The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper deals with an interesting topic of reviewing the potential of specific macroalgae Kappaphycus alvareziii for fermentation and related biorefinery abilities. The idea of the paper seems attractive and interesting, however there are some comments that need to be addressed for the betterment of the paper from the reader’s point of view.
1. Abstract: Author has mentioned that “Kappaphycus alvarezii has been introduced to the Brazilian southern coastline in 20th century” which is obfuscating, was the macroalgae introduced there? or it was identified then? or increasingly attracted and subjected for research then? Better to replace it appropriately.
2. The second-half of the abstract has just mentioned about the contents included in the review paper rather than highlighting the insights obtained from the review paper and hence it requires revision.
3. Introduction: The section initiates with the need for alternative green energy along with discussing the environmental deterioration associated with the conventional fuels. However, no established reports were included empirically. Better to add the current carbon foot print data along with the expected rise in near future which will convey the severity of the adversities associated with conventional fuels to the readers.
4. In section, 2, authors have talked about renewable materials. It would be interesting to know the authors take on why macroalgae need to be preferred as alternative source for fossil-based fuels while microalgae are available with various better potentials, for example: Many types of fuels, value added products and food applications. The following studies are available for reference:
Integrated microalgal biorefinery – Routes, energy, economic and environmental perspectives. Journal of Cleaner Production, 2022, 348: 131245.
Photobioreactor configurations in cultivating microalgae biomass for biorefinery. Bioresource Technology, 2024, 394: 130208.
5. Section 2.3 deals with the Kappaphycus alvarezii species, however a table detailing the elemental/macromolecular composition of Kappaphycus alvarezii could better describe the nutritional quality and its scope of various applications. In addition, in the same section, adding the structure of carrageenan (apart from fig. 5) could be vital along with a brief content about the structural aspect would be better as it is also carrageenan aspect of the review paper.
6. As the paper mainly deals with the potential of K. alvarezii, a brief section before the conclusion can be added which can include how K. alvarezzi is a better feedstock for fermentation and fuel application perspectives as compared to other macroalgae.
7. As the author suggest this macroalgal based fermentation would be sustainable as like other biomass-based energy conversion processes, how about the process economy.? It would be a wholistic view if the author able to include the possible economic potential of the processes at the end of the manuscript.
Comments on the Quality of English Language
Minor editing of English language required
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors1. Line 66 "seaweed rich in the galactan polysaccharide carrageenan "
The sentence is hard to follow. Please rewrite the sentence.
2. Line 85: "The removal of HMF could be carried out using activated charcoal [20,22], and overliming processes [22–25] "
HMF could not removed from the hydrolysate by overliming. Lime is used to neutralize acidic hydrolysate, but it doesn't affect this inhibitor's concentration. Please correct the text.
3. Line 86-88 : "Alternatively, HMF can be removed directly from the hydrolysates, applying reactive extraction via zeolites as catalysts or using absorption in an organic phase in slug flow reactors or stripping columns [26] "
The reference doesn't match the text. Please correct the sentence or the add new reference.
4. Lines 108-111: "This paper aims at presenting up to date procedures developed for K. alvarezii processing for fermentation processes, revising emergent enzymatic degradation technology, and bringing to light potential products that can be obtained from the algal biomass via fermentation."
The sentence is hard to follow; please rewrite it.
5. Lines 113-120 "Biomasses, like biofuels, are classified in generations according to their origin. First- 113 generation (1G) biomass is essentially food products, extracted from agricultural crops such as sugarcane, wheat, corn etc. Second-generation (2G) biomass is lignocellulosic material obtained from waste agricultural products, or other biomass not used for human consumption. Algal biomass, being it microalgae or macroalgae, is defined as third generation (3G) biomass [3,30]. The literature mentions also a fourth-generation biomass, which consists of microorganisms, such as cyanobacteria, with genetically engineered metabolic pathways, that can convert sunlight directly into biofuels and other desired products [30–32] "
The References [3,30] don't match the text. In cited literature, different generations of biofuels are mentioned, but not the different generations of biomass. The authors should provide new references or change the text according to the cited papers.
6. Lines 140-142 "Most 1G and 2G biomasses require in-land cultivation, which is identified as one of the contributors to carbon gas emissions in the form of nitrous oxide, methane, and carbon dioxide "
The burning of fossil fuels produces NOx, and carbon dioxide, not biomass. The authors should correct it accoringly.
7. Line 147 : "Replacement of natural ecosystems by cropland … "
Since the ecosystem is a biological community of interacting organisms and their physical environment, one ecosystem cannot be replaced with another. The authors probably meant the replacement of wild flora with specific crop/s. Please correct accordingly.
8. Line 148: "cropland can create a long a deficit in carbon fixation "
Replacing wild flora with industrial crops will not necessarily decrease the carbon fixation by photosynthesis. Please rewrite the sentence.
9. Line 304-312: "Another reported method for HMF removal is that of overliming, which consists of 304 addition of calcium oxide (CaO) [23], or calcium hydroxide (Ca(OH)2) [22,24,25]. Chi et al. 305 [24] studied overliming processes for sugar mixtures obtained by pyrolysis, affirming that 306 preparing a solution for ethanolic fermentation with E. coli, using an addition of 18.5 g/L 307 of CA(OH)2 required 8-16 hours of treatment at 20°C, and a 1-4 hours treatment period when temperatures were maintained at 60°C. Mohgheghi et al. [25] used a shorter treatment period (30 minutes), showing that pH and temperature are key factors for efficient removal of HMF. In a recent study, K. alvarezii hydrolysates containing 24 g/L were reduced to 9 g/L using addition of Ca(OH)2 elevating pH values to over 11 for 30 minutes 312 [22]. "
In cited literature overliming is not mentioned as method for reducing the HMF: Mohagheghi, A., Ruth, M., & Schell, D. J. (2006). Conditioning hemicellulose hydrolysates for fermentation: Effects of overliming pH on sugar and ethanol yields. Process Biochemistry, 41(8), 1806–1811. doi:10.1016/j.procbio.2006.03.028
Chi, Z., Rover, M., Jun, E., Deaton, M., Johns not cell wall ton, P., Brown, R. C., … Jarboe, L. R. (2013). Overliming detoxification of pyrolytic sugar syrup for direct fermentation of levoglucosan to ethanol. Bioresource Technology, 150, 220–227. doi:10.1016/j.biortech.2013.09.13
Tabacof, V. Calado, N. Pereira, Third Generation Lactic Acid Production by Lactobacillus pentosus from the Macroalgae Kappaphycus alvarezii Hydrolysates, Fermentation 9 (2023) 319. https://doi.org/10.3390/fermentation9040319.
Although adding lime improved the fermentation efficiency due to the neutralization of acid used in carbohydrate (cellulose, hemicellulose) hydrolysis, the addition of lime did not affect the HMF concentration, nor did the studies mentioned above confirmed that.
10. Line 420: "In the Leloir pathway, D-galactose is transported passively through the cell wall with a galactose permease galP. "
Substrates are transported via transport proteins through the cell membrane, not the cell wall, from the extracellular to the cytoplasmic side. Please correct.
In general, the authors mentioned HMF as the only type of inhibitor derived from biomass by acid at higher temperatures. Except for the HMF, is there any other inhibitor present in seaweed hydrolysate?
11. Line 315: "The HMF tolerance varies depending on the species of the fermenting agents "
The reviewer assumes that when the authors wrote about "fermenting agents," they meant microorganisms. This term is commonly used in microbiology for fermenting organisms. Please correct accordingly.
12. Line 320 "either short term processes where cells retain protective proteins "
The authors should write the names of "protective proteins" and briefly describe their function.
13. Line 191: "Macroalgae have a high saccharide content "
Line 223: "Like most seaweeds, K. alvarezii is composed mostly of saccharides"
Which type of saccharide (monosaccharide, disaccharide, polysaccharide)? Please correct the text accordingly
14. Lines 239-241 : "Fermentation can be carried out as a separate process, jointly with saccharification e.g. simultaneous saccharification and co-fermentation (SSCF). The products are then separated from the fermented media, prepared and packed for commercialization. "
The sentence is hard to follow. The fermentation and hydrolysis process can be conducted separately or simultaneously. Please correct the sentence.
15. Lines 259-260: "K. alvarezii biomass to prolonged acid hydrolysis results in the formation of HMF in a 1/4 ratio to galactose "
The sentence is unclear. The authors should rewrite the sentence.
16. Incorrect names of enzyme preparation. The enzyme names are "Celluclast® 1.5 L "and "Cellic CTec2 ". The authors should correct the text.
17. Table 1. Instead of "cellulase "the authors should write the name of the enzyme (commercial or prepared enzyme) used in hydrolysis.
18. Lines 230-231: "The interior part, made of linear cellulose, has a 70% glucose content, 14% galactose, about 4% of mannose and 1% of xylose [57]. "
In reference [57] Lechat et al. described the composition of hydrosoluble extract and hydro-insoluble residue. The composition of hydro-insoluble residue corresponds to the composition of the "interior part" that the authors of this review gave. Please correct the text according to the reference [57].
19. Line 431: "1st and 2nd generation raw materials. "
It should be written "biomass "instead of "raw materials ".
The authors should use the term "Bacterial Cellulose "instead of "Bacteriological Cellulose "(Table 5)
20. Line 537-538: "For dilute-acid hydrolysis, the overliming and activated charcoal methods were shown to be efficient in HMF removal for enabling fermentation. "
HMF can not be removed by overliming. Please correct the sentence.
Comments on the Quality of English LanguageThe manuscript needs extensive revision for language, grammar and spelling errors. Furthermore, the clarity of the sentences (especially long ones) should also be improved.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
