SEPA
United States
Environmental Protection
Agency
EPA/540/SR-96/505
August 1997
SUPERFUND INNOVATIVE
TECHNOLOGY EVALUATION
Emerging Technology
Summary
Innovative Methods for Bioslurry
Treatment
The tests reported herein were con-
ducted by IT Corporation (IT), Knox-
ville, TN, to investigate the feasibility
of combined biological and chemical
treatments to treat polycyclic aromatic
hydrocarbons (PAHs). Bioslurry treat-
ment of PAH-contaminated soils was
demonstrated under the Superfund In-
novative Technology Evaluation -
Emerging Technology Program (SITE
ETP) as an extension of research pre-
viously funded by IT (Brown and
Sanseverino 1993) and additional in-
vestigations supported by the U.S. En-
vironmental Protection Agency (U.S.
EPA) (Davila et al. 1994). All testing
was initiated in September 1994.
During the demonstration, IT oper-
ated two 60-liter (L) TEKNO Associates
bioslurry reactors (Salt Lake City, UT)
and a 10-L reactor in series under
semicontinuous, plug-flow mode for a
7-month period. The first 60-L reactor
received fresh feed daily and supple-
ments of salicylate and succinate to
enhance PAH biodegradation.
Slurry from the first reactor was fed
to the second 10-L reactor, where
Fenton's reagent (Fe*N-H2O ) was added
to accelerate chemical oxidation of 4
to 6-ring PAHs. The third reactor in
series was used to biologically oxidize
contaminants remaining following ad-
dition of Fenton's reagent. This reactor
received no additions of salicylate and
succinate and was aerated, nutrient
amended, and pH adjusted only.
During operation, the reactor system
demonstrated total PAH and carcino-
genic PAH (CPAH) transformation up
to 95% and 84%, respectively.
This Summary was developed by
EPA's National Risk Management Re-
search Laboratory, Cincinnati, OH, to
announce key findings of the SITE
emerging technology project that is
fully documented In a separate report
of the same title (see ordering informa-
tion at back).
Introduction
Among the types of organic contami-
nants present in Superfund soils, complex
PAH constitute one of the more challeng-
ing class to remediate. Sites that contain
PAH contamination include coal gasifica-
tion sites, wood-treating facilities, petro-
chemical facilities, and coke plants. Soil
contamination by PAH is a large environ-
mental problem in the United States and,
using traditional biological and chemical
treatments separately, desirable target lev-
els for PAH cleanup may not be reached
in a timely desirable manner. Therefore,
this investigation focussed on increasing
the rate and extent of PAH oxidation us-
ing a combination of biological and chemi-
cal treatments.
PAHs are characterized by high organic
partition coefficients, low aqueous solubil-
ity, and low vapor pressures. These char-
acteristics result in the highly sorptive
nature of PAH and their subsequent lim-
ited availability to microbial populations.
Printed on Recycled Paper
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Us past experience with PAH-contami-
nated soils indicated that contaminant de-
sorptbn from soil is the rate limiting factor
in bioremediation (Sanseverino et al.
1993). Manipulation of parameters such
as pH, agitation, and temperature, as well
as the addition of surfactants or solvents,
can be used to enhance the rate of des-
orptfon, increasing the rate of biodegrada-
tfon.
Bioslurry reactors can provide rapid bio-
degradation of contaminants due to en-
hanced mass transfer rates and increased
contaminant-to-microorganism contact.
These units are capable of treating high
concentrations of organic contaminants in
soils and sludges, with demonstrated bio-
degradation of selected contaminant con-
centrations ranging from 2,500 to 250,000
milligrams per kilogram (mg/kg). In gen-
eral, the percent removal of PAH in these
systems ranges from 70% to 95%, with
30% to 80% reduction of the carcinogenic
PAH (CPAH) fraction (EPA 1990). The
primary objective of this SITE project was
to achieve greater than 80% CPAH trans-
formation with subsequent increases in
PAH transformation.
Process Description
The primary objective of reactor 1 (R1)
operation was to increase the biological
transformation of organic carbon. Salicy-
late was used to induce the naphthalene
degradation operon on NAH plasmids. It
was assumed that NAH plasmids were
naturally occurring in microbial populations
indigenous to subject soils. This operon
has also been shown to degrade phenan-
threna and anthracene (Sanseverino et
al. 1993), The naphthalene pathway may
also play a role in CPAH metabolism.
Succinate, a by-product of naphthalene
metabolism, served as a general carbon
source in R1 which removed easily de-
gradable carbon and increased biological
activity against more recalcitrant PAH (i.e.,
4-ring compounds and higher).
Slurry from R1 was fed to reactor 2
(R2) where Fenton's reagent was continu-
ously introduced, resulting in chemical oxi-
dation being the primary mechanism for
PAH transformation in this reactor. The
pH in R2 was maintained at 2.0 following
the addition of R1 slurry. Fenton's reagent
(hydrogen peroxide in the presence of re-
duced iron salts) produces free radicals,
effective in extensively oxidizing multiring
aromatic hydrocarbons in both soil and
water systems (Gauger et al. 1991; Kelley
et al. 1991; Elizardo 1991; Frisbie 1992).
Because hydroxylation of high-molecular-
wetght PAHs is generally the rate-limiting
step in biological oxidation, the objective
of adding Fenton's reagent was hydroxy-
lation of PAH, not mineralization. Reactor
3 (R3) was used for biological oxidation of
R2 slurry. R3 received no additions of
salicylate and succinate. The reactor was
aerated, nutrient amended, and pH ad-
justed following the introduction of R2 feed.
This study differs from other investiga-
tions in the reactor configuration which
allowed for evaluation of biological treat-
ment followed by chemical oxidation, and
chemical oxidation followed by biological
treatment. In addition, the introduction of
organic supplements to increase biologi-
cal oxidation of PAH, and minimization of
Fenton's reagent addition were evaluated.
Bioslurry reactors can aerobically bio-
degrade aqueous slurries created through
the mixing of soils or sludges with water.
The most common state of bioslurry treat-
ment is batch; however, continuous-flow
operation can be achieved. Continuous-
flow operation is preferred over batch due
to the maintenance of a higher average
reaction rate (Benefield and Randall 1985).
Aeration is provided through floating or
submerged aerators or compressors and
spargers. Mixing may be achieved through
aeration alone or in conjunction with me-
chanical mixers. Nutrient addition and pH
adjustment are accomplished through me-
tered chemical addition to the reactor. Fol-
lowing aeration, the treated slurry is
dewatered via standard dewatering equip-
ment, such as clarifiers or filter presses.
The residual streams created during
bioslurry treatment include treated solids,
process water, and possible air emissions.
The process water collected during the
solids/liquid separation phase is recycled
for influent waste stream slurrying or dis-
charged under permit. Air emissions may
be controlled through air pollution control
devices.
Full-scale commercial bioslurry units re-
quire approximately 0.5 to 1 acre per mil-
lion gallons of reactor volume (EPA 1990).
Reactor size is determined based on the
hydraulic retention time (HRT) required
for treatment. Retention times are estab-
lished based on the biodegradability of
the waste, level of treatment required, in-
fluent contaminant concentration, and
physical/chemical nature of the waste.
Major issues of concern during bioslurry
treatment system design include reducing
system HRT and increasing the rate and
extent of contaminant biodegradation.
Test Procedures
PAH and CPAH-contaminated soils were
collected from a southeastern wood-treat-
ment facility. The soils, sand (30%) with
silt and clay (70%), were wet-sieved on
site through a 30 mesh screen and sub-
mitted to IT's Biotechnology Applications
Center (BAG) located in Knoxville, TN, for
bioslurry testing. Oversized material was
disposed of on site. Blended slurry PAH
and CPAH maximum concentrations were
6,120 and 434 mg/kg, respectively. Wet
sieving the soils increased the uniformity
of the slurry, thereby reducing the poten-
tial for sampling variability.
All reactors were loaded to establish
40% total solids (TS) slurry of the screened
soil and operated as batch reactors for 2
weeks prior to the initiation of
semicontinuous flow. The 40% solids load-
ing was required to suspend the sandy
slurry. Following 3 months of operation,
the initial 40% solids loading was de-
creased to a set point of 30% to reduce.
the total mass of organic carbon loaded to
the system. Additionally, in order to main-
tain suspension of the slurry, the silt and
clay content of the feed was increased to
greater than 75% by blending clean, clayey
soils with the highly impacted screened
material.
No microbial cultures were added to the
reactors during the project. All reactors
were operated at approximately 25°C. Dur-
ing treatment, several operational difficul-
ties were encountered, including significant
foaming and tar ball formation. To reduce
foaming, antifoaming agents were manu-
ally added, diffused air sparging de-
creased, and mixing intensity reduced. Tar
balls were physically removed prior to con-
tinuous-flow operation.
During semicontinuous flow, 6 L/day of
influent slurry was manually introduced to
R1, resulting in a HRT of 10 days. To
induce biological activity for PAH biodeg-
radation, salicylate and succinate were
added on a weekly basis to achieve final
reactor concentrations of 86 and 4.4 mg/
L, respectively. This addition rate was in-
creased to 3 times per week after 4 weeks •
of operation to increase PAH transforma-
tion in R1. Salicylate and succinate addi-
tion was discontinued following four
months of operation to evaluate its effect
on biological oxidation and to reduce the
amount of organic carbon in the system.
The second reactor in series (R2) re-
ceived dilute concentrations of Fenton's
reagent to accelerate chemical oxidation
of PAH. Fenton's reagent was prepared
by mixing a 1:1 volumetric ratio of 30%
hydrogen peroxide and 8.4 millimolar (mM)
iron sulfate solution. The iron sulfate solu-
tion and the hydrogen peroxide were indi-
vidually applied to R2 below the slurry
surface at a combined rate of 2.0 L/day.
The introduction of Fenton's components
was simultaneous. The influent feed rate
to R2 was 6 L/day, resulting in a substrate
HRT of 1 day. The addition of Fenton's
reagent maintained the reactor pH at 2.
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The last reactor in series (R3) was used
for the transformation of any partially oxi-
dized contaminants remaining following
addition of Fenton's reagent. PAH and
CPAH concentrations following chemical
oxidation in R2 averaged 947 and 140
mg/kg, respectively. The system received
8.0 L/day of influent feed from R2, result-
ing in an HRT of 7.5 days. All operational
parameters were maintained similar to R1,
however, no salicylate and succinate ad-
ditions were made. The pH was buffered
in the reactor due to the addition of R2
contents. Table 1 illustrates the initial op-
erating set points for all reactors.
After four months of operation, the HRT
in R1 and R3 was increased to 20 days.
R2 HRT was increased to 2 days. This
change was initiated to decrease the
amount of total carbon introduced to the
reactors and extend the microorganism-
to-contaminant contact time.
Reactor pH, dissolved oxygen, ammo-
niacal nitrogen, and ortho-phosphate con-
centrations were monitored weekly during
system operation. During operation, slurry
in R1, R2, and R3 was monitored weekly
for TS, volatile solids (VS) concentrations,
and slurry density. Microbial enumeration
of total heterotrophs and naphthalene de-
graders was also conducted on the same
schedule, using spread plate techniques.
Aqueous- and solid-phase PAH concen-
trations in R1, R2, and R3 were also moni-
tored once per week. Grab slurry samples
were collected from the middle sample
port on each reactor, centrifuged, and the
solid- and aqueous-phases submitted for
analysis using high performance liquid
chromatography. Volatilization of constitu-
ents was monitored monthly in R1 off-gas
Table 1. Initial Operational Setpoints
Parameter
through the analysis of XAD sorbent me-
dia. All PAH concentrations in aqueous
phase, solid phase, or in sorbent media
were determined using modified EPA
Method 8310.
Results
The bioslurry reactor system demon-
strated up to 95% and 84% transforma-
tion of PAH and CPAH, respectively.
Transformation was determined through
comparison of influent and effluent PAH
and CPAH concentrations adjusted for
abiotic losses. Figure 1 illustrates the over-
all reduction in concentration of all PAH
compounds. Overall, the biologically ac-
tive reactors (R1 and R3) illustrated a
decreasing effectiveness in PAH transfor-
mation as a function of compound mo-
lecular weight.
Prior to operational changes initiated in
March (following 5 months of treatment),
R1 demonstrated 62% ± 15% transforma-
tion of PAH, with approximately 27% ±
18% transformation of CPAH. R2 demon-
strated comparable transformation of PAH
and CPAH (approximately 40% ± 16%),
as expected during chemical oxidation.
R3 CPAH and PAH transformations aver-
aged approximately 25% ± 19% and 34%
±. 24%, respectively. The total system PAH
and CPAH transformations averaged 85%
± 6% and 65% ± 8%, respectively. No
significant volatilization of PAH was evi-
dent in R1.
Following operational changes initiated
in March 1995, including increasing the
HRT from 10 to 20 days, overall PAH and
CPAH transformation rates increased up
to 95% and 84%. R1 demonstrated 87%
± 1% transformation of PAH, with 65% ±
R1
R2
R3
Feed Flow
Hydraulic Retention Time
Temperature
Dissolved Oxygen
PH
Agitation
Working Volume
Ammoniacal Nitrogen
o-phosphate
Sodium Salicylate
Sodium Succinate
Fenton's Reagent Addition
6 L/day
1 0 days
25°C ± 5°C
3 mg/L
7.0 ± 0.5
500 rpm
57 L
50 mg/L
10 mg/L
1 00 mg/L
10 mg/L
—
6 L/day
1 day
24°C + 5°C
—
<5.0
250 rpm
6L
—
—
—
—
2 L/day
8 L/day
7.5 days
25°C ± 5°C
3 mg/L
7.0 + 0.5
500 rpm
57 L
—
10 mg/L
—
—
—
4% transformation of CPAH. R2 demon-
strated comparable transformation of PAH
and CPAH (greater than 45%), as ex-
pected during chemical oxidation. R3 PAH
and CPAH transformations were de-
creased averaging 4% ±. 6% and 0.4% ±
1%, respectively. The total system PAH
and CPAH transformations increased to
91% ± 4% and 75% ± 11%, respectively.
All data is presented in Tables 2 and 3.
During optimal operation, the influent
PAH concentration was decreased from
6,210 mg/kg to 325 mg/kg. Influent CPAH
concentrations were decreased from 422
mg/kg to 65 mg/kg.
R1 and R3 pH averaged 7.0, with R2
pH reduced to an average of 2. Dissolved.
oxygen in R1 and R3 varied depending
on foaming and mechanical upsets; how-
ever, during optimal periods of operation
the dissolved oxygen concentrations in
these reactors were greater than 2 mg/L.
Ammoniacal nitrogen was added to R1 to
maintain an average concentration
throughout the system of approximately
80 mg/L. Phosphate addition to the sys-
tem was not required due to the naturally
high concentrations in the soil. The aver-
age ortho-phosphate concentration in all
reactors was 150 mg/L. Total heterotrophic
counts in R1 and R3 ranged from 10e to
109 colony forming unit per milliliter (CPU/
mL). Despite severe conditions maintained
in R2, total microbial counts ranged up to
104CFU/mL
The naphthalene-degrading bacterial
population in the influent slurry was 105
CFU/mL slurry at the initiation of the dem-
onstration. Following 7 months of treat-
ment, the influent feed population had
decreased to 104 CFU/mL. Populations in
R1 and R3 measured 106 and 10" CFU/
mL slurry, respectively.
Conclusions
The investigation illustrated the poten-
tial effectiveness of combined bioslurry
treatment and chemical oxidation for the
treatment of PAH-impacted soils. Overall,
the following conclusions were made.
The 80% CPAH transformation goal had
been achieved with CPAH transformation
following operational changes ranging up
to 84%.
An HRT of approximately 20 days in-
creased system performance.
Due to the increased transformation of
PAH in R1 and R2 during optimal perfor-
mance, transformation rates in R3 were
significantly decreased. This result may
indicate that R3 is not required for effec-
tive treatment and biological treatment fol-
lowing chemical oxidation may be
preferred.
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52 a
SAMPLE DATE
•PAH
CPAH
CPAH GOAL
Flgun 1. Percent reduction - overall.
Ttbl* 2. PAH Removal Efficiencies (Percent)
Date
Reactor 1
Reactor 2
Reactor 3
Overall
10/10
10/19
10/26
11/2
11/9
11/16
11/22
12/2
12/8
12/15
12/30
1/5
1/26
2/2
2/16
3/2
319
3/16
3/30
4/13
4m
Total Operational
Period Average
Average Prior
to 3/2
Average
Following 3/2
85
65
29
55.5
59.2
61.7
55.3
30.4
56.9
68.6
71.6
63.6
69.3
71.6
79.8
85.9
87.9
86
85.8
88.6
88.3
69.17 ±17.9
61 .5 ±15.45
87.08 ±1.32
0
58.2
45.2
37.7
26.7
41
42.9
62
44.1
22
51.1
64
44.8
38
22.3
33
39.1
37
65.9
37
55.4
41.4 + 15.7
40 ±16.93
44.6 ±13.04
36.1
67
59.3
42
44.8
59.6
69.5
28.9
5.7
31.7
6.5
44.8
12.1
1.2
0
0
0
10.3
0
12.9
0
25.35 ± 24.81
33.95 ± 24.27
3.87 ± 6.05
72.6
95.2
84.2
83.9
83.5
90.9
92.2
81
77.2
83.3
87
92.8
85.1
82.6
81.3
82.8
90.4
92
91.9
93.8
94.7
86.59 + 6.17
84.85 + 6.05
90.93 + 4.26
Recommendations
Continued investigation under the pro-
cess set points maintained during the final
month of system operation is recom-
mended to consistently achieve greater
than 80% CPAH transformation. As dem-
onstrated by the increase in PAH and
CPAH transformation during this period,
reduced solids loading, increased clay con-
tent, and extended HRTset points proved
beneficial to the treatment process.
Modifications to the pilot-scale reactor
design should be investigated to decrease
the incidence of foaming. Additional opti-
mization of the addition of Fenton's re-
agent to reduce foaming is also
recommended. Process foaming, particu-
larly when operating on the full-scale, will
result in poor system performance, reac-
tor overflow, and the inability to effectively
aerate the system.
During the spring of 1995 results of this
project were presented at the following
conferences.
• Brown, K.L., B. Davila, and J.
Sanseverino. "Combined Chemical
and Biological Oxidation of Slurry-
Phase Polycyclic Aromatic Hydrocar-
bons." Proceedings of HAZMACON
'95, San Jose, CA, 1995.
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Table 3. CPAH Removal Efficiencies (Percent)
Date
Reactor 1
Total Operational
Period Average 37.65 ± 23.61
Reactor 2
Reactor 3
Overall
10/10
10/19
10/26
11/2
11/9
11/16
11/22
12/2
12/8
12/15
12/30
1/5
1/26
2/2
2/16
3/2
3/9
3/16
3/30
4/13
4/19
60
28.2
0
2.5
0
9.3
30.4
10.4
30.9
32.7
43.5
38
30.7
37.6
44.4
66.4
71
59.9
60.9
69
64.8
0
56.9
48.5
40.5
23.9
50
33.4
51
48.3
15
43.3
59
35.3
40
23.3
34
46.3
44
69
44
55.9
40.9
49
33.9
26
44
44.5
51.2
26.3
0
36.1
12.9
0
14.4
0
0
0
0
2.5
0
0
0
62.2
84.3
60.5
56.9
56.9
74.7
77.4
67.7
61.8
63.4
72.1
67.7
61.6
59.4
55.2
54.2
74.7
78
76.6
82.6
84
41.03 ±15.86
18.18+ 19.84
Average Prior
to 3/2 26.57 ±18.16
37.89 ±16.44
25.28 ±19.31
Average
Following 3/2
• Brown, K.L., B. Davila, J. Sanseverino,
M. Thomas, C. Lang, K. Hague, and
T. Smith. "Combined Chemical and
Biological Oxidation of Slurry-Phase
Polycyclic Aromatic Hydrocarbons."
Proceedings of the Third International
In Situ and On-Site Bioreclamation
Symposium, San Diego, CA, 1995.
• Brown, K.L., B. Davila, and J.
Sanseverino. "Combined Chemical
and Biological Oxidation of Slurry-
Phase Polycyclic Aromatic Hydrocar-
bons." Proceedings of the Air and
Waste Management Association 88th
Annual Meeting and Exhibition, San
Antonio, TX, 1995.
The full report was submitted by IT in
fulfillment of Cooperative Agreement No.
CR821186-01-01 under the sponsorship
of the EPA.
68.19 ±9.92
65.45 ± 8.42
65.33 ± 4.39
48.87 ±12.08
0.42 ± 1.02
75.02 ± 10.79
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77i/s Emerging Technology Summary was prepared by the staff of IT Corpora-
tion, San Bernardino, CA 92408.
Brunllda Davlla is the EPA Project Manager (see below).
The complete report, entitled "Innovative Methods for Bioslurry Treatment,"
(Order No. PB97-176820; Cost: $21.50, subject to change) will be avail-
able only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Manager can be contacted at:
National Risk Management Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
Official Business
Penalty for Private Use
$300
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EPA
PERMIT No. G-35
EPA/540/SR-96/505
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