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JOURNAL OF BIOSCIENCE AND BIOENGINEERING

Vol. 96, No. 5, 429433. 2003

Bioremediation of 2,4,6-Trinitrotoluene Contaminated Soil


in Slurry and Column Reactors
CHULHWAN PARK,1,3 TAK-HYUN KIM,1 SANGYONG KIM,1*
JINWON LEE,2 AND SEUNG-WOOK KIM3
Industrial Ecology National Research Laboratory, Korea Institute of Industrial Technology, Chonan 330-825, Korea,1
Department of Chemical Engineering, Kwangwoon University, Seoul 139-710, Korea,2 and Department of
Chemical and Biological Engineering, Korea University, Seoul 136-701, Korea3
Received 9 October 2002/Accepted 1 August 2003

The bioremediation of 2,4,6-trinitrotoluene (TNT) contaminated soil was performed on a laboratory scale. To compare bioremediation methods, a soil slurry reactor and a soil column reactor
were operated and the effects of supplemental sources were investigated. Optimal conditions for
the two bioremediation systems for the removal of TNT were obtained. In the soil slurry reactor,
about 60% of the 1000 mg/kg TNT contaminated soil was degraded after 10 d, nearly complete
biodegradation (> 99%) was achieved within 25 d, and the microorganisms grew and reached a
maximum of 9.5 109 CFU/ml at 15 d. In the soil column reactor, about 50% of the 1000 mg/kg
TNT contaminated soil was degraded after 25 d and nearly complete biodegradation (>99%) was
achieved within 60 d. Microorganisms grew and reached a maximum of 9.8 1010 CFU/g soil at
40 d. These results should help in determining the best bioremediation method and improving the
design and operation of large scale clean up of contaminated sites by bioremediation systems.
[Key words: TNT, soil slurry reactor, soil column reactor, biodegradation, Pseudomonas putida]

pollution, and disposal on land may lead to soil and groundwater contamination that will affect aquatic life forms, humans, and animals. Bioremediation holds the promise of being a cost effective and environmentally safe practice. Biological removal of TNT and other explosives has been
proven to be feasible (1113). Many studies have also investigated the biodegradation of nitroaromatic compounds
including TNT, but only a few reports have been published
on the bioremediation of TNT contaminated soils (14, 15).
Therefore, to treat TNT contaminated soil, the isolated microorganisms were inoculated and the required nutrients
were supplied into the polluted soil, such as in our previous
studies (7, 16). In the present study, we described bioremediation methods using a soil slurry reactor (the effect of
soil/water ratio and mixing condition) and a soil column reactor (the effect of addition of the isolated microorganisms
and various energy sources) and the optimization of these
two remediation systems for the removal of TNT. In addition, the results from this work should help in determining
the best bioremediation method and improving the design
and operation of large scale clean up of contaminated sites
by bioremediation systems.

Nitroaromatics form an important group of recalcitrant


xenobiotics. Only a few aromatic compounds, bearing one
nitro group as a substituent of the aromatic ring, are produced as secondary metabolites by microorganisms. The
majority of nitroaromatic compounds in the biosphere are
industrial chemicals such as explosives, dyes, polyurethane
foams, herbicides, insecticides, and solvents (1). These compounds are generally recalcitrant to biological treatment and
remain in the biosphere, where they constitute a source of
pollution, producing both toxic and mutagenic effects in
humans, fish, algae, and microorganisms (2, 3). However, a
few microorganisms have been described as being able to
use nitroaromatic compounds, such as explosives, as carbon
and/or nitrogen energy sources (49).
The best-known nitroaromatic compound is the explosive
2,4,6-trinitrotoluene (TNT). Though most munitions waste
sites date back from the past, many are still a serious environmental hazard. Sites contaminated with TNT range from
ammunition plants and testing facilities to military zones
and battlefields. Although this contamination is known to be
widespread, little information is available in the literature
documenting these sites and their degree of contamination
(1). Disposal of munition wastes in the past occurred by
dumping in the deep sea, dumping at specified landfill
areas, and sometimes incineration, when quantities were
small (10). All of these methods can potentially have very
serious effects on the ecosystem. Incineration causes air

MATERIALS AND METHODS


Soil and microorganism
Contaminated soil samples were
collected near an ammunition plant. The concentration of TNT in
the contaminated soils ranged from 1200 to 1500 mg/kg. The samples were mixed with noncontaminated soil to achieve a final TNT
concentration of 1000 mg/kg. This soil was dried at 30C for 5 d
before experimentation. The soil size distribution is shown in

* Corresponding author. e-mail: sykim@kitech.re.kr


phone: +82-41-589-8356 fax: +82-41-589-8330
429

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J. BIOSCI. BIOENG.,

PARK ET AL.

TABLE 1. Soil size distribution


Opening (mm)
Over 2
12
Under 1

Percent retained (%)


14
79
7

Table 1.
Pseudomonas putida KP 201 was isolated from a TNT-contaminated site and added to the reactors as the 1 ml inoculating microorganism at 2.4 108 CFU/ml. For inoculation in the reactors, P.
putida KP 201 was cultured in a modified heterotrophic medium
consisting of the following components (g/l of distilled water):
K2HPO4, 7; KH2PO4, 3; MgSO4, 0.1; NaCl, 0.1; (NH4)Cl, 0.25;
corn steep liquor (CSL), 10; Tween 80, 1 at pH 7.0. One milliliter
of soil slurry and 1 g of soil sample were prepared to determine the
growth of microorganisms in the case of the soil slurry reactor and
soil column reactor, respectively. Each sample was mixed with
9 ml of sterile phosphate buffer and vortexed for 2 min to detach
the microorganisms from the soil samples. The treated liquid was
serially diluted and poured onto tryptic soy agar (TSA) (Difco,
Sparks, MD, USA) plates. Standard total plate count methods were
followed with agar plates at 35C for 48 h, as described by Brock
and Madigan (17). The amount of microorganisms in the soil reactor was measured every 5 d.
Soil slurry experiments
The volume of the soil slurry reactor was 1000 ml and the working volume of the reactor was
500 ml. Reactors were controlled for temperature and rotation rate.
Each reactor was operated at 30C and air was supplied through a
diffuser. A schematic diagram of the soil slurry reactor is shown in
Fig. 1. The reactors were started with 10%, 20%, 30%, 40%, and
50% (w/v) of TNT contaminated soil. CSL (1.0%), NH4Cl (0.025%),
and Tween 80 (0.1%) were added to the reactors and the information concerning these supplemental sources was gained from our
earlier studies for the biodegradation of TNT (7). The soil slurry
was mixed at the rates of 0, 20, 40, 60, 80, and 100 rpm using a
stirring motor. The TNT concentration, microorganism growth,
pH, and dissolved oxygen (DO) were measured periodically.
Soil column experiments
The volume of the soil column reactor was about 370 ml and the reactor was operated with a working volume of 350 g of soil. Each reactor had an inlet and outlet for
supplemental sources and air, and was set up by adding 350 g of
TNT contaminated soil. P. putida KP 201 was cultured in a modified heterotrophic medium and 1 ml of microorganism (2.4 108
CFU/ml) was centrifuged at 3000 rpm for 5 min. The centrifuged
microorganism was the mixed with soil. The sampling point is described in Fig. 2 and sampling was performed in the top 3 cm of

FIG. 2. Schematic diagram of soil column reactor.

the column. The experiment was performed in a series of four soil


column reactors. Soil columns 1 and 3 were treated without P.
putida KP 201 inoculation, while soil columns 2 and 4 were
treated with P. putida KP 201 inoculation. Soil columns 1 and 2
were operated without, while soil columns 3 and 4 were operated
with supplemental sources. Soil columns 1 and 2 received 0.07 ml
of aerated distilled water and soil columns 3 and 4 received 0.07
ml of aerated supplemental sources (1.0% CSL, 0.025% NH4Cl,
and 0.1% Tween 80) per minute from the bottom through silicon
tubing by a multichannel peristaltic pump (MasterFlex L/S; ColeParmer Instrument, Barrington, IL, USA) and this flow rate represents the approximate cumulative pore space of the soil in each
column per day. Aerated distilled water and aerated supplemental
sources were delivered during the initial 8 d (total volume, 800
ml). Only wet air was supplied into the soil column after 8 d. It is
believed that the inside conditions of soil columns 3 and 4 would
become facultative anaerobic for 8 d, and would then gradually
change into aerobic conditions after 8 d.
Analysis The soil sample was dried and TNT and its metabolites were extracted with 10 ml of acetonitrile from 1 g of soil.
The whole mixture was sonicated for 24 h using an ultrasonic
cleaning system (Branson, Danbury, CT, USA). After sonication,
5 ml of soil slurry was mixed with 5 ml of 5 g/l calcium chloride
solution. The whole mixture was then centrifuged at 5000 rpm for
10 min. The supernatant was used for the high performance liquid
chromatography (HPLC) analysis of TNT and its intermediates.
HPLC analysis of the samples was conducted with a Varian liquid
chromatograph equipped with a model 230 ternary gradient pump,
model 410 autosampler, data module, and model 310 UV-VIS
detector set at 254 nm (Varian, Palo Alto, CA, USA). The mobile
phase was methanol : water (50:50, v/v) and 10 ml samples were
injected onto a Restek C-18 column (Restek, Bellefonte, PA, USA)
at 30C. The flow rate of the solvent was 1.0 ml/min and the detection limit was 0.01 mg TNT/g soil. Standard methods for examination of water and wastewater (18) were used for the estimation of
pH and DO. The pH and DO were measured using a pH meter
(model 420+; ThermoOrion, Beverly, MA, USA) and a DO meter
(model 50175; HACH, Loveland, CO, USA).

RESULTS AND DISCUSSION

FIG. 1. Schematic diagram of soil slurry reactor.

Soil slurry reactor


The soil slurry experiments were
operated for 30 d. The concentration of the soil slurry and
the rotation rate are the most important variables of the soil
slurry reactor. Therefore, the effects of these two variables
on the treatment of contaminated soil were examined using

VOL. 96, 2003

FIG. 3. Biodegradation of TNT in soil slurry reactor at 30 d.

the soil slurry reactors. Supplemental sources (CSL, NH4Cl,


and Tween 80) were also added to the soil slurry reactor to
reduce degradation time. Our previous studies have demonstrated that the efficiency of biodegradation of TNT was enhanced by supplemental sources such as CSL or molasses
(7). In addition, the concentrations of these supplemental
sources used in this study were also obtained from our earlier study (16).
Most of the TNT (> 99%) was removed in 1030% soil
slurries and the removal ratio of TNT decreased in soil slurries over 40% (Fig. 3). These results indicate that the soil
had to be properly slurried with water and then treated in the
reactor, where the conditions for bioremediation were enhanced. These results are believed to be because the mass
transfer of substrate was limited under these operating conditions. TNT was adequately removed in the 30% soil slurry
when the rotation rate of the mixing impeller was 40 to 60
rpm. When the rotation rate was increased to over 80 rpm,
the removal ratio of TNT decreased. The removal ratio also
decreased when the rotation rate was below 20 rpm. This
was attributed to the fact that the soil was adequately mixed.
The percent removal of TNT decreased because rapid mixing at more than 80 rpm caused an increase in foam and pulverized the soil. These results suggest that the optimal concentration of soil was 30% and the optimal rotation rate of

FIG. 4. Biodegradation of TNT in soil slurry reactor at 30% soil


slurry and 40 rpm rotation rate. Symbols: circles, residual TNT; triangles, biomass growth; squares, DO; diamonds, pH.

BIODEGRADATION OF ENERGETIC MATERIALS

431

the mixing impeller was 40 rpm in the soil slurry reactor.


Figure 4 shows the changes in residual TNT, biomass
growth, DO, and pH in a 30% soil slurry at a rotation rate of
40 rpm. About 60% of the TNT was degraded after 10 d and
nearly complete biodegradation (>99%) was achieved within
25 d. Microorganisms grew and reached a maximum of
9.5 109 CFU/ml at 15 d. In addition, the biodegradation of
TNT commenced immediately after reaction with no apparent lag phase. The initial pH of the soil slurry was 7.4 and
the final pH (after 25 d) was 5.6. This is believed to be due
to the accumulation of fatty acids from metabolism of CSL
by the microorganisms. Boopathy et al. also reported similar results for the metabolism of molasses (19). This pH
change did not affect the bacterial ability to degrade TNT
because the microorganisms maintain a high degradation
ability at this pH range. In the experimental reactor, the concentration of DO dropped from 8.7 mg/l to 0.5 mg/l and
stayed in this concentration range (0.51.2 mg/l). The values of DO after 25 d increased (data not shown). These
trends for the various factors were similar to those observed
in our previous study on wastewater treatment (20). In addition, it was observed that the viscosity of the soil slurry
increased during operation. This phenomenon seemed to
occur due to the presence of some viscous materials from
the microbial cells such as polysaccharides and/or from soil
such as colloidal particles. In the case of treatment of soil
slurry, the biomass may not increase to a high density because the culture conditions are not optimal compared with
the fermentation process. Therefore, the viscosity problem
in this operation would be attributed mainly to soil properties. Nevertheless, the microorganisms may have partly influenced the increase in viscosity of the soil slurry. This is
demonstrated by the fact that the settling velocity of the soil
slurry after operation was much lower than that in the initial
period.
Soil column reactor
The soil column experiment was
also operated for 60 d to compare with the treatment in the
soil slurry reactor. The concentrations of TNT in soil samples taken from soil columns 14 during the experiment are
shown in Fig. 5. The natural attenuation control (soil column 1) showed no removal of TNT. This fact emphasized

FIG. 5. Biodegradation of TNT in soil column reactor. Symbols:


circles, soil column 1; triangles, soil column 2; squares, soil column 3;
diamonds, soil column 4.

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J. BIOSCI. BIOENG.,

PARK ET AL.

FIG. 6. Biomass growth in soil column reactor. Symbols: circles,


soil column 1; triangles, soil column 2; squares, soil column 3; diamonds, soil column 4.

the necessity of proper treatment methods. In soil column 2,


about 40% removal of TNT was achieved, demonstrating
the removal ability of the inoculated microorganisms. The
TNT in soil column 3 slowly decreased low range (about
20% removal of TNT). In soil column 4, about 50% of the
TNT was degraded after 25 d and nearly complete biodegradation (> 99%) was achieved within 60 d. The microorganisms grew and reached a maximum of 9.8 1010 CFU/g soil
at 40 d. Soil columns 3 and 4 also showed that the use of
CSL and Tween 80 could improve the in situ bioremediation
of TNT contaminated soil. The removal rate of TNT was the
fastest in soil column 4, which was operated using CSL,
NH4Cl, and Tween 80 with aeration and inoculation of P.
putida KP 201. This column reactor removed TNT very
efficiently. This result demonstrated that the isolated microorganism was very effective and that adapted microorganisms added to the soil could enhance the process efficiency.
This enhancement also occurred because of the complex
composition of CSL or molasses which contain sugars,
amino acids, proteins, vitamins, minerals, and organic nitrogen, made it conducive to the growth of microorganisms in
the soil column that could degrade TNT (21). The growth
of microorganisms was investigated several times from soil
columns 14 as bacterial plate counts (Fig. 6). The microorganisms in soil columns 1, 2, 3, and 4 grew and reached
a maximum of 3.6 105 CFU/g soil, 5.3 106 CFU/g soil,
3.1 106 CFU/g soil, and 9.8 1010 CFU/g soil during the experiments, respectively. The bacterial population densities
in soil column 4 were three to five orders of magnitude
higher than those in the other three soil columns. Indigenous
microorganisms would be inhibited by high concentrations
of TNT in soil columns 1 and 3, and the supplemental
sources added to soil column 3 would not be able to support
a high level of growth of indigenous microorganisms. However, they would be able to support a high level of growth of
inoculated microorganisms in soil column 4.
Various supplemental sources used in the soil slurry reactor and the soil column reactor could improve the efficiency of treatment. CSL and molasses in particular are
known as effective and cheap carbon sources that enhance
the transformation rate of TNT significantly over other car-

bon sources studied. Tween 80 can desorb contaminant


from soil in aqueous medium and the addition of Tween 80
alone did not enable the microorganisms to completely remove TNT and its metabolites. CSL supported a well balanced degradation with the removal of TNT. Boopathy et al.
showed that TNT was not removed in an abiotic control
where the soil was autoclaved (19) and Boopathy also demonstrated that the addition of molasses and Tween 80 adequately balanced the biodegradation with removal of both
TNT and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT) (22).
In any investigation of the biological degradation of TNT, it
is also important to track the production, accumulation, and
removal of metabolic intermediates within the experimental
system. The predominant intermediates by mass concentration observed were 4-A-2,6-DNT, 2-amino-4,6-dinitrotoluene
(2-A-4,6-DNT), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT).
Conclusion
The soil slurry reactor and the soil column reactor for TNT-contaminated soils described above is
very effective. The soil slurry reactor can provide faster
treatment than the soil column reactor, but it will be expensive to apply in the field because of the high labor, soil extraction, and operation costs of the soil slurry reactor and
the high post treatment cost. The rate of bioremediation is
relatively slow in the soil column reactor, but the method is
very cheap. The soil slurry reactor is suitable when fast
treatment is required and the concentration of the contaminant is relatively low, and in situ bioremediation is also suitable when the area of contamination is widespread. In addition, this study suggests that the treatment ability could be
improved by adopting isolated inoculating microorganisms
and adding supplemental sources.
ACKNOWLEDGMENTS
This work was supported by the National Research Laboratory
Program of the Korea Ministry of Science and Technology.

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