Bio-Flocculation: A Green Tool in Biorefineries for Recovering High Added-Value Compounds from Microalgae
, Alba MarÃa Gutiérrez-Diánez 2 and Encarnación DÃaz-Santos 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Review for the paper “Bio-flocculation: a green tool in biorefineries for recovering high added-value compounds from microalgae” by Luis G. Heredia-Martínez and co-authors submitted to “Phycology”.
The authors of this review conducted an analysis of the current methodologies employed in the sustainable production of high-value compounds using microalgae, highlighting the increasing concentration on biofuels, lipids, and pigments such as carotenoids and phycobilin. They found that despite the promising potential of microalgae as a renewable feedstock, significant challenges persist in establishing economically and environmentally sustainable microalgal biorefineries. Specifically, the research emphasizes the need for improved technologies that address both cost-effectiveness and energy efficiency in microalgal processing. The results of this study may have important implications for the future of biotechnological applications in sustainable production.
The paper is well organized and coherently written. Only minor revisions are required to improve the text.
Recommendations:
Introduction.
L 45. The authors should provide a definition for the term "biorefinery". They should mention what distinguishes microalgae-based biorefineries from traditional fossil fuel-based production methods.
L 46. The authors should provide more detail on the "high photosynthetic efficiency" of microalgae. What specific mechanisms contribute to this efficiency compared to other forms of biomass?
L 53. The authors mention that microalgae are considered "third-generation biofuels." They should mention some data on how microalgae compare with first and second-generation biofuels in terms of sustainability and impact.
Flocculation Methods
Section 2.1. The authors should mention about the comparative efficiency of flocculation in microalgae harvesting relative to other harvesting techniques such as centrifugation, air flotation, or gravity sedimentation.
Section 2.2. pH-based flocculation has been described as an efficient alternative to chemical flocculation. It would be useful to provide more information about the economic feasibility of pH-based methods at industrial scales. How do costs compare with traditional chemical flocculants?
Section 2.3. The authors should discuss the potential scalability challenges for bio-flocculation in large-scale microalgae harvesting operations, and compare this approach with other flocculation methods in terms of energy consumption, cost, and environmental sustainability.
L 237-244. The authors should supplement the text with relevant citations.
L 256. Consider replacing “In several studies have identified” with “Several studies have identified”. The authors mention “several studies” but cite only one of them.
L 320. The authors should discuss how zeta potential is related to cell size and flocculation efficiency in different microalgal species. Are there methods to artificially alter the zeta potential to improve flocculation?
In this section, authors should discuss the potential influence of culture conditions. They should discuss how environmental conditions (CO2 availability, temperature and mixing) affect EPS production, flocculation efficiency and downstream biomass recovery.
L 373-376. The authors mention that microalgal species contribute to nutrient removal efficiency in wastewater treatment systems. Are there specific microalgal species that perform better under nutrient-rich or challenging wastewater conditions?
L 412. Consider replacing “As previously describe in 2.3.5” with “As previously described in 2.3.5”. It is unclear why the authors mention the parent section for this sub-section. They should change the text, for example as follows: “As mentioned above”.
L 437. The authors mentioned CRISPR technology in flocculation. Could they provide information on the specific gene targets within microalgal genomes that can be modified using CRISPR-Cas systems to enhance flocculation potential?
Valuable microalgae compounds and bio-flocculation implications
L 475-478. The authors should clarify the main economic costs associated with current methods of harvesting microalgal biomass.
Section 3.2. The authors should report the specific biosynthetic pathways involved in the production of carotenoids in microalgae. It would be useful to compare different co-culture strategies (e.g., filamentous fungi with microalgae vs. flocculent vs. non-flocculent microalgae co-cultures) in terms of efficiency and yield for carotenoid production.
L 627. The authors should specify the nutritional profile of microalgae. What are the specific essential amino acids found in microalgae? How do they compare to amino acid profiles of traditional protein sources such as soy, chicken, or fish?
L 640. Consider replacing “Microalgae have been identified as a promising organism” with “Microalgae have been identified as promising organisms”
L 656. In the authors' opinion, what are the best cultivation practices for maximizing the protein content of microalgae?
L 693. It would be useful to provide some data to quantify the impact of microalgal biofertilizers on crop yield and soil health compared to traditional fertilizers.
A table or figure summarizing the main results described in section 3 should be included in the paper to improve data visualization and interpretation.
Author Response
Response to Reviewer 1 Comments
Summary
We would like to sincerely thank the reviewers for their time, effort, and insightful comments on our manuscript titled "Bio-flocculation: a green tool in biorefineries for recovering high added-value compounds from microalgae." Their valuable suggestions and constructive feedback have greatly contributed to improving the quality and clarity of our review. We truly appreciate their dedication to helping us enhance our work.
Point-by-point response to Comments and Suggestions for Authors
The authors of this review conducted an analysis of the current methodologies employed in the sustainable production of high-value compounds using microalgae, highlighting the increasing concentration on biofuels, lipids, and pigments such as carotenoids and phycobilin. They found that despite the promising potential of microalgae as a renewable feedstock, significant challenges persist in establishing economically and environmentally sustainable microalgal biorefineries. Specifically, the research emphasizes the need for improved technologies that address both cost-effectiveness and energy efficiency in microalgal processing. The results of this study may have important implications for the future of biotechnological applications in sustainable production.
The paper is well organized and coherently written. Only minor revisions are required to improve the text.
Recommendations:
Introduction.
Comment 1: L 45. The authors should provide a definition for the term "biorefinery". They should mention what distinguishes microalgae-based biorefineries from traditional fossil fuel-based production methods.
Response 1: Thank you for your valuable comment. In response, we have added a clear definition of the term "biorefinery" in the introduction section of the manuscript. Additionally, we have highlighted the main differences between microalgae-based biorefineries and traditional fossil fuel-based production methods. We believe these additions enhance the clarity and context of our review.
L 46-51: “In this context, biorefineries—facilities that integrate biomass conversion processes and equipment to produce fuels, power, heat, and value-added chemicals from biomass—represent a sustainable alternative to traditional fossil fuel-based industries. Unlike conventional refineries, which rely on finite and polluting resources, biorefineries utilize renewable biological resources, thereby significantly lowering greenhouse gas emissions [6].”
Comment 2: L 46. The authors should provide more detail on the "high photosynthetic efficiency" of microalgae. What specific mechanisms contribute to this efficiency compared to other forms of biomass?
Response 2: Thank you very much for pointing this out. We have included a paragraph detailing the photosynthetic efficiency aspects in relation with microalgae cells.
L 52-58: “Among the various types of biorefineries, microalgae-based biorefineries are particularly promising due to the exceptional characteristics of microalgae. These include a higher photosynthetic efficiency compared to terrestrial plants, as microalgae can convert solar energy into biomass, in some cases, ten times more than the land plants [7]. This efficiency is primarily attributed to their simple cellular structure, direct access to nutrients in aquatic environments, and the ability to grow in photobioreactors under optimized conditions.”
Comment 3: L 53. The authors mention that microalgae are considered "third-generation biofuels." They should mention some data on how microalgae compare with first and second-generation biofuels in terms of sustainability and impact.
Response 3: Thank you for your insightful suggestion. In response, we have expanded the discussion at lines 58-64 by adding comparative on the sustainability and environmental impact of microalgae-based (third-generation) biofuels relative to first- and second-generation biofuels. Specifically, we highlight aspects such as land and water use, greenhouse gas emissions, and biomass productivity
L 58-64: “Moreover, microalgae are capable of capturing COâ‚‚ directly from industrial emissions, can be cultivated using wastewater as culture medium and unlike terrestrial crops, the sources of first-generation and second-generation biofuels, microalgae do not compete with food production and can be cultivated on non-arable land. Microalgae biofuel productions are recognized as third-generation biofuels, positioning these organisms as one of the most promising feedstocks [8,9] contributing both to carbon mitigation and sustainable production of biofuels and high-value compounds.”
Flocculation Methods
Comment 4: Section 2.1. The authors should mention about the comparative efficiency of flocculation in microalgae harvesting relative to other harvesting techniques such as centrifugation, air flotation, or gravity sedimentation.
Response 4: Thank you for your helpful comment. In response, we have revised Section 2.1 to include a paragraph describing a comparison of the efficiency of flocculation for microalgae harvesting relative to other commonly used techniques such as centrifugation, air flotation, and gravity sedimentation.
L 165-173: “Compared to other physical harvesting methods, flocculation offers significant advantages in terms of energy efficiency and operational costs. For instance, while centrifugation enables rapid separation and high recovery rates, it involves high ener-gy consumption and maintenance costs. Dissolved air flotation is effective for low-density microalgae but requires the addition of reagents and more complex equipment. Gravity sedimentation, although low-cost, is a slow and inefficient process for small-sized microalgae. In contrast, flocculation enables rapid cell aggregation and subsequent separation with lower energy input, making it a more viable option for large-scale applications, especially when natural or low-cost flocculants are employed [12,19,20,38].”
Comment 6: Section 2.2. pH-based flocculation has been described as an efficient alternative to chemical flocculation. It would be useful to provide more information about the economic feasibility of pH-based methods at industrial scales. How do costs compare with traditional chemical flocculants?
Response 6: We thank the reviewer for this insightful comment. In response, we have expanded Section 2.2 to include additional discussion regarding the economic feasibility of pH-based flocculation methods, especially at industrial scales.
L 221-229: “Several techno-economic assessments have indicated that pH-induced flocculation can be competitive at scale. For example, Lu et al., in 2022 reported that the energy input for pH-based flocculation was around 0.389 kWh/m³, significantly lower than many chemical or mechanical harvesting methods [38]. Additionally, the cost of reagents (e.g., NaOH or lime) used for pH adjustment is generally lower than that of high-purity chemical flocculants such as alumimum or polyaluminum chloride, especially when applied in closed-loop systems that allow for reuse and pH recovery. However, the economic performance of this method can vary depending on the water hardness, initial pH, and buffering capacity of the culture medium.”
Comment 7: Section 2.3. The authors should discuss the potential scalability challenges for bio-flocculation in large-scale microalgae harvesting operations, and compare this approach with other flocculation methods in terms of energy consumption, cost, and environmental sustainability.
Response 7: Thank you for this important suggestion. In response, we have expanded Section 2.3 to discuss the potential scalability challenges associated with bio-flocculation in large-scale microalgae harvesting operations. We have also included a comparative analysis between bio-flocculation and other flocculation methods, focusing on aspects such as energy consumption, operational costs, and environmental sustainability. These additions aim to provide a clearer understanding of the practical implications of implementing bio-flocculation at an industrial scale.
L 244-264: “Despite its promise, the scalability of bio-flocculation for large-scale microalgae harvesting remains a significant challenge. Key limitations include variability in flocculation efficiency depending on species, culture conditions, and microbial community dynamics. Maintaining stable bioflocculant-producing consortia in large open systems can be difficult, and the time required for effective aggregation is often longer compared to chemical flocculants. In contrast, conventional chemical flocculation offers rapid and consistent cell aggregation but relies on costly and potentially toxic reagents, such as aluminum or ferric salts, which can contaminate the biomass and complicate downstream processing [70,71]. Electroflocculation, while more sustainable in terms of chemical inputs, involves high energy consumption and infrastructure costs, making it less viable for low-margin applications like bulk biomass production [72].
From an energy and cost perspective, bio-flocculation is significantly more favorable, particularly when integrated into low-input systems. It requires no external energy input or chemical additives and can potentially leverage native microbial communities. However, scaling bio-flocculation requires optimization of culture parameters, co-cultivation strategies, and real-time monitoring to ensure consistent performance. Environmentally, bio-flocculation is the most sustainable option, as it avoids secondary pollution and aligns with green processing goals. Therefore, while not yet fully optimized for industrial use, bio-flocculation holds considerable promise as a low-cost, low-impact alternative, especially when coupled with other harvesting techniques in hybrid systems designed for biorefinery applications [73,74].”
Comment 8: L 237-244. The authors should supplement the text with relevant citations.
Response 8: Thank you for your observation. We have reviewed lines 237–244 and supplemented the text with relevant citations to support the statements made. These references strengthen the discussion and provide readers with sources for further information.
L 282-290: “Fungi have the ability to form filaments or, in some cases, undergo self-pelletization, which can be leveraged for the harvesting of microalgae cells [82]. Certain fungi from the Basidiomycetes, Aspergillus and Phanerochaete species can facilitate coagulation by forming pellets or aggregates through the presence of spores [83]. In contrast, non-coagulative fungi from the Mucor, Rhizopus and Penicillium species can aid in trapping algal cells by developing extensive hyphae [84]. To promote bio-flocculation, fungi and microalgae can sometimes be co-cultivated. However, the effectiveness of this process is often uncertain, as fungi may outgrow microalgae due to their faster growth rates and the concurrent competition for available carbon sources [84,85].”
Comment 9: L 256. Consider replacing “In several studies have identified” with “Several studies have identified”. The authors mention “several studies” but cite only one of them.
Response 9: L 301-302 Done. “Other study has identified”
Comment 10: L 320. The authors should discuss how zeta potential is related to cell size and flocculation efficiency in different microalgal species. Are there methods to artificially alter the
Response 10: We appreciate the reviewer’s valuable comment. In response, we have expanded the relevant section to further discuss the role of zeta potential in flocculation efficiency.
L 378-384: “Zeta potential is a key indicator of cell surface charge, and lower (less negative) values reduce electrostatic repulsion between cells, favoring aggregation. Differences in zeta potential among species can partly explain variability in natural flocculation behavior. Moreover, zeta potential can be artificially modified to improve flocculation efficiency—for example, by adjusting pH, ionic strength, or adding multivalent cations, which neutralize surface charges and promote aggregation [106].”
Comment 11: In this section, authors should discuss the potential influence of culture conditions. They should discuss how environmental conditions (CO2 availability, temperature and mixing) affect EPS production, flocculation efficiency and downstream biomass recovery.
Response 11: Thank you for your valuable suggestion. In response, we have expanded the section.
L 359-368: “These extracellular substances can be found either attached to the cells or dissolved in the surrounding environment. The synthesis and production of these substances are complex processes, influenced by factors such as the specific microalgal strain and various cultivation conditions, including nutrient availability, salinity, temperature, and light intensity. Research on extracellular polymeric substances production has been conducted in various microalgal species (Porphyridium cruentum, Chlorella vulgaris, Spirulina sp., and Nostoc commune). But compared to polymeric substances derived from seaweeds, terrestrial plants, fungi, or non-photosynthetic microorganisms, these processes have been less studied in cyanobacteria and microalgae [103].”
Comment 12: L 373-376. The authors mention that microalgal species contribute to nutrient removal efficiency in wastewater treatment systems. Are there specific microalgal species that perform better under nutrient-rich or challenging wastewater conditions?
Response 12: We thank the reviewer for this relevant observation. In response, we have added further discussion regarding the performance of specific microalgal species under nutrient-rich or challenging wastewater conditions.
L 445-452: "Certain microalgal species have shown superior nutrient removal efficiency in wastewater treatment systems due to their tolerance to high nutrient loads and fluctuating environmental conditions. For example, Chlorella vulgaris and Scenedesmus obliquus are widely studied for their robust growth in nutrient-rich wastewater and their ability to efficiently uptake nitrogen and phosphorus. Similarly, Tetraselmis sp. and Picochlorum sp. are known for their high adaptability to saline and variable wastewater environments, making them promising candidates for treatment of more challenging effluents."
This addition highlights the importance of species selection in optimizing microalgae-based wastewater treatment systems.
Comment 13: L 412. Consider replacing “As previously describe in 2.3.5” with “As previously described in 2.3.5”. It is unclear why the authors mention the parent section for this sub-section. They should change the text, for example as follows: “As mentioned above”.
Response 13: Done in line 486.
Comment 14: L 437. The authors mentioned CRISPR technology in flocculation. Could they provide information on the specific gene targets within microalgal genomes that can be modified using CRISPR-Cas systems to enhance flocculation potential?
Response 14: We thank the reviewer for this pertinent comment. We have revised the manuscript to briefly mention specific gene targets associated with a possible flocculation in microalgae.
L 517-520: “Some of these genes could be those involved in cell wall biosynthesis (e.g., cellulose synthase), extracellular polysaccharide production (e.g., beta-glucan synthase), and surface charge regulation, as they could influence cell aggregation behavior [29,133].”
Valuable microalgae compounds and bio-flocculation implications
Comment 15: L 475-478. The authors should clarify the main economic costs associated with current methods of harvesting microalgal biomass.
Response 15: Thank you for your helpful comment. In response, we have clarified the main economic costs associated with current methods of harvesting microalgal biomass in lines 555–561.
L 555-561: “The harvesting of microalgae biomass is challenging due to the highly diluted nature of the culture, which typically has a cell density of less than 1.0 g/L. Consequently, a substantial volume of water must be removed during the microalgae harvesting process. The inherent properties of microalgal cells, namely their diminutive size and colloidal stability within the culture medium, contribute to the complexity of the harvesting process [146]. For this reason, the economic costs associated with microalgal biomass harvesting have emerged as a significant barrier to the market's expansion for microalgae-derived products.”
Comment 16: Section 3.2. The authors should report the specific biosynthetic pathways involved in the production of carotenoids in microalgae. It would be useful to compare different co-culture strategies (e.g., filamentous fungi with microalgae vs. flocculent vs. non-flocculent microalgae co-cultures) in terms of efficiency and yield for carotenoid production.
Response 16: We appreciate the reviewer’s insightful suggestion. In response, we have added an schematic illustration of a general carotenogenesis pathway in a microalgae cell. Figure 3. Moreover, we have added a comparison of different co-culture strategies in lines 726-731.
L 726-761: In terms of efficiency and yields for carotenoids recovery, co-cultures of filamentous fungi and microalgae generally achieve higher harvesting efficiencies and carotenoid yields compared to flocculent and non-flocculent microalgae co-cultures. Flocculent microalgae co-cultures show intermediate performance, while non-flocculent species typically require additional flocculation aids, resulting in lower overall efficiency and carotenoid recovery [167,168].
Comment 17: L 627. The authors should specify the nutritional profile of microalgae. What are the specific essential amino acids found in microalgae? How do they compare to amino acid profiles of traditional protein sources such as soy, chicken, or fish?
Response 17: Thank you for the helpful comment. We have addressed it by specifying the essential amino acids found in microalgae.
L 743-747: “Microalgae have attracted great interest among food technologists due to their distinct advantages over traditional protein sources. They have been found to be rich in essential amino acids (EAA), such as leucine, arginine, lysine, isoleucine, phenylalanine, threonine and valine [171] and many authors have studied amino acid profiles of conventional meat sources with microalgal proteins.”
Comment 18: L 640. Consider replacing: “Microalgae have been identified as a promising organism” with “Microalgae have been identified as promising organisms”
Response 18: It has been done in line 769.
Comment 19: L 656. In the authors' opinion, what are the best cultivation practices for maximizing the protein content of microalgae?
Response 19: Thank you for this interesting and relevant comment. In response, our perspective is the implementation of the best cultivation practices for maximizing the protein content of microalgae. We believe that key factors such as optimizing nitrogen availability, since sufficient nitrogen levels promote protein synthesis; maintaining appropriate light intensity and photoperiods to support optimal growth; controlling temperature to ensure conditions favorable for biomass accumulation; and selecting fast-growing strains known for their high protein yield, such as some species of Chlorella could significantly enhance the protein productivity of microalgal cultures.
Comment 20: L 693. It would be useful to provide some data to quantify the impact of microalgal biofertilizers on crop yield and soil health compared to traditional fertilizers.
Response 20: Thank you for this valuable suggestion. In response, we have introduced a new table, Table 2, in the manuscript where specific examples and data from different tudies are summarized.
Comment 21: A table or figure summarizing the main results described in section 3 should be included in the paper to improve data visualization and interpretation.
Response 21: Thank you very much for the suggestions. Some tables and figures have been added.
Figure 2 and Table 2
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
The manuscript is very interesting.
Introduction, some paragraphs are too long, I recommend to divide into several paragraphs.
Also, I recommend to give a general subsection about harvesting microalgae, to give the reader some information: why Flocculation is important.
The manuscript is very well structured, however, I recommend the addition of tables to synthesize information. And restructure the text to be less difficult to read.
Author Response
Response to Reviewer 2 Comments
Summary
We would like to sincerely thank the reviewers for their time, effort, and insightful comments on our manuscript titled "Bio-flocculation: a green tool in biorefineries for recovering high added-value compounds from microalgae." Their valuable suggestions and constructive feedback have greatly contributed to improving the quality and clarity of our review. We truly appreciate their dedication to helping us enhance our work.
Point-by-point response to Comments and Suggestions for Authors
Comments and Suggestions for Authors
The manuscript is very interesting.
Recommendations:
Comment 1: Introduction, some paragraphs are too long, I recommend to divide into several paragraphs.
Response 1: Thank you for your helpful observation. In response, we have revised the Introduction section by dividing overly long paragraphs into shorter, more focused ones. This restructuring improves the readability and flow of the text, making it easier for readers to follow the key ideas presented.
Comment 2: Also, I recommend to give a general subsection about harvesting microalgae, to give the reader some information: why Flocculation is important.
Response 2: Thank you for this valuable suggestion. In response, we have added a new general subsection on microalgae harvesting concretely focussed on bio-flocculation strategy, as it is the main topic of the Review.
L 112-126: “Bio-flocculation is an emerging low-cost and eco-friendly technique that leverages microbial interactions to aggregate suspended particles, particularly microalgal cells, without the need for synthetic chemicals. This natural flocculation process, often mediated by bacteria, filamentous fungi or microalgae, offers several environmental and economic advantages. It reduces reliance on energy-intensive harvesting methods such as centrifugation or chemical flocculants, which are often expensive, resource-intensive, and may introduce contaminants [26]. From an environmental perspective, bio-flocculation minimizes secondary pollution and aligns with circular bio-economy principles by promoting sustainable biomass recovery. In the context of microalgae harvesting, bio-flocculation is especially important due to the small size and dilute concentration of microalgal cultures, which make harvesting a major bottleneck in large-scale production. Efficient harvesting through bio-flocculation enhances the overall feasibility of microalgae-based biorefineries, lowering operational costs and preserving the biochemical integrity of valuable compounds for downstream pro-cessing, highlighting the applicability of bio-flocculation in integrated biorefinery frameworks [27].”
Comment 3: The manuscript is very well structured, however, I recommend the addition of tables to synthesize information. And restructure the text to be less difficult to read.
Response 3: Thank you for your positive feedback and constructive suggestions. In response, we have introduced a new table in section 3 to synthesize key information and improve clarity. Additionally, we have restructured parts of the text by breaking down long sentences and paragraphs, making the manuscript easier to read and follow, mainly in the Introduction section. We believe these changes enhance both the accessibility and the overall presentation of our work. All the modifications throughout the manuscript are highlighted in red.
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
The work is interesting due to the importance of using biological processes for microalgae flocculation, whose advantages lie in reducing costs and maintaining the quality of the products of interest. The chapter on bioflocculation methods is well presented, with a thorough review of the processes.
However, I suggest the following corrections to be taken into account:
1. Introduction: The topic of biofuels is widely studied, discussed, and analyzed in thousands of articles, which show their low viability, and there are many treatises on biomass harvesting and lipid production for biofuel. It is better to provide an introduction justifying bioflocculation in terms of obtaining current or future commercial products, such as food, secondary metabolites, genetically engineered products, etc. This demonstrates the importance of this review topic in current commerce.
2. The chapter "Valuable Microalgae Compounds and Bioflocculation Implications" focuses on the description of compounds and very superficially presents the implications of bioflocculation. Again, it is repetitive to talk about compounds in general terms; there is a large body of literature; the implications of bioflocculation methods should be explored in more depth.
Author Response
Response to Reviewer 3 Comments
Summary
We would like to sincerely thank the reviewers for their time, effort, and insightful comments on our manuscript titled "Bio-flocculation: a green tool in biorefineries for recovering high added-value compounds from microalgae." Their valuable suggestions and constructive feedback have greatly contributed to improving the quality and clarity of our review. We truly appreciate their dedication to helping us enhance our work.
Point-by-point response to Comments and Suggestions for Authors
Comments and Suggestions for Authors
The work is interesting due to the importance of using biological processes for microalgae flocculation, whose advantages lie in reducing costs and maintaining the quality of the products of interest. The chapter on bioflocculation methods is well presented, with a thorough review of the processes.
However, I suggest the following corrections to be taken into account:
Comment 1: 1. Introduction: The topic of biofuels is widely studied, discussed, and analyzed in thousands of articles, which show their low viability, and there are many treatises on biomass harvesting and lipid production for biofuel. It is better to provide an introduction justifying bioflocculation in terms of obtaining current or future commercial products, such as food, secondary metabolites, genetically engineered products, etc. This demonstrates the importance of this review topic in current commerce.
Response 1: We sincerely appreciate the reviewer’s insightful suggestion. Following the recommendation, we have revised the Introduction section to better emphasize the relevance of bioflocculation not only for biofuel production but also for its significant potential in the commercial development of valuable products. These include food ingredients or high-value secondary metabolites. We have modified and restructured different parts in the Introduction. This broader perspective highlights the topic of the review and the biotechnological interests. We believe that this change strengthens the justification for the review and better reflects its relevance to current field. The modifications can be found in the revised Introduction section, in red.
L 65-73: “Beyond their applications in energy production, microalgae are also recognized for their capacity to synthesize a wide range of high-value compounds with potential uses in food, pharmaceuticals, and nutraceuticals. These include proteins with balanced amino acid profiles, essential fatty acids such as omega-3 and omega-6, pigments like chlorophylls, carotenoids (e.g., astaxanthin, lutein), and phycobiliproteins, as well as vitamins, antioxidants, and bioactive polysaccharides. Their biochemical versatility, combined with fast growth and minimal resource requirements, makes microalgae a highly attractive platform for the sustainable production of bioactive compounds that can contribute to human health and food security [12].”
L 112-126: “Bio-flocculation is an emerging low-cost and eco-friendly technique that leverages microbial interactions to aggregate suspended particles, particularly microalgal cells, without the need for synthetic chemicals. This natural flocculation process, often mediated by bacteria, filamentous fungi or microalgae, offers several environmental and economic advantages. It reduces reliance on energy-intensive harvesting methods such as centrifugation or chemical flocculants, which are often expensive, resource-intensive, and may introduce contaminants [26]. From an environmental perspective, bio-flocculation minimizes secondary pollution and aligns with circular bio-economy principles by promoting sustainable biomass recovery. In the context of microalgae harvesting, bio-flocculation is especially important due to the small size and dilute concentration of microalgal cultures, which make harvesting a major bottleneck in large-scale production. Efficient harvesting through bio-flocculation enhances the overall feasibility of microalgae-based biorefineries, lowering operational costs and preserving the biochemical integrity of valuable compounds for downstream processing, highlighting the applicability of bio-flocculation in integrated biorefinery frameworks [27].”
Comment 2: 2. The chapter "Valuable Microalgae Compounds and Bioflocculation Implications" focuses on the description of compounds and very superficially presents the implications of bioflocculation. Again, it is repetitive to talk about compounds in general terms; there is a large body of literature; the implications of bioflocculation methods should be explored in more depth.
Response 2: We appreciate the reviewer’s valuable feedback. In response, we have substantially revised the section "Valuable Microalgae Compounds and Bio-flocculation Implications" to minimize the general description of compounds and focus more deeply on the specific implications of bio-flocculation methods. We have summarized some parts of the manuscript to give a simpler point of view of these kinds of compounds, necessary to understand the context in which bio-flocculation could be involved.
To enhance clarity and make the information more accessible, we have also added new tables and figures that summarize key data and highlight the relationships between bio-flocculation techniques and product recovery. Specifically, Figure 2 and Table 2 have been included to provide a clearer and more visual representation of the information.
Please see the attachment.
Author Response File: Author Response.pdf
