Integration of the polyphenol and Maillard reactions into a unified abiotic pathway for humification

16 Pages·2010·0.86 MB·English

Integration of the polyphenol and Maillard reactions into a
unified abiotic pathway for humification in nature:
the role of δ -
MnO
2

A. Jokica,M.C. Wangb,C. Liua,A.I. Frenkelc,P.M. Huanga,*
aDepartment of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8 bDepartment and
Graduate Institute of Environmental Engineering and Management, Chaoyang University of Technology, Taichung County,
Taiwan
cPhysics Department, Yeshiva University, New York, NY 10016, USA

Received 31 August 2003; accepted 25 January 2004
(returned to author for revision 31 August 2003)

Abstract

The Maillard reaction involving condensation reactions between sugars and amino acids is considered to be an
important pathway in natural humification processes. Polyphenols in terrestrial and aquatic environments are regarded
as important precursors in the formation of humic substances. In nature it is most likely that these two processes do
not occur separately but rather interact with each other. We report that the ubiquitous soil mineral d-MnO
2
significantly accelerated humification processes in a system containing glucose,glycine,and catechol,at temperatures
and a pH typical of natural environments. The data obtained indicate the significance of linking the polyphenol and
Maillard reactions as promoted by d-MnO into an integrated humification pathway.
2
# 2004 Elsevier Ltd. All rights reserved.

1. Introduction nese oxides are powerful oxidizing agents (Shindo and
Huang,1982, 1984) and are considered the most
In soils and sediments,humification processes are of important abiotic redox-active minerals found in many
principal importance in the transformation of organic soils and sediments (Risser and Bailey,1992). Their
molecules originating from organized structures typical effiectiveness as electron acceptors in a very wide range
of organisms (e.g.,carbohydrates and proteins) to ran- of redox reactions is unique among common soil
domly polymerized,heterogeneous humic substances. minerals (McBride,1989).
Soil mineral colloids are significant in turnover of The Maillard reaction involving condensation reac-
organic matter (Huang,1995; Torn et al.,1997) and tions between sugars and amino acids (Maillard,1913)
manganese (Mn) oxides are ubiquitous and widely leading to the formation of melanoidins is perceived to
occurring in nature (McKenzie,1989). They frequently be an important pathway in natural humification pro-
occur in conjunction with Fe oxides (Childs,1975; cesses (Ikan et al.,1996). The great appeal of the Mail-
Sidhu et al.,1977; McKeague et al.,1986),as discrete lard reaction in humification processes lies in the two
particles or partial coatings or films on soil and sedi- proposed precursors (sugars and amino acids) being
ment particles (Burns and Burns,1977; Giovanoli and among the most abundant constituents of terrestrial and
Balmer,1981; Post et al.,1982; Manceau and Combes, aquatic environments (Anderson et al.,1989). Further
1988),or as suspended particulates in water columns support is lent by the presence of humic substances in
(Larson and Hufnal,1980; McKenzie, 1989). Manga- marine environments where carbohydrates and proteins,
because of their abundance,are more probable pre-
* Corresponding author. Fax: [phone]. cursors of humic substances than are lignin or phenolic
E-mail address: [email protected] (P.M. Huang). polymers (Nissenbaum and Kaplan,1972; Hedges and

0146-6380/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.orggeochem.2004.01.021
748 A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762
Parker,1976; Ikan et al.,1996),although autooxidative (Risser and Bailey,1992). However,naturally occurring
crosslinking reactions between adjacent polyunsaturated d-MnO probably contains significant amounts of
2
fatty acids in fat molecules exposed to sunlight at the adsorbed metal ions and/or organic compounds which
ocean surface may also contribute to the formation of could interfere in studies on the abiotic transformations
humic substances in seawater (Harvey et al.,1983; Har- of organic structures. Therefore,a synthetic d-MnO ,
2
vey and Boran,1985). Humic substances isolated from prepared according to McKenzie (1971) and known to
natural environments,e.g.,Lake Hula (Israel) and Lake be free of contaminants and chemicals other than those
Haruna (Japan) humic acids,have extremely similar 13C used in its preparation,was utilized in the experiments.
NMR spectra to synthetic melanoidins (Ikan et al., X-ray absorption near-edge structure (XANES) spec-
1986,1996). A major criticism of the Maillard reaction troscopic analysis of the d-MnO showed that it con-
2
has been that it is very slow under ambient conditions tained predominantly Mn(IV) but with a significant
(Hedges,1988). However,it has recently been demon- content of Mn(III) (Jokic et al.,2001). Catechol (mini-
strated that the action of birnessite (d-MnO2),under mum 99%),glucose (ACS reagent grade), 14C uniformly
ambient environmental conditions,significantly accel- labelled g lucose [UL-14C d-glucose (HO14CH2[14-
erates the Maillard reaction and merits attention as an CHOH]41 4CHO)] and glycine (Sigma Ultra grade pur-
important abiotic pathway for the formation of humic ity) were obtained from Sigma Chemical Co. (St. Louis,
substances (Jokic et al.,2001). MO). Distilled,deionized water (henceforth referred to
Polyphenols present in terrestrial and aquatic envir- as water) was used in all experiments.
onments are regarded as important precursors in the The effiect of d-MnO was investigated by incubating
2
formation of humic substances via processes of oxida- sealed 250 mL flasks,each containing 2.50 g d-MnO
2
tive polymerization which take place in soils and sedi- suspended in a solution containing an equi-molar mix-
ments (Pal et al.,1994; Stevenson,1994; Sparks, 1995; ture of catechol,glucose and glycine (0.05 M each) or
Huang,2000). The polyphenol model of abiotic catechol and glycine (0.05 M each) or glucose and gly-
(mineral catalyzed) formation of humic substances has cine (0.05 M each) in an oscillating water bath. The
been well documented (Huang,1995,2000) and the cat- concentrations of glucose and glycine used were as
alytic action of d-MnO on mixtures of polyphenols and recommended by Hedges (1978),Benzing-Purdie et al.
2
amino acids leading to the formation of nitrogen con- (1985),and Taguchi and Sampei (1986),in investiga-
taining polycondensates has been demonstrated (Shindo tions of the Maillard reaction. The concentration of
and Huang,1984; Wang and Huang,1987). catechol used was therefore set to equal that of the glu-
Hence,both the Maillard reaction and the polyphenol cose and glycine. The pH of the system was adjusted to
model are separate,significant abiotic pathways for the an environmentally relevant initial pH of 7.00,and the
formation of humic polycondensates. In nature how- final volume of the solution was set to 100 ml. The
ever,it is most likely that these two pathways do not reaction mixture was incubated for 15 d at 45 C. 45 C
occur separately,but rather interact closely with each is the approximate temperature of the soil surface on a
other,since sugars,amino acids,and polyphenols coex- day when the ambient air temperature is 25 C (Baver,
ist in soil solutions and natural waters. It is therefore,a 1956; Jury et al.,1991),and is frequently encountered in
logical step to attempt to unify the two separate path- tropical and subtropical regions,and even in temperate
ways,i.e.,the Maillard reaction and polyphenol model, regions during the summer. Some experiments were also
into one combined humification pathway incorporating conducted at 25 C with the incubation time extended to
carbohydrate,amino acid and polyphenol moieties into 60 d. Experiments using UL-14C glucose were per-
the humic structure as would likely occur in nature. formed only at 45 C (15 d). Control systems
The objective of the present research was to examine containing
the catalytic effiect of d-MnO on the process of humifi- no d-MnO only had a liquid phase since the reaction
2 2
cation in the catechol–glucose–glycine system and to products remained in solution. Systems containing
analyze humic acids (HAs) isolated from the glucose– manganese had a solid phase,initially consisting only of
glycine–d-MnO2 (representative of the mineral colloid- d-MnO2,but at the end of the reaction period,the solid
Maillard reaction pathway),glycine–catechol– d-MnO2 phase consisted of unreacted d-MnO2 and adsorbed
(representative of the mineral colloid-polyphenol path- reaction products.
way),and glucose–glycine–catechol– d-MnO systems Sterile conditions were maintained by autoclaving all
2
(representative of the mineral colloid-unified pathway). apparatus,water and d-MnO2 used,and by adding
thimerosal (0.02% w/v,final volume). The absence of
aerobic microbial growth was verified by culturing
2. Experimental methods aliquots from reaction mixtures on Trypticase Soy Agar
(TSA) plates (Dandurand and Knudsen,1997). In
Mn oxides are considered the most important abiotic addition the absence of anaerobic microorganisms was
redox-active minerals found in many soils and sediments tested by culturing aliquots from selected reaction
mixtures on TSA plates in a BBL (Cockeysville,
A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762 749
Maryland) GasPak 150TM Large Anaerobic System,i.e., 15 d,15 mg; from system (3) the glycine–catechol–d-
in a sealed container from which all the oxygen was MnO system at 45 C,15 d,325 mg; and from system
2
removed. (4) the glucose–glycine–catechol–d-MnO system at
2
The procedure for experiments incorporating UL-14C 45 C,15 d,35 mg. The greatest yield of HA was
glucose was as follows. The volume of the aqueous obtained from the glycine–catechol–d-MnO system at
2
solution of UL-14C glucose was 333 ml with radioactivity 45 C,15 d. However,the glucose–glycine–catechol–d-
of 0.15 mCi/ml. The specific activity of this solution was MnO system provides for the incorporation of carbo-
2
7.1 mCi/mmol. The original solution was diluted to 50 hydrate,amino acid and polyphenol moieties into the
ml with water. The radioactivity of the diluted solution humic structure as would likely occur in nature. Control
was 50 mCi. In the experiment there were four diffierent systems containing no d-MnO yielded much less HA.
2
reaction systems (i.e.,glucose–glycine–catechol–d- For example,at 45 C,the glucose–glycine system yielded
MnO2,glucose–glycine– d-MnO2,glucose–glycine–cate- less than 1 mg HA,and the glucose–glycine–catechol
chol,and glucose–glycine) with three replicates each. To control produced less than 5 mg HA. The glycine–
each of the three replicates was added 2 ml of the dilu- catechol system was not run as a control.
ted solution with radioactivity of 2 mCi (0.2817 mmol).
The experimental procedure then followed that given 2.1.1. Visible absorbance spectroscopy
above for the experiments using no radioactive chemi- The formation of humification products was mon-
cals with one diffierence. An autoclaved polyethylene itored by measuring changes in absorbance of the
tube (of sufflcient dimensions that it leaned against the supernatants of the glucose–glycine,glucose–glycine–d-
wall of the reaction vessel without danger of its contents MnO,glucose–glycine–catechol and the glucose–gly-
2
mixing with those of the vessel) containing 10 ml of cine–catechol–d-MnO systems (25 C,60 d),and the
2
1/3M KOH was placed in each reaction flask,prior to glucose–glycine,glucose–glycine–d-MnO,glucose–
2
closing,in order to absorb any 14CO2 released. glycine–catechol,glucose–glycine–catechol– d-MnO2 and
the glycine–catechol–d-MnO systems (45 C,15 d).
2
2.1. Characterization of reaction products and isolation After centrifugation at 25 000g for 40 min,spectra were
and analysis of humic acids recorded in the visible range from 400 to 600 nm using a

Beckman DU 650 microprocessor-controlled spectro-
At the end of the reaction period,the solid residue photometer (Fullerton,CA). The pH and Eh of sus-
from systems which contained d-MnO2 was separated pensions of the systems were measured and the Mn
from the supernatant by centrifugation at 25,000g for 40 content of the supernatants was determined at 279.5 nm
min. The residue was repeatedly washed,dialysed by atomic absorption (AA) spectroscopy.
against distilled,deionized water [dialysis tubing with The HAs obtained from the glucose–glycine–cate-
molecular weight cut-offi (MWCO) 1000] and then lyo- chol–d-MnO system (25 C,60 d) and the glucose–gly-
2
philized. Humic acids (HAs) were isolated after 60 d cine–catechol–d-MnO and the glycine–catechol–d-
2
reaction at 25 C from the (1) the glucose–glycine–cate- MnO systems (45 C,15 d) were also analyzed by visi-
2
chol–d-MnO2 system (representing the mineral colloid- ble absorption spectrophotometry. The ratio of the
unified pathway) and also after 15 d reaction at 45 C absorbance at 465 nm to that at 665 nm (E/E ratio)
4 6
from (2) the glucose–glycine–d-MnO2 (representing the was calculated because it is commonly used to char-
mineral colloid-Maillard reaction pathway),(3) the gly- acterise humic substances (Stevenson,1994). Salt con-
cine–catechol–d-MnO2 (representing the mineral col- centrations can affiect absorbance and E4/E6 ratios
loid-polyphenol pathway),and (4) the glucose–glycine– (Ghosh and Schnitzer,1979) as can the solvent used
catechol–d-MnO2 (representing the mineral colloid-uni- (Baes and Bloom,1990) and the E4/E6 ratio is also pH
fied pathway) systems. The supernatant combined with dependent (Chen et al.,1977). Therefore,2 mg of each
water extracts of the sediment,after centrifugation at HA were dissolved in 10 ml of 0.05 M NaHCO. This
3
25 000g for 40 min,was acidified to pH 1.0 by the addi- results in a pH of approximately 8.0 and a fixed salt
tion of 6 M HCl to precipitate the HA. After standing concentration as recommended by Chen et al. (1977).
for 24 h,any precipitate was separated from the super-
natant by centrifugation at 2000g for 20 min. The HA 2.1.2. Fourier Transform Infrared (FTIR) spectroscopy
polycondensates were transferred to dialysis tubing The FTIR spectra of the lyophilised solid residues of the
(MWCO 1000) using water and were dialyzed against glucose–glycine–catechol–d-MnO system (25 C,60 d),
2
water until a negative chloride test (10% w/v aqueous and the glycine–catechol–d-MnO ,the glucose–glycine–
2
silver nitrate) was obtained. After dialysis was com- catechol–d-MnO,and the glucose–glycine–d-MnO
2 2
pleted,the precipitates were lyophilized. The quantity of systems (all at 45 C,15 d) were obtained using KBr
HA isolated from system (1) the glucose–glycine–cate- disks containing 0.5% w/w sample on a Biorad 3240
chol–d-MnO2 system at 25 C,60 d was 7 mg; from SPS microprocessor-controlled spectrometer (Cambridge,
system (2) the glucose–glycine–d-MnO2 system at 45 C, MA). Samples were flushed with dry N2 gas for 10–15
750 A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762
min prior to analysis in order to remove atmospheric 2.1.6. Atomic Force Microscopy (AFM)
CO2 and moisture. The HAs obtained from the glucose–glycine–d-MnO2,
The FTIR spectra of HA samples from the glycine– glucose–glycine–catechol–d-MnO and the glycine–cate-
2
catechol–d-MnO2,the glucose–glycine–catechol–d- chol–d-MnO2 systems (45 C,15 d) were compared with
MnO2,and the glucose–glycine–d-MnO2 systems (all at the standard soil HA sample of the International Humic
45 C,15 d) were obtained using KBr disks containing Substances Society (1S102H). Five milligrams of each
0.5% w/w sample as above. HA was dispersed in 22 ml water using ultrasonification
(Sonifier Model 350,Danbury,CT) at 40 kHz and 150
2.1.3. X-ray absorption near-edge structure (XANES) watts for 2 min in an ice bath. The suspension was
spectroscopy adjusted to pH 5.0 with 0.01 M NaOH and HNO and
3
XANES spectroscopy was employed to follow the diluted to 25 ml. One drop of the suspension was
changes in the speciation of Mn in d-MnO2 containing deposited on a watch glass and air-dried overnight at
systems. A sample of untreated,pure d-MnO2 was used room temperature (23.5 0.5 C). The watch glass was
as control and the solid phases of the glucose–glycine– then mounted on a magnetized stainless steel disk (12
catechol–d-MnO2 systems (treated at 25 C,60 d and mm diameter) with double-sided tape. The 3-dimen-
45 C,15 d) were examined. XANES experiments were sional AFM images were obtained under ambient con-
conducted in transmission mode at the Materials ditions using the contact mode of a NanoScopeTMIII
Research Laboratory (MRL) University of Illinois— atomic force microscope (Digital Instruments,Inc.,
Agere beam line X16-C at the National Synchrotron Santa Barbara,CA). The imaging area measured was 5
Light Source,Brookhaven National Laboratory,Upton, mm 5 mm. The scanner type was 1881E and scanner size
New York. The X-ray energy varied from 200 eV below was 15 mm. A silicon nitride cantilever with a spring
to 1000 eV above the absorption K-edge of Mn (Ek= constant of 0.12 N/m was used in the contact mode. The
6540 eV) using a Si 111 double-crystal monochromator. scanning rate was 2.2 Hz. To protect against experi-
mental artifacts,the AFM cantilever was changed fre-
2.1.4. 1H solution Nuclear Magnetic Resonance (NMR) quently. The scanning area and angle were also changed
spectroscopy often in order to detect artifacts caused by adhesion of
1H solution NMR spectra were obtained for HAs particles to the AFM tip. The particle size was estimated
isolated from the glycine–catechol–d-MnO,the glu- using Section analysis based on 30 particles after the
2
cose–glycine–d-MnO,and the glucose–glycine–cate- image was enlarged.
2
chol–d-MnO systems (all at 45 C,15 d) on a Bruker
2
Avance 500 spectrometer (Bruker Analytik,Rhein- 2.2. Determination of amount of 14CO2 released from
stetten/Karlsruhe Germany) operating at 500.13 MHz oxidation of UL-14C glucose
for 1H. The samples were dissolved in 2.0 ml 0.1 M
NaOD/D O under nitrogen. 256 or 2400 scans were At the end of the reaction period in the experiments
2
used in obtaining the spectra,with a delay time of 1 s, incorporating UL-14C glucose,the KOH solution in
and line broadening of 0.30 Hz. The water peak was each reaction flask was analyzed for 14CO2 content
used as internal standard with deuterated chloroform as using a Beckman LS3801 Liquid Scintillation Counter.
external standard.

2.1.5. 13C Cross Polarization Magic Angle Spinning 3. Results
(CPMAS) Nuclear Magnetic Resonance (NMR)
Spectroscopy 3.1. Solution and solid phase reaction products
A sample of HA from the glycine–catechol–d-MnO
2
system (45 C,15 d) was analyzed by 13C CPMAS 3.1.1. Supernatants
NMR spectroscopy on a Bruker Avance 360 DRX In all the systems studied the presence of culturable,
spectrometer (Bruker Analytik,Rheinstetten/Karlsruhe aerobic or anaerobic micro-organisms was not detected,
Germany) operating at 90.57 MHz for 13C. Optimum indicating that all reactions were abiotic in nature.
spectral conditions were obtained using a contact time Visible absorbance spectra of diluted solutions of the
of 3.0 ms and relaxation delay of 2.0 s. Spinning the supernatants of the systems were recorded and the values
sample at 12 kHz eliminated sidebands but did not sig- obtained were subsequently multiplied by the dilution
nificantly alter spectral characteristics compared to a factor. The units are arbitrary absorbance units. The
dissolved sample analyzed by liquid 13C NMR. A 4 mm data clearly show the significant increase in browning
Bruker probe and phase stabilized zirconium rotor were exhibited by systems containing d-MnO (both at 25
2
used. Chemical shifts are expressed relative to tetra- and 45 C) compared to non-d-MnO containing
2
methylsilane. No background rotor effiects were systems (Table 1). The browning was most
observed under these experimental conditions. pronounced in systems that also contained catechol.
A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762 751
The addition of d-MnO caused an increase in pH in MnO system,the spectral characteristics of the glycine–
2 2
all systems studied (Table 2). In contrast,the pH value of catechol–d-MnO system at 45 C (not shown)
2
the control glucose–glycine–catechol systems (both at 25 were very similar.
or 45 C) was lower than that of glucose–glycine systems. At the qualitative level the FTIR spectra of the freeze-
The pE+pH value of the glucose-glycine–catechol–d- dried residues of the glucose–glycine–catechol–d-MnO
2
MnO system at 25 C was considerably lower than that system (25 C,60 d) (Fig. 2a),the glycine–catechol–d-
2
of the glucose–glycine–catechol control (Table 2). MnO system (45 C,15 d) (Fig. 2b),and the glucose–
2
glycine–catechol–d-MnO system (45 C,15 d) (Fig. 2c),
2
3.1.2. Lyophilized residues are very similar and diffier from the FTIR spectrum of
X-ray absorption near-edge structure (XANES) the glucose–glycine–d-MnO system (45 C,15 d)
2
spectroscopy was employed to follow the changes in the (Fig. 2d). The absence of a band at approximately 1714
speciation of Mn in d-MnO2. Fig. 1 shows the Mn K-edge cm 1 (undissociated carboxyl) in (Fig. 2a–d) is con-
XANES spectra obtained,indicating Mn speciation in sistent with the system pH’s being near or above 7
the diffierent experimental systems: (a) pure d-MnO , (Wang and Huang,1992) and therefore,the carboxyl
2
and the glucose–glycine–catechol–d-MnO system (b) at groups are present in the dissociated form as absorption
2
25 C (60 d),and (c) at 45 C (15 d) all at initial pH bands at 1394–1410 and 1585–1593 cm 1 (Fig. 2a–d and
7.00. Compared with the glucose–glycine–catechol–d- Table 3).

Table 1
Visible absorbances of supernatants in a series of reaction systemsa

Reaction system Absorbance

400 nm 600 nm

25 C, 60 d
Glucose–glycine 0.077 0.004 0.021 0.001
Glucose–glycine–d-MnO 0.367 0.006 0.067 0.002
2
Glucose–glycine–catechol 10.8 0.6 3.8 0.2
Glucose–glycine–catechol–d-MnO 90.4 3.8 37.6 1.7
2
45 C, 15 d
Glucose–glycine 0.342 0.018 0.103 0.006
Glucose–glycine–d-MnO 14.7 0.68 1.50 0.10
2
Glucose–glycine–catechol 5.60 0.35 2.13 0.08
Glucose–glycine–catechol–d-MnO 73.7 1.3 24.7 1.5
2
Glycine–catechol–d-MnO 242.0 14.7 115.0 6.9
2
a The glycine–catechol–d-MnO system was not studied at 25 C.
2

Table 2
Final pH,redox status,and Mn content of supernatants in a series of reaction systems

Treatmenta Mn (mg mL 1) pH Eh (mV) pE+pH

25 C, 60 d

Glucose–glycine 0 7.11 0.05 411 12 14.03 0.23
Glucose–glycine–d-MnO2 2839 71 7.68 0.09 343 10 13.47 0.26
Glucose–glycine–catechol 0 6.42 0.08 296 10 11.42 0.25
Glucose–glycine–catechol–d-MnO 3010 174 7.60 0.09 87 15 9.07 0.34
2
45 C, 15 d
Glucose–glycine 0 7.01 0.09 NDb ND
Glucose–glycine–d-MnO 7803 74 7.98 0.11 ND ND
2
Glucose–glycine–catechol 0 5.92 0.09 ND ND
Glucose–glycine–catechol–d-MnO 3222 120 7.21 0.13 ND ND
2
Glycine–catechol–d-MnOc 3281 141 7.66 0.15 ND ND
2
a The systems were at an initially adjusted pH of 7.00.
b ND—not determined.
c The glycine-catechol-d-MnO2system was not studied at 25 C.
752 A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762

Fig. 1. XANES spectra of (a) control sample of pure d-MnO,and the solid phase of the glucose–glycine–catechol–d-MnO system
2 2
after treatment at (b) 60 d at 25 C and (c) 15 d at 45 C. The XANES spectrum of the glycine–catechol–d-MnO system (15 d at 45 C)
2
which is not shown is very similar to spectra (b) and (c).

Fig. 2. FTIR spectra of the solid phase of (a) the glucose–glycine–catechol–d-MnO system following reaction at 25 C for 60 d,(b)
2
the glycine–catechol–d-MnO,(c) the glucose–glycine–catechol–d-MnO and (d) the glucose–glycine–d-MnO systems all following
2 2 2
reaction at 45 C for 15 d. Systems (a)–(d) had an initially adjusted pH of 7.00.
Table 3
Assignations of FTIR absorption bands of the solid phase of the glucose–glycine–catechol–d-MnO2,the glycine–catechol–d-MnO2,and the glucose–glycine–d-MnO2 systemsa

(a) Glucose–glycine–catechol-d-MnO2 (b) Glycine–catechol–d-MnO2 (45 C)c (c) Glucose–glycine–catechol–d-MnO2 (d) Glucose-glycine-d-MnO2 (45 C)c
(25 C)b (45 C)c

Wavenumber (cm 1) Relative Wavenumber (cm 1) Relative Wavenumber (cm 1) Relative Wavenumber (cm 1) Relative

intensity intensity intensity intensity

576 (vibrations of MnO Weak 576 (vibrations of MnO Broad 591 (vibrations of MnO Weak 725 (carbohydrate Weak
6 6 6
octahedra from unreacted octahedra from unreacted octahedra from unreacted interactions)

d8s1e-51mM61 4in(c c(iOarCcr2-lb)Oe o s hstytrrdeetrtcachthe) orfi ng WWeeaakk d8s1e-61mM014 in(c c(iOarCcr2-lb)eO o s hstytrredetrtcachthe) orfi ng WWeeaakk d8s1e-61mM01 9in(c c(iOarCcr2-lb)Oe o s hstytrrdeetrtcachthe) orfin g WWeeaakk 8s1e60m07 7i(c c(iarCcr-lbOeo s hstytrrdeetrtcachthe) orfi ng IsBnhrtaoeranpds ,e , A J eo.kic
c1pa2lar7nb3eo (hCCy--dOOr- asHtter be) tecnhd ainngd o ifn - Strong c1pa2lar7nb0eo (hCCy--dOOr- asHtter be) tecnhd ainngd o inf - Strong c1pa2lar7nb0eo (hCCy--dOOr- asHtter be) tecnhd ainngd o ifn - Strong c1sta4rr1eb0toc (hhC)y OdOra-t esy) mmetric mShooduelrdaetre a / Oltr.g
CphOeOnoHls,C) -O stretch of CphOeOnoHl,sC) -O stretch of CphOeOnoHl,sC) -O stretch of Ganic
1394 (OH deformation of Strong 1402 (OH deformation of Strong 1398 (OH deformation of Strong 1456 (asymmetrical bending Strong eo
c
poab1ne4fh nt8eCid5nsHy io(nmsl2gsy m, amCbnea-mdtHnr eiCd ctdr )His ect3faro,lCer tNmOchHOaitn3 +i og n) Strong poab1ne4fh nt8eCid6snHy io(nmsl2gsy m, amCbnea-mdtHnr eiCd ctdr )His ect3faro,le r CtNmcOhHaOitn3+i o g n) Strong poab1ne4fh nt9eCid0snHy io(nmsl2gsy m ,amCbnea-mdtHnr eiCd ctd)r H siect3far,oeClr tNmOchHOaitn3 +io g n) Strong vC1st5irHb8er82tac dt(hiAeo,afnroso yrommmf aamCttiiHecot rn3C i cab= nCaCdnO/ dorO)ir n g Strong tr 3 (y25hemis
stretch) 00
1st5r8e7tc (hA,arsoymmamtiect rCic= CCO rOin g Strong 1as5y8m5 m(Aertroimc CatOicO C = sCtr,e tch) Strong 1st5r9e3tc (hA,arsoymmamtiect rCic= CCO rOin g Strong 2st9r2e5tc (ha)s ymmetric CH2 Weak 7474)
stretch) stretch) –7
3342 (OH stretch) Broad, 3396 (OH stretch) Broad, 3342 (OH stretch) Broad, 3376 (OH stretch) Broad, 62
strong strong strong strong
a The assignations are based on Colthup et al. (1990),Stevenson (1994) and Silverstein and Webster (1998).
b The system was treated for 60 d at 25 C and was at an initially adjusted pH of 7.00.
c The systems were treated for 15 d at 45 C and were at an initially adjusted pH of 7.00.
7
5
3
754 A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762
3.2. Isolated humic acids At 45 C the presence of glucose markedly increases the
E/E ratio in HA isolated from the glucose–glycine–
4 6
The highest yield of HA was obtained from the gly- catechol–d-MnO compared with the HA isolated from
2
cine–catechol–d-MnO system (45 C,15d; 325 mg). The the glycine–catechol–d-MnO system (Table 4).
2 2
polyphenol-amino acid system is known to yield sig-
nificant amounts of humic polycondensates (Shindo and 3.2.2. FTIR analysis
Huang,1984). The glucose–glycine–d-MnO system had FTIR analysis was performed on HAs precipitated
2
the lowest yield of HA (45 C,15 d; 15 mg),however,it is from the supernatants of the glycine–catechol–d-MnO2
known that a substantial amount of the humified and the glucose–glycine–catechol–d-MnO systems.
2
material in this system remains in the supernatant and Principal bands identified include those belonging to

can be isolated as fulvic acid (Jokic et al. 2001). The phenols,COOH groups, and NH3 + from amino acids.
presence of glucose in the glucose–glycine–catechol–d- The results are shown in Fig. 3 and Table 5.
MnO2 system (45 C,15 d) greatly reduces the yield of
HA (35 mg),although it is quite possible that sub- 3.2.3. 1H NMR analysis
stantial humified material occurs in the form of fulvic 1H solution NMR spectra were obtained from HAs
acid and remains in solution. isolated from (a) the glucose–glycine–catechol–d-MnO2
system,(b) the glycine–catechol–d-MnO system,and (c)
2
3.2.1. Visible absorption spectroscopy the glucose–glycine–d-MnO system (all at 45 C,15 d).
2
The E4/E6 ratio is considered to be an indicator of The aromatic region (6–8 ppm) of the glycine–cate-
the degree of humification of a system,i.e., a low ratio chol–d-MnO system is particularly pronounced relative
2
may indicate a higher content of aromatic structures or to other signals in the spectrum. Principal components
a higher degree of condensation,and a high ratio a of interest in the spectra include aliphatic,carbohydrate
greater content of aliphatic structures (Stevenson,1994). and aromatic structures (Fig. 4 and Table 6).

Table 4
Visible absorbances and E/E ratios for humic acids obtained from the glucose–glycine–catechol–d–MnO systems,and from the
4 6 2
glycine–catechol–d-MnO system
2

Systema A A A A E /E
400 465 600 665 4 6
Glucose–glycine–catechol–d-MnO (25 C)b 1.62 0.98 0.35 0.22 4.46
Glucose–glycine–catechol–d-MnO2 (45 C)c 1.91 1.04 0.28 0.14 7.43
2
Glycine–catechol–d-MnO (45 C)c 9.00 6.70 3.20 2.30 2.91
2
a The systems were at an initially adjusted pH of 7.00. Two milligrams of humic acid were dissolved in 10 mL of 0.05 M NaHCO .
3
b The system was treated for 60 d.
c The system was treated for 15 d.

Fig. 3. FTIR spectra of isolated humic acids from: (a) the glycine–catechol–d-MnO2,and (b) the glucose–glycine–catechol–d-MnO2
systems. The humic acids were isolated from systems with an initially adjusted pH of 7.00 and following reaction at 45 C for 15 d.
A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762 755
Table 5
Assignations of FTIR absorption bands of humic acids isolated from the glycine-catechol-d-MnO2 and the glucose–glycine–catechol–
d-MnO systemsa
2

(a) Glycine–catechol–d-MnO humic acid (b) Glucose–glycine–catechol–d-MnO humic acid
2 2

Wavenumber (cm 1) Relative Wavenumber (cm 1) Relative
intensity intensity

1206 (C-O stretch of phenol) Shoulder
1284 (C-O stretch and in-plane C-O-H Strong 1276 (C-O stretch and in-plane C-O-H Strong
bending of COOH,C-O stretch of phenols) bending of COOH,C-O stretch of phenols)
1373 (OH deformation of phenols,C-H Shoulder 1384 (OH deformation of phenols,C-H Moderate
deformation of CH2 and CH3,COO deformation of CH2 and CH3,COO
antisymmetric stretching) antisymmetric stretching)
1437 (OH bending of COOH) Weak 1457 (asymmetrical bending vibration of CH3 Weak
and/or CH2 deformation band)
1501 (symmetrical NH+3 bending band, Moderate 1514 (symmetrical NH+3 bending band, Weak
aromatic C=C stretch) aromatic C=C stretch)
1601 (aromatic C=C ring stretch) Strong 1600 (aromatic C=C ring stretch) Strong
1706 (asymmetric C=O stretch of COOH) Shoulder
3000–2000 (NH+3 stretch) Very broad 3000–2000 (NH+3 stretch) Very broad
3213 (OH stretch) Broad, 3382 (OH stretch) Broad,
strong strong

a The humic acids were prepared from systems with an initially adjusted pH of 7.00 following reaction at 45 C for 15 d. The
assignations are based on Colthup et al. (1990),Stevenson (1994) and Silverstein and Webster (1998).

3.2.4. 13C CPMAS NMR analysis 3.3. Systems containing UL-14C glucose
The spectrum obtained from the HA isolated from
the glycine–catechol–d-MnO system (45 C) is shown in 3.3.1. Evolution of 14CO released by oxidation of
2 2
Fig. 5. The spectrum broadly consists of three regions: UL-14C glucose
(1) Small peaks are seen in the region < 110 ppm (ali- The presence of d-MnO2 or catechol in the UL
phatic including carbohydrate); (2) strong,broad peaks 14C-labelled glucose–glycine–catechol system markedly
are found in the region of 110–160 ppm (aromatic and increased the evolution of 14CO (Table 7). The sequence
2
aromatic heterocylic); and (3) relatively minor peaks are of the amounts of 14CO released from the reaction
2
found at > 160 ppm (carboxyl and carbonyl including systems was glucose–glycine–catechol–d-MnO > glucose–
2
amide carbon). glycine – d - MnO > glucose–glycine–catechol > glucose–
2
glycine and the diffierences were significant (Table 7).
3.2.5. AFM analysis
The HAs obtained from the supernatants of the gly-
cine–catechol–d-MnO2,glucose–glycine–d-MnO2,and 4. Discussion
glucose–glycine–catechol–d-MnO systems (45 C,15 d)
2
appear as spheroidal particles in the AFM images 4.1. Solution and solid phase reaction products
(Fig. 6b,c and d),and are very similar to the surface
features of the IHSS standard soil HA (Fig. 6a). The 4.1.1. Supernatants
diameter of the IHSS standard soil HA particles ranges The ability of d-MnO to accelerate the polymeri-
2
from ca. 50 to ca. 150 nm with an average of 85 nm (Liu zation of polyphenols and polycondensation of poly-
and Huang,1999). The particle size of the HAs formed phenols and amino acids is well known (e.g., Shindo and
in the glycine–catechol–d-MnO2 (Fig. 6b),glucose–gly- Huang,1982,1984). Its role in accelerating the Maillard
cine–d–MnO (Fig. 6c),and glucose–glycine–catechol– (polycondensation) reaction between sugars and amino
2
d-MnO2 (Fig. 6d) systems in the present study,are, acids has recently been noted (Jokic et al.,2001).
respectively,in the range of ca. 60–140 nm with an Therefore,in the glucose–glycine–catechol–d-MnO
2
average of 94 nm,in the range of ca. 50–130 nm with system a complex process apparently occurs,i.e.,a
an average of 76 nm,and in the range of ca. 60–150 nm simultaneous polymerisation reaction involving catechol
with an average of 98 nm. The standard errors of the and polycondensation reactions of (i) glucose and gly-
measurements are 40.27 nm. cine,(ii) catechol and glycine and (iii) glucose,glycine,
756 A. Jokic et al. / Organic Geochemistry 35 (2004) 747–762
and catechol. Reduction of Mn(IV) and Mn(III) (from apparent inhibiting effiect of glucose on polymerization/
d-MnO) to Mn(II) and release of Mn(II) into the polycondensation reactions in the glucose–glycine–cate-
2
supernatant also occurs (Table 2). The polymerization chol–d-MnO system,and/or the increased content of
2
and polycondensation products (humic substances) aliphatic structures in the glucose–glycine–catechol–d-
formed in the glycine–catechol–d-MnO system had the MnO system which absorb less intensely at 400 and 600
2 2
highest absorbances in the visible range 400–600 nm of nm. The products formed in the glycine–catechol–d-
the systems studied (Table 1). This is attributed to the MnO system are therefore apparently more aromatic in
2
nature (cf. Section 3.2.1 visible absorption spectroscopy,
Table 4; Section 3.2.3 1H NMR analysis, Fig. 4 and
Table 6; Section 3.2.4 13C CPMAS NMR spectroscopic
data, Fig. 5).
Since the pE+pH value is a function of O concen-
2
tration,it is a measure of the redox status of a system
(Lindsay,1979). Polymerization of catechol generates
protons whereas the reduction of Mn(IV) and Mn(III)
consumes protons. Polycondensation reactions produce
water. In the absence of d-MnO the pH in the glucose-
2
glycine-catechol systems both at 25 and at 45 C
was lower (Table 2). In the presence of d-MnO the
2
pH at the end of the reaction increases,because of
the net effiect of these concomitant
processes,i.e.,reduction of Mn and the
polymerisation/polycondensation reactions. At pH
7,the amino group of glycine is still partially
protonated (isoelectric point for glycine is 6.06). As the
pH in the systems increases during the course of reac-
tion there is increasing deprotonation of the glycine
molecules which may also provide protons for reduction
of Mn(IV) and Mn(III). Reduction of Mn with sub-
sequent consumption of protons and increase in pH was
the dominant process in all systems containing d-MnO .
2
At 45 C the amount of Mn released to the
solution from the glucose–glycine–d-MnO system was
2
the high- est of all the systems containing d-MnO
2
(Table 2). This may be due to partial adsorption of
Mn(II) on the sur- face of the solid phase of the
glycine–catechol–d-MnO and glucose–glycine–catechol–
2
d-MnO systems. Adsorp- tion of reaction products on
2
the surface of the glycine– catechol–d-MnO and
2
glucose–glycine–catechol–d- MnO
2
systems could also prevent further polymerisa-
tion/polycondensation reactions from occurring.

4.1.2. Lyophilized residue
Several features in the XANES spectra in the energy
region between 6545 and 6565 eV (Fig. 1) indicate dis-
tinct changes in the oxidation state of Mn in samples (b)
and (c) compared to sample (a). The energies corres-
ponding to the positions of Mn(II),Mn(III), and
Mn(IV),indicated by the assignations (2+),(3+) and
Fig. 4. 1H solution NMR spectra of isolated humic acids from: (4+),are in good agreement with those obtained from
(a) the glucose–glycine–catechol–d-MnO system (20 mg),(b)
2 several reference Mn-containing compounds (Manceau
the glycine–catechol–d-MnO system (20 mg),and (c) the glu-
2 et al.,1992). The XANES data indicate that there was
cose–glycine–d-MnO system (10 mg). All samples were dis-
2 substantial reduction of Mn(IV) to lower Mn oxidation
solved in 2.0 ml NaOD/DO. All humic acids were precipitated
2 states,Mn(II) and Mn(III), (Fig. 1b and c). This sup-
from systems with an initially adjusted pH of 7.00 and treated
at 45 C for 15 d. ports the atomic absorption (AA) spectroscopic data
(Table 2) which indicates dissolution of a substantial
amount of Mn in the liquid phase of the glucose–gly-