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Removal of xylene by a mixed culture of Pseudomonas sp. NBM21 and Rhodococcus sp. BTO62 in biofilter
Journal of Bioscience and Bioengineering VOL. 108 No. 2, 136 – 141, 2009 www.elsevier.com/locate/jbiosc
Removal of xylene by a mixed culture of Pseudomonas sp. NBM21 and Rhodococcus sp. BTO62 in biofilter Euisoon Jeong,⁎,§ Mitsuyo Hirai, and Makoto Shoda Chemical Resources Laboratory, Tokyo Institute of Technology 4259, Nagatsuta, Midori-ku, Yokohama 226-8503, Japan Received 19 March 2008; accepted 10 March 2009
Xylene (a mixture of o-, m-, p-xylenes and ethylbenzene) gas removal was conducted in the a biofilter inoculated with a mixture of the m- and p-xylene-degraders, Pseudomonas sp. NBM21 and an o-xylene degrader, Rhodococcus sp. BTO62 under non-sterile conditions at 20 °C. Elimination capacities of o-, m-, and p-xylenes obtained were 180 g/m3/h at 20 °C and 100 g/m3/h at 10 °C, which were significantly higher than the 60–78 g/m3/h of previously reported biofilters, indicating that the two bacteria inoculated exhibited an almost total ability to remove xylene although only present in low numbers in the biofilter. In the sterile biofilter, carbon mass balance showed that 11.6% of the removed xylene was converted to cell mass. Among the xylene components, o-xylene was the most resistant to microbial degradation in spite of the low component ratio. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Xylene; Pseudomonas sp.; Rhodococcus sp.; Biofilter; Elimination capacity]
Xylene is a chemical that occurs naturally in crude oil, but is supplemented by additional amounts in the refining process. It is used in many common products, such as paints, rubber, adhesives, plastic bottles, and clothing ((1); Forsyth, C. S., and Faust, R. A. Toxicity summary for xylene, Oak Ridge Reservation Environmental Restoration Program, managed by Martin Marietta Energy Systems Inc., USA [http://risk.lsd.ornl. gov] [1994]). It is toxic to the liver, kidneys, and the central nervous system when it enters the body by skin contact or breathing (2). In order to protect the environment, air quality, and health, it needs to be eliminated or reduced from gaseous effluents prior to their release into the atmosphere. The biological utilization of xylene has recently received attention because it can be completely degraded under aerobic conditions (3). Biofiltration, used as a one of the biological gas treatments, has some advantages in comparison to other purifying techniques because it does not give rise to further environmental problems, it has lower investment and operational costs, and it possesses good operational stability under properly operated conditions (4). The performance of biofiltration depends on the biodegradation effectiveness and selectivity of the microbial population, composition of the waste gas stream, degree of recalcitrance of the contaminants, structure and composition of the packing material, its operating conditions, and the bioreactor design. Accordingly, it is crucial to
⁎ Corresponding author. Tel.: +82 32 590 3604; fax: +82 32 590 3600. E-mail address: [email protected] (E. Jeong). § Present address: Odor Management Team, 2-dong, Environmental Management Corporation, Environmental Research Complex, Kyungseo-dong, Seo-gu, Incheon, 404708, Korea.
understand the various aspects of the process so as to improve the efficiency of biofilters. Studies on xylene degradation in flasks have been carried out by several groups of researchers. The ability of Pseudomonas spp. as mand p-xylene degrading microorganisms (5–13) has been intensively investigated. As o-xylene degrading microorganisms, that is the most recalcitrant isomer of xylene to microbes microbial attack, Corynenbacterium strain C125 (14), Pseudomonas stutzeri OX1 (15), Rhodococcus strain B3 (16), Rhodococcus sp. strain YU6 (17), and Rhodococcus opacus TKN14 (18) have been reported. These studies focus on identification, metabolism, proposition pathway, and cloning of genes for xylene-degrading enzymes. Only a few investigations focused on the performance of gas treatment in the removal of xylene as the sole pollutant in the air flow (1, 19–21). We have isolated two bacteria that are xylene-degraders, Pseudomonas sp. NBM21 and Rhodococcus sp. BTO62 and reported the characteristics of NBM21 (22) and BTO62 (23). In this study, using a mixed culture of NBM21 and BTO62, which showed excellent removal activity for p- and o-xylene, respectively, in pure culture in the previous study, xylene gas (a mixture of three xylene isomers and ethylbenzene) removal was investigated in a non-sterile biofilter to show whether the bacteria exhibit their abilities under nonsterile conditions. In addition, degradation of each xylene isomer in the biofilter was evaluated at three temperatures. Finally, carbon mass balance in the biofilter was calculated under sterile conditions. MATERIALS AND METHODS Media composition and microorganisms The nutrient broth (NB) (Eiken Chemical Co. LTD., Tokyo) medium used in preculture contained 3 g meat extract, 10 g
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FIG. 1. Schematic diagram of biofilter. 1, air compressor; 2, xylene generator; 3, mixing chamber; 4, flow meter with needle valve; 5, inlet sampling port; 6, column; 7, outlet sampling port; 8, leachate and nutrient; 9, peristaltic pump; 10, activated carbon tower.
Polypepton, and 5 g NaCl in 1 L of distilled water. Nutrient agar (NA) containing 15 g agar in 1 L NB medium was used for counting cell number. Basal mineral (BM) medium consisted of 1.55 g K2HPO4, 0.85 g NaH2PO4∙2H2O, 2.0 g (NH4)2SO4, 0.1 g MgCl2∙6H2O, and 1 mL trace mineral solution (TMS) in 1 L distilled water. TMS was composed of 10 g EDTA, 2.0 g ZnSO4∙7H2O, 1.0 g CaCl2∙2H2O, 5.0 g FeSO4∙7H2O, 0.2 g Na2MoO4∙2H2O, 0.2 g CuSO4∙5H2O, 0.4 g CoCl2∙6H2O, and 1.0 g MnCl2∙4H2O in 1 L of distilled water. The pHs of all media were adjusted at 7.0 ± 0.2. The characteristics of the two strains of a gram-negative bacterium, NBM21, as m-, and p-xylene degraders and a gram-positive bacterium, BTO62 as an o-xylene degrader were described previously (22, 23). Biofilter set-up A laboratory-scale biofilter was set up as shown in Fig. 1. It consisted of a glass column (5 cm diameter × 30 cm long), a xylene generator, nutrient supply and water content maintenance system, and several flow control devices. Three columns were operated under the same conditions except at different temperatures. The operating conditions of the biofilter are shown in Table 1. Each column was packed along a 20-cm length with packing material, Biosol (180 g), which is made from foamed waste glass mixed with corrugated cardboard (Nishinihon Engineering Co., Shibushi). The physical properties of the Biosol are described in a previous study (22). Xylene vapor was generated by flowing air from a compressor into a generation bottle containing xylene solution, diluted by a secondary air stream from a compressor to an appropriate concentration, and was supplied to the biofilter by down flow. To keep the moisture content of the biofilter at 45% and to supply nutrients to the biofilter, 500 mL of BM medium was circulated from the top of the column using a peristaltic pump and the medium was replaced every 2–3 days. The xylene removal test was performed in a small size filter (1.5 cm diameter × 10 cm long) without inoculation of strains NBM21 and BTO62 and was shown to be less than 0.1 g/m3/h. Inoculation to biofilter Inocula for the biofilter were prepared as follows. Two 500 mL shaking flasks containing 200 mL of NB medium were inoculated with strains NBM21 and BTO62, and 100 µL of pure p- or o-xylenes were added, respectively. They were incubated for 72 h at 30 °C with 120 strokes per min. Then, each cultured broth was collected by centrifugation at 10,000 rpm for 30 min at 4 °C and the cells were washed twice with sterile deionized water. The washed cells were suspended in 100 mL of BM medium and mixed with Biosol in a sterile beaker just before packing it into a biofilter on a clean bench. The viable cell numbers of NBM21 and BTO62 at the beginning of experiment were approximately 9.0 × 106 and 7.5 × 106 CFU/g dry packing material, respectively.
Carbon mass balance To analyze carbon mass balance, the biofilter inoculated with a mixed culture of strains NBM21 and BTO62 was operated under sterile conditions where xylene vapor (mixture of xylene isomers and ethylbenzene) was supplied, after filtering through a 0.2 μm filter (Tokyo Roshi Kaisha Ltd., Advantech), for 20 days with an average loading of 51 g/m3/h. The amount of carbon incorporated into the cell mass was calculated from the relationship between the increased cell number in the packing material and dry cell weight per unit cell number, which was obtained from the cell number and the carbon content of the dried cells in drain water and the data of carbon content obtained by an elemental analyzer (MT-5, Yanaco, Japan). Analytical methods Inlet and outlet xylene concentrations sampled with syringes were determined using a gas chromatography (GC; GC-14A, Shimadzu). The GC was equipped with a flame ionization detector (FID) and a capillary column (URBON HR-1; 0.53 mm in inner diameter × 30 m in length). The GC conditions were as described in a previous study (22). Used xylene vapor contained 9% of ethylbenzene, 81% of m- and p-xylenes, and 10% of o-xylene, ethylbenzene, and each isomer concentration was measured and xylene concentration was expressed as the sum of the three isomers and ethylbenzene, unless otherwise stated. Because the retention times of m- and p-xylenes are the same with GC under these GC analytical conditions, the concentration of the sum of m- and pxylenes was used. Viable cell number in the packing material was determined as follows: about 3 gwet weight of Biosol was homogenized (EX-3 homogenizer, Nihon Seiki LTD.) in 10 mL of sterilized distilled water at 10,000 rpm for 3 min and the homogenized suspension was serially diluted with sterilized distilled water and spread on NA plates. After incubation at 30 °C for 72 h, the number of colonies that grew on the plates was counted and expressed as colony forming units (CFU) per g dry weight of Biosol.
TABLE 1. Operating condition of biofilter. Packing material Initial viable cell number (CFU/g) Flow rate (mL/min) Space velocity (SV) (h− 1) Linear velocity (LV) (m/h) Empty bed retention time (EBRT) (s) Inlet concentration (g/m3) Inlet load (g/m3/h) Temperature (°C)
Biosol 7.3 × 106 500 76 15 47 0.4–7.5 33–570 10 ± 2, 20 ± 2, 30 ± 2
FIG. 2. Biofilter performances using a mixture of strains NBM21 and BTO62 for xylene removal at 20 °C. Closed squares, inlet xylene concentration and inlet loading rate; open squares, outlet xylene concentration; open triangles, xylene removal efficiency.
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J. BIOSCI. BIOENG., TABLE 2. Amount of carbon converted to cells of xylene degradation in biofilter. Item of measured carbon Removal xylene Biomass a
FIG. 3. Elimination capacity of xylene against inlet loading rate of xylene at 20 °C in the biofilter inoculated with a mixture of strains NBM21 and BTO62. Closed triangles, sum of o-, m-, p-xylenes, and ethylbenzene; open circles, sum of o-, m-, and p-xylenes.
Strains NBM21 and BTO62 were distinguishable from the contaminants by their morphology and color. Each column was operated under three different temperatures. For each column, data deviations, such as xylene in outlet, were less than 10%.
RESULTS Biofilter performance Xylene, the sum of o-, m-, p-xylenes and ethylbenzene was supplied to the biofilter in the concentration range of 0.4–7.5 g/m3, corresponding to an inlet load of 30–530 g/ m3/h. The gas flow rate was fixed at 500 mL/min (corresponding 47 s of empty bed retention time (EBRT) and 75 h− 1 of space velocity (SV)). Fig. 2 shows xylene removal under non-sterile conditions at 20 °C for 18 days. The initial inlet load of xylene was set at 30 g/m3/h and more than 90% xylene removal efficiency was achieved. At 3, 7, 13, and 16 days, inlet load was increased to 90, 170, 290, and 530 g/m3/h, respectively, but the removal efficiency was decreased from 100% to 60%. The relation between elimination capacity of xylene and inlet loading of xylene in the biofilter is shown in Fig. 3. Maximum xylene elimination capacity to maintain
FIG. 4. Gas chromatograms of leachate extract from the biofilter under 100% xylene removal (A) and 80% removal (B) efficiencies. Peaks of 6.1, 6.4, and 10.5 min: m- and p-xylenes, o-xylene and m- and p-toluic acids, respectively, other peaks: unknown.
Amount of carbon (g-C/m3/h) 42.4 (100%) a 4.9 (11.6%)
Parenthesis: percentage against removal xylene.
greater than 90% removal efficiency was 220 g/m3/h (solid line). The elimination capacity of the sum of o-, m- and p-xylenes was 180 g/m3/h (dotted line). The viable cell numbers of strains NBM21 and BTO62 at the end of the experiment were estimated to be 1.3 × 109 and 2.4 × 108 CFU/g, respectively, which were about 28% and 5% of total cell numbers (4.6 × 109 CFU/g), respectively. Analysis by GC of the leachate extract from the biofilter under 100% removal and 80% removal efficiencies are shown in Figs. 4A and B, respectively. The few peaks corresponding to the metabolic intermediates of xylene are shown in Fig. 4A. This indicates that xylene was degraded to low molecular weight compounds or non-aromatic compounds such as pyruvate, acetaldehyde and 1-propanol in the biofilter. Some studies have reported metabolites of xylene (24, 25). At a removal efficiency of 80%, several peaks were detected as shown in Fig. 4B. Peaks at 3.6, 3.8, 4.2, and 10.5 min in Fig. 4B were identified as ethylbenzene, m- and p-xylenes, o-xylene, and m- and p-toluic acids, respectively, but other peaks were not identified. Carbon mass balance The carbon mass balance from a mixture of NBM21 and BTO62 was calculated under sterile conditions for 3 days after steady state of xylene removal (17 days) as shown in
FIG. 5. Biofilter performances using a mixture of strains NBM21 and BTO62 for xylene removal at 10 °C (A), and 30 °C (B). Closed squares, inlet xylene concentration and inlet loading rate; open squares, outlet xylene concentration; open triangles, xylene removal efficiency.
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experiment in Fig. 2. As the inlet loading rate of xylene was increased, removal efficiencies of m-, p- and o-xylenes decreased, however, ethylbenzene was completely removed, mainly because ethylbenzene was preferentially utilized at 20 °C (Fig. 8A). o-Xylene removal efficiency was the lowest among all components in spite of its low loading rate, indicating that o-xylene is the least biodegradable in biofiltration. The overall removal efficiency of xylenes was maintained at above 90%, and o-xylene removal efficiency was 65%. This result indicates that majority of xylene removal was m-, and pxylenes, but 65% o-xylene removal efficiency contributed to the high overall efficiency of xylenes. Elimination capacity to maintain above 90% o-xylene removal efficiency was 150 g/m3/h (Fig. 7), which was higher than those of other reports shown in Table 3. Fig. 8 shows the removal efficiencies of xylene components at different temperatures.
FIG. 6. Relationship between elimination capacity and inlet loading rate of xylene at 10 °C (closed squares), 20 °C (closed triangles), and 30 °C (open circles).
Table 2. Average inlet and outlet loadings were 51 and 4.2 g/m3/h, corresponding to carbon of 46.2 and 3.8 g-C/m3/h, respectively, indicating that the removed carbon of xylene was 42.4 g-C/m3/h. The carbon content of the dry cell mass was determined to be 42.3% by elemental analysis indicating that the amount of the carbon incorporated into the cells was 4.9 g/m3/h. Of the removed xylene, 11.6% was used for cell growth. Temperature effect The effect of the temperatures on the xylene removal efficiency was investigated at 10 °C and 30 °C under the same conditions as for 20 °C. Fig. 5 shows xylene removal at 10 °C (A) and 30 °C (B). At 10 °C, in spite of low inlet loading rate (90 g/m3/h), removal efficiency was 89% and when inlet loading was increased to 530 g/m3/h, removal efficiency was decreased to 43%. At 30 °C, xylene removal efficiency was maintained at more than 90% up until 170 g/m3/h of the inlet loading rate, which was lower than that at 20 °C. Fig. 6 shows elimination capacities against the inlet loading rate of xylene in biofilters at three different temperatures. Xylene (sum of o-, m- and p-xylenes) elimination capacities to guarantee more than 90% removal ratios are 100, 180, and 170 g/m3/h at 10 °C, 20 °C, and 30 °C, respectively. Xylene removal at 20 °C and 30 °C were similar, but at 10 °C the elimination capacity was considerably reduced. Removal efficiency of xylene isomers Fig. 7 presents the removal efficiency of xylene isomers and ethylbenzene against inlet loading rate of xylene. Concentrations of each component were determined at the inlet and the outlet of the biofilter in the
FIG. 7. Removal efficiency of xylene isomers and ethylbenzene against inlet loading rate of xylene at 20 °C. Open triangles, ethylbenzene; open circles, m- and p-xylenes; open squares, o-xylene; closed triangles, elimination capacity of xylene.
FIG. 8. Removal efficiencies of xylene isomers and ethylbenzene against the inlet loading rate of xylene at 10 °C (open triangles), 20 °C (open squares) and 30 °C (closed circles) in the biofilter. (A) Ethylbenzene, (B) m- and p-xylenes, (C) o-xylene.
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J. BIOSCI. BIOENG., TABLE 3. Elimination capacity (EC) for xylene reported previously in biofilter.
EBRT a (s) 56–150 67 63–157 Not shown 47 a b c
Packing materials (diameter)
Seed
Inlet loading (g m− 3 h− 1)
EC (g m− 3 h− 1)
References
Peat Peat balls (5–10 mm) Peat Pall rings Biosol (4–6 mm)
Microbial consortium Microbial consortium Microbial consortium Microbial consortium NBM21 and BTO62
34–95 25–195 10–110 20–120 33–570
61 (93% of RE b) 60 (maximum) 67 (maximum) 78 (67% of RE) c 180 (at 20 °C) (90% of RE)
1 19 20 21 This study
EBRT: empty bed retention time. RE: removal efficiency. The value excepted ethylbenzene among the xylene components.
Removal efficiencies of m-, and p-xylenes (Fig. 8B), and o-xylene (Fig. 8C) at 20 °C and 30 °C were almost similar, however, that of ethylbenzene (Fig. 8A) was 100% at 20 °C, and decreased significantly at 10 °C and 30 °C. DISCUSSION Three non-sterile biofilters of a mixed culture of NBM21 and BTO62 were run to investigate xylene degradation under three different temperatures and one sterile biofilter was operated to calculate the carbon mass balance of xylene degradation. The xylene elimination capacity of the sum of o-, m-, p-xylenes, and ethylbenzene of a mixed culture was 220 g/m3/h. Elimination capacity (180 g/m3/h) of o-, m-, and p-xylenes was considerably higher than the 60–78 g/m3/h of previously reported biofilters that used microbial consortium such as horticultural peat-soil, garden soil, and the activated sludge of waste treatment plants (Table 3). The p-xylene elimination capacity for more than 90% removal efficiency by the strain NBM21 was 160 g/m3/h (22), and that of o-xylene by the strain BTO62 was 160 g/m3/h (23). From the data, it is speculated that the two strains exhibited their full degradation abilities in the biofilter, although the proportion of the two strains was not high. Strains that have a high conversion from volatile organic compounds (VOCs) to CO2 and a low conversion to cell mass are the best candidates for xylene removal because the excessive production of biomass in a filter causes clogging of the filter bed. However, there are no reports on the conversion ratio of p- or oxylene to biomass by xylene-degrading bacteria. In this study, the amount of carbon converted to cells in the biofilter was calculated as 11.6%. These values were similar to values in the sealed flask test: cells of 7.3% for p-xylene removal by strain NBM21; cells of 12.7% for o-xylene removal by strain BTO62 (Tables 2 and 4). Therefore, the use of the two strains would be beneficial for the treatment of xylene because packing or clogging in the cell-supporting materials will be minimized in the biofilter. Although operating times were short, up to 18 days, percentages of NBM21 and BTO62 viable cell number decreased due to an increase of other strains, but viable cell numbers of NBM21 and BTO62 were constantly maintained until the end of operation. The xylene (sum of o-, m- and p-xylenes) maximum elimination capacity to maintain above 90% removal efficiency was 180 g/m3/h at 20 °C, which was higher than those of 100 g/m3/h at 10 °C and 170 g/m3/h at 30 °C. This means that optimum temperature was
TABLE 4. Amount of carbon converted to cells of p-xylene degradation by NBM21 and o-xylene degradation by BTO62 in sealed flasks. Items of measured carbon Degraded xylene Biomass a
Amount of carbon (g-C/m3) p-xylene by NBM21
o-xylene by BTO62
835 (100%) a 61 (7.3%)
771 (100%) 98 (12.7%)
Percentage against degraded xylene.
between 20–30 °C. The data indicate that the performance of the biofilter will decrease by about 45% in winter and this must be considered in future biofilter design. From GC patterns, the amount of xylene degradation products decreased to about 170 g/m3/h of the inlet loading rate at 100% removal. This also must be considered in the design of a biofilter.
References 1. Elmrini, H., Bredin, N., Shareefdeen, Z., and Heitz, M.: Biofiltration of xylene emissions: bioreactor response to variations in the pollutant inlet concentration and gas flow rate, Chem. Eng. J., 100, 149–158 (2004). 2. Yoon, I. and Park, C.: Effects of gas flow rate, inlet concentration and temperature on biofiltration of volatile organic compounds in a peat-packed biofilter, J. Biosci. Bioeng., 93, 165–169 (2002). 3. Agteren, M. H., Keuning, S., and Janssen, D. B.: Handbook on Biodegradation and Biological Treatment of Hazardous Organic Compounds, 2nd ed. Academic Press, Dordrecht, 1998. 4. Zilli, M., Converti, A., Lodi, A., Borghi, M. D., and Ferraiolo, G.: Phenol removal from waste gases with a biological filter by Pseudomonas putida, Biotechnol. Bioeng., 41, 693–699 (1993). 5. Omori, T., Horiguchi, S., and Yamada, K.: Studies on the utilization of hydrocarbons by microorganisms: X. Screening of aromatic hydrocarbon-assimilating microorganisms and p-toluic acid formation from p-xylene, Agr. Biol. Chem., 31, 1337–1342 (1967). 6. Davis, R. S., Hossler, F. E., and Stone, R. W.: Metabolism of p- and m-xylene by species of Pseudomonas, Can. J. Microbiol., 14, 1005–1009 (1968). 7. Nozaka, J. and Kusunose, M.: Metabolism of hydrocarbon in microorganism: I. Oxidation of p-xylene and toluene by cell free enzyme preparation of Pseudomonas aeruginosa, Agr. Biol. Chem., 32, 1033–1039 (1968). 8. Davey, J. F. and Gibson, D. T.: Bacterial metabolism of para- and meta-xylene: oxidation of a methyl substituent, J. Bacteriol., 119, 923–929 (1974). 9. Worsey, M. J. and Williams, P. A.: Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid, J. Bacteriol., 124, 7–13 (1975). 10. Skriabin, G. K., Ganbarov, K. H. G., Golovleva, L. A., Chervin, I. I., and Adanin, V. M.: Peripheral metabolism of isomeric xylenes by Pseudomonas aeruginosa, Mikrobiologiia, 45, 951–954 (1976). 11. Golovleva, L. A., Golovlev, E. L., Panchak, N. V., and Ganbarov, K. G.: Variability in the enzyme properties of Pseudomonas aeruginosa strain 2X oxidizing p-xylene, Biol. Bull. Acad. Sci. USSR, 6, 459–463 (1979). 12. Cruden, D. L., Wolfram, J. H., Rogers, R. D., and Gibson, D. T.: Physiological properties of a Pseudomonas strain which grows with p-xylene in a twophase (organic–aqueous) medium, Appl. Environ. Microbiol., 58, 2723–2729 (1992). 13. Bramucci, M. G., McCutchen, C. M., Singh, M., Thomas, S. M., Larsen, B. S., Buckholz, J., and Nagarajan, V.: Pure bacterial isolates that convert p-xylene to terephthalic acid, Appl. Microbiol. Biotechnol., 58, 255–259 (2002). 14. Schraa, G., Bethe, B. M., van Neerven, A. R. W., van den Tweel, W. J. J., van der Wende, E., and Zehnder, A. J. B.: Degradation 1, 2-dimethylbenzene by Corynebacterium strain C125, Antonie van Leeuwenhoek, 53, 159–170 (1987). 15. Baggi, G., Barbieri, P., Galli, E., and Tollari, S.: Isolation of a Pseudomonas stutzeri strain that degrades o-xylene, Appl. Environ. Microbiol., 53, 2129–2132 (1987). 16. Bickerdike, S. R., Holt, R. A., and Stephens, G. M.: Evidence for metabolism of o-xylene by simultaneous ring and methyl group oxidation in a new soil isolate, Microbiology, 143, 2321–2329 (1997). 17. Jang, J. Y., Kim, D., Bae, H. W., Choi1, K. Y., Chae, J. C., Zylstra, G. J., Kim, Y. M., and Kim, E.: Isolation and characterization of a Rhodococcus species strain able to grow on ortho- and para-xylene, J. Microbiol., 43, 325–330 (2005). 18. Maruyama, T., Ishikura, M., Taki, H., Shindo, K., Kasai, H., Haga, M., Inomata, Y., and Misawa, N.: Isolation and characterization of o-xylene oxygenase genes
VOL. 108, 2009 from Rhodococcus opacus TKN14, Appl. Environ. Microbiol., 71, 7705–7715 (2005). 19. Bibeau, L., Kiared, K., Brzezinski, R., Viel, G., and Heitz, M.: Treatment of air polluted with xylenes using a biofilter reactor, Water Air Soil Pollut., 118, 377–393 (2000). 20. Jorio, H., Bibeau, L., Viel1, G., and Heitz, M.: Effects of gas flow rate and inlet concentration on xylene vapors biofiltration performance, Chem. Eng. J., 76, 209–221 (2000). 21. Gloria, T. A., Sergio, R., and Ricardo, L. O.: Hydrodynamic characterization of a trickle bed air biofilter, Chem. Eng. J., 113, 145–152 (2005).
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22. Jeong, E., Hirai, M., and Shoda, M.: Removal of p-xylene with Pseudomonas sp. NBM21 in biofilter, J. Biosci. Bioeng., 102, 281–287 (2006). 23. Jeong, E., Hirai, M., and Shoda, M.: Removal of o-xylene using biofilter inoculated with Rhodococcus sp. BTO62, J. Hazard. Mater., 152, 140–147 (2008). 24. Gibson, D. T.: Microbial degradation of organic compounds, Microbiology Series, vol. 13, Marcel Dekker, New York, 2002, pp. 197–198 (2002). 25. Gennaro, P. D., Rescalli, E., Galli, E., Sello, G., and Bestetti, G.: Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o-xylene isolated from a polycyclic aromatic hydrocarbon-contaminated soil, Res. Microbiol., 152, 641–651 (2001).
Removal of xylene by a mixed culture of Pseudomonas sp. NBM21 and Rhodococcus sp. BTO62 in biofilter Euisoon Jeong,⁎,§ Mitsuyo Hirai, and Makoto Shoda Chemical Resources Laboratory, Tokyo Institute of Technology 4259, Nagatsuta, Midori-ku, Yokohama 226-8503, Japan Received 19 March 2008; accepted 10 March 2009
Xylene (a mixture of o-, m-, p-xylenes and ethylbenzene) gas removal was conducted in the a biofilter inoculated with a mixture of the m- and p-xylene-degraders, Pseudomonas sp. NBM21 and an o-xylene degrader, Rhodococcus sp. BTO62 under non-sterile conditions at 20 °C. Elimination capacities of o-, m-, and p-xylenes obtained were 180 g/m3/h at 20 °C and 100 g/m3/h at 10 °C, which were significantly higher than the 60–78 g/m3/h of previously reported biofilters, indicating that the two bacteria inoculated exhibited an almost total ability to remove xylene although only present in low numbers in the biofilter. In the sterile biofilter, carbon mass balance showed that 11.6% of the removed xylene was converted to cell mass. Among the xylene components, o-xylene was the most resistant to microbial degradation in spite of the low component ratio. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Xylene; Pseudomonas sp.; Rhodococcus sp.; Biofilter; Elimination capacity]
Xylene is a chemical that occurs naturally in crude oil, but is supplemented by additional amounts in the refining process. It is used in many common products, such as paints, rubber, adhesives, plastic bottles, and clothing ((1); Forsyth, C. S., and Faust, R. A. Toxicity summary for xylene, Oak Ridge Reservation Environmental Restoration Program, managed by Martin Marietta Energy Systems Inc., USA [http://risk.lsd.ornl. gov] [1994]). It is toxic to the liver, kidneys, and the central nervous system when it enters the body by skin contact or breathing (2). In order to protect the environment, air quality, and health, it needs to be eliminated or reduced from gaseous effluents prior to their release into the atmosphere. The biological utilization of xylene has recently received attention because it can be completely degraded under aerobic conditions (3). Biofiltration, used as a one of the biological gas treatments, has some advantages in comparison to other purifying techniques because it does not give rise to further environmental problems, it has lower investment and operational costs, and it possesses good operational stability under properly operated conditions (4). The performance of biofiltration depends on the biodegradation effectiveness and selectivity of the microbial population, composition of the waste gas stream, degree of recalcitrance of the contaminants, structure and composition of the packing material, its operating conditions, and the bioreactor design. Accordingly, it is crucial to
⁎ Corresponding author. Tel.: +82 32 590 3604; fax: +82 32 590 3600. E-mail address: [email protected] (E. Jeong). § Present address: Odor Management Team, 2-dong, Environmental Management Corporation, Environmental Research Complex, Kyungseo-dong, Seo-gu, Incheon, 404708, Korea.
understand the various aspects of the process so as to improve the efficiency of biofilters. Studies on xylene degradation in flasks have been carried out by several groups of researchers. The ability of Pseudomonas spp. as mand p-xylene degrading microorganisms (5–13) has been intensively investigated. As o-xylene degrading microorganisms, that is the most recalcitrant isomer of xylene to microbes microbial attack, Corynenbacterium strain C125 (14), Pseudomonas stutzeri OX1 (15), Rhodococcus strain B3 (16), Rhodococcus sp. strain YU6 (17), and Rhodococcus opacus TKN14 (18) have been reported. These studies focus on identification, metabolism, proposition pathway, and cloning of genes for xylene-degrading enzymes. Only a few investigations focused on the performance of gas treatment in the removal of xylene as the sole pollutant in the air flow (1, 19–21). We have isolated two bacteria that are xylene-degraders, Pseudomonas sp. NBM21 and Rhodococcus sp. BTO62 and reported the characteristics of NBM21 (22) and BTO62 (23). In this study, using a mixed culture of NBM21 and BTO62, which showed excellent removal activity for p- and o-xylene, respectively, in pure culture in the previous study, xylene gas (a mixture of three xylene isomers and ethylbenzene) removal was investigated in a non-sterile biofilter to show whether the bacteria exhibit their abilities under nonsterile conditions. In addition, degradation of each xylene isomer in the biofilter was evaluated at three temperatures. Finally, carbon mass balance in the biofilter was calculated under sterile conditions. MATERIALS AND METHODS Media composition and microorganisms The nutrient broth (NB) (Eiken Chemical Co. LTD., Tokyo) medium used in preculture contained 3 g meat extract, 10 g
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FIG. 1. Schematic diagram of biofilter. 1, air compressor; 2, xylene generator; 3, mixing chamber; 4, flow meter with needle valve; 5, inlet sampling port; 6, column; 7, outlet sampling port; 8, leachate and nutrient; 9, peristaltic pump; 10, activated carbon tower.
Polypepton, and 5 g NaCl in 1 L of distilled water. Nutrient agar (NA) containing 15 g agar in 1 L NB medium was used for counting cell number. Basal mineral (BM) medium consisted of 1.55 g K2HPO4, 0.85 g NaH2PO4∙2H2O, 2.0 g (NH4)2SO4, 0.1 g MgCl2∙6H2O, and 1 mL trace mineral solution (TMS) in 1 L distilled water. TMS was composed of 10 g EDTA, 2.0 g ZnSO4∙7H2O, 1.0 g CaCl2∙2H2O, 5.0 g FeSO4∙7H2O, 0.2 g Na2MoO4∙2H2O, 0.2 g CuSO4∙5H2O, 0.4 g CoCl2∙6H2O, and 1.0 g MnCl2∙4H2O in 1 L of distilled water. The pHs of all media were adjusted at 7.0 ± 0.2. The characteristics of the two strains of a gram-negative bacterium, NBM21, as m-, and p-xylene degraders and a gram-positive bacterium, BTO62 as an o-xylene degrader were described previously (22, 23). Biofilter set-up A laboratory-scale biofilter was set up as shown in Fig. 1. It consisted of a glass column (5 cm diameter × 30 cm long), a xylene generator, nutrient supply and water content maintenance system, and several flow control devices. Three columns were operated under the same conditions except at different temperatures. The operating conditions of the biofilter are shown in Table 1. Each column was packed along a 20-cm length with packing material, Biosol (180 g), which is made from foamed waste glass mixed with corrugated cardboard (Nishinihon Engineering Co., Shibushi). The physical properties of the Biosol are described in a previous study (22). Xylene vapor was generated by flowing air from a compressor into a generation bottle containing xylene solution, diluted by a secondary air stream from a compressor to an appropriate concentration, and was supplied to the biofilter by down flow. To keep the moisture content of the biofilter at 45% and to supply nutrients to the biofilter, 500 mL of BM medium was circulated from the top of the column using a peristaltic pump and the medium was replaced every 2–3 days. The xylene removal test was performed in a small size filter (1.5 cm diameter × 10 cm long) without inoculation of strains NBM21 and BTO62 and was shown to be less than 0.1 g/m3/h. Inoculation to biofilter Inocula for the biofilter were prepared as follows. Two 500 mL shaking flasks containing 200 mL of NB medium were inoculated with strains NBM21 and BTO62, and 100 µL of pure p- or o-xylenes were added, respectively. They were incubated for 72 h at 30 °C with 120 strokes per min. Then, each cultured broth was collected by centrifugation at 10,000 rpm for 30 min at 4 °C and the cells were washed twice with sterile deionized water. The washed cells were suspended in 100 mL of BM medium and mixed with Biosol in a sterile beaker just before packing it into a biofilter on a clean bench. The viable cell numbers of NBM21 and BTO62 at the beginning of experiment were approximately 9.0 × 106 and 7.5 × 106 CFU/g dry packing material, respectively.
Carbon mass balance To analyze carbon mass balance, the biofilter inoculated with a mixed culture of strains NBM21 and BTO62 was operated under sterile conditions where xylene vapor (mixture of xylene isomers and ethylbenzene) was supplied, after filtering through a 0.2 μm filter (Tokyo Roshi Kaisha Ltd., Advantech), for 20 days with an average loading of 51 g/m3/h. The amount of carbon incorporated into the cell mass was calculated from the relationship between the increased cell number in the packing material and dry cell weight per unit cell number, which was obtained from the cell number and the carbon content of the dried cells in drain water and the data of carbon content obtained by an elemental analyzer (MT-5, Yanaco, Japan). Analytical methods Inlet and outlet xylene concentrations sampled with syringes were determined using a gas chromatography (GC; GC-14A, Shimadzu). The GC was equipped with a flame ionization detector (FID) and a capillary column (URBON HR-1; 0.53 mm in inner diameter × 30 m in length). The GC conditions were as described in a previous study (22). Used xylene vapor contained 9% of ethylbenzene, 81% of m- and p-xylenes, and 10% of o-xylene, ethylbenzene, and each isomer concentration was measured and xylene concentration was expressed as the sum of the three isomers and ethylbenzene, unless otherwise stated. Because the retention times of m- and p-xylenes are the same with GC under these GC analytical conditions, the concentration of the sum of m- and pxylenes was used. Viable cell number in the packing material was determined as follows: about 3 gwet weight of Biosol was homogenized (EX-3 homogenizer, Nihon Seiki LTD.) in 10 mL of sterilized distilled water at 10,000 rpm for 3 min and the homogenized suspension was serially diluted with sterilized distilled water and spread on NA plates. After incubation at 30 °C for 72 h, the number of colonies that grew on the plates was counted and expressed as colony forming units (CFU) per g dry weight of Biosol.
TABLE 1. Operating condition of biofilter. Packing material Initial viable cell number (CFU/g) Flow rate (mL/min) Space velocity (SV) (h− 1) Linear velocity (LV) (m/h) Empty bed retention time (EBRT) (s) Inlet concentration (g/m3) Inlet load (g/m3/h) Temperature (°C)
Biosol 7.3 × 106 500 76 15 47 0.4–7.5 33–570 10 ± 2, 20 ± 2, 30 ± 2
FIG. 2. Biofilter performances using a mixture of strains NBM21 and BTO62 for xylene removal at 20 °C. Closed squares, inlet xylene concentration and inlet loading rate; open squares, outlet xylene concentration; open triangles, xylene removal efficiency.
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FIG. 3. Elimination capacity of xylene against inlet loading rate of xylene at 20 °C in the biofilter inoculated with a mixture of strains NBM21 and BTO62. Closed triangles, sum of o-, m-, p-xylenes, and ethylbenzene; open circles, sum of o-, m-, and p-xylenes.
Strains NBM21 and BTO62 were distinguishable from the contaminants by their morphology and color. Each column was operated under three different temperatures. For each column, data deviations, such as xylene in outlet, were less than 10%.
RESULTS Biofilter performance Xylene, the sum of o-, m-, p-xylenes and ethylbenzene was supplied to the biofilter in the concentration range of 0.4–7.5 g/m3, corresponding to an inlet load of 30–530 g/ m3/h. The gas flow rate was fixed at 500 mL/min (corresponding 47 s of empty bed retention time (EBRT) and 75 h− 1 of space velocity (SV)). Fig. 2 shows xylene removal under non-sterile conditions at 20 °C for 18 days. The initial inlet load of xylene was set at 30 g/m3/h and more than 90% xylene removal efficiency was achieved. At 3, 7, 13, and 16 days, inlet load was increased to 90, 170, 290, and 530 g/m3/h, respectively, but the removal efficiency was decreased from 100% to 60%. The relation between elimination capacity of xylene and inlet loading of xylene in the biofilter is shown in Fig. 3. Maximum xylene elimination capacity to maintain
FIG. 4. Gas chromatograms of leachate extract from the biofilter under 100% xylene removal (A) and 80% removal (B) efficiencies. Peaks of 6.1, 6.4, and 10.5 min: m- and p-xylenes, o-xylene and m- and p-toluic acids, respectively, other peaks: unknown.
Amount of carbon (g-C/m3/h) 42.4 (100%) a 4.9 (11.6%)
Parenthesis: percentage against removal xylene.
greater than 90% removal efficiency was 220 g/m3/h (solid line). The elimination capacity of the sum of o-, m- and p-xylenes was 180 g/m3/h (dotted line). The viable cell numbers of strains NBM21 and BTO62 at the end of the experiment were estimated to be 1.3 × 109 and 2.4 × 108 CFU/g, respectively, which were about 28% and 5% of total cell numbers (4.6 × 109 CFU/g), respectively. Analysis by GC of the leachate extract from the biofilter under 100% removal and 80% removal efficiencies are shown in Figs. 4A and B, respectively. The few peaks corresponding to the metabolic intermediates of xylene are shown in Fig. 4A. This indicates that xylene was degraded to low molecular weight compounds or non-aromatic compounds such as pyruvate, acetaldehyde and 1-propanol in the biofilter. Some studies have reported metabolites of xylene (24, 25). At a removal efficiency of 80%, several peaks were detected as shown in Fig. 4B. Peaks at 3.6, 3.8, 4.2, and 10.5 min in Fig. 4B were identified as ethylbenzene, m- and p-xylenes, o-xylene, and m- and p-toluic acids, respectively, but other peaks were not identified. Carbon mass balance The carbon mass balance from a mixture of NBM21 and BTO62 was calculated under sterile conditions for 3 days after steady state of xylene removal (17 days) as shown in
FIG. 5. Biofilter performances using a mixture of strains NBM21 and BTO62 for xylene removal at 10 °C (A), and 30 °C (B). Closed squares, inlet xylene concentration and inlet loading rate; open squares, outlet xylene concentration; open triangles, xylene removal efficiency.
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experiment in Fig. 2. As the inlet loading rate of xylene was increased, removal efficiencies of m-, p- and o-xylenes decreased, however, ethylbenzene was completely removed, mainly because ethylbenzene was preferentially utilized at 20 °C (Fig. 8A). o-Xylene removal efficiency was the lowest among all components in spite of its low loading rate, indicating that o-xylene is the least biodegradable in biofiltration. The overall removal efficiency of xylenes was maintained at above 90%, and o-xylene removal efficiency was 65%. This result indicates that majority of xylene removal was m-, and pxylenes, but 65% o-xylene removal efficiency contributed to the high overall efficiency of xylenes. Elimination capacity to maintain above 90% o-xylene removal efficiency was 150 g/m3/h (Fig. 7), which was higher than those of other reports shown in Table 3. Fig. 8 shows the removal efficiencies of xylene components at different temperatures.
FIG. 6. Relationship between elimination capacity and inlet loading rate of xylene at 10 °C (closed squares), 20 °C (closed triangles), and 30 °C (open circles).
Table 2. Average inlet and outlet loadings were 51 and 4.2 g/m3/h, corresponding to carbon of 46.2 and 3.8 g-C/m3/h, respectively, indicating that the removed carbon of xylene was 42.4 g-C/m3/h. The carbon content of the dry cell mass was determined to be 42.3% by elemental analysis indicating that the amount of the carbon incorporated into the cells was 4.9 g/m3/h. Of the removed xylene, 11.6% was used for cell growth. Temperature effect The effect of the temperatures on the xylene removal efficiency was investigated at 10 °C and 30 °C under the same conditions as for 20 °C. Fig. 5 shows xylene removal at 10 °C (A) and 30 °C (B). At 10 °C, in spite of low inlet loading rate (90 g/m3/h), removal efficiency was 89% and when inlet loading was increased to 530 g/m3/h, removal efficiency was decreased to 43%. At 30 °C, xylene removal efficiency was maintained at more than 90% up until 170 g/m3/h of the inlet loading rate, which was lower than that at 20 °C. Fig. 6 shows elimination capacities against the inlet loading rate of xylene in biofilters at three different temperatures. Xylene (sum of o-, m- and p-xylenes) elimination capacities to guarantee more than 90% removal ratios are 100, 180, and 170 g/m3/h at 10 °C, 20 °C, and 30 °C, respectively. Xylene removal at 20 °C and 30 °C were similar, but at 10 °C the elimination capacity was considerably reduced. Removal efficiency of xylene isomers Fig. 7 presents the removal efficiency of xylene isomers and ethylbenzene against inlet loading rate of xylene. Concentrations of each component were determined at the inlet and the outlet of the biofilter in the
FIG. 7. Removal efficiency of xylene isomers and ethylbenzene against inlet loading rate of xylene at 20 °C. Open triangles, ethylbenzene; open circles, m- and p-xylenes; open squares, o-xylene; closed triangles, elimination capacity of xylene.
FIG. 8. Removal efficiencies of xylene isomers and ethylbenzene against the inlet loading rate of xylene at 10 °C (open triangles), 20 °C (open squares) and 30 °C (closed circles) in the biofilter. (A) Ethylbenzene, (B) m- and p-xylenes, (C) o-xylene.
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J. BIOSCI. BIOENG., TABLE 3. Elimination capacity (EC) for xylene reported previously in biofilter.
EBRT a (s) 56–150 67 63–157 Not shown 47 a b c
Packing materials (diameter)
Seed
Inlet loading (g m− 3 h− 1)
EC (g m− 3 h− 1)
References
Peat Peat balls (5–10 mm) Peat Pall rings Biosol (4–6 mm)
Microbial consortium Microbial consortium Microbial consortium Microbial consortium NBM21 and BTO62
34–95 25–195 10–110 20–120 33–570
61 (93% of RE b) 60 (maximum) 67 (maximum) 78 (67% of RE) c 180 (at 20 °C) (90% of RE)
1 19 20 21 This study
EBRT: empty bed retention time. RE: removal efficiency. The value excepted ethylbenzene among the xylene components.
Removal efficiencies of m-, and p-xylenes (Fig. 8B), and o-xylene (Fig. 8C) at 20 °C and 30 °C were almost similar, however, that of ethylbenzene (Fig. 8A) was 100% at 20 °C, and decreased significantly at 10 °C and 30 °C. DISCUSSION Three non-sterile biofilters of a mixed culture of NBM21 and BTO62 were run to investigate xylene degradation under three different temperatures and one sterile biofilter was operated to calculate the carbon mass balance of xylene degradation. The xylene elimination capacity of the sum of o-, m-, p-xylenes, and ethylbenzene of a mixed culture was 220 g/m3/h. Elimination capacity (180 g/m3/h) of o-, m-, and p-xylenes was considerably higher than the 60–78 g/m3/h of previously reported biofilters that used microbial consortium such as horticultural peat-soil, garden soil, and the activated sludge of waste treatment plants (Table 3). The p-xylene elimination capacity for more than 90% removal efficiency by the strain NBM21 was 160 g/m3/h (22), and that of o-xylene by the strain BTO62 was 160 g/m3/h (23). From the data, it is speculated that the two strains exhibited their full degradation abilities in the biofilter, although the proportion of the two strains was not high. Strains that have a high conversion from volatile organic compounds (VOCs) to CO2 and a low conversion to cell mass are the best candidates for xylene removal because the excessive production of biomass in a filter causes clogging of the filter bed. However, there are no reports on the conversion ratio of p- or oxylene to biomass by xylene-degrading bacteria. In this study, the amount of carbon converted to cells in the biofilter was calculated as 11.6%. These values were similar to values in the sealed flask test: cells of 7.3% for p-xylene removal by strain NBM21; cells of 12.7% for o-xylene removal by strain BTO62 (Tables 2 and 4). Therefore, the use of the two strains would be beneficial for the treatment of xylene because packing or clogging in the cell-supporting materials will be minimized in the biofilter. Although operating times were short, up to 18 days, percentages of NBM21 and BTO62 viable cell number decreased due to an increase of other strains, but viable cell numbers of NBM21 and BTO62 were constantly maintained until the end of operation. The xylene (sum of o-, m- and p-xylenes) maximum elimination capacity to maintain above 90% removal efficiency was 180 g/m3/h at 20 °C, which was higher than those of 100 g/m3/h at 10 °C and 170 g/m3/h at 30 °C. This means that optimum temperature was
TABLE 4. Amount of carbon converted to cells of p-xylene degradation by NBM21 and o-xylene degradation by BTO62 in sealed flasks. Items of measured carbon Degraded xylene Biomass a
Amount of carbon (g-C/m3) p-xylene by NBM21
o-xylene by BTO62
835 (100%) a 61 (7.3%)
771 (100%) 98 (12.7%)
Percentage against degraded xylene.
between 20–30 °C. The data indicate that the performance of the biofilter will decrease by about 45% in winter and this must be considered in future biofilter design. From GC patterns, the amount of xylene degradation products decreased to about 170 g/m3/h of the inlet loading rate at 100% removal. This also must be considered in the design of a biofilter.
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