Chlorophenols degradation by pentachlorophenol degrading bacteria Sphingomonas chlorophenolica in batch reactor Chu-Fang Yang*, Chi-Mei Lee* and Chun-Chin Wang* *Department of Environmental Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC (E-mail:
[email protected];
[email protected];
[email protected])
ABSTRACT Chlorophenols are common environmental contaminants which have been using as the major compositions of wide-spectrum biocides in industry and agriculture. Many chlorophenols tend to persist in the environment, and they may become public health hazards. Among chlorophenols, pentachlorophenol (PCP) is a priority pollutant that has been using widely as a general biocide in commercial wood treatment. PCP is toxic to all forms of life since it is an inhibitor of oxidative phosphorylation. This research had studied the ability of PCP degrading bacterium Sphingomonas chlorophenolica to degrade and dechlorinate other chlorophenols. In addition, the characteristics of Sphingomonas chlorophenolica had also been investigated. The results indicated that Sphingomonas chlorophenolica was able to completely degrade 2,3,6-trichlorophenol (2,3,6-TCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) and PCP within 38.1, 15.1, 11.8 and 11.8 hours, and to release concentration 50.1, 60.9, 63.7 and 58.5 mg/l chloride at the same period of time. Furthermore, the results showed that four kinds of chlorophenols had been dechlorinated approximately 100% by the cell suspensions. In the presence of supplementary carbon sources (concentration 300 mg/l glucose, pyruvate, sodium acetate and concentration 150 mg/l 2,4,6-TCP, PCP), PCP removal efficiency increased with the presence of glucose or pyruvate, but the removal efficiency of 75 mg/l 2,4-dichlorophenol did not raise with the supplement of carbon sources.
KEYWORDS Biodegradation; Chlorophenols; PCP; Sphingomonas chlorophenolica; Dechlorination INTRODUCTION Chlorophenols are common environmental contaminants which have been using as the major compositions of wide-spectrum biocides in industry and agriculture. In general, chlorophenols are contained in some direct industrial waste (Vallecillo et al., 1999). Chlorophenols compounds are quite toxic. The toxicity of them increases with their degree of chlorination (Liu et al., 1982). Many chlorophenols tend to persist in the environment, and they may become public health hazards. Among chlorophenols, pentachlorophenol (PCP) is a priority pollutant which has been using widely as a general biocide in commercial wood treatment (Crosby, 1981). PCP is toxic to all forms of life since it is an inhibitor of oxidative phosphorylation. Moreover, its degradation is difficult because of its stable aromatic ring system and the highest chloride content (Okeke et al., 1997). To treat chlorophenols, biological methods are superior to physicochemical methods, because the latter ones have higher treatment costs and possibilities of causing a secondary pollution. Aerobic degradation of polychlorinated phenols had been studied extensively during the last few years (Häggblom, 1992). Several strains of bacteria that were able to completely mineralize chlorophenols had been isolated, such as Pseudomonas sp. (Radehaus and Schmidt,
1992), Alcaligenes sp. (Valenzuela et al., 1997), Azotobacter sp. (Li et al., 1991), Novosphingobium sp. (Tiirola et al., 2002) and Arthrobacter sp. (Edgehill, 1996). These aerobes were more efficient at degrading toxic compounds because they grew faster than anaerobes and usually transformed organic compounds to inorganic compounds. In Taiwan, owing to the rapid industrial growth in the past four decades, serious soil and water pollutions by chlorophenols had been reported. The purpose of this research was to study the ability of PCP degrading bacterium Sphingomonas chlorophenolica to degrade and dechlorinate other chlorophenols. In addition, the characteristics of Sphingomonas chlorophenolica had also been investigated.
MATERIALS AND METHODS Organism, media and culture conditions Sphingomonas chlorophenolica that was used in all tests had been obtained from PCP contaminated soils in Taiwan (Yang et al., 2003). It had been isolated from the acclimated mixed culture and was purified and maintained on R2A agar (Reasoner and Geldreich, 1985). All microbial experiments were performed in inorganic culture media. The compositions of inorganic culture media and trace element solution were listed in Table 1 and Table 2. The pH of inorganic culture media was adjusted to 7.5. Each batch was conducted with a series of batch reactors. The batch reactor contained inorganic culture media with PCP induced or uninduced Sphingomonas chlorophenolica cells. Chlorophenols in each reactor served as the sole carbon and energy source. Table 1. The Compositions of inorganic culture media Composition
Concentration (g/l)
Table 2. The Compositions of trace element solution Composition
Concentration (mg/l)
MgSO4⋅7H2O
0.2
FeSO4⋅7H2O
300
CaCl2⋅2H2O
0.02
MnSO4⋅H2O
50
K2HPO4
0.5
CoCl2⋅6H2O
106
KH2PO4
0.5
Na2MoO4⋅2H2O
34
NH4NO3
0.5
ZnSO4⋅7H2O
40
10 ml/l
CuSO4⋅5H2O
50
Trace element solution
To prepare PCP induced cell suspensions, Sphingomonas chlorophenolica cells grew initially on R2A agar. After 2 days of incubation at 30°C, the cells from R2A agar plates were transferred into a 2-liter flask which contained inorganic culture media with 100~150 mg/l PCP. The 2-liter flask was incubated by shaking (120 rpm) in dark at 30°C. After approximately 18~25 hours of incubation and induction, the cells were harvested by centrifugation (6000 × g at 4°C for 14 minutes). The bacterial pellet was washed twice with fresh inorganic culture media and then re-suspended in appropriate amount of fresh inorganic culture media prior to use. PCP un-induced cell suspensions were prepared following the same procedures for preparation of PCP induced cell suspensions, but cells were not induced by PCP. Degradation of various chlorophenols by PCP degrading bacteria To evaluate the degradation ability of PCP degrading bacteria, several kinds of chlorophenols were
prepared (2-chlorophenol (2-CP), 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), 2,3-dichlorophenol (2,3-DCP), 2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,6-dichlorophenol (2,6-DCP), 3,4-dichlorophenol (3,4-DCP), 3,5-dichlorophenol (3,5-DCP), 2,3,5-trichlorophenol (2,3,5-TCP), 2,3,6trichlorophenol (2,3,6-TCP), 2,4,5-trichlorophenol (2,4,5-TCP), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)). The experiment was conducted with a series of 250 ml batch reactors, and each reactor contained 100 ml of un-induced cell suspensions. The amounts of pure culture of bacteria were 107 cells/ml. 75 mg/l various chlorophenols were added to serve as the sole carbon and energy source. The reactors were sealed with cotton stoppers and shaken at 120 rpm in dark at 30°C to observe chlorophenols and phenol removal under aerobic conditions. For each substrate, the stability of compounds in the medium was tested in un-inoculated flasks (blanks). Dechlorination of chlorophenols by PCP degrading bacteria In order to find out whether chlorides were released during degrading process, various chlorophenols were prepared (2,3,6-trichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and PCP). A series of 125 ml batch reactors were used to proceed the experiments. Each reactor contained 40 ml of PCP induced cell suspensions. The amounts of pure culture of bacteria were 107 cells/ml. 100 mg/l of 2,3,6-trichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and PCP mixture was added to serve as the sole carbon and energy source. The reactors were sealed with cotton stoppers and shaken at 120 rpm in dark at 30°C and then sampled periodically to observe chlorophenols removal and chloride release under aerobic conditions. PCP and 2,4-dichlorophenol degradation in the presence of various supplementary carbon sources For testing the effects of various supplementary carbon sources on 2,4-DCP and PCP degradation by Sphingomonas chlorophenolica, the experiment was conducted with a series of 250 ml batch reactors, and each reactor contained 100 ml of PCP induced cell suspensions. After adjusting the amounts of pure culture of bacteria to 107 cells/ml and adding 75 mg/l of 2,4-DCP and appropriate concentration of various supplementary carbon sources (300 mg/l glucose, pyruvate, sodium acetate and concentration 150 mg/l 2,4,6-TCP, PCP), the reactors were sealed with cotton stoppers and shaken at 120 rpm in dark at 30°C. The flasks were sampled periodically to measure chlorophenols removal, growth of cells and variation of pH under aerobic conditions. The PCP removal in the presence of supplementary carbon sources was investigated using the same setup with the exception that the initial concentration of PCP was 150 mg/l. Analytical methods Chlorophenols and phenol were analyzed by high performance liquid chromatography (HPLC) with ultraviolet detector (UV detector). The cell suspensions were clarified by centrifugation at 8000 rpm for 3 minutes. Moreover, the cell-free supernatant fraction was also analyzed by HPLC with UV detector. HPLC was performed with a Hitachi system equipped with a Merck Lichrospher 100 PR-18 endcapped (5 µm) column at a flow rate of 0.8 ml/min. The ratio of solvent system was acetonitrile: water: phosphoric acid= 65: 35: 0.1. The UV detector absorbency wavelength was fixed at 284 nm. Chloride ion concentrations of culture fluids were detected by an ion chromatography equipped with a 2740 column and guard column (Hitachi), and an eluent of 2.0 mM 4-hydroxybenzoic acid and 2.3 mM triethylamine. The pH and O.D. were measured using a pH meter and Spectrophotometer at 600 nm, respectively.
RESULTS AND DISCUSSION Degradation of various chlorophenols by PCP degrading bacteria
Table 3 showed the results of various chlorophenols removal in the presence and absence of Sphingomonas chlorophenolica cells. The cells were capable of degrading 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6-TeCP, but could not degrade 2-CP, 3-CP, 4-CP, 2,3-DCP, 2,4-DCP, 2,5-DCP, 2,6-DCP, 3,4DCP, 3,5-DCP, 2,3,5-TCP and 2,4,5-TCP (the reductions of chlorophenols in blanks were caused by evaporation). Banerjee et al. reported that chlorophenols are less readily biodegradable than phenol. The rate of chlorophenols biodegradation decreases with increasing number of chlorine substituents on the aromatic ring (Banerjee et al., 1984). Steiert et al. tested the ability of a PCP degrading Flavobaterium sp. to dechlorinate other chlorinated phenols. The result indicated that chlorinated phenols with chlorine atoms at positions 2 and 6 of phenol ring were dechlorinated completely, and substitution patterns of chlorine atoms on phenol ring appeared to be important regulators of dechlorination and mineralization of chlorophenol compounds by Flavobaterium sp. (Steiert et al., 1987). However, according to the results showed in Table 3, the ability of Sphingomonas chlorophenolica cells to degrade various chlorophenols was not related to the chlorine ring substitution patterns of specific compounds. The reason may be explained by finding out the metabolic pathway of this strain. The removal of 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6-TeCP in the presence and absence of cell suspensions of Sphingomonas chlorophenolica was shown in Figure 1. When the initial concentration of 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6-TeCP was 75 mg/l, Sphingomonas chlorophenolica cells could degrade these chlorophenols completely within 3 days. After re-adding 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6-TeCP served as carbon source, these chlorophenols could still be degraded completely within another 2 days. Based on the results showed in Figure 1 and Table 3, Sphingomonas chlorophenolica cells could degrade not only PCP but also 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6-TeCP. Dechlorination of chlorophenols by PCP degrading bacteria The removal and dechlorination of four kinds of chlorophenols by PCP induced cells in 125-ml flasks Table 3. The degradation of various chlorophenols and phenol by Sphingomonas chlorophenolica* Substrate
Suspension with cells of Sphingomonas chlorophenolica
Suspension without cells of Sphingomonas chlorophenolica
Substrate remaining (%)
Substrate remaining (%)
Chlorophenol 234-
70 100 104
72 105 104
Dichlorophenol 2,32,42,52,63,43,5-
82 73 93 85 104 99
89 75 91 92 103 101
Trichlorophenol 2,3,52,3,62,4,52,4,6-
94 0 94 0
96 99 95 99
Tetrachlorophenol 2,3,4,6-
0
97
Pentachlorophenol
0
103
* Each experiment was conducted with a series of 250 ml batch reactors. The reactor contained 100 ml inorganic culture media with/without Sphingomonas chlorophenolica cells (≈107 cells/ml) and 75 mg/l substrate. Each reactor was shaken at 120 rpm in dark at 30°C under aerobic conditions. After 7 days, the flask was sampled to measure substrate removal.
Chlorophenols conc. (mg/l)
120 100 80 60 40 20 0 0
1
2
3
4
5
6
7
8
9
time (day)
Figure 1. The removal of 2,3,6-TCP (, ), 2,4,6-TCP (,
) and 2,3,4,6-TeCP (S, U) in the presence (closed symbol) and absence (open symbol) of cell suspensions of Sphingomonas chlorophenolica. Arrow sign indicates the resupplement time of chlorophenols. was shown in Figure 2. The results indicated PCP induced cells were able to completely degrade 100 mg/l 2,3,6-TCP, 2,4,6-TCP, 2,3,4,6-TeCP and PCP within 38.1, 15.1, 11.8 and 11.8 hours, and to release concentration 50.1, 60.9, 63.7 and 58.5 mg/l chloride at the same period of time. The rates of degrading PCP, 2,3,4,6-TeCP and 2,4,6-TCP by Sphingomonas chlorophenolica were better than the rate of degrading 2,3,6-TCP. Furthermore, after calculating the dechlorination of 2,3,6-TCP, 2,4,6-TCP, 2,3,4,6-TeCP and PCP, four kinds of chlorophenols were dechlorinated approximately 100% by PCP induced cells. CPs conc. (mg/l)
120 100 80 60 40 20
Cl- conc. (mg/l)
0 0
10
0
10
20
30
40
20
30
40
70 60 50 40 30 20 10 0
time (hr)
Figure 2. The removal and dechlorination of four kinds of chlorophenols by PCP induced cell suspensions in 125-ml flasks. Symbols: (), 2,3,6-TCP; (), 2,4,6-TCP; (z), 2,3,4,6-TeCP; (S), PCP. Chlorophenols degrading in the presence of various supplementary carbon sources In the presence of two or more substrates, the interaction between bacteria and substrates became more complex. The mutual inhibition between two different substrates may slow down the removal rate of any individual substrate. Strains may utilize one substrate to increase biomass furthermore to remove other substrates. Substrates that are not served as a source of energy or nutrients to microorganisms may be oxidized and decomposed through cometabolism. In this research, the effect of adding various additional substrates was studied for enhancing the removal of 2,4-DCP and PCP.
In the part of 2,4-DCP, 2,4-DCP (75 mg/l) was used as the primary substrate; glucose (300 mg/l), pyruvate (300 mg/l), sodium acetate (300 mg/l) 2,4,6-TCP (150 mg/l) and PCP (150 mg/l) were used as the secondary substrates. The removal of 2,4-DCP in the presence of various supplementary carbon sources by PCP induced cells was shown in Figure 3. Sphingomonas chlorophenolica was not capable of decomposing 2,4-DCP with or without the addition of various supplementary carbon sources (evaporation of 2,4-dichlorophenol was the main cause for its reduction in flasks). Moreover, no significant growth of cells was observated, and 150 mg/l of 2,4,6-TCP and PCP were not degraded by Sphingomonas chlorophenolica (data not shown). The result demonstrated 75 mg/l of 2,4-DCP was inhibitive to the growth and activity of cells. Figure 4 showed the removal of PCP in the presence of various supplementary carbon sources by PCP induced cells. In the presence of supplementary carbon sources (concentration 300 mg/l glucose, pyruvate, sodium acetate), the additions of glucose or pyruvate as the supplementary carbon sources were noted to enhance the removal of PCP. The PCP was removed more rapidly in the presence of glucose or pyruvate, and 150 mg/l PCP was degraded completely within 14.4 hours. However, there was no effect in the presence of sodium acetate for PCP induced cells to degrade 150 mg/l PCP. The growth of cells in the presence of supplementary carbon sources increased obviously, especially in the presence of glucose.
O.D.× 10
pH
2,4-DCP conc. (mg/l)
From the results of this research, the metabolism of PCP by Sphingomonas chlorophenolica was facilitated by adding glucose or pyruvate. The first possible reason was suggested that the metabolic pathway of PCP and the metabolic pathway of glucose or pyruvate were partially the same. According to the former reports of PCP degradation pathway by Sphingomonas chlorophenolica (Orser et al., 1993; Orser and Lange, 1994; Xun and Orser, 1991; Xun et al., 1992; Xu et al., 1999; Cai and Xun, 2002), PCP could be biotransformed to 2,6-dichlorohydroquinone (2,6-DCHQ). Subsequently, 2,6-DCHQ entered the ring cleavage pathway to form maleylacetate, and then maleylacetate entered TCA cycle which was an important metabolic pathway of glucose and pyruvate. Thus Sphingomonas chlorophenolica might use some induced enzymes that produced by metabolizing glucose or pyruvate to metabolize PCP. The second possible reason was that the metabolism of glucose and pyruvate could provide more NADH to facilitate the degradation of PCP. The former reports showed the degradation of PCP need some NADH to perform the frist step. Therefore, Sphingomonas chlorophenolica might use NADH that produced by metabolizing glucose or pyruvate to metabolize PCP. The final conjecture 80 60 40 20 0 7.5 7 6.5 6
0
30
60
90
120
150
180
0
30
60
90
120
150
180
0
30
60
90
120
150
180
1.5 1 0.5
time (hr) none pyruvate
glucose 2,4,6-TCP
NaOAc PCP
Figure 3. The removal of 2,4-DCP in the presence of various supplementary carbon sources by PCP induced cells in 125-ml flasks.
PCP conc. (mg/l) pH O.D.× 10
160 120 80 40 0
8 7.5 7 6.5 6
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
4.5 2.5 0.5
time (hr) none NaOAc
glucose pyruvate
Figure 4. The removal of PCP in the presence of various supplementary carbon sources by PCP-induced cell suspensions in 125-ml flasks. could be that Sphingomonas chlorophenolica used glucose and pyruvate, which were easily biodegraded, to increase the biomass and thus increasing the total activity for metabolizing PCP. Further tests should be performed to find the real reason.
CONCLUSIONS AND PERSPECTIVES The purpose of this research was to study the ability of PCP degrading bacterium Sphingomonas chlorophenolica to degrade and dechlorinate other chlorophenols. Moreover, the characteristics of Sphingomonas chlorophenolica had also been investigated. The conclusions derived from this research were stated as follows: 1) Sphingomonas chlorophenolica cells were capable of degrading 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6TeCP, but could not degrade 2-CP, 3-CP, 4-CP, 2,3-DCP, 2,4-DCP, 2,5-DCP, 2,6-DCP, 3,4-DCP, 3,5-DCP, 2,3,5-TCP and 2,4,5-TCP. 2) The Sphingomonas chlorophenolica was able to completely degrade 2,3,6-TCP, 2,4,6-TCP, 2,3,4,6TeCP and PCP within 38.1, 15.1, 11.8 and 11.8 hours, and to release concentration 50.1, 60.9, 63.7 and 58.5 mg/l chloride at the same period of time. Furthermore, the results showed that four kinds of chlorophenols had been dechlorinated approximately 100% by the cell suspensions of Sphingomonas chlorophenolica. 3) In the presence of supplementary carbon sources (concentration 300 mg/l glucose, pyruvate, sodium acetate and concentration 150 mg/l 2,4,6-TCP, PCP), no significant amounts of 2,4-DCP were removed during the incubation, and 2,4,6-TCP and PCP also could not be degraded by Sphingomonas chlorophenolica. The result indicated that 75 mg/l of 2,4-dichlorophenol was inhibitive to the activity of cells. 4) The additions of glucose or pyruvate as the supplementary carbon sources were noted to enhance the removal of PCP. PCP was removed more rapidly in the presence of glucose or pyruvate, and 150 mg/l PCP was degraded completely within 14.4 hours. REFERENCES
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