a simple method for disrupting the cell walls of different microorganisms [43]. However, Figure 3B indicates that the BM treatment did not effectively disrupt the microalgal cells. The biomass submitted to BM treatment showed strong agglomeration. In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with vigorous agitation. Due to shear force, kinetic energy is transferred to biomass to break the cells. This behavior hinders the dye's permeation in the medium, making differentiating intact and damaged cells impossible, and because of this, it was not possible to determine the percentage of ruptured cells. Furthermore, Bunge et al. [44] observed complete cell disruption of the bacterium Arrhrobacter sp. in a stirred BM, in which the enzymes were released without any degradation by using small grinding balls.

The high-pressure homogenization (HPH) of the microalgal cells revealed significant differences between the treatment and control samples (Figure 3C) and broken fragments (parts with a redder color) of the cell wall. The percentage of ruptured cells reached 78.51 ± 1.97%. In the HPH process, cell disruption is achieved through the high-pressure impact (shear forces) of the accelerated fluid jet on the homogenizer stationary valve surface and hydrodynamic cavitation from the pressure drop-induced shear stress [10]. Thus, HPH is especially suitable for emulsification processes.

Dias et al. [12] used HPH, followed by solvent extraction, to simultaneously extract lipids and carotenoids from Rhodosporidium toruloides biomass without any harvesting technique. The combined use of HPH and solvent extraction promoted higher extraction than conventional solvent extraction. Günerken et al. [10] argue some disadvantages of the HPH process, such as nonselective intracellular compound releases, difficulties in breaking rigid cell walls, generation of superfine cell debris, not a mild technique, and inability to isolate fragile functional compounds. However, among the different cell disruption techniques, HPH is easily scalable.

Figure 3D shows that the ultrasound (US) treatment exhibited the highest number of red cells compared to the BM and HPH treatments, suggesting that US effectively disrupted the microalgal biomass. The percentage of ruptured cells was 80.17 ± 0.54%. Ultrasound is a physical treatment based on bubble cavitation that uses sound waves to propagate pressure fluctuations, induces cavitation, and promotes nonspecific cell-surface barrier disruption [45]. The wall structure and size of the cells are critical factors affecting cell disruption efficacy in ultrasonic processing. Concerning the wall structure, microalgae with cellulose carbohydrate-based cell walls typically show more resistance against ultrasound than cell walls mainly composed of hydroxyproline-rich glycoproteins [46]. According to Do Carmo Cesário et al. [33], the T. obliquus cell wall is filled predominantly with fibrous material and has three well-defined layers, making breaking down even more challenging.

Spiden et al. [47] used the cell counting technique to evaluate Saccharomyces cerevisiae cell disruption after HPH treatment. According to the authors, an interval between 10 and 30 min was necessary to perform the cell count, depending on the sample volume and the cell concentration. Due to cell debris, reliable automated cell counting is not always possible. These observations show that a study of cell counting is insufficient to understand cell disruption, and an effective technique for monitoring and measuring the impact of treatment on microalgae cell disruption is necessary. Thus, protein extraction yield under different rupture treatments is another parameter to evaluate cell breaking.

 

• Evaluation of the cell disruption level through the amount of extracted soluble protein

The resistance of cell walls to disruption is a barrier hindering the efficient removal of intracellular components and may interfere with the accuracy of compound quantification [48]. Thus, the more intracellular compounds recovered after cell breaking, the greater the efficiency of the disruption method. According to Safi et al. [49], the amount of protein in the aqueous supernatant was a technique appropriate for evaluating the degree of cell disruption in three species of microalgae: M. aeruginosa, C. pyrenoidosa, and C. reinhardtii.

According to Figure 4, the number of extraction steps positively affected protein recovery. The ultrasound treatment resulted in the highest total protein extraction yield (19.95%). This result agrees with the behavior of cell disruption observed by optical microscopy after US treatment, in which most cells were red-colored, and the number of disrupted cells (80.17%) was the highest. Therefore, the efficiency of US treatment is evident in disrupting the cell wall, allowing protein extraction from microalgae cells.

The US treatment for cell disruption is based on the emission of high-frequency wave sounds (up to 15–20 kHz) in liquid. These sound waves create gas bubbles that achieve a critical size, collapsing and releasing large amounts of energy [50]. Cells adjacent to collapsing cavitation bubbles are broken, while cells located farther away from bubble cavitation also experience a smaller local energy flux [51]. Thus, the cell disruption power extends beyond the effect on the cell wall, reaching other microalgae cell organelles.

Furthermore, it is emphasized that acoustic cavitation occurs by increasing the local temperature [50], and this temperature rise might destroy target compounds, especially proteins. As proteins can be used as a techno-functional ingredient in food systems, the protein extraction process must occur without drastic conditions that could make them nonfunctional [9]. González-Fernandez et al. [52] reported temperatures up to 85 °C when 100.7 MJ/kg of energy was supplied to Scenedesmus biomass through US treatment for 15 min. The authors suggested that thermal effects might have accounted for cell disruption of Scenedesmus biomass. US treatment needs to be optimized to avoid thermal overexposure of biomass; once cell disruption is achieved, more energy is absorbed or scattered by the cell debris [53]. Cooling is usually used to prevent overexposure during or after ultrasound operation.

Recently, Delran et al. [11] verified that a low-frequency ultrasound of 20 kHz was adequate for breaking Tetraselmis suecica cells at a power of 120 W and 60 min ultrasonication, allowing the extraction of 90% of the total proteins. Compared with the literature, the low protein extraction (19.95%) observed in our US study could be explained by the intensity of cell rupture. Do Carmo Cesário et al. [33] observed the presence of proteins in the nucleus and cytoplasmic regions of T. obliquus by histochemistry tests. Therefore, mechanical treatments should collapse different parts of microalgae cells to achieve protein extraction.

Lupatini et al. [54] studied the US-assisted extraction of algal proteins from Spirulina platensis. The authors found that sonication degraded the cell wall entirely or partially, providing a valuable technique to extract proteins and carbohydrates. The optimized percentage of protein extracted was 75.76%. The ultrasound bath conditions used were 30 °C, 37 Hz, and 100% sonication amplitude for 35 min of sonication. Keris-Sem et al. [55] applied US treatment to disintegrate microalgal cells and extract components other than protein. The authors achieved higher protein, carbohydrate, and lipid extraction efficiency using an ultrasonic energy intensity of 0.4 kWh/L. Soto Sierra et al. [56] achieved a maximized yield of Chlamydomonas reinhardtii protein using autolysin coupled with sonication.

The results observed in Figure 4 show that the amounts of protein extracted from T. obliquus with HPH and BM treatments were lower than that achieved with US treatment. The low extraction yields of soluble proteins with BM can be due to the use of dry biomass instead of wet biomass. The energy for cell rupture is well utilized when the disintegration rate correlates with a specific energy. Schuller et al. [48] used BM to extract lutein and β-carotene from wet and lyophilized biomass of Tetraselmis sp. The break with wet biomass glass beads best responded to the extractive treatments. However, the literature also reported promising results for BM mechanical disruption with dried biomass [13,16].

Katsimichas et al. [57] reported protein extraction from Chlorella pyrenoidosa after HPH treatment. The application of 800 bar of pressure and a four-pass treatment caused maximization of protein recovery of 382.0 mg proteins/g dry biomass. In our study, the pressure was lower, at 350 bar. Safi et al. [49] noted HPH as a more efficient cell disruption technique than manual grinding, ultrasonication, and alkaline treatment for extracting proteins from Nannochloropsis oculata, Chlorella vulgaris, and Hematococcus pluvialis, a green microalgae with rigid cell walls. The biomass at 2% dry weight was disrupted by HPH working with two passes at 2700 bar.

In our tests, HPH effectively broke the T. obliquus cell wall, as demonstrated by optical microscopy (Figure 3C) and by disrupted counting (78,51%). Additionally, the HPH protein extract showed a remarkable light color (Table 1), showing potential for food ingredient application. Most likely, fewer chlorophyll pigments were extracted in the HPH mechanical treatment. HPH allowed greater protein extraction and was ineffective in breaking other microalgae organelles, suggesting that higher pressures can contribute to greater protein recovery efficiency [49].

Zhang et al. [14] studied the effect of US, HPH, and their combination on the efficiency of biomolecule extraction from Parachlorella kessleri. The authors verified that applying a preliminary US treatment with 10% dry matter and a final HPH treatment with 1% dry matter increases extraction efficiency and decreases energy consumption.

Since parts of the cellular fragments came together and formed large aggregates, it was not possible to quantify the disruption effect with ball mill treatment using the cell counting method; however, it was possible to determine the disruption effects through the extraction of soluble proteins. The protein extraction yield of T. obliquus with BM treatment was 14.11% (Figure 4), in which the protein showed particles with larger sizes and irregular shapes, including sheet-like structures, as seen in Figure 5. The changes in morphology could significantly affect the functional properties of microalgae protein extract. Despite the low yield found, ball milling and HPH are the preferred methods for industrial-scale microalgal cell disruption [10].

 

Now (lines 402-552):

4.3. Cell rupture and protein extraction from T. obliquus

• Evaluation of the cell disruption level through cell counting

Optical microscopic examination of cells is a common technique for measuring cell breakage. Figure 3A shows the control sample used in the disruption studies. T. obliquus cells not subjected to any disruption treatment were used; these cells were also referred to as original T. obliquus cells in the present work. The control sample exhibited an ellipsoidal shape, with a concave or linear fusiform contour, and cells were grouped into groups of 4 or 8 cenobia. Even the control sample showed the presence of cells with a reddish color, suggesting that some of the cells may have been damaged or ruptured. This behavior is probably due to cell disintegration and membrane permeabilization of microalgal species with rigid cell walls, such as T. obliquus [37], during freeze-drying [43]. The freeze-drying process causes some damage to the cell membrane, increasing its porosity; however, the cell wall is not ruptured [44–46]. It is important to highlight that all disruption methods were carried out with freeze-dried biomass; thus, the eventual damage caused by freeze-drying in the cell wall was the same for all the treatments evaluated.

A ball mill (BM) is a simple method for disrupting the cell walls of different microorganisms [47] because it provides good cell wall disintegration efficiency and does not use chemical reagents. The operating conditions of the method were based on previous studies that evaluated the occurrence of cell rupture in microalgae [17,21,30]. However, as shown in Figure 3B, the cell counting methodology was not appropriate for determining the level of microalgal cell disruption following BM treatment because the treatment promoted strong agglomeration. In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with a high level of stirring (617 strokes per minute) within the closed chamber to facilitate effective milling. Due to shear force, kinetic energy is transferred to biomass to break the cells. This behavior hinders dye permeation in the medium, making differentiating intact and damaged cells impossible; moreover, it was impossible to determine the percentage of ruptured cells. In contrast, Bunge et al. [48] observed complete cell disruption of the bacterium Arrhrobacter sp. in stirred BM, in which the enzymes were released without any degradation using small grinding balls. Thus, the type of biomass determines the adequacy of some techniques for analyzing cell breaks.

The high-pressure homogenization (HPH) of the microalgal cells revealed significant differences between the treatment and control samples (Figure 3C) and broken fragments (parts with a redder color) of the cell wall. The percentage of ruptured cells reached 78.51 ± 1.97%. In the HPH process, cell disruption is achieved through the high-pressure impact (shear forces) of the accelerated fluid jet on the homogenizer stationary valve surface and hydrodynamic cavitation from the pressure drop-induced shear stress [15]. After the HPH process, the cells were more disaggregated (Figure 3C); they exhibited more globular, almost swollen shapes, with a possible loss of wall resistance. Broken fragments (lighter parts) of the cell wall can also be observed, increasing the contact surface area and dye permeation.

Additionally, the dye (erythrosin B) permeated into cells that sustained critical damage to their plasma membranes [31], which explains the high level of rupture observed for this method. However, Günerken et al. [15] argue for several disadvantages of the HPH process, such as the release of nonselective intracellular compounds, the difficulty of breaking rigid cell walls, the generation of superfine cell debris, the absence of a mild technique, and the inability to isolate fragile functional compounds. In our study, the suspensions in the homogenizer were cooled to avoid compound degradation due to the temperature increase during each pass.

Figure 3D shows that, compared with the BM and HPH treatments, the ultrasound (US) treatment produced the highest number of red cells, suggesting that the US treatment effectively disrupted the microalgal biomass. The percentage of ruptured cells was 80.17 ± 0.54%. Ultrasound is a physical treatment based on bubble cavitation that uses sound waves to propagate pressure fluctuations, induces cavitation, and promotes nonspecific cell-surface barrier disruption [49]. The wall structure and size of cells are critical factors affecting cell disruption efficacy during ultrasonic processing. Concerning the wall structure, microalgae with cellulose carbohydrate-based cell walls typically show more resistance against ultrasound than cell walls mainly composed of hydroxyproline-rich glycoproteins [50]. According to Do Carmo Cesário et al. [37], the T. obliquus cell wall is filled predominantly with fibrous material and has three well-defined layers, making breaking down even more challenging.

Spiden et al. [51] used the cell counting technique to evaluate Saccharomyces cerevisiae cell disruption after HPH treatment. According to the authors, an interval between 10 and 30 min was necessary to perform the cell count, depending on the sample volume and the cell concentration. Due to cell debris, reliable automated cell counting is not always possible. These observations show that cell counting is insufficient for understanding cell disruption, and an effective technique for monitoring and measuring the impact of treatment on microalgal cell disruption is necessary. Thus, the protein extraction yield under different rupture treatments is another parameter for evaluating cell breakage.

 

• Evaluation of the cell disruption level through the amount of extracted soluble protein

The resistance of cell walls to disruption is a barrier hindering the efficient removal of intracellular components and may interfere with the accuracy of compound quantification [52]. The recovery efficiency of intracellular compounds from microalgae also depends on different factors, such as the type of target compound [17], the specific characteristics of the microalgal strain analyzed [15], and the chosen disruption and recovery methods [26,44]. According to Safi et al. [53], the amount of protein in the aqueous supernatant was appropriate for evaluating the degree of cell disruption in three species of microalgae: M. aeruginosa, C. pyrenoidosa, and C. reinhardtii.

According to Figure 4, an increase in the number of extraction steps positively affected protein recovery, and ultrasound treatment promoted the highest total protein extraction yield (19.95%). This result agrees with the cell disruption behavior observed by optical microscopy after US treatment, in which most cells were red, and the number of disrupted cells (80.17%) was the highest. However, this value is lower than the protein content remaining in the biomass (80.05%) after US treatment. The specific characteristics of the microalgal strains analyzed could explain this behavior. The microalgal cell wall coating components are species-specific and affect disruption efficiency. Do Carmo Cesário et al. [37] observed the presence of proteins in the nucleus and cytoplasmic regions of T. obliquus by histochemistry tests. Thus, mechanical treatments should collapse different parts of T. obliquus microalgal cells to achieve protein extraction.

The US treatment for cell disruption is based on the emission of high-frequency wave sounds (up to 15–20 kHz) in liquid. These sound waves create gas bubbles that achieve a critical size, collapsing and releasing large amounts of energy [54]. Cells adjacent to collapsing cavitation bubbles are broken, while cells located further away from bubble cavitation also experience a smaller local energy flux [55]. Thus, the cell disruption power extends beyond the effect on the cell wall, reaching other microalgal cell organelles. Furthermore, acoustic cavitation occurs by increasing the local temperature [54], and this temperature increase might destroy target compounds, especially proteins. As proteins can be used as techno-functional ingredients in food systems, the protein extraction process must occur without drastic conditions that could make them nonfunctional [9]. González-Fernandez et al. [56] reported temperatures up to 85 °C when 100.7 MJ/kg of energy was supplied to Scenedesmus biomass through US treatment for 15 min. The authors suggested that thermal effects might have accounted for cell disruption of the Scenedesmus biomass. US treatment needs to be optimized to avoid thermal overexposure of biomass; once cell disruption is achieved, additional energy is absorbed or scattered by the cell debris [57]. In our study, the cells were disrupted at 40 °C while cooling using an ice bath to avoid overheating the system.

Moreover, increasing the exposure time of cells to US treatment can affect the protein recovery rate. A longer treatment time allows for increased cell disruption due to additional energy input [58]. Delran et al. [11] verified that a low-frequency ultrasound of 20 kHz was adequate for breaking Tetraselmis suecica cells at a power of 120 W and 60 min of ultrasonication, allowing 90% of the total proteins to be extracted. Lupatini et al. [59] studied the US-assisted extraction of algal proteins from Spirulina platensis. The authors found that sonication degraded the cell wall entirely or partially, providing a valuable technique for extracting proteins and carbohydrates. The optimized percentage of protein extracted was 75.76% after 35 min of sonication at 30 °C, 37 Hz, and 100% sonication. In our study, US treatment was the most efficient treatment but failed to reverse more than 20% of the proteins produced by T. obliquus, indicating that additional extraction time is required to completely disrupt the macrostructure of these organisms; thus, additional energy is needed.

As shown in Figure 4, the amounts of protein extracted from T. obliquus following HPH and BM treatment were lower than those following US treatment. The literature on HPH shows that a high working pressure followed by cycling has the most positive effect on cell disruption efficiency [12,14,30,51,53,60]. In our tests, HPH effectively disrupted the T. obliquus cell wall, as demonstrated by optical microscopy (Figure 3C), and disrupted counting (78,51%). The HPH protein extract also showed a remarkable light color (Table 1), indicating potential for food ingredient application. The protein recovery was 15.68% when using a low working pressure of 350 bar due to the homogenizer's operation limits, the high number of passes of the suspensions in the homogenizer (25), and the pH of the microalgal suspension being 10. Katsimichas et al. [60] reported protein extraction from Chlorella pyrenoidosa after HPH treatment, in which an 800 bar of pressure and a four-pass treatment caused maximization of protein recovery of 382.0 mg proteins/g dry biomass at pH 13. Safi et al. [53] noted that HPH is a more efficient cell disruption technique than manual grinding, ultrasonication, or alkaline treatment for extracting proteins from Nannochloropsis oculata, Chlorella vulgaris, and Hematococcus pluvialis, which are green microalgae with rigid cell walls. The microalgal biomasses at 2% dry weight were disrupted by HPH working with two passes at 2700 bar and a pH of 12. There was a difference in the protein recovery methods used in the studies.

The cell counting method was not applied to quantify the disruption effect of ball mill treatment since parts of the cellular fragments came together and formed large aggregates; thus, the disruption effects were evaluated using soluble protein extraction data. The yield of protein extracted from T. obliquus following BM treatment was 14.11% (Figure 4), in which the protein showed particles of larger sizes and irregular shapes, including sheet-like structures, as shown in Figure 5. Changes in morphology could significantly affect the functional properties of microalgal protein extracts. The low extraction yields of soluble proteins with BM could be due to the use of dry biomass instead of wet biomass. The energy needed for cell rupture is well utilized when the disintegration rate correlates with a specific energy. Schuller et al. [52] used BM to extract lutein and β-carotene from wet and lyophilized biomass of Tetraselmis sp. The break with wet biomass glass beads best responded to the extractive treatments. However, the literature has also reported promising results for mechanical disruption of BM with dry biomass [13,17].

 

Q17. Line 311-313: "Thus T. obliquus biomass is a strategic ingredient for the food industry, both in nutritional terms and as a raw material for obtaining protein isolates and concentrates".  To detail the nutritional aspect of the proteins and not the whole microalga since the article deals with the extraction/valorisation of these compounds. Response: Thank you. The sentence was revised as follows.

 

Before (Lines 311-313): Thus, T. obliquus biomass is a strategic ingredient for the food industry, both in nutritional terms [3] and as a raw material for obtaining protein isolates and concentrates [18,19].

 

Now (lines 350-333): Thus, T. obliquus biomass is a strategic ingredient for the food industry since it is a potential alternative raw material for obtaining protein isolates and concentrates [20,21] that can be cultivated in large quantities for industrial feed applications in the food industry.

 

Q18. Line 319-320: "The observed low lipid content (4.23 ± 0.21%), compared with reported literature for T. obliquus lipid contents [13,16,18], is probably due to the culture media composition and harvest conditions": The links with the harvest conditions should be explained. Response: The sentence was modified as follows.

 

Before (Lines 319-321): The observed low lipid content (4.23 ± 0.21%), compared with reported literature for T. obliquus lipid contents [13,16,18], is probably due to the culture media composition and harvest conditions [13].

 

Now (lines 359-371): The observed low lipid content (4.23 ± 0.21%), compared with that reported in the literature for T. obliquus [13,17,20], is probably due to the culture conditions and nutrients used [25]. The cultivation process affects the quantity of metabolites, such as lipids and proteins, that accumulate in cells [23-25]. Some relevant factors to the cultivation process include the culture conditions, temperature, pH, light intensity, and photoperiod to increase biomass and metabolite production; nutrient optimization, which should be adjusted during the different macroalgal growth phases; carbon sources, since CO₂ supplementation can increase growth and protein content [24]; and stress induction, as nitrogen starvation can trigger microalgae to accumulate more protein as a survival mechanism [7]. Researchers and cultivators often consider and manipulate these environmental parameters to increase microalgal productivity under controlled conditions. For example, nutrients such as nitrogen and phosphorus at balanced ratios promote protein synthesis to the detriment of the synthesis of other metabolites.

 

Q19. Line 366-367: "A ball mill (BM) is a simple method for disrupting the cell walls of different microorganisms [43]. However, Figure 3B indicates that the BM treatment did not effectively disrupt the microalgal cells.": Results must be discussed facing with the operating conditions and scaling-up. Why did you choose these operating conditions because under other conditions, there would have been destruction? So, it is necessary to discuss the results facing with the operating conditions. Response: The statement was corrected as follows.

 

Before (Lines 367-368): However, Figure 3B indicates that the BM treatment did not effectively disrupt the microalgal cells.

 

Now (lines 417-432): A ball mill (BM) is a simple method for disrupting the cell walls of different microorganisms [47] because it provides good cell wall disintegration efficiency and does not use chemical reagents. The operating conditions of the method were based on previous studies that evaluated the occurrence of cell rupture in microalgae [17,21,30]. However, as shown in Figure 3B, the cell counting methodology was not appropriate for determining the level of microalgal cell disruption following BM treatment because the treatment promoted strong agglomeration. In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with a high level of stirring (617 strokes per minute) within the closed chamber to facilitate effective milling. Due to shear force, kinetic energy is transferred to biomass to break the cells. This behavior hinders dye permeation in the medium, making differentiating intact and damaged cells impossible; moreover, it was impossible to determine the percentage of ruptured cells. In contrast, Bunge et al. [48] observed complete cell disruption of the bacterium Arrhrobacter sp. in stirred BM, in which the enzymes were released without any degradation using small grinding balls. Thus, the type of biomass determines the adequacy of some techniques for analyzing cell breaks.

 

Q20. The discussion focuses on the disruption methods but should integrate a part on the recovery method of proteins. Proteins could be released from the cells but not totally recovered because embedded in aggregates. So, it's necessary to discuss the couple "disruption method + recovering method" and not just the disruption method. Response: Thank you for the recommendation. This text was modified in lines 310-323.

 

Now (lines 306-323):  Table 1 shows the protein mass yield (%) (g of protein from powder extract/100 g of dried microalgae), % of protein from the extracts (g of protein from powder extract/100 g of extract), the visual appearance of the protein extracts, and colorimetric parameters of the protein extracts. The protein mass yields of the US, HPH, and BM treatments were 20.8%, 17.1%, and 16.1%, respectively. Notably, this value represents only the extraction of aqueous soluble protein at pH 10; therefore, a large portion of the insoluble proteins could not be recovered from the biomass. The total protein content (w/w) of the T. obliquus biomass determined in our study by the Kjeldahl method was 40.29 ± 0.24%, but the protein mass yields found in your study were lower than 20.8%. Thus, insoluble proteins were not extracted under the process conditions studied and remained in the residual biomass (pellet) during the centrifugation step; proteins can be released from cells but not fully recovered because they are incorporated into aggregates. Other extraction conditions, such as temperature and pH [29], surfactant [30], and solvent type [9,27], could be used to improve protein extraction yields; however, these conditions were not in the scope of the article. Suarez Garcia et al. [36] reported that 72% of proteins are associated with insoluble cellular structures and can be extracted only using surfactants. In our study, we verified that the content of extracted soluble protein was a reliable indirect index for measuring the extent of cell disruption and comparing different mechanical treatments.

 

Q21. Line 369: "In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with vigorous agitation": To be placed to the material and methods part and to be more precise. "vigorous agitation": what does it means?  This applies to the entire document that means it is necessary to set the values of each operating condition. Response: The operating conditions of the ball mill were incorporated into the text, as can be seen in the answer to question 09. The term "vigorous agitation" was removed from the text and changed to a high level of stirring (617 strokes per minute).

 

Before (Lines 369-370):  In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with vigorous agitation.

 

Now (lines 423-425): In the BM treatment, the microalgal biomass was placed inside a closed chamber in the presence of small steel spheres with a high level of stirring (617 strokes per minute) within the closed chamber to facilitate effective milling.

Q22. Line 382: "Thus, HPH is especially suitable for emulsification processes": To be detailed or to be deleted because it does not seem to be the topic of this article. Response: The sentence was deleted.

 

Q23. Line 384-390: "Dias et al. [12] used HPH, followed by solvent extraction, to simultaneously extract lipids and carotenoids from Rhodosporidium toruloides biomass without any harvesting technique. The combined use of HPH and solvent extraction promoted higher extraction than conventional solvent extraction. Günerken et al. [10] argue some disadvantages of the HPH process, such as nonselective intracellular compound releases, difficulties in breaking rigid cell walls, generation of superfine cell debris, not a mild technique, and inability to isolate fragile functional compounds. However, among the different cell disruption techniques, HPH is easily scalable.": solvent extraction is not the topic of this article. Moreover, the HPH results of the literature should be discussed facing with those obtained in this work. What about selectivity facing with proteins obtained in the present work, what about cell size debris compared to the present work. Response: Thank you. The paragraph was rewritten.

 

Before (Lines 384-391):  Dias et al. [12] used HPH, followed by solvent extraction, to simultaneously extract lipids and carotenoids from Rhodosporidium toruloides biomass without any harvesting technique. The combined use of HPH and solvent extraction promoted higher extraction than conventional solvent extraction. Günerken et al. [10] argue some disadvantages of the HPH process, such as nonselective intracellular compound releases, difficulties in breaking rigid cell walls, generation of superfine cell debris, not a mild technique, and inability to isolate fragile functional compounds. However, among the different cell disruption techniques, HPH is easily scalable.

 

Now (lines 433-450): The high-pressure homogenization (HPH) of the microalgal cells revealed significant differences between the treatment and control samples (Figure 3C) and broken fragments (parts with a redder color) of the cell wall. The percentage of ruptured cells reached 78.51 ± 1.97%. In the HPH process, cell disruption is achieved through the high-pressure impact (shear forces) of the accelerated fluid jet on the homogenizer stationary valve surface and hydrodynamic cavitation from the pressure drop-induced shear stress [15]. After the HPH process, the cells were more disaggregated (Figure 3C); they exhibited more globular, almost swollen shapes, with a possible loss of wall resistance. Broken fragments (lighter parts) of the cell wall can also be observed, increasing the contact surface area and dye permeation.

Additionally, the dye (erythrosin B) permeated into cells that sustained critical damage to their plasma membranes [31], which explains the high level of rupture observed for this method. However, Günerken et al. [15] argue for several disadvantages of the HPH process, such as the release of nonselective intracellular compounds, the difficulty of breaking rigid cell walls, the generation of superfine cell debris, the absence of a mild technique, and the inability to isolate fragile functional compounds. In our study, the suspensions in the homogenizer were cooled to avoid compound degradation due to the temperature increase during each pass.

 

Q24. Line 416: "Thus, the more intracellular compounds recovered after cell breaking, the greater the efficiency of the disruption method": Not quite true. What about the efficiency of the recovery method? There are two things, the disruption method but also the recovery method. The disruption could be effective but generate a juice that would be impossible to centrifuge. Response: Thank you.   The text was revised as follows.

Before (Lines 416-417): Thus, the more intracellular compounds recovered after cell breaking, the greater the efficiency of the disruption method.

 

Now (lines 473-478): The resistance of cell walls to disruption is a barrier hindering the efficient removal of intracellular components and may interfere with the accuracy of compound quantification [52]. The recovery efficiency of intracellular compounds from microalgae also depends on different factors, such as the type of target compound [17], the specific characteristics of the microalgal strain analyzed [15], and the chosen disruption and recovery methods [26,44].

 

Conclusions

Q25. "The content of extracted soluble protein was a reliable indirect index to measure the level of cell disruption": it's better to discuss the couple disruption plus recovery method than disruption method alone. Response: Thank you. The text was modified as follows.

 

Now (lines 554-567):  The content of extracted soluble protein was a reliable indirect index for measuring the extent of cell disruption. However, defining an efficient method of protein recovery is still necessary to propose a route to extract proteins from T. obliquus biomass for industrial applications, particularly regarding the recovery of aqueous insoluble proteins. Optical cell counting is not an optimal indicator for monitoring cell disruption, although it is a common technique for determining cell concentration in microbial cultures. The microalgal disruption caused by different mechanical treatments could be evaluated by evaluating the extracted protein content combined with the cell counting technique. Ultrasound treatment prevented microalgal cells from breaking better than high-pressure homogenization and ball mill treatments. However, the efficiency of the extraction of protein must be improved. Combining conventional techniques, e.g., ultrasonication with high-pressure homogenization, may reduce the energy demand of mechanical disruption methods and provide better protein extraction yields. Additionally, purification steps may be employed for further protein refinement to obtain a high purity.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

no comments

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your revised version