Degradation of chlorobenzene and 2-Chlorotoluene by immobilized bacteria strains comamonas testosterone KT5 and bacillus subtilis DKT

TAP CHI SINH HOC 2019, 41(4): 131–138 DOI: 10.15625/0866-7160/v41n4.13684 131 DEGRADATION OF CHLOROBENZENE AND 2-CHLOROTOLUENE BY IMMOBILIZED BACTERIA STRAINS Comamonas testosterone KT5 AND Bacillus subtilis DKT Ha Danh Duc * , Nguyen Thi Oanh Dong Thap University, Cao Lanh City, Dong Thap Province, Viet Nam Received 13 March 2019, accepted 5 October 2019 ABSTRACT Chlorobenzenes and chlorotoluenes have been used to produce a number of industrial products. They are toxic and widely detected in environments due to human contributory negligence. In this article, the mixed culture of a toluenes-degrading bacterial strain, Comamonas testosterone KT5 (a Gram-positive, catalase-positive bacterium) and a chlorobenzenes-degrading bacterial strain, Bacillus subtilis DKT (a Gram-negative soil bacterium) effectively degraded both chemical compounds co-contaminating in liquid media. In addition, the degradations of mixed compounds by biofilm, bacteria immobilized in polyurethane foam (PUF) and alginate were determined. The results showed that the degradation of both compounds by cells in alginate was significantly higher than that by suspended cells. Moreover, cells immobilized in these materials showed lower adverse effects than those of non-immobilized cells for long-term storage. For examples, the degradation rates for chlorobenzine and 2-chlorotoluene by resting cells reduced by 39.5% and 37.3% after storage for 4 months at 4°C, while the degradation rates by immobilized cells decreased by from 16.3% to 19.8% respectively. Keywords: Bacillus subtilis DKT, Comamonas testosterone KT5, chlorobenzenes, chlorotoluenes, degradation, immobilized cells. Citation: Ha Danh Duc, Nguyen Thi Oanh, 2019. Degradation of chlorobenzene and 2- chlorotoluene by immobilized bacteria strains Comamonas testosterone KT5 and Bacillus subtilis DKT. Tap chi Sinh hoc (Journal of Biology), 41(4): 131–138. https://doi.org/10.15625/0866-7160/v41n4.13684. *Corresponding author email: hadanhduc@gmail.com ©2019 Vietnam Academy of Science and Technology (VAST) Ha Danh Duc, Nguyen Thi Oanh 132 INTRODUCTION Chlorobenzenes and chlorotoluenes are aromatic components widely used to produce a number of industrial products. For examples, chlorobenzenes are used to synthesize solvent, pesticides, dyes, pharmaceuticals, rubbers, plastics and disinfectants (Yadav et al., 1995; Zhang et al., 2005). Chlorotoluenes are components of agro-chemicals, flame retardants, dyes, pesticides, varnish and pigments, textile additives, pharmaceuticals, adhesives, polymers and resins, drain cleaners (Dobslaw & Engesser 2012). Meanwhile, they harmfully affect people and other species in food chains. Hence, these compounds are identified as priority pollutants by the U.S. Environmental Protection Agency (Zhang et al., 2011; Oh & Bartha, 1994). Both chlorobenzenes and chlorotoluenes are commonly detected and persistent in environments. They are main components detected in river water, lake water, seawater and treated wastewater in Greece (Nikolaou et al., 2002). In other investigations, benzenes and toluenes co-contaminate water bodies (Slaine & Barker 1990; Nikolaou et al., 2002; Groschen et al., 2004; Mottaleb et al., 2003; Robinson et al., 2004; Waddell et al., 2004). Biodegradation is the main mechanism for the removal of organic aromatic compounds contaminated environments because this method is an effectively, ecofriendly and cheap approach. A number of reports indicate that toluene and benzene inhibit to other during biodegradation processes (Chang et al., 1993; Oh et al., 1994; Bielefeldt & Stensel 1999; Reardon et al., 2000; Lin et al., 2007). In a previous report, the use of dual-species biofilm of Comamonas testosterone KT5 and Bacillus subtilis DKT could overcome this inhibition (Nguyen & Ha 2019). Comamonas testosterone KT5 effectively degraded some toluene compounds (Duc 2017), while B. subtilis DKT degraded a wide range of chlorobenzenes (Nguyen & Ha 2019). The dual-species biofilm of these isolates enhanced the degradation of both chlorobenzine and 2- chlorotoluene (Nguyen & Ha 2019). For degradation of toxic substrates by microorganisms, cell immobilization is a preferred method because it may enhance degradation rates, reduces cell leakage and reactor volume. Some common immobilization methods such as biofilm, using alginate and PUF have been widely applied. This study describes the degradation of monochlorobenzene and 2-chlorotoluene by immobilized mixed-species of C. testosterone KT5 and B. subtilis BKT. MATERIALS AND METHODS Culture media The mineral medium (MM medium) components were described by Duc (2017) supplemented with 0.1% yeast extract (w/v, MMY medium). The media were autoclaved at 121 o C for 15 min. Monochlorobenzene and 2-chlorotoluene (purity > 99.5%) were purchased from Sigma–Aldrich (Singapore). These compounds were dissolved in methanol as stock solutions (0.1 M) prior to use. Immobilization methods Comamonas testosterone KT5 and Bacillus subtilis BKT were cultured in the MMY medium supplemented with 0.25 mM monochlorobenzene and 0.5 mM 2- chlorotoluene for 24 hours. The incubation was carried out at room temperature (30 o C) and 150 rpm. Cells were harvested by centrifuging at 8.000 rpm for 5 min at 4 o C. The cell pellets were then washed twice with saline (0.85% NaCl) and resuspended in MMY medium (resting cell culture, 5×10 11 CFU/ml) and used for immobilization. The alginate entrapment of bacteria was performed according to a previous study (Ha Danh Duc & Bui Minh Triet 2017). Bacterial cell suspension of the mixed culture was added to a sterilized alginate solution and mixed by stirring with a magnetic stirrer to give a final alginate concentration of 3%. This mixture was dropped using a syringe to a cold sterile CaCl2 (0.2 M) solution to obtain beads with 3 mm in diameter. The cell numbers with approximately 2×10 8 CFUs/bead were yielded. The beads were washed twice with Degradation of chlorobenzene and 2- chlorotoluene 133 saline and then hardened by submerging in a fresh sterile CaCl2 solution for 8 hours in a static condition. The PUF immobilization was carried out by the method described by Ha Danh Duc & Bui Minh Triet (2017) with modification. The PUF was cut into 5 mm cubes, placed in the packed reactor up to 70 cm 3 . 130 mL of resting cell culture were added to the reactor. The reactor was operated for 1 day allowing bacteria to immobilize in the PUF cubes. For cell immobilization in ceramic rings, the carriers made from Jiangxi Yiyuan Industrial Co., Ltd, China were used. Each ceramic ring has a height × outer diameter of 1.5 × 1.5 cm, specific surface of 330 m 2 /m 3 , void volume of 0.7 m 3 /m 3 , bulk density of 690 kg/m 3 . Ceramic rings were placed in the packed reactor up to 70 cm 3 . 130 mL of resting cell culture were added into the reactor. The reactor was operated for 1 day for biofilm formation. Set up and operational conditions of a packed reactor A cylindrical, lab-scale packed reactor was set up using a glass column with the schematic illustrated in fig. 1. All reactor parameters were shown in table 1. The feed was pumped into the reactor on the top, while filter sterile air was introduced to the bottom through a ceramic diffuser. The packed reactor was operated at room temperature (30 o C) in batch reactor. After 24 hours for each cycle, the spent medium was removed and the carriers were clean with sterile water twice. A new sterile MMY medium was then added for the next cycle. The chemical degradation was carried out by immobilized and resting cell culture of mixed species of C. testosterone KT5 and B. subtilis BKT. MMY medium with chlorobenzine (0.5 mM) and 2- chlorotoluene (1.0 mM) was used in all experiments. Table 1. Main features of the packed reactor. Attribute Packed reactor Height 15 cm Diameter 5.5 cm (inside) Liquid volume 130 cm 3 Aeration 0.75 mL air/mL liquid medium/min (Habibi & Vahabzadeh 2013) Packed volume 70.0 cm 3 Figure 1. Ceramic rings used for cell immobilization Figure 2. Schematic diagram of the lab-scale packed reactor Ha Danh Duc, Nguyen Thi Oanh 134 Storage conditions The immobilized cells were used to degrade chlorobenzine and 2-chlorotoluene for 3 cycles in the reactor when cells on the carriers were saturated. These carriers with immobilized cells and resting cells were stored at 4 o C and room temperature in a dark condition for 4 months. Analytical method Chlorobenzine and 2-chlorotoluene were measured using reverse phase high performance liquid chromatography (HPLC) (LC-10AD, Shimadzu, Japan) with a C18 column (5 µm, 250 mm×4.6 mm; Hyperclone, Phenomenex, USA) at absorbance of 240 nm. A mixture of acetonitrile and ultrapure water (7: 3, v/v) served as a mobile phase at a flow rate of 1 mL/min. Cell numbers of each strain were determined by counting CFUs on solid MMY medium after 24 hours of incubation. C. testosterone KT5 formed round and convex colonies, while colonies of B. subtilis DKT were irregular, large size and undulate margin. Statistical analysis All experiments were conducted at least in triplicate. Data are shown as the mean ± one standard deviation. The SPSS software program version 22.0 was used to analyze variance, and significant differences (p < 0.05) were calculated using Duncan’s test. RESULTS AND DISCUSSION Chemical degradation by mixed culture of Comamonas testosterone KT5 and Bacillus subtilis DKT in a packed reactor The degradation of chlorobenzine and 2- chlorotoluene by resting cell culture and cells immobilized in alginate were compared. The results showed that the degradation percentages of chlorobenzine by immobilized cells and suspended cells for 24 hours were 84.4 ± 5.4 and 60.2% ± 7.1%, while data for 2-chlorotoluene were 92.2 ± 2.3 and 68.0 ± 6.1% in spite of the equal cell numbers (10 9 CFUs/ml), respectively. The alginate layer protected immobilized bacteria from chemical toxicity resulting in higher degradation compared to counterparts. Figure 3. Degradation of chlorobenzine ( ) and 2-chlorotoluene ( ) by mixed-species biofilm of C. testosterone KT5 and B. subtilis DKT on ceramic rings. Chlorobenzine and 2-chlorotoluene were used at 0.5 and 1.0 mM, respectively. Different small letters (a, b, c) indicate statistically significant differences (p < 0.05) in chlorobenzine degradation, while capital letters (A, B, C) indicate statistically significant differences in 2-chlorotoluene degradation Degradation of chlorobenzene and 2- chlorotoluene 135 The degradation rates of chlorobenzine and 2-chlorotoluene by several types of immobilized cell mixture were compared. In our previous report, B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm on carriers (Nguyen & Ha 2019). The degradation performances of both compounds by biofilm on ceramic rings were low in the first cycle (Fig. 3) probably due to low biofilm formation. However, the degradation increased in following cycles because more cells attached to the carrier. The degradation rates were not statistically different at the 3 rd , 4 th , 5 th and 6 th cycle (Fig. 3) probably because cell numbers in biofilm forming on the carriers did not change. The reductions of available O2 and nutrients in inner biofilm layer were the reasons that limit attached cell numbers (Lazarova & Manem, 1995). Similar to the degradation by cell immobilized on ceramic rings, the degradation of chlorobenzine and 2-chlorotoluene by mixed-species culture in PUF was low at the first cycle (Fig. 4). Meanwhile, the degradation rates in other cycles were not statistically different. The degradation rates of these substrates by cell immobilized in PUF were higher than those in ceramic rings about 20% though both carriers had the same volume. The high porosity of PUF probably accounted for higher cell numbers immobilized in this material. Figure 4. Degradation of chlorobenzine ( ) and 2-chlorotoluene ( ) by mixed-species of C. testosterone KT5 and B. subtilis DKT immobilized in PUF. Chlorobenzine and 2-chlorotoluene were used at 0.5 and 1.0 mM, respectively. Different small letters (a, b, c) indicate statistically significant differences (p < 0.05) in chlorobenzine degradation, while capital letters (A, B, C) indicate statistically significant differences in 2-chlorotoluene degradation For degradation by bacteria immobilized in alginate, the degradation rates were similar to those by bacteria immobilized in PUF. However, the rates were reduced in two last cycles (Fig. 5). The decrease of degradation occurred because some beads were broken and/or dissolved in solution resulting higher cell leakage (Table 1). The poor instability of alginate beads was also reported in a previous study (Ha Danh Duc & Bui Minh Triet, 2017). During operation processes, bacteria detached from biofilm on ceramic rings, moved from PUF and alginate beads into liquid medium. The determination of cell numbers in liquid media with immobilization carriers from the first to third cycles were: ceramic rings ≈ CFU > alginate, at the fourth cycle were: ceramic rings ≈ CFU ≈ alginate, and at the fifth and sixth cycles were: ceramic rings ≈ CFU < alginate. Bacterial cell Ha Danh Duc, Nguyen Thi Oanh 136 numbers in liquid medium with ceramic rings and PUF were similar in all cycles, while those with alginate increased from the first to the 6 th cycle (table 2). The alginate layer reduced bacteria to move into liquid media in early cycles. The determination of cell numbers in the mixture in liquid media showed that cell numbers of each strain were similar (data were not shown). The previous study was also showed that both strains did not inhibit to other during the incubation (Nguyen & Ha 2019). Figure 5. Degradation of chlorobenzine ( ) and 2-chlorotoluene ( ) by mixed-species of C. testosterone KT5 and B. subtilis DKT immobilized in alginate. Chlorobenzine and 2-chlorotoluene were added at 0.5 and 1.0 mM, respectively. Different small letters (a, b, c) indicate statistically significant differences in chlorobenzine degradation, while capital letters (A, B, C) indicate statistically significant differences in 2-chlorotoluene degradation (p < 0.05) Table 2. Bacteria in liquid media in the reactor after 24 hours of operation. Materials Cell in liquid media (×108 CFUs/mL)* 1st cycle 2nd cycle 3rd cycle 4th cycle 5th cycle 6th cycle Ceramic rings 0.72 ± 0.10aA 0.78 ± 0.17aA 0.81 ± 0.16aA 0.83 ± 0.16aA 0.80 ± 0.16aA 0.84 ± 0.16aA PUF 0.71 ± 0.09aA 0.71 ± 0.11aA 0.74 ± 0.12aA 0.75 ± 0.14aA 0.73 ± 0.13aA 0.69 ± 0.12aA Alginate 0.41 ± 0.05bA 0.48 ± 0.03bA 0.60 ± 0.06bAB 0.74 ± 0.12aA 1.14 ± 0.15bC 1.25 ± 0.17bC Note: *: Different small letters (a and b) indicate statistically significant differences (p < 0.05) in the same columns, while capital letters (A, B and C) indicate statistically significant differences in the same lines. Storage stability of free cells and immobilized bacterial cells in degrading chlorobenzine and 2-chlorotoluene After 4-month storage at room temperature and 4°C, the biodegradation percentages of both chlorobenzine and 2- chlorotoluene were determined and shown in Figure 6. The decrease of degradation by immobilized cells was lower than resting cells. This result indicated that the immobilized cells showed lower reduction of the adverse effects compared to free cells. For storage at room temperature, the degradation rates of these compounds by resting cells reduced by 47.7% and 50.5%, respectively. The degradation rates of both compounds by immobilized cells decreased by from 28.1 to 33.3%, and these reductions of immobilized cells on each carrier were similar. Degradation of chlorobenzene and 2- chlorotoluene 137 For storage at 4°C, the degradation rates of chlorobenzine and 2-chlorotoluene by resting cells reduced by 39.5 and 37.3%, respectively. The degradation rates of immobilized cells decreased by from 16.3 to 19.8%. Similar to the storage at room temperature, the degradation by any type of immobilized cells was similar. Figure 6. Degradation of chlorobenzine ( ) and 2-chlorotoluene ( ) by suspended and immobilized cells of the mixed-species of C. testosterone KT5 and B. subtilis DKT after 4-month storage at room temperature (1) and 4 o C (2). Chlorobenzine and 2-chlorotoluene were added at 0.5 and 1.0 mM, respectively. Different small letters (a, b, c) indicate statistically significant differences in chlorobenzine degradation, while capital letters (A, B, C) indicate statistically significant differences in 2-chlorotoluene degradation (p < 0.05) These results indicated that the immobilized cells showed better degradation in any condition and reduced adverse effects for long-term storage. Although a number of studies investigating the degradation of chlorobenzine and 2- chlorotoluene have been reported, no publication described the degradation by cells immobilized in PUF or alginate. CONCLUTION This study investigated the degradation of chlorobenzine and 2-chlorotoluene by mixed culture of Comamonas testosterone KT5 and Bacillus subtilis DKT. The degradation rates for these compounds of immobilized cells were higher than those of suspended cells. The immobilized cells also reduced adverse effects during storage. The results in this work showed a potential for application of immobilized cells as well as the mixed species for degradation of chlorobenzine and 2- chlorotoluene. REFERENCES Bielefeldt A. R, Stensel H. D., 1999. Modeling competitive inhibition effects during biodegradation of BTEX mixtures. Water Res., 33: 707–714. Chang M. K, Voice T. C., Criddle C. S., 1993. Kinetics of competitive inhibition and co- metabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates. Biotechnol. Bioeng., 41: 1057–1065. Dobslaw D., Engesser K. H., 2012. Degradation of 2-chlorotoluene by Rhodococcus sp. OCT 10. Appl. Microbiol. Biotechnol., 93: 2205–2214. Duc H. D., 2017. Degradation of chlorotoluenes by Comamonas testosterone KT5. Appl. Biol. Chem., 60(4): 457–465. Groschen G. E., Arnold T. L., Harris M. A., Dupre D. H., Fitzpatrick F. A., Scudder B. C., Morrow W. S., Terrio P. J., Warner K. L., Elizabeth A., 2004. Murphy water quality in the upper Illinois River Basin, Illinois, Indiana, and Wisconsin, 1999-2001. U.S. Geological Survey, Reston, Virginia. Ha Danh Duc, Nguyen Thi Oanh 138 Ha Danh Duc, Bui Minh Triet, 2017. Biodegradation of aniline by freely suspended and immobilized Pseudomonas moraviensis AN-5. Tap chi Sinh hoc (Journal of Biology), 39(3): 303–308. https://doi.org/10.15625/0866-7160/ v39n3.9630. Habibi A., Vahabzadeh F., 2013. Degradation of formaldehyde in packed-bed bioreactor by kissiris-immobilized Ralstonia eutropha. Biotechnol. Bioprocess Eng., 18(3): 455–464. Lazarova V., Manem J., 1995. Biofilm characterization and activity analysis in water and wastewater treatment. Water Research, 29: 2227–2245. Lin C. W., Cheng Y. W., Tsai S. L., 2007. Multi-substrate biodegradation kinetics of MTBE and BTEX mixtures by Pseudomonas aeruginosa. Process Biochem., 42(8): 1211–1217. Mottaleb M. A., Abedin M. Z., Islam M. S., 2003. Determination of benzene, toluene, ethylbenzene and xylene in river water by solid-phase extraction and Gas chromatography. Anal. Sci., 19(10): 1365–1369. Nguyen T. O., Ha D. D., 2019. Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT. Ann. Microbiol., 69: 267–277. Nikolaou A. D., Golfinopoulos S. K., Kostopoulou M. N., Kolokythas G. A., Lekkas T. D., 2002. Determination of volatile organic compounds in surface waters and treated wastewater in Greece. Water Res., 36(11): 2883–2890. Oh Y. S., Bartha R., 1994. Design and performance of a trickling air biofilter for chlorobenzene and o-dichlorobenzene vapors. Appl. Environ. Microbiol., 60: 2717–2722. Oh Y. S., Shareefdeen Z., Baltzis B. C., Bartha R., 1994. Interactions between benzene, toluene and p-xylene (BTX) during their biodegradation. Biotechnol. Bioeng., 44: 533–538. Reardon K. F., Mosteller D. C., Rogers J. D. B., 2000. Biodegradation kinetics of benzene, toluene and phenol as single and mixed substrates for Pseudomonas putida F1. Biotechnol. Bioeng., 69: 385–400. Robinson K., Flanagan S., Ayotte J., Campo K., Chalmers A., Coles J. F., Cuffney T. F. 2004. Water quality in the New England coastal basins, Maine, New Hampshire, Massachusetts, and Rhode Island, 1999-2001. U.S. Geological Survey, Reston, Virginia. Slaine D. D., Barker J. F., 1990. The detection of naturally occurring BTX during a hydrogeologic investigation. Groundwater Monitoring & Remediation, 10: 89–94. Waddell K. M., Gerner S., Thiros S. A., Giddings E. M., Baskin R. L., Cederberg J. R., Albano C. M., 2004. Water quality in the Great Salt Lake Basins, Utah, Idaho, and Wyoming, 1998-2001. U.S. Geological Survey, Reston, Virginia. Yadav J. S., Wallace R. E., Reddy C. A., 1995. Mineralization of mono- and dichlorobenzenes and simultaneous degradation of chloro- and methyl- substitued benzenes by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol., 61: 677–680. Zhang J., Zhao W., Pan J., Qiu L., Zhu Y., 2005. Tissue-dependent distribution and accumulation of chlorobenzenes by vegetables in urban area. Environ. Int., 31: 855–860. Zhang L. L., Leng S. Q., Zhu R. Y., Chen J.M., 2011. Degradation of chlorobenzene by strain Ralstonia pickettii L2 isolated from a biotrickling filter treating a chlorobenzene- contaminated gas stream. Appl. Microbiol. Biotechnol., 91(2): 407–415.