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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.
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J. BIOSCI. BIOENG.,
PARK ET AL.
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
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J. BIOSCI. BIOENG.,
PARK ET AL.
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