Environmentally Friendly Chelation for Enhanced Algal Biomass Deashing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

REVIEW of Algae Ash Leaching Paper (numbers refere to lines in the draft)

Abstract is a bit long and could be reduced a little.

55 to 62:  the ash content due to inorganic nutrient uptake is usually not the main source of ash content in microalgae, but, rather due to precipitation of salts (e.g. Ca, Mg, Fe, P, etc) from the cultivation medium due to high pH caused by the algae cultures, as well as a ash deposited from outside sources.  Also silicates due to diatoms present in the algal biomass is often a major source of ash.  This should be noted in this context.  Note that Table 1 demonstrates that the Scenedesmus has the lowest ash content, which is from the inorganic nutrients.  Indeed, the low removal rate for  the ATS samples suggests that this ash may not be ameanable to leaching (e.g. sand, silicates). 

Table 1, it is not clear if any total weight (e.g. ash-free component) was lost in the process. 

41 .  change “consumable” to “commodity”

45 ‘feedstock’ to “feedstocks”

49. Delete word “lately”

52. “removal” to “utilization”

52-56. Recommend editing From: Cultivating algae on an extensive scale proves challenging considering the high production costs. This is one of the main challenges of utilizing algae to produce an alternative energy source. On the other hand, the environ-mental conditions of its habitat, including salinity, light, temperature, pH, availability of nutrients, and water contamination, influence its growth rate and productivity [11-13].

TO: Cultivating algae on an extensive scale is challenging due to the high production costs. This is one of the main challenges of utilizing algae to produce an alternative energy source. On the other hand, the environmental conditions of its habitat, including salinity, light, temperature, pH, availability of nutrients, and water contamination, influence its growth rate and productivity [11-13].

61. Delete sentence: The algae absorb these metals as they mature, thus increasing their ash content. NOTE: algae accumulate these elements throughout their growth.

71. accelerate ash generation This is poor choice of words as the ash is not generated. Change to : “result in ash volatilization and deposition” or something similar (and better). 

73. can be manufactured from algae amid conversion procedures  ALSO poor choice of words. Change to “can be recovered from algal biomass conversion processes “ or something similar.

74, 75.  Add the word “biomass” in there as this is not specific for algae.

80 “of the algae”  To ‘’from algae biomass”

100. Add word ‘found that’ before NTA

102 At end of sentence add: “through hydrothermal liquefaction”

109  change “paramount focuses”  to “objectives”

115-117.  remove word exists at end and state and before state “no data has been published” instead of ‘no published data’

117 “The findings aim to bring out new knowledge”  This is poor wording. Suggest “ These findings contribute to our knowledge “ or similar.

128 “These samples were left at room temperature overnight”.  It is not clear what this refers to

141 “metal plate”  Not correct terminology use “Stirring plate”

148 to 159 . FIGURE 1.  Legend to Figure 1 needs to be expanded.  Note the temperature of the algae sample. The Figure explanation in the text is not very clear.  How does Stage 3 differ? It seems that between Stage 2 and 3 there is a further processing?     From Section 3.2.1. line 349 it appears that the three stages are not sequential but distinct.  The arrows between the stages suggest  sequential processes.  This should be clarified and better explained.

158-59.  Suggest that the reference to the NREL report be condensed, to mainly citation and a page number.

162. Word ‘carefully’ is not needed. ALSO in this paragraph the Figure 2 should be referenced.

166. Explain how it was heated to 130oC without boiling off the water or pressurizing.

Figure 2.  Legend needs more explanation and detail on the process.

188-191  It is not correct to state that Equation 1 accounts for the ash content in the “algae”. We do not know what the algae content actually is. The ‘algae’ should be defined as organic matter, or ‘ash-free dry weight’ (as the simplest definition).  The equation includes both loss of biomass and ash together, and these may not be lost in the same proportion.  This can be problematic as differential loss of one or the other would skew results.  Thus, for an example, with an initial ash content of 50%, and an 20% loss of biomass, but no loss of ash, the total weight of the sample would then be 0.9 of the original, with the ash content increasing from 50% to 55.5%, showing an apparent gain of 5.5%, while actual ash content actually did not change.  Conversely, loosing no biomass but 20% ash, the ash content decreases to 44.4%, an apparent loss of only 5.6%, while the loss was actually 20% of the initial ash content.     Although organic C and N were determined in the analysis and can be used to estimate organic matter in these samples (see below) the above equation cannot be used as such for estimation of ash losses. This is relevant to Table 1.

Table 1. The table legend does not state what process is actually used (e.g. from Figure 1).  Also some information should be provided of the amount of total dry weight lost, e.g. weight recovered, after the processing of the biomass, even if the actual ‘algae’ content (e.g. ash free dry weight) is not available. This could also be provided in the legend.  This is particularly of interest in the case of the Scenedesmus biomass, which is the focus of the remainder of this paper. 

 Figure 4.  Same comment as above: more detail in the legend.  ALSO: the total removal is shown at maximum of 56%, vs 75% in Table 1.  The difference is apparently due to the higher temperatures (see Figure 5), that should be stated in the Table 1 Figure legend.

372-381 An argument is made that the added 2% ash removal due to the last DI treatment is beneficial in the overall process.  Seems a better statement would be that the small marginal added effect would not be worth the added trouble of doing this in practice.

Section 3.3  Lines 413 to 436.  The increase in ash content in Figure 6 cannot be easily explained as the final ash content is higher than the initial one, at least that appears to be the case. This is likely due to the above mentioned problem of loss of biomass (organic dry weight) during the processing. This should be discussed in this context.    

Section 3.4.1. Line 438.  In light of the above the following CHN analysis needs explanation. C (or H) content is not significantly affected, but N is reduced by 15%.  Again, the question arises of the total biomass actually recovered in these protocols (e.g. Stages 1 and 2, with 3 being rather minor as noted above).  Also this section could use some rewrites, such as line 465  to 471, for examples.

Section 3.4.2.  Table 3, line 483 The content of K at only 4.3 ppm seems low by almost a factor of a thousand!  Ca is also very low.  Microalgae contain typically well over 1000 ppm of both, and more K than Ca (depending on growth conditions).  Maybe these are supposed to be in %?

Sections 3.4.3  and 3.4.4.  This section on N content and protein can be deleted.  The protein content is only approximate and there is no real need to emphasize it in this publication.  The argument that washing of Scenedesmus biomass will improve its feed quality is not that relevant to feed quality.  The paper is already too long and could use some trimming.  Same can be said about Section 3.4.4 and carbohydrate contents.  The information provided in these two sections could be summarized in a short paragraph, if that, and combined into the next section.

Section 3.4.5.  Figure 9 is somewhat problematic. What does it mean g/ml??  Note that the DI wash shows about 0.07 mg/ml N and 0.09 g/ml of C.  This C:N  ratio is off by a factor of about five-fold from that given earlier, of C:N of about six (table 2).  Can this be explained?

IN brief Section 3.4 can be significantly reduced, while, explaining the above issues, as necessary..

This paper makes a valuable contribution to this field and should be published with the above cited revisions (or reasons for why the edits/revisions are not being made or required explained).

Author Response

Please find the attached Word document for the response to the comments.

Many thanks.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors evaluated a green chelation strategy using NTA and deionised water to purify Scenedesmus algae, which was selected among the tested species for its superior ash removal potential. Below are comments/concerns:

1. The process relies on elevated temperatures (up to 130 °C), which can lead to higher energy consumption. This may reduce the overall environmental benefit and economic feasibility at an industrial scale, particularly when processing large biomass volumes.
2. The method involves multiple sequential steps—DI prewash, NTA chelation, and NTA+DI post-wash—which may complicate scaling up due to increased equipment, time, and operational control needs.
3. Frequent use of deionized water and chelators like NTA may add to the process cost, making it less economically viable without an efficient recycling system or alternative cheaper reagents.
4. Although the biochemical integrity (CHN content) was preserved immediately post-treatment, the study does not discuss long-term impacts on biomass storage, microbial contamination, or conversion efficiency during downstream processing.

Author Response

Reviewer #1

1. The process relies on elevated temperatures (up to 130 °C), which can lead to higher energy consumption. This may reduce the overall environmental benefit and economic feasibility at an industrial scale, particularly when processing large biomass volumes.

Response: We appreciate the reviewer's valid concern regarding energy consumption at 130 °C. However, we would like to inform you that almost all downstream processing of microalgae for fuels and bioproducts is expected to adopt thermochemical and hydrothermal liquefaction (HTL) processes. The U.S. Department of Energy (DOE) has placed significant investment in hydrothermal liquefaction (HTL) as a promising conversion pathway for algae and other wet organic waste feedstocks (Ref: https://www.energy.gov/eere/bioenergy/articles/integrated-strategies-enable-lower-cost-biofuels).

It is evidenced by their several versions of State of Technology (SOT) reports over the last 10 years. HTL and other thermochemical processes are primarily operated above 300 °C, which provides an opportunity for waste heat recovery integration to the proposed ash removal temperature. Therefore, there may not be a requirement for additional heat.

2. The method involves multiple sequential steps—DI prewash, NTA chelation, and NTA+DI post-wash—which may complicate scaling up due to increased equipment, time, and operational control needs.

Response: DI water was used for research to minimize ambiguity in interpreting experimental results. In any scaled-up/industrial operations, process water is expected to be used for the proposed desalination process. In other words, DI pre/post-wash is a lab-scale descriptor. Industrially, this translates to the standard use of process water in industrial applications. The rinsed water saturated with ash/inorganic elements will be recycled in the algae farm, which can partly supplement the macro- and micro-nutrient needs in algae cultivation.

• Operation Control and Complexity: The NTA chelation process is a simple chemical treatment step, with rinses serving as standard solid-liquid separation (e.g., filtration), and pH/temperature monitoring for NTA chelation is a routine practice in any process industry.
• Scalability: The process can be adapted to continuous counter-current flow reactors (e.g., plug-flow or CSTR designs) with in-line filtration, eliminating "sequential" batch operations. Pilot-scale algal biorefineries already use configurations requiring no exotic equipment.

3. Frequent use of deionized water and chelators like NTA may add to the process cost, making it less economically viable without an efficient recycling system or alternative cheaper reagents.

Response: We agree that process economics are crucial for industrial adoption. However, the process can remain economically viable due to the following reasons:

• Industrial water use: Deionized water is not required in industrial settings. Instead, recycled process water or filtered municipal water can be used for washing steps, significantly reducing water costs.
• NTA recyclability: NTA is readily recoverable and recyclable after use, which is one of the significant findings of this study. Established methods such as pH adjustment, membrane filtration, or ion exchange allow for efficient NTA recovery and reuse, minimizing chemical consumption and operational costs.

4. Although the biochemical integrity (CHN content) was preserved immediately post-treatment, the study does not discuss long-term impacts on biomass storage, microbial contamination, or conversion efficiency during downstream processing.

Response: We appreciate the reviewer's insightful comment regarding long-term biomass stability and downstream impacts. Our work was limited to evaluating NTA's efficacy in deashing algal biomass while preserving CHN integrity, a critical prerequisite for downstream conversion. There are several studies on biomass storage and microbial contamination challenges. The following is one of the examples.

Cite: Smith, W. A., Wendt, L. M., Bonner, I. J., & Murphy, J. A. (2020). Effects of storage moisture content on corn stover biomass stability, composition, and conversion efficacy. Frontiers in Bioengineering and Biotechnology, 8, 716.

Reviewer 3 Report

Comments and Suggestions for Authors

This work is important; it addresses a significant problem in the production of biofuels from algal biomass.

The experimental design and methodology analyze different variables to determine ash reduction and the effects on the biomass to ensure its subsequent use in biorefineries.

However, the authors did not elaborate on some of the results:
The decrease in protein content is a positive result, as it favors the conversion of algal biomass into biocrude.

Furthermore, the Ca, K, Cu, Zn, and Pb ions analyzed are few, and these results do not allow for conclusions as described in the article, but only approximate estimates regarding chelating capacity and double treatment.

I recommend that the authors further discuss the quality of the biomass at the end of the treatments according to the type of biofuel that can be used (biodiesel, biocrude, etc.).

The authors should specify their conclusions regarding the use of low-ash biomass, such as Scenedesmus, but not applicable to biomasses with higher ash concentrations or algae cultures with culture media very rich in heavy metals.

Comments on the Quality of English Language

The English is good.

Author Response

Please refer to the attached Word document for the response to the comments. Many thanks

Author Response File: Author Response.pdf