Effect of Green Light on Citrate-Coated Gold Nanoparticles and Their Effect on the Growth of Cellulolytic Fungi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study investigates the effect of green light irradiation (520 nm) on the physicochemical properties of citrate-stabilized gold nanoparticles and their biological activity toward the cellulolytic fungus Chaetomium globosum at environmentally relevant concentrations (0.098–0.49 µg/mL Au). The work addresses an important scientific problem concerning the environmental fate and biological effects of gold nanoparticles after their release from biomedical and technical applications into ecosystems. Understanding how visible light exposure modifies AuNP surface chemistry, plasmonic properties, and biological reactivity is relevant for environmental risk assessment, optimization of photodynamic antimicrobial therapies, and evaluation of impacts on beneficial soil microorganisms involved in organic matter decomposition. The practical significance lies in providing data for environmental safety guidelines, developing light-activated nanoparticle systems for controlled antimicrobial applications, and creating biotechnological tools for regulating fungal cellulase activity in biowaste treatment and biofuel production, thus contributing to the "safe-by-design" paradigm in nanotechnology.

However, the manuscript cannot be accepted in its current form due to several reasons:

1. The authors claim to investigate a novel approach combining green light irradiation of citrate-AuNPs with biological testing on environmental fungi. However, the novelty is poorly defined and inadequately justified in the Introduction. The same authors published similar work in 2016 [http://dx.doi.org/10.1016/j.ijpharm.2016.04.004] investigating visible light effects on gold nanoparticles and Chaetomium globosum. The current manuscript fails to differentiate the present study from this earlier publication. The Introduction lacks: (1) explicit statement of what remains unknown after previous work; (2) justification for why green light at 520 nm specifically advances knowledge; (3) clearly stated hypothesis.
2. GC and GGL have different chemical histories (aged vs aged+irradiated), lack dark/thermal controls, and no temperature monitoring during irradiation. Observed differences could be aging or thermal artifacts rather than photochemical effects. Experimental redesign with proper controls is mandatory.
3. The Discussion provides extensive mechanistic explanations (ROS generation, antioxidant enzyme inhibition, photocatalytic activity, phosphate secretion) entirely without experimental evidence—none of these proposed mechanisms were measured. Contradictory size data (TEM shows decrease, UV-Vis suggests nucleation, dark-field shows dimers) lack coherent explanation. Circular reasoning: citrate detachment enables dimers, yet free citrate supposedly stabilizes electrostatically. Hormesis interpretation unsupported by dose-response modeling.
4. The Conclusions overstate findings and present speculation as established fact. Line 428-429 claims oxide formation "could influence photocatalytic activity"—but photocatalytic activity was never measured. Line 434-436 describes "presumed increased photocatalytic activity"—acknowledging it's speculative yet drawing conclusions from it. Line 437-441 suggests "new biotechnological tools could be developed"—this is aspirational, not a data-driven conclusion.

Author Response

•  

  Comments 1: The authors claim to investigate a novel approach combining green light irradiation of citrate-AuNPs with biological testing on environmental fungi. However, the novelty is poorly defined and inadequately justified in the Introduction. The same authors published similar work in 2016 [http://dx.doi.org/10.1016/j.ijpharm.2016.04.004] investigating visible light effects on gold nanoparticles and Chaetomium globosum. The current manuscript fails to differentiate the present study from this earlier publication. The Introduction lacks: (1) explicit statement of what remains unknown after previous work; (2) justification for why green light at 520 nm specifically advances knowledge; (3) clearly stated hypothesis

  Response 1: Thank you for pointing this out. The 2016 article investigated the effects of different ranges of visible light on gold nanoparticles before their administration to fungal cultures, focusing on another fungal decomposer, Phanerochaete chrysosporium (belonging to a different biological class than Chaetomium globosum), in which some biochemical changes were observed without following its actual growth.

  The present study brings novel elements:

  (i) the irradiation is performed at a wavelength of 520 nm, corresponding to the spectral maximum of the LSPR band of gold nanoparticles, which allows for a selective and controlled photochemical interaction with the surface structure of the particles;

  (ii) the nanoparticle surface reactivity was investigated electrochemically by cyclic voltammetry, absent in the 2016 paper;

  (iii) the current study focuses on the systematic comparison of dose-response profiles of irradiated versus non-irradiated nanoparticles at ecologically relevant concentrations.

  The working hypothesis is that the irradiation with green light at the LSPR wavelength modifies the surface properties of gold nanoparticles (citrate distribution, surface oxide layer structure), which leads to measurable changes in their biological reactivity towards cellulolytic fungi.

  Comments 2: GC and GGL have different chemical histories (aged vs aged+irradiated), lack dark/thermal controls, and no temperature monitoring during irradiation. Observed differences could be aging or thermal artifacts rather than photochemical effects. Experimental redesign with proper controls is mandatory.

  Response 2: We acknowledge that we did not explain that AuNPs were irradiated with green light under dark conditions (in an isolated room without light) and in the presence of a thermal control.

  Under the experimental conditions used, the incident power of the irradiation source was 26.3 W/m², and the exposure sessions alternated with 5-minute breaks and 20-minute light exposure time, to limit sample heating. The suspension temperature did not exceed the threshold of 30 °C during the irradiation, according to the recordings made with the PMA 2130 sensor. However, we acknowledge that, in the absence of an explicit thermal control (sample maintained at the same temperature, without light irradiation) and a dark control (sample maintained at room temperature, without light), we cannot definitely exclude the contribution of thermal or differential aging effects.

  We added in the sub-section Light exposure of AuNPs :

  "The temperature of the suspension was monitored throughout the irradiation sessions using the PMA 2130 sensor and did not exceed 30 °C, owing to the alternating light-pause cycle (20 min exposure / 5 min pause). No thermal control (dark sample maintained at equivalent temperature) was included in the present experimental design."

  A complete redesign of the experiment will address this limitation in future research by adding the two aforementioned controls, which is now beyond the current capabilities of the study and will be further developed.

  Comment 3. The Discussion provides extensive mechanistic explanations (ROS generation, antioxidant enzyme inhibition, photocatalytic activity, phosphate secretion) entirely without experimental evidence—none of these proposed mechanisms were measured. Contradictory size data (TEM shows decrease, UV-Vis suggests nucleation, dark-field shows dimers) lack coherent explanation. Circular reasoning: citrate detachment enables dimers, yet free citrate supposedly stabilizes electrostatically. Hormesis interpretation unsupported by dose-response modeling

  Response 3. Regarding the ROS, we rephrased in the manuscript accordingly (at Discussion section):

  "It may be hypothesized that, at high concentrations, the tendency of GGL nanoparticles to form dimers could reduce their effective reactive surface area, potentially limiting the generation of reactive oxygen species (ROS) and thus exerting less inhibitory effect on fungal growth. However, ROS levels were not measured in the present study, and this interpretation remains speculative."

  Regarding the apparent contradiction between the dimensional data:

  -the decrease in the average diameter in TEM distribution as well as the increase in the spectral band intensity for GGL compared to GC reflects the redistribution of the population of individual particles due to the continuation of nucleation under the action of light with generation of mostly small particles, together with some other particle dimmerization (evidenced by dark-field microscopy) and highlighted by DLS results.

   

   Regarding the apparent circular reasoning regarding citrate:

  - the partial release of citrate from the AuNP surface under the action of light allows the particles to approach each other and form dimmers; free citrate in solution contributes additionally to the electrostatic stabilization of the collective system, including already formed dimmers.

   

  In the Discussion section we wrote:

  "The decrease of the mean diameter in TEM distribution as well as the increase in the spectral band intensity for GGL suspension compared to GC one reflects the redistribution of the population of individual particles due to the continuation of nucleation under the action of light with generation of mostly small particles, while some other particles experienced dimmerization (evidenced by dark-field microscopy) and highlighted by DLS results.

  We can state that irradiation can support the continuation of the nucleation process, after the completion of the thermal synthesis, even if the reduction of Au³⁺ ions with citrate can be considered theoretically complete for the reaction parameters used (30 minutes at 101°C, citrate/Au molar ratio of 3.5).

  However traces of incompletely reduced gold or citrate ions may persist, especially during the rapid cooling phase [19] conferring the dynamic equilibrium character of the reaction environment.

  The increase in LSPR band intensity observed in the GGL sample compared to GC was interpreted as a continuation of nucleation under light, possibly by photochemical reactions of these residual species. However, this interpretation is indirect, not being directly validated by a method for dosing residual gold ions (e.g., inductively coupled plasma mass spectrometry, ICP-MS).

  The partial release of citrate from the AuNP surface under the action of light allows the particles to approach each other and form dimmers; free citrate in solution contributes additionally to the electrostatic stabilization of the collective system including already formed dimmers."

  We abandon the claim about hormesis, admitting that hormesis-like behavior did not occur in our experiments, even if we would have expected it to happen.

  Comment 4. The Conclusions overstate findings and present speculation as established fact. Line 428-429 claims oxide formation "could influence photocatalytic activity"—but photocatalytic activity was never measured. Line 434-436 describes "presumed increased photocatalytic activity"—acknowledging it's speculative yet drawing conclusions from it. Line 437-441 suggests "new biotechnological tools could be developed"—this is aspirational, not a data-driven conclusion.

  Response 4. Indeed, the Conclusions section went beyond the factual framework of the experimental data presented in some points. The Conclusions were revised in their entirety to strictly reflect what was measured and demonstrated in the study.

  The statements regarding photocatalytic activity (lines 428-429 and 434-436) were reformulated as future research perspectives, not as results of the present study. The suggestion regarding potential biotechnological tools (lines 437-441was included to the research perspectives, formulated as a direction for further investigation  clearly delimited from the actual conclusions.

   

  "Gold nanoparticles synthesized with sodium citrate in the presence of sodium hydroxide, as well as those resulting from photochemical synthesis, i.e. after irradiation of the first product, presented small colloidal particles, both for the physical diameter, obtained by TEM, and for the hydrodynamic diameter given by DLS, as well as distinct plasmonic properties. The Zeta potential is negative in both samples reflecting the electrostatic stability of the colloidal suspensions. Cyclic voltammetry indicated the formation of gold oxide on the surface of nanoparticles exposed to green light, which could influence the reactivity needed for AuNP biomedical or environmental applications.

  Microbiological tests focused on the growth of cellulolytic fungal cultures revealed some differences in the effect of adding AuNPs to the culture medium for pure particles and those exposed to green light on fungal growth, especially for relatively high concentrations of irradiated nanoparticles, such as 1000 µL/L, which induced higher growth than non-irradiated AuNPs. These indicate a relative biocompatibility with beneficial fungi in the biosphere and rather suggest possible biotechnological applications related to the stimulation of fungal growth for the controlled degradation of cellulosic residues of natural or anthropogenic origin."

   Thank you very much for your help!

   

   

  • Comments 1: Lines 137, 138 - How were the gold solutions adsorbed onto the electrodes? Why were different concentrations of nanoparticles used?

  Response 1. Here we provide some detailed explanations.

  We wrote in the Materials and Methods section:

  "The deposition of samples on the electrodes was performed by the drop-casting method: precise volumes of 3 µL and, respectively, 9 µL of the GC and GGL suspensions were applied directly onto the surface of the glassy carbon electrode, being allowed to dry at room temperature for approximately 30 minutes, to form a stable adsorbed film. The use of two different volumes (3 µL and 9 µL) aimed to investigate the dependence of the voltammetric response on the amount of material deposited on the electrode, allowing the evaluation of the AuNP film thickness and its effect on the observed anodic/cathodic currents."

  • Comments 2: Lines 165-169 - Why is the average size of irradiated gold nanoparticles smaller than that of non-irradiated ones? According to the Reviewer, this difference is within the margin of error.

  Response 2: The difference between the two mean values is small but an order of magnitude over the standard error. Mean size values were of 16.4 nm and 15.8 nm with standard errors of 0.11 nm for GC and 0.10 nm for GGL, as obtained from Gaussian fitting of the size histograms, which means about 0.6% while the difference between the two mean values is approx. 3.6%.

  We wrote in the Manuscript:

  "From a mechanistic point of view, a slight reduction in the mean diameter of individual particles in the GGL sample could be explained by the detachment and partial rearrangement of citrate on the particle surface under the action of green light at LSPR resonance [19]). Also, the nucleation continuation under the irradiation effect is supposed to lead to new small particles formation as long as the LSPR band intensity increased. Although the spectral maximum remained at the same position however the spectral band narrows towards shorter wavelengths (Figure 2). Mean size values were of 16.4 nm and 15.8 nm with standard errors of 0.11 nm for GC and 0.10 nm for GGL, as obtained from Gaussian fitting of the size histograms, which means about 0.6% while the difference between the two mean values is approx. 3.6%."

  • Comment 3. Lines 182-184 – Once the synthesis process is complete, does this mean that there are still non-reduced gold ions present in the solution?

  Response 3. This is due to the dynamic equilibrium characterizing the chemical species in the reaction medium after the completion of the thermal synthesis.

  In the Discussion section we wrote:

  "We can suppose that irradiation can support the continuation of the nucleation process, after the completion of the thermal synthesis, when the reduction of Au³⁺ ions with citrate can be considered theoretically complete for the reaction parameters used (30 minutes at 101°C, citrate/Au molar ratio of 3.5). However traces of incompletely reduced gold or citrate ions may persist, especially during the rapid cooling phase [19] conferring the dynamic equilibrium character of the reaction environment. The increase in LSPR band intensity observed in the GGL sample compared to GC can be interpreted as a continuation of nucleation under light impact, due to photochemical reactions of these residual species. However, this interpretation is indirect, not being directly validated by a method for dosing residual gold ions (e.g., inductively coupled plasma mass spectrometry, ICP-MS)."

  • Comment 4. Lines 227-230 – Why is the hydrodynamic diameter of irradiated nanoparticles smaller?

  Response 4. We hypothesized that the rearrangement of the citrate layer after irradiation may modify the interaction of colloidal nanoparticles with the surrounding liquid layers, not forgetting that the physical diameter determined by TEM analysis was also statistically reduced.

  We wrote in the manuscript at Results section:

  "The diameter measured by DLS reflects the hydrodynamic size of the particle together with the coating shell and the solvation layer interacting with it. It should be noted that the hydrodynamic diameter is larger than the TEM diameter for both samples (DLS: ~61 nm for GGL, with a mean size of about 15.8 nm in TEM and ~66 nm for GC with a mean size of about 16.4 nm in TEM). The smaller value of the hydrodynamic diameter in GGL compared to GC can be attributed to the rearrangement or partial release of the citrate layer under the action of light irradiation, thus reducing the size of the solvation layer. This interpretation is correlated with the dark-field microscopy data, which suggest a change in the citrate distribution at the surface of the GGL particles."

  • Comment 5. Lines 237-239 - If the nanoparticles undergo dimerisation when exposed to light, why is their polydispersity lower?

  Response 5. We assume that this apparent discrepancy is due to the fact that dimmers are a minor sub-population of nanostructures in the analyzd system.

  We wrote at the Discussion section:

  "A higher polydispersity index, PDI = 0.604, for GC sample with a mean value of approximately 66 nm, compared to PDI = 0.505 for GGL sample with a mean value of approximately 61 nm, together with a smaller standard deviation of the hydrodynamic size for GC (0.12 nm) than for GGL (0.24 nm - reflecting the contribution of the minority dimer subpopulation ~1000 nm), may be related to the fact that standard deviation is an absolute measure while PDI is a relative measure of dispersion, the results depending on instrument settings for acquisition or averaging time of the measurement, and this may come into play generating the counterintuitive result."

  • Comment 6. Lines 262-264 - Were the solutions exposed to light during cyclic voltammetry? If not, why do the authors mention a photocurrent?

  Cyclic voltammetry was performed without active light irradiation during the measurement GC and GGL samples were deposited on the electrodes under standard laboratory conditions (ambient lighting), without photoexposure. The use of the term "photocurrent" in lines 262-264 was imprecise and may be misleading. What was intended to be expressed is that the GGL sample, which previously underwent photochemical surface modifications (rearrangement of citrate, formation of a superficial gold oxide layer), exhibits a different anodic current than the GC sample, as a result of these irradiation-induced structural modifications. The correct term would be "anodic current of the preirradiated sample" or similar.

  Response 6. The term "photocurrent" was replaced with a precise formulation that reflects that the cyclic voltammetry was performed without active irradiation, and the observed current difference is attributed to surface changes previously induced by irradiation (e.g.: "anodic current of the electrode covered with the pre-irradiated GGL sample").

  Indeed there is a writing mistake.

  We wrote by rephrasing and adding:

  "Regarding the cyclic voltammetry results we need to mention that the oxide film which is formed in the positive scan is not readily reduced in the reverse scan as seen by the smaller reduction peak (Figure 6). This indicates the formation of a thick oxide layer on the AuNPs since higher activation energy is required for its reduction compared to non-irradiated nanoparticles. The smaller reduction peak observed for GGL sample compared to GC one suggests that the oxide layer formed on irradiated nanoparticles exhibits modified reduction kinetics — likely due to a more disordered or hydrated oxide structure — rather than simply a thicker layer. A systematic scan-rate analysis would be required to quantify the oxide charge and confirm this interpretation [21]. Figure 6b illustrates the typical cyclic voltammetry curves for different amount of GGL adsorbed on the electrode surface, and evaluated in sulfuric acid, but scanning the potential on a shorter range. Similar to Figure 6a, the two oxidation peaks are present, and the height of the peak increases with the amount of GGL. The position of peaks is slightly shifted to more positive value as expected for a larger amount of nanoparticles that require a higher potential for oxidation (regarded as the driving force for electronic transfer).

  For the irradiated GGL sample, the anodic current increases steeply starting from approximately +0.85 V — a positive shift of ~80 mV relative to GC — suggesting that the oxidation of gold surface atoms requires a higher overpotential after irradiation, consistent with a modified surface layer induced by light exposure. At equal deposited volumes (3 µL), GGL shows reduced electrochemical surface area compared to GC, reflecting restructuring of the adsorbed citrate layer and partial surface oxidation. This does not imply lower biological activity, but rather a different electrochemical profile.

  Regarding the cathodic peak, the less pronounced reduction signal observed for GGL compared to GC may reflect altered reduction kinetics of the surface oxide layer rather than a thinner oxide — a more hydrated or disordered oxide formed under irradiation conditions may be reduced at a slightly different potential and with a broader current distribution [24]. A systematic scan-rate dependent analysis would be needed to quantify the oxide charge and confirm this interpretation, and is proposed as a direction for future work.

  The chemisorbed gold oxide species identified by cyclic voltammetry may be relevant to the surface reactivity of AuNPs in biological environments; however, their potential role in photocatalytic processes was not directly assessed in the present study and remains a hypothesis for further investigation."

  • Comment 7. Lines 356-360 – Why the exposure to green light led to the release of some citrate groups from the AuNP surface, which further determined the formation of dimmers?

  Response 7. We tried to explain this picture of the citrate coating dynamics in more detail.

  We wrote at the Discussion section:

  "The underlying mechanism could be based on phenomena described in [19] regarding the photomodification of AuNPs irradiated at the plasmonic resonance wavelength. Upon LSPR excitation, the photonic energy absorbed by the nanoparticle can induce hotspot electron transfers to the surface, which can weaken the coordination bonds of the citrate carboxylate groups with the gold surface.

  Thus, it could be said that exposure to green light led to the release of some citrate groups from the AuNP surface in the irradiated suspension, leaving uncovered spots on the particle surface that attract each other and lead to the formation of dimmers, and the DF microscopy results confirm this by the twofold higher intensity of the plasmonic profile (Figure 3a) compared to the unassociated monomers (Figure 3a).

  The decrease in the mean diameter in TEM distribution as well as the increase in the spectral band intensity for GGL sample compared to GC one reflects the redistribution of the population of individual particles due to the continuation of nucleation under the action of light with generation of mostly small particles, together with some other particle dimmerization (evidenced by dark-field microscopy) and highlighted by DLS results.

  The partial release of citrate from the AuNP surface under the action of light allows the particles to approach each other and form dimmers; free citrate in solution contributes additionally to the electrostatic stabilization of the collective system including already formed dimmers."

  • Comment 8. Line 361 – 365 - During cyclic voltammetry, if a thicker oxide layer forms under anodic polarisation, a more noticeable reduction peak should appear during the reversible process.

  Response 8.

  Indeed, a thicker oxide layer should correspond to a cathodic (reduction) peak of higher amplitude. Analysis of the cyclic voltammograms presented in Figure 6 shows that the cathodic peak of the GGL sample does indeed exhibit a slight shift and shape change compared to the GC sample, although the amplitude difference is not larger. A possible explanation is that the irradiation-induced surface modification (rearrangement of the citrate layer, formation of a superficial Au₂O/Au(OH)₃ layer) also modifies the kinetics of the oxide reduction process, not only the layer thickness. In practice, a more hydrated or porous oxide layer could be reduced at slightly different potentials and with a different current distribution. We acknowledge that a more detailed analysis (e.g., scan rate dependence, integration of the reduction charge) would clarify this issue, and we mention it as a perspective.

  We wrote by rephrasing and adding:

  "Regarding the cyclic voltammetry results we need to mention that the oxide film which is formed in the positive scan is not readily reduced in the reverse scan as seen by the smaller reduction peak (Figure 6). The smaller reduction peak observed for GGL compared to GC suggests that the oxide layer formed on irradiated nanoparticles exhibits modified reduction kinetics — likely due to a more disordered or hydrated oxide structure — rather than simply a thicker layer. A systematic scan-rate analysis would be required to quantify the oxide charge and confirm this interpretation [21]. Figure 6b illustrates the typical cyclic voltammetry curves for different amount of GGL adsorbed on the electrode surface, and evaluated in sulfuric acid, but scanning the potential on a shorter range. Similar to Figure 6a, the two oxidation peaks are present, and the height of the peak increases with the amount of GGL. The position of peaks is slightly shifted to more positive value as expected for a larger amount of nanoparticles that require a higher potential for oxidation (regarded as the driving force for electronic transfer).

  For the irradiated GGL sample, the anodic current increases steeply starting from approximately +0.85 V — a positive shift of ~80 mV relative to GC — suggesting that the oxidation of gold surface atoms requires a higher overpotential after irradiation, consistent with a modified surface layer induced by light exposure. At equal deposited volumes (3 µL), GGL shows reduced electrochemical surface area compared to GC, reflecting restructuring of the adsorbed citrate layer and partial surface oxidation. This does not imply lower biological activity, but rather a different electrochemical profile.

  As for the cathodic peak, the less pronounced reduction signal observed for GGL compared to GC may reflect altered reduction kinetics of the surface oxide layer rather than a thinner oxide — a more hydrated or disordered oxide formed under irradiation conditions may be reduced at a slightly different potential and with a broader current distribution [24].A systematic scan-rate dependent analysis would be needed to quantify the oxide charge and confirm this interpretation, and is proposed as a direction for future work.

  The chemisorbed gold oxide species identified by cyclic voltammetry may be relevant to the surface reactivity of AuNPs in biological environments; however, their potential role in photocatalytic processes was not directly assessed in the present study and remains a hypothesis for further investigation."

  Many thanks for your very helpful analysis.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This article focuses on synthesis of gold nanoparticles (AuNP) using a conventional method, as well as assessing the impact of green light irradiation on the size, zeta potential, and biological properties of citrate-stabilised gold nanoparticles.

While reviewing the manuscript, the Reviewer had some questions and made the following comments, such as:

• Lines 137, 138 - How were the gold solutions adsorbed onto the electrodes? Why were different concentrations of nanoparticles used?
• Lines 165-169 - Why is the average size of irradiated gold nanoparticles smaller than that of non-irradiated ones? According to the Reviewer, this difference is within the margin of error.
• Lines 182-184 – Once the synthesis process is complete, does this mean that there are still non-reduced gold ions present in the solution?
• Lines 227-230 – Why is the hydrodynamic diameter of irradiated nanoparticles smaller?
• Lines 237-239 - If the nanoparticles undergo dimerisation when exposed to light, why is their polydispersity lower?
• Lines 262-264 - Were the solutions exposed to light during cyclic voltammetry? If not, why do the authors mention a photocurrent?
• Lines 356-360 – Why the exposure to green light led to the release of some citrate groups from the AuNP surface, which further determined the formation of dimmers?
• Line 361 – 365 - During cyclic voltammetry, if a thicker oxide layer forms under anodic polarisation, a more noticeable reduction peak should appear during the reversible process.

Author Response

 

Comments 1: The authors claim to investigate a novel approach combining green light irradiation of citrate-AuNPs with biological testing on environmental fungi. However, the novelty is poorly defined and inadequately justified in the Introduction. The same authors published similar work in 2016 [http://dx.doi.org/10.1016/j.ijpharm.2016.04.004] investigating visible light effects on gold nanoparticles and Chaetomium globosum. The current manuscript fails to differentiate the present study from this earlier publication. The Introduction lacks: (1) explicit statement of what remains unknown after previous work; (2) justification for why green light at 520 nm specifically advances knowledge; (3) clearly stated hypothesis

Response 1: Thank you for pointing this out. The 2016 article investigated the effects of different ranges of visible light on gold nanoparticles before their administration to fungal cultures, focusing on another fungal decomposer, Phanerochaete chrysosporium (belonging to a different biological class than Chaetomium globosum), in which some biochemical changes were observed without following its actual growth.

The present study brings novel elements:

(i) the irradiation is performed at a wavelength of 520 nm, corresponding to the spectral maximum of the LSPR band of gold nanoparticles, which allows for a selective and controlled photochemical interaction with the surface structure of the particles;

(ii) the nanoparticle surface reactivity was investigated electrochemically by cyclic voltammetry, absent in the 2016 paper;

(iii) the current study focuses on the systematic comparison of dose-response profiles of irradiated versus non-irradiated nanoparticles at ecologically relevant concentrations.

The working hypothesis is that the irradiation with green light at the LSPR wavelength modifies the surface properties of gold nanoparticles (citrate distribution, surface oxide layer structure), which leads to measurable changes in their biological reactivity towards cellulolytic fungi.

Comments 2: GC and GGL have different chemical histories (aged vs aged+irradiated), lack dark/thermal controls, and no temperature monitoring during irradiation. Observed differences could be aging or thermal artifacts rather than photochemical effects. Experimental redesign with proper controls is mandatory.

Response 2: We acknowledge that we did not explain that AuNPs were irradiated with green light under dark conditions (in an isolated room without light) and in the presence of a thermal control.

Under the experimental conditions used, the incident power of the irradiation source was 26.3 W/m², and the exposure sessions alternated with 5-minute breaks and 20-minute light exposure time, to limit sample heating. The suspension temperature did not exceed the threshold of 30 °C during the irradiation, according to the recordings made with the PMA 2130 sensor. However, we acknowledge that, in the absence of an explicit thermal control (sample maintained at the same temperature, without light irradiation) and a dark control (sample maintained at room temperature, without light), we cannot definitely exclude the contribution of thermal or differential aging effects.

We added in the sub-section Light exposure of AuNPs :

"The temperature of the suspension was monitored throughout the irradiation sessions using the PMA 2130 sensor and did not exceed 30 °C, owing to the alternating light-pause cycle (20 min exposure / 5 min pause). No thermal control (dark sample maintained at equivalent temperature) was included in the present experimental design."

A complete redesign of the experiment will address this limitation in future research by adding the two aforementioned controls, which is now beyond the current capabilities of the study and will be further developed.

Comment 3. The Discussion provides extensive mechanistic explanations (ROS generation, antioxidant enzyme inhibition, photocatalytic activity, phosphate secretion) entirely without experimental evidence—none of these proposed mechanisms were measured. Contradictory size data (TEM shows decrease, UV-Vis suggests nucleation, dark-field shows dimers) lack coherent explanation. Circular reasoning: citrate detachment enables dimers, yet free citrate supposedly stabilizes electrostatically. Hormesis interpretation unsupported by dose-response modeling

Response 3. Regarding the ROS, we rephrased in the manuscript accordingly (at Discussion section):

"It may be hypothesized that, at high concentrations, the tendency of GGL nanoparticles to form dimers could reduce their effective reactive surface area, potentially limiting the generation of reactive oxygen species (ROS) and thus exerting less inhibitory effect on fungal growth. However, ROS levels were not measured in the present study, and this interpretation remains speculative."

Regarding the apparent contradiction between the dimensional data:

-the decrease in the average diameter in TEM distribution as well as the increase in the spectral band intensity for GGL compared to GC reflects the redistribution of the population of individual particles due to the continuation of nucleation under the action of light with generation of mostly small particles, together with some other particle dimmerization (evidenced by dark-field microscopy) and highlighted by DLS results.

 

 Regarding the apparent circular reasoning regarding citrate:

- the partial release of citrate from the AuNP surface under the action of light allows the particles to approach each other and form dimmers; free citrate in solution contributes additionally to the electrostatic stabilization of the collective system, including already formed dimmers.

 

In the Discussion section we wrote:

"The decrease of the mean diameter in TEM distribution as well as the increase in the spectral band intensity for GGL suspension compared to GC one reflects the redistribution of the population of individual particles due to the continuation of nucleation under the action of light with generation of mostly small particles, while some other particles experienced dimmerization (evidenced by dark-field microscopy) and highlighted by DLS results.

We can state that irradiation can support the continuation of the nucleation process, after the completion of the thermal synthesis, even if the reduction of Au³⁺ ions with citrate can be considered theoretically complete for the reaction parameters used (30 minutes at 101°C, citrate/Au molar ratio of 3.5).

However traces of incompletely reduced gold or citrate ions may persist, especially during the rapid cooling phase [19] conferring the dynamic equilibrium character of the reaction environment.

The increase in LSPR band intensity observed in the GGL sample compared to GC was interpreted as a continuation of nucleation under light, possibly by photochemical reactions of these residual species. However, this interpretation is indirect, not being directly validated by a method for dosing residual gold ions (e.g., inductively coupled plasma mass spectrometry, ICP-MS).

The partial release of citrate from the AuNP surface under the action of light allows the particles to approach each other and form dimmers; free citrate in solution contributes additionally to the electrostatic stabilization of the collective system including already formed dimmers."

We abandon the claim about hormesis, admitting that hormesis-like behavior did not occur in our experiments, even if we would have expected it to happen.

Comment 4. The Conclusions overstate findings and present speculation as established fact. Line 428-429 claims oxide formation "could influence photocatalytic activity"—but photocatalytic activity was never measured. Line 434-436 describes "presumed increased photocatalytic activity"—acknowledging it's speculative yet drawing conclusions from it. Line 437-441 suggests "new biotechnological tools could be developed"—this is aspirational, not a data-driven conclusion.

Response 4. Indeed, the Conclusions section went beyond the factual framework of the experimental data presented in some points. The Conclusions were revised in their entirety to strictly reflect what was measured and demonstrated in the study.

The statements regarding photocatalytic activity (lines 428-429 and 434-436) were reformulated as future research perspectives, not as results of the present study. The suggestion regarding potential biotechnological tools (lines 437-441was included to the research perspectives, formulated as a direction for further investigation  clearly delimited from the actual conclusions.

 

"Gold nanoparticles synthesized with sodium citrate in the presence of sodium hydroxide, as well as those resulting from photochemical synthesis, i.e. after irradiation of the first product, presented small colloidal particles, both for the physical diameter, obtained by TEM, and for the hydrodynamic diameter given by DLS, as well as distinct plasmonic properties. The Zeta potential is negative in both samples reflecting the electrostatic stability of the colloidal suspensions. Cyclic voltammetry indicated the formation of gold oxide on the surface of nanoparticles exposed to green light, which could influence the reactivity needed for AuNP biomedical or environmental applications.

Microbiological tests focused on the growth of cellulolytic fungal cultures revealed some differences in the effect of adding AuNPs to the culture medium for pure particles and those exposed to green light on fungal growth, especially for relatively high concentrations of irradiated nanoparticles, such as 1000 µL/L, which induced higher growth than non-irradiated AuNPs. These indicate a relative biocompatibility with beneficial fungi in the biosphere and rather suggest possible biotechnological applications related to the stimulation of fungal growth for the controlled degradation of cellulosic residues of natural or anthropogenic origin."

 Thank you very much for your help!

 

 

• Comments 1: Lines 137, 138 - How were the gold solutions adsorbed onto the electrodes? Why were different concentrations of nanoparticles used?

Response 1. Here we provide some detailed explanations.

We wrote in the Materials and Methods section:

"The deposition of samples on the electrodes was performed by the drop-casting method: precise volumes of 3 µL and, respectively, 9 µL of the GC and GGL suspensions were applied directly onto the surface of the glassy carbon electrode, being allowed to dry at room temperature for approximately 30 minutes, to form a stable adsorbed film. The use of two different volumes (3 µL and 9 µL) aimed to investigate the dependence of the voltammetric response on the amount of material deposited on the electrode, allowing the evaluation of the AuNP film thickness and its effect on the observed anodic/cathodic currents."

• Comments 2: Lines 165-169 - Why is the average size of irradiated gold nanoparticles smaller than that of non-irradiated ones? According to the Reviewer, this difference is within the margin of error.

Response 2: The difference between the two mean values is small but an order of magnitude over the standard error. Mean size values were of 16.4 nm and 15.8 nm with standard errors of 0.11 nm for GC and 0.10 nm for GGL, as obtained from Gaussian fitting of the size histograms, which means about 0.6% while the difference between the two mean values is approx. 3.6%.

We wrote in the Manuscript:

"From a mechanistic point of view, a slight reduction in the mean diameter of individual particles in the GGL sample could be explained by the detachment and partial rearrangement of citrate on the particle surface under the action of green light at LSPR resonance [19]). Also, the nucleation continuation under the irradiation effect is supposed to lead to new small particles formation as long as the LSPR band intensity increased. Although the spectral maximum remained at the same position however the spectral band narrows towards shorter wavelengths (Figure 2). Mean size values were of 16.4 nm and 15.8 nm with standard errors of 0.11 nm for GC and 0.10 nm for GGL, as obtained from Gaussian fitting of the size histograms, which means about 0.6% while the difference between the two mean values is approx. 3.6%."

• Comment 3. Lines 182-184 – Once the synthesis process is complete, does this mean that there are still non-reduced gold ions present in the solution?

Response 3. This is due to the dynamic equilibrium characterizing the chemical species in the reaction medium after the completion of the thermal synthesis.

In the Discussion section we wrote:

"We can suppose that irradiation can support the continuation of the nucleation process, after the completion of the thermal synthesis, when the reduction of Au³⁺ ions with citrate can be considered theoretically complete for the reaction parameters used (30 minutes at 101°C, citrate/Au molar ratio of 3.5). However traces of incompletely reduced gold or citrate ions may persist, especially during the rapid cooling phase [19] conferring the dynamic equilibrium character of the reaction environment. The increase in LSPR band intensity observed in the GGL sample compared to GC can be interpreted as a continuation of nucleation under light impact, due to photochemical reactions of these residual species. However, this interpretation is indirect, not being directly validated by a method for dosing residual gold ions (e.g., inductively coupled plasma mass spectrometry, ICP-MS)."

• Comment 4. Lines 227-230 – Why is the hydrodynamic diameter of irradiated nanoparticles smaller?

Response 4. We hypothesized that the rearrangement of the citrate layer after irradiation may modify the interaction of colloidal nanoparticles with the surrounding liquid layers, not forgetting that the physical diameter determined by TEM analysis was also statistically reduced.

We wrote in the manuscript at Results section:

"The diameter measured by DLS reflects the hydrodynamic size of the particle together with the coating shell and the solvation layer interacting with it. It should be noted that the hydrodynamic diameter is larger than the TEM diameter for both samples (DLS: ~61 nm for GGL, with a mean size of about 15.8 nm in TEM and ~66 nm for GC with a mean size of about 16.4 nm in TEM). The smaller value of the hydrodynamic diameter in GGL compared to GC can be attributed to the rearrangement or partial release of the citrate layer under the action of light irradiation, thus reducing the size of the solvation layer. This interpretation is correlated with the dark-field microscopy data, which suggest a change in the citrate distribution at the surface of the GGL particles."

• Comment 5. Lines 237-239 - If the nanoparticles undergo dimerisation when exposed to light, why is their polydispersity lower?

Response 5. We assume that this apparent discrepancy is due to the fact that dimmers are a minor sub-population of nanostructures in the analyzd system.

We wrote at the Discussion section:

"A higher polydispersity index, PDI = 0.604, for GC sample with a mean value of approximately 66 nm, compared to PDI = 0.505 for GGL sample with a mean value of approximately 61 nm, together with a smaller standard deviation of the hydrodynamic size for GC (0.12 nm) than for GGL (0.24 nm - reflecting the contribution of the minority dimer subpopulation ~1000 nm), may be related to the fact that standard deviation is an absolute measure while PDI is a relative measure of dispersion, the results depending on instrument settings for acquisition or averaging time of the measurement, and this may come into play generating the counterintuitive result."

• Comment 6. Lines 262-264 - Were the solutions exposed to light during cyclic voltammetry? If not, why do the authors mention a photocurrent?

Cyclic voltammetry was performed without active light irradiation during the measurement GC and GGL samples were deposited on the electrodes under standard laboratory conditions (ambient lighting), without photoexposure. The use of the term "photocurrent" in lines 262-264 was imprecise and may be misleading. What was intended to be expressed is that the GGL sample, which previously underwent photochemical surface modifications (rearrangement of citrate, formation of a superficial gold oxide layer), exhibits a different anodic current than the GC sample, as a result of these irradiation-induced structural modifications. The correct term would be "anodic current of the preirradiated sample" or similar.

Response 6. The term "photocurrent" was replaced with a precise formulation that reflects that the cyclic voltammetry was performed without active irradiation, and the observed current difference is attributed to surface changes previously induced by irradiation (e.g.: "anodic current of the electrode covered with the pre-irradiated GGL sample").

Indeed there is a writing mistake.

We wrote by rephrasing and adding:

"Regarding the cyclic voltammetry results we need to mention that the oxide film which is formed in the positive scan is not readily reduced in the reverse scan as seen by the smaller reduction peak (Figure 6). This indicates the formation of a thick oxide layer on the AuNPs since higher activation energy is required for its reduction compared to non-irradiated nanoparticles. The smaller reduction peak observed for GGL sample compared to GC one suggests that the oxide layer formed on irradiated nanoparticles exhibits modified reduction kinetics — likely due to a more disordered or hydrated oxide structure — rather than simply a thicker layer. A systematic scan-rate analysis would be required to quantify the oxide charge and confirm this interpretation [21]. Figure 6b illustrates the typical cyclic voltammetry curves for different amount of GGL adsorbed on the electrode surface, and evaluated in sulfuric acid, but scanning the potential on a shorter range. Similar to Figure 6a, the two oxidation peaks are present, and the height of the peak increases with the amount of GGL. The position of peaks is slightly shifted to more positive value as expected for a larger amount of nanoparticles that require a higher potential for oxidation (regarded as the driving force for electronic transfer).

For the irradiated GGL sample, the anodic current increases steeply starting from approximately +0.85 V — a positive shift of ~80 mV relative to GC — suggesting that the oxidation of gold surface atoms requires a higher overpotential after irradiation, consistent with a modified surface layer induced by light exposure. At equal deposited volumes (3 µL), GGL shows reduced electrochemical surface area compared to GC, reflecting restructuring of the adsorbed citrate layer and partial surface oxidation. This does not imply lower biological activity, but rather a different electrochemical profile.

Regarding the cathodic peak, the less pronounced reduction signal observed for GGL compared to GC may reflect altered reduction kinetics of the surface oxide layer rather than a thinner oxide — a more hydrated or disordered oxide formed under irradiation conditions may be reduced at a slightly different potential and with a broader current distribution [24]. A systematic scan-rate dependent analysis would be needed to quantify the oxide charge and confirm this interpretation, and is proposed as a direction for future work.

The chemisorbed gold oxide species identified by cyclic voltammetry may be relevant to the surface reactivity of AuNPs in biological environments; however, their potential role in photocatalytic processes was not directly assessed in the present study and remains a hypothesis for further investigation."

• Comment 7. Lines 356-360 – Why the exposure to green light led to the release of some citrate groups from the AuNP surface, which further determined the formation of dimmers?

Response 7. We tried to explain this picture of the citrate coating dynamics in more detail.

We wrote at the Discussion section:

"The underlying mechanism could be based on phenomena described in [19] regarding the photomodification of AuNPs irradiated at the plasmonic resonance wavelength. Upon LSPR excitation, the photonic energy absorbed by the nanoparticle can induce hotspot electron transfers to the surface, which can weaken the coordination bonds of the citrate carboxylate groups with the gold surface.

Thus, it could be said that exposure to green light led to the release of some citrate groups from the AuNP surface in the irradiated suspension, leaving uncovered spots on the particle surface that attract each other and lead to the formation of dimmers, and the DF microscopy results confirm this by the twofold higher intensity of the plasmonic profile (Figure 3a) compared to the unassociated monomers (Figure 3a).

The decrease in the mean diameter in TEM distribution as well as the increase in the spectral band intensity for GGL sample compared to GC one reflects the redistribution of the population of individual particles due to the continuation of nucleation under the action of light with generation of mostly small particles, together with some other particle dimmerization (evidenced by dark-field microscopy) and highlighted by DLS results.

The partial release of citrate from the AuNP surface under the action of light allows the particles to approach each other and form dimmers; free citrate in solution contributes additionally to the electrostatic stabilization of the collective system including already formed dimmers."

• Comment 8. Line 361 – 365 - During cyclic voltammetry, if a thicker oxide layer forms under anodic polarisation, a more noticeable reduction peak should appear during the reversible process.

Response 8.

Indeed, a thicker oxide layer should correspond to a cathodic (reduction) peak of higher amplitude. Analysis of the cyclic voltammograms presented in Figure 6 shows that the cathodic peak of the GGL sample does indeed exhibit a slight shift and shape change compared to the GC sample, although the amplitude difference is not larger. A possible explanation is that the irradiation-induced surface modification (rearrangement of the citrate layer, formation of a superficial Au₂O/Au(OH)₃ layer) also modifies the kinetics of the oxide reduction process, not only the layer thickness. In practice, a more hydrated or porous oxide layer could be reduced at slightly different potentials and with a different current distribution. We acknowledge that a more detailed analysis (e.g., scan rate dependence, integration of the reduction charge) would clarify this issue, and we mention it as a perspective.

We wrote by rephrasing and adding:

"Regarding the cyclic voltammetry results we need to mention that the oxide film which is formed in the positive scan is not readily reduced in the reverse scan as seen by the smaller reduction peak (Figure 6). The smaller reduction peak observed for GGL compared to GC suggests that the oxide layer formed on irradiated nanoparticles exhibits modified reduction kinetics — likely due to a more disordered or hydrated oxide structure — rather than simply a thicker layer. A systematic scan-rate analysis would be required to quantify the oxide charge and confirm this interpretation [21]. Figure 6b illustrates the typical cyclic voltammetry curves for different amount of GGL adsorbed on the electrode surface, and evaluated in sulfuric acid, but scanning the potential on a shorter range. Similar to Figure 6a, the two oxidation peaks are present, and the height of the peak increases with the amount of GGL. The position of peaks is slightly shifted to more positive value as expected for a larger amount of nanoparticles that require a higher potential for oxidation (regarded as the driving force for electronic transfer).

For the irradiated GGL sample, the anodic current increases steeply starting from approximately +0.85 V — a positive shift of ~80 mV relative to GC — suggesting that the oxidation of gold surface atoms requires a higher overpotential after irradiation, consistent with a modified surface layer induced by light exposure. At equal deposited volumes (3 µL), GGL shows reduced electrochemical surface area compared to GC, reflecting restructuring of the adsorbed citrate layer and partial surface oxidation. This does not imply lower biological activity, but rather a different electrochemical profile.

As for the cathodic peak, the less pronounced reduction signal observed for GGL compared to GC may reflect altered reduction kinetics of the surface oxide layer rather than a thinner oxide — a more hydrated or disordered oxide formed under irradiation conditions may be reduced at a slightly different potential and with a broader current distribution [24].A systematic scan-rate dependent analysis would be needed to quantify the oxide charge and confirm this interpretation, and is proposed as a direction for future work.

The chemisorbed gold oxide species identified by cyclic voltammetry may be relevant to the surface reactivity of AuNPs in biological environments; however, their potential role in photocatalytic processes was not directly assessed in the present study and remains a hypothesis for further investigation."

Many thanks for your very helpful analysis.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All my comments have been taken into account and the corresponding corrections have been incorporated into the text. The article in its current form is suitable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the Authors for their valuable contributions.
They have addressed the comments in great detail and revised the manuscript accordingly.