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Journal of J. Appl. Biomed.

APPLIED 3: 15·24, 2006


BIOMEDICINE ISSN 1214.-
0287

REVIEW

Humic substances which elements still unknown structure:


Product's application in agriculture and industry.

I 2 ,
Eladia M. Pen a-Mendez , Josef Havel, Jifi Patocka
I
Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, University of La
Laguna, Spain
2
,Department of Analytical Chemistry, Faculty of Science, Masaryk University, Bmo, Czech Republic
Department of Toxicolog y, Military Medical Academy, Bradec Knilove and Department of Radiology,
Faculty of Health and Social Studies, University of South Bohemia, Ceske Budejovice, Czech Republic

th
Received 20 September 2004.
th
Revised 20 October 2004. Published

Summary
Humic substances as part of humus -soil organic matter - are compounds arising from the physical,
chemical and microbiological transformation (humification) of biomolecules. They are important
because they constitute the most ubiquitous source of non -living organic material that nature knows.
Approximately 80% of the total carbon in terrestrial media and 60% of the carbon dissolved in aquatic
media are made up of humic substances. Humic substances have important roles in soil fertility, and are
considered to have primal relevance for the stabilization of soil aggregates. They can be divided into
three components according to their solubility: humic acids, fulvic acids and humin. Humic acids are the
most explored group of humic substances. Beyond their relev ance for life these substances have
industrial applications in the development of absorbents to be used at the sources of metal -poisoning.
Being natural substances, their purification process is cheaper than the synthesis of any other sorbent
and, moreover, due to their high operability, they absorb more than the absorbents used to date, such as
active charcoals or clays. The specific properties of humic acid products enable their application in
industry, agriculture, environmental and biomedicine.

Keywords: humification - humin - humic acids - fulvic acids - fugavic acids

INTRODUCTION
e Eladia M. Pena-Mendez, Department of Humic substances (HS) are the most widely -spread
Analytical Chemistry, Nutrition and Food Science, natural complexing ligands occurring in nature. The
Faculty of Chemistry, University of La Laguna, presence of HS in soils have also been detected, even
38071-La Laguna, Spain in the Antarctic continent where the humification
process under Antarctic conditions is very specific
and different from the other
Peila-Mendez et at.: Peat substances

continents (Gajdolova et al. 2001, Pacheco and Havel WHERE DO HUMIC SUBSTANCES COME
2002, Gajdolova et al. 2003). They make up the bulk FROM?
of organic matter, because they represent most of the
organic materials of soil, peat, lignites, brown coals, Although the formation process of HS has been
sewage, natural waters and their sediments. Humic studied hard and for a long time, their formation is
substances can be divided into three components: still the subject of long-standing and continued
group of fulvic acids such as fulvic acid (F As) and research. Some theories have lasted for years; for
fugavic acid (FgAs) , humic acids (HAs) and humin. example, the "sugar-amine condensation' theory, the
One of the most important parts of HS is HAs. Humic "lignin' theory or the "polyphenol' theory. A review of
acids and F As represent alkali-soluble humus such theories can be found in a monograph of Davies
fragments, humin represents the insoluble residue. and Ghabbour 1999. Nowadays, most investigators
Because of their molecular structure, they provide suppose that humic substances originated in lignin
numerous benefits to crop production. They help break (Oglesby et al. 1967).
up clay and compacted soils, assist in transferring
micronutrients from the soil to the plant, enhance Polyphenols come mostly from lignin during its
water retention, increase seed germination rates and biodegradation, and probably play a key role in the
penetration, and stimulate the development of formation process. Polyphenols are also regarded as
microflora populations in soils (Senesi et al. 1991). the main agents in the formation of humic substances
The remarkable properties of humic acids have from some plants that do not contain much lignin
attracted the attention of many investigators. The and/or from non-lignin containing plants. Polyphenols
results over the years have brought new knowledge on can be considered as humic acid precursors. They
their structure and physicochemical properties and themselves possess enough reactive sites to permit
pointed to the use of these interesting natural further transformations, for example some
compounds in many practical applications. The aim of condensation reactions.
this article is to review the current state of knowledge
and to present current applications in agriculture, The humic substances system is created. by the
industry, the environment, and biomedicine (see, e.g. association of various components present in the
Ziechmann 1994). humification process, such as amino acids, lignins,
pectins or carbohydrates, through intermolecular
forces (donor-acceptor, ionic, hydrophilic, and
hydrophobic) It is evident that the mechanisms of the
HISTORY formation of humic substances can be slightly
different, depending on geographical, climatic,
physical and biological circumstances, respectively.
The term "humus" originates from the Romans, when These compounds can be formed in several ways, and
it was familiarly used to signify the entire soil. Later the role of lignin is important in the majority of these
the term was used to denominate soil organic matter processes (Burdon 200 I, Davies et al. 200 I). Burdon
and compost or for different parts of this organic (200 I) proposed that humic organic matter consists
matter, as well as for complexes created by chemical mainly of a mixture of plant and microbial
agent treatments to a wide palette of organic constituents plus the same constituents in various
substances. The principal defmition of humus, as stages of decomposition (i.e. plant/microbial mixtures
decomposed organic matter, originates from 1761 of carbohydrates, proteins, lipids and partially
(Stevenson 1982). The first relevant study of the origin degraded lignins, tannins, melanins, etc.).
and chemical nature of HS was worked out by
Sprengel (1839). His comprehensive study on the
acidic nature of HAs is thought to be his most
important benefit to humus chemistry. Research on the CHARACTERIZATION OF HUMIC SUBSTANCES
chemical properties of HS was extended by the
Swedish researcher Berzelius, whose main
contribution was the isolation of two light-
yellowcoloured HS from mineral water and slimy mud As already pointed out, all the humic substances can
rich in iron oxides (Berzelius 1839). be divided into components according to their
solubility in different media (Fig. I). Humic ecids and
Enormous advances have been made during the last F As group represent alkali-soluble humus fragments;
decade thanks to modem physicochemical methods. HAs are commonly extracted using diluted alkali and
Nevertheless, the structural chemistry of lignin and HS precipitated with an acid, and so are separated from
did not advance so fast as the chemistry of animal- the soluble FAs group and then acidfication solution
originated biopolymers. to separate F As and FgAs . Humin represents the
insoluble residue (Thorn 1996). From a geological
point of view, humic substances are chemical
intermediates between plants and fossils. The
chemical nature of soils,

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Pena-Mendez et a/ : Humic substances

sludge and sediments can subsequently, continually (VPO) (Marinsky et al. 1990), ultrafiltration (Aiken
and selectively vary via the conversion and and Malcom (I 987), conductometry and
degradation of organic matter (Ziechman 1993). spectrophotometry in combination with factor
Extinct vegetation represents the main source of analysis (pefla-Mendez et al. 2004). HAs consist of
organic matter transforming to humic substances in a mixture of molecules with much lower molecular
the environment. Humification is a continuous weight than proposed before in the literature. HAs
historical process, and soil humus is a dynamic components are low molecular weight compounds
system of both chemically active lPld passive but they aggregate step by step to give higher
components (Gonzalez et al. 2003). The amount of molecular weight aggregates and supramolecules of
organic carbon in the Earth in the form of humic higher molecular weight. Hosse and Wilkinson (200
substances exceeds that which makes up living I), using fluorescence correlation spectroscopy
organisms (Engel and Macko 1993). observed that the aggregation of HAs is due to the
Humic acids, one of the most important formation of dimers and trimers at low pH.
components of HS, help break up clay and The characterization of complex mixtures of F
compacted soils, assist in transferring As and HAs is considered to be one of the most
micronutrients from soil to plants, enhance water important items in HS research nowadays. Thanks
retention, increase seed germination rates, and to the development of analytical techniques and
stimulate the development of microflora populations computer technologies, great efforts have been
in soils. Humic acids also slow down water made to elucidate the molecular structures of F As
evaporation from soils. This is especially important and HAs. From the work of Stevenson (1982),
in soils where clay is present at low concentration or Burne et al. (1977) to more recent models of
not at all, in arid areas, and in sandy soils without Shulten (2002, 2003), Kujawinski et al. (2002a,
the capability to hold water. Humic acids provide 2002b) and Stenson et al. (2002, 2003), several
also sites for microflora to colonize. Bacteria secrete molecular structures describing the structure of
enzymes which act as catalysts, liberating calcium humic acids have been proposed. For a long time, it
and phosphorous from insoluble calcium phosphate, has been suggested using different analytical
and iron and phosphorous from insoluble iron techniques that HAs are high molecular weight
phosphate. The chemical structure of HAs is very compounds. However, five years ago, we proved by
complicated and depends on their source. CE that HAs are low molecular weight compounds.
The elemental composition of different F As and This was announced for the frrst time at the 9 th
HAs shows that the major elements in their International Meeting of the IHSS, Adelaide
composition are C, H, 0, N, and S. These major Australia, September 20-25, 1998, IHSS, Atlanta
elements are always present regardless their origin (Havel et al. 2001) and in a subsequent publication.
and country or continent (Gajdo~ova et al. 200 I, Mass spectra of HAs (Fig. 4) obtained by LDI- TOF
Tan 2003, Kurkova et al. 2004). Moreover, besides MS shows the presence of low molecular weight
elemental composition, group composition is used molecules from low m/z values to higher. The
to characterize HS as it gives information about the analysis of the mass spectra obtained for different
chemistry and structural properties of HS (Purdue HAs showed that several m/z values are the 'same
1988, Tan et al. 2000). Fulvic acids contain more for all the HAs, suggesting that some compounds
functional groups of an acidic nature, particulary - are the same and that they are present in HAs from
eOOH. The total acidities of fulvic acids (900- very different origins and sources. The latest results
1400mmol/100g) are considerably higher than for from mass spectrometry (MS) combined with other
humic acids (400-87Ommol/100g). Another analytical techniques have confirmed our previous
important difference is that while the oxygen in results. As for fulvic acids, several thousand
fulvic acids is largely in known functional groups (- compounds have been identified, and are
eOOH, -0H, -e=0), with a high oxygen content, the co'nsidered to be mostly derived from lignin
acidity and degree of polymerisation all change (Kujawinski et al. 2002a, Stenson et al. 2003).
systematically with increasing molecular weight. The chemical formulas of individual fulvic acids
The proportion of oxygen in humic acids seems to have been determined (Stenson et al. 2003). In the
occur as a structural component of the nucleus. case of HAs, interesting results have been achieved
The formation of aggregates within solutions of recently applying Electrospray Ionization (ESI)
humic acids was studied by capillary electrophoresis (Kujawinski et al. 2002a, Brown and Rice 2002,
for the frrst time by Fetsch et al. (l998a, I 998b). Kujawinski et al. 2002b, Stenson et al. 2002,
Humic acids (HAs) aggregation has also been Stenson et al. 2003) coupled with Fourier Transform
studied in aqueous solution by high performance ICR mass spectrometry (FT-ICR) and Laser
size exclusion chromatography (peuravuori and Desorption/Ionization time of flight (LDITOF)
Pihlaja 1997), light scattering (Manning et al. 2000), (Pokorna et al. 1999, Havel et al. 1999, Gajdo~ova
vapor pressure osmometry et al. 2000, Gajdo~ova et al. 2003). Based on the
latest experimental results obtained by LDI- rOF
MS and also on the base of the

15
I'

.I /

Pena-Mendez et al . Humic substances

isotopic patterns observed for compounds present in 2002a, Brown and Rice 2002, Kujawinski et a!.
HAs (Fig. 4), Pacheco and Havel (2004) have 2002b, Stenson et a!. 2002, Stenson et a!. 2003).
suggested the latest empirical fonnula for HAs; the
results from their model are in a good agreement
with those obtained by ESI (Kujawinski et a!.

01!gtinic compounds of
soil

Living Soil organic


organismus matter
Alkaline precipitation
(pH=1<J.12)

Alkali-soluble Insoluble
humus fragment residue HUMIN
Acidic precipitation
(pH=6)
Insoluble residue Soluble group
HUMIC ACIDS FULVIC ACIDS

Fig. I. Scheme of division of humic substances in dependence of their solubility.

APPLICATIONS OF HUMIC SUBSTANCES important for soil fertility (Lotosh 1991, Zhang and
He 2004).
Humus represents one of the greatest carbon reservoirs Currently, humic materials are used as additives
on Earth. So far, industrial applications of humus and in fertilizers (Garcia et aI. 1994, Madejon et al. 200 I,
humus-derived products are rare. On the contrary, the A Ibiach et al. 200 I, Kerek et al. 2003, Arancon et al
usage of coal was more abundant and essentially, it 2004). Different salts of humic s ubstances, such as
constituted the basis of the chemical industry in the calcium humate, were used to increase soil fertility
second half of the 19th century and the fIrSt half of (e.g. Buckau et aI. 2000). The fertilizing effect of
the 20th century. Petroleum was also an application sodium, humate on plant leaves has been described.
and it was regarded as the main raw mate rial for the Ammonium humate was also found to have a
significant growth-stimulating effect (Lotosh 1991).
chemical industry of the 20 lb century. Nowadays,
The characteristics of and applications for humic
applications of HS can be divided into four main acids extracted from different compost have also
categories: agriculture, industry, environment and been studied (Ceppi et al. 1999, Madejon et al. 2001,
biomedicine. Arancon et aI2004).
The growth-promoting effect of humic substances
has been observed by many investigators and
Agriculture applications humates are often part of different preparations for
growth-improvement of plants. Productivity of soil is
HS play an important role from the agronomical increased by different ways in the presence of humic
point of view. They influence significantly the materials. The indirect effects of humic substances
quality and productivity of the soil. In addition to the are very important as they integrate iron to the
improvement of the soil's physical properties and chelates and make it available to plants. Another role
moisture conditions mentioned above, HS also show of humic substances lies in the enhancement of the
a high base exchange capacity, which is quality of

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Pena-Mendez et al.: Humic substances

soils when they are very poor in organic matter. Furthermore, humic materials have found
Recent research shows that humic acid can be used application in the production of plastics, especially
as fann animal feed thanks to its growth-promoting as dyes for coloring Nylon 6 or PVC plastics,
effect (e.g. Kocabagli et al. 2002). hardeners of polyurethane foams or as plasticizer
ingredients for PVC (Majakova and Proskurjakov
Industrial applications 1972).
Humic materials found numerous applications in
Humus and humus-containing materials have been the paper industry too. They are included in
used in large-scale building, for instance, as different manufacturing procedures, for example in
additives to control the setting rate of concrete. the production of electricity conducting paper sheets
Humic materials found use also in the preparation or in the manufacture of high tensile strength paper,
of leather. Initially, they were used as a leather dye, and also in the recycling of paper. Other industrial
later on as an agent for tanning leather and, fmallY' applications can be mentioned: as an ion exchanger,
as an ingredient of a solution to fmish leather. as a source of synthetic hydrocarbons and fuel oils
The woodworking industry is another field (Duncan et al. 1981), in foodprocessing or to
where HS have been applied. They were used to enhance the extraction of uranium from its ores
prepare a "natural indigo" to dye wood veneer. In (Schmeide et al. 2000). Humic substances have a
addition to this use, humic materials appeared to be large capacity to retain transition metals, forming
suitable agents as a component of water-soluble metalorganic complexes, which cause these metals
stains for wood furniture. to be more or less available for plants which include
In the ceramic industry, humic substances were then them into the food chain.
employed mainly as additives to enhance the Interesting possibilities are the manufacture of
mechanical strength of unprocessed ceramics, to humic substances from the waste processing
improve the casting properties of ceramics industry. Results showed that the conversion of
(Waksman 1938), to color clay tiles and among cellulose into humic acids is clearly underway in the
many other uses they were also applied in the production of recycled liquid packaging board
preparation of earthenware. (LPB) bales (Koivula and Hanninen 1999)

HC=O

(sugar)
I
(HG-OH) 4

I
HC=O OH
I
H 0

R
i"
! 0
COOH

COOH
o
HO HNy
R HN~O
. /0 OH

..,
V
COOH H
(peptide)

Fig. 2. Model structure of humic acid according to Stevenson (1982); R can be alkyl, aryl or aralkyl.

ENVIRONMENTAL APPLICATIONS humic substances in natural waters can influence


the uptake of radionuclides by natural solids and
Natural organic colloids (humic and fulvic acids) thus their migration to surface and ground waters
are important because they form water-soluble (Bondietti 1982, Samanidou et al. 1991). The main
complexes with many metals including task of humic substances in environmental
radionuclides (Lubal et al. 1998, Lubal et al. 2000, chemistry is to remove toxic metals, anthropogenic
Pacheco and Havel 2001, Ghabbour et al. 2001). organic chemicals and other pollutants from water.
These organics may therefore be important as lon-exchange materials based on calcium humate
radionuclide transport agents through the were found suitable for the removal of such heavy
environment. It is known that the presence of metals as iron, nickel, mercury, cadmium and

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Pena-Mendez et a/.: Humic substances

copper from water and also to remove radioactive for sewage purification, with many applications. The
elements from water discharges from nuclear power filters are useful to clean chromate smelter
plants. Their selective binding capabilities are also wastewater, to remove oil and dyes from wastewaters
exploited for the destruction of munitions and and aquatic systems (Versraete and Devliegher 1997),
chemical warfare agents (Ghabbour and Davies to filter urban and industrial
(999). Humus-based filters have been developed

Haac
Haac

a
Fig. 3. Model structure group of fulvlc acid according to Buffle et a!. (1977)

wastewaters, to remove pesticides from sewage and cumulative production of acetate during this process
to remove phenol from water. seems to be energetically advantageous for fermenting
Humus-containing materials have been also bacteria (Benz et al. 1998). Utilization of humics as
utilized for sorbing gases, e.g. the removal of waste the energy supply for specific bacteria, fungi and
gases from an animal-carcass rendering plant. Slightly higher microorganisms was studied by many
modified humates can be applied to remove hydrogen investigators (Bhardwaj and Gaur 1971) but it was
sulfide and mercaptans from municipal gas supplies, noted that they can not exploit humic materials as a
and sulfur dioxide from stack gases (Green and food source.
Manahan (981).
Different groups of compounds such as
herbicides, fungicides, insecticides, nematicides,
BIOMEDICAL APPLICATIONS
dioxins and also some pharmaceutical products like
estrogenic compounds were determined as possible
Humic substances produced on a commercial scale
environmental endocrine disruptors. Thanks to their are used in veterinary and human medicine. Several
ability to adsorb organic pollutants from the studies of the medicinal properties of humic materials
environment, humic substances were found to be have been reported (Mund-Hoym 1981, Brzozowski
useful to remove those contaminants from water, soil et al. 1994). It was found that humic acids
and sewage sludges (Shin et al. 1999, Lofredo et al. administered prophylactically to rats decreased
2000). The complex nature of the interaction between significantly the extension of gastric damage induced
HS and xenobiotics and their influence in the by ethanol. TPP administered to rats with
environmental quality (water, soil, and atmosphere) experimental gastric and duodenal ulcers significantly
has been studied by different authors. The study of the accelerated the healing process (Brzozowski et al.
acida-basic and complexation properties of HAs with 1994)
several inorganic and organic compounds has Pflug and Ziechrnan (1982) reported that humic
attracted increased attention due to their influence on acids are able to interact with the bacterium
many aspects of soil and water quality, and industrial Micrococcus luteus. In this case humic materials
processes. Pacheco et al. (2003) found that some protected the organism against cell-wall disruption by
inorganic and/or organic pollutants were strongly the enzyme lysozyme. Thiel et al. (1981), who found
complexed (bound) only with some of the HAs that preincubation of cell cultures with ammonium
components. As a consequence of such interaction humate avoided infection by the herpes virus, noted
quite stable entities of the supramolecular kind were the function of humic substances as protectors of the
formed. organism.
The interactions between humic materials and In the last decade there has been an increasing
microorganisms have been intensively studied for the interest in the employment of humic materials in
past 30 years. It was found that fermenting bacteria medicine and biology. The possibility of soil humus
could reduce humic substances. This fact has extract with amino acid complexes and vitamin B
significant implications for the autecology of analogues being a candidate as a base of
anaerobic bacteria in soils and sediments. The
-----------------------------------------------------------------
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Pena-Mendez et al.: Humic substances

cosmetic and pharmaceutical products has been complexes with heavy metals (such as cadmium)
studied. The main reason for the increasing enable them to be used for the elimination of heavy
attention devoted to humic acids can be explain by metals from living organisms (KIOcking 1992).
their antiviral, profibrinolytic, anti-inflammatory Humic materials in aquatic systems and water
and estrogenic activities (Yamada et al. 1998). The sediments have been observed to be closely
potential of humic substances to form chelate connected with efficacy of hydrotherapy and
129.0 360.2
100

90

557.4
111D
8
0 827.4
7 429.1
0
6 413.2
sa;
0
.0
301.2
.-.E
~ 50 1050
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ MalllCharge
4
0 1 5.0 36 .2
3
0
2
0 17
827.4
1
0
0
100 200 300 400 500 70 80 90 100
600 0 0 0 0
Mass/Charge
Fig. 4. LDI TOF MS mass spectra ofDAs soil (IHSS).

balneotherapy (Gadzhieva et al. 1991, Hampl et al. On the other hand, humic acid has been shown
1994). to be a toxic factor for many mammalian cells, but
Antibacterial (Ansorg 1978, Skliar et al. 1998) the specific mechanism of its cytotoxicity remains
and antiviral (KIOcking and Sprossig 1972, Thiel et unclear. Its redox properties make humic acid
al. 1977 1981, Schiller et al. 1979, Klicking 1991, capable of reducing iron(III) to iron(II) in aqueous
KIOcking et al. 2002) properties of humic conditions over a broad range of pH values (from
substances represent new possibilities for their 4.0 to 9.0) and of reducing and releasing iron from
medical application. ferritin, but this process is partially inhibited by
Of great interest is that hospital studies show superoxide scavengers. Subsequently, the iron
that difficult viral respiratory illnesses common in released from ferritin has been shown to accelerate
children are readily resolved with fulvic acid the humic acid-induced lipid peroxidation. Humic
dietary supplementation. Fulvic acid is a humic acids have the ability to reduce and release iron
extract common to rich organic humus soil and also from ferritin storage as well as to promote lipid
certain ancient plant deposits. Many medical studies peroxidation. Therefore, HAs coupled with released
show that humic substances, especially fulvic acids, iron can disturb the redox balance and elicit
have the power to protect against cancer and related oxidative stress within a biological system. This
cancer-causing viruses. Studies often show reversal may be one of the most important mechanisms for
of deadly cancers and tumors using special humic HA-induced cytotoxicity (Ho et al. 2003).
substance therapies (Schneider et al. 1996, van Humus represents one of the greatest carbon
Rensburg et al. 2002, Joone et al. 2003). reservoirs on the Earth. Although industrial
applications of humus and humus-derived products

1
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Pena-Mendez et al.: Humic substances

were a relatively low scale, usage of coal was more REFERENCES


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