1. Introduction
Soil organic matter (SOM) constitutes one of the key indicators of soil
quality, due to its significant role in many interconnected chemical and
biochemical processes occurring in the soil. The transformation of SOM,
related to its decomposition and humification, depends on many
environmental factors that may modify the ongoing changes. Soils that
are considered to be the largest carbon pool contain more organic carbon
than is contained in the atmosphere and vegetation together (Minasny et
al 2013). Therefore, the stabilization of SOM may greatly affect carbon
capture and immobilization to efficiently limit global climate change
(Bhattacharyya et al., 2022; Liu et al., 2022; Pham and Katayama, 2018;
Spaccini et al., 2002). Due to the complicated matrix and heterogeneity
of SOM, the recognition of the chemical diversity of its individual
components is significant in the processes of controlling its
transformations (Kibblewhite et al. 2008, Rabot et al. 2018, Wiesmeier
et al. 2019).
Decades of research have shown that to recognize SOM’s role in the
environment, it is necessary to separate it into components with
different properties (Hayes et al. 2017, 2020). Although more than 230
years have passed since the first extraction of humic substances (HS) by
Achard, the nature, structure and fractionation of organic matter are
still the subject of scientific discussion, and today there is no
consensus on which approach should be applied (Gerke, 2018; Janzen,
2019; Olk et al., 2019a and 2019b; Schnitzer and Monreal, 2011; Weber et
al., 2018). Recently, several new concepts concerning SOM isolation and
fractionation have appeared, including physical separation methods
(Cambardella and Elliott, 1992; Lavallee et al., 2020) that base on size
and density. The last decade has brought more and more heated debate
after Lehmann and Kleber (2015) called into question several established
views and concepts regarding SOM, including the occurring and the
formation of HS. This concept is controversial and does not have the
approval of many SOM researchers (De Nobili, 2019; Hayes and Swift,
2020; Olk et al., 2019a, 2019b). Obviously, humic fractionation, like
any procedure, has limitations; however, any procedure can and does
provide useful results (Olk et al., 2019a; Zaccone et al., 2018).
According to the widely accepted proposal of Hayes and Swift (1978),
which is in line with the classical approach, “SOM is a heterogeneous
mixture of all organic components found in soil, which can be subdivided
into” (1) unaltered materials and (2) transformed products which
consist of both: (2a) HS and (2b) non-humic substances. According to the
International Humic Substances Society, HS “”are complex and
heterogeneous mixtures of polydispersed materials formed in soils,
sediments, and natural waters by biochemical and chemical reactions
during the decay and transformation of plant and microbial remains”
(https://humic-substances.org/what-are-humic-substances-2/). Some
researchers (Piccolo, 2002; Sutton and Sposito, 2005) are inclined to
the view that “humic substances are collections of diverse, relatively
low molecular mass components forming dynamic associations stabilized by
hydrophobic interactions and hydrogen bonds”. HS are divided into three
main groups: humic acids (HA), fulvic acids (FA), and humin fraction
(HM). The HA and FA can be extracted from the soil with a strong alkali,
then the HA is separated by adding a strong acid, which causes their
precipitation. The HM can HM can be extracted with neither a strong base
nor a strong acid (Aiken et al., 1985; Hayes et al., 2017; Rice and
MacCarthy, 1988), which is why some researchers do not include this
fraction in HS, while others do. Thus, HM research could be the ”bridge”
between more traditional and newer theories of SOM investigation.
The HM usually constitutes more than 50% of HS, but the resistant
chemical nature of this fraction as well as its lack of solubility
contribute to a significant difficulty in its investigating. Thus,
although it is a main component of SOM, the composition and properties
of the HM have rarely been studied. Since its structure is close to that
of HA, but slightly more aliphatic (Hatcher et al., 1985; Tan, 2014),
some authors claim that the HM is formed as a mixture of highly
condensed HA bonded to fine mineral soil particles (clay minerals),
melanin of fungal origin, paraffinic compounds (Stevenson, 1994), lignin
from plants or peptidoglycan structures from microbial cells (Simpson et
al., 2007). Hayes et al. (2017) indicated that the backbone of humin is
predominantly composed of “aliphatic hydrocarbon functionalities,
especially those found in lipids, waxes, cuticular materials, cutin,
cutan, suberin, and suberin”. However, the HM is strongly bonded
physically and chemically to HA and FA fractions associated with “a
disordered matrix entrapping a significant proportion of non-polar
compounds” (Almendros and González-Vila, 1987; Song et al., 2011).
Generally, the HM revealed a particle diameter smaller than the FA and
HA, and according Ukalska-Jaruga et al. (2021) “a more spherical shape
caused by the higher intermolecular forcing between the particles”. In
addition, the aromatic-aliphatic carbon core structure of the HM also
depends on its origin and soil depth (Tatzber et al., 2007). Simpson et
al. (2007) showed that all HM species “are present as macromolecules
(or stable aggregate species)” and belong to “five main categories of
components, namely, peptides, aliphatic species, carbohydrates,
peptidoglycan and lignin”. Song et al. (2011) isolated and fractionated
the HM with alkaline urea and acidified dimethyl sulfoxide (DMSO),
suggesting that the recalcitrant HM consists of molecules originating
“from plants and the microbial population, that are likely to be
protected from degradation by their hydrophobic moieties and by sorption
to the soil clays”.
Despite the difficulties in HM studies due to their insolubility, there
has been growing attention to the issues related to its genesis and role
in soil and carbon sequestration (Pham et al., 2021; Schnitzer and
Monreal, 2011, Tang et al., 2022; Zhang and Katayama, 2012). To assess
this, it is necessary to obtain as much data as possible on the
properties of the HM of soils derived from different parent materials
under diverse vegetation and climatic conditions. HM is most often
extracted from soil material by organic solvents, whereas in our work we
did not extract this fraction by dissolution but isolated it by
eliminating other organic and mineral components from the soil material.
As a consequence, investigated humin as a strong organo-mineral
complexes reflects the properties of this fraction that naturally
occurres in the soil environment. The main aim of the research was to
assess the molecular properties of the HM obtained from various arable
soils in the temperate climate zone, using spectroscopic methods
(13C CP MAS NMR, FTIR, and EPR), HPLC, SEM-EDX, and
elemental composition analyses.