Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue7-8.2000:
Organic Geochemistry 31 (2000) 679±695
www.elsevier.nl/locate/orggeochem
Encapsulation of protein in humic acid from a histosol
as an explanation for the occurrence of organic nitrogen
in soil and sediment
Xu Zang, Jasper D.H. van Heemst, Karl J. Dria, Patrick G. Hatcher *
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA
Abstract
Recent work suggests that nitrogen in humic acids exists primarily as amide functional groups that mirror those in
protein. However, the mode for the existence of such labile materials as protein still remains unclear. With the combined applications of NMR spectroscopy, tetramethylammonium hydroxide (TMAH) thermochemolysis, pyrolysis/
GC/MS, and elemental analysis, we propose that the survival of proteins in humic acids is carried out by encapsulation
into hydrophobic domains of humic acids. To test this hypothesis, we simulated encapsulation of 15N-labeled protein
extracts into humic acids and demonstrated that complete hydrolysis of the protein is prevented by the encapsulating
humic acid. Results from this study constitute evidence to support the encapsulation mechanism involved in the formation of refractory organic nitrogen during sediment diagenesis. # 2000 Published by Elsevier Science Ltd. All rights
reserved.
Keywords: Refractory organic nitrogen; Protein survivability; Encapsulation; Humic acid; TMAH GC/MS; Pyrolysis GC/MS; Solidstate NMR
1. Introduction
The profound in¯uence of humic materials on the
distribution, bioavailability, and ultimate fate of sedimentary organic nitrogen in various environmental
sediments has been recognized in the past decade (Barancikova et al., 1997; Schulten and Schnitzer, 1997;
Lichtfouse et al., 1998; Stankiewicz and van Bergen,
1998). Sedimentary organic nitrogen (SON) refers to the
sum-total of all nitrogen-containing substances in sediments. SON provides most of the nitrogen necessary for
the terrestrial bioproductivity. It primarily results from
the microbial decay of proteins and peptides in bioorganic residues and is part of the nitrogen cycle in the
biosphere. During early diagenesis, most of the labile
nitrogen-containing materials, such as peptide, protein,
and chitin, are quickly degraded and mineralized by
microbial and/or enzymatic degradation (Codispoti and
Christensen, 1985; Blackburn and Sorensen, 1988; Endo
et al., 1995; Nguyen and Harvey, 1998; Walton, 1998).
* Corresponding author.
However, recent studies have shown that part of these
labile nitrogen-containing materials are incorporated
into refractory organic components in humic substances, where they are protected from biodegradation
(Hedges and Keil, 1995; Henrichs, 1995; Knicker and
Hatcher, 1997; Nguyen and Harvey, 1998; Knicker et
al., 1999). This incorporated organic nitrogen has been
removed from the active nitrogen pool of the nitrogen
cycle and is, therefore, no longer available for biological
production (Knicker and Hatcher, 1997). Estimates of
refractory organic nitrogen in humic substances range
from 30 to 50% of total nitrogen (Schulten and Schnitzer, 1997). It is essential to understand the mechanisms
and processes involved in the stabilization of labile
organic nitrogen in humic substances to improve our
knowledge of the chemical composition of the refractory
organic nitrogen and understanding of the nitrogen
cycling in the biosphere.
A number of analytical techniques have been applied
to reveal the identity of the refractory organic nitrogen
in humic substances. Due to the chemically complex and
physically heterogeneous system, it has been dicult to
0146-6380/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00040-1
680
X. Zang et al. / Organic Geochemistry 31 (2000) 679±695
obtain accurate structural information on the organic
nitrogen in humic substances. The advent of modern
analytical methodologies, namely solid state nuclear
magnetic resonance (NMR) spectroscopy, pyrolytic
methods coupled to gas chromatography and mass
spectrometry (Py/GC/MS), and the tetramethylammonium
hydroxide (TMAH) thermochemolytic methods (TMAH/
GC/MS), have provided a wealth of new structural
information on the dierent types of organic moieties in
humic substances (Knicker et al., 1996; Schulten and
Schnitzer, 1997; del Rio et al., 1998). Recent applications of solid-state 15N NMR to the study of dierent
types of organic matter in soil systems at natural 15N
abundance levels have shown that the main functionality of the refractory organic nitrogen in humic acid
exists primarily as amide functional groups, probably
from proteins (Knicker et al., 1996; Knicker and Hatcher,
1997; Derenne et al., 1998). The amide functional group
persistently exists even after intense chemical and
microbial degradation. Pyrolytic methods coupled to
analytical techniques, such as gas chromatography and
mass spectrometry, have oered additional structural
information and have identi®ed proteinaceous pyrolysis
products from humic acid (Bracewell and Robertson,
1984; Zsolnay and Harvey, 1985; Gadel and Bruchet,
1987; Schulten and Schnitzer, 1997). Furthermore, the
application of TMAH thermochemolysis for the structural analysis of the peptide-like refractory organic
nitrogen in humic substances has resulted in a more
comprehensive evaluation of structural entities of the
amide-nitrogen and con®rmed that it is derived from
proteinaceous material (Knicker and Hatcher, 1997).
Heterocyclic nitrogen-containing compounds, such as
pyrrole and indole, are also part of the refractory
organic nitrogen, but they only constitute minor proportions (less than 5% of the total refractory organic
nitrogen) (Schulten and Schnitzer, 1997).
Although various studies have revealed that the
nitrogen-bearing compounds in humic acid are most
likely proteins, less well understood and far more controversial are the roles played by various mechanisms in
the association of humic acid and refractory organic
nitrogen. The depolymerization-recondensation hypothesis
was proposed to explain the existence of the refractory
organic nitrogen (Tissot and Welte, 1984; Bada, 1998).
This mechanism speculates that the formation of
organic nitrogen matter involves an initial degradation of
cellular components and a subsequent recondensation of
these degradation species with other low molecular
weight compounds, resulting in new substances, which
cannot easily be related to their biological precursors. In
this process, naturally occurring macromolecules such
as proteins and carbohydrates are enzymatically degraded to oligomers and monomers, which for the most
part are mineralized. A small fraction of these oligomers
and monomers may condense or polymerize into
organic rich colloids by chemical (Maillard, 1912; Flaig
et al., 1975; Hedges, 1978; Keil and Kirchman, 1994) or
photochemically initiated crosslinking reactions (Harvey
et al., 1983). A well known example is the condensation
reaction between amino acids and carbohydrates to
form Schi bases, which subsequently undergo Amadori
rearrangements to form dark colored melanoidins
(Maillard, 1912). However, solid-state 15N NMR studies
do not con®rm the presence of melanoidins as the major
nitrogen-containing compounds in humic substances
(Knicker et al., 1996) excluding this mechanism as the
primary preservation mechanism. In contrast to the
depolymerization-recondensation mechanism, the selective
preservation mechanism claims that refractory organic
matter is a fraction of cell components which have not
been degraded due to the inherent resistance of the
compound itself to enzymatic or chemical attack, whereas
the other more labile constituents are mineralized in the
water column and the upper layer of sediments (Hatcher
et al., 1983; Moers et al., 1994; Derenne and Largeau,
1998; Lichtfouse et al., 1998). Therefore, macromolecular
structures such as aquatic kerogen and algaenan have
been selectively preserved during early diagenesis and
have survived microbial decomposition. Nevertheless,
proteins, the largest compartment of nitrogen in most
cells, and thus the greatest potential contributor of
nitrogen to environmental sediments, are rapidly lost
during early diagenesis and, therefore, are an unlikely
substrate for a selective preservation mechanism.
Adsorption of intrinsically labile organic matter to
mineral surfaces, which are inaccessible to bacterial
extracellular enzymes (
www.elsevier.nl/locate/orggeochem
Encapsulation of protein in humic acid from a histosol
as an explanation for the occurrence of organic nitrogen
in soil and sediment
Xu Zang, Jasper D.H. van Heemst, Karl J. Dria, Patrick G. Hatcher *
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA
Abstract
Recent work suggests that nitrogen in humic acids exists primarily as amide functional groups that mirror those in
protein. However, the mode for the existence of such labile materials as protein still remains unclear. With the combined applications of NMR spectroscopy, tetramethylammonium hydroxide (TMAH) thermochemolysis, pyrolysis/
GC/MS, and elemental analysis, we propose that the survival of proteins in humic acids is carried out by encapsulation
into hydrophobic domains of humic acids. To test this hypothesis, we simulated encapsulation of 15N-labeled protein
extracts into humic acids and demonstrated that complete hydrolysis of the protein is prevented by the encapsulating
humic acid. Results from this study constitute evidence to support the encapsulation mechanism involved in the formation of refractory organic nitrogen during sediment diagenesis. # 2000 Published by Elsevier Science Ltd. All rights
reserved.
Keywords: Refractory organic nitrogen; Protein survivability; Encapsulation; Humic acid; TMAH GC/MS; Pyrolysis GC/MS; Solidstate NMR
1. Introduction
The profound in¯uence of humic materials on the
distribution, bioavailability, and ultimate fate of sedimentary organic nitrogen in various environmental
sediments has been recognized in the past decade (Barancikova et al., 1997; Schulten and Schnitzer, 1997;
Lichtfouse et al., 1998; Stankiewicz and van Bergen,
1998). Sedimentary organic nitrogen (SON) refers to the
sum-total of all nitrogen-containing substances in sediments. SON provides most of the nitrogen necessary for
the terrestrial bioproductivity. It primarily results from
the microbial decay of proteins and peptides in bioorganic residues and is part of the nitrogen cycle in the
biosphere. During early diagenesis, most of the labile
nitrogen-containing materials, such as peptide, protein,
and chitin, are quickly degraded and mineralized by
microbial and/or enzymatic degradation (Codispoti and
Christensen, 1985; Blackburn and Sorensen, 1988; Endo
et al., 1995; Nguyen and Harvey, 1998; Walton, 1998).
* Corresponding author.
However, recent studies have shown that part of these
labile nitrogen-containing materials are incorporated
into refractory organic components in humic substances, where they are protected from biodegradation
(Hedges and Keil, 1995; Henrichs, 1995; Knicker and
Hatcher, 1997; Nguyen and Harvey, 1998; Knicker et
al., 1999). This incorporated organic nitrogen has been
removed from the active nitrogen pool of the nitrogen
cycle and is, therefore, no longer available for biological
production (Knicker and Hatcher, 1997). Estimates of
refractory organic nitrogen in humic substances range
from 30 to 50% of total nitrogen (Schulten and Schnitzer, 1997). It is essential to understand the mechanisms
and processes involved in the stabilization of labile
organic nitrogen in humic substances to improve our
knowledge of the chemical composition of the refractory
organic nitrogen and understanding of the nitrogen
cycling in the biosphere.
A number of analytical techniques have been applied
to reveal the identity of the refractory organic nitrogen
in humic substances. Due to the chemically complex and
physically heterogeneous system, it has been dicult to
0146-6380/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00040-1
680
X. Zang et al. / Organic Geochemistry 31 (2000) 679±695
obtain accurate structural information on the organic
nitrogen in humic substances. The advent of modern
analytical methodologies, namely solid state nuclear
magnetic resonance (NMR) spectroscopy, pyrolytic
methods coupled to gas chromatography and mass
spectrometry (Py/GC/MS), and the tetramethylammonium
hydroxide (TMAH) thermochemolytic methods (TMAH/
GC/MS), have provided a wealth of new structural
information on the dierent types of organic moieties in
humic substances (Knicker et al., 1996; Schulten and
Schnitzer, 1997; del Rio et al., 1998). Recent applications of solid-state 15N NMR to the study of dierent
types of organic matter in soil systems at natural 15N
abundance levels have shown that the main functionality of the refractory organic nitrogen in humic acid
exists primarily as amide functional groups, probably
from proteins (Knicker et al., 1996; Knicker and Hatcher,
1997; Derenne et al., 1998). The amide functional group
persistently exists even after intense chemical and
microbial degradation. Pyrolytic methods coupled to
analytical techniques, such as gas chromatography and
mass spectrometry, have oered additional structural
information and have identi®ed proteinaceous pyrolysis
products from humic acid (Bracewell and Robertson,
1984; Zsolnay and Harvey, 1985; Gadel and Bruchet,
1987; Schulten and Schnitzer, 1997). Furthermore, the
application of TMAH thermochemolysis for the structural analysis of the peptide-like refractory organic
nitrogen in humic substances has resulted in a more
comprehensive evaluation of structural entities of the
amide-nitrogen and con®rmed that it is derived from
proteinaceous material (Knicker and Hatcher, 1997).
Heterocyclic nitrogen-containing compounds, such as
pyrrole and indole, are also part of the refractory
organic nitrogen, but they only constitute minor proportions (less than 5% of the total refractory organic
nitrogen) (Schulten and Schnitzer, 1997).
Although various studies have revealed that the
nitrogen-bearing compounds in humic acid are most
likely proteins, less well understood and far more controversial are the roles played by various mechanisms in
the association of humic acid and refractory organic
nitrogen. The depolymerization-recondensation hypothesis
was proposed to explain the existence of the refractory
organic nitrogen (Tissot and Welte, 1984; Bada, 1998).
This mechanism speculates that the formation of
organic nitrogen matter involves an initial degradation of
cellular components and a subsequent recondensation of
these degradation species with other low molecular
weight compounds, resulting in new substances, which
cannot easily be related to their biological precursors. In
this process, naturally occurring macromolecules such
as proteins and carbohydrates are enzymatically degraded to oligomers and monomers, which for the most
part are mineralized. A small fraction of these oligomers
and monomers may condense or polymerize into
organic rich colloids by chemical (Maillard, 1912; Flaig
et al., 1975; Hedges, 1978; Keil and Kirchman, 1994) or
photochemically initiated crosslinking reactions (Harvey
et al., 1983). A well known example is the condensation
reaction between amino acids and carbohydrates to
form Schi bases, which subsequently undergo Amadori
rearrangements to form dark colored melanoidins
(Maillard, 1912). However, solid-state 15N NMR studies
do not con®rm the presence of melanoidins as the major
nitrogen-containing compounds in humic substances
(Knicker et al., 1996) excluding this mechanism as the
primary preservation mechanism. In contrast to the
depolymerization-recondensation mechanism, the selective
preservation mechanism claims that refractory organic
matter is a fraction of cell components which have not
been degraded due to the inherent resistance of the
compound itself to enzymatic or chemical attack, whereas
the other more labile constituents are mineralized in the
water column and the upper layer of sediments (Hatcher
et al., 1983; Moers et al., 1994; Derenne and Largeau,
1998; Lichtfouse et al., 1998). Therefore, macromolecular
structures such as aquatic kerogen and algaenan have
been selectively preserved during early diagenesis and
have survived microbial decomposition. Nevertheless,
proteins, the largest compartment of nitrogen in most
cells, and thus the greatest potential contributor of
nitrogen to environmental sediments, are rapidly lost
during early diagenesis and, therefore, are an unlikely
substrate for a selective preservation mechanism.
Adsorption of intrinsically labile organic matter to
mineral surfaces, which are inaccessible to bacterial
extracellular enzymes (