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HUMIC SUBSTANCES
Natural organic matter (NOM) represents a complex heterogeneous pool of active organic carbon compounds (Hertkorn et al., 2007 ; Woods et al., 2011 ; Buffle, 1984, 1990 ; Amon and Benner, 1996); it consists of living organisms (plants, animals, microorganisms), their secreted matters, as well as decomposed residues (plant debris, bacteria…) through biological processes that include physical breakdown and biochemical transformation of molecules such as cellulose, fats, waxes, tannin, lignin, carbohydrate and proteins (Juma, 1999). They are group into volatile (VOC) and non-volatile (NVOC) organic matter (Atkins and Jones, 1998), the latter being divided into dissolved organic carbon (DOC) and particulate organic carbon (POC), based on filtration typically using a 0.45 or 0.22μm filter (Azam and Malfatti, 2007).
Depending on their chemical properties, they are separated into identifiable molecules that have been synthesized directly from plants or other living organisms and non-identifiable complex structures (Figure. Ι-1) that are not easily used by many micro-organisms as an energy source and persist in the environment for a relatively long time (Tipping, 2002 ; Jones and Bryan, 1998):
Non- Humic Substances: are identifiable compounds that belong to specific biochemical classes (Thurman, 1985 ; Christman and Gjessing, 1983 ; Perdue et al., 1990 ; Beck et al., 1993) where they can be represented by distinct chemical formula such as carbohydrates (mono-, Oligo- and polysaccharides), cellulose and hemicellulose, lipids and Amino Acids/proteins (Piccolo, 2001 ; McDonald et al., 2004). In general, the microorganisms rapidly degrade those compounds because of the simplicity of their chemical nature. As a consequence, they do not persist for a long period in the environment. Other chemicals such as resins, waxes, and lignin, due to their complex structures, are more difficult for microorganisms to break down.
Humic Substances (HS): are non-identifiable naturally occurring, yellow to black, biogenic organic substances of high molecular weight (Wall and Choppin, 2003 ; Lead et al., 1994 ; Redwood et al., 2005) known for being heterogeneous refractory (Gjessing, 1976 ; Aiken et al., 1985 ; Huang et al., 1986) since they resist the decomposition and breakdown (Sparks, 2003). They may persist in nature for long periods of time and are produced as byproducts of microbial metabolisms, physicochemical degradation of organic materials (plant debris, bacteria), and condensation of small organic molecules Humic Nano-Colloids-General Context that cannot be categorized to a definite chemical or distinctive structural group nor a unique functional term (do not carry any specific biochemical function) (Schnitzer, 1978 ; Tadros and Gregory, 2013 ; Rowell, 1994).
Definitions and Terminologies
The scarcity of an explicit definition for humic substances is a problematic issue in this field, where the different terminologies are not used in a compatible manner. Some soil scientists use the term humus synonymously with soil organic matter (SOM) that is composed of both the humic and non-humic substances without the undecayed or partially decayed animal and plant debris, as well as the soil living organisms (Stevenson, 1994). Concurrently, in soil science, this term (i.e. humus) is used to refer only to the humic substances within which various constituents such as polysaccharides and amino acids that are usually linked with the humic materials during extraction are removed (Tan, 2011, 2014 ; MacCarthy, 2001). However, the segregation of these constituents, or excluding the partially decayed products from the isolated humic substances, is practically tedious if not impossible as it is very challenging during the decomposition process to identify the level of humification (Knicker et al., 1997 ; Wershaw, 1994). In other terms we have a pool of humic substances, where part is completely formed, some is undergoing formation, and the remaining is still subjecting to breaking down and decomposition.
Despite the discrepancies in definitions, humic substances are considered as an amorphous mixture, composed of heterogeneous biochemical substances (proteins, carbohydrates, lipids…) degraded to a greater or lesser extent by microorganisms and physicochemical processes (Stevenson, 1994). This diversity results in a poorly understood structure where they lack a definite or regular structural form. These substances are highly refractory, so they can oppose microorganisms degradation and their breaking down is slower than that of other simpler organic components (e.g. biopolymers of polysaccharides, proteins) (MacCarthy, 2001).
Classification of Humic Substances
The complex nature of humic substances raises a conceptual paradox when classification is considered. The lack of a unique biochemical content makes it difficult to categorize them into functional or chemical terms as any other chemical compounds (e.g. proteins, lignin, etc.) (Xing, 1998 ; Steelink, 1963). Instead, grouping is based on their solubility behavior in aqueous media (pH-dependent) that is applied during their extraction process (Rosa et al., 2000 ; Moraes and Rezende, 2008). Humic substances are then classified into three major fractions: humic acid (HA), fulvic acid (FA) and humin.
Humic Acid
Humic acid corresponds to colloidal fraction of humic substances that is insoluble under acidic conditions (pH < 2), which implies that they can be found in aqueous system (Hayes and Clapp, 2001 ; Chilom and Rice, 2009 ; Thorn et al., 1996). They behave as a weak acid and represent the main extractable fraction of soil humic substances. Humic acids are dark brown to black, contain essentially aromatic and aliphatic structures (Chien and Bleam, 1998), that made up 35% and 65% of their molecular composition respectively (Pettit, 2004). That gives them active surface property, allowing the establishment of hydrophobic interactions (Chen and Schnitzer, 1978 ; Engebretson and von Wandruszka, 1994). In addition, other functional groups such as carboxylic acids and phenolic give them their hydrophilic property (Ritchie and Perdue, 2003). The surface active properties are influenced by the hydrophobic and hydrophilic structures, i.e. hydrophobic/hydrophilic ratio =HB/HI, (Quadri et al., 2008 ; Adani et al., 2010 ; Salati et al., 2011 ; Quagliotto et al., 2006).
The size of humic acids have been determined by various techniques, such as transmission electron microscopy (TEM), atomic force microscopy (AFM), small angle X-ray and neutron scattering (SAXS and SANS) (Balnois et al., 1999 ; Wilkinson et al., 1999 ; Kawahigashi et al., 1995 ; Pranzas et al., 2003 ; Osterberg et al., 1993). All of these methods suggest that humic acids are nanoscale structures with a diameter between 1 and 2 nm (Lyvén et al., 2003; Baalousha et al., 2005). They are ascribed for being a high molecular weight fraction, ranging from 10,000 to greater than 1,000,000 dalton (Da) for soil-extracted materials (Piccolo et al., 2002) and about 2000 to 3000 Da in the case of aquatic origin (Flaig and Beutelspacher, 1968 ; Malcolm, 1990 ; Aiken and Wershaw, 1985). This size can vary depending on the pH: at basic pH, they present an extended configuration due to electrostatic repulsions, whereas small aggregates begin to form with pH˂5 (Lead et al., 1999 ; Plaschke et al., 1999). In addition, humic acids are known to have a high cation exchange capacity and metal-chelating properties, which is crucial to understand their coagulation with cationic and inorganic minerals species (Pettit, 2004 ; El Samrani et al., 2004).
Fulvic Acid
Fulvic acids are the light yellow to yellow-brown fraction of humic substances, soluble in water under all pH conditions (Nam and Kim, 2002 ; Kim and Osako, 2004). They remain in solution when the humic acids are removed by acidification. In aquatic systems, they make up to 30-50% of the natural organic matter (Abbt-Braun and Frimmel, 2002 ; Reemtsma and These, 2005). Their size is smaller compared with humic acid, i.e. 1.5nm using conductimetry, 0.95nm using DLS and 0.1-2nm by AFM (Roger et al., 2010), with molecular weight ranging from 1500 to 2000 Da in streams and 1000 to 10,000 in soil-derived matter (Flaig and Beutelspacher, 1968 ; MacCarthy, 2001 ; Thurman et al., 1982 ; Plancque et al., 2001). They demonstrate colloidal properties due to their relatively small size (Tombácz et al., 1999 ; Fetsch et al., 1998), which in turn increases their availability to plants; fulvic acids are considered as mineral and trace elements chelating agent, making them very vital components for soil quality.
The high oxygen contents in the form of carboxyl (-COOH) and hydroxyl (-OH), in addition to ketonic and carbonyl groups, render them highly chemically active products, and enhance their solubility in the aquatic systems (Dixon et al., 1989 ; Lead et al., 2000a ; Hosse and Wilkinson, 2001). In addition, we can also find aliphatic chains and aromatic groups. These various constituents give rise to the amphiphilic nature, where analogy to surface-active agents can be anticipated (Leenheer et al., 2003), since they exhibit the capability of linking various hydrophobic and hydrophilic compounds (Schulten and Schnitzer, 1995 ; Pompe et al., 1996)
Humin
The fraction of humic substances that is not soluble in water at any pH value is defined as Humin (Rice, 2001 ; Rice and Maccarthy, 1989a); humin is what is left after humic and fulvic acids extraction (Schnitzer and Khan, 1972 ; MacCarthy et al., 1990). They are black in color, long regarded as macro-organic due to their high molecular weight approximately ranging from 100,000 to 10,000,000 Da (Rice and MacCarthy, 1988 ; Rice and MacCarthy, 1989b). Due to the resemblance in the nature of functional chemical groups and similar elemental compositions (Rice and MacCarthy, 1991), humin has been viewed as humic acid linked to inorganic matter as clay (Theng, 1979 ; Shah et al., 1975a ; Shah et al., 1975b ; Banerjee, 1979 ; Cloos et al., 1981). Humin exhibits high resistance to degradation than the other fractions of humic substances and play a significant role in the sequestration of anthropogenic organic chemicals (pesticides, herbicides…) (Rice, 2001).
The biogeochemical alterations and the fate of humin in the environment are not well known, since scientific researchers gave less interest toward such studies, although humin comprises more than 50% and 70% of organic carbon in soils (Kononova, 1966) and in sediments, respectively (Peters et al., 1981 ; Durand and Nicaise, 1980 ; Hatcher et al., 1985 ; Vanderbrouke et al., 1985; Ishiwatari, 1985 ; Hedges and Keil, 1995 ; Mayer, 1994). According to C-14 dating, humin is the oldest of humic substances that may eventually convert into coal or kerogen (Tissot and Welte, 1978). It has been regarded as an intermediate product in the transition of peat into coal (Fischer and Schrader, 1921 ; Funasaka and Yokokawa, 1953 ; Van Krevelen, 1963 ; Stach, 1975), also as a precursor of kerogen that may eventually transform to petroleum in sedimentary rocks (Breger, 1960 ; Welte, 1973 ; Cane, 1976 ; Huc and Durand, 1977 ; Vanderbrouke et al., 1985).
Comparison of Humic Substance Fractions (Humic Acid, Fulvic Acid and Humin)
The different fractions of humic substances are relatively similar but not completely identical, since they can be distinguished by their solubility in aqueous solutions. Even when the physico-chemical properties are investigated, analogy can be found in between them. Although the molecular weight, elemental compositions, degree of polymerization and the number and distribution of functional chemical groups can help to differentiate the fractions, no obvious boundary can be drawn, but rather a gradual evolution of properties should be considered when passing from one fraction of the other (Giannissis, 1987).
The higher oxygen content in fulvic acids is attributed to the fact that they are richer in acidic functional groups such as carboxylic acid, phenolic and ketonic groups, and their content in aromatic and aliphatic moieties is less than humic acids (Schulten and Schnitzer, 1995 ; Hosse and Wilkinson, 2001 ; Lead et al., 2000a) except for aquagenic fulvic acids with more aliphatic chains than humic acids (Table I-2) (Malcolm, 1990). This accounts for the solubility of fulvic acid at any pH value and the insolubility of humic acids (being more aliphatic and aromatic, and poorer in carboxylic and phenolic) at low pH where deionization and protonation of their carboxylic groups takes place to render them more hydrophobic (Pompe et al., 1996).
The amount of the various humic fractions varies noticeably from one type of soil to another. In forest soils, there is a higher content of fulvic than humic acids; whereas in peat and grassland soils, humic acids are considerably higher (Figure. Ι-3).
HUMIC SUBSTANCE GENESIS
Humic substances have been a subject of interest for centuries (Schnitzer and Khan, 1972 ; Frimmel et al., 1988). Their complex nature is consented by anyone who works with these substances, as mixture of plant residues, components of microorganism and their decomposition by-products, which make it very challenging for writing a unique molecular formula representing their structures. But these humic substances are kind of similar regardless of their origin because the biochemical processes on Earth are similar (Burdon, 2001). Two modes of formation of HS have been suggested (Hayes et al., 1989a ; Hayes and Swift, 1990):
Purely biological or Degradative concept: the breakdown and the transformation (basically lignin) of biological macromolecules under microbial (biotic) and chemical (abiotic) processes lead to humic substances that have related features to the components from which they were degraded (Hatcher and Spiker, 1988 ; Largeau et al., 1984).
Biological processes followed by chemical reactions (abiotic) or Synthetic concept: aggregation and condensation through polymerization of smaller organic compounds released from metabolism and molecular degradation (e.g. Proteins, polysaccharides and phenols) (Flaig et al., 1975 ; Flaig, 1988 ; Hedges, 1988).
The first pathway is based on oxidation and depolymerisation, whereas the second pathway leads to the production of novel molecules that will also be subjected to oxidative degradation. Considering abiotic processes alone for the formation of humic substances is not possible since plant components are not capable of reacting with themselves, which implies that a biological step should precede the route of formation. These two concepts comprise three different pathways (Figure. Ι-4); the lignin theory (degradative concept) (Waksman and Iyer, 1932 ; 1933), the polyphenol theory (Hänninen et al., 1987), and the sugar-amine theory also called the “Maillard Reaction” (synthesis concept) (Stevenson, 1994).
This theory considers lignin as the primary precursor of humic substances (Waksman, 1932) because of its high molecular weight (Goring, 1971) and its resistance to decomposition (Marcusson, 1926). Lignin is incompletely degraded by microorganisms and undergoes a series of modifications where it loses, by demethylation, methoxy groups (-OCH3) to produce ortho-hydroxyphenol and produces carboxyl groups (-COOH) through the oxidation of terminal ends of aliphatic chains (Figure. Ι-5) (Wershaw, 1993). The ortho-hydroxyphenol parts can produce quinones by further oxidation that is capable of reacting with amino compounds and -NH3 by-product of N-containing organic compounds through condensation (pathway 4 in Figure. Ι-4).
In addition to lignin, other plant residues have been considered as key-components in humification (Zech et al., 1997 ; Amalfitano et al., 1992). Carbohydrates (cellulose and hemicelluloses) (Detmer, 1871 ; Rose and Lisse, 1917), cutin (Zech et al., 1990 ; Lähdesmäki and Piispanen, 1988) or wax (Nip et al., 1986), have also been linked as potential precursors of humic substances (tannin are not taken into consideration since it is not found in all plants) (Haider et al., 1975 ; Senesi and Loffredo, 2001). Because these substances (Carbohydrates) are rapidly degraded, they cannot solely link as humic substances precursors, then interaction between these degraded matter and synthesis of new products might contribute to the formation of humic substances.
However, this pathway has been criticized because, if lignin was the only precursor, then the structure of humic substance would have been similar to that of lignin and we would have had enough knowledge of their composition. Furthermore, the presence of various moieties that cannot be generated from lignin such as phenolic groups, nitrogen, simple sugars and amino acids (Anderson et al., 1989), were found to be released from hydrolysis experiments of humic substances (Cheshire, 1979 ; Parsons, 1989). Thus, the humic substances produced from lignin degradation alone do not account for the existence of these groups. Therefore, the degradation process should be followed by synthesis reactions, where those compounds interact with the backbone and incorporate into the structure, as the lingo-protein model in which the amino groups of proteins interact with the carboxyl groups of the modified lignin that is responsible for the presence of these groups in humic material (Jensen, 1931 ; Hobson, 1925 ; Bennett, 1949).
The Poly-Phenol Theory (Biological Processes Followed by Chemical Reaction “Abiotic” or Synthetic Concept)
This theory considers Polyphenols as precursors of humic substances (pathway 2 and 3 in Figure. Ι-4). Here, both the decomposition of plant biopolymers (e.g. cellulose) and the microbial synthesis lead to polyphenol production through oxidation and demethylation. Moreover, lignin can be a source of polyphenols under microbial degradation to produce phenolic aldehydes and phenolic acids that are converted into polyphenols by enzymatic reactions (Saiz-Jimenez et al., 1975 ; Haider et al., 1965). The Polyphenols are then converted into quinones by Polyphenol-oxidase that will also react with N-containing organic compounds and polymerize into humic substances (Figure. Ι-6) (Flaig, 1964 ; Martin et al., 1980).
Table of contents :
CHAPTER I: Humic Nano-Colloids “General Context”
Ι.1 HUMIC SUBSTANCES
Ι.1-1 Definitions and Terminologies
Ι.1-2 Classification of Humic Substances
Ι.1-2-1 Humic Acid
Ι.1-2-2 Fulvic Acid
Ι.1-2-3 Humin
Ι.1-2-4 Comparison of Humic Substance Fractions (Humic Acid, Fulvic Acid and Humin)
Ι.2 HUMIC SUBSTANCE GENESIS
I.2-1 Lignin-Protein Theory (Biological or Degradative Concept)
I.2-2 The Poly-Phenol Theory (Biological Processes Followed by Chemical Reaction “Abiotic” or Synthetic Concept)
I.2-3 Sugar-Amine Theory “Maillard Reaction” (Biological Processes Followed by Chemical Reaction “Abiotic” or Synthetic Concept)
Ι.3 ISOLATION/EXTRACTION AND FRACTIONATION
I.3-1 Isolation
Ι.3-2 Drawback of Isolation Processes
Ι.4 CHARACTERIZATION: CHEMICAL AND PHYSICAL PROPERTIES
I.4-1 Elemental Analyses
I.4-2 Functional Group Composition
I.4-3 Optical Properties
I.4-3-1 UV/Vis Spectrophotometry
I.4-3-2 Fluorescence Spectroscopy
I.4-4 Molecular Conformation (Size and shape) and Molecular Weight
I.4-5 Structural Organization of Humic Substances
I.4-5-1 Humic Substances as Polymers (Macromolecule)
I.4-5-2 Humic Substances Membrane-Like Micelles
I.4-5-3 Humic Substances as Supramolecular Associations
I.5 MODELING REPRESENTATION OF HUMIC SUBSTANCE
Ι.6 ROLES OF HUMIC SUBSTANCES
I.7 SURFACTANTS
І.7-1 Definition and Classification
І.7-2 Applications
І.7-3 Interaction between Surfactants-Polymers and Surfactants-Humic Substances
CHAPTER II: Materials and Experimental Methods
ІІ.1 MATERIALS
ІІ.1-1 Humic Substances and Dissolved Organic Matter (DOM)
ІІ.1-2 Surfactants
ІІ.2 PREPARATION OF HS/SURFACTANT COMPLEXES
ІІ.3 METHODS OF CHARACTERIZATION
ІІ.3-1 Turbidity
ІІ.3-2 Dynamic Light Scattering
ІІ.3-3 Surface Tension
ІІ.3-4 Electrophoretic Mobility and Zeta Potential
ІІ.3-5 Cryogenic Transmission Electron Microscopy (Cryo-TEM)
ІІ.3-5-1 Sample Preparation for Cryo-TEM Observation
ІІ.3-5-2 Cryo-TEM Imaging Procedure
ІІ.3-6 Small Angle Neutron Scattering (SANS)
ІІ.3-7 Ultraviolet/Visible Spectrophotometer
ІІ.3-8 Fluorescence Spectroscopy
CHAPTER III: Spontaneous Vesicles in the Suwannee River Fulvic Acid/DTAC System: Implications for the Supramolecular Organization of Humic Substances
III.1 PREFACE
III.2 PROBING THE ORGANIZATION OF FULVIC ACID USING A
III.2-1 Abstract
III.2-2 Introduction
III.2-3 Experimental Section
III.2-3-1 Materials
III.2-3-2 Sample Preparation
III.2-3-3 Characterization of SRFA/DTAC Complexes
III.2-4 Results
III.2-4-1 Turbidity Measurements
III.2-4-2 Electrophoretic Mobility, DLS and Surface Tension Measurements.
III.2-4-3 Cryo-TEM Observations
III.2-5 Discussion
III.2-5-1 Self-assemblies of Fulvic Acid and DTAC
III.2-5-2 Towards Average Geometric Characteristics of SRFA Constituents
III.2-6 Conclusions
