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The Fate of Hydrogen Peroxide as an Oxygen
Source for Bioremediation Activities within
Saturated Aquifer Systems
Mark Zappi a , Kenneth White b , Huey-Min Hwang c , Rakesh Bajpai d & Mohammad
Qasim e
a Department of Chemical Engineering , Mississippi State University
b Georgia Department of Natural Resources
c School of Science and Technology , Jackson State University , Jackson , Mississippi ,
USA
d Department of Chemical Engineering , University of Missouri-Columbia
e Environmental Laboratory, USAE Waterways Experiment Station , Vicksburg ,
Mississippi , USA
Published online: 27 Dec 2011.
To cite this article: Mark Zappi , Kenneth White , Huey-Min Hwang , Rakesh Bajpai & Mohammad Qasim (2000) The Fate
of Hydrogen Peroxide as an Oxygen Source for Bioremediation Activities within Saturated Aquifer Systems, Journal of
the Air & Waste Management Association, 50:10, 1818-1830, DOI: 10.1080/10473289.2000.10464207
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Zappi et al.
TECHNICAL PAPER
ISSN 1047-3289 J. Air & Waste Manage. Assoc. 50:1818-1830
Copyright 2000 Air & Waste Management Association
The Fate of Hydrogen Peroxide as an Oxygen Source for
Bioremediation Activities within Saturated Aquifer Systems
Mark Zappi
Department of Chemical Engineering, Mississippi State University
Kenneth White
Georgia Department of Natural Resources
Huey-Min Hwang
School of Science and Technology, Jackson State University, Jackson, Mississippi
Rakesh Bajpai
Department of Chemical Engineering, University of Missouri–Columbia
Mohammad Qasim
Environmental Laboratory, USAE Waterways Experiment Station, Vicksburg, Mississippi
ABSTRACT
priate biological activity. Hydrogen peroxide has been
commonly used as an oxygen source because of the lim-
ited concentrations of oxygen that can be transferred into
the groundwater using above-ground aeration followed
by reinjection of the oxygenated groundwater into the
aquifer or subsurface air sparging of the aquifer. Because
of several potential interactions of H2O2 with various aqui-
fer material constituents, its decomposition may be too
rapid, making effective introduction of the H2O2 into tar-
geted treatment zones extremely difficult and costly.
Therefore, a bench-scale study was conducted to deter-
mine the fate of H2O2 within subsurface aquifer environ-
ments. The purpose of this investigation was to identify
those aquifer constituents, both biotic and abiotic, that
are most active in controlling the fate of H2O2. The de-
composition rates of H2O2 were determined using both
equilibrated water samples and soil slurries. Results showed
H2O2 decomposition to be effected by several commonly
found inorganic soil components; however, biologically
mediated catalytic reactions were determined to be the
most substantial.
In situ bioremediation is an innovative technique for the
remediation of contaminated aquifers that involves the
use of microorganisms to remediate soils and
groundwaters polluted by hazardous substances. During
its application, this process may require the addition of
nutrients and/or electron acceptors to stimulate appro-
IMPLICATIONS
In situ bioremediation is an innovative treatment process
that results in the cleanup of both groundwater and aqui-
fer solids. The process typically involves the addition of
oxygen and nutrients for simulation of the natural bacte-
rial populations within the aquifer that ultimately use the
pollutant(s) as a food source. The benefits of this process
are reduced costs, treatment of contaminated aquifers
under existing structures, and low site worker exposure
to the pollutants.
This study focused on the fate of injected H2O2 within
various aquifer matrices. Hydrogen peroxide was utilized
as an oxygen source for in situ bioremediation. The re-
sults indicate that the bacterial populations themselves
had the greatest impact of H2O2 degradation. Naturally
occurring Fe had the next greatest impact, albeit at a much
lower rate than the biomass-related reactions. The pri-
mary conclusion is that prior to injection of H2O2 into pol-
luted subsurface environments, careful evaluation of po-
tential scavengers needs to be addressed, especially ori-
ented toward biotic reactions.
INTRODUCTION
Many aquifer systems in the United States have become
contaminated due to past military and industrial activi-
ties.1,2 Many of these contamination problems are com-
posed of highly biodegradable chemicals such as
1818 Journal of the Air & Waste Management Association
Volume 50 October 2000

Zappi et al.
petroleum hydrocarbons and wood-preserving products.3
In general, remediation techniques may be categorized
into two applications-based techniques: invasive and
noninvasive technologies. Invasive techniques are ex situ
treatment processes that require the excavation of soil
prior to application. Examples include incineration, so-
lidification/stabilization, composting, and bioslurry treat-
ment.4,5,6 Invasive technologies utilize highly engineered
reactor units that offer elevated levels of process control
and relatively rapid soil throughputs. The use of engi-
neered reactors with such high levels of process control
results in a treatment process that is kinetically more rapid
and complete in terms of contaminant destruction than
is typically afforded with noninvasive techniques. How-
ever, soil excavation and handling along with high capi-
tal and operational costs make invasive techniques more
expensive than noninvasive approaches. Also, the poten-
tial for worker and community exposure to the contami-
nants is greatly increased with the use of invasive
techniques.
Noninvasive techniques are inclusive of both in situ
and pump-and-treat systems. They are usually more cost-
effective than invasive techniques because of reduced
capital and operational costs. Noninvasive techniques
also offer an additional benefit in that they can be ap-
plied at facilities with existing structures, such as build-
ings or runways, that cannot be demolished to facilitate
soil excavation. Pump-and-treat systems involve ground-
water collection with subsequent treatment using wa-
ter treatment processes, such as carbon adsorption,
chemical oxidation, and activated sludge,4,7 followed by
reinjection of the groundwater effluents back into the
aquifer to facilitate aquifer flushing or disposal of the
treated water off-site. Pump-and-treatment systems are
sometimes costly and time-intensive because of prohibi-
tively slow removal of the contaminants due to sorp-
tive limitations associated with many organic
contaminants, resulting in potentially extended
remediation times.5,8 In situ systems essentially convert
portions of the aquifers into subsurface reactors. The
most common form of in situ treatment is biotreatment;6
however, soil flushing and vapor extraction have also
been successfully used.9,10,11
ticed form of in situ biotreatment involves the use of in-
digenous aerobic microbes.5,6,17 Typically, aerobic
biotreatment is used to degrade organic contaminants
within polluted aquifers because of rapid removal rates
and the achievable extent of contaminant degradation.6,13
During aerobic degradation, free molecular oxygen
accepts electrons released by an electron donor (typically
the contaminant), which is reduced to a lower oxidation
state. Oxygen, if not present in adequate concentrations
within biologically active zones, will limit the ability of
aerobic microorganisms to actively degrade contami-
nants.13,17 The rate of biotransformation, and thus con-
taminant persistence, has been reported to be controlled
by the transport of oxygen into the contaminated ground-
water, indicating that oxygen is usually the most limiting
factor within contaminated aquifer systems.13,18 The im-
portance of adequately supplying oxygen into targeted,
aerobic, biologically active zones cannot be overstated.
Unfortunately, the oxygen demands of an active in situ
biotreatment system can be quite high.
Fogel et al.19 estimated that 3 lb of oxygen are required
for every lb of petroleum product degraded. Goldsmith
and Balderson20 estimated a stoichiometric requirement
of 8.6 mol oxygen for every mol of diesel fuel degraded.
Bajpai and Zappi15 estimated a typical requirement of
0.5–1 g oxygen/g hydrocarbon biodegraded. They further
stated that oxygen to hydrocarbon dosing (w/w) require-
ments exceeding 4:1 are possible. Huling et al.18 estimated
a 5:1 ratio of oxygen to gasoline (w/w) requirement based
on laboratory column testing. It can easily be seen from
these references that the effective introduction of oxygen
into highly biologically active zones is paramount in the
design and maintenance of an effective in situ
biotreatment system.
There are several techniques that may be used for
effectively introducing oxygen into targeted biologically
active zones within an in situ bioremediation system
treating a saturated aquifer. Potential oxygen applica-
tion options include down-hole air sparging, above-
ground aeration/reinjection, or injection of H2O2 directly
into the aquifer.5,6,12 Dissolved oxygen, when introduced
via aeration, has a limited solubility in aqueous solu-
tions (~9 mg/L at 25 °C and 11 mg/L at 5°C). This solu-
bility limitation can severely hinder establishment of
areas of high biological activity due to the suppressed
oxygen levels associated with the inherent mass trans-
fer problems found with aeration-based equilibrium con-
centrations. Alternatively, oxygen may be delivered to
the subsurface in the form of H2O2, which is a clear,
odorless liquid that is readily available in large quanti-
ties and is fully immersible in water. The natural de-
composition of H2O2 provides the molecular oxygen
needed for aerobic microbial metabolic activity.21 Within
In situ biotreatment appears to offer the most prom-
ise of all of the in situ techniques under development for
remediation of saturated aquifers because of the ease of
application and the state of technology maturity. In situ
bioremediation utilizes microorganisms to remediate con-
taminated aquifers in biologically active zones established
within the contaminated soil structure.12,13,14,15 In most
cases, the stimulation of microorganisms within the sub-
surface requires the addition of electron acceptors and/or
nutrients.13,15,16 The most developed and commonly prac-
Volume 50 October 2000
Journal of the Air & Waste Management Association 1819

Zappi et al.
subsurface systems, H2O2 typically dissociates to produce
1/2 mol dissolved oxygen/mol H2O2, as shown in the
scheme illustrated below:18
that chelating and/or sequestering agents have been used
within H2O2 amending solutions to curb cationic reac-
tions.
Bajpai and Zappi15 suggested the use of phosphate
stabilizers to enhance the transportability of H2O2
through subsurface systems. Aggarwal et al.30 reported
limited success in stabilizing H2O2 solutions from both
biotic and abiotic reactions within a soil system.
Schumb29 reported significant decomposition of H2O2 at
both low (<3) and high (>7) pH values. This is not sur-
prising when considering the increased disassociation
of H2O2 at higher pH values (i.e., pKa = 11.6).31 However,
most research efforts that evaluated the fate of H2O2
within biologically active systems agreed that biologi-
cally based reactions (i.e., catalase) were the primary
mechanism for H2O2 degradation. Reaction of H2O2 with
naturally occurring organic matter is also possible based
on reactions reported between H2O2 and organically rich
soils;30,32 plus, the reaction of O3 with humic acids is well
documented as an oxidizer sink during water treatment,
indicating the susceptibility of most chemical oxidizers
to reaction with natural organic matter.33 Clearly, exces-
sive losses of H2O2 to nonbeneficial reactions (i.e., reac-
tion with soil constituents or excessive biotic reactions)
can result in significant increases in the overall cost of
remediation using in situ biotreatment.
An improved understanding of what aquifer sys-
tems, in terms of geobiochemical composition, are best
suited for the use of H2O2 as an oxygen source is key to
the design of effective aerobic in situ biotreatment sys-
tems. Therefore, a series of laboratory experiments was
performed to assess what H2O2 sinks within aquifer sys-
tems undergoing aerobic biotreatment most control
H2O2 fate within these subsurface environments. This
was accomplished through the study of the reaction of
H2O2 with various biologically (biotic) and
nonbiologically (abiotic) active soil types that were
considered characteristic of various geochemical con-
stituents found in aquifer systems.
H2O2 + H2O → 0.5 O2 + 2 H2O
(1)
Molecular oxygen, which can be supplied using H2O2
or pure oxygen sparging, has a solubility of 40–50 mg/L,22
representing at least a 4-fold increase in available oxy-
gen within a saturated aquifer environment. Other at-
tributes of H2O2 as an oxygen source for in situ
biotreatment are that H2O2 is (1) reasonably inexpen-
sive; (2) nonpersistent; (3) a stable liquid, which elimi-
nates problems with storage and introduction into the
aquifer; and (4) generally environmentally benign.21
Zappi et al.5 reported that some in situ systems initially
using groundwater injected back into the aquifer and
charged with oxygen via above-ground aeration were
forced to convert to H2O2 injection due to the inability
to keep up with the high oxygen demands of the sub-
surface biomass. Several reports on the use of H2O2 in-
jection to supply oxygen into subsurface biologically
active zones indicated various degrees of suc-
cess.17,19,22,23,24,25 Fogel et al.19 reported the successful
remediation of a petroleum hydrocarbon-contaminated
aquifer using H2O2 injection. Flathman et al.26 evaluated
H2O2 injection using a series of column experiments and
reported a high potential for the use of H2O2 as an oxy-
gen source for in situ treatment.
The primary problem with the use of H2O2 as an oxy-
gen source is the excessive reaction of the H2O2 with vari-
ous components of the aquifer environment, which can
dramatically impede the effective transport of the H2O2
into the biologically active zone.5,15,18,23 Spain et al.23 re-
ported significant losses of H2O2 attributed to catalase
degradation via the indigenous microorganisms that were
present within the aquifer. Catalase is a biological enzyme
(hemetin-containing) with an average molecular weight
of 240,000 that is produced by living cells as a removal
mechanism for excessive H2O2 produced during aerobic
respiration.27,28 Hinchee et al.17 reported pseudo-first-
order H2O2 decomposition rate constants ranging from
0.10 to 0.01 min–1 during field tests of H2O2 injection.
They reported that the predominant H2O2 sink appeared
to be biologically mediated catalase decomposition.
Reaction of H2O2 with naturally occurring cations
within soils has been reported.18,22,29 Transition metals
appear to be the most reactive metallic species. Schumb29
reported that Mn and Fe are very reactive with concen-
trated H2O2 solutions. Morgan and Watkinson22 reported
that Fe-based reactions between H2O2 and soil particles
have been problematic in terms of effectively transport-
ing the H2O2 into targeted treatment zones. They reported
MATERIALS AND METHODS
The general approach to this study was to divide the aqui-
fer environment into two separate physical compartments
(groundwater and soil) that contain naturally occurring
biological and chemical species that may react with H2O2.
The reactants found in either compartment in most in situ
biotreatment application scenarios were taken to be natu-
ral soil chemical constituents (cations, high pH, and or-
ganic matter) and active bacterial populations. In order to
segregate soil constituent effects based on a single predomi-
nant soil constituent, soils were collected from across the
United States based solely on their having a characteristic
chemical constituent that was dramatically higher than the
1820 Journal of the Air & Waste Management Association
Volume 50 October 2000

Zappi et al.
other constituents commonly found in U.S. soils.15 The
rationale for evaluating many soil constituents was to de-
velop an empirical database that can be used to assess the
relative difficulty that may be encountered if H2O2 injec-
tion was attempted at a given site. By using a suite of con-
stituents and respective soil types, this database would have
a universal appeal for use by environmental engineers when
designing in situ biotreatment systems.
plus, this specimen had average constituent lev-
els compared to the characteristics of the reported
composition of U.S. soils.15
(5) AVG (Warren County, MS)—another soil consid-
ered generally representative of an “average” U.S.
soil.
(6) HI-PH (Custer County, OK)—evaluation of both
elevated pH and high biomass density.
Materials
Analytical and Microbiological Methods
Water used in all experiments was distilled and deionized
(DDI water) prior to use. Solutions of H2O2 were formu-
lated from a 50% (w/w) solution (Fisher Scientific). A stock
solution of H2O2 was made by diluting the 50% H2O2 so-
lution with the DDI water to formulate an aqueous solu-
tion of 10,000 mg/L H2O2 (w/w). Several serial dilutions
of known concentrations of H2O2 were made on an as-
needed basis from this stock solution during testing.
Several soil samples, each having a unique character-
istic geochemical component that dominated its compo-
sition, were selected based on review of U.S. Geological
Survey databases. The soil samples were collected from
their native environments and shipped to the laboratory,
where they were air-dried and sieved through a No. 4 sieve
to remove debris and oversized detritus. Table 1 lists grada-
tion data for the soil specimens used in this study. Table 2
lists additional details on the geochemical and biological
characteristics of the soil samples used in this study. Table
3 provides a listing of the dominant soil characteristic
and respective sample ID coding used throughout this
paper. The rationale for including each soil specimen in
the experimental design and the original location of the
sample are detailed below.
A variety of analytical techniques for H2O2 were evalu-
ated. The reasons for evaluating several methods were
that poor light transmittance, complex soil chemical
matrix, and the rapid reactions associated with the soil-
water slurries used during this study made analysis of
H2O2 using traditional colorimetric techniques difficult
at best. A reflective colorimetric measuring system mar-
keted as the RQFlex Reflectometer by EM Scientific Inc.
was selected for water-phase H2O2 analysis because of ease
of operation, accuracy of results, and flexibility in terms
of water color/turbidity variation. This system uses a
color-change reaction based on H2O2 concentration that
is reflected off of a reactive strip into the detector of a
hand-held colorimeter with a preset wavelength emis-
sion band. The soil-water slurries were phase-separated
using centrifugation, and the H2O2 concentration within
the liquid phase was measured using the RQFlex system.
Due to the rapid decomposition of H2O2 within some of
the experimental matrices tested in this study, the cen-
trifugation and analysis of liquid centrates were per-
formed using an organized protocol of rapid
centrifugation and analysis that only took a few min-
utes to complete and that resulted in H2O2 concentra-
tions reflective of actual sampling time increments.
Microbial enumerations were determined using the
acridine orange direct count method, which gives total
bacterial populations using epifluorescent microscopy.
Epifluorescent microscopy allowed the direct observation
and total enumeration of viable versus nonviable micro-
organisms within the soil specimens in less time than that
required for other culturing methods (i.e., total heterotro-
phs). This technique is admittedly not as truly reflective
of the total microbiological character and population den-
sity present in each soil specimen as are other bacterial
enumeration techniques, such as fatty acid/phospholipid
analysis using a gas chromatography system; however, it
was considered a good choice for its intended use within
the framework of this study because it provided a com-
parative assessment of bacterial activity and relative popu-
lations within a rapid analytical time frame and an easy,
well-established laboratory procedure.
(1) Control (purchased from U.S. Silica Inc.)—a non-
reactive soil that allows for evaluation of experi-
mental losses.
(2) HI-FE (Pope County, AR)—evaluation of elevated
levels of both Fe and Mn.
(3) HI-TOC (Newton County, MS)—evaluation of re-
action with organic matter and associated micro-
bial consortia.
(4) HI-NA (LeFlore County, MS)—evaluation of Na,
Table 1. Soil specimen gradation data.
Soil Specimena
% Sand
% Silt
% Clay
Sand Control
Tellico Loam
Gessie
96
38
48
13
8
4
0
40
46
65
76
24
22
6
Aligator Clay
WES
22
16
20
The bulk mineralogy and the clay minerals content in
the five test soils (excluding the Ottawa sand used as a
nonreactive control) were determined by X-ray diffraction
Crot
56
aSpecimens are named after the location from which they were collected.
Volume 50 October 2000
Journal of the Air & Waste Management Association 1821

Zappi et al.
Table 2. Soil chemical and microbiological data.
Soil Specimen
Mineral
Content
Microbial
Elemental
Other Analytes (mg/kg)
Enumeration (#/mL)
Analysis (mg/kg)
Sand Control
Tellico Loam
Quartza
–
Very Low
Very Low
pH 6.8
Quartza
Hematiteb
7.0 × 106
51600
3850
671
Fe
Mn
P
6033
11
TOC
CEC
pH 6.6
580
K
416
Ca
Na
Fe
Ca
K
22
Gessie
Aligator Clay
WES
Quartza
1.4 × 107
2.0 × 107
2.1 × 107
1.4 × 108
17900
13300
983
14296
15
TOC
CEC
K-Feldspara
Na-Feldsparb
Dolomiteb
pH 7.2
655
P
647
Mn
Na
Fe
Na
Ca
K
42
Quartza
K-Feldsparb
Na-Feldsparb
16400
7503
2560
1560
514
7227
17
TOC
CEC
pH 5.5
P
462
Mn
Fe
Ca
K
Quartza
K-Feldsparb
Na-Feldspar2
21100
1440
1140
606
5320
11
TOC
CEC
pH 5.3
P
449
Mn
Na
Ca
Fe
Na
K
29
Crot
Quartza
K-Feldsparb
Na-Feldsparb
Amphiboleb
Calciteb
59500
13500
5570
4470
514
4746
14
TOC
CEC
pH 10.0
P
255
Mn
Note: Soil pH measured using slurry technique; aPrimary mineral component; bSecondary mineral component.
(XRD) analysis. This investigation was an attempt to obtain
as much information as possible, with an emphasis on link-
ing the observed mineralogy to the chemical properties of
the soils. In preparation for XRD of the bulk sample, a por-
tion of each sample was ground in a mortar and pestle to
pass through a 45-µm (no. 325) mesh sieve. For analysis of
the clay-size fraction, an aqueous slurry of the powder was
made, placed on a substrate, and allowed to air-dry over-
night at room temperature (23 °C). An XRD pattern was
collected on glycol for each sample. Bulk sample, random
powder mounts were analyzed using XRD to determine the
mineral constituents present in each soil specimen.
Table 3. Summary of dominant soil specimen characteristics.
Soil Specimen
Dominant Feature
Sample ID
The Fate of H2O2 in the Groundwater
Compartment
Sand
Clean Media
Fe and Mn
TOC
Control
HI-FE
HI-TOC
HI-NA
AVG
The reaction of H2O2 with solubilized chemical constituents
from each soil specimen was evaluated by monitoring the
reaction of H2O2 with waters equilibrated with each soil
sample. This allows for evaluation of any chemical solute
that derived from the soil phase that may degrade H2O2 [i.e.,
cations, humics, or enzymes (both inter- and intracellular
Tellico Loam
Gessie
Aligator Clay
WES
Na
Average Soil
pH/Ca
Crot
HI-PH
1822 Journal of the Air & Waste Management Association
Volume 50 October 2000

Zappi et al.
enzymes)]. Equilibrated waters were produced by mixing
20 g of each soil specimen with 80 mL DDI water for 24
hr in a 250-mL plastic bottle, and were mixed using a
recipritating shaker table. After 24 hr, the samples were
removed and centrifuged at 13,000 × g for 15 min, and
the centrate was filtered through a 0.45-µm membrane
filter. A second set of bacterial-free equilibrated water
samples were prepared just as the first set of samples, ex-
cept that these samples were autoclaved at 121 °C for 25
min after equilibration. Autoclaving was found by Spain
et al.23 to provide very efficient deactivation of catalase
activity within aquifer soils. Their results indicated that
effective deactivation was provided by autoclaving on a
similar scale to that achieved using HgCl2 amending.
Aggarwal et al.30 reported that autoclaving was more effi-
cient in deactivating catalase activity within soils as op-
posed to HgCl2 amending.
Hydrogen peroxide was dosed into the equilibrated
waters by adding a sufficient amount to achieve a final
dose of 20 mg/L. These tests were performed in triplicate
using 250-mL plastic bottles (autoclaved where appropri-
ate). Mixing within the reaction bottles was accomplished
by agitating the bottles using the reciprocating table.
Samples were collected at various intervals, depending on
observed rate of H2O2 depletion and the respective H2O2
concentrations measured during the previous sampling
event (none exceeded 20 min lapsed reaction time).
using batch shake experiments. Using the reflectometric
technique, the H2O2 was analyzed. The source of the bio-
mass, as measured by volatile suspended solids (VSS) ac-
cording to Standard Methods (1995), was an activated
sludge chemostat operated during a separate, yet concur-
rent study which involved acclimation of an aerobic con-
sortia to acetone to be used for the biodegradation of high
levels of ketones from a contaminated groundwater source.
Waste sludge taken from the chemostat was diluted to
targeted concentrations of VSS using distilled water. Re-
action with various levels of VSS were reacted with 200
mL of a 20-mg/L H2O2 solution contained in 250 mL plas-
tic bottles that were agitated using the shaker table.
Reaction with catalase was evaluated by dosing various
enzyme units of bovine catalase (Aldrich Chemicals Inc.)
into a 20-mg/L H2O2 solution contained in a 250-mL plastic
bottle also agitated using shaker table. During the active bio-
mass, VSS, and the catalase reaction experiments, H2O2 lev-
els within the liquid samples were monitored over time using
the reflectometric technique described above. Both experi-
ments were performed in triplicate.
RESULTS AND DISCUSSION
Soil Characterization
Tables 1–3 present information concerning the bio-
geochemical characteristics of the soil specimens used in
this study. From Table 1, it can be seen the specimens
ranged from well to uniformly graded soils. Most of the
soils were generally a silty clay, except for the sand con-
trol. Table 2 lists various information about each speci-
men. From the list in Table 2, Table 3 was drafted to
summarize the perceived predominant characteristic of
each specimen and to list the specimen ID codes that will
be used herein during discussion of the various soils. As
seen in Table 2, all specimens had nominal biomass den-
sities that are typical of most healthy, non-contaminated
soils.34 Not surprisingly, quartz was the main building-
block mineral, with several other minor minerals, for the
soil specimens used in this study (Table 2).
Fate of Hydrogen Peroxide within the Soil
Compartment
The fate of H2O2 within the soil compartment was evalu-
ated by reacting H2O2 with the various soil specimens us-
ing 25% (w/w) soil slurries. Soil slurries were formulated
by adding 20-g dry weight of each soil sample to 80 mL of
a sterilized 20-mg/L H2O2/DDI water solution. Two sets of
experiments were performed—one using the prepared soil
samples as is and a second set using soil samples that were
autoclaved prior to slurrying. This was done to segregate
the impact of active biodegradation from abiotic reactions.
The soil slurries were shaken for 24 hr using the recipro-
cating table. The H2O2 was monitored over time via peri-
odic analysis (<45 min for total experimental run time)
of the aqueous phase of the slurries for H2O2 concentra-
tion. Aqueous samples were collected from each bottle,
centrifuged, then filtered through a 0.45-µm membrane
filter, and the filtrate was analyzed for H2O2 concentra-
tions using the RQFlex system. These experiments were
also performed in triplicate.
Degradation of Hydrogen Peroxide in
Equilibrated Water Solutions
Table 4 lists the results of chemical analysis of the equili-
brated waters that were used during H2O2 fate experiments
([H2O2]o = 20 mg/L) within the groundwater compartment.
The concentrations shown are generally reflective of the
primary constituents of the soil specimens used in the
equilibration experiments (see Table 2).
The results of these experiments are presented in Fig-
ures 1 and 2 for the nonautoclaved and autoclaved water
samples, respectively. From both figures, only the HI-PH
water that was not autoclaved resulted in appreciable H2O2
degradation (Figure 1); however, this removal appears to
Reaction of Hydrogen Peroxide with
Biocatalysts
The fate of H2O2 during reaction with both viable bacterial
cells (biomass) and pure catalase enyzme was evaluated
Volume 50 October 2000
Journal of the Air & Waste Management Association 1823

Zappi et al.
Table 4. Chemical analysis of equilibrated waters.
Analyte
Control
HI-FE
HI-TOC
HI-NA
AVG
HI-PH
Ca
1.92
0.21
ND
1.51
0.034
0.025
1.09
99.3
0.10
2.13
64.2
5.25
24.9
2.76
15.4
86.4
0.683
0.7
7.27
60.4
9.52
4.28
275
63.8
712
Fe
Mn
0.096
0.286
11.8
9.52
442
Na
2.95
<10
Alka
NH3-N
Total-P
TOC
pH
64.2
0.562
0.239
65.9
7.20
2590
0.345
1.51
97.6
10.0
<0.02
<0.2
58.1
6.81
0.066
<0.2
0.08
2.01
75.2
5.52
50.7
Test Time, min
6.58
Figure 2. Reaction of H2O2 with equilibrated water (autoclaved).
be biotic in nature because this extent of removal was no
longer active after the sample was autoclaved (Figure 2).
This extent of reaction with the biocatalysts in the HI-PH
water is attributed to the microbial consortia that are
present in this highly alkaline soil. Skujins (1976) concluded
that bacteria living in alkaline systems have high catalase
activity, which is supported by the high reactivity
of the H2O2 observed with the HI-PH water experiments.
The other equilibrated waters resulted in negligible H2O2
degradation. The AVG soil equilibrated water that was not
autoclaved indicated a slight H2O2 removal potential (5
mg/L) that was expended within the first 5 min of test-
ing. A review of the microbial enumeration data presented
in Table 2 indicates the HI-PH soil contained bacterial
populations that were an order of magnitude higher than
the next highest populations of bacteria enumerated in
the other soil specimens. The AVG soil had the next high-
est bacterial counts of the other soil specimens, indicat-
ing a high reactivity of the H2O2 with the active biomass
(or enzymes) that was desorbed from the soil into the
equilibrated waters.
These results indicate that chemical constituents
(minerals) that were desorbed from the soil specimens were
present at levels that were not adversely reactive to the
dosed H2O2. Therefore, the reaction of H2O2 with chemi-
cal constituents found in typical groundwater appears to
be minimal, except for the presence of biologically active
agents such as viable bacterial cells.
It is realized that the organic contaminants within
the groundwater and soil compartments also may pose
an H2O2 sink. However, without some form of degrada-
tion into OH, H2O2 is not very reactive with most organic
contaminants.35 Zappi et al.36 evaluated the fate of H2O2
as an oxygen source for a suspended growth bioreactor
treating groundwater contaminated with high levels of
benzene. Their results indicated that the H2O2 was very
reactive to the biomass, but hardly reactive with the high
levels of benzene present in the bioreactor. Benzene has
been traditionally considered a very oxidizable organic
compound37 using powerful chemical oxidizers, such as
O3 and OH, but not H2O2, which has a much lower oxida-
tion potential. Therefore, based on these past efforts, most
of the organic contaminant(s) present in the aquifer sys-
tem undergoing treatment can be considered a minor fate
mechanism. However, a careful review of the reactivity of
the contaminant is recommended using methods de-
scribed by Zappi et al.36 for each contaminant type under
investigation for a given site or a series of simple reactiv-
ity experiments performed using actual site samples to
assess the overall reactivity of the total contaminant ma-
trix present in the aquifer system with H2O2. One inter-
esting aspect to the fate of H2O2 within a contaminated
aquifer system is that Alyea and Pace38 report that the
presence of organic chemicals, such as phenol, catechol,
and p-cresol, all had a stabilizing effect on H2O2 reaction
with catalase. Therefore, it is possible that some stabiliz-
ing effect may be realized when supplying H2O2 into con-
taminated aquifers (albeit minor, as witnessed by the
Test Time, min
Figure 1. Reaction of H2O2 with equilibrated water (not autoclaved).
1824 Journal of the Air & Waste Management Association
Volume 50 October 2000

Zappi et al.
reports discussed earlier concerning excessive H2O2 losses
during actual in situ bioremediation activities).
Degradation of Hydrogen Peroxide within the
Soil Compartment
Results of the experiments evaluating H2O2 fate within
the soil compartment are presented as Figures 3 and 4.
Figure 3 presents the results of H2O2 reactivity studies us-
ing soil samples that were not autoclaved, making them
biologically active. These data indicate that the HI-FE and
HI-TOC soils provided the most rapid H2O2 sinks of all
the soils tested. Both resulted in complete removal of H2O2
within 8 min of testing. The HI-FE soil was expected to
provide a significant sink due to the Fe-H2O2 reactions.
Based on the results from the equilibrated water experi-
ments, the high levels of H2O2 reactivity observed with
HI-TOC soil were surprising.
However, Aggarwal et al.30 reported that soil contain-
ing high TOC levels provides conditions conducive to the
growth of microorganisms with high catalase-production
capability. The reason that this level of activity was not
observed in the equilibrated water experiments is likely
due to strong sorptive bonds of the enzymes on the el-
evated humic fractions found on soils containing high
levels of organic matter.39,40 The HI-PH soil provided the
next highest level of H2O2 degradation by achieving com-
plete removal within 12 min of reaction. The ability of
the HI-PH soil specimen to degrade the H2O2 supports the
data generated during the H2O2 fate experiments using
the nonautoclaved equilibrated waters, in which the HI-
PH soil was the only sample to exhibit an appreciable H2O2
loss. The HI-NA and AVG soils were the next most reac-
tive, respectively. The only soil sample not to show sig-
nificant H2O2 decay was the sand control. This is not
surprising, considering the lack of reactive sites on a quartz
sand particle.
Test Time, min
Figure 4. Reaction of H2O2 with soil (autoclaved).
The soil specimens were then reacted with the H2O2
after autoclaving to eliminate the presence of biologically
active agents. These data are shown in Figure 4. The im-
pact of autoclaving (i.e., deactivation of biocatalysts) on
the extent and rate of H2O2 degradation was dramatic. The
HI-FE soil exhibited the least change, yet the time required
to remove the H2O2 to below detection limits was increased
from 8 min to over 15 min reaction time (see Figures 3
and 4). With the other soil specimens, the impact of auto-
claving on H2O2 removal appeared to be even more signifi-
cant. This strongly suggests that a large fraction of the H2O2
loss observed in the nonautoclaved soils is attributable to
biotic mechanisms. This observation is supported by those
of others who evaluated the fate of H2O2 within soil sys-
tems.23,41,42 Unfortunately, none of these studies reported
rate constants or rigorously separated soil compartment
reactions. However, their reported conclusions do support
the present conclusion that biotic reactions appear to domi-
nate H2O2 fate within soil systems.
Table 5 lists the pseudo-first-order rate constants cal-
culated from the data presented in Figures 3 and 4. Clearly,
deactivation of the biological activity via autoclaving dra-
matically reduced, but not eliminated, the H2O2 decay
reactions. Using the assumption that autoclaving fully
deactivated all of the biological agents, it can be stated
that Fe clearly appears to most impact the abiotic fate of
H2O2 within soil matrices. This observation is not surpris-
ing, given that the reaction of H2O2 with ferrous iron to
form ferric iron is well-documented as “Fenton’s reaction,”
shown below, which has been used for water and soil treat-
ment due to the production of OH as an advanced oxida-
tion mechanism.43,44,45
Fe2+ + H2O2 → Fe3+ + OH. + HO–
Fe3+ + H2O2 → HO2. + H+ + Fe2+
Fe2+ + OH. → OH– + Fe3+
(2)
(3)
(4)
Test Time, min
Figure 3. Reaction of H2O2 with soil (not autoclaved).
Volume 50 October 2000
Journal of the Air & Waste Management Association 1825

Zappi et al.
expected that the reduced iron (Fe2+)
present in the HI-FE soil would also
be oxidized to Fe3+, resulting in the
loss of a Fenton’s reactant, if auto-
claving provided that significant of
an oxidative step, which was not the
case, as seen on review of the HI-FE
data in Figure 3.
Table 5. First-order rate constants for soil-slurry experiments.
Soil System
Rate Constant, min–1
Rate Constant, min–1
dK a (min–1)
(r2) Autoclaved
(r2) Not Autoclaved
HI-FE
HI-TOC
HI-NA
HI-PH
AVG
0.26
0.075
0.043
0.043
0.0092
0.0061
(0.99)
(0.98)
(0.97)
(0.95)
(0.63)
(0.98)
0.51
0.38
(0.89)
(0.99)
(0.99)
(0.87)
(0.77)
(0.93)
0.25
0.305
0.087
0.297
0.131
–0.0004
0.13
The evaluation of the H2O2 re-
0.34
action data showed that the degra-
dation of H2O2 follows first-order
kinetics (i.e., all r2 > 0.9). Therefore,
assuming that the degradation of
H2O2 within a soil slurry follows first-
0.14
Control
0.0057
adK = Knot-auto – Kauto
.
order kinetics for both biotic and abiotic reactions, the
removal of H2O2 can be presented as
HO2. + Fe3+ → O2 + H+ + Fe2+
H2O2 + OH. → H2O + HO2.
(5)
(6)
(7)
Fenton’s reaction is maximized at an acidic pH be-
cause of increased solubility of soluble, reduced Fe
species (i.e., Fe2+) that initiate radical production. This re-
action does occur at neutral to slightly basic pH and is
not limited to acidic pH; however, the rate is reduced be-
cause of lower aqueous phase, reduced Fe species concen-
trations. The above reactions highlight several H2O2 fate
mechanisms associated with Fenton’s reaction that are
possible within soil media. Several sources of naturally
occurring Fe species capable of mediating these reactions
that can be attributed to H2O2 degradation have been de-
tailed in the literature.46,47,48 Candidate species reported
include simple ferrous iron (Fe2+), geothite
(a-FeOOH), and ferrous sulfates (e.g., FeSO4). Soils typi-
cally serve as a good reservoir of Fe compounds, as dis-
cussed by Dragun and Chiasson.49 They list Fe
concentrations within U.S. soils ranging from 100 to over
100,000 mg/kg. Additionally, OH produced via Fenton’s
reagent could also degrade organically bound Fe, which,
in turn, once liberated can further serve as a scavenger for
more H2O2 as shown in the above reactions.
where Chp is [H2O2], mg/L; Kbiotic is the first-order rate con-
stant for biotic reactions, min–1; and Kabiotic is the first-
order rate constant for abiotic reactions, min–1.
The above equation can be rearranged as follows:
(8)
and
(9)
where K = Kbiotic + Kabiotic. Therefore, the rate constant for
the abiotic reaction can be calculated from the first-order
constants calculated from each H2O2 fate experiment us-
ing the following relationship:
Kbiotic = K – Kabiotic
(10)
By assuming autoclaving inactivated all or most of
the biological activity, for the nonautoclaved soil
The most dramatic change in H2O2 decay reactions
was found to be with the HI-TOC and HI-PH soils [Kno-auto
vs. Kauto (see Table 5)]. Based on a review of Table 2, it can
be seen that the HI-PH soil had the highest biomass con-
centration of all the soil specimens by almost a full order
of magnitude; therefore, a dramatic change in H2O2 de-
cay rate due to biocatalyst inactivation is not unexpected.
A similar fate must have been experienced by the
biocatalysts associated with the HI-TOC soil. It is also
possible that the high heat and oxidizing conditions
present within the autoclave may have oxidized much of
the oxidizable TOC within the soil, resulting in the re-
moval of a significant oxidizer sink. However, it would be
Kno-auto = K = Kbiotic + Kabiotic
(11)
Thus, the first order rate constant for the experiments
using the autoclaved soil can be expressed as for each soil
specimen as
Kauto = Kabiotic
(12)
The difference between the rate constants generated
during the autoclaved and nonautoclaved soil specimens
can be assumed to be Kbiotic for each soil specimen. These
values are listed as “dK” in Table 5. The values of Kbiotic cal-
culated using this method are relatively consistent between
1826 Journal of the Air & Waste Management Association
Volume 50 October 2000

Zappi et al.
each soil specimen, ranging from 0.087 to 0.305 min–1.
These values indicate that the biomass present in all soil
specimens had significant activity toward H2O2. To evalu-
ate if biomass density (microorganism populations) cor-
related directly with H2O2 activity, regardless of the
possible difference in microbial consortia physiological
composition, a plot of acridine orange counts versus H2O2
degradation rate constant was drafted as Figure 5. The data
imply that the soil microbial consortia established under
the unique conditions associated with that soil ecosys-
tem can have vastly differing H2O2 degradation activity
(assumed to be catalase activity). This agrees with some
of the published efforts indicating soil ecology can have
an impact on the magnitude of catalase activity.31,50
Based on a review of the rate constants listed in Table 5,
it can be stated that biotic mechanisms appear to be the
major mechanism of H2O2 degradation. As stated earlier,
this conclusion is supported by other studies. The rapid
degradation of H2O2 in the HI-FE soil indicates that the
degradation of H2O2 via Fe-based catalysis (likely a Fenton’s
reaction) also is a major removal mechanism when com-
pared with results obtained for the other soil specimens.
The presence of high TOC (HI-TOC) appears to have an
equal impact with high pH (HI-PH) on H2O2 degradation
(see Table 5). The HI-NA and AVG soil specimens resulted
in rate constants of approximately one-third that for the
other soils, but within the range reported by Hinchee and
Downey.51 The lesser degree of H2O2 degradation observed
with the HI-NA and AVG soil specimens is probably more
realistic for most soil undergoing biotreatment, since the
other soil specimens were selected because they represent
extremes in terms of at least one of the soil constituents.
Therefore, the H2O2 degradation rate constants calculated
for the HI-NA and AVG soils can be used as good esti-
mates when designing in situ biotreatment systems for
soil of “average” composition, if testing on the actual site
soil is not planned.
Test Time, min
Figure 5. Degradation rate of H2O2 vs. soil biomass level.
of this plot, which was calculated to be 0.539
L/(min*enzyme units). This method of kinetic data analy-
sis is described in more detail by Kuo et al.52
Figure 8 presents the reaction data for H2O2 with vari-
ous levels of active biomass (represented as VSS). The rate
constants for VSS levels of 20, 50, 100, and 200 mg/L VSS
were calculated to be 0.027, 0.078, 0.11, and 0.27 min–1,
respectively. The correlation of fit constants for the semi-
log plot regressions were all in excess of 0.9, indicating
acceptable fit. Figure 9 presents an X-Y plot of VSS versus
respective H2O2 rate constant. The calculated overall rate
constant is 0.0013 L/(min*mg).
These data present pseudo-first-order H2O2 degrada-
tion rate constants of similar magnitude to those estimated
for the biotic activity observed with the soil specimens.
These data collectively indicate the high level of reactiv-
ity that biomass and associated enzymes possess for H2O2
degradation. It is interesting to ponder that the biochemi-
cal reactions deemed of targeted interest (i.e., biotic utili-
zation of the oxygen from the H2O2) are also the same
reactions that can account for excessive degradation of
Reaction with Biocatalysts
Figure 6 presents the results from the reactivity experi-
ments of H2O2 with bovine catalase. These data indicate
that the higher the catalase concentration, the more rapid
the degradation of H2O2. From these data, first-order rate
constants of 0.54, 0.051, and 0.0092 min–1 for catalase
concentrations of 1, 0.1, and 0.01 enzyme units, respec-
tively, were calculated with correlation of fit values all in
excess of 0.9, indicating acceptable statistical fit. These
rate constants were then plotted on an X-Y plot of cata-
lase concentration versus rate constant (Figure 7). The
straight line indicates that the overall reaction of H2O2
with catalase is a second-order reaction (first order with
respect to both catalase and H2O2 concentrations) with
the overall kinetic rate constant represented by the slope
Test Time, min
Figure 6. Reaction of H2O2 with catalase (CAT).
Journal of the Air & Waste Management Association 1827
Volume 50 October 2000

Zappi et al.
Catalase Concentration, enzyme units/L
Figure 7. Determination of overall rate constant. Reaction of H2O2 with catalase.
the H2O2 upon introduction into biologically active zones
within an in situ biotreatment system.
organisms when used in large concentrations (i.e., greater
than 2500 mg/L).12 Our findings are supported by previous
studies pertaining to H2O2 decomposition. Biological sinks
attributed to high catalase activity pose the greatest impedi-
ment to H2O2 transport. Therefore, as biomass production is
stimulated within the targeted subsurface treatment zones,
the difficulties with maintaining effective oxygen tensions
will heighten as the oxygen levels exceed solubility (~40 mg/
L), causing excessive molecular oxygen losses via produc-
tion of bubbles, which tend to float upward into useless aqui-
fer zones overlying the targeted active areas. This loss prevents
the oxygen from being beneficial
ENGINEERING SIGNIFICANCE
Understanding the fate of H2O2 in the subsurface environ-
ment is essential to its efficient use as an oxygen source in
the in situ biodegradation process. A substantial amount of
literature exists on the use of H2O2 as an oxygen source for
bioremediation of a variety of contaminants.15,18,21,36,51 How-
ever, many of these sources express concern over the rapid
decomposition of H2O2 as well as its toxic effects on micro-
to downstream bacterial popula-
tions. In essence, as oxygen de-
mand increases with increased
biomass, so does the reactivity of
the H2O2 with the biotic compo-
nent of the aquifer matrix.
In terms of abiotic reactivity,
Fe appears to be the most reac-
tive species evaluated, followed
closely by TOC, pH, and
monovalent cations. The im-
pact of these chemical constitu-
ents on H2O2 fate generally
followed the impacts of these
constituents on the fate of O3
(another oxidizer) within water
Test Time, min
matrices containing similar
chemical species.33
Figure 8. Reaction of H2O2 with biomass.
1828 Journal of the Air & Waste Management Association
Volume 50 October 2000

Zappi et al.
Biomass (VSS) Concentration, mg/L
Figure 9. Determination of overall rate constant. Reaction of H2O2 with biomass.
extended to Messrs. Daniel Averett and Norman
Francingues, WES, for their support and encouragement
during this effort. Also, the technical discussions with Dr.
Herb Fredrickson, WES, concerning the evaluation of these
data are deeply appreciated. Permission to publish this
information was granted by the Chief of Engineers.
CONCLUSIONS
The results of this study indicate that the groundwater
compartment of an aquifer system will generally have a
small impact on the fate of H2O2, except when large quan-
tities of suspending bacteria are present. The soil-slurry
experiments clearly indicate that biotic mechanisms are
the predominant fate mechanism for H2O2. Reaction with
Fe and, although not directly, other studied reactive cat-
ions, such as Mn, also appears to be a significant sink for
those soils containing high levels of these cations. The
degradation of H2O2 within all of the systems tested were
adequately modeled using the pseudo-first-order kinetic
model. The reaction of H2O2 with biocatalysts was found
to be an overall second-order reaction that is first order
with respect to either reactant. The overall implication
of this study was that reaction with biomass will greatly
impact the transport of H2O2 as compared with those
abiotic reactions associated with soil chemical constitu-
ents, with the possible exception of reaction with the
cations.
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About the Authors
Mark Zappi (corresponding author; e-mail:
zappi@che.msstate.edu) is a professor of Chemical
Engineering at the Dave C. Swalm School of Chemi-
cal Engineering at Mississippi State University. Ken-
neth White is an environmental scientist at the Geor-
gia Department of Natural Resources. Huey-Min
Hwang is an associate professor of Biology at Jack-
son State University in Jackson, MS. Rakesh Bajpai
is a professor of chemical engineering at the Univer-
sity of Missouri, Columbia. Mohammad Qasim is a
research scientist at the USAE Waterways Experiment
Station, Vicksburg, MS.
1830 Journal of the Air & Waste Management Association
Volume 50 October 2000