- Email: [email protected]
Improved Biodegradation of 1,2,4-Trichlorobenzene by Adapted Microorganisms in Agricultural Soil and in Soil Suspension Cultures
Pedosphere 21(4): 423–431, 2011 ISSN 1002-0160/CN 32-1315/P c 2011 Soil Science Society of China Published by Elsevier B.V. and Science Press
Improved Biodegradation of 1,2,4-Trichlorobenzene by Adapted Microorganisms in Agricultural Soil and in Soil Suspension Cultures∗1 SONG Yang1 , WANG Fang1 , F. O. KENGARA2 , BIAN Yong-Rong1 , YANG Xing-Lun1 , LIU Cui-Ying1 and JIANG Xin1,∗2 1 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China) 2 Department of Chemistry, Maseno University, Maseno 40105 (Kenya)
(Received October 14, 2010; revised April 15, 2011)
ABSTRACT Inoculating soil with an adapted microbial community is a more effective bioaugmentation approach than inoculation with pure strains in bioremediation. However, information on the potential of different inocula from sites with varying contamination levels and pollution histories in soil remediation is lacking. The objective of the study was to investigate the potential of adapted microorganisms in soil inocula, with different contamination levels and pollution histories, to degrade 1,2,4-trichlorobenzene (1,2,4-TCB). Three different soils from chlorobenzene-contaminated sites were inoculated into agricultural soils and soil suspension cultures spiked with 1,2,4-TCB. The results showed that 36.52% of the initially applied 1,2,4-TCB was present in the non-inoculated soil, whereas about 19.00% of 1,2,4-TCB was present in the agricultural soils inoculated with contaminated soils after 28 days of incubation. The soils inoculated with adapted microbial biomass (in the soil inocula) showed higher respiration and lower 1,2,4-TCB volatilization than the non-inoculated soils, suggesting the existence of 1,2,4-TCB adapted degraders in the contaminated soils used for inoculation. It was further confirmed in the contaminated soil suspension cultures that the concentration of inorganic chloride ions increased continuously over the entire experimental period. Higher contamination of the inocula led not only to higher degradation potential but also to higher residue formation. However, even inocula of low-level contamination were effective in enhancing the degradation of 1,2,4-TCB. Therefore, applying adapted microorganisms in the form of soil inocula, especially with lower contamination levels, could be an effective and environment-friendly strategy for soil remediation. Key Words:
bioaugmentation, chlorobenzenes, contaminated soil, dechlorination, inoculation, volatilization
Citation: Song, Y., Wang, F., Kengara, F. O., Bian, Y. R., Yang, X. L., Liu, C. Y. and Jiang, X. 2011. Improved biodegradation of 1,2,4-trichlorobenzene by adapted microorganisms in agricultural soil and in soil suspension cultures. Pedosphere. 21(4): 423–431.
INTRODUCTION Chlorobenzenes (CBs) are used as starting materials and additives in the production of insecticides, fungicides, herbicides, dyes, pharmaceuticals, disinfectants, rubbers, plastics and electric goods, and they have become ubiquitous pollutants (Rapp, 2001; Schroll et al., 2004). 1,2,4-trichlorobenzene (1,2,4TCB), the most widely used chlorobenzene, has been ∗1
detected in soil (Zolezzi et al., 2005), groundwater (Boyd et al., 1997), wastewater (Oliver and Nicol, 1982; Monferr´ an et al., 2005), sewage sludge (Freitag et al., 1985), sediment (Gaffney, 1976) and vegetables (Zhang et al., 2005). Due to the persistence, toxicity, and bioaccumulation potential (Adebusoye et al., 2007), elimination of 1,2,4-TCB from contaminated sites is of worldwide concern (Sander et al., 1991; Rapp and Timmis, 1999).
Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KZCX2-EW-QN403), the National Natural Science Foundation of China (Nos. 41030531, 4092106, and 20707028), and the Jiangsu Provincial Natural Science Foundation of China (No. BK2010608). ∗2 Corresponding author. E-mail: [email protected].
424
Y. SONG et al.
Bioremediation has been widely applied in the biological treatment of contaminated sites due to the advantages of cost-effectiveness and environmentalfriendliness. Bioaugmentation, a commonly used bioremediation technique, involves the addition of external microbial strains (indigenous or exogenous) which have the ability to degrade the target toxic molecules (Odokuma and Dickson, 2003; Hamdi et al., 2007). Under laboratory conditions, biodegradation of 1,2,4TCB in soil could be enhanced by inoculating adapted bacteria such as Pseudomonas sp. P51 (van der Meer et al., 1987) and Bordetella sp. F2 (Wang et al., 2007). However, when the microorganisms are applied to contaminated sites for in-situ remediation, their ability to metabolize target compounds is not unequivocal (Tchelet et al., 1999). Compared with inoculating pure strains into soil, inoculation of an adapted microbial community seems to be more effective. Wang et al. (2010) reported that soil inoculated with a microbial community showed a higher 1,2,4-TCB mineralizing rate than pure strains. However, the process of isolating the microbial community is time consuming (Wang et al., 2007, 2010). Schroll et al. (2004) reported that the transfer of contaminated soil with adapted microbial population into an agricultural soil significantly enhanced the mineralization of 1,2,4-TCB in the agricultural soil, indicating that the transferred microbial population in soil inocula survived and maintained its degradation ability in the new microbial ecosystem. However, using soil inocula for bioaugmentation poses the problem of introducing further contamination to the soil under remediation. Report on the potential of different inocula, from sites with low or high concentration of pollutants, in
soil remediation is lacking (D´ıaz, 2004; Singh, 2008; Cao et al., 2009). The effect of pollution history on the effectiveness of the inocula is also rarely reported (Aelion et al., 1987). This work sought to fill in these mising gaps. Three soils with low and high contamination levels and different pollution histories were selected for inoculation into 1,2,4-TCB-spiked soils. The aim of the study was to test the bioaugmentation potential of the adapted microbes in the different soil inocula. The hypothesis was that soil inocula with longer contamination history and higher concentration would have higher degradation efficiency. If found to be environmentally sustainable, using soil inocula would offer an efficient, cost-effective and easy method for introducing adapted microbes to polluted sites. MATERIALS AND METHODS Chemicals Standards of monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3DCB), 1,4-dichlorobenzene (1,4-DCB) and 1,2,4-TCB with purity > 99.5% were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Solvents and other chemicals (Nanjing Chemical Factory, Nanjing, China) were of analytical grade. Anhydrous sodium sulfate was oven-dried at 400 ◦ C for 4 h. Soil preparation Four different soil samples, AS, CS1, CS2 and CS3, were used in this study (Table I). Soil AS was uncontaminated while soils CS1, CS2 and CS3 were contaminated with CBs. Soil AS was used for incubation and the other three contaminated soils were used as inocula. Soil AS was collected from an agricultural field in
TABLE I Physico-chemical properties of soils and the concentrations of chlorobenzenes (CBs) Soila) AS CS1 CS2 CS3 a)
pH
OMb)
6.97 8.00 7.47 8.06
% 3.19 2.13 1.45 3.50
C:N 9.30 13.89 12.76 17.63
Clay
Silt
31.01 9.36 25.68 2.37
% 57.54 52.12 58.87 13.29
Sand 11.45 38.52 15.45 84.34
1,2-DCBc) e)
ND 0.03 0.38 3.54
1,3-DCB ND 0.02 0.11 1.50
1,4-DCB μg g−1 ND 0.04 0.35 2.74
Soil AS was uncontaminated, while soils CS1, CS2 and CS3 were contaminated with CBs. Organic matter. c) DCB = dichlorobenzene. d) TCB = trichlorobenzene. e) Not detected. b)
1,2,4-TCBd) 0.004 0.15 0.81 22.77
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE
Baguazhou, Nanjing, China (32◦ 12 4.6 N, 118◦ 50 112.3 E). Soil CS1 was collected from arable land near Yangnong Chemical Factory, Yangzhou, China (32◦ 21 57.5 N, 119◦ 25 14.8 E) and had been contaminated for about 15 years due to dry-wet deposition. Soils CS2 and CS3 were collected in the garden of Haichen Chemical Factory, Jiangdu, China (32◦ 34 14.5 N, 119◦ 44 34.8 E) and had been contaminated for about 40 years due to the dumping of waste materials and dry-wet deposition. All the soils were sampled from the depth of 0–20 cm, sieved through a 2-mm mesh and stored at 4 ◦ C. Prior to the start of the experiments, the soil samples were adjusted to a water content of 280 mg g−1 by oven-dry weight and equilibrated for one week at 30 ± 1 ◦ C in dark. The concentrations of CBs in the four soils are listed in Table I. Biodegradation of 1,2,4-TCB in soil 400 μg of 1,2,4-TCB dissolved in 400 μL acetone was applied to an aliquot of 10 g (dry weight) of soil AS in a 50-mL glass beaker. After evaporation of acetone, the soil aliquot was mixed and transferred into a glass beaker which already contained 180 g (dry weight) of equilibrated soil AS and 10 g (dry weight) of soil inocula. For the non-inoculated treatment, the spiked 10 g of soil aliquot was mixed with 190 g of soil AS. The 200 g of soil was mixed by stirring carefully and thoroughly with a spatula, transferred to a 1 000-mL incubation flask, and adjusted to −15 kPa of soil water potential. An aliquot of 5 g of soil was sampled with a stainless steel soil borer to determine the initial concentration of 1,2,4-TCB in soil. The remaining soil was compacted to a volume equivalent to 1.3 g cm−3 of soil density (Schroll et al., 2006). The flask was closed tightly with a rubber plug and incubated at 30 ± 1 ◦ C for 56 days in the dark. Using a closed laboratory trapping system (Schroll et al., 2004), the flasks were aerated once per week with humidified CO2 -free air for 20 min at an exchange rate of 0.4 L min−1 to flush out the produced CO2 and the volatile compounds from the incubation flasks to the traps filled with 10 mL 1 mol L−1 NaOH and 15 mL n-hexane, respectively. After aeration, an aliquot of 5 g of soil was sampled for 1,2,4-TCB and its possible dechlorinated products analysis. There were a total of 4 treatments: AS + 1,2,4-TCB (NIS), AS + 1,2,4-TCB + inocula CS1 (ICS1), AS + 1,2,4-TCB + inocula CS2 (ICS2), and AS + 1,2,4-TCB + inocula CS3 (ICS3). An additional set containing 200 g of soil AS only (blank) was used as the control for soil respiration. All treatments were conducted in triplicate.
425
Biodegradation of 1,2,4-TCB in suspension cultures To further confirm the biodegradation potential of the three contaminated soils CS1, CS2 and CS3, a parallel experiment was conducted with soil suspension cultures. 300-mL sealed incubation flasks (Schroll et al., 2004, modified) containing 100 mL sterilized mineral medium (Wang et al., 2007) were separately inoculated with 5 g (oven-dry weight) of CB-contaminated soils CS1, CS2 or CS3, respectively. 2 000 μg 1,2,4TCB dissolved in 50 μL acetone was spiked into all flasks. Filters (0.2 μm, Sartorius Midisart-2000, Germany) were installed at both the air inlet and outlet tubes of the glass flasks. Flasks were incubated on an orbital shaker at 150 r min−1 and 30 ± 1 ◦ C in the dark and aerated twice per week by the same trapping system mentioned above. After aeration, 0.5 and 1.0 mL soil suspension were sampled for analysis of 1,2,4-TCB plus its possible dechlorination products and chloride release, respectively. 100 μL of suspension were removed for determination of cell forming units. In order to keep the TCB concentration in the suspension culture nearly constant, 2 000 μg 1,2,4-TCB was reapplied as above after each aeration up to day 28, after which there was no further application. An additional set containing only mineral medium and 1,2,4-TCB (blank) was incubated as above and used as control for measurement of chloride release. All treatments were conducted in triplicate. Analytical methods Residues of CBs in soils were extracted with hexane/acetone (4:1, v/v) at a temperature of 100 ◦ C and a pressure of 10 MPa by accelerated solvent extraction (ASE 200, Dionex, USA). The concentrated extract was purified on a silica gel/anhydrous sodium sulfate column with 15 mL elution of hexane/dichloromethane (9:1, v/v). The eluate was concentrated to 1 mL for gas chromatograph (GC) analysis. During the experimental period the volatile fraction of CBs was trapped with hexane, dried with anhydrous sodium sulfate and concentrated to 1 mL for subsequent GC analysis. The concentrations of CBs were measured by a gas chromatograph (Agilent 6890, USA) equipped with a DB-5 capillary column (30 m length × 0.32 mm inside diameter × 0.25 μm film thickness), a 63 Ni electron capture detector and an HP 7683 auto-sampler. Nitrogen (purity > 99.999%) was used as the carrier gas at a flow rate of 1.5 mL min−1 . The column tempera-
426
ture was programmed from 60 ◦ C (1 min) to 140 ◦ C at a rate of 20 ◦ C min−1 and then to 280 ◦ C (5 min) at a rate of 8 ◦ C min−1 . The injector and detector temperatures were 220 and 300 ◦ C, respectively. The injection volume was 1 μL in the splitless mode. To quantify the respiration of the soils and the soil suspensions, CO2 absorbed by NaOH from the soil experiments and the soil suspension cultures were transferred from the traps into triangular flasks, and then titrated with 1 mol L−1 HCl (Haney et al., 2008). Inorganic chloride was used as an indicator of 1,2,4-TCB degradation. The concentration of inorganic chloride in the soil suspension cultures was detected by HNO3 -AgNO3 method (Bidlan and Manonmani, 2002). Cell counting in soil suspension cultures was performed by spreading serial dilutions of the liquid culture on Nutrient Broth (Shanghai Science Biotech Company, China) agar plates, and incubating at 30 ◦ C. Colony forming units (cfu) were determined after 48 h incubation.
Y. SONG et al.
treatments (Fig. 1a, Table II). The dissipation of 1,2,4TCB in soils was followed for a period of 56 days (Fig. 1). The possible dechlorination products of 1,2,4TCB (MCB, 1,2-DCB, 1,3-DCB and 1,4-DCB) were screened for by GC, but were not detected in any treatment except for ICS3, in which 1,2-DCB, 1,3-DCB and 1,4-DCB were present at the start of the experiment (Table I). 1,2,4-TCB concentrations decreased rapidly in all the treatments in the first 14 days (Fig. 1). The degradation curve of treatment NIS leveled off in 14 days at 36.52% of the initial concentration, after which no further significant degradation could be observed (P > 0.05). However, the inoculated treatments decreased up to day 28 (P < 0.05). After 56 days, a much higher amount of 1,2,4-TCB was eliminated from treatment ICS3 than in the other two inoculated treatments (Table II), indicating that ICS3 was more effective in degradation. The dissipation of 1,2,4-TCB fitted into
Quality control and data analysis To estimate the recoveries of CBs residues in soil and trapped CBs by hexane, a recovery study was carried out by spiking a mixture of CBs (each 20 ng) to 10 g of soil AS or 15 mL of hexane in the trap. The extraction and purificiation of the samples were performed using the same procedure mentioned above and recoveries were 60.76%–91.56% in soil extraction and 72.63%–99.94% in trapping. The degradation of 1,2,4-TCB in the soil was evaluated by fitting the data with modified first-order kinetics equation: Ct = C0 [λ + (1 − λ)e−kt ] where C0 and Ct are the concentrations of 1,2,4-TCB in soil at start time (0) and time t, respectively; λ is the coefficient of non-available fraction of 1,2,4-TCB in soil; and k is the first-order rate constant for degradation. All statistical data analysis was done with Statistical Product and Service Solutions (SPSS 13.0) and the significance level was P < 0.05. RESULTS Residues of 1,2,4-TCB in soils The initial concentration of 1,2,4-TCB in treatment ICS3 was nearly double that of the other three
Fig. 1 (a) Degradation kinetics (dots: measured data; curves: modified first-order kinetics model fitted) and (b) percentage residues of 1,2,4-trichlorobenzene (1,2,4-TCB) in non-inoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3). Error bars represent the standard deviations of the means (n = 3).
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE
427
TABLE II Initial and eliminated amounts of 1,2,4-trichlorobenzene (1,2,4-TCB) in non-inoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3) and the regression equations of degradation fitted into the modified first-order kinetics model Treatment
NIS ICS1 ICS2 ICS3
Initial amount
Eliminated amount
Freshly spiked
Aged in inocula
193.80bb) 243.95a 249.90a 230.34a
μg 0.00c 1.46b 8.10b 227.66a
Modified first-order kinetics modela) Regression equation
123.01c 198.02b 207.98b 297.51a
Ct Ct Ct Ct
= 0.98(0.37 + 0.63e−0.122t ) = 1.28(0.15 + 0.85e−0.080t ) = 1.30(0.19 + 0.81e−0.133t ) = 2.34(0.32 + 0.68e−0.063t )
R2
t1/2
0.98 0.96 0.99 0.97
d 12.83b 10.97b 7.20c 21.24a
Ct = concentration of 1,2,4-TCB in soil (μg g−1 ) at time t; t1/2 = half-life time (d). Means in a column followed by the same letter are not significantly different at P < 0.05 by Fisher’s protected least significant difference. a)
b)
the modified first-order kinetics model (Fig. 1a), with the longest and the shortest half-lives observed in treatments ICS3 and ICS2, respectively (Table II). Estimation of biodegradation of aged 1,2,4-TCB in soil CS3 The high concentration of 1,2,4-TCB in soil CS3 (Table I) resulted in a nearly similar amount of aged and freshly spiked 1,2,4-TCB in treatment ICS3 at the beginning (Table II). Therefore, it was necessary to estimate the biodegradation of aged 1,2,4-TCB in treatment ICS3. This could be done based on the degradation results of treatment ICS2 since soil inocula CS2 and CS3 were sampled from the same site and had the same pollution history, but the aged 1,2,4-TCB residues in soil CS2 were negligible compared to soil CS3. In treatment ICS2, due to the negligible amount of aged 1,2,4-TCB introduced by soil CS2, 1,2,4-TCB residues (19.38%) at the end of the experiment (Fig. 1b) should have come from the freshly spiked 1,2,4-TCB. Since soil inocula CS3 was more effective than CS2, freshly spiked 1,2,4-TCB in treatment ICS3 remaining at the end of the experiment should not have been higher than 19.38% (44.69 μg). However, 1,2,4-TCB remaining in treatment ICS3 at the end of the experiment was 160.49 μg. Therefore, 115.80 μg of 1,2,4-TCB remaining in treatment ICS3 should have come from the 10 g of inocula CS3, corresponding to 50.85% of the introduced 1,2,4-TCB. Thus, 49.14% of aged 1,2,4-TCB residues in soil CS3 were degraded. Production of CO2 As shown in Fig. 2a, CO2 production in the inocu-
Fig. 2 Time course of cumulative CO2 production in noninoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3) (a) and in suspension cultures (b). Blank was non-spiked, non-inoculated agricultural soil. 1,2,4-trichlorobenzene (1,2,4-TCB) was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
428
lated soils was much higher than that in treatment NIS (P < 0.05). Compared with the blank, a significant increase of CO2 production was observed in treatment NIS (P < 0.05), indicating that spiking of 1,2,4-TCB stimulated soil respiration. As shown in Fig. 2b, CO2 production in the suspension cultures showed a nearly linear increase with time in the first 28 days, indicating that microbial activity was stimulated by the mineral nutrients and continuous addition of 1,2,4-TCB. When no further 1,2,4-TCB was added after 28 days, CO2 production decreased.
Y. SONG et al.
release dynamics were observed in the different inoculated treatments. A lag phase of ten days was observed followed by a continuous Cl− release in treatment ICS1. In treatments ICS2 and ICS3, there was no lag phase and chloride release showed a rapid, slow and rapid increase over the experimental period. Among the inoculated treatments, ICS3 had the highest chloride release (P < 0.05). Although no further 1,2,4TCB was added to the flasks after day 24, the chloride concentration continued increasing with time in both treatments ICS1 and ICS3 (Fig. 4).
Microbial growth in suspension cultures Bacterial cell numbers in the three soil suspension cultures increased rapidly until day 17 (Fig. 3). The population in treatment ICS2, however, decreased rapidly after day 17. Treatment ICS2 was therefore stopped on day 24 because the experiment was meant to monitor the increase in microbial population. Treatment ICS3 showed a significantly higher amount of microorganisms than treatment ICS1 over the whole incubation period (P < 0.05), though they both exhibited similar growth trends. Fig. 4 Time course of chloride (Cl− ) release from spiked mineral medium (blank) and from suspension cultures inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3). 1,2,4-trichlorobenzene (1,2,4-TCB) was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
Volatilization of 1,2,4-TCB from soils and suspension cultures
Fig. 3 Time course of cell density in suspension cultures inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3). 1,2,4-trichlorobenzene (1,2,4-TCB) was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
Chloride release in suspension cultures To evaluate the degradation of 1,2,4-TCB in soil suspension cultures, chloride release was determined over the incubation period. Generally, the release of chloride increased in all the treatments over the whole incubation period (Fig. 4). However, different chloride
A rapid volatilization of 1,2,4-TCB from the soils was observed in the first week in all the treatments (Fig. 5a). Treatment NIS showed the highest volatilization at each sampling point and thus led to the highest volatilization losses. In contrast, the lowest volatilization was detected in treatment ICS3. Volatilization of treatments ICS1 and ICS2 ranged in between. The volatilization of 1,2,4-TCB from the suspension culture in treatment ICS3 was also much lower than that in treatments ICS1 and ICS2 (P < 0.05) (Fig. 5b). It was observed that higher degradation resulted in lower volatilization in the soil and soil suspension cultures. DISCUSSION Biodegradation of long-term aged contaminants in
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE
Fig. 5 Time course of cumulative 1,2,4-trichlorobenzene (1,2,4-TCB) volatilization from non-inoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3) (a) and that in suspension cultures (b). 1,2,4-TCB was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
soil is a great challenge (Mancera-L´opez et al., 2008). Inoculation with contaminated soils significantly enhanced the degradation of 1,2,4-TCB in the agricultural soil (Schroll et al., 2004), which showed that adapted microbes in soil inocula enhanced the degradation of freshly-spiked 1,2,4-TCB. However, in this study, soil inocula resulted in the enhanced degradation of both freshly spiked and aged 1,2,4-TCB residues. The increased degradation, respiration and chloride emission all pointed to the presence of adapted microbes in soil inocula of CS1, CS2 and CS3. The enhanced degradation of aged 1,2,4-TCB might be due to the increased nutrients, supplied by the agricultural soil, which stimulated the microorganism in the soil inocula. This indicated that bioaugmentation with soil inocula is an applicable and effective strategy for enhancing the breakdown of aged contaminants in soil.
429
The higher degradation and dechlorination of 1,2,4TCB corresponded to lower volatilization (Figs. 4 and 5), indicating that enhanced degradation of 1,2,4-TCB results in reduced volatilization losses (Wang et al., 2007). A possible explanation is that microorganisms acted as a sink for 1,2,4-TCB during the biodegradation process, thereby reducing its volatilization. Therefore, inoculated degraders could effectively degrade the bioavailable 1,2,4-TCB and thereby decrease its environmental risk, even if the concentration of 1,2,4-TCB remains high in the contaminated soil. Dechlorination is an important process during the degradation of chlorinated compounds. Generally in aerobic degradation, in contrast to reductive dechlorination, very few or no chlorine atoms are eliminated by microorganisms before the aromatic ring cleavage (Commandeur and Parsons, 1990; van der Meer et al., 1991; Marco-Urrea et al., 2009). The fact that lower-chlorinated benzenes (MCB, 1,2-DCB, 1,3-DCB and 1,4-DCB) were not detected could mean that ring cleavage preceded dechlorination, as was the case in other studies (van der Meer et al., 1991; Marco-Urrea et al., 2009). The concentration of chloride ions with time was measured to find out whether dechlorination of the aliphatic chain, which was formed after ring cleavage, occurs (Spiess et al., 1995). As can be seen from Fig. 4, chloride concentration increased with time, indicating that dechlorination of the aliphatic chain occurred (Spiess et al., 1995). To study the effect of contamination history on the degradation capacity of the inocula, the results of treatments ICS1 and ICS2, which had been contaminated for 15 and 40 years, respectively, were compared. The similar degradation kinetics and residues of 1,2,4-TCB in the spiked soils ICS1 and ICS2 (Fig. 1) indicated a similar dissipation pattern. This is further supported by the similar respiration and volatilization in the two treatments (Figs. 2 and 5). All these indicated that contamination period may not be a critical factor in determining the activity and efficacy of the soil inocula. This could mean that the time span of 15 years may be long enough for complete microbial adaptation, hence a longer time span could not contribute more to biodegradation capacity (Aelion et al., 1987). To check for the effect of contaminant concentration on the degradation efficacy of the inocula, the results of treatments ICS2 and ICS3, both of which had been contaminated for about 40 years, were compared. Inocula CS3 had a 1,2,4-TCB concentration 28 times
430
higher than CS2 (Table I). The higher cell counts (Fig. 3), higher dechlorination (Fig. 4) and lower volatilization (Fig. 5) in ICS3 indicated a higher microbial activity in CS3 in the soil suspension culture. This was further indicated by the fact that more 1,2,4TCB was removed from the CS3 inoculated soil (Fig. 1, Table II). However, residues of 1,2,4-TCB in the ICS3 were much higher than those in the ICS2, which could be attributed to the different aged residues in the inocula (Table II). Inocula CS2 had a lower contaminant concentration, whereas nearly half of the 1,2,4-TCB in the ICS3 was aged. Of the aged residues, 50.85% were unavailable, thus leading to the higher residues in the ICS3. Therefore, the inocula with lower contamination level might be more environment-friendly in enhancing degradation of 1,2,4-TCB in soil, considering the larger amount of aged residues that would be introduced by using higher-contaminated soils as inocula. CONCLUSIONS Inoculation of soil from previously contaminated sites could enhance the degradation and limit volatilization of 1,2,4-TCB in artificially contaminated soils, thus resulting in reduced environmental risk. Long-term contaminated soils contained the adapted microorganisms able to metabolize 1,2,4-TCB with subsequent chloride release. The concentration of contaminants, rather than contamination period, seems to be the more important factor influencing the activity of soil inocula. Large amounts of aged residues in the contaminated soil inocula were also degraded. Although highly contaminated soil inocula were more effective in degradation, bioaugmentation with lowcontaminated soil inocula is more recommended for insitu bioremediation of 1,2,4-TCB polluted soils. This is because of the environmental cost due to the possible secondary contamination introduced by using highlycontaminated soils as inocula. REFERENCES Adebusoye, S. A., Picardal, F. W., Ilori, M. O., Amund, O. O., Fuqua, C. and Grindle, N. 2007. Aerobic degradation of di- and trichlorobenzenes by two bacteria isolated from polluted tropical soils. Chemosphere. 66: 1939–1946. Aelion, C. M., Swindoll, C. M. and Pfaender, F. K. 1987. Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer. Appl. Environ. Microbiol. 53: 2212–2217. Bidlan, R. and Manonmani, H. K. 2002. Aerobic degrada-
Y. SONG et al. tion of dichlorodiphenyltrichloroethane (DDT) by Serratia marcescens DT-1P. Process Biochem. 38: 49–56. Boyd, E. M., Killham, K., Wright, J., Rumford, S., Hetheridge, M., Cumming, R. and Meharg, A. A. 1997. Toxicity assessment of xenobiotic contaminated groundwater using lux modified Pseudomonas fluorescens. Chemosphere. 35: 1967–1985. Cao, B., Nagarajan, K. and Loh, K. C. 2009. Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches. Appl. Microbiol. Biot. 85: 207–228. Commandeur, L. C. M. and Parsons, J. R. 1990. Degradation of halogenated aromatic compounds. Biodegradation. 1: 207–220. D´ıaz, E. 2004. Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility. Int. Microbiol. 7: 173–180. Freitag, D., Ballhorn, L., Geyer, H. and Korte, F. 1985. Environmental hazard profile of organic chemicals: An experimental method for the assessment of the behaviour of organic chemicals in the ecosphere by means of simple laboratory tests with 14 C labelled chemicals. Chemosphere. 14: 1589–1616. Gaffney, P. E. 1976. Carpet and rug industry case-study II: biological effects. J. Water Pollut. Con. F. 48: 2731–2737. Hamdi, H., Benzarti, S., Manusadˇzianas, L., Aoyama, I. and Jedidi, N. 2007. Bioaugmentation and biostimulation effects on PAH dissipation and soil ecotoxicity under controlled conditions. Soil Biol. Biochem. 39: 1926–1935. Haney, R. L., Brinton, W. F. and Evans, E. 2008. Soil CO2 respiration: Comparison of chemical titration, CO2 IRGA analysis and the Solvita gel system. Renew. Agr. Food. Syst. 23: 171–176. Mancera-L´ opez, M., Esparza-Garc´ıa, F., Ch´ avez-G´ omez, B., Rodr´ıguez-V´ azquez, R., Saucedo-Casta˜ neda, G. and Barrera-Cort´es, J. 2008. Bioremediation of an aged hydrocarbon-contaminated soil by a combined system of biostimulation-bioaugmentation with filamentous fungi. Int. Biodeter. Biodegr. 61: 151–160. Marco-Urrea, E., Perez-Trujillo, M., Caminal, G. and Vicent, T. 2009. Dechlorination of 1,2,3- and 1,2,4trichlorobenzene by the white-rot fungus Trametes versicolor. J. Hazard. Mater. 166: 1141–1147. Monferr´ an, M. V., Echenique, J. R. and Wunderlin, D. A. 2005. Degradation of chlorobenzenes by a strain of Acidovorax avenae isolated from a polluted aquifer. Chemosphere. 61: 98–106. Odokuma, L. O. and Dickson, A. A. 2003. Bioremediation of a crude oil polluted tropical mangrove environment. J. Appl. Sci. Environ. Manag. 7: 23–29. Oliver, B. G. and Nicol, K. D. 1982. Chlorobenzenes in sediments, water, and selected fish from Lakes Superior, Huron, Erie, and Ontario. Environ. Sci. Technol. 16: 532–536.
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE Rapp, P. 2001. Multiphasic kinetics of transformation of 1,2,4-trichlorobenzene at nano- and micromolar concentrations by Burkholderia sp. strain PS14. Appl. Environ. Microbiol. 67: 3496–3500. Rapp, P. and Timmis, K. N. 1999. Degradation of chlorobenzenes at nanomolar concentrations by Burkholderia sp. strain PS14 in liquid cultures and in soil. Appl. Environ. Microbiol. 65: 2547–2552. Sander, P., Wittich, R. M., Fortnagel, P., Wilkes, H. and Francke, W. 1991. Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Appl. Environ. Microbiol. 57: 1430–1440. Schroll, R., Becher, H. H., D¨ orfler, U., Gayler, S., Grundmann, S., Hartmann, H. P. and Ruoss, J. 2006. Quantifying the effect of soil moisture on the aerobic microbial mineralization of selected pesticides in different soils. Environ. Sci. Technol. 40: 3305–3312. Schroll, R., Brahushi, F., D¨ orfler, U., Kuhn, S., Fekete, J. and Munch, J. C. 2004. Biomineralisation of 1,2,4trichlorobenzene in soils by an adapted microbial population. Environ. Pollut. 127: 395–401. Singh, D. K. 2008. Biodegradation and bioremediation of pesticide in soil: concept, method and recent developments. Indian J. Microbiol. 48: 35–40. Spiess, E., Sommer, C. and Gorisch, H. 1995. Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1. Appl. Environ. Microbiol. 61: 3884–3888. Tchelet, R., Meckenstock, R., Steinle, P. and van der Meer, J. R. 1999. Population dynamics of an introduced bacterium degrading chlorinated benzenes in a soil column and in sewage sludge. Biodegradation. 10: 113–125.
431 van der Meer, J. R., Roelofsen, W., Schraa, G. and Zehnder, A. J. B. 1987. Degradation of low concentrations of dichlorobenzenes and 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 in nonsterile soil columns. FEMS Microbiol. Lett. 45: 333–341. van der Meer, J. R., Van Neerven, A. R. W., De Vries, E. J., De Vos, W. M. and Zehnder, A. J. B. 1991. Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro- and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51. J. Bacteriol. 173: 6–15. Wang, F., D¨ ofler, U., Schmid, M., Fischer, D., Kinzel, L., Scherb, H., Munch, J. C., Jiang, X. and Schroll, R. 2010. Homogeneous inoculation vs. microbial hot spots of isolated strain and microbial community: What is the most promising approach in remediating 1,2,4-TCB contaminated soils? Soil Biol. Biochem. 42: 331–336. Wang, F., Grundmann, S., Schmid, M., D¨ ofler, U., Roherer, S., Charles, M. J., Hartmann, A., Jiang, X. and Schroll, R. 2007. Isolation and characterization of 1,2,4-trichlorobenzene mineralizing Bordetella sp. and its bioremediation potential in soil. Chemosphere. 67: 896–902. Zhang, J. Y., Zhao, W., Pan, J., Qiu, L. M. and Zhu, Y. M. 2005. Tissue-dependent distribution and accumulation of chlorobenzenes by vegetables in urban area. Environ. Int. 31: 855–860. Zolezzi, M., Cattaneo, C. and Tarazona, J. V. 2005. Probabilistic ecological risk assessment of 1,2,4-trichlorobenzene at a former industrial contaminated site. Environ. Sci. Technol. 39: 2920–2926.
Improved Biodegradation of 1,2,4-Trichlorobenzene by Adapted Microorganisms in Agricultural Soil and in Soil Suspension Cultures∗1 SONG Yang1 , WANG Fang1 , F. O. KENGARA2 , BIAN Yong-Rong1 , YANG Xing-Lun1 , LIU Cui-Ying1 and JIANG Xin1,∗2 1 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China) 2 Department of Chemistry, Maseno University, Maseno 40105 (Kenya)
(Received October 14, 2010; revised April 15, 2011)
ABSTRACT Inoculating soil with an adapted microbial community is a more effective bioaugmentation approach than inoculation with pure strains in bioremediation. However, information on the potential of different inocula from sites with varying contamination levels and pollution histories in soil remediation is lacking. The objective of the study was to investigate the potential of adapted microorganisms in soil inocula, with different contamination levels and pollution histories, to degrade 1,2,4-trichlorobenzene (1,2,4-TCB). Three different soils from chlorobenzene-contaminated sites were inoculated into agricultural soils and soil suspension cultures spiked with 1,2,4-TCB. The results showed that 36.52% of the initially applied 1,2,4-TCB was present in the non-inoculated soil, whereas about 19.00% of 1,2,4-TCB was present in the agricultural soils inoculated with contaminated soils after 28 days of incubation. The soils inoculated with adapted microbial biomass (in the soil inocula) showed higher respiration and lower 1,2,4-TCB volatilization than the non-inoculated soils, suggesting the existence of 1,2,4-TCB adapted degraders in the contaminated soils used for inoculation. It was further confirmed in the contaminated soil suspension cultures that the concentration of inorganic chloride ions increased continuously over the entire experimental period. Higher contamination of the inocula led not only to higher degradation potential but also to higher residue formation. However, even inocula of low-level contamination were effective in enhancing the degradation of 1,2,4-TCB. Therefore, applying adapted microorganisms in the form of soil inocula, especially with lower contamination levels, could be an effective and environment-friendly strategy for soil remediation. Key Words:
bioaugmentation, chlorobenzenes, contaminated soil, dechlorination, inoculation, volatilization
Citation: Song, Y., Wang, F., Kengara, F. O., Bian, Y. R., Yang, X. L., Liu, C. Y. and Jiang, X. 2011. Improved biodegradation of 1,2,4-trichlorobenzene by adapted microorganisms in agricultural soil and in soil suspension cultures. Pedosphere. 21(4): 423–431.
INTRODUCTION Chlorobenzenes (CBs) are used as starting materials and additives in the production of insecticides, fungicides, herbicides, dyes, pharmaceuticals, disinfectants, rubbers, plastics and electric goods, and they have become ubiquitous pollutants (Rapp, 2001; Schroll et al., 2004). 1,2,4-trichlorobenzene (1,2,4TCB), the most widely used chlorobenzene, has been ∗1
detected in soil (Zolezzi et al., 2005), groundwater (Boyd et al., 1997), wastewater (Oliver and Nicol, 1982; Monferr´ an et al., 2005), sewage sludge (Freitag et al., 1985), sediment (Gaffney, 1976) and vegetables (Zhang et al., 2005). Due to the persistence, toxicity, and bioaccumulation potential (Adebusoye et al., 2007), elimination of 1,2,4-TCB from contaminated sites is of worldwide concern (Sander et al., 1991; Rapp and Timmis, 1999).
Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KZCX2-EW-QN403), the National Natural Science Foundation of China (Nos. 41030531, 4092106, and 20707028), and the Jiangsu Provincial Natural Science Foundation of China (No. BK2010608). ∗2 Corresponding author. E-mail: [email protected].
424
Y. SONG et al.
Bioremediation has been widely applied in the biological treatment of contaminated sites due to the advantages of cost-effectiveness and environmentalfriendliness. Bioaugmentation, a commonly used bioremediation technique, involves the addition of external microbial strains (indigenous or exogenous) which have the ability to degrade the target toxic molecules (Odokuma and Dickson, 2003; Hamdi et al., 2007). Under laboratory conditions, biodegradation of 1,2,4TCB in soil could be enhanced by inoculating adapted bacteria such as Pseudomonas sp. P51 (van der Meer et al., 1987) and Bordetella sp. F2 (Wang et al., 2007). However, when the microorganisms are applied to contaminated sites for in-situ remediation, their ability to metabolize target compounds is not unequivocal (Tchelet et al., 1999). Compared with inoculating pure strains into soil, inoculation of an adapted microbial community seems to be more effective. Wang et al. (2010) reported that soil inoculated with a microbial community showed a higher 1,2,4-TCB mineralizing rate than pure strains. However, the process of isolating the microbial community is time consuming (Wang et al., 2007, 2010). Schroll et al. (2004) reported that the transfer of contaminated soil with adapted microbial population into an agricultural soil significantly enhanced the mineralization of 1,2,4-TCB in the agricultural soil, indicating that the transferred microbial population in soil inocula survived and maintained its degradation ability in the new microbial ecosystem. However, using soil inocula for bioaugmentation poses the problem of introducing further contamination to the soil under remediation. Report on the potential of different inocula, from sites with low or high concentration of pollutants, in
soil remediation is lacking (D´ıaz, 2004; Singh, 2008; Cao et al., 2009). The effect of pollution history on the effectiveness of the inocula is also rarely reported (Aelion et al., 1987). This work sought to fill in these mising gaps. Three soils with low and high contamination levels and different pollution histories were selected for inoculation into 1,2,4-TCB-spiked soils. The aim of the study was to test the bioaugmentation potential of the adapted microbes in the different soil inocula. The hypothesis was that soil inocula with longer contamination history and higher concentration would have higher degradation efficiency. If found to be environmentally sustainable, using soil inocula would offer an efficient, cost-effective and easy method for introducing adapted microbes to polluted sites. MATERIALS AND METHODS Chemicals Standards of monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3DCB), 1,4-dichlorobenzene (1,4-DCB) and 1,2,4-TCB with purity > 99.5% were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Solvents and other chemicals (Nanjing Chemical Factory, Nanjing, China) were of analytical grade. Anhydrous sodium sulfate was oven-dried at 400 ◦ C for 4 h. Soil preparation Four different soil samples, AS, CS1, CS2 and CS3, were used in this study (Table I). Soil AS was uncontaminated while soils CS1, CS2 and CS3 were contaminated with CBs. Soil AS was used for incubation and the other three contaminated soils were used as inocula. Soil AS was collected from an agricultural field in
TABLE I Physico-chemical properties of soils and the concentrations of chlorobenzenes (CBs) Soila) AS CS1 CS2 CS3 a)
pH
OMb)
6.97 8.00 7.47 8.06
% 3.19 2.13 1.45 3.50
C:N 9.30 13.89 12.76 17.63
Clay
Silt
31.01 9.36 25.68 2.37
% 57.54 52.12 58.87 13.29
Sand 11.45 38.52 15.45 84.34
1,2-DCBc) e)
ND 0.03 0.38 3.54
1,3-DCB ND 0.02 0.11 1.50
1,4-DCB μg g−1 ND 0.04 0.35 2.74
Soil AS was uncontaminated, while soils CS1, CS2 and CS3 were contaminated with CBs. Organic matter. c) DCB = dichlorobenzene. d) TCB = trichlorobenzene. e) Not detected. b)
1,2,4-TCBd) 0.004 0.15 0.81 22.77
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE
Baguazhou, Nanjing, China (32◦ 12 4.6 N, 118◦ 50 112.3 E). Soil CS1 was collected from arable land near Yangnong Chemical Factory, Yangzhou, China (32◦ 21 57.5 N, 119◦ 25 14.8 E) and had been contaminated for about 15 years due to dry-wet deposition. Soils CS2 and CS3 were collected in the garden of Haichen Chemical Factory, Jiangdu, China (32◦ 34 14.5 N, 119◦ 44 34.8 E) and had been contaminated for about 40 years due to the dumping of waste materials and dry-wet deposition. All the soils were sampled from the depth of 0–20 cm, sieved through a 2-mm mesh and stored at 4 ◦ C. Prior to the start of the experiments, the soil samples were adjusted to a water content of 280 mg g−1 by oven-dry weight and equilibrated for one week at 30 ± 1 ◦ C in dark. The concentrations of CBs in the four soils are listed in Table I. Biodegradation of 1,2,4-TCB in soil 400 μg of 1,2,4-TCB dissolved in 400 μL acetone was applied to an aliquot of 10 g (dry weight) of soil AS in a 50-mL glass beaker. After evaporation of acetone, the soil aliquot was mixed and transferred into a glass beaker which already contained 180 g (dry weight) of equilibrated soil AS and 10 g (dry weight) of soil inocula. For the non-inoculated treatment, the spiked 10 g of soil aliquot was mixed with 190 g of soil AS. The 200 g of soil was mixed by stirring carefully and thoroughly with a spatula, transferred to a 1 000-mL incubation flask, and adjusted to −15 kPa of soil water potential. An aliquot of 5 g of soil was sampled with a stainless steel soil borer to determine the initial concentration of 1,2,4-TCB in soil. The remaining soil was compacted to a volume equivalent to 1.3 g cm−3 of soil density (Schroll et al., 2006). The flask was closed tightly with a rubber plug and incubated at 30 ± 1 ◦ C for 56 days in the dark. Using a closed laboratory trapping system (Schroll et al., 2004), the flasks were aerated once per week with humidified CO2 -free air for 20 min at an exchange rate of 0.4 L min−1 to flush out the produced CO2 and the volatile compounds from the incubation flasks to the traps filled with 10 mL 1 mol L−1 NaOH and 15 mL n-hexane, respectively. After aeration, an aliquot of 5 g of soil was sampled for 1,2,4-TCB and its possible dechlorinated products analysis. There were a total of 4 treatments: AS + 1,2,4-TCB (NIS), AS + 1,2,4-TCB + inocula CS1 (ICS1), AS + 1,2,4-TCB + inocula CS2 (ICS2), and AS + 1,2,4-TCB + inocula CS3 (ICS3). An additional set containing 200 g of soil AS only (blank) was used as the control for soil respiration. All treatments were conducted in triplicate.
425
Biodegradation of 1,2,4-TCB in suspension cultures To further confirm the biodegradation potential of the three contaminated soils CS1, CS2 and CS3, a parallel experiment was conducted with soil suspension cultures. 300-mL sealed incubation flasks (Schroll et al., 2004, modified) containing 100 mL sterilized mineral medium (Wang et al., 2007) were separately inoculated with 5 g (oven-dry weight) of CB-contaminated soils CS1, CS2 or CS3, respectively. 2 000 μg 1,2,4TCB dissolved in 50 μL acetone was spiked into all flasks. Filters (0.2 μm, Sartorius Midisart-2000, Germany) were installed at both the air inlet and outlet tubes of the glass flasks. Flasks were incubated on an orbital shaker at 150 r min−1 and 30 ± 1 ◦ C in the dark and aerated twice per week by the same trapping system mentioned above. After aeration, 0.5 and 1.0 mL soil suspension were sampled for analysis of 1,2,4-TCB plus its possible dechlorination products and chloride release, respectively. 100 μL of suspension were removed for determination of cell forming units. In order to keep the TCB concentration in the suspension culture nearly constant, 2 000 μg 1,2,4-TCB was reapplied as above after each aeration up to day 28, after which there was no further application. An additional set containing only mineral medium and 1,2,4-TCB (blank) was incubated as above and used as control for measurement of chloride release. All treatments were conducted in triplicate. Analytical methods Residues of CBs in soils were extracted with hexane/acetone (4:1, v/v) at a temperature of 100 ◦ C and a pressure of 10 MPa by accelerated solvent extraction (ASE 200, Dionex, USA). The concentrated extract was purified on a silica gel/anhydrous sodium sulfate column with 15 mL elution of hexane/dichloromethane (9:1, v/v). The eluate was concentrated to 1 mL for gas chromatograph (GC) analysis. During the experimental period the volatile fraction of CBs was trapped with hexane, dried with anhydrous sodium sulfate and concentrated to 1 mL for subsequent GC analysis. The concentrations of CBs were measured by a gas chromatograph (Agilent 6890, USA) equipped with a DB-5 capillary column (30 m length × 0.32 mm inside diameter × 0.25 μm film thickness), a 63 Ni electron capture detector and an HP 7683 auto-sampler. Nitrogen (purity > 99.999%) was used as the carrier gas at a flow rate of 1.5 mL min−1 . The column tempera-
426
ture was programmed from 60 ◦ C (1 min) to 140 ◦ C at a rate of 20 ◦ C min−1 and then to 280 ◦ C (5 min) at a rate of 8 ◦ C min−1 . The injector and detector temperatures were 220 and 300 ◦ C, respectively. The injection volume was 1 μL in the splitless mode. To quantify the respiration of the soils and the soil suspensions, CO2 absorbed by NaOH from the soil experiments and the soil suspension cultures were transferred from the traps into triangular flasks, and then titrated with 1 mol L−1 HCl (Haney et al., 2008). Inorganic chloride was used as an indicator of 1,2,4-TCB degradation. The concentration of inorganic chloride in the soil suspension cultures was detected by HNO3 -AgNO3 method (Bidlan and Manonmani, 2002). Cell counting in soil suspension cultures was performed by spreading serial dilutions of the liquid culture on Nutrient Broth (Shanghai Science Biotech Company, China) agar plates, and incubating at 30 ◦ C. Colony forming units (cfu) were determined after 48 h incubation.
Y. SONG et al.
treatments (Fig. 1a, Table II). The dissipation of 1,2,4TCB in soils was followed for a period of 56 days (Fig. 1). The possible dechlorination products of 1,2,4TCB (MCB, 1,2-DCB, 1,3-DCB and 1,4-DCB) were screened for by GC, but were not detected in any treatment except for ICS3, in which 1,2-DCB, 1,3-DCB and 1,4-DCB were present at the start of the experiment (Table I). 1,2,4-TCB concentrations decreased rapidly in all the treatments in the first 14 days (Fig. 1). The degradation curve of treatment NIS leveled off in 14 days at 36.52% of the initial concentration, after which no further significant degradation could be observed (P > 0.05). However, the inoculated treatments decreased up to day 28 (P < 0.05). After 56 days, a much higher amount of 1,2,4-TCB was eliminated from treatment ICS3 than in the other two inoculated treatments (Table II), indicating that ICS3 was more effective in degradation. The dissipation of 1,2,4-TCB fitted into
Quality control and data analysis To estimate the recoveries of CBs residues in soil and trapped CBs by hexane, a recovery study was carried out by spiking a mixture of CBs (each 20 ng) to 10 g of soil AS or 15 mL of hexane in the trap. The extraction and purificiation of the samples were performed using the same procedure mentioned above and recoveries were 60.76%–91.56% in soil extraction and 72.63%–99.94% in trapping. The degradation of 1,2,4-TCB in the soil was evaluated by fitting the data with modified first-order kinetics equation: Ct = C0 [λ + (1 − λ)e−kt ] where C0 and Ct are the concentrations of 1,2,4-TCB in soil at start time (0) and time t, respectively; λ is the coefficient of non-available fraction of 1,2,4-TCB in soil; and k is the first-order rate constant for degradation. All statistical data analysis was done with Statistical Product and Service Solutions (SPSS 13.0) and the significance level was P < 0.05. RESULTS Residues of 1,2,4-TCB in soils The initial concentration of 1,2,4-TCB in treatment ICS3 was nearly double that of the other three
Fig. 1 (a) Degradation kinetics (dots: measured data; curves: modified first-order kinetics model fitted) and (b) percentage residues of 1,2,4-trichlorobenzene (1,2,4-TCB) in non-inoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3). Error bars represent the standard deviations of the means (n = 3).
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE
427
TABLE II Initial and eliminated amounts of 1,2,4-trichlorobenzene (1,2,4-TCB) in non-inoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3) and the regression equations of degradation fitted into the modified first-order kinetics model Treatment
NIS ICS1 ICS2 ICS3
Initial amount
Eliminated amount
Freshly spiked
Aged in inocula
193.80bb) 243.95a 249.90a 230.34a
μg 0.00c 1.46b 8.10b 227.66a
Modified first-order kinetics modela) Regression equation
123.01c 198.02b 207.98b 297.51a
Ct Ct Ct Ct
= 0.98(0.37 + 0.63e−0.122t ) = 1.28(0.15 + 0.85e−0.080t ) = 1.30(0.19 + 0.81e−0.133t ) = 2.34(0.32 + 0.68e−0.063t )
R2
t1/2
0.98 0.96 0.99 0.97
d 12.83b 10.97b 7.20c 21.24a
Ct = concentration of 1,2,4-TCB in soil (μg g−1 ) at time t; t1/2 = half-life time (d). Means in a column followed by the same letter are not significantly different at P < 0.05 by Fisher’s protected least significant difference. a)
b)
the modified first-order kinetics model (Fig. 1a), with the longest and the shortest half-lives observed in treatments ICS3 and ICS2, respectively (Table II). Estimation of biodegradation of aged 1,2,4-TCB in soil CS3 The high concentration of 1,2,4-TCB in soil CS3 (Table I) resulted in a nearly similar amount of aged and freshly spiked 1,2,4-TCB in treatment ICS3 at the beginning (Table II). Therefore, it was necessary to estimate the biodegradation of aged 1,2,4-TCB in treatment ICS3. This could be done based on the degradation results of treatment ICS2 since soil inocula CS2 and CS3 were sampled from the same site and had the same pollution history, but the aged 1,2,4-TCB residues in soil CS2 were negligible compared to soil CS3. In treatment ICS2, due to the negligible amount of aged 1,2,4-TCB introduced by soil CS2, 1,2,4-TCB residues (19.38%) at the end of the experiment (Fig. 1b) should have come from the freshly spiked 1,2,4-TCB. Since soil inocula CS3 was more effective than CS2, freshly spiked 1,2,4-TCB in treatment ICS3 remaining at the end of the experiment should not have been higher than 19.38% (44.69 μg). However, 1,2,4-TCB remaining in treatment ICS3 at the end of the experiment was 160.49 μg. Therefore, 115.80 μg of 1,2,4-TCB remaining in treatment ICS3 should have come from the 10 g of inocula CS3, corresponding to 50.85% of the introduced 1,2,4-TCB. Thus, 49.14% of aged 1,2,4-TCB residues in soil CS3 were degraded. Production of CO2 As shown in Fig. 2a, CO2 production in the inocu-
Fig. 2 Time course of cumulative CO2 production in noninoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3) (a) and in suspension cultures (b). Blank was non-spiked, non-inoculated agricultural soil. 1,2,4-trichlorobenzene (1,2,4-TCB) was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
428
lated soils was much higher than that in treatment NIS (P < 0.05). Compared with the blank, a significant increase of CO2 production was observed in treatment NIS (P < 0.05), indicating that spiking of 1,2,4-TCB stimulated soil respiration. As shown in Fig. 2b, CO2 production in the suspension cultures showed a nearly linear increase with time in the first 28 days, indicating that microbial activity was stimulated by the mineral nutrients and continuous addition of 1,2,4-TCB. When no further 1,2,4-TCB was added after 28 days, CO2 production decreased.
Y. SONG et al.
release dynamics were observed in the different inoculated treatments. A lag phase of ten days was observed followed by a continuous Cl− release in treatment ICS1. In treatments ICS2 and ICS3, there was no lag phase and chloride release showed a rapid, slow and rapid increase over the experimental period. Among the inoculated treatments, ICS3 had the highest chloride release (P < 0.05). Although no further 1,2,4TCB was added to the flasks after day 24, the chloride concentration continued increasing with time in both treatments ICS1 and ICS3 (Fig. 4).
Microbial growth in suspension cultures Bacterial cell numbers in the three soil suspension cultures increased rapidly until day 17 (Fig. 3). The population in treatment ICS2, however, decreased rapidly after day 17. Treatment ICS2 was therefore stopped on day 24 because the experiment was meant to monitor the increase in microbial population. Treatment ICS3 showed a significantly higher amount of microorganisms than treatment ICS1 over the whole incubation period (P < 0.05), though they both exhibited similar growth trends. Fig. 4 Time course of chloride (Cl− ) release from spiked mineral medium (blank) and from suspension cultures inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3). 1,2,4-trichlorobenzene (1,2,4-TCB) was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
Volatilization of 1,2,4-TCB from soils and suspension cultures
Fig. 3 Time course of cell density in suspension cultures inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3). 1,2,4-trichlorobenzene (1,2,4-TCB) was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
Chloride release in suspension cultures To evaluate the degradation of 1,2,4-TCB in soil suspension cultures, chloride release was determined over the incubation period. Generally, the release of chloride increased in all the treatments over the whole incubation period (Fig. 4). However, different chloride
A rapid volatilization of 1,2,4-TCB from the soils was observed in the first week in all the treatments (Fig. 5a). Treatment NIS showed the highest volatilization at each sampling point and thus led to the highest volatilization losses. In contrast, the lowest volatilization was detected in treatment ICS3. Volatilization of treatments ICS1 and ICS2 ranged in between. The volatilization of 1,2,4-TCB from the suspension culture in treatment ICS3 was also much lower than that in treatments ICS1 and ICS2 (P < 0.05) (Fig. 5b). It was observed that higher degradation resulted in lower volatilization in the soil and soil suspension cultures. DISCUSSION Biodegradation of long-term aged contaminants in
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE
Fig. 5 Time course of cumulative 1,2,4-trichlorobenzene (1,2,4-TCB) volatilization from non-inoculated agricultural soil (NIS) and agricultural soils inoculated with contaminated soils CS1 (ICS1), CS2 (ICS2) or CS3 (ICS3) (a) and that in suspension cultures (b). 1,2,4-TCB was spiked in the suspension cultures after each sampling before day 24 while no more 1,2,4-TCB was spiked after day 24. Error bars represent the standard deviations of the means (n = 3).
soil is a great challenge (Mancera-L´opez et al., 2008). Inoculation with contaminated soils significantly enhanced the degradation of 1,2,4-TCB in the agricultural soil (Schroll et al., 2004), which showed that adapted microbes in soil inocula enhanced the degradation of freshly-spiked 1,2,4-TCB. However, in this study, soil inocula resulted in the enhanced degradation of both freshly spiked and aged 1,2,4-TCB residues. The increased degradation, respiration and chloride emission all pointed to the presence of adapted microbes in soil inocula of CS1, CS2 and CS3. The enhanced degradation of aged 1,2,4-TCB might be due to the increased nutrients, supplied by the agricultural soil, which stimulated the microorganism in the soil inocula. This indicated that bioaugmentation with soil inocula is an applicable and effective strategy for enhancing the breakdown of aged contaminants in soil.
429
The higher degradation and dechlorination of 1,2,4TCB corresponded to lower volatilization (Figs. 4 and 5), indicating that enhanced degradation of 1,2,4-TCB results in reduced volatilization losses (Wang et al., 2007). A possible explanation is that microorganisms acted as a sink for 1,2,4-TCB during the biodegradation process, thereby reducing its volatilization. Therefore, inoculated degraders could effectively degrade the bioavailable 1,2,4-TCB and thereby decrease its environmental risk, even if the concentration of 1,2,4-TCB remains high in the contaminated soil. Dechlorination is an important process during the degradation of chlorinated compounds. Generally in aerobic degradation, in contrast to reductive dechlorination, very few or no chlorine atoms are eliminated by microorganisms before the aromatic ring cleavage (Commandeur and Parsons, 1990; van der Meer et al., 1991; Marco-Urrea et al., 2009). The fact that lower-chlorinated benzenes (MCB, 1,2-DCB, 1,3-DCB and 1,4-DCB) were not detected could mean that ring cleavage preceded dechlorination, as was the case in other studies (van der Meer et al., 1991; Marco-Urrea et al., 2009). The concentration of chloride ions with time was measured to find out whether dechlorination of the aliphatic chain, which was formed after ring cleavage, occurs (Spiess et al., 1995). As can be seen from Fig. 4, chloride concentration increased with time, indicating that dechlorination of the aliphatic chain occurred (Spiess et al., 1995). To study the effect of contamination history on the degradation capacity of the inocula, the results of treatments ICS1 and ICS2, which had been contaminated for 15 and 40 years, respectively, were compared. The similar degradation kinetics and residues of 1,2,4-TCB in the spiked soils ICS1 and ICS2 (Fig. 1) indicated a similar dissipation pattern. This is further supported by the similar respiration and volatilization in the two treatments (Figs. 2 and 5). All these indicated that contamination period may not be a critical factor in determining the activity and efficacy of the soil inocula. This could mean that the time span of 15 years may be long enough for complete microbial adaptation, hence a longer time span could not contribute more to biodegradation capacity (Aelion et al., 1987). To check for the effect of contaminant concentration on the degradation efficacy of the inocula, the results of treatments ICS2 and ICS3, both of which had been contaminated for about 40 years, were compared. Inocula CS3 had a 1,2,4-TCB concentration 28 times
430
higher than CS2 (Table I). The higher cell counts (Fig. 3), higher dechlorination (Fig. 4) and lower volatilization (Fig. 5) in ICS3 indicated a higher microbial activity in CS3 in the soil suspension culture. This was further indicated by the fact that more 1,2,4TCB was removed from the CS3 inoculated soil (Fig. 1, Table II). However, residues of 1,2,4-TCB in the ICS3 were much higher than those in the ICS2, which could be attributed to the different aged residues in the inocula (Table II). Inocula CS2 had a lower contaminant concentration, whereas nearly half of the 1,2,4-TCB in the ICS3 was aged. Of the aged residues, 50.85% were unavailable, thus leading to the higher residues in the ICS3. Therefore, the inocula with lower contamination level might be more environment-friendly in enhancing degradation of 1,2,4-TCB in soil, considering the larger amount of aged residues that would be introduced by using higher-contaminated soils as inocula. CONCLUSIONS Inoculation of soil from previously contaminated sites could enhance the degradation and limit volatilization of 1,2,4-TCB in artificially contaminated soils, thus resulting in reduced environmental risk. Long-term contaminated soils contained the adapted microorganisms able to metabolize 1,2,4-TCB with subsequent chloride release. The concentration of contaminants, rather than contamination period, seems to be the more important factor influencing the activity of soil inocula. Large amounts of aged residues in the contaminated soil inocula were also degraded. Although highly contaminated soil inocula were more effective in degradation, bioaugmentation with lowcontaminated soil inocula is more recommended for insitu bioremediation of 1,2,4-TCB polluted soils. This is because of the environmental cost due to the possible secondary contamination introduced by using highlycontaminated soils as inocula. REFERENCES Adebusoye, S. A., Picardal, F. W., Ilori, M. O., Amund, O. O., Fuqua, C. and Grindle, N. 2007. Aerobic degradation of di- and trichlorobenzenes by two bacteria isolated from polluted tropical soils. Chemosphere. 66: 1939–1946. Aelion, C. M., Swindoll, C. M. and Pfaender, F. K. 1987. Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer. Appl. Environ. Microbiol. 53: 2212–2217. Bidlan, R. and Manonmani, H. K. 2002. Aerobic degrada-
Y. SONG et al. tion of dichlorodiphenyltrichloroethane (DDT) by Serratia marcescens DT-1P. Process Biochem. 38: 49–56. Boyd, E. M., Killham, K., Wright, J., Rumford, S., Hetheridge, M., Cumming, R. and Meharg, A. A. 1997. Toxicity assessment of xenobiotic contaminated groundwater using lux modified Pseudomonas fluorescens. Chemosphere. 35: 1967–1985. Cao, B., Nagarajan, K. and Loh, K. C. 2009. Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches. Appl. Microbiol. Biot. 85: 207–228. Commandeur, L. C. M. and Parsons, J. R. 1990. Degradation of halogenated aromatic compounds. Biodegradation. 1: 207–220. D´ıaz, E. 2004. Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility. Int. Microbiol. 7: 173–180. Freitag, D., Ballhorn, L., Geyer, H. and Korte, F. 1985. Environmental hazard profile of organic chemicals: An experimental method for the assessment of the behaviour of organic chemicals in the ecosphere by means of simple laboratory tests with 14 C labelled chemicals. Chemosphere. 14: 1589–1616. Gaffney, P. E. 1976. Carpet and rug industry case-study II: biological effects. J. Water Pollut. Con. F. 48: 2731–2737. Hamdi, H., Benzarti, S., Manusadˇzianas, L., Aoyama, I. and Jedidi, N. 2007. Bioaugmentation and biostimulation effects on PAH dissipation and soil ecotoxicity under controlled conditions. Soil Biol. Biochem. 39: 1926–1935. Haney, R. L., Brinton, W. F. and Evans, E. 2008. Soil CO2 respiration: Comparison of chemical titration, CO2 IRGA analysis and the Solvita gel system. Renew. Agr. Food. Syst. 23: 171–176. Mancera-L´ opez, M., Esparza-Garc´ıa, F., Ch´ avez-G´ omez, B., Rodr´ıguez-V´ azquez, R., Saucedo-Casta˜ neda, G. and Barrera-Cort´es, J. 2008. Bioremediation of an aged hydrocarbon-contaminated soil by a combined system of biostimulation-bioaugmentation with filamentous fungi. Int. Biodeter. Biodegr. 61: 151–160. Marco-Urrea, E., Perez-Trujillo, M., Caminal, G. and Vicent, T. 2009. Dechlorination of 1,2,3- and 1,2,4trichlorobenzene by the white-rot fungus Trametes versicolor. J. Hazard. Mater. 166: 1141–1147. Monferr´ an, M. V., Echenique, J. R. and Wunderlin, D. A. 2005. Degradation of chlorobenzenes by a strain of Acidovorax avenae isolated from a polluted aquifer. Chemosphere. 61: 98–106. Odokuma, L. O. and Dickson, A. A. 2003. Bioremediation of a crude oil polluted tropical mangrove environment. J. Appl. Sci. Environ. Manag. 7: 23–29. Oliver, B. G. and Nicol, K. D. 1982. Chlorobenzenes in sediments, water, and selected fish from Lakes Superior, Huron, Erie, and Ontario. Environ. Sci. Technol. 16: 532–536.
BIODEGRADATION OF 1,2,4-TRICHLOROBENZENE Rapp, P. 2001. Multiphasic kinetics of transformation of 1,2,4-trichlorobenzene at nano- and micromolar concentrations by Burkholderia sp. strain PS14. Appl. Environ. Microbiol. 67: 3496–3500. Rapp, P. and Timmis, K. N. 1999. Degradation of chlorobenzenes at nanomolar concentrations by Burkholderia sp. strain PS14 in liquid cultures and in soil. Appl. Environ. Microbiol. 65: 2547–2552. Sander, P., Wittich, R. M., Fortnagel, P., Wilkes, H. and Francke, W. 1991. Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Appl. Environ. Microbiol. 57: 1430–1440. Schroll, R., Becher, H. H., D¨ orfler, U., Gayler, S., Grundmann, S., Hartmann, H. P. and Ruoss, J. 2006. Quantifying the effect of soil moisture on the aerobic microbial mineralization of selected pesticides in different soils. Environ. Sci. Technol. 40: 3305–3312. Schroll, R., Brahushi, F., D¨ orfler, U., Kuhn, S., Fekete, J. and Munch, J. C. 2004. Biomineralisation of 1,2,4trichlorobenzene in soils by an adapted microbial population. Environ. Pollut. 127: 395–401. Singh, D. K. 2008. Biodegradation and bioremediation of pesticide in soil: concept, method and recent developments. Indian J. Microbiol. 48: 35–40. Spiess, E., Sommer, C. and Gorisch, H. 1995. Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1. Appl. Environ. Microbiol. 61: 3884–3888. Tchelet, R., Meckenstock, R., Steinle, P. and van der Meer, J. R. 1999. Population dynamics of an introduced bacterium degrading chlorinated benzenes in a soil column and in sewage sludge. Biodegradation. 10: 113–125.
431 van der Meer, J. R., Roelofsen, W., Schraa, G. and Zehnder, A. J. B. 1987. Degradation of low concentrations of dichlorobenzenes and 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 in nonsterile soil columns. FEMS Microbiol. Lett. 45: 333–341. van der Meer, J. R., Van Neerven, A. R. W., De Vries, E. J., De Vos, W. M. and Zehnder, A. J. B. 1991. Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro- and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51. J. Bacteriol. 173: 6–15. Wang, F., D¨ ofler, U., Schmid, M., Fischer, D., Kinzel, L., Scherb, H., Munch, J. C., Jiang, X. and Schroll, R. 2010. Homogeneous inoculation vs. microbial hot spots of isolated strain and microbial community: What is the most promising approach in remediating 1,2,4-TCB contaminated soils? Soil Biol. Biochem. 42: 331–336. Wang, F., Grundmann, S., Schmid, M., D¨ ofler, U., Roherer, S., Charles, M. J., Hartmann, A., Jiang, X. and Schroll, R. 2007. Isolation and characterization of 1,2,4-trichlorobenzene mineralizing Bordetella sp. and its bioremediation potential in soil. Chemosphere. 67: 896–902. Zhang, J. Y., Zhao, W., Pan, J., Qiu, L. M. and Zhu, Y. M. 2005. Tissue-dependent distribution and accumulation of chlorobenzenes by vegetables in urban area. Environ. Int. 31: 855–860. Zolezzi, M., Cattaneo, C. and Tarazona, J. V. 2005. Probabilistic ecological risk assessment of 1,2,4-trichlorobenzene at a former industrial contaminated site. Environ. Sci. Technol. 39: 2920–2926.