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Enhancement of styrene removal efficiency in biofilter by mixed cultures of Pseudomonas sp. SR-5
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 102, No. 1, 53–59. 2006 DOI: 10.1263/jbb.102.53
© 2006, The Society for Biotechnology, Japan
Enhancement of Styrene Removal Efficiency in Biofilter by Mixed Cultures of Pseudomonas sp. SR-5 Jong Hee Jang,1 Mitsuyo Hirai,1 and Makoto Shoda1* Chemical Resources Laboratory, Tokyo Institute of Technology, R1-29-4259 Nagatsuta, Midori-Ku, Yokohama 226-8503, Japan1 Received 26 January 2006/Accepted 18 April 2006
The styrene-degrading bacterium Pseudomonas sp. SR-5 exhibited a high styrene removability in a biofilter. However, the styrene removal efficiency (RE) of SR-5 decreased with time. We carried out styrene gas removal in a biofilter inoculated with mixed cultures of SR-5 and other microorganisms to determine the possibility of obtaining an enhanced RE for a long period. The following three inocula were carried out: (i) styrene-degrading bacteria, strains 1 and 3, (ii) a benzoic acid-degrading bacterium Raoultella sp. A, and (iii) wastewater from a chemical company dealing with styrene. These biofilters with mixed SR-5 showed an enhanced RE compared with those with a single culture of SR-5. The complete styrene elimination capacities for ensuring 100% styrene removal in those mixed cultures were 151, 108 and 124 g/m3/h, compared with a single culture of SR-5. [Key words: styrene, Pseudomonas sp. SR-5, biofilter, mixed culture]
We isolated Pseudomonas sp. SR-5 as a styrene-degrading bacterium, and the characteristics of SR-5 have been described in our previous paper (14). In biofilters inoculated with SR-5, the maintenance of stable styrene removal is a problem. In this study, styrene gas removal was carried out in a biofilter inoculated with a mixed culture of Pseudomonas sp. SR-5 and different microorganisms such as strains 1 and 3, Raoultella sp. A or wastewater from a chemical company, and biofilter performance and styrene elimination capacity were compared between mixed cultures and a single culture of SR-5.
Styrene is a volatile organic compound (VOC) used in large quantities in the chemical industry. However, it is malodorous, potentially toxic and carcinogenic (1); its metabolism has been investigated in humans and other mammals. Biofiltration is considered to be an effective means of treating VOCs because some microorganisms capable of degrading VOCs have capacities to mineralize VOCs. Styrenedegrading-microorganisms, such as Pseudomonas sp. (2–4), Xanthobacter sp. (5), Rhodococcus rhodochros (6), Exophiala jeanselmei (7), and fungi (8) have been isolated, and different styrene degradation mechanisms have been proposed (9, 10). Biofiltration is a process based on the use of microbial consortia (mixed culture) (11). Although mixed cultures invariably involve interactions among species of various populations, a consortium is viewed as a single functional population, and its kinetics is studied under the assumption that a consortium is homogeneous and stable. Oh et al. (12) have reported results of BTX (benzene, toluene, and xylene) vapor removal by both a pure culture and a consortium, showing that in liquid cultures benzene and toluene are completely mineralized, but xylene is only transformed to some organic intermediates. When the same consortium was used in a biofilter, BTX removal initially occurred and then virtually ceased, probably due to the accumulation of stable organic products of xylene degradation, and the inhibition of BTX removal by the consortium in the supported biofilm. However, by the proper selection of microorganisms, the complete mineralization of BTX constituents is feasible (12, 13).
MATERIALS AND METHODS Microorganisms and inoculum preparation The styrene degradation characteristics of Pseudomonas sp. SR-5 have been described in our previous study (14). New-styrene-degrading microorganisms were isolated from peat biofilters for styrene degradation where wastewater from a chemical company dealing with styrene was inoculated and operation was carried out for 90 d. After 90 d, the peat diluted with sterilized water was spread on nutrient broth (NB) and colonies appeared were randomly picked up. NB was composed 5 g of meat extract, 10 g of peptone, and 5 g of NaCl in the 1 l of deionized water. Styrene degradation by isolates in a liquid mineral medium containing 0.5% (v/v) styrene was confirmed. Strains 1 and 3 that appeared on nutrient broth agar plates were selected. Styrene concentration in the headspace was determined by gas chromatography (GC). Strain A was also isolated from the same peat biofilter. SR-5, or strains 1, 3, or A was cultured in NB devoid of 15 g/l agar in nutrient agar (NA) medium at 30°C at 120 strokes per minute (spm) for 15 h. The culture was centrifuged for 20 min at 9200×g and the cells were washed twice with sterile deionized water. The cell suspension was used as inoculum. The wastewater used for inoculation was supplied by a chemical com-
* Corresponding author. e-mail: [email protected] phone: +81-(0)45-924-5274 fax: +81-(0)45-924-5976 53
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TABLE 1. Experimental conditions of three types of operation Run no. Packing materials Control 1 Ceramics Strain SR-5b 2 Peat + ceramics (1: 1)a Strain SR-5 3 Peat + ceramics (1: 1)a Waste water a Ratio on dry weight basis of each packing material. b Strains SR-5, 1 and 3 are styrene-degrading microorganisms. c Initial ratio of cell number of each microbial source inoculated to each packing material. d Strain A is a benzoic acid-degrading microorganism.
pany where styrene is used as a synthetic material, and 4 ×107 cfu/ml of microbial count was detected in the original wastewater. The specific styrene degradation rates of SR-5, and strains 1 and 3 were determined in a flask under aerobic conditions by the same method previously reported (14). Biofilter setup and operation The mixing ratios of each microorganism as inoculum to the biofilter and two packing materials used in biofilter are shown in Table 1. In run 1 of ceramic biofilter, a mixed culture of SR-5, and strains 1 and 3 at a ratio of 2:1: 1 on a cell number basis was inoculated. In this experiment we aimed to enhance removal efficiency by mixing the three styrene-degrading microorganisms. In run 2 of the biofilter carrying a mixed packing material of peat and ceramic at a ratio of 1 to 1 on a dry weight basis, inoculation of a mixed culture of SR-5 and strain A at a ratio of 1 to 1 on a cell number basis was intended to increase removal efficiency of SR-5 by degrading benzoic acid, which is inhibitory to SR-5 growth. In run 3 of the biofilter packed with the same packing material as in run 2, a mixed culture of SR-5 and wastewater at a ratio of 1 to 3 on a cell number basis was used. This experiment was intended to improve removal efficiency using the activities of the microbial community indigenous to wastewater. Control experiment was carried out using a single culture of strain SR-5 for runs 1 and 2, or wastewater for run 3 under the same conditions of the mixed cultures. Two parallel laboratory-scale biofilters shown in Fig. 1 were used in this study, where one column was for the control, and the other column was for the mixed culture. Glass columns (5 cm inner diameter × 37 cm length) were packed with ceramics (Kubota, Tokyo) for run 1 or a mixed packing material of peat (Takahashi
Mixed culture Strain SR-5 : strain 1b : strain 3b (2:1: 1)c SR-5 : strain Ad (1: 1)c Strain SR-5 : waste water (1: 3)c
Peat Moss, Hokkaido) and ceramics for runs 2 and 3 to a height of 17 cm. The physical and chemical properties of peat and ceramics have been described previously (14). The dry weights of a single packing material of ceramics, and a mixed packing material of peat and ceramic were 80 g and 74 g, respectively. Peat was neutralized with 0.4 M Ca(OH)2/kg-dry peat before use. Each packing material was sterilized by autoclaving four times at 120°C for 60 min. The initial moisture contents of the ceramic, and the mixed packing material of peat and ceramic were adjusted to about 50% and 64%, respectively. To maintain moisture content and pH, and supply nutrients to the biofilter, 100 ml of mineral medium buffered with 50 mM sodium phosphate (pH 7.0) was supplied daily from the top of the column using a peristaltic pump. The mineral medium consisted of 1.55 g of K2HPO4, 0.85 g of NaH2PO4 ⋅ 2H2O, 2.0 g of (NH4)2SO4, 0.1 g of MgCl2 ⋅ 6H2O, 10.0 mg of EDTA, 2.0 mg of ZnSO4 ⋅ 7H2O, 1.0 mg of CaCl2 ⋅ 2H2O, 5.0 mg of FeSO4 ⋅ 7H2O, 1.0 mg of Na2MoO4 ⋅ 2H2O, 0.2 mg of CuSO4 ⋅ 5H2O, 0.4 mg of CoCl2 ⋅6H2O, and 1 mg of MnCl2 ⋅ 4H2O in 1 l of deionized water (pH 7.0). Styrene gas was supplied to the columns by passing air from a compressor through a broad-neck bottle in which a reagent bottle containing 15 ml of styrene was placed. The inlet styrene load was changed by adjusting styrene concentration (0.1–3.5 g/m3) and air flow rate (30–500 ml/min). The experiment was performed at room temperature, 20 ±3°C. Analytical methods Styrene concentration sampled with a gas-tight syringe (MS-GANX00; Ito Co., Tokyo) at the inlet and outlet of the biofilter was determined by GC (Shimadzu GC 14A; Shimadzu, Kyoto) equipped with a capillary column (0.53 mm diameter×30 m length; URBON HR-1) and a flame ionization detector (FID). The analytical conditions of GC were as follows: injection and detection temperatures, 150°C; column temperature, 60°C to 120°C, increased at 2°C/min; and flow rate of He as carrier gas, 15 ml/min. Total styrene mass in a flask was calculated using Henry’s partition coefficient (HC) (15). The removal efficiency (RE) and elimination capacity (EC) of styrene were calculated using; RE (%) = ((Cin − Cout)/Cin) × 100 EC (g/m3/h) = (Cin − Cout) × F/V
FIG. 1. Schematic diagram of biofilter system. 1, Air compressor; 2, flow meter; 3, styrene chamber; 4, air filter; 5, three-way cock; 6, peristaltic pump; 7, mixed culture column; 8, single culture column; 9, drain flask. Solid arrows indicate styrene gas flow and dotted arrows indicate liquid medium flow.
where Cin is the inlet styrene concentration (g/m3), Cout, the outlet styrene concentration (g/m3), F, the gas flow rate (m3/h), and V, the packing volume (m3). Viable cell number was determined using NA plates. One gram (wet weight) of packing material was homogenized (EX-3 homogenizer; Nihon Seiki, Tokyo) in 9 ml of sterilized distilled water at 9200×g for 5 min for the ceramics or 10 min for a mixed packing material of peat and ceramics. The homogenate was serially diluted with sterilized distilled water and spread on NA plates. After 48 h of incubation at 30°C, viable cell number was determined and expressed as colony forming units (cfu) per g-dry packing material. The homogenate was also used for pH measurement. The moisture content of the mixed packing material was determined after drying for 12 h at 105°C. Drain water from a biofilter was extracted with diethyl ether after removing the cells by centrifugation at 9200×g for 20 min,
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TABLE 2. Specific styrene degradation rate of the strains SR-5, 1, and 3 Microorganisms SR-5 1 3
Specific styrene degradation rate (µmol/h/cell) 3.1 ×10–9 5.4 ×10–9 9.3 ×10–10
and dried by adding sodium sulfate. Then, the solvent was evaporated and the residue was re-dissolved in ethyl acetate and subjected to GC to analyze microbial styrene degradation metabolites. The analytical conditions of GC were the same as those described above. The amounts of CO2 at the inlet and outlet were measured using an infrared CO2 analyzer (EX-1562-1; ABLE, Tokyo) for 7 d and 10 d to calculate carbon mass balance in the biofilter in runs 1 and 3, respectively. Carbon mass (C-mass) balance of styrene degradation C-mass balance was conducted to estimate the amount of styrene converted to low-molecular-weight compounds including CO2 during 7 d and 10 d of operation of the styrene removal biofilter for runs 1 and 3, respectively. During this period, the amount of styrene loaded to biofilter, amount of styrene at the outlet, the amount of cell growth in the biofilter, and degradation intermediates in the drain water were calculated on a carbon basis.
RESULTS Performance of biofilter inoculated with SR-5, and strains 1 and 3 (run 1) The specific styrene degradation rates of strains 1, 3, and SR-5 were calculated from the slope of the time course of styrene degradation and the initial viable cell number (Table 2). Strain 1 colonies were glossy orange and round on NA (5 g of meat extract, 10 g of peptone, 5 g of NaCl, and 15 g of agar in 1 l distilled water [pH 7.0]) plate, and the growth rate of strain 1 was higher than that of SR-5. Strain 3 colonies were lusterless orange and round on the NA plate and the growth rate of strain 3 was higher than that of SR-5. Strains 1 and 3 have not yet been identified. The styrene concentrations at the inlet and outlet, and styrene REs at different space velocities for 31 d of operation are shown in Fig. 2. In the control biofilter inoculated with SR-5 (Fig. 2A), more than 90% RE was obtained at 1 to 45 g/m3/h inlet styrene loads for 15 d, but RE decreased to 60% with an increase in inlet styrene load to 132 g/m3/h on day 30. However, the mixed-culture biofilter inoculated with SR-5, strains 1, and 3 (Fig. 2B) showed more than 90% styrene RE even by increasing the styrene load to 151 g/m3/h. The relationship between inlet styrene load and the EC of the two biofilters is shown in Fig. 3. The complete elimination capacities for guaranteeing 100% removability were 44 and 151 g/m3/h in the control and mixed-culture biofilters, respectively. The results of C-mass balance in the biofilters for a 7-d period from days 17 to 23 of the experiment are shown in Table 3. In the mixed-culture biofilter, 2125 g-C/m3 styrene was loaded to the ceramic biofilter, and 113 g-C/m3 styrene was detected at the outlet and 0.7 g-C/m3 styrene was detected in the drain water because styrene in the packing material was washed by the mineral medium. Phenylacetic acid
FIG. 2. Performance of biofilter carrying ceramics as a packing material inoculated with a single culture of Pseudomonas sp. SR-5 (A) and a mixed culture of strains SR-5, 1, and 3 (B). Symbols: closed circles, inlet styrene concentration; open circles, outlet styrene concentration; triangles, removal efficiency.
FIG. 3. Relationship between styrene load and elimination capacity for run 1. Symbols: open circles, single culture; closed circles, mixed culture. Dotted line shows 100% removal.
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TABLE 3. Carbon mass balance of run 1 biofilter inoculated with strains SR-5, 1, and 3 using ceramic as a packing material for the 7 d operation period Item of measured carbon Total input styrene Output styrene Degradation productsa
Amount of carbon (g-C/m3) Single culture Mixed culture (SR-5) (SR-5 +1+ 3) 2246 2125 113 (5.32%) 1271 (56.6%)c PAA, 9.0 (0.40%) Styrene, 0.7 (0.02%) 2-PE, 4.9 (0.22%) PAA, 6.4 (0.30%) 137 (6.1%) 27.5 (1.29%) 824 (36.7 %) 1977 (93. 0%)
Biomass Low molecular weight substancesb a PAA, Phenylacetic acid; 2-PE, 2-phenylethanol. b Low molecular weight substances = total input styrene − output styrene − degradation products − biomass c Percentage against total input styrene.
(6.4 g-C/m3) was detected by GC in the drain water. Styrene (27.5 g-C/m3) was converted to the cell mass of SR-5. The data show that 1.59% of the loaded styrene was converted to styrene-degradation intermediates and cell mass. This indicates that 93% of the loaded styrene was converted to lowmolecular-weight compounds including CO2. However, in the control biofilter, 2246 g-C/m 3 styrene was loaded and 1271 g-C/m3 was detected at the outlet. The loaded styrene (6.7%) was converted to styrene-degradation intermediates (phenylacetic acid and 2-phenylethanol) and cell mass. This indicates that 36.7% of the loaded styrene was converted to low-molecular-weight compounds including CO2. Biofilter performance inoculated with SR-5 and strain A (run 2) Strain A was isolated from peat biofilters for styrene degradation inoculated with wastewater by the same method as above. Strain A exhibited a higher benzoic acid degradability than styrene degradability. Benzoic acid is a styrene degradation intermediate produced by SR-5 that inhibits SR-5 growth. Strain A colonies were glossy beige and round on the NA plate, and the growth rate of strain A was higher than that of SR-5. Strain A was identified as Raoultella sp. by the 16S rDNA homology test by the National Collections of Industrial, Food and Marine Bacteria, Japan (16). The styrene concentrations at the inlet and outlet, and styrene REs at different space velocities for 45 d are shown in Fig. 4 in a biofilter with a packing material of peat and ceramic. In the single-culture biofilter inoculated with strains SR-5 (Fig. 4A), more than 90% RE was obtained at 1 to 66 g/m3/h styrene loads for 19 d of operation. Then, RE decreased to 21% mainly because inlet styrene load was increased to 189 g/m3/h. In the mixed-culture biofilter inoculated with SR-5 and A (Fig. 4B), more than 90% RE was obtained at 1 to 58 g/m3/h styrene loads for 19 d of operation. Thereafter, styrene load increased gradually to 158 g/m3/h, and a decrease in RE was observed. However, RE recovered and finally reached 90% at 111 g/m3/h styrene load after fluctuation of RE. The relationship between inlet styrene load and EC in both the control and mixed-culture biofilters is shown in Fig. 5. The complete elimination capacities were 66 and 108 g/m3/h in the control and mixed culture, respectively. This result suggests that strain A that degrades benzoic acid removes benzoic acid that accumu-
FIG. 4. Performance of biofilter carrying a mixed packing material of peat and ceramics inoculated with a single culture of Pseudomonas sp. SR-5 (A) and a mixed culture of SR-5 and strain A (B). Symbols: closed circles, inlet styrene concentration; open circles, outlet styrene concentration; triangles, removal efficiency.
FIG. 5. Relationship between styrene load and elimination capacity for run 2. Symbols: open circles, single culture; closed circles, mixed culture. Dotted line shows 100% removal.
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TABLE 4. Carbon mass balance of run 3 biofilter inoculated with strain SR-5 and waste water using a mixed packing material for 10 d operation period Amount of carbon (g-C/m3) Single culture Mixed culture (Waste water) (SR-5 + waste water) Total input styrene 2751 2604 8.6 (0.30%) Output styrene 360 (13.1%)a NDc Degradation products PAAb 0.4 (0.01%) Biomass 99.8 (3.63%) 51.3 (2.0%) 2077 (75.5%) 2391 (91.8%) CO2 emission 213 (7.75%) 153 (5.90%) Unidentified carbond a Percentage against input styrene. b PAA, Phenylacetic acid. c Not detected. d Unidentified carbon presumed to be extracellular products and polymer. Item of measured carbon
FIG. 6. Performance of biofilter carrying a mixed packing material of peat and ceramics inoculated with wastewater (A) and a mixed culture of SR-5 and wastewater (B). Symbols: closed circles, inlet styrene concentration; open circles, outlet styrene concentration; triangles, removal efficiency.
lates in the biofilter, resulting in the maintenance of SR-5 activity for degrading styrene. The fluctuation of RE may be attributed to styrene inhibition to strain A during change space velocities because strain A has no ability to degrade styrene. Performance of biofilter inoculated with SR-5 and wastewater (run 3) The styrene concentrations at the inlet and outlet, and styrene REs at different space velocities for 51 d are shown in Fig. 6. The packing material was the same as that in run 2. In the control biofilter inoculated with wastewater (Fig. 6A), more than 87% RE was obtained in the range of 1 to 48 g/m3/h inlet styrene loads for 45 d of operation. However, RE decreased rapidly to 60% with an increase in styrene load to 66 g/m3/h. In the mixed-culture biofilter of SR-5 and wastewater (Fig. 6B), more than 85% RE was obtained at 1 to 161 g/m3/h inlet styrene loads for 35 d of operation, but RE decreased to 63% with an increase in styrene load to 245 g/m3/h. Complete elimination capacities of 42 and 124 g/m3/h were obtained in the control and the mixed culture in the biofilter, respectively (Fig. 7). The results of carbon mass balance in the biofilters for 10 d of operation during the biofilter operation are shown in Table 4. In the mixed culture, 2604 g-C/m 3 styrene was loaded to the biofilter and 8.6 g-C/m3 was detected at the outlet. Styrene degradation intermediates were not detected by GC in the drain water. The amount of styrene converted to cell mass was 51.3 g-C/m3 styrene. Detected amount of CO2 by using a CO2 analyzer was 2391 g-C/m3 CO2. However, 153 g-C/m3 input styrene carbon was unidentified. This shows that 91.8% of the input styrene was completely degraded to CO2 by the mixed culture of SR-5 and wastewater. In the control, 2751 g-C/m3 styrene was loaded and 360 g-C/m3 was detected at the outlet. The amount of the styrene-degradation intermediates and cell mass converted from the loaded styrene was 3.64%, and 75.5% of the loaded styrene carbon was converted to CO2. The unidentified amount of carbon was 7.75% of the loaded styrene. DISCUSSION
FIG. 7. Relationship between styrene load and elimination capacity for run 3. Symbols: open circles, wastewater; closed circles, mixed culture. Dotted line shows 100% removal.
Styrene degradation in a biofilter was compared using different combinations of microorganisms, operation conditions, and packing materials. In run 1, an improvement in
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styrene removal efficiency was obtained by mixing three styrene-degrading-microorganisms. When peat as a packing material was used under the same conditions as those for run 1, operation had to be stopped within 1 week because pressure drop was significantly enhanced presumably because of a rapid cell growth in peat (data not shown). Actually, strains 1 and 3 grew faster than SR-5 in peat-suspended and mineral media (data not shown). Therefore, ceramics was used as a packing material. In the ceramic biofilter, the high styrene removal efficiency of a mixed culture lasted for a long period and was significantly higher than that a single culture of SR-5 because ceramics as a packing material has better characteristics such as a lower compaction ability and a lower nutrient content than peat, and it minimized the growth of the mixed culture. SR-5 produces benzoic acid as degradation intermediate (Jang, J.H., Ph.D. thesis, Tokyo Institution of Technology, 2004), which inhibited the styrene degradation activity of SR-5. This was one of the reasons for the quick deterioration of biofilter performance. As shown in Table 2, the specific styrene degradation rates of strains 1 and 3 were similar to that of SR-5, and no benzoic acid was produced by the degradation of styrene by strains 1 and 3 (data not shown). However, it is not clear whether the activities of strains 1 and 3 are inhibited by benzoic acids. Although the initial inoculation ratio of the three stains, SR-5, 1 and 3 was fixed, the distribution of the three strains on 31 d was no clearly determined. In a single inoculation of strain 1 or strain 3 to biofilters, the removal efficiency of the two strains deteriorated significantly at the load of more than 50 g/m3/h. The mixed culture inoculation enhanced the complete removal capacity to 151 g/m 3/h. This clearly indicates the advantages of the mixed culture rather than a single culture, although details of interactions among three strains are not clear yet. As the strain SR-5 has properties to be resistant to high load of styrene (14), the strain SR-5 may have played a role to shorten the initial acclimation period and alleviate the quick increase of styrene load during operation period. In run 2, the mixed culture of strains SR-5 and A showed a higher removal efficiency than the control, presumably because strain A removed accumulated benzoic acid derived from SR-5 styrene degradation in the biofilter. In run 3, a high removal efficiency from the start of operation was observed in the mixture of wastewater with strain SR-5. By the single inoculation of wastewater, the acclimation period to become stable removal of styrene took more than 1 month and removal efficiency decreased to 44% at more than 65 g/m3/h. However, the mixture of wastewater with the strain SR-5 shortened the acclimation period to 2 weeks and complete removal capacity increased to 124 g/m3/h. This also indicates that strain SR-5 played the role in the two effects in the styrene removal. In this study, complete elimination capacities in the mixedculture biofilter in runs 1, 2, and 3 were 151, 108, and 124 g/m3/h, respectively. These were higher than or equivalent to the reported values of 90 (17), 62 (18), 79 (19), and 140 g/m3/h (20). Hwang et al. (21) reported the degradation of ethyl acetate and toluene mixtures in a biofilter using different com-
binations of bacterial cultures and changes in the degradabilities of ethyl acetate and toluene induced by substrate competition and microbial community competition. Cho et al. (22) reported an enhanced removability of odorous sulfur-containing gases such as hydrogen sulfide (H2S), methanethiol (MT), dimethyl sulfide (DMS), and dimethyl disulfide (DMDS) by a mixed culture of purified bacteria. The mixed culture seemed to be effective if a proper microbial combination is selected. In this study, the difference in removal efficiency in a mixed culture among runs 1, 2 and 3 was small, suggesting that SR-5 can be applied to any combinations of microorganisms to enhance styrene removal efficiency. The stability of this biofilter for longer operation periods is under investigation.
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© 2006, The Society for Biotechnology, Japan
Enhancement of Styrene Removal Efficiency in Biofilter by Mixed Cultures of Pseudomonas sp. SR-5 Jong Hee Jang,1 Mitsuyo Hirai,1 and Makoto Shoda1* Chemical Resources Laboratory, Tokyo Institute of Technology, R1-29-4259 Nagatsuta, Midori-Ku, Yokohama 226-8503, Japan1 Received 26 January 2006/Accepted 18 April 2006
The styrene-degrading bacterium Pseudomonas sp. SR-5 exhibited a high styrene removability in a biofilter. However, the styrene removal efficiency (RE) of SR-5 decreased with time. We carried out styrene gas removal in a biofilter inoculated with mixed cultures of SR-5 and other microorganisms to determine the possibility of obtaining an enhanced RE for a long period. The following three inocula were carried out: (i) styrene-degrading bacteria, strains 1 and 3, (ii) a benzoic acid-degrading bacterium Raoultella sp. A, and (iii) wastewater from a chemical company dealing with styrene. These biofilters with mixed SR-5 showed an enhanced RE compared with those with a single culture of SR-5. The complete styrene elimination capacities for ensuring 100% styrene removal in those mixed cultures were 151, 108 and 124 g/m3/h, compared with a single culture of SR-5. [Key words: styrene, Pseudomonas sp. SR-5, biofilter, mixed culture]
We isolated Pseudomonas sp. SR-5 as a styrene-degrading bacterium, and the characteristics of SR-5 have been described in our previous paper (14). In biofilters inoculated with SR-5, the maintenance of stable styrene removal is a problem. In this study, styrene gas removal was carried out in a biofilter inoculated with a mixed culture of Pseudomonas sp. SR-5 and different microorganisms such as strains 1 and 3, Raoultella sp. A or wastewater from a chemical company, and biofilter performance and styrene elimination capacity were compared between mixed cultures and a single culture of SR-5.
Styrene is a volatile organic compound (VOC) used in large quantities in the chemical industry. However, it is malodorous, potentially toxic and carcinogenic (1); its metabolism has been investigated in humans and other mammals. Biofiltration is considered to be an effective means of treating VOCs because some microorganisms capable of degrading VOCs have capacities to mineralize VOCs. Styrenedegrading-microorganisms, such as Pseudomonas sp. (2–4), Xanthobacter sp. (5), Rhodococcus rhodochros (6), Exophiala jeanselmei (7), and fungi (8) have been isolated, and different styrene degradation mechanisms have been proposed (9, 10). Biofiltration is a process based on the use of microbial consortia (mixed culture) (11). Although mixed cultures invariably involve interactions among species of various populations, a consortium is viewed as a single functional population, and its kinetics is studied under the assumption that a consortium is homogeneous and stable. Oh et al. (12) have reported results of BTX (benzene, toluene, and xylene) vapor removal by both a pure culture and a consortium, showing that in liquid cultures benzene and toluene are completely mineralized, but xylene is only transformed to some organic intermediates. When the same consortium was used in a biofilter, BTX removal initially occurred and then virtually ceased, probably due to the accumulation of stable organic products of xylene degradation, and the inhibition of BTX removal by the consortium in the supported biofilm. However, by the proper selection of microorganisms, the complete mineralization of BTX constituents is feasible (12, 13).
MATERIALS AND METHODS Microorganisms and inoculum preparation The styrene degradation characteristics of Pseudomonas sp. SR-5 have been described in our previous study (14). New-styrene-degrading microorganisms were isolated from peat biofilters for styrene degradation where wastewater from a chemical company dealing with styrene was inoculated and operation was carried out for 90 d. After 90 d, the peat diluted with sterilized water was spread on nutrient broth (NB) and colonies appeared were randomly picked up. NB was composed 5 g of meat extract, 10 g of peptone, and 5 g of NaCl in the 1 l of deionized water. Styrene degradation by isolates in a liquid mineral medium containing 0.5% (v/v) styrene was confirmed. Strains 1 and 3 that appeared on nutrient broth agar plates were selected. Styrene concentration in the headspace was determined by gas chromatography (GC). Strain A was also isolated from the same peat biofilter. SR-5, or strains 1, 3, or A was cultured in NB devoid of 15 g/l agar in nutrient agar (NA) medium at 30°C at 120 strokes per minute (spm) for 15 h. The culture was centrifuged for 20 min at 9200×g and the cells were washed twice with sterile deionized water. The cell suspension was used as inoculum. The wastewater used for inoculation was supplied by a chemical com-
* Corresponding author. e-mail: [email protected] phone: +81-(0)45-924-5274 fax: +81-(0)45-924-5976 53
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TABLE 1. Experimental conditions of three types of operation Run no. Packing materials Control 1 Ceramics Strain SR-5b 2 Peat + ceramics (1: 1)a Strain SR-5 3 Peat + ceramics (1: 1)a Waste water a Ratio on dry weight basis of each packing material. b Strains SR-5, 1 and 3 are styrene-degrading microorganisms. c Initial ratio of cell number of each microbial source inoculated to each packing material. d Strain A is a benzoic acid-degrading microorganism.
pany where styrene is used as a synthetic material, and 4 ×107 cfu/ml of microbial count was detected in the original wastewater. The specific styrene degradation rates of SR-5, and strains 1 and 3 were determined in a flask under aerobic conditions by the same method previously reported (14). Biofilter setup and operation The mixing ratios of each microorganism as inoculum to the biofilter and two packing materials used in biofilter are shown in Table 1. In run 1 of ceramic biofilter, a mixed culture of SR-5, and strains 1 and 3 at a ratio of 2:1: 1 on a cell number basis was inoculated. In this experiment we aimed to enhance removal efficiency by mixing the three styrene-degrading microorganisms. In run 2 of the biofilter carrying a mixed packing material of peat and ceramic at a ratio of 1 to 1 on a dry weight basis, inoculation of a mixed culture of SR-5 and strain A at a ratio of 1 to 1 on a cell number basis was intended to increase removal efficiency of SR-5 by degrading benzoic acid, which is inhibitory to SR-5 growth. In run 3 of the biofilter packed with the same packing material as in run 2, a mixed culture of SR-5 and wastewater at a ratio of 1 to 3 on a cell number basis was used. This experiment was intended to improve removal efficiency using the activities of the microbial community indigenous to wastewater. Control experiment was carried out using a single culture of strain SR-5 for runs 1 and 2, or wastewater for run 3 under the same conditions of the mixed cultures. Two parallel laboratory-scale biofilters shown in Fig. 1 were used in this study, where one column was for the control, and the other column was for the mixed culture. Glass columns (5 cm inner diameter × 37 cm length) were packed with ceramics (Kubota, Tokyo) for run 1 or a mixed packing material of peat (Takahashi
Mixed culture Strain SR-5 : strain 1b : strain 3b (2:1: 1)c SR-5 : strain Ad (1: 1)c Strain SR-5 : waste water (1: 3)c
Peat Moss, Hokkaido) and ceramics for runs 2 and 3 to a height of 17 cm. The physical and chemical properties of peat and ceramics have been described previously (14). The dry weights of a single packing material of ceramics, and a mixed packing material of peat and ceramic were 80 g and 74 g, respectively. Peat was neutralized with 0.4 M Ca(OH)2/kg-dry peat before use. Each packing material was sterilized by autoclaving four times at 120°C for 60 min. The initial moisture contents of the ceramic, and the mixed packing material of peat and ceramic were adjusted to about 50% and 64%, respectively. To maintain moisture content and pH, and supply nutrients to the biofilter, 100 ml of mineral medium buffered with 50 mM sodium phosphate (pH 7.0) was supplied daily from the top of the column using a peristaltic pump. The mineral medium consisted of 1.55 g of K2HPO4, 0.85 g of NaH2PO4 ⋅ 2H2O, 2.0 g of (NH4)2SO4, 0.1 g of MgCl2 ⋅ 6H2O, 10.0 mg of EDTA, 2.0 mg of ZnSO4 ⋅ 7H2O, 1.0 mg of CaCl2 ⋅ 2H2O, 5.0 mg of FeSO4 ⋅ 7H2O, 1.0 mg of Na2MoO4 ⋅ 2H2O, 0.2 mg of CuSO4 ⋅ 5H2O, 0.4 mg of CoCl2 ⋅6H2O, and 1 mg of MnCl2 ⋅ 4H2O in 1 l of deionized water (pH 7.0). Styrene gas was supplied to the columns by passing air from a compressor through a broad-neck bottle in which a reagent bottle containing 15 ml of styrene was placed. The inlet styrene load was changed by adjusting styrene concentration (0.1–3.5 g/m3) and air flow rate (30–500 ml/min). The experiment was performed at room temperature, 20 ±3°C. Analytical methods Styrene concentration sampled with a gas-tight syringe (MS-GANX00; Ito Co., Tokyo) at the inlet and outlet of the biofilter was determined by GC (Shimadzu GC 14A; Shimadzu, Kyoto) equipped with a capillary column (0.53 mm diameter×30 m length; URBON HR-1) and a flame ionization detector (FID). The analytical conditions of GC were as follows: injection and detection temperatures, 150°C; column temperature, 60°C to 120°C, increased at 2°C/min; and flow rate of He as carrier gas, 15 ml/min. Total styrene mass in a flask was calculated using Henry’s partition coefficient (HC) (15). The removal efficiency (RE) and elimination capacity (EC) of styrene were calculated using; RE (%) = ((Cin − Cout)/Cin) × 100 EC (g/m3/h) = (Cin − Cout) × F/V
FIG. 1. Schematic diagram of biofilter system. 1, Air compressor; 2, flow meter; 3, styrene chamber; 4, air filter; 5, three-way cock; 6, peristaltic pump; 7, mixed culture column; 8, single culture column; 9, drain flask. Solid arrows indicate styrene gas flow and dotted arrows indicate liquid medium flow.
where Cin is the inlet styrene concentration (g/m3), Cout, the outlet styrene concentration (g/m3), F, the gas flow rate (m3/h), and V, the packing volume (m3). Viable cell number was determined using NA plates. One gram (wet weight) of packing material was homogenized (EX-3 homogenizer; Nihon Seiki, Tokyo) in 9 ml of sterilized distilled water at 9200×g for 5 min for the ceramics or 10 min for a mixed packing material of peat and ceramics. The homogenate was serially diluted with sterilized distilled water and spread on NA plates. After 48 h of incubation at 30°C, viable cell number was determined and expressed as colony forming units (cfu) per g-dry packing material. The homogenate was also used for pH measurement. The moisture content of the mixed packing material was determined after drying for 12 h at 105°C. Drain water from a biofilter was extracted with diethyl ether after removing the cells by centrifugation at 9200×g for 20 min,
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TABLE 2. Specific styrene degradation rate of the strains SR-5, 1, and 3 Microorganisms SR-5 1 3
Specific styrene degradation rate (µmol/h/cell) 3.1 ×10–9 5.4 ×10–9 9.3 ×10–10
and dried by adding sodium sulfate. Then, the solvent was evaporated and the residue was re-dissolved in ethyl acetate and subjected to GC to analyze microbial styrene degradation metabolites. The analytical conditions of GC were the same as those described above. The amounts of CO2 at the inlet and outlet were measured using an infrared CO2 analyzer (EX-1562-1; ABLE, Tokyo) for 7 d and 10 d to calculate carbon mass balance in the biofilter in runs 1 and 3, respectively. Carbon mass (C-mass) balance of styrene degradation C-mass balance was conducted to estimate the amount of styrene converted to low-molecular-weight compounds including CO2 during 7 d and 10 d of operation of the styrene removal biofilter for runs 1 and 3, respectively. During this period, the amount of styrene loaded to biofilter, amount of styrene at the outlet, the amount of cell growth in the biofilter, and degradation intermediates in the drain water were calculated on a carbon basis.
RESULTS Performance of biofilter inoculated with SR-5, and strains 1 and 3 (run 1) The specific styrene degradation rates of strains 1, 3, and SR-5 were calculated from the slope of the time course of styrene degradation and the initial viable cell number (Table 2). Strain 1 colonies were glossy orange and round on NA (5 g of meat extract, 10 g of peptone, 5 g of NaCl, and 15 g of agar in 1 l distilled water [pH 7.0]) plate, and the growth rate of strain 1 was higher than that of SR-5. Strain 3 colonies were lusterless orange and round on the NA plate and the growth rate of strain 3 was higher than that of SR-5. Strains 1 and 3 have not yet been identified. The styrene concentrations at the inlet and outlet, and styrene REs at different space velocities for 31 d of operation are shown in Fig. 2. In the control biofilter inoculated with SR-5 (Fig. 2A), more than 90% RE was obtained at 1 to 45 g/m3/h inlet styrene loads for 15 d, but RE decreased to 60% with an increase in inlet styrene load to 132 g/m3/h on day 30. However, the mixed-culture biofilter inoculated with SR-5, strains 1, and 3 (Fig. 2B) showed more than 90% styrene RE even by increasing the styrene load to 151 g/m3/h. The relationship between inlet styrene load and the EC of the two biofilters is shown in Fig. 3. The complete elimination capacities for guaranteeing 100% removability were 44 and 151 g/m3/h in the control and mixed-culture biofilters, respectively. The results of C-mass balance in the biofilters for a 7-d period from days 17 to 23 of the experiment are shown in Table 3. In the mixed-culture biofilter, 2125 g-C/m3 styrene was loaded to the ceramic biofilter, and 113 g-C/m3 styrene was detected at the outlet and 0.7 g-C/m3 styrene was detected in the drain water because styrene in the packing material was washed by the mineral medium. Phenylacetic acid
FIG. 2. Performance of biofilter carrying ceramics as a packing material inoculated with a single culture of Pseudomonas sp. SR-5 (A) and a mixed culture of strains SR-5, 1, and 3 (B). Symbols: closed circles, inlet styrene concentration; open circles, outlet styrene concentration; triangles, removal efficiency.
FIG. 3. Relationship between styrene load and elimination capacity for run 1. Symbols: open circles, single culture; closed circles, mixed culture. Dotted line shows 100% removal.
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TABLE 3. Carbon mass balance of run 1 biofilter inoculated with strains SR-5, 1, and 3 using ceramic as a packing material for the 7 d operation period Item of measured carbon Total input styrene Output styrene Degradation productsa
Amount of carbon (g-C/m3) Single culture Mixed culture (SR-5) (SR-5 +1+ 3) 2246 2125 113 (5.32%) 1271 (56.6%)c PAA, 9.0 (0.40%) Styrene, 0.7 (0.02%) 2-PE, 4.9 (0.22%) PAA, 6.4 (0.30%) 137 (6.1%) 27.5 (1.29%) 824 (36.7 %) 1977 (93. 0%)
Biomass Low molecular weight substancesb a PAA, Phenylacetic acid; 2-PE, 2-phenylethanol. b Low molecular weight substances = total input styrene − output styrene − degradation products − biomass c Percentage against total input styrene.
(6.4 g-C/m3) was detected by GC in the drain water. Styrene (27.5 g-C/m3) was converted to the cell mass of SR-5. The data show that 1.59% of the loaded styrene was converted to styrene-degradation intermediates and cell mass. This indicates that 93% of the loaded styrene was converted to lowmolecular-weight compounds including CO2. However, in the control biofilter, 2246 g-C/m 3 styrene was loaded and 1271 g-C/m3 was detected at the outlet. The loaded styrene (6.7%) was converted to styrene-degradation intermediates (phenylacetic acid and 2-phenylethanol) and cell mass. This indicates that 36.7% of the loaded styrene was converted to low-molecular-weight compounds including CO2. Biofilter performance inoculated with SR-5 and strain A (run 2) Strain A was isolated from peat biofilters for styrene degradation inoculated with wastewater by the same method as above. Strain A exhibited a higher benzoic acid degradability than styrene degradability. Benzoic acid is a styrene degradation intermediate produced by SR-5 that inhibits SR-5 growth. Strain A colonies were glossy beige and round on the NA plate, and the growth rate of strain A was higher than that of SR-5. Strain A was identified as Raoultella sp. by the 16S rDNA homology test by the National Collections of Industrial, Food and Marine Bacteria, Japan (16). The styrene concentrations at the inlet and outlet, and styrene REs at different space velocities for 45 d are shown in Fig. 4 in a biofilter with a packing material of peat and ceramic. In the single-culture biofilter inoculated with strains SR-5 (Fig. 4A), more than 90% RE was obtained at 1 to 66 g/m3/h styrene loads for 19 d of operation. Then, RE decreased to 21% mainly because inlet styrene load was increased to 189 g/m3/h. In the mixed-culture biofilter inoculated with SR-5 and A (Fig. 4B), more than 90% RE was obtained at 1 to 58 g/m3/h styrene loads for 19 d of operation. Thereafter, styrene load increased gradually to 158 g/m3/h, and a decrease in RE was observed. However, RE recovered and finally reached 90% at 111 g/m3/h styrene load after fluctuation of RE. The relationship between inlet styrene load and EC in both the control and mixed-culture biofilters is shown in Fig. 5. The complete elimination capacities were 66 and 108 g/m3/h in the control and mixed culture, respectively. This result suggests that strain A that degrades benzoic acid removes benzoic acid that accumu-
FIG. 4. Performance of biofilter carrying a mixed packing material of peat and ceramics inoculated with a single culture of Pseudomonas sp. SR-5 (A) and a mixed culture of SR-5 and strain A (B). Symbols: closed circles, inlet styrene concentration; open circles, outlet styrene concentration; triangles, removal efficiency.
FIG. 5. Relationship between styrene load and elimination capacity for run 2. Symbols: open circles, single culture; closed circles, mixed culture. Dotted line shows 100% removal.
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TABLE 4. Carbon mass balance of run 3 biofilter inoculated with strain SR-5 and waste water using a mixed packing material for 10 d operation period Amount of carbon (g-C/m3) Single culture Mixed culture (Waste water) (SR-5 + waste water) Total input styrene 2751 2604 8.6 (0.30%) Output styrene 360 (13.1%)a NDc Degradation products PAAb 0.4 (0.01%) Biomass 99.8 (3.63%) 51.3 (2.0%) 2077 (75.5%) 2391 (91.8%) CO2 emission 213 (7.75%) 153 (5.90%) Unidentified carbond a Percentage against input styrene. b PAA, Phenylacetic acid. c Not detected. d Unidentified carbon presumed to be extracellular products and polymer. Item of measured carbon
FIG. 6. Performance of biofilter carrying a mixed packing material of peat and ceramics inoculated with wastewater (A) and a mixed culture of SR-5 and wastewater (B). Symbols: closed circles, inlet styrene concentration; open circles, outlet styrene concentration; triangles, removal efficiency.
lates in the biofilter, resulting in the maintenance of SR-5 activity for degrading styrene. The fluctuation of RE may be attributed to styrene inhibition to strain A during change space velocities because strain A has no ability to degrade styrene. Performance of biofilter inoculated with SR-5 and wastewater (run 3) The styrene concentrations at the inlet and outlet, and styrene REs at different space velocities for 51 d are shown in Fig. 6. The packing material was the same as that in run 2. In the control biofilter inoculated with wastewater (Fig. 6A), more than 87% RE was obtained in the range of 1 to 48 g/m3/h inlet styrene loads for 45 d of operation. However, RE decreased rapidly to 60% with an increase in styrene load to 66 g/m3/h. In the mixed-culture biofilter of SR-5 and wastewater (Fig. 6B), more than 85% RE was obtained at 1 to 161 g/m3/h inlet styrene loads for 35 d of operation, but RE decreased to 63% with an increase in styrene load to 245 g/m3/h. Complete elimination capacities of 42 and 124 g/m3/h were obtained in the control and the mixed culture in the biofilter, respectively (Fig. 7). The results of carbon mass balance in the biofilters for 10 d of operation during the biofilter operation are shown in Table 4. In the mixed culture, 2604 g-C/m 3 styrene was loaded to the biofilter and 8.6 g-C/m3 was detected at the outlet. Styrene degradation intermediates were not detected by GC in the drain water. The amount of styrene converted to cell mass was 51.3 g-C/m3 styrene. Detected amount of CO2 by using a CO2 analyzer was 2391 g-C/m3 CO2. However, 153 g-C/m3 input styrene carbon was unidentified. This shows that 91.8% of the input styrene was completely degraded to CO2 by the mixed culture of SR-5 and wastewater. In the control, 2751 g-C/m3 styrene was loaded and 360 g-C/m3 was detected at the outlet. The amount of the styrene-degradation intermediates and cell mass converted from the loaded styrene was 3.64%, and 75.5% of the loaded styrene carbon was converted to CO2. The unidentified amount of carbon was 7.75% of the loaded styrene. DISCUSSION
FIG. 7. Relationship between styrene load and elimination capacity for run 3. Symbols: open circles, wastewater; closed circles, mixed culture. Dotted line shows 100% removal.
Styrene degradation in a biofilter was compared using different combinations of microorganisms, operation conditions, and packing materials. In run 1, an improvement in
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styrene removal efficiency was obtained by mixing three styrene-degrading-microorganisms. When peat as a packing material was used under the same conditions as those for run 1, operation had to be stopped within 1 week because pressure drop was significantly enhanced presumably because of a rapid cell growth in peat (data not shown). Actually, strains 1 and 3 grew faster than SR-5 in peat-suspended and mineral media (data not shown). Therefore, ceramics was used as a packing material. In the ceramic biofilter, the high styrene removal efficiency of a mixed culture lasted for a long period and was significantly higher than that a single culture of SR-5 because ceramics as a packing material has better characteristics such as a lower compaction ability and a lower nutrient content than peat, and it minimized the growth of the mixed culture. SR-5 produces benzoic acid as degradation intermediate (Jang, J.H., Ph.D. thesis, Tokyo Institution of Technology, 2004), which inhibited the styrene degradation activity of SR-5. This was one of the reasons for the quick deterioration of biofilter performance. As shown in Table 2, the specific styrene degradation rates of strains 1 and 3 were similar to that of SR-5, and no benzoic acid was produced by the degradation of styrene by strains 1 and 3 (data not shown). However, it is not clear whether the activities of strains 1 and 3 are inhibited by benzoic acids. Although the initial inoculation ratio of the three stains, SR-5, 1 and 3 was fixed, the distribution of the three strains on 31 d was no clearly determined. In a single inoculation of strain 1 or strain 3 to biofilters, the removal efficiency of the two strains deteriorated significantly at the load of more than 50 g/m3/h. The mixed culture inoculation enhanced the complete removal capacity to 151 g/m 3/h. This clearly indicates the advantages of the mixed culture rather than a single culture, although details of interactions among three strains are not clear yet. As the strain SR-5 has properties to be resistant to high load of styrene (14), the strain SR-5 may have played a role to shorten the initial acclimation period and alleviate the quick increase of styrene load during operation period. In run 2, the mixed culture of strains SR-5 and A showed a higher removal efficiency than the control, presumably because strain A removed accumulated benzoic acid derived from SR-5 styrene degradation in the biofilter. In run 3, a high removal efficiency from the start of operation was observed in the mixture of wastewater with strain SR-5. By the single inoculation of wastewater, the acclimation period to become stable removal of styrene took more than 1 month and removal efficiency decreased to 44% at more than 65 g/m3/h. However, the mixture of wastewater with the strain SR-5 shortened the acclimation period to 2 weeks and complete removal capacity increased to 124 g/m3/h. This also indicates that strain SR-5 played the role in the two effects in the styrene removal. In this study, complete elimination capacities in the mixedculture biofilter in runs 1, 2, and 3 were 151, 108, and 124 g/m3/h, respectively. These were higher than or equivalent to the reported values of 90 (17), 62 (18), 79 (19), and 140 g/m3/h (20). Hwang et al. (21) reported the degradation of ethyl acetate and toluene mixtures in a biofilter using different com-
binations of bacterial cultures and changes in the degradabilities of ethyl acetate and toluene induced by substrate competition and microbial community competition. Cho et al. (22) reported an enhanced removability of odorous sulfur-containing gases such as hydrogen sulfide (H2S), methanethiol (MT), dimethyl sulfide (DMS), and dimethyl disulfide (DMDS) by a mixed culture of purified bacteria. The mixed culture seemed to be effective if a proper microbial combination is selected. In this study, the difference in removal efficiency in a mixed culture among runs 1, 2 and 3 was small, suggesting that SR-5 can be applied to any combinations of microorganisms to enhance styrene removal efficiency. The stability of this biofilter for longer operation periods is under investigation.
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