Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review the results presented in Figure 1, sections e, f, r, and s. It does not show any results or is not explained in the main text.

In the entire experimental section, the authors should formally cite the underlying method used for each method.

In Table 2, review the title.

The authors discuss the results at the macroscopic and operational levels. I consider it highly relevant for lectures on microbiotechnology to explain the different mechanisms from a molecular perspective.

The authors should include a process flow diagram with the optimal conditions for each stage.

In Figure 2, SEM images, they should be enhanced by indicating magnification and scale.

The manuscript title should be revised based on the results and main objective.

Author Response

Comment 1: Review the results presented in Figure 1, sections e, f, r, and s. It does not show any results or is not explained in the main text.

Response 1: The authors are grateful for the reviewer’s observations. Figure 1 “e, f, r and s” are empty because no fungal pellets were achieved under those culture conditions. This has been explained in the results and discussion on Page 9, in section 3.1.3, Lines 358 – 359.

“No distinct pellets were achieved under inoculum size 50 and 500x. Hence, the graphs shown in Figure 1 e, f, r and s were empty”.

Comment 2: In the entire experimental section, the authors should formally cite the underlying method used for each method.

Response 2: Thank you for this suggestion. Citations have been added to various sections of the methodology where citations were previously omitted.

Page 4, section 2.4.4, line 171 – “Four distinct minimal media formulations were prepared, with sucrose, glucose, fructose, and galactose as the primary carbon sources [12]”.

Page 5, section 2.6, line 201 – “These flasks were then agitated in an orbital shaker (VWR, USA) to facilitate interaction between the fungal fluffs and the microalgal cells [15]”.

Page 6, section 2.8.1.5, line 273 – “The element distribution of samples was obtained using an energy-dispersive X-ray (EDS) spectroscope [17]”.

Comment 3: In Table 2, review the title.

Response 3: The authors are grateful for the reviewer’s keen observations. Table 2 title has been revised.

Page 14, section 3.5.3, Line 539 – “Comparison among systems using microalgae and fungi co-cultivation treating various wastewater”

Comment 4: The authors discuss the results at the macroscopic and operational levels. I consider it highly relevant for lectures on microbiotechnology to explain the different mechanisms from a molecular perspective.

Response 4: We sincerely thank the reviewer for the insightful suggestion regarding the inclusion of molecular-level mechanisms to support the observed results. We agree that a molecular perspective would provide a deeper understanding of the interactions involved in fungal pellet formation and microalgae-fungi consortia. However, the current study was designed to focus primarily on the macroscopic, operational, and performance-based aspects of microalgae harvesting and wastewater nutrient removal. During the experiment, the authors observed the performance of Chlorella sp. cells in cocoa-pulp wastewater due to high sulfur content and decided to investigate that further. Thus, in a subsequent study, the effect of sulfur on Chlorella sorokiniana was further examined at the molecular level. The manuscript is under preparation. However, due to limitations in time and scope, molecular-level analyses such as gene expression or protein profiling were not within the objectives of this work.

Comment 5: The authors should include a process flow diagram with the optimal conditions for each stage.

Response 5: A process flow diagram has been included as Figure 5. Reference to this figure is given on Page 15, Section 3.6, Line 550 - 551

Comment 6: In Figure 2, SEM images, they should be enhanced by indicating magnification and scale.

Response 6: The authors appreciate the reviewer’s keen observations. The image in Figure 2a has been enlarged to show the scale and magnification. The figure caption has also been revised; Page 11, Section 3.3.2 Line 422-425

“Figure 2. (a) SEM images of entrapped fungal mycelia showing rough fungal hyphae ((i) 100 µm, mag. 1000x); ((ii) 2 µm, mag. 50,000x); and harvested microalgae entrapped in fungal mycelia ((iii) 20 µm, mag. 5000x) and ((iv) 50 µm, mag. 2000x). (b) Harvesting efficiency of isolate T10 and FP”.

Comment 7: The manuscript title should be revised based on the results and main objective.

Response 7: The authors are grateful for your kind suggestions about revising the manuscript title. Based on this suggestion, the manuscript title has been revised.

Page 1, Lines 1-3 -  "Integrating Fungal-Assisted Microalgal Harvesting for Sustainable Treatment and Resource Recovery from Wastewater".

Reviewer 2 Report

Comments and Suggestions for Authors

1. This article focused on combination of algae and fungal consortia for treatment of cocoa pulp wastewater.
2. Basically, it is skeptical the significant enhancement of treatment efficiency of this system from the data, seen in 4 and Table 2.
3. Application of fungal consortia seems to be new, but the long term operation may change the consortia, leading to the inefficient treatment.
4. The experiment time is rather short to answer the question in no.3.

 Some experimental procedures are not shown in results.

5. Experimental procedures may not conducted in cocoa pulp wastewater ?

It should be cleared.

6. Figures 2 and 4 need more accurate explanation. Colors in Figure 4 are not clear. In text Figure 2Ai and ii?
7. Figure 4. Why control is so active in the treatment?
8. Figure 1.What is the unit of pellet numbers ?
9. Table 2.The advantage of fungal consortium is not clear compared with other single fungal system. What is N/A?
10. Text is rather redundant. More refined text should be prepared.

Comments on the Quality of English Language

The more clear explanation in the text and figure, Tables is needed. 

Author Response

Comment 1: This article focused on combination of algae and fungal consortia for treatment of cocoa pulp wastewater.

Response 1: The authors thank the reviewer for their comments on the manuscript. Yes, the article focused on the combination of microalgae and fungal consortia for the treatment of cocoa pulp wastewater. However, the process of obtaining the fungal pellets needed optimization. Thus, the first part of the manuscript focused on screening several fungal isolates in synthetic culture media (as shown in Supplementary materials, Table S1), prior to introducing the fungal pellets into real wastewater. This was clearly communicated in the experimental design; Page 3, section 2.3, Lines 126 – 136.

Comment 2: Basically, it is skeptical the significant enhancement of treatment efficiency of this system from the data, seen in 4 and Table 2.

Response 2: The authors thank the reviewer for their comment regarding the importance of the treatment efficiency of the microalgae-fungal system proposed in this study. The authors wish to highlight the aim of this study, which was to optimize the process of pellet formation for the sustainable production of a cost-effective bioflocculant for microalgae harvesting. The fungal isolates screened in this study were capable of producing pellets even under stationary and neutral pH conditions, making their application in real wastewater more straightforward and feasible.

Comment 3: Application of fungal consortia seems to be new, but the long term operation may change the consortia, leading to the inefficient treatment. The experiment time is rather short to answer the question in no.3.

Response 3: The authors thank the reviewer for their candid opinion on the implications of long-term fungal consortium operation. The short experiment time was not within the author's control, but rather the nature of the microbes, which were capable of forming pellets within a short period. Subsequently, the extensive network of fungal hyphae resulted in high harvesting efficiencies in the selected microbes, which also occurred within a short period. The microbe’s intrinsic capabilities determined the duration of the experiment.

Comment 4: Some experimental procedures are not shown in results.                                         

Response 4: The authors thank the reviewer for this observation. The manuscript has been perused repeatedly, and the authors would be grateful if the reviewer could point to specific areas for the authors to address.

Comment 5: Experimental procedures may not conducted in cocoa pulp wastewater? It should be cleared.

Response 5: We sincerely appreciate the observation and comment made by the reviewer. The authors had stated this on Page 3, in section 2.3, lines 125 - 151, that pellet formation from fungal isolates was conducted in a synthetic culture medium (minimal medium) while real wastewater (cocoa pulp wastewater) was introduced later in the experiment to demonstrate their nutrient removal efficiencies.

Comment 6: Figures 2 and 4 need more accurate explanation. Colors in Figure 4 are not clear. In text Figure 2Ai and ii?

Response 6: The authors are grateful for the comment on the figures. Figure 2 has been enlarged for better visibility, and the caption has been revised. Page 11, section 3.3, Line 422 - 425

“Figure 2. (a) SEM images of entrapped fungal mycelia showing rough fungal hyphae ((i) 100 µm, mag. 1000x); ((ii) 2 µm, mag. 50,000x); and harvested microalgae entrapped in fungal mycelia ((iii) 20 µm, mag. 5000x) and ((iv) 50 µm, mag. 2000x). (b) Harvesting efficiency of isolate T10 and FP”.

Comment 7: Figure 4. Why control is so active in the treatment?

Response 7: The authors are grateful for the insightful question about Figure 4. In the batch cultures, the control group had no living cells, such as microalgae or fungi, added. However, there was some nutrient reduction, although slower than the other treatment groups. This is due to chemical and physical processes such as chemical precipitation or volatilization. Thus, the higher nutrient reduction efficiency of the other treatment groups proved that the excess nutrient removal was due to the presence of microalgae and fungi. The consistency of the results in all scenarios, such as the higher nutrient removal of the microalgae-fungi group, showcases the synergistic effect of the consortia compared to mono-algal or mono-fungal treatment groups

Comment 8: Figure 1. What is the unit of pellet numbers ?

Response 8: The authors thank the reviewer for the question. The pellet numbers are ordinary numbers; therefore, no unit is attached to the graphs in Figure 1.

Comment 9: Table 2. The advantage of fungal consortium is not clear compared with other single fungal system. What is N/A? Text is rather redundant. More refined text should be prepared.

Response 9: The advantage of the fungal consortium over the single fungal system in this study was the pelleting ability under stationary conditions within 48 hours. No agitation was required to produce the fungal fluffs during the pre-treatment stage, as well as the ability of the selected fungal isolate to produce pellets at acidic, neutral and alkaline pH conditions. These characteristics were attributed to the fungal consortium in comparison to single fungal systems outlined in the manuscript.

N/A (not applicable) was used in Table 2 to refer to studies that did not use wastewater in their experiment. This has been revised to “none”. The title of Table 2 has also been revised accordingly

Table 2. “Comparision among systems using microalgae and fungi co-cultivation treating various wastewater” (Page 14, section 3.5, Line 530)

Fungi	Microalgae	pH	HT (h) /Eff* (%)	WW type	ref
A. niger	C. vulgaris	5.0 – 8.0	72 / >80	None	[24]
A. niger	Chlorella sp.	5.0	72 / >98.4	Litopenaeus vannamei cultures	[22]
A. niger	S. subsala	6.0	24 / > 61	None	[43]
Penicillum	Chlorella sp.	5.9	72 / ~100	None	[30]
Fungi consortium	Chlorella sp.	5.0 – 7.0	24 /~100	Cocoa pulp WW	This study

Comment 10: Text is rather redundant. More refined text should be prepared.

Response 10: The text in the revised manuscript has been refined.     

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript was improved based on the comments and can be accepted under this criterion.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript was improved and properly written.