Atlas Journal of Biology 2 (2): 116–124, 2012 doi: 10.5147/ajb.2012.0093 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A New Strain of Bacteria Degrading TNT and 2,4/2,6-DNT From Explosives - Contaminated Soil Jinsoo Kim1, Yong-Kju Yu2, Fei Yan3, John Bang4, Taek You5, and Sang-Seob Lee1* 1 Department of Life Science, Kyonggi University, Suwon, Korea 442-760; 2 Department of Biological En- gineering, Kyonggi University, Suwon, Korea 442-760; 3 Department of Chemistry, North Carolina Central University, NC, USA; 4 Department of Environmental, Earth, and Geospatial Sciences, North Carolina Central University, Durham, NC 27707, USA; 5 Department of Biological sciences, Campbell University, Buies Creek, NC 27506, USA Received: May 17, 2012 / Accepted: June 17, 2012 __________________________________________________ * Corresponding author: sslee@kyonggi.ac.kr 116 Abstract The 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), and Toluene derived from a nitroaromatic compound(NAC) cause high toxicity and mutagenicity to environment. One of best methods to remove their toxicity from contaminated soil is known biological remediation with soil bacteria. Total of 235 strains of explosives-removing soil bacteria were iso- lated from the shooting gallery at a military base in Korea. They were identified as Acinetobacter, Agrobacterium, Alca- ligenes, Flavobacterium, Klebsiella, Pseudomonas, Serratia, and Citrobacter species. After further screened for the bet- ter removal efficiencies, KT22 identified as Serratia sp., KD4 identified as Klebsiella sp., and KD6 identified also as Kleb- siella sp. showed the highest removal efficiency for TNT, 2,4- DNT, and 2,6-DNT, respectively. The optimal removal condi- tions were shown as follows: cell concentration 1 g/L, pH 7, and temperature 25-30°C. In a Luria-Bertani (LB) medium containing 100 mg/L of TNT, KT22 strain could remove TNT over 99% and showed a good biomass growth after 6 h of incubation. Furthermore, when the KT22 strain was mixed with a Bacillus sp., the mixed culture showed the improved TNT removal efficiency. Conclusively, this new strain could be most effective to remove NAC toxicity with rhizosphere remediation system on contaminated soil. Introduction 2,4,6-trinitrotoluene (TNT) is a nitroaromatic compound (NAC) commonly used in explosives, and 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) are two of the six DNT isomers that are used primarily as chemical intermediates in the pro- duction of toluene diamines and diisocyanates, dyes, explosives, and propellants. Two products derived from TNT are 2,4-dini- trotoluene (DNT) and toluene (Spanggord et al., 1991; Duque et al., 1993). These toxic compounds are often the main contami- nants of soil and groundwater at their manufacturing, process- ing, and disposal facilities (Kalafut et al., 1998). Especially, TNT has been widely used during and even after WWII, resulting in widespread soil contamination (Scheibner et al., 1997), and was also used throughout the entire Korean peninsula during the Korean War. The high toxicity and mutagenicity of TNT and some of its metabolites have led to a harmful effect in their fates in the environment. Numerous cases of munitions workers who have de- veloped liver damage and anemia owing to the TNT exposure have been documented (Voegtlin et al., 1919; Hamilton, 1921; Bridge et al., 1942; Sax, 1963). In addition, TNT has been shown to have toxic effects in rats and mice (Dale, 1921; Channon et al., 1994), fish, algae, and oyster larvae (Smock et al., 1976; Won et al., 1976). The International Agency for Research on Cancer (IARC) has also determined that 2,4- and 2,6-DNT are possibly carcinogenic to humans. In addition, 2,6-DNT showed hepatocarcinogenic effects in Fischer-344 rats, and exposure to A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 117 high levels of DNTs in animals caused lowered numbers of sperm and reduced fertility (Popp, 1983; Richart, 1984). The most practical approach toward the remediation of explosives is currently soil incineration, but it can be a costly, energy-intensive process that destroys much of the soil, leaving ash as the primary residue with estimates approaching $800/ ton (Funk et al., 1993). Chemical treatment, on the other hand, requires the exercise of rigid controls to avoid the discharge of unreacted materials and is not practical in many situations. It is believed that biological remediation of explosives is the most economical and reliable method of response to this problem at a cost that ranges $30-$150/yd3 (Montemagno and Irvine, 1990; Preslan et al., 1993). Composting, one of the biological remediation methods applied to explosives, has been proven to be effective and is compatible with the incineration method (Williams et al., 1992). Bio-treatment with bacteria has focused on less expensive method of bioremediation, because many dif- ferent species of bacteria potentially reduce nitro groups on the aromatic ring in soil with no requirement for additional nutri- ents. Recently researchers have tried to hybrid more than two techniques to overcome weak points of each technique, and im- prove efficiency in the remediation fields. For instance, phytore- mediation, which is a combined technique with bioremediation using soil bacteria and plant, was applied to remediate the con- taminated soil. For NAC group is fairly toxic and combined one is applied to various environmental conditions, bacteria which could degrade TNT under aerobic and/or anaerobic condition are required to grow well with other organisms. To develop an enhanced rhizosphere remediation system with NAC removing soil bacteria, we were isolated and identi- fied them in this study. Then, they were further screened for high removal efficiencies toward TNT and 2,4/2,6-DNT, using batch tests. Effects of pure vs. mixed culture with common soil microbes on the removal efficiencies were also evaluated. Materials and Methods Isolation, Cultivation, and Identification of Explosives-Removing Soil Bacteria All the samples for the microbial isolation were obtained from the shooting gallery at a military base in Kyonggi-province, Korea. Samples were obtained with the bottom soil, and the pH and temperature were 5.6, 9.5-10.50C, respectively. Four different types of media, i.e., nutrient broth, Medium 1, Medium 2, and King’s B medium, were used as the growth medium for culturing TNT and 2,4/2,6-DNT removing bacteria. The composition of each medium was as follows per liter of de- ionized water. Nutrient broth: Bacto beef extract 3.0g, Bacto peptone 5.0g, TNT 0.1 g; Medium 1: K2HPO4 7.0 g, KH2PO4 3.0 g, MgSO4 0.1 g, NaCl 0.1 g, NH4Cl 0.25 g, peptone 0.5 g, yeast extract 0.1 g, succinate 5.0 g, and TNT 0.1 g; Medium 2: MgSO4 0.1 g, K2HPO4 3.5 g, KH2PO4 1.5 g, TNT 0.1 g, yeast extract 1.0 g, and trace element solution 1.0 mL, consisting of ethylenediaminetetraacetic acid (EDTA) 0.25 g, FeSO4•7H2O 0.1 g, and trace element solution SL-6 1.0 mL; King’s B medium: peptone 20.0 g, K2HPO4 1.5 g, and MgSO4•7H2O 1.5 g. Agar at 1.5% (w/v) was added for the solid medium. The explosive stock solution was prepared by dissolving 0.5 g TNT in 50 mL N,N-dimethylformamide to have a final concentration of 10,000 mg/L. The final TNT concentration of test media was adjusted to 100 mg/L using stock solution. In case of aerobic cultivation, 20 mL cultivation solution was added in a 100 mL flask, inoculated, and then cultivated. Facul- tative anaerobic cultivation was performed using test tubes full of the medium. Cultures were incubated at 28±20C. For the identification of isolates, Gram staining and bio- chemical tests were performed. Cell size, motility, and morphol- ogy were determined microscopically (x1,000) under the light microscope (Olympus BH-2, Japan). Chracteristics of the results were compared with the properties described in the Bergey’s Manual of Systematic Bacteriology (Krieg et al., 1994). For ge- netic characteristics, the 16S rRNA gene was amplified by PCR with 27F/ 1492R primers (universal primers for bacteria) in 35 amplification cycles at 940C for 45 sec, 550C for 60 sec, and 720C for 60 sec. For purification of PCR products, unincorpo- rated PCR primers and dNTPs from PCR products were removed by using the Montage PCR Clean up kit (Millipore Co., USA). The purified PCR products were sequenced by using 27F/ 1492R primers. Sequencing was performed by using Big Dye terminator cycle sequencing kit v.3.1 (Applied BioSystems, USA). Sequenc- ing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSys- tems, USA) at the Macrogen, Inc., Seoul, Korea. The nearly com- plete sequence of the 16S rRNA gene (1427nt) was compiled with SeqMan software (DNASTAR Inc.). The 16S rRNA gene se- quences of the related taxa were obtained from GenBank. Screening of Highly Efficient Explosives-Removing Strains Each of those 235 isolated strains of soil bacteria was screened for the TNT and 2,4/2,6-DNT removal efficiencies, us- ing a Luria-Bertani (LB) medium containing 100 mg/L of TNT or 2,4/2,6-DNT, under aerobic conditions. After 12 and 18 h of cul- tivation, concentrations of TNT and 2,4/2,6-DNT were measured on a gas chromatograph (HP-6890, USA) equipped with the electron capture detector (GC-ECD) and RTX-TNT column. ECD has been widely used for the determination of NACs (Walsh, 2001). Nitrogen was used as a carrier gas at the flow rate of 60 mL/min. Injector and detector temperatures were 250 and 300°C, respectively. The column temperature was programed as follows: held for 1 min at 80°C; temperature increased to 180°C at the ratio of 10°C/min; temperature increased to 300°C at the ratio of 30°C/min; and held for 3 min at 300°C. Batch Experiments for High Explosives Removal Efficiencies In order to determine the optimum culture conditions for the explosives-removing bacteria, the batch culture was performed under various conditions. For the effect of explosive concentra- tion on bacterial growth rate, the bacterial cell concentration was set at 1.0 g WCW (Wet Cell Weight)/L. TNT and 2,4/2,6- DNT concentrations in LB medium and basal mineral medium A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 118 were modified to 10, 100, and 200 mg/L, respectively. For the optimum cell concentration, TNT and 2,4/2,6-DNT concentra- tions in LB medium and basal mineral medium were set at 100 mg/L but the bacterial cell concentrations were modified to 0.5, 1.0, and 1.5 g WCW/L. For the optimum pH and temperature, pH was modified to 6, 7, 8, and 9 and temperature to 15, 20, 25, and 30°C. In addition, to evaluate the possibility of even higher removal efficiencies for TNT and 2,4/2,6-DNT, the mixed cultures consisting of isolates mixed with either Bacillus strains, purple non-sulfur bacterial strains, or Pseudomonas strains, were compared with single cultures in terms of the TNT removal ef- ficiency. Results and Discussion Bacterial Isolation and Identification In total, 235 strains of soil bacteria were isolated from the military shooting gallery and they were identified as follows, according to the classification keys of Bergey’s Manual of Sys- tematic Bacteriology (Krieg et al., 1994): Acinetobacter (51 strains), Agrobacterium (2), Alcaligenes (2), Azorhizobium (15), Citrobacter (14), Flavobacterium (27), Hafnia (9), Klebsiella (8), Klyvera (16), Pantoea (5), Proteus (18), Pseudomonas (36), Ser- ratia (22), and Yersinia (10). Among 235 isolates, 168 strains could remove TNT and were identified as Pseudomonas (24), Flavobacterium (13), Citrobacter (14), Proteus (12), Yersina (6), Hafnia (4), Klyvera (15), Pantoea (4), Klebsiella (4), Serratia (21), Azorhizobium (9), and Acineto- bacter (42). Fifty one strains removed 2,4-DNT and were iden- tified as Pseudomonas (11), Flavobacterium (13), Proteus (4), Agrobacterium (2), Yersina (4), Hafnia (3), Klyvera (1), Pantoea (1), Klebsiella (4), Serratia (1), Azorhizobium (1), and Acineto- bacter (6). Sixteen strains removed 2,6-DNT and were identified as Acinetobacter (3), Alcaligenes (2), Azorhizobium (5), Flavobac- terium (1), Hafnia (2), Proteus (2), and Pseudomonas (1) (Table 1). The other 77 strains were not shown explosives-removal ef- ficiency, but could survive. The strains were tolerant to explosives as bacteriostatics. For the screening of strains with high explosives removal ef- ficiencies, 147 isolates were tested for TNT and 2,4/2,6-DNT removal. Results showed that 113 strains removed TNT at the average removal efficiency of 84.4%, 27 strains removed 2,4- DNT at the average efficiency of 89.8%, and 7 strains removed 2,6-DNT at the average efficiency of 72.6%. Among these iso- lated tested, KT22 strain showed the highest removal efficiency for TNT (100%, after 6 h), KD4 strain for 2,4-DNT (100%, after 18 h), and KD6 strain for 2,6-DNT (97.7%, after 18 h), respec- tively (Table 2). High efficiency bacteria KT22 was identified as Serratia sp., and KD4 and KD6 were identified as Klebsiella sp. (Figure 1, Table 3). On the basis of the 16S rRNA gene sequences revealed that strain KT22 showed the high sequence similarity to Serratia marcescens (100%), strain KD4 and KD6 showed the high sequence similarity to Klebsiella oxytoca (99%) as Blast searching results. (a) (b) (c) Figure 1. Micrographs of the selected bacterial strains (x 1,000). (a) KT22, Serratia sp., TNT remov- ing strain; (b) KD4, Pseudomonas sp., 2,4-DNT removing strain; (c) KD6, Pseudomonas sp., 2,6-DNT removing strain. 119 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Ta bl e 1. M or ph ol og ic al a nd b io ch em ic al c ha ra ct er ist ic s of is ol at es f ro m th e m ili ta ry s ho ot in g ga lle ry . Iso late s* 51 2 2 15 14 27 9 8 16 5 18 36 22 10 Ide ntif ica tion Ac ine tob act er Ag rob act eri um Alc alig ene s Az orh izo biu m Cit rob act er Fla vob act eri um Ha fnia Kle bsi ella Kly ver a Pan toe a Pro teu s Pse udo mo nas Ser rat ia Ye rsin ia Mo rph olo gic al Ch ara cte rist ics Gra m s tain ing - - - - - - - - - - - - - - Sha pe Rod Rod / C occ i Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Siz e 0.8 x 1 .5- 2.5 0.5 -0. 8 x 1.0 -1. 5 1.0 x 1 .5- 2.0 0.8 -1. 0 x 1.5 -2. 0 1.0 x 1 .5- 2.0 0.8 -1. 0 x 1.5 -2. 0 0.8 -1. 0 x 1.5 -2. 0 0.8 -1. 0 x 1.5 -2. 5 0.8 -1. 0 x 1.0 -1. 5 0.8 -1. 0 x 1.5 -2. 5 0.8 -1. 0 x 1.5 -2. 0 0.5 -0. 8 x 1.5 -2. 0 0.5 -0. 8 x 1.2 -2. 0 0.8 -1. 0 x 1.0 -1. 5 Re plic atio n B B B B B B B B B B B B B B Mo tilit y - - + - - - + - - + - + - - End osp ore fo rm ing - - - - - - - - - - - - - - Bio che mi cal C har act eri stic s Ca tala se + + + + + + + + + + + + + + Ox ida se - + + + - + - - - - - + - - Nit rat e r edu cta se - + - - + - + + + + + + + + Ca sei n - - - - - - + - - - - - V - Sta rch hy dro lys is - - + - - - - - - - - - - - Ure a - + - - + - - + - - + - - + Ge lati n h ydr oly sis - - + - - - - - - - - V + - MR V - - V + - + - + + + + + + VP - - - - - (+) + - - + V - + - Ind ole - - - - - - - - - - - - - - Ga s fr om glu cos e - V - - + - - - V - + - - - 120 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Table 2. Results of screening tests for selected isolates. Table 3. Morphological and biochemical characteristics of the selected isolates (KT22, KD4, and KD6) with the highest explosives removal efficiencies. Explosive Strain No. Initial Conc. (mg/L) Final Conc. (mg/L) R.E. (%) Strain No. Initial Conc. (mg/L) Final Conc. (mg/L) R.E. (%) TNT KT22 107.7 0.0 100.0 KT60 107.7 41.7 61.3 KT23 107.7 4.2 96.1 KT88 144.5 71.5 50.5 KT 92 107.7 11.2 89.6 KT147 107.7 59.8 44.5 KT76 107.7 19.7 81.7 KT43 107.7 72.4 32.8 KT104 107.7 32.0 70.3 KT110 107.7 95.2 11.6 2,4-DNT KD4 68.1 0.0 100.0 KD27 125.0 46.0 63.0 KD9 125.0 0.5 99.6 KD14 125.0 84.5 32.4 KD28 125.0 0.6 99.5 KD3 68.1 49.1 27.9 KD23 125.0 17.7 85.8 KD13 125.0 104.2 16.6 2,6-DNT KD6 103.9 2.4 97.7 KD53 71.4 30.9 56.7 KD59 103.9 4.3 95.9 KD55 103.9 64.0 38.4 KD64 103.9 12.7 87.8 KD62 103.9 79.8 23.2 KD58 103.9 30.0 71.1 KD63 103.9 97.3 6.4 KD54 103.9 36.3 65.1 KD52 71.4 75.3 - R.E.: removal efficiency. Strain KT22 KD4 KD6 Shape Rod Rod Rod Size (μm) 0.8 X 2.0 0.8 X 2.0-2.5 0.8 X 1.5 Gram - - - Reproduction B B B Motility + + + Endospore forming - - - Catalase + + + Oxidase - + + Nitrate reductase + + + Casein - - - Starch - - + Urea - + - Gelatin - - MR (+) - VP + - - Indole - - Gas from glucose B: binary replication, V: various results, +: positive reaction, -: negative reaction, and weak reaction is given in the parentheses. Batch Culture Three bacterial strains (KT22, KD4, and KD6) which showed the highest explosives removal efficiencies were selected for the batch experiments in order to determine optimal culture condi- tions. Effects of various factors, including cell concentrations, ex- plosives concentrations, medium composition, temperature, pH, and pure vs. mixed cultures, on explosives removal efficiencies were evaluated. Determination of Optimal Cell Concentration In order to determine the optimum cell concentration for the removal of TNT, different cell concentrations (0.5, 1.0, 1.5, and 2.0 g WCW/L) of KT22 strain and the Stanier’s basal mineral medium were used. As shown in Table 4, the removal efficien- cy for TNT used as sole carbon source was 38.8, 51.2, 49.5, and 52.5% at the cell concentration of 0.5, 1.0, 1.5, and 2.0 g WCW/L, respectively, after 6 h of incubation. Therefore, the optimum cell concentration was determined as 1 g/L because the higher amount of biomass at 2.0 g/L did not show signifi- cantly higher TNT removal efficiency. Effects of Explosives Concentrations on Bacterial Growth Rates Using the cell concentration of 1 g WCW/L, effects of explo- sives concentrations on bacterial growth rates were evaluated. Three different concentrations (10, 100, 200 mg/L) of TNT and A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 121 2,4/2,6-DNT in the basal mineral medium were used. Results showed the higher bacterial growth rate and TNT removal ef- ficiency at the lower TNT concentration (Figure 2a and Table 5). Likewise, 2,4-DNT showed similar effects for KD4 strain (Figure 2b). However, in case of 2,6-DNT, KD6 strain showed similar growth rates regardless of 2,6-DNT concentrations (Figure 2c). Effect of Medium Composition on Explosives Removal Efficiency To determine the effect of medium composition on the ex- plosive removal efficiency, LB medium and Stanier’s basal min- eral medium were compared, using KT22, KD4, and KD6 strains. The TNT removal efficiency for KT22 was 99.7% in LB medium and 51.2% in Stanier’s basal medium, and the 2,4/2,6-DNT re- moval efficiency for KD4 and KD6 was 99.1% and 92.7% in LB medium and 63.0% and 34.5% in Stanier’s basal medium, respectively. Therefore, each selected strain showed higher re- moval efficiency for the corresponding explosive in LB medium which is a rich medium containing additional carbon and nitro- gen sources such as peptone and yeast extract, compared to Stanier’s basal mineral medium which contained explosives as sole carbon sources. These results are also in good agreements with previously published studies on the effects of supplemental nitrogen sources and carbon sources on the biodegradation of TNT (Won et al., 1974; Boopathy et al., 1997; Park et al., 2003). These stud- ies showed that in the presence of other carbon sources except aspirate, Pseudomonas putida U-T202 could degrade TNT over 92% and showed a good biomass growth after 30 h of incuba- tion (Park et al., 2003; Won et al., 1974) also reported that the TNT oxidation required the addition of glucose or nitrog- enous substances to achieve accelerated transformation and in a medium supplemented with 0.5% yeast extract, 100 mg/L of TNT was completely transformed to intermediates such as monoamino- dinitrotoluene (MADNT), diaminomononitrotoluene, or azoxy compound. Cell Conc. (g (WCW)/L) 0 h (mg/L) 6 h (mg/L) R.E. (%) 0.5 84.7 51.9 38.8 1.0 84.7 41.5 51.2 1.5 84.7 42.8 49.5 2.0 84.7 40.2 52.5 WCW: Wet Cell Weight, R.E.: removal efficiency. Table 4. Effect of cell concentrations of KT22 strain on TNT removal efficiency. Explosive Conc. (mg/L) TNT 2,4-DNT 2,6-DNT 0 h (mg/l) 6 h (mg/l) R.E. (%) 0 h (mg/l) 12 h (mg/l) R.E.(%) 0 h (mg/l) 18 h (mg/l) R.E. (%) 10 9.6 5.1 46.9 9.1 0.0 100.0 12.5 8.1 35.5 100 76.5 37.9 50.3 89.4 33.1 63.0 103.9 68.0 34.5 200 149.4 121.2 18.9 166.7 74.7 55.2 192.0 172.7 10.1 R.E.: removal efficiency. Table 5. Effects of explosives concentrations on removal efficiencies. Determination of Optimum Temperature and pH For the ranges of temperature (15-30°C) and pH (5-8) test- ed in this study, the explosive removal efficiencies for those three selected strains were the highest at around 25°C and at pH 7, as shown in Figures 3 and 4. Explosive Removal Efficiency for Mixed Culture To further investigate the effect of the presence of pure cul- ture (containing one type of explosive-removing strain) vs. mixed culture (containing more than one type of explosive-removing strains) on the explosive removal efficiency, KT22 strain which showed a high TNT removal efficiency was used as a represen- tative TNT-removing strain. For the mixed culture, KT22 strain was mixed with Bacillus or Pseudomonas strain, both commonly occurring soil bacteria, or with purple non-sulfur bacterial strain capable of degrading some recalcitrant organic compounds. As shown in Figure 5, TNT removal was the highest for the mixed culture consisting of KT22 and Bacillus sp., with the removal ef- ficiency of 76.6% after 6 h, compared to 51.2% when KT22 strain used alone. This study carries several significant scientific and industrial application merits. Firstly, Serratia sp. has been rarely reported for its efficiency in TNT biodegradation. Moreover, three bacte- rial strains (Serratia sp. KT22, Klebsiella sp. KD4, and Klebsiella sp. KD6) showed higher removal efficiencies on the explosives than the efficiencies reported in other previous studies. Finally, Serratia sp. KT 22 showed synergistic removal efficiency in a mixed culture with Bacillus sp. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Figure 2. Effects of explosives concentrations on bacterial growth rates. (a) Effect of TNT concentra- tions on growth of KT22; (b) Effect of 2,4-DNT concentrations on growth of KD4; (c) Effect of 2,6-DNT concentrations on growth of KD6. (a) (b) (c) (a) (b) (c) Figure 3. Effect of temperature on explosive removal efficiency. 122 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Despite the significant level of advancement made in the en- vironmental remediation techniques by using chemical, physical and biological media, numerous side effects induced by chemi- cals used or the underlying challenges in identifying the bacteria with high removal efficiency always have been limiting factors. 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