BioLumCity: 3D-Printed Bioluminescent Urban Tiles Employing Aliivibrio fischeri Bioink as Passive Urban Light

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper titled "BioLumCity: 3D Printed Bioluminescent Urban Tiles Employing Aliivibrio Fischeri Bioink as Passive Urban Light" offers a sustainable, stand-alone, bioluminescent urban screen system employing Aliivibrio fischeri CECT 524 bioink on 3D-printed customized scaffolds to act as bioreceptive panel design based on fields-diffusion pattern to host the bioluminescent bacterial bioink. Overall, I think this paper is well written, and the conclusions are well-supported and interesting, but some minor revisions are necessary before acceptance:
(1) The abstract is too long, 200-250 words are enough, include reseach background, method, main conclusions.
(2) Line 60, "For example, [1] reported the high cost of ..." should be "For example, Atasu et al, 2021 reported the high cost of ...", please check the whole manscript.
(3) In introduction, the summary of biotechnology is not enough. It is recommended to summarize the recently popular MICP technology. The following papers are worth reading and analyzing: Biogeotechnics, 2023, 1(2), 100018; Environmental Science and Ecotechnology, 2021, 6, 100096; Journal of Materials in Civil Engineering, 2021, 33(2), 04020455. Frontiers in Materials, 2019, 6, 126.   Coastal Engineering, 2024, 189, 104489.
(4) Line 368, the citation (https://www.creality.com/products/creality-ender-3-s1-pro-fdm-3d-printer) should appear in the references rather than in the text, please check the whole manscript.
(5)While the paper criticizes the shortcomings of traditional energy systems, it does not directly compare the luminous efficiency of this study with conventional LED lighting or other bioluminescent systems. The authors are advised to provide additional comparisons (even if only by orders of magnitude) to highlight potential advantages or areas for improvement. 
(6) Although Figure 5 shows the results of different topological structures, it is not very easy to interpret and requires the reader to understand it in conjunction with the text. It is recommended to improve the figure annotation (add quantitative values ​​of light intensity or normalization curves) and reduce the over-reliance on text descriptions.
(7) The discussion is also too long, Please shorten it to improve readability.
(8) It is recommended that the conclusion be listed in bullet points rather than a paragraph.

Comments on the Quality of English Language

The paper titled "BioLumCity: 3D Printed Bioluminescent Urban Tiles Employing Aliivibrio Fischeri Bioink as Passive Urban Light" offers a sustainable, stand-alone, bioluminescent urban screen system employing Aliivibrio fischeri CECT 524 bioink on 3D-printed customized scaffolds to act as bioreceptive panel design based on fields-diffusion pattern to host the bioluminescent bacterial bioink. Overall, I think this paper is well written, and the conclusions are well-supported and interesting, but some minor revisions are necessary before acceptance:
(1) The abstract is too long, 200-250 words are enough, include reseach background, method, main conclusions.
(2) Line 60, "For example, [1] reported the high cost of ..." should be "For example, Atasu et al, 2021 reported the high cost of ...", please check the whole manscript.
(3) In introduction, the summary of biotechnology is not enough. It is recommended to summarize the recently popular MICP technology. The following papers are worth reading and analyzing: Biogeotechnics, 2023, 1(2), 100018; Environmental Science and Ecotechnology, 2021, 6, 100096; Journal of Materials in Civil Engineering, 2021, 33(2), 04020455. Frontiers in Materials, 2019, 6, 126.   Coastal Engineering, 2024, 189, 104489.
(4) Line 368, the citation (https://www.creality.com/products/creality-ender-3-s1-pro-fdm-3d-printer) should appear in the references rather than in the text, please check the whole manscript.
(5)While the paper criticizes the shortcomings of traditional energy systems, it does not directly compare the luminous efficiency of this study with conventional LED lighting or other bioluminescent systems. The authors are advised to provide additional comparisons (even if only by orders of magnitude) to highlight potential advantages or areas for improvement. 
(6) Although Figure 5 shows the results of different topological structures, it is not very easy to interpret and requires the reader to understand it in conjunction with the text. It is recommended to improve the figure annotation (add quantitative values ​​of light intensity or normalization curves) and reduce the over-reliance on text descriptions.
(7) The discussion is also too long, Please shorten it to improve readability.
(8) It is recommended that the conclusion be listed in bullet points rather than a paragraph.

Author Response

Comment (1): The abstract is too long, 200-250 words are enough, include research background, method, main conclusions.

Reply (1): Done, the abstract was reduced to 300 words. 

Comment (2): Line 60, "For example, [1] reported the high cost of ..." should be "For example, Atasu et al, 2021 reported the high cost of ...", please check the whole manuscript.

Reply (2): Done, the manuscript was revised for Grammar and style. 

Comment (3): In the introduction, the summary of biotechnology is not enough. It is recommended to summarize the recently popular MICP technology. The following papers are worth reading and analyzing: Biogeotechnics, 2023, 1(2), 100018; Environmental Science and Ecotechnology, 2021, 6, 100096; Journal of Materials in Civil Engineering, 2021, 33(2), 04020455. Frontiers in Materials, 2019, 6, 126. Coastal Engineering, 2024, 189, 104489.

Reply (3): The current work focuses on the specific topic of integrating bioactive microorganisms in bioreactors and/or materials for the production of light and/or electricity. Thus, the introduction analysed literature that falls under this tight scope, specifically, bioluminescent bacteria and fungi in bioreactors and bioreceptive systems and materials, or employment of fungi, algae, and bacteria in biobatteries such as microbial fuel cells to generate electricity. Thus, the wider scope proposed by the reviewer in this comment falls out of the scope of the current work, as the proposed papers have more focus on bio-based or natural materials for civil engineering applications or as building materials, not as bioluminescent materials. 

Experimental study on the reinforcement mechanism and wave thumping resistance of EICP reinforced sand slopes. Biogeotechnics, 2023, 1(2), 100018; https://doi.org/10.1016/j.bgtech.2023.100041 

MICP as a potential sustainable technique to treat or entrap contaminants in the natural environment: A review; Environmental Science and Ecotechnology, 2021, 6, 100096, https://doi.org/10.1016/j.ese.2021.100096.

An Experimental Investigation on Shear Strength, Deformation, and Particle Breakage of Carbonate Sand–Concrete Interface through Direct Shear Tests Subjected to Monotonic–Cyclic Thermal Loadings; Journal of Materials in Civil Engineering, 2021, 33(2), 04020455.

While the other two proposed references: Frontiers in Materials, 2019, 6, 126. Coastal Engineering, 202papers9, 104489, were found as full journals or volumes and not specific papers. 

Thus, due to the irrelevance of the proposed references by the reviewer to the specific scope of the current study, the authors can not add these references since they are not relevant and don´t serve the review, objectives, or methods of the current work. 

Comment (4): Line 368, the citation (https://www.creality.com/products/creality-ender-3-s1-pro-fdm-3d-printer) should appear in the references rather than in the text, please check the whole manscript.

Reply (4): The referred link https://www.creality.com/products/creality-ender-3-s1-pro-fdm-3d-printer is not a citation; it is the website of the manufacturer and distributor of the 3D printer used in the current study. It is mandatory to mention the supplier or manufacturer of all the materials and tools used in the current work. Thus, this link is not a reference; it is the manufacturer of this tool used within the materials and methods of the current work. However, the link was replaced by the name of the manufacturer and their location.  The same correction was applied to the following two links in lines 389 and 398, which were replaced by the name of the company and its location.  

Comment (5): While the paper criticizes the shortcomings of traditional energy systems, it does not directly compare the luminous efficiency of this study with conventional LED lighting or other bioluminescent systems. The authors are advised to provide additional comparisons (even if only by orders of magnitude) to highlight potential advantages or areas for improvement. 

Reply (5): There are two points to reply to this comment; first, the study criticized the conventional energy systems in terms of their sustainability and centrality, with focus on their harmful environmental impact, their high cost, and their dependence on centralized management and distribution, not in terms of the light intensity. The proposed alternative bioenergy system proposed in the current study will require further studies to analyze, quantify, categorize, and improve its light intensity for addressing various functions. Second, the quantification of the light intensity and duration of the proposed system in the current study requires a separate detailed study with the main focus on testing various measuring methods, quantification of the light intensity and comparison to control as well as comparison to conventional energy systems considering all the other aspects as the cost, duration, maintenance, etc. which cannot be included in the current study due to legnth and scope. Whilst the current work proposes a proof of concept for the proposed use of bioluminescent bacteria in a bioluminescent bioink on a bioreceptive scaffold as a stand-alone, affordable, and easily maintained system to provide naturally emitted light in the current phase. Explaining what the components of the system are and how it is assembled and operated. The performance of the evaluation, including light intensity and comparisons, will be published in a future study. 

Comment (6): Although Figure 5 shows the results of different topological structures, it is not very easy to interpret and requires the reader to understand it in conjunction with the text. It is recommended to improve the figure annotation (add quantitative values ​​of light intensity or normalization curves) and reduce the over-reliance on text descriptions.

Reply (6): Done. Figure 5 was enhanced in terms of annotation by adding a bar chart showing a comparison between the four parts of the tile (P1, P2, P3, and P4) and their quantitative differentiation in terms of topology, including the number of peaks and the number of strips. 

Comment (7): The discussion is also too long. Please shorten it to improve readability.

Reply (7): The discussion length currently is approximately four pages, including mandatory parts to justify the objectives, methods, and results of the current study, especially since it is proposing a novel concept of application of bioluminescent bacterial bioink on an architectural and urban scale.  Therefore, the authors would like to maintain the current form of the discussion section, as it includes mandatory comparisons with the literature and justifications of the methods and results. Nevertheless, the discussion section was revised to reduce any unnecessary parts where applicable, as well as the entire manuscript to comply with the reviewer comments. 

Comment (8): It is recommended that the conclusion be listed in bullet points rather than a paragraph.

Reply (8): The authors believe that this comment is of a formatting aspect; thus, the authors would like to comply with the common formatting for the conclusion section, which is usually in a paragraph rather than a bullet point list. 

Reviewer 2 Report

Comments and Suggestions for Authors

Line 33. «…bacterial bioink Furthermore…». Probably there is a missing «.» before «Furthermore». Check it.
Lines 256-258. «To define their efficiency as domestic and urban lights and their functional efficiency. As part of the funded research project BioLumCity funded by the Fritz und Trude Fortmann Stiftung». Both sentences seem to be incomplete in meaning. Please check it.
Lines 366-367. «…with 300% printing speed per tile…» this phrase is not clear and requires clarification.
Line 430. An explanation of the parameter «Abs» should be added in the manuscript or in the Title of Figure 4.
Lines 435-436. Title«Table 1. The urban screen panels bioluminescent activity cultured on MB vs. LB measured by absorbance at 600 nm in 5, 3 and 2 hours respectively.» must be placed before the Table. Also, in Table 1 it is necessary to indicate the units of measurement of the values ​​that are indicated in this way (probably for the parameter "absorbance").
It is not clear from the text of the manuscript for which architectural elements the proposed development can be used.
The manuscript should have added data on the durability of the proposed bioluminescent coating in the proposed urban operating conditions.

Author Response

Comment 1: Line 33. «…bacterial bioink Furthermore…». Probably there is a missing «.» before «Furthermore». Check it.

Reply (1): Done, The authors couldn´t find the part referred to in lines 32, 33, and 34: ¨P1 achieved the highest performance in terms of visible blue-green light and duration of 10 days active bioluminescence without the need for refile.¨ in the abstract. It is probably in lines 35 and 36. The sentence was missing punctuation, and a full stop was added at the end of "bioluminescent bacterial bioink". 

Comment 2: Lines 256-258. «To define their efficiency as domestic and urban lights and their functional efficiency. As part of the funded research project BioLumCity funded by the Fritz und Trude Fortmann Stiftung». Both sentences seem to be incomplete in meaning. Please check it.

Reply (2): Done, the sentence "This study is conducted as "was added before "As part of the funded research project BioLumCity funded by the Fritz und Trude Fortmann Stiftung". While the first sentence is complete is corrected in punctuation to be a part of the previous sentence: " To prove the economic feasibility and competence of this system on the architecture and urban scale as well as paving the way for the following step in this research project, which is measuring the spatial bioluminescence light intensity from various spatial points in the interior space and in the exterior, to define their efficiency as domestic and urban lights and their functional efficiency".

Comment 3: Lines 366-367. «…with 300% printing speed per tile…» this phrase is not clear and requires clarification.

Reply (3): " The bioreceptive tile design is adequate for mass production with affordable 3D printing tools and materials, with a moderate fabrication time of 4 hours with 300% 3D printing speed per tile." This sentence explains a parameter of the fabrication tool and the time for one bioreceptive tile. The 300% 3D printing speed is an option available in the majority of 3D printing machines to reduce the time of a printing job. In the current study, the printing speed was tripled to reduce the printing time. This explanation was added to the text: "To reduce the printing time per tile by increasing the speed of the 3D printing process, which proves the compatibility of the tile´s design with high definition, the accurate printing settings, and the high resolution and precision of the employed 3D printer in the current study".  

Comment 4: Line 430. An explanation of the parameter «Abs» should be added in the manuscript or in the Title of Figure 4.

Reply (4): Done, the following expansion was added to Figure 4, caption: "The unit (Abs) is 'Absorbance, a quantity that measures how much light a substance absorbs at a particular wavelength. It's a logarithmic measure, often called optical density, with higher Abs values indicating more light absorption by the sample and less light passing through". 

Comment 5: Lines 435-436. Title«Table 1. The urban screen panels bioluminescent activity cultured on MB vs. LB measured by absorbance at 600 nm in 5, 3 and 2 hours respectively.» must be placed before the Table. Also, in Table 1 it is necessary to indicate the units of measurement of the values ​​that are indicated in this way (probably for the parameter "absorbance").

Reply (5): Done, the title of the table was moved to be placed before the table, and the parameter "absorbance" was added next to Abs in the table to indicate the units of measurement. 

Comment 6: It is not clear from the text of the manuscript for which architectural elements the proposed development can be used.

Reply (6): The architecture element application of the proposed bioluminescent tiles is cladding that can be employed exteriorly (in urban applications as coverage to walls) or interiorly (in architecture applications). Further explanation was added to the introduction and discussion sections, stating that the developed bioluminescent tiles are proposed for use as cladding: "Which can be used as cladding on urban and architectural scale".

Comment 7: The manuscript should have added data on the durability of the proposed bioluminescent coating in the proposed urban operating conditions.

Reply (7): The manuscript currently includes data on the durability of the bioluminescent tiles, which last for 10 days without the need for refilling or recharging the media, when incubated at room temperature for domestic use on an architectural scale. Further data on the durability of the proposed bioluminescent tiles under varied urban conditions, including orientation, varied temperature, environmental light type, darkness levels, humidity, aeration levels, and other relevant parameters, will be included in a future study. This will focus on quantification and measurement of the emitted light intensity from the bioluminescent ties under varied environmental and spatial conditions. 

Reviewer 3 Report

Comments and Suggestions for Authors   This manuscript represents an innovative and timely contribution to the field of sustainable bio-based lighting systems. The authors propose the development of 3D-printed, bioreceptive urban panels using Aliivibrio fischeri bioink to provide a decentralized, low-cost, and environmentally friendly passive light source for architectural and urban applications. The scope of the study is ambitious, combining microbiology, biotechnology, materials science, and architectural design.   While the introduction is overly long, the article is well-structured, detailed, and provides a clear explanation of the research rationale, methods, and results. Integrating bioluminescent microorganisms with architectural materials is an emerging field with great potential, and the manuscript represents an important step towards its practical implementation. This study goes beyond previous pilot or scale models by attempting to create real-scale urban panels. Its focus on scalability and feasibility is its main strength.   The experimental design is thoroughly described, including scaffold design, culture media optimization, and inoculation procedures. The authors also provide a detailed rationale for the chosen solutions. Including cost estimates for both 3D printing and bacterial cultivation enhances the practical relevance of the study.   While the authors acknowledge that standard bioassays have limitations, the study would benefit from a more systematic quantitative assessment of light intensity, even if only relative measurements. A comparison with conventional light sources (e.g., LEDs) would help contextualize the results. The study reports light emission lasting up to 10 days, but information on long-term efficiency, charging frequency, and bacterial survival under real-world environmental conditions is lacking.   Further work should focus on multiple media replenishment cycles and light flux stability. Although A. fischeri is a nonpathogenic bacterium, the manuscript should include a section explicitly addressing biosafety, potential risk of environmental release, and regulatory issues. Some figures (e.g., Figures 5 and 6) are essential for understanding the results but would benefit from clearer labeling, higher resolution, and quantitative scales.   While the text is generally clear, some sections are excessively long and could be simplified for better readability. Careful grammatical editing and text consistency are recommended.   I recommend publishing the manuscript after minor revisions.

Author Response

 Comment 1: While the authors acknowledge that standard bioassays have limitations, the study would benefit from a more systematic quantitative assessment of light intensity, even if only relative measurements. A comparison with conventional light sources (e.g., LEDs) would help contextualize the results.

Reply (1): There are two points to reply to this comment; first, the study criticized the conventional energy systems in terms of their sustainability and centrality, with focus on their harmful environmental impact, their high cost, and their dependence on centralized management and distribution, not in terms of the light intensity. The proposed alternative bioenergy system presented in the current study requires further investigation to analyze, quantify, categorize, and optimize its light intensity for addressing various functions. The point of its advantage over other bioenergy systems is the ease of use and affordable maintenance, as well as its application on an architectural and urban scale, not just on the pilot scale experimental attempts reported in the literature and presented in the introduction. Thus, in comparison to similar bioenergy systems that propose the use of bioluminescent microorganisms for passive lighting, the current study represents an unprecedented advancement, specifically in terms of scale of application, rather than light intensity, as the majority of the literature in this field does not report light intensity either.  Second, the quantification of the light intensity and duration of the proposed system in the current study requires a separate detailed study with the main focus on testing various measuring methods, quantification of the light intensity and comparison to control as well as comparison to conventional energy systems considering all the other aspects as the cost, duration, maintenance, etc. which cannot be included in the current study due to legnth and scope. Whilst the current work proposes a proof of concept for the proposed use of bioluminescent bacteria in a bioluminescent bioink on a bioreceptive scaffold as a stand-alone, affordable, and easily maintained system to provide naturally emitted light in the current phase. Explaining what the components of the system are and how it is assembled and operated. The performance of the evaluation, including light intensity and comparisons, will be published in a future study. 

Comment 2: The study reports light emission lasting up to 10 days, but information on long-term efficiency, charging frequency, and bacterial survival under real-world environmental conditions is lacking. Further work should focus on multiple media replenishment cycles and light flux stability.

Reply (2): The current work proposes a proof of concept for the proposed use of bioluminescent bacteria in a bioluminescent bioink on a bioreceptive scaffold as a stand-alone, affordable, and easily maintained system to provide naturally emitted light in the current phase. Explaining what the components of the system are and how it is assembled and operated. The duration of light emission without the need for recharge is 10 days, as stated in the results and discussion in the current work, where the system is adopted for domestic use at room temperature. Nevertheless, a more detailed study of the different architectural and urban environmental conditions and their effect on the bioluminescence performance of the system, including light intensity and durability, will be published in a future study, due to the length and scope of the current work. 

Comment (3): Although A. fischeri is a nonpathogenic bacterium, the manuscript should include a section explicitly addressing biosafety, potential risk of environmental release, and regulatory issues.

Reply (3): Done, A paragraph highlighting the non-pathogenic nature of Alivibrio fischeri and the biosafety evaluation of the system has been added to the end of the discussion section, as shown in yellow. 

Comment (4): Some figures (e.g., Figures 5 and 6) are essential for understanding the results but would benefit from clearer labeling, higher resolution, and quantitative scales.  

Reply (4): Done. Figure 5 has been modified to include a bar chart for quantifying morphological parameters of the four different parts (P1, P2, P3, and P4) of the bioreceptive scaffold (tile), specifically the number of peaks and the number of strips. Figure 6 presents a summary of the study's experimental procedures and the bioluminescent tile P1 resulting from this experimental work. 

Comment (5): While the text is generally clear, some sections are excessively long and could be simplified for better readability.

Reply (5): Done, the manuscript was revised, and some sections were reduced. The abstract was reduced to 330 words. Other sections were also reduced where applicable. 

Comment (6): Careful grammatical editing and text consistency are recommended.  

Reply (6): Done, the manuscript was revised for Grammar and style, and corrections were made where applicable. 

Comment (7): I recommend publishing the manuscript after minor revisions.

Reply (7): The authors thank the reviewer for their constructive comments. 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript can be accepted in present form