Humic Substances: From Supramolecular Aggregation to Fractal Conformation—Is There Time for a New Paradigm?

Round 1

Reviewer 1 Report

The manuscript by Angelico and coworkers summarizes recent advances in characterization and understanding of the aggregation of humic substances into complex structural forms. To that purpose the Authors prepared a comprehensive review in which main models in the field and the corresponding experimental techniques along with their limitations were discussed. The variety of experimental approaches to determine structural properties of the humic aggregates was illustrated by the representative examples, including AFM, SEM and TEM images and ultrasonic velocity plots. Other experimental techniques were also described (e.g. spectroscopic measurements) and collected in a separate table with suitable references provided. The manuscript is clearly written and its conclusions are supported by the gathered research material. This review can be helpful in designing and controlling aggregation of humic substances to optimize structural and chemical properties. In summary , the manuscript can be accepted provided the following minor points have been addressed:

 

1) The Authors discuss fractal dimension of the humic aggregates without providing formal definition of this parameter. A short definition with some graphical simple illustration  would be helpful here.

2) Can the Authors comment on the possibility of formation of ordered assemblies by the considered humic substances? Have such structures ever been observed in reality?

3)  Are there any examples of the use of adsorption-based methods (e.g. BET isotherms) to determine structural properties of the humic aggregates (including specific are and porosity)?

4) In the second line of Abstract it should be “chemical bonds”.

Author Response

Reviewer 1

Comments and Suggestions for Authors

The manuscript by Angelico and coworkers summarizes recent advances in characterization and understanding of the aggregation of humic substances into complex structural forms. To that purpose the Authors prepared a comprehensive review in which main models in the field and the corresponding experimental techniques along with their limitations were discussed. The variety of experimental approaches to determine structural properties of the humic aggregates was illustrated by the representative examples, including AFM, SEM and TEM images and ultrasonic velocity plots. Other experimental techniques were also described (e.g. spectroscopic measurements) and collected in a separate table with suitable references provided. The manuscript is clearly written and its conclusions are supported by the gathered research material. This review can be helpful in designing and controlling aggregation of humic substances to optimize structural and chemical properties. 

Answer. We are very grateful to the referee for the positive comments which reward the hard work behind writing this review.

1) The Authors discuss fractal dimension of the humic aggregates without providing formal definition of this parameter. A short definition with some graphical simple illustration would be helpful here.

Answer. We are grateful to the referee for this helpful constructive observation and consequently a new text has been inserted at line 765, containing the formal definition of fractal dimension together with a new bibliographic reference. Moreover, Figure 6 has been modified to include a graphical correlation between the fractal dimension and the spatial organization of the clusters.

2) Can the Authors comment on the possibility of formation of ordered assemblies by the considered humic substances? Have such structures ever been observed in reality?

Answer. Unlike conventional linear polymer chains, which under certain conditions can generate ordered self-assembled structures such as, e.g., liquid crystals, the heterogeneous assemblies of HS could hardly give rise to ordered supramolecular aggregates, which indeed have never been observed at the best of our knowledge.

3) Are there any examples of the use of adsorption-based methods (e.g. BET isotherms) to determine structural properties of the humic aggregates (including specific are and porosity)?

Answer. The application of BET method to determine the specific area and porosity of humic substances is very poorly documented in literature. Some works report studies of BET isotherms conducted on humin extracts from Leonardite and carbonized at various temperatures to investigate their properties as green adsorbents (see, e.g. “Porous Carbon Adsorbent from Humin Derived from Thai Leonardite for Methylene Blue Dye Adsorption”, Curr. Appl. Sci. Tech. Vol. 19, 2019, DOI: 10.14456/cast.2019.1).

4) In the second line of Abstract it should be “chemical bonds”.

Answer. The typo has been corrected, thanks for reporting.

Reviewer 2 Report

Humic substances: from supramolecular aggregation to fractal 2 conformation. Is there
time for a new paradigm?
Authors: Ruggero Angelico , Claudio Colombo, Erika Di Iorio, Martin Brtnický, Jakub Fojt and Pellegrino Conte
The aim of the paper is to review the evolution of the models describing
physicochemical properties of humic substances, from hydrogel-like structure
comprised by a hydrophobic core of aromatic residues surrounded by polar and
amphiphilic molecules, to either mass or surface fractals.
The models are critically examined in terms of their ability to describe the structural
organization of humic substances. In this study, authors intend to understand how
much is known about humic substances in the light of environmental problems such as
global climatic changes.
Introduction includes definitions of particulate organic matter (POM), of natural
organic matter (NOM) and dissolved organic matter (DOM), their provenance and use
in the study of ecological processes. In this context, humic substances (HS) are
introduced as part of DOM, underlining that their chemical structure and their
production is a matter of discussion. Debates about chemical nature of HS are listed,
together with the corresponding reference.
In Section 2, authors overviewed the current models in literature, on the HS structure:
The Ghosh model of rigid sphero-colloids, the first hypothesis of molecular aggregation
of Wershaw, the micelle-like behavior of HS, the formation of aggregates in solutions of
HS. In parallel, the paper shows criticisms against micellar model, summarizing some
questions. The models based on fractal theory are also developed and described in
extenso: the concept of small heterogeneous molecules with self-organize
supramolecular aggregates with hierarchical
or the model of hydrophobic inner core stabilized by layers of amphilic and polar
molecules, metachemical hydrogels embedded within water dispersible porous
physical hydrogel scaffolds .
In Section 3, details on the recent scientific results of the modern experimental
techniques, on the HS structure were presented: extraction and fractionation procedure
of HS, solid-state investigations : SEM, TEM and AFM indicating supramolecular
arrangements, nuclear magnetic resonance techniques together to its limits in humic
chemistry and also, results showing that metal ions play a crucial role in aggregate
formation and stability. Other methods described: scattering techniques : dynamic light
scattering which permits the study of the fractal dynamics of HA colloids and
aggregation kinetics, light/X-Ray /neutron scattering techniques which lead to fractal
dimension determination of humic substances, generally found between 2 and 3, mass
or surface fractals, ultrasonic velocimetry.
Section 4 is dedicated to the future challenges and conclusions. Table 1 is a very
interesting way to show in a structured way the most scientific reports cited in the
review and the corresponding models.
Authors shows that there are still questions without answer, related to HS and its
influence on surrounded water molecules; answering these questions help to
understand the role of HS in biogeochemical cycles and include HS and their dynamics
in carbon sequestration strategies.
Also, the authors showed that fractal formalism lead to several open questions about
the genesis, structural nature, surface reactivity, and aggregation-dispersion
phenomena of HS in the environment.
The paper is in extenso documented and explained; the article is an important tool for
deepening the study of humic substances.
The paper is very interesting and offers access to an extremely vast scientific chapter,
structuring a lot of information, offering results, critical point of view and indicating
also scientific directions to study in the future, open questions that must be
investigated, the importance of the domain of HS related to carbon sequestration and
environmental problems.
Some minor issues:
Figures 3 and 4: I did not understand if there are authors contributions or there is a link
to a reference?
Some type-errors must be consider (for example, row 805 it is D2O not D20)
For ease of reading, because the material is extremely dense, a few-more sub-chapters
would be desirable.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Comments and Suggestions for Authors

The aim of the paper is to review the evolution of the models describing physicochemical properties of humic substances, from hydrogel-like structure comprised by a hydrophobic core of aromatic residues surrounded by polar and amphiphilic molecules, to either mass or surface fractals. The models are critically examined in terms of their ability to describe the structural organization of humic substances. In this study, authors intend to understand how much is known about humic substances in the light of environmental problems such as global climatic changes. Introduction includes definitions of particulate organic matter (POM), of natural organic matter (NOM) and dissolved organic matter (DOM), their provenance and use in the study of ecological processes. In this context, humic substances (HS) are introduced as part of DOM, underlining that their chemical structure and their production is a matter of discussion. Debates about chemical nature of HS are listed, together with the corresponding reference. In Section 2, authors overviewed the current models in literature, on the HS structure: The Ghosh model of rigid sphero-colloids, the first hypothesis of molecular aggregation of Wershaw, the micelle-like behavior of HS, the formation of aggregates in solutions of HS. In parallel, the paper shows criticisms against micellar model, summarizing some questions. The models based on fractal theory are also developed and described in extenso: the concept of small heterogeneous molecules with self-organize supramolecular aggregates with hierarchical or the model of hydrophobic inner core stabilized by layers of amphilic and polar molecules, metachemical hydrogels embedded within water dispersible porous physical hydrogel scaffolds. In Section 3, details on the recent scientific results of the modern experimental techniques, on the HS structure were presented: extraction and fractionation procedure of HS, solid-state investigations: SEM, TEM and AFM indicating supramolecular arrangements, nuclear magnetic resonance techniques together to its limits in humic chemistry and also, results showing that metal ions play a crucial role in aggregate formation and stability. Other methods described: scattering techniques: dynamic light scattering which permits the study of the fractal dynamics of HA colloids and aggregation kinetics, light/X-Ray /neutron scattering techniques which lead to fractal dimension determination of humic substances, generally found between 2 and 3, mass or surface fractals, ultrasonic velocimetry. Section 4 is dedicated to the future challenges and conclusions. Table 1 is a very interesting way to show in a structured way the most scientific reports cited in the review and the corresponding models. Authors shows that there are still questions without answer, related to HS and its influence on surrounded water molecules; answering these questions help to understand the role of HS in biogeochemical cycles and include HS and their dynamics in carbon sequestration strategies. Also, the authors showed that fractal formalism lead to several open questions about the genesis, structural nature, surface reactivity, and aggregation-dispersion phenomena of HS in the environment. The paper is in extenso documented and explained; the article is an important tool for deepening the study of humic substances. The paper is very interesting and offers access to an extremely vast scientific chapter, structuring a lot of information, offering results, critical point of view and indicating also scientific directions to study in the future, open questions that must be investigated, the importance of the domain of HS related to carbon sequestration and environmental problems.

Answer. We greatly appreciate the positive comments of the reviewer who rightly underlined the importance of this review in the context of a complex research field such as the study of humic substances.

Some minor issues: Figures 3 and 4: I did not understand if there are authors contributions or there is a link to a reference?

Answer. Yes, they are new AFM figures provided by one of the coauthors (C. Colombo).

Some type-errors must be consider (for example, row 805 it is D2O not D20)

Answer. The mentioned typo, as well as several others, have been corrected.

For ease of reading, because the material is extremely dense, a few-more sub-chapters would be desirable.

Answer. According to the referee's advice, we have added two new sub-paragraphs: 2.1 “The micellar model” and 2.2 “The supramolecular aggregate model”.