Environ. Sci. Technol.
2003, 37, 4221-4227
Inclusionof PersistentOrganic
Pollutantsin Humification
Processes: Direct Chemical
Incorporationof Phenanthrenevia
OxidativeCoupling
W A L T E R J . W E B E R , JR . * A N D
QINGGUOH UANG
Environ mental and Water Resources Engineering Progra m,
The University of Michigan, Ann Arbor, Michigan 48109-2125
The participation of phenanthrene in phenol-coupling
reactions mediated by horseradish peroxidase was
investigated. Aqueous-phase concentrations of phenanthrene
were observed to decrease dramatically with phenol as
a result of the formation of precipitated products, suggesting
a potential means for simultaneous treatment of phenolic
contaminants and polycyclic aromatic hydrocarbons
(PAHs) using peroxidase-mediated oxidative coupling
processes. The studies reveal that phenanthrene removal
from the aqueous phase occurs by a combination of
sorption by and chemical bonding to precipitated reaction
products. In that the oxidative coupling reactions of
phenolics comprise an important step in the initiation of
humification processes, the results obtained provide insights
to potential roles that natural humification may play in
the sorption, sequestration, and environmental fate of PAHs
and other organic xenobiotics of similar nature.
Introduction
Biogeochemical turnover of natural organic matter is believed
to be governed by two primary and counter-directional
transformation pathways (i.e., degradation and humification)
(1). As depicted schematically in Figure 1, degradation is
destructive in nature, fractionating large natural macromol-
ecules into smaller and more labile molecules that can be
more readily mineralized. Conversely, hu mification is a
process in which small molecules are aggregated chemically
to form macromolecular hu mic substances. Both transfor-
mation pathways comprise potential bases for processes that
can be applied for prevention of pollution and / or mitigation
of environ mental contamination by organic xenobiotics,
either in engineered reactor systems or under enhanced in-
situ conditions. The humification pathway has received much
less attention than the degradation and mineralization
pathway in this regard, although the oxidative coupling of
phenols and anilines is one prominent exam ple of a
hu mification reaction that has attracted significant recent
interest for both pollutio n prevention and contaminated site
re m e diatio n ( 2-16). Oxidative coupling reactio ns co nstitute
one of the most im portant classes of reactions involved in
natural hu mification processes ( 1). In soil systems, these
reactions lead to incorporation of phenolic and anilinic
contaminants into soil organic matter (SOM), thereby
* Corresponding author e- mail: wjwjr@u mich.edu; telephone:
(734)763-2274; fax: (313)936-4391.
10.1021/es030330u CCC: $25.00  2003 American Chemical Society
Published on Web 08/07/2003
effecting an ultimate state of immobilization and detoxifi-
cation ( 2-9). In aqueous systems in which SOM is absent,
coupling reactions lead to the polymerization of phenolic
contaminants, forming precipitated products that can be easily
removed by sedimentation or filtration ( 10-16).
Peroxidases com prise an im portant class of catalysts for
the oxidative coupling of a broad spectru m of phenolic and
anilinic substrates in the presence of hydrogen peroxide ( 17-
19). The substrates are generally converted via enzymatic
mediation to radicals, which can then either couple with
each other (self-couple) or bind to other reactive substances
present in the system (cross-couple). The catalysis dynamics
of the horseradish peroxidase (HRP) are well-established ( 14,
20), and this specific enzyme has been extensively used as
a model catalyst in oxidative coupling studies ( 20), including
our own investigations of phenol-coupling reactions in the
presence of differe nt geosorbent materials ( 21).
Oxidative cross-coupling reactions between two differe nt
active substrates m ay occur thro ugh radical -radical bin ding
or nucleophilic addition mech anisms ( 4-6). It is also possible
for cross-coupling reactions to occur between an active
substrate and an inert chemical if free radicals generated
from active substrates transfer to the inert chemical via
hydrogen abstraction or free-radical addition. Cross-coupling
reactions between two active substrates containing phenolic
or a nilinic m oieties are well-docu m e nted ( 4-6, 22-24), but
there have been few investigatio ns of cross-coupling between
active substrates and inert chemicals. Removal of polychlo-
rinated biphenyls (PCBs) from the aqueous phase in per-
oxidase-catalyzed phenol-coupling reactions was reported
in one study ( 10). The investigator postulated, without
definitive experimental evidence, that the observed PCB
removal may have been attributable to one or both of two
possible mechanisms, i.e.: (i) coprecipitation of PCBs via
cross-coupling reactions with phenoxy radicals enzymatically
generated from phenolic substrates; and / or, (ii) adsorption
of PCBs by precipitated polymeric products resulting from
phenol self-coupling reactions. The goal of the work pre-
sented here was to develop more rigorous mechanistic
information to facilitate a better understanding of the
influe nces of natural hu mification processes on the envi-
ron mental behavior and fate of such persistent organic
pollutants as PAHs, PCBs, and pesticides.
Incorporation of PAHs into soil organic matter to form bound
residuals has in fact been observed empirically during
contaminant degradation and hu mification processes in soil and
sediment systems ( 25-30). The formation of bound residuals
in these cases was attributed to cross-coupling reactions between
the SOM and the PAH derivatives rather
than the parent PAH co m pounds the mselves ( 27-30), it being
generally presumed that hydroxylation of PAHs during partial
degradation processes was a prerequisite step in the cross-
coupling phe no m e n a observed ( 28-30). That the parent
PAHs may also have reacted with SOM via direct cross-
coupling was not precluded by the results of those studies,
but elucidation of actual reaction pathways was problematic
given the inherent com plexities of the soil systems and
processes involved. To circu mvent these com plexities, we
investigated phenanthrene transformation in HRP-mediated
phenol-coupling reaction systems operated under carefully
designed semi-batch conditions. The approach allowed us
to examine phenanthrene transformation mechanisms in
phenol-coupling processes in a manner that facilitates the
drawing of insights and conclusions regarding the potential
direct reactive inclusion of PAHs and other similar organic
xenobiotics in hu mification processes.
VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4221
FIGURE 1. Biogeochemical turnover of natural and anthropogenic organic matter.
Experimental Section
Materials. Extracellular horseradish peroxidase (type-I, RZ
) 1.3), hydrogen peroxide (30.8%, ACS reagent), 2,2 - azinobis-
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (98%, in di-
ammoniu m salt form), phenol-UL- 14C (51.4 mCi/ mmol),
phenanthrene, and phenanthrene-9- 14C (14 mCi/ mmol) were
obtained from Sigma Chemical Co. (St. Louis, MO). Phenol
(99+%, biochemical grade) was from Acros Organics (Bel-
giu m, NJ), and the carbon-14 cocktail for Harvey Biological
Oxidizer was from R. J. Harvey Instru ment Co. (Hillsdale,
NJ). ScintiSafe Plus 50% liquid scintillation cocktail and
methanol (HPLC grade) were obtained from Fisher Scientific
(Fairlawn, NJ).
Enzymatic Coupling Reactions. A phenanthrene stock
solution was prepared by dissolving a crystalline form of the
reagent in 10 m M phosphate buffer (p H 7.0). The solution was
passed through a 0.2- µm membrane filter (Nalgen Nunc
International, Rochester, NY) and stored in an amber glass jar at 4
C. The phenanthrene concentration in the resulting stock solution,
which was used for all experiments described unless otherwise
specified, was determined to be 105.5 µg/L.
Enzymatic coupling reactions were performed at room tem
perature in seru m bottles wrapped in alu minu m foil, and the
solutions were stirred constantly using glass-sealed magnetic bars.
Each reactor initially contained 50 mL of the phenanthrene
solution. Concentrated stock solutions of phenol, HRP, and
hydrogen peroxide were sequentially injected into the reactor at
volu mes of 0.1, 0.2, and 0.1 mL, respectively, to obtain the
concentration for each reagent desired at the outset of the reaction.
The initial concentrations of the reaction reagents determine the
strength of the enzymatic coupling reactions, and the standard
initial reaction condition examined in this study involved a
combination of 250 µM phenol, 250 µM H2O2, and 1 unit / mL
HRP. Experim ents were performed at other reaction strengths as
well by changing the concentrations of the reagent stocks, but the
concentration ratios among the three reactants were maintained at
constant values. Preliminary tests indicted
that nearly complete phenol conversion was obtained within a
half-hour reaction time at this ratio of reactants, with H 2O2 and
HRP being nearly com pletely consu med in the same reaction
period.
4222 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 18, 2003
Each reaction was allowed to proceed for 30 min, after which
time the three reacting reagents were again added to the reactor in
the same amounts to repeat the reaction. The reactions were
repeated in this manner 5 times a day for 4 consecutive days. At
the end of the fifth reaction of each day, two 1- mL sam ples
were taken from the resultant product mixture using glass pipets.
The reactor systems were thus operated in semi-batch mode in
the sense that the total volu me of the stock solutions added each
day was equal to the total volu me of sam ples withdrawn. The
reactors were stored at 4 C overnight during the 4-d experiment,
and fresh stock solutions of HRP and hydrogen peroxide were
prepared every day.
The two 1- mL sam ples taken after the fifth reaction each day
were processed immediately after sam pling and in differe nt
manners for differe nt analytical purposes; that is, one sample
was used for measuring phenanthrene concen- tration remaining
in the aqueous phase, and the other was used for analyzing the
concentrations of phenanthrene and phenol that were extractable
with 50% methanol. The sample used for measuring aqueous-
phase phenanthrene was centrifuged at 1300 g for 30 min to
separate liquid and solid phases. Sam ples for HPLC analysis
were then prepared by transferring 0.5 mL of the supernatant into
an HPLC vial and mixing with 0.5 mL of methanol preplaced in
the vial. The other 1- mL sam ple was combined with 1 mL of
methanol, shaken for a 0.5-h extraction period, and then
centrifuged for 30 min at 1300 g. The supernatant was then sam
pled for HPLC analysis. All concentrations for phenol and
phenan- threne were reported as measured in the original reaction
solutions before dilution with methanol. Preliminary tests
indicated that phenol sorption on precipitated reaction products
was negligible under the experimental conditions em ployed.
Phenol concentrations measured in the 50% methanol extracts
were very close to those remaining in the aqueous phase, each
representing the total phenol concen- trations remaining after
reaction.
An Agilent 1100 series HPLC system equipped with a
Phenomenex C18 colu m n (250 × 2.0 mm, 5 µm particle size)
was em ployed for sam ple analyses. The mobile phase
consisted of an acetonitrile com ponent (A) and an aqueous
com ponent (B), each containing 1% acetic acid. To facilitate
analysis, phenanthrene and phenol concentrations were
measured in separate HPLC analyses using differe nt mobile-
phase conditions and detectors. Phenanthrene was analyzed
using a 0.6 mL/ min flow of mobile phase consisting of 55%
A and 45% B and a fluorescence detector at 270-n m excitation
and 410-n m emission wavelengths, and phenol was analyzed
with a 0.4 mL/ min flow of mobile phase consisting of 35%
A and 65% B and a UV detector at 270 n m.
Each reaction corresponding to a specific combination
of reactant concentrations was examined in triplicate.
Because constant ratios among reactants were used for
differe nt reaction strengths, we describe reaction strengths
directly in terms of phenol concentrations. One blank control
test was performed with phenol and H 2O2 (i.e., no HRP),
both repeatedly supplied at 250 µM to a reactor system
containing a phenanthrene solution. The same schedule as
that used for the HRP- mediated reaction experiments was
followed with the blank control. Both phenanthrene and
phenol were quantitatively recovered at the end of the test,
indicating that physical losses of these reactants during the
reaction experiments were negligible. Another blank test was
conducted with H 2O2 and HRP (no phenol) repeatedly
supplied, at 250 µM and 1 unit / mL, respectively; no evidence
of phenanthrene transformation was observed.
Sorption of Phenanthrene by Precipitated Phenol-
Coupling Products. A representative sam ple of phenol-
coupling products was prepared by carrying out the enzy-
matic coupling reactions with additions of 250 µM phenol
in the absence of phenanthrene. The sam ple was collected
from the solution by filtration of the reaction solution through
a 0.2-µm membrane filter (Nalgen Nunc International,
Rochester, NY) and freeze-dried prior to use in the sorption
experiments. These experiments were then performed by
adding predetermined amounts of the precipitate sam ple to
50- mL aliquots of the 105.5 µg/L phenanthrene solution and
mixing continuously for 4 d, after which two 1- mL aliquots
of the mixture were taken from each reactor. One aliquot
was used for measuring phenanthrene concentrations re-
maining in the aqueous phase, and the other was used for
measuring concentrations of phenanthrene extractable with
50% methanol by the method described earlier.
Estimates of Total Precipitate Formation. To examine
methods for estimating quantities of precipitate formed in
enzymatic reaction systems, the reaction was conducted with
radiolabeled phenol at the 250 µM strength following the
procedure described above but in an amber glass bottle containing
500 mL of phenanthrene-free solution. At the end of the 4-d
experiment, 2 mL of the product solution was centrifuged, and 1
mL of the supernatant was sam pled for HPLC analysis to
measure the concentration of phenol remaining in the aqueous
phase. Another 0.5 mL of the supernatant was mixed with 3 mL
of a ScintiSafe Plus 50% liquid scintillation cocktail, and the 14C
radioactivity was measured using a Beckman LS6500 liquid
scintillation counter (Beckman Instru ments, Inc.) and expressed
as a phenol equivalent concentration. Total phenol conversion
was then quantified by subtracting the quantity of phenol
remaining from the total phenol added to the reactor. Total 14C
conversion was calculated in the same manner. The remainder of
the product solution was filtered through a 0.2-µm membrane
filter, and the precipitate was collected, freeze-dried, and weighed
to determine its mass.
Analysis of Precipitate. To analyze the distribution of
phenanthrene between sorbed and chemically incorporated forms
in the precipitate, the enzymatic reaction was con- ducted at the
250 µM strength in an amber glass bottle containing 500 mL of
105.5 µg/L radiolabeled-phenanthrene solution. At the end of the
4-d experiment, the resultant product was mixed with 500 mL of
methanol for 30 min, and the precipitate was then collected by
filtration through a 0.2-µm membrane filter. After freeze-drying,
the precipitate
was weighed and partitioned into sam ples for subsequent
analyses.
Triplicate precipitate sam ples of approximately 30 mg
each were analyzed for total radioactivity by combustion,
and other triplicate samples were extracted using a methanol
saponification extraction (MSE) method. The MSE method
has been shown to “loosen” SOM matrixes via cleavage of
certain ether and ester bond linkages through saponification
(27, 31). The method was selected on the basis of its superior
performance in preliminary studies to several other solvent
and Soxhlet extraction methods in terms of recovery and
reproducibility. The MSEs were performed in capped 13 ×
100- mm test tubes containing 7 mL of alkaline methanol
(m etha n ol:2 N KOH, 14:1 v:v) heated to 75 -80 C in a water
bath for 2 h. After the sam ples were cooled and centrifuged, a
0.5- mL aliquot of the extract was sam pled and mixed with 3
mL of the ScintiSafe Plus 50% liquid scintillation cocktail
for analysis of radioactivity, and another 1- mL sam ple was
taken for HPLC analysis. The remaining solve nt was carefully
withdrawn using a glass pipet to leave the residual solids
undisturbed in the test tube. Washing of these solids was
then done by repeated 1- mL fresh methanol additions,
mixing, centrifugation, and methanol withdrawal. This was
done until no radioactivity significantly above background
could be measured in the solvent. The 14C radioactivity of
the solid was then measured via combustion.
A Harvey Biological Material Oxidizer (R. J. Harvey
Instru ment Co., Hillsdale, NJ) was em ployed to combust the
solid sam ples, and the resulting 14CO2 was absorbed into a
15- mL volu me of carbon-14 cocktail. 14C radioactivity was
measured using a Beckman LS6500 liquid scintillation
counter (Beckman Instru ments, Inc.).
To com pare phenanthrene distributions in solid phenol-
coupling products resulting from sorption or mixing systems
in the absence of enzymatic coupling reactions, two control
sam ples were analyzed following the procedure described
above. One sam ple was prepared in a sorption system
containing 225 mg of precipitate prepared by phenol-
coupling reactions in the absence of phenanthrene mixed
with 500 mL of the radiolabeled phenanthrene solution. After
4 d of equilibration, the solution was mixed with 500 mL of
methanol for extraction, and the solid com ponent was
collected by filtration and freeze-dried prior to analysis.
Another sam ple was prepared by immersing 225 mg of the
precipitate formed in the absence of phenanthrene in 0.5
mL of methanol solution containing 80 mg/L radiolabeled
phenanthrene. The sam ple was shaken periodically to allow
the methanol to evaporate and to ensure thorough mixing
of phenanthrene and the precipitate.
Results and Discussion
Phenanthrene Transformation in the Reaction System.
Figure 2 illustrates both phenol conversion and phenanthrene
transformation from a dissolved state to a solids-associated state
in an enzymatic coupling system initiated at a reaction
strength of 250 µM; i.e., addition of 250 µM phenol, 250 µM
H2O2, and 1 unit / mL of HRP to the reaction system at the
initiation of each round of reaction. Five repetitions of the
250 µM reaction each day provided a cu m ulative amount of
5000 µM phenol to the reaction system over the 4-d course
of the experiment. Phenol concentrations remaining at the
end of each daily reaction set were measured by HPLC. Phenol
conversion was calculated by subtracting the measured
quantity of phenol remaining from the total quantity of
phenol added to the system to the point of sam pling and
measurement. It is evident in Figure 2 that phenol was nearly
com pletely converted under the experimental conditions
em ployed and that essentially linear phenol conversion
occurred over the 20 reactions steps of the 4-d experiment.
It is also evident in Figure 2 that the concentration of
VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4223
FIGURE 2. Phenanthrene transformation and cumulative phenol
conversionduring semi-batch operation ofan HRP-mediatedphenol-
coupling reaction system. Reactions repeated 5 times a day at 250
µM strength (i.e., 250µM phenol, 250µM H2O2, and 1 unit/mL HRP
added to the system for each round of reaction). Data points are FIGURE 3. Concentrations of phenanthrene extractable with 50%
the means of triplicate experiments with 1 SD error bars. methanol from semi-batch phenol-coupling systems of different
reaction strengths. Semi-batch operations were conducted 5 times
phenanthrene remaining in the aqueous phase dropped sharply a day as indicated in the Experimental Section. The dashed line
from 105.5 to 7.8 µg/L on the first day of the experiment and represents an estimate ofdilution effects caused by thesemi-batch
remained at or lower than that level for the remainder of the operation. Data points are themeans of triplicate experiments with
experiment. Conversely, concentrations of phenanthrene 1 SD error bars.
extractable with 50% methanol were m uch higher than the
aqueous-phase phenanthrene concentrations and continued to
decline steadily over the 4-d duration of the experiment. A blank
test with repeated additions of only
250 µM H2O2 and 1 unit / mL HRP (i.e., no phenol) was
performed in parallel to the reaction experiment. No ap- preciable
phenanthrene transformation was observed in this blank test,
indicating that HRP was not able to directly catalyze
phenanthrene oxidation under the experimental conditions em
ployed. Thus, although PAHs have been reported to function as
active substrates for certain lignin- degrading fungal peroxidases (
32-34), it can be concluded that the phenanthrene transformation
observed in the HRP- mediated reaction system studied here
occurred via phenol
conversion-associated mechanisms.
Similar trends with respect to decreasing levels of extractable
phenanthrene were observed in experiments involving a series of
phenol-coupling systems having different reaction strengths.
Figure 3 reveals that decreases of extract- able phenanthrene
correlate with reaction strength; i.e., the higher the reaction
strength, the greater the amount of unextractable phenanthrene.
With the experimental method em ployed, 2- mL aliquots of
reactants were added and 2- mL sam ples were withdrawn from FIGURE 4. Concentrations of dissolved phenanthrene remaining in
the 50- mL reaction solution in the semi-batch reactor each day of the aqueous phase in semi-batch phenol-coupling systems of
an experiment, yielding a 96% dilution effect per day. This different reaction strengths. Semi-batch operations were conducted
dilution effect, represented in Figure 3 by the blank symbols and 5 times a day as indicated in the Experimental Section. Data points
dashed line, can be seen to virtually coincide in terms of are the means of triplicate experiments with 1 SD error bars.
measured phenanthrene decrease with the decrease measured in a
control system absent of any phenol-coupling reaction (i.e., zero aqueous phase during phenol-coupling reactions; (ii) such
reaction strength), confirming that physical losses of removal is likely in large measure attributable to sorption of
phenanthrene over the 4-d experiment were negligible. phenanthrene on precipitated phenol-coupling products; (iii)
Phenanthrene concentrations remaining in the aqueous phases of a fraction of the solids-associated phenanthrene is converted
systems of differe nt reaction strength are shown in Figure 4. The into a form that cannot be recovered by 50% methanol
fact that the concentrations of phenanthrene remaining in the solution extraction; and (iv) the converted fraction of solids-
aqueous phase shown in Figure 4 are consistently much lower associated phenanthrene correlates directly with the phenol-
than the corresponding methanol extractable concentrations coupling reaction strength.
shown in Figure 3 indicates that significant reversible sorption of Sorption of Phenanthrene on Precipitated Phenol-
phenanthrene on precipitated phenol-coupling products likely Coupling Products. To quantify the extent of phenanthrene
occurred. sorption by precipitated phenol-coupling products, an experiment
The data presented in Figures 2 -4 lead to the conclusions using precipitate prepared by enzymatic coupling reactions in the
that (i) phenanthrene can be effectively removed from the absence of phenanthrene was performed. As described in the
Experim ental Section, the preformed

4224 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 18, 2003
 TABLE2. Distributions of Phenanthrenein Phenol-Coupling
Precipitates Prepared in Different Systems
concn concn of concn of
and (SD) 14C before concn in MSE extracts 14C after
systema units MSEb 14Cb phenanthrenec MSEb
reaction µg/g 102.1 88.9 69.2 5.7
(µg/g)d 1.0 1.6 1.3 0.1
%e 100.0 87.0 67.8 5.6
(%)d 1.0 1.6 1.3 0.1
sorption µg/g 19.6 18.5 18.7 6.9E-04
(µg/g)d 0.6 0.4 0.7 5.2E-03
%e 100.0 94.2 95.4 0.00
(%)e 2.8 1.9 3.5 0.03
mixture µg/g 116.3 112.7 111.2 -1.9E-03
(µg/g)d 2.4 0.4 2.6 5.1E-02
FIGURE 5. Comparisons of residual dissolved and extractable %e 100.0 96.9 95.6 0.00
(%)d 2.1 0.3 2.2 0.04
phenanthrene in the sorption and reaction systems.
a Systems and sample preparations are described in detail in

te Experimental Section. b 14C concentrations expressed as

h
TABLE 1. Estimates of Precipitated Product Formataion phenanthrene equivalent concentrations. c Measured by HPLC
and expressed as
concentrations in the precipitate. d Standard deviations calculated
concne SDe from measurements of triplicate samples. e Percentage of total
method (mg/L) (mg/L) radioactivity.
phenol conversion b 448.9 0.32
14C conversion b,c 432.9 0.26
d
a small faction of precipitated product was lost in the semi-
mass of precipitate 404.5 4.6 batch operation of the reactor systems. Given the results listed in
Table 1, it can be concluded that both phenol
a Data obtained for a reaction system involving 250 µM phenol
measured by weighing are somewhat lower than those estimated from
as described in the Experimental Section. b Calculated from mass the 14C conversion, probably because
balances. c Expressed as phenol equivalent concentrations. d
Measured by weigh- ing. e Standard deviations calculated from
triplicate experiments.

precipitate was added quantitatively to aliquots of phenan- threne

solution in differe nt amounts designed to give varying sorbent/
water ratios, and the concentrations of phenanthrene remaining in
the aqueous phase were measured after a 4-d period of
equilibration. The residual aqueous phase con- centrations of
phenanthrene, represented in Figure 5 as open
diamonds, decreased sharply as the sorbent/ solution ratio
increased, indicative of phenanthrene sorption by the
precipitated solids. The percentage of total phenanthrene
extractable from the sorption system with 50% methanol,
represented in Figure 5 as open squares, was substantial
(i.e., more than 90% recovery over the entire range of sorbent/
solution ratios).
It is evident then that phenanthrene sorption by pre- cipitated
products formed in HRP mediated phenol-coupling systems can
contribute to phenanthrene disappearance from the aqueous
phase. To evaluate this contribution more quantitatively requires
estimation of the quantities of pre- cipitate formed in the reaction.
Several means for doing so were examined in experiments
involving enzymatic coupling at the 250 µM reaction strength, as
described in the
Experim ental Section. The data for phenol and 14C conver-
sions obtained in this experiment are listed in Table 1 along
with masses of precipitate measured by weighing. Phenol
conversion can provide an estimate of precipitate formation
if it can be assu med that all of the phenol in a system is
converted to a precipitated form. This measure is likely to
overestimate actual precipitate formation because certain
polyphe nol-coupling products of appropriate molecular size
and configuration are soluble and thus not recoverable by
filtratio n. In this sense, 14C conversion should provide a closer
estimate because it excludes dissolved products. The esti-
mates based on phenol conversion given in Table 1 can be
observed to be only slightly greater than those based on 14C
conversion, indicating that most of the products were in fact
driven to a precipitate form under the reaction conditions
em ployed. As shown in Table 1, the actual precipitate quantities
VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4225
conversion and 14C conversion provide reasonable bases for
estimation of precipitate formation under the conditions
studied.
The phenanthrene transformation data obtained in the
phenol-coupling reaction systems and presented previously
in Figures 2 -4 can thus be re-evaluated using estimates of
precipitate quantity based on phenol conversion data. These
re-evaluated data are presented in Figure 5 as solid symbols.
The relative phenanthrene concentrations were calculated by
dividing the measured phenanthrene concentrations by the
total phenanthrene concentrations, with the dilution effect
shown by the dashed line in Figure 2 taken into account. It is
evident in Figure 5 that the phenanthrene concentrations
remaining in the aqueous phase and those extractable with
50% methanol are both m uch lower in the phenol-coupling
reaction systems than those in sorption systems having
essentially the same precipitate/ solution ratios. Therefore,
the phenanthrene removal from aqueous phase observed in
the reaction systems may involve more mechanisms than sim
ply sorption.
Phenanthrene Distributions in Precipitated
Products. A further understanding of the mechanisms
of phenan- threne transformation in the phenol-
coupling reaction systems can be obtained from
analyses of phenanthrene distributions in the
precipitated products. To this end and as described in
the Experim ental Section, precipitated products after
50% methanol extraction were obtained from a
coupling system run with radiolabeled phenanthrene at
the 250 µM strength. The total 14C concentrations in the
precipitated product samples were determined by
combus- tion followed by 14C radioactivity analyses.
Parallel sam ples were subjected to a strong extraction
procedure (methanol saponification extraction or MSE),
and 14C and phenanthrene concentrations in the extracts
were determined respectively by liquid scintillation
counting and HPLC analyses. 14C concentrations
remaining in the precipitates after MSE were measured
by combustion and radioactivity analyses. The 14C and
phenanthrene concentrations so measured in the
precipitates are presented in Table 2 as phenanthrene
equivalent concentrations. Two control sam ples, one
pre- pared by sorption of phenanthrene on pre-
precipitated products formed in a phenanthrene-free
reaction system and the other by sim ply mixing
phenanthrene with these pre- precipitated products,
were analyzed following the above

4226 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 18, 2003
procedure. To differe ntiate between the two control sam ples
and the precipitate sam ple prepared in the phenanthrene-
containing reaction system, the latter is referred to as
“reaction” sam ple in Table 2; the control prepared by
phenanthrene sorption is designated as the “sorption”
sam ple; and the other control is designated as the “mixture”
sam ple.
Of particular note in the data presented in Table 2 is the
fact that the 14C concentration in the reaction sam ple solid
phase (102.1 µg/ g) is m uch greater than that in the sorption
sam ple solid phase (19.6 µg/ g). The reaction sam ple was
prepared in a phenol-coupling reaction system at the 250
µM strength level, which yielded 448.9 mg/L precipitate as
estimated from the phenol conversion data, and the sorption
sam ple was prepared in a system containing 450 mg/L of
precipitate. The two systems were thus com parable in terms
of solid / solution ratios, and because the same phenanthrene
solution was used, the initial phenanthrene concentrations
were com parable as well. Eve n under these very similar
solution conditions, the precipitate in the reaction system
contains approximately 5 times as m uch phenanthrene as
that in the sorption system.
As also shown in Table 2, 94.2% of the total 14C in the
sorption sample was recovered in the MSE extract, phenan-
threne accounts for 95.4% of the total 14C, and no 14C was
measured in the precipitate after MSE. This indicates that
the 14C was nearly com pletely recovered as phenanthrene in the
MSE extracts. Similar results are observed for the mixture sam
ple. Conversely, 87.0% of the 14C in the reaction sam ple was
recovered in the MSE extract, the phenanthrene accounts for only
67.8% of the total 14C, and 5.7% 14C remains in the precipitate
after MSE. The significant differe nce between the 14C and
phenanthrene concentrations in the MSE extracts (i.e., 19.2%)
indicates chemical incorporation of phenan-
threne in the precipitated products. The 5.7% of 14C that
remains in the precipitate after MSE further supports that a
fraction of the phenanthrene is covalently bonded to the
precipitate matrix. Peroxidases catalyze phenol-coupling
reactions through the conversion of phenol substrates into
phenoxy radicals, which then polymerize through radical -
radical coupling processes. While phenanthrene itself may
be inert with respect to HRP catalysis, the phenoxy radicals
generated from phenolic substrates in the enzymatic reaction
are highly reactive. These radicals may indiscriminately attack
phenanthrene molecules and convert them into radical form
via hydrogen abstraction and / or free radical addition, which
would allow the phenanthrene to participate in the polym-
erization process and thus be chemically incorporated into
precipitated products. While such a radical transfer pathway
may not be kinetically favorable as com pared to alternative
phenoxy radical reaction pathways, its occurrence could
become evident under conditions of massive phenol con-
version, such as in the semi-batch reactor operations
em ployed in this research. These reaction conditions may
also be met in natural hu mification processes in which
phenolic substrates are cu m ulatively converted into soil
organic matter at relatively low reaction strengths over
geochemical time scales.
The fact that 67.8% of the total 14C in the reaction sam ple
was recovered as phenanthrene in the MSE extract suggests that
physical sorption is still the primary mechanism of phenanthrene
association with precipitated coupling prod- ucts and that
chemical incorporation contributes a m uch smaller irreversible
component. It is likely that the phenan- threne sorption process is
affected by the chemical bonding process, in that phenol-coupling
products are formed and growing in size while sorption occurs. It
can be rationalized that this dynamic sorption process thus
involves several unique features of sorbate distribution in
precipitated products. First, because the precipitate particles grow
in size
during the course of sorption, it is likely that phenanthrene tends
to concentrate in the core of the precipitate mass and becomes
more dilute toward its fresh outer surfaces. This would result in a
sorbate distribution profile diametrically opposite to that which
occurs in a general sorption process. Second, the configurations
of individual precipitate particles may go through significant
chemical and physical changes during their formation and growth
via phenol-coupling reactions. These reformation processes can
result in phenan- threne distributions discordant with site energy
distributions. Some phenanthrene molecules initially residing at
low-energy sites may thus become trapped in high-energy
microenvi- ron ments because of precipitate reformation process.
The potential effects of the aforementioned sorbent
growth and reformation process in dynamic sorbent systems
may be somewhat analogous to the aging effects frequently
observed in more static sorbent systems. In the latter type
of system, an organic sorbate tends initially to concentrate
on the sorbent surface and slowly migrate inward to the
sorbent core, thus manifesting a decreasing spatial gradient.
Provided an appropriate “aging time”, the sorbate diffuses
from lower energy sites at the outer surface to sites of higher
energy in the core. This aging effect usually leads to increased
sorption and decreased extractability of the sorbate ( 35, 36).
The same effective results may be obtained m uch more
rapidly in systems involving sim ultaneous dynamic sorbent
growth and reformation processes, and this may explain the
greater sorption and lower extractability of phenanthrene in
the actively precipitating reaction systems than in the
preformed precipitate sorption system (Figure 5). It is possible
that simultaneous sorbent growth and reformation processes
also influe nce other relevant system properties that are
governed by or related to sorbate distributions (e.g., enhanced
contaminant sequestration and reduced bioavailability) ( 37,
38). As illustrated in Figure 1, soil organic matter in natural
environ ments continues slowly but steadily to grow and
undergo reformation during hu mification processes. On the
basis of the findings reported here, it is possible that these
processes may exert unique influe nces on contaminant sorption,
sequestration, and bioavailablity.
Acknowledgments
We thank Sung Ho Kim for his diligent laboratory assistance and
valuable contributions to the experimental phase of the work and
Thomas Yavaraski for his assistance with instru- mentation. The
research was supported in part by the Environ mental
Management Science Program of the United States Department
of Energy (DOE) through Grant DE-FG07- 02ER63488 and in
part by Research Grant P42ES04911-14 from the National
Institutes for Environ mental and Health Sciences. The content of
this paper does not necessarily represent the views of either
funding agency.
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Received for review January 22, 2003. Revised manuscript
received June 18, 2003. Accepted June 25, 2003.
ES030330U