Min ZHANG, Xiangwei WU, Fuhai ZHANG*

1. Anhui Environmental Monitoring Center, Hefei 230061, China

2. College of Resources and Environment, Anhui Agricultural University, Hefei 230036, China

Phthalate esters, also known as phthalic acid esters, abbreviated as PAEs,, are a major class of important environmental hormone pollutants that are commonly used as plasticizers. At present, phthalate esters have been called as the"second global PCB pollutant" and are distributed widely in the world. In China,the biodegradation of phthalate esters has been investigated since 1986 when Cheng[1]isolated two din-butyl phthalate-degrading strains from the soil. Subsequently, two PAEdegrading strains were isolated from coke sludge[2-4]. In 2005, Qin et al.[5]isolated a high-efficiency bis-(2-ethylhexyl) phthalate (DEHP)-degrading bacterium from the activated sludge of a chemical plant and identified the bacterium as a Cellulomonas species.In 2005, Li et al.[6]isolated a dibutyl phthalate (DBP)-degrading bacterium from the soil samples in a landfill and identified the bacterium as Rhodococcus ruber. However, current studies are mainly focused on screening high-efficiency phthalate-degrading strains from activated sludge and sediments of nonbiodegradable petrochemical wastewater that generally biodegrade single species of phthalates. At present, in biological treatment of phthalate pollutants,high-efficiency obligate or facultative degrading bacteria should be screened to improve the biodegradation efficiency. In this study, a highefficiency PAE-degrading bacterium was isolated from the sediments of Chaohu Lake. Furthermore, the isolated high-efficiency bacterium was identified, and the biodegradation rate of different concentrates of mixed PAE systems by the high-efficiency bacterium was investigated, aiming at providing a theoretical basis for the treatment of PAEs contamination by high-efficiency degrading bacteria.

Materials and Methods

Materials

The shallow sediments from Chaohu Lake, activated sludge from Huposhanzhuang Sewage Treatment Plant in Hefei City, and sewage outlet sediments from Guangming Industrial Area in Hefei City were collected as experimental materials.

Preparation of medium[7-9]

(1) Basal medium (inorganic salt medium). MgSO4·7H2O 0.2 g, FeCl30.01 g,KH2PO41.0 g,(NH4)2SO41.0 g,CaCl20.1 g, double distilled water 1 L,pH 7.0.

(2) Enrichment medium. NaCl 5.0 g, peptone 10.0 g, beef extract 4.0 g,distilled water 1 000 ml,pH 7.0.

(3) Isolation medium. NaCl 5.0 g,peptone 10.0 g, beef extract 4.0 g,distilled water 1000 ml, pH 7.0; agar 18.0 g.

Domestication, isolation and identification of PAE-degrading bacteria

In accordance with enrichment culture techniques,10 g of sludge was collected,transferred to a 250 ml flask,added with 100 ml of 50 μg/ml PAE broth, incubated in a 30 ℃water bath with shaking at 120 r/min for 7 d (30 min × 3 times/d), and inoculated to fresh medium with an inoculum concentration of 10%; the concentration of PAEs gradually increased to 700 μg/ml. Subsequently, after domestication, the diluted mixed bacterial suspension was isolated and purified repeatedly with 10-fold plate dilution method. After smear microscopy, the purified bacteria were inoculated to slant culture medium and identified by morphological observation and physiological and biochemical tests (physiological and biochemical tests were performed in the Institute of Microbiology,Chinese Academy of Sciences).

Extraction of PAEs from microbial broth

Firstly, 5 ml of petroleum ether was transferred into a test tube containing 3 ml of PAE broth,added with a small amount of anhydrous sodium sulfate, extracted with shaking for 5 min, and placed at room temperature for liquid separation. The upper petroleum ether layer was transferred into a 10 ml volumetric flask. Subsequently, the above extraction procedure was repeated with 5 ml of petroleum ether. The extracts were combined and added with petroleum ether to a final volume of 10 ml for gas chromatography.

Chromatographic conditions

HP6890N gas chromatograph;63Ni electron capture detector (ECD);HP5 column (30 m × 250 μm × 0.25 μm); column temperature raised from 100 ℃to 270 ℃ (retention time: 10 min) by 10 ℃/min; inlet temperature:250 ℃; detector temperature: 300℃; carrier gas: high purity nitrogen(99.999% ); column flow rate: 1.0 ml/min.

Biodegradation of phthalate esters by the high-efficiency degrading bacterium

A certain amount of suspended bacterial liquid was added into different concentrations of PAE broth and incubated at 30 ℃in a shaker at 120 r/min(30 min×3 times/d)in the dark.Samples were collected at different time points for analysis; PAE broth without the addition of bacterial liquid was used as a control.

The biodegradation trends of five phthalate esters by the high-efficiency phthalate ester-degrading bacterium were fitted with first-order kinetic equations:

Where, k indicates the biodegradation rate constant; C0indicates the initial concentration of PAEs; Ctindicates the residual concentration of PAEs at a certain time(t);T1/2indicates the biodegradation half-life.

Results and Analysis

Sources of high-efficiency PAE-degrading bacteria

Microorganisms collected from different sources were domesticated and cultured for 6 d. The remaining amount of PAEs was determined by gas chromatography to compare the degradation effects of different microorganisms. As shown in Table 1,sediments from Chaohu Lake,activated sludge from a sewage treatment plant and sediments from an industrial area exhibited varying degradation effects on PAEs. To be specific, sediments from Chaohu Lake exhibited the best degradation effects on PAEs,which indicated that there were active PAE-degrading bacteria in sediments from Chaohu Lake.

Table 1 Effects of different microorganisms on the biodegradation of PAEs

Determination of the high-efficiency degrading bacterium

After domestication and culture,ten PAE-degrading bacterial strains were isolated from the sediments of Chaohu Lake with 10-fold plate dilution method, which were named , DM1,DM2, DM3, DM4, DM5, DM6, DM7,DM8, DM9, and DM10. Under the same conditions, mixed PAEs (the concentration of each phthalate ester was 20 mg/L) were degraded and incubated for 38 h with shaking before extraction. According to the results of gas chromatography,there were three high-efficiency degrading bacteria in the sediments from Chaohu Lake, including DM1, DM3 and DM5. Specifically, DM1 strain exhibited the best degradation effects.

Identification of the high-efficiency phthalate ester-degrading bacterium

Morphological characteristics Under a microscope,DM1 bacterium was rod-shaped, 0.6-1.0 μm in size, forming round colonies in irregular clusters,which was Gram-negative, capsulefree, non-spore-forming and non-flag-ellum-forming, with wrinkles on the surface.

Physiological and biochemical characteristics According to the results of physiological and biochemical tests, DM1 strain could grow at below 41 ℃; the appropriate growth and metabolism temperature was 25 -35℃,the appropriate growth time was 2-4 d, and the appropriate pH was 6.0-8.0.However, DM1 strain exhibited no growth at pH 4.5.DM1 strain was positive to catalase and oxidase, which could not produce arginine dihydrolase and lecithinase, hydrolyze gelatin and starch,reduce nitrate or grow anaerobically by using acetate, but could grow anaerobically by using nitrate. In addition,DM1 strain could grow by using glucose, xylose, sucrose and tartrate. DM1 strain was heterotrophic,aerobic and positive to oxidase and catalase, which could grow in liquid medium with ammonia as the sole nitrogen source and utilize most of carbohydrates. The physiological and biochemical characteristics of DM1 strain were shown in Table 2.

Based on the morphological,physiological and biochemical characteristics, in accordance with previous literature[10-12]and Bergey’s Manual of Determinative Bacteriology[13], phthalate ester-degrading bacterium DM1 was identified as Burkholderia pickettii, which was finally named B. pickettii.z-1.

Table 2 Physiological and biochemical characteristics of DM1 strain

Biodegradation of five mixed systems of phthalate esters with different initial concentrations by B.pickettii.z-1 strain

Five mixed systems of phthalate esters with initial concentrations of 100 mg/L (the concentration of each phthalate ester was 20 mg/L), 250 mg/L(the concentration of each phthalate ester was 50 mg/L)and 500 mg/L(the concentration of each phthalate ester was 100 mg/L) were biodegraded by B. pickettii.z-1 strain. The results indicated that biodegradation trends of five phthalate esters by B. pickettii.z-1 strain were in accordance with the first-order kinetic equation: Ct=C0·e-kt.Biodegradation kinetic parameters of different concentrations of PAEs by B. pickettii.z-1 strain were shown in Table 3.

As shown in Table 3, as the initial concentration of PAEs increased,the biodegradation rate constants of different concentrations of PAEs by B. pickettii.z-1 strain were reduced,while half-life increased. The initial concentration of PAEs significantly affected the degradation capacity of short-side-chain DMP and DEP but exhibited no significant effects on the degradation capacity of long-sidechain DBP and DEHP. In the biodegradation of several mixed systems of PAEs, B. pickettii.z-1 strainonly exhibited strong degradation effects on a certain concentration of PAEs. As the concentration of PAEs increased the degradation rate of PAEs by B. pickettii.z-1 strain was reduced.

Phthalate esters are a class of compounds with different number of alkyl chains and significantly different chemical properties. As shown in Table 3, B. pickettii.z-1 strains exhibited remarkably different degradation effects on various PAEs.To be specific,short-side-chain DMP and DEP were degraded rapidly, while longside-chain DBP and DEHP were degraded slowly.The biodegradation capacity of PAEs was reduced as the alkyl side chain length of PAEs increased.

Table 3 Biodegradation kinetic parameters of different concentrations of PAEs by B.pickettii.z-1 strain

Discussion

In this study, the degradation rate of 500 mg/L PAEs reached the lowest, but B. pickettii.z-1 strain exhibited good degradation effects on higher concentrations (250 and 500 mg/L) of PAEs, which might be due to the specific domestication. Therefore, in the treatment of wastewater containing high concentrations of PAEs, B. pickettii.z-1 strain could be used as a starting strain to be domesticated in high-concentration wastewater before use.

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