Mineralization of chlorpyrifos by co-culture of Serratia ... - Springer Link

Biotechnol Lett (2007) 29:1469–1473 DOI 10.1007/s10529-007-9444-0

ORIGINAL RESEARCH PAPER

Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp. Gangming Xu Æ Yingying Li Æ Wei Zheng Æ Xiang Peng Æ Wen Li Æ Yanchun Yan

Received: 27 April 2007 / Revised: 31 May 2007 / Accepted: 31 May 2007 / Published online: 4 July 2007 Springer Science+Business Media B.V. 2007

Abstract A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l 1 within 7 days. Cocultures completely mineralized 50 mg chlorpyrifos l 1 within 18 h at 308C and pH 8 using a total inocula of 0.15 g biomass l 1. Keywords Chlorpyrifos Mineralization Serratia sp. Trichosporon sp. 3,5,6-Trichloro-2-pyridinol

Introduction Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is one of the most widely

G. Xu Y. Li W. Zheng X. Peng W. Li State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, P.R. China G. Xu Y. Li W. Zheng X. Peng W. Li College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, P.R. China Y. Yan (&) Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China e-mail: [email protected]

used organophosphate insecticides. It is effective against a broad spectrum of agricultural and household pests. It has moderate toxicity, low water solubility and high soil sorption (Singh and Walker 2006). Excessive use of chlorpyrifos has contaminated several ecosystems (Environmental Protection Agency 1995). Its half-life in soil varies from 10 to 120 days, with 3,5,6-trichloro-2-pyridinol (TCP) as the major degradation product. TCP is moderately mobile due to its greater water solubility, leading to widespread contamination of soils, sediments and water (Li et al. 2007). The accumulation of TCP, which has anti-microbial properties, prevents the proliferation of chlorpyrifos-degrading microbes, so normally enhanced degradation does not occur (Singh and Walker 2006). A few chlorpyrifos-degrading bacteria, including Enterobacter strain B-14, Stenotrophomonas sp. YC-1, and Sphingomonas sp. Dsp-2, have been described (Singh et al. 2004; Yang et al. 2006; Li et al. 2007). Several chlorpyrifos-degrading fungi, such as Phanerochaete chrysosporium, Aspergillus terreus, and Verticillium sp. DSP have also been reported (Bumpus et al. 1993; Omar 1998; Yu et al. 2006). Unfortunately, bacterial degradation was only partial and TCP accumulated in the medium without further metabolism. Only one TCP-mineralizing bacterium (a Pseudomonas sp.) has been described so far (Feng et al. 1997), a little information is available on the microbial metabolism of TCP. A chlorpyrifos- and TCP-degrading Alcaligenes faecalis strain was also

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reported (Yang et al. 2005) but was not efficient enough for chlorpyrifos removal. A co-culture of a bacterium and a fungus that is capable of mineralizing chlorpyrifos has been tested in this study. A Serratia sp. (isolate TCR) that can transform chlorpyrifos to TCP, and a Trichosporon sp. (isolate TCF) that is capable of mineralizing TCP as well as chlorpyrifos were isolated, characterized, and co-cultivated in liquid medium. The optimal conditions for efficient chlorpyrifos degradation by this co-culture were also investigated.

Materials and methods Reagents Chlorpyrifos (>98% purity) was obtained from Shandong Tiancheng Pesticides Co., Ltd, China. 3,5,6-trichloro-2-pyridinol standard (93.5% purity) was purchased from Dr. Ehrenstorfer, Augsburg, Germany. All other chemicals used were of analytical grade and commercially available. Isolation and identification of chlorpyrifos and TCP degraders Activated sludge was obtained from Tiancheng Pesticide Co. in Shandong, China. An enrichment culture was prepared using mineral salts medium (Singh et al. 2004) containing 25 mg CP or TCP l 1, and cultured in a continuous reactor system. The culture was maintained at 308C for 2 months with increase of chlorpyrifos and TCP, respectively, from 25 to 400 mg l 1. After the enrichment of the activated sludge, the culture was spread onto a solid minimal medium containing 100 mg chlorpyrifos l 1 or TCP, and incubated at 308C. Several apparently different colonies were selected and sub-cultured several times to isolate pure cultures. The bacterial isolate was identified based on morphological and biochemical characteristics and by analysis of the 16S rRNA gene (Holt et al. 1994). At the same time, the fungal strain was identified by its colony morphology on PDA plates and by analysis of its 18S rRNA gene (Yu et al. 2006). The rRNA partial sequence of strain TCR and TCF were deposited in the GenBank database under accession numbers EF070125 and EF091819, respectively.

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Biotechnol Lett (2007) 29:1469–1473

Microbial growth and biodegradation assays of two isolates and their co-culture Strains were pre-cultivated (at 308C, 200 rpm, in the dark) in nutrient broth medium containing chlorpyrifos or TCP at 50 mg l 1. Microbial growth was determined by the dry weight biomass (g l 1). After full growth, the culture was harvested and inoculated into minimal medium with 50 mg chlorpyrifos l 1 or TCP as the sole carbon source. The biodegradation assays for each pure isolate and their co-culture were conducted at 308C and pH 7 in the dark. The coculture of strain TCR and TCF were used to determine the optimal incubation conditions. A series of experiments were carried out with different degradation time (0*7 day), initial chlorpyrifos concentration (25*400 mg l 1), total inocula biomass amount (0.05*0.25 g l 1) each in half, incubation temperature (15*358C), pH (5*9) and additional carbon source (glucose or sucrose, 0*20 g l 1). Cultures without inoculation were used as abiotic controls. All the experiments were performed in triplicate. Chromatographic analysis For the analysis of residual chlorpyrifos and its metabolites, whole cultures were harvested at different time intervals and cell-free liquors analyzed by GC with a flame photometric detector and by GC-MS (with a DB-5-MS capillary column) as described in details previously (Yu et al. 2006). TCP was analyzed by HPLC (using a Zorbax SB-C18 column) with detection at 230 nm (Feng et al. 1997) and with array detection from 200 to 600 nm to identify its possible metabolites. CO2 concentration in gas samples withdrawn from the flask headspace was analyzed by GC equipped with thermal conductivity detector (Soares et al. 2005).

Results and discussion Isolation and identification of chlorpyrifos and TCP degrading microorganisms Several chlorpyrifos-degrading bacteria and one TCP-mineralizing fungus were isolated by enrichment culture technique from the activated sludge. The


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bacterial strain (coded TCR) was Gram-negative, aerobic, catalase and oxidase positive. It was a motile rod (0.6*0.8 mm by 1.0*2.0 mm) that formed occasional filaments. Its colony was lustrous and red. Sequence analysis its 16S rRNA gene showed that strain TCR was most closely related to Serratia marcescens. The structure and shape of the fungal colonies indicated that strain TCF was related to Trichosporon species. It grew rapidly on PDA plates and yielded irregularly folded or verrucosed colonies. The partial sequence of its 18S rRNA gene placed the isolate within the genus Trichosporon. Several strains from Serratia sp. have previously been reported to degrade methyl parathion and synthetic pyrethroid (Grant and Betts 2004; Pakala et al. 2007). A few Trichosporon spp. were also obtained to degrade chlorinated biphenyl and phenol (Boszczyk et al. 2002; Sietmann et al. 2006). However, no chlorpyrifos or TCP degrading microorganism has been reported from these two genera. Growth and degrading ability of Serratia sp. TCR and Trichosporon sp. TCF Degradation patterns of strain TCR and TCF were studied in the liquid culture medium with chlorpyrifos or TCP as the sole carbon source. The time course of chlorpyrifos metabolism by Serratia sp. TCR is shown in Fig. 1. Complete disappearance of 50 mg chlorpyrifos l 1 was observed within 4 days. TCP (retention time 3.7 min by HPLC) was detected as the only major metabolites of chlorpyrifos degradation,

Fig. 2 Fungal growth and mineralization of TCP (50 mg l 1) by Trichosporon sp. TCF cultivated with mineral salts medium. Fungal growth (•), TCP inoculated (m) and TCP control (D) in the culture medium. Values are means ± standard deviations of three replicates

and it accumulated in the medium without further metabolism. Strain TCF grew rapidly when TCP was provided as the sole carbon source and 50 mg TCP l 1 could be degraded within 5 days (Fig. 2a). No intermediate was detected in the medium. The CO2 levels increased with the decrease of TCP, showing the capability of mineralizing TCP. Strain TCF could also degrade chlorpyrifos but to a lesser extent; 50 mg l 1 was degraded within 7 days (Fig. 2b). Transient accumulation of TCP was detected, but its concentration decreased rapidly and disappeared finally. The growths of two individual isolates, TCR and TCF, were slightly inhibited as shown in Figs. 1 and 2. Mineralization of chlorpyrifos by co-culture of isolates TCR and TCF

Fig. 1 Bacterial growth and degradation of chlorpyrifos (50 mg l 1) by Serratia sp. TCR cultivated in mineral salts medium. Bacterial growth (•), chlorpyrifos inoculated (j), chlorpyrifos control (h), and TCP (m) in the culture medium. Values are means ± standard deviations of three replicates

No growth inhibition was observed in the co-culture of strain TCR and TCF, complete biodegradation of 50 mg chlorpyrifos l 1 was achieved by the

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which is resistant to microbial attack (Singh and Walker 2006). To our knowledge, this co-culture has the fastest mineralizing rate among the chlorpyrifosdegrading microbes reported. Optimal conditions for biodegradation of chlorpyrifos by the co-culture

Fig. 3 Microbial growth and mineralization of chlorpyrifos (50 mg l 1) by the co-culture of Serratia sp. TCR and Trichosporon sp. TCF cultivated with mineral salts medium. Microbial growth (•), chlorpyrifos inoculated (j), chlorpyrifos control (h), and TCP (m) in the culture medium. Values are means ± standard deviations of three replicates

co-culture within 18 h (Fig. 3). This was much faster than the rate achieved by pure fungal isolate, which is stimulated by a synergistic interaction with the bacterial strain TCR isolated from the same sludge sample. The clearly lower concentrations of accumulated TCP in the co-culture could be in part responsible for the significantly enhanced biodegradation of chlorpyrifos. The increase of CO2 level in the headspace indicated the mineralization of chlorpyrifos (data not shown). Throughout all studies, the degradation was accompanied by microbial growth as monitored by the dry weight biomass. Under all experimental conditions, chlorpyrifos and TCP removal was never greater than 10% in the abiotic controls. In most cases described to date, the degrading bacteria tend to transform chlorpyrifos by hydrolysis to produce diethylthiophosphoric acid and TCP, which in turn accumulate in the culture medium without further metabolism. Several soil fungi can apparently mineralize chlorpyrifos as well as TCP,

Fig. 4 Effects of inocula biomass amount (a), temperature (b), pH (c) and additional carbon source (d) on biodegradation of chlorpyrifos (50 mg l 1) over 12 h. Total inocula biomass

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Slower degradation rate was observed due to increased toxicity at higher chlorpyrifos concentrations; 100 mg chlorpyrifos l 1 required 24 h for complete mineralization. As shown in Fig. 4a, b and c, the optimal conditions for chlorpyrifos degradation by the co-culture were determined to be 308C, pH 8 and total inocula biomass of 0.15 g dry wt l 1 for an assay time of 12 h in the medium containing 50 mg chlorpyrifos l 1. Figure 4d shows that biodegradation of chlorpyrifos was greatly enhanced by the addition of sucrose. Biodegradation occurring in an environment exposed to the chemical pollutant is a complex process in which many different metabolically active microbial communities take part. Several co-cultivated or mixed cultures have been reported for efficient mineralization of resistant or complex contaminants (Dejonghe et al. 2003; Bazot et al. 2007). The interaction between different microbial species on biodegradation of chlorpyrifos is an important consideration in developing bioremediation strategies for chlorpyrifos removal. In this study, we isolated a chlorpyrifos-degrading bacterial strain TCR, identified as Serratia sp.; and an effective TCP-mineralizing fungal strain TCF, identified as Trichosporon sp., this co-culture could rapidly mineralize chlorpyrifos. Degradation was possible under a broad range of temperatures and pH values, showing that microbial remediation should be possible under many environmental conditions. To clarify the mechanism of

amount (0.05*0.25 g l 1), temperature (15*358C), pH (5*9) and sucrose (0*20 g l 1). Values are means ± standard deviations of three replicates


chlorpyrifos and TCP degradation, purification and characterization of the genes and enzymes involved in this process is underway in our lab. More studies on the interaction between different microorganisms and effects of different environmental factors on biodegradation are essential. Acknowledgments This study was sponsored by the Hitech Research and Development Program of China (No.2006AA10Z401). We are indebted to Min Xia, Xinxin Wang, and Xiaoman Zhang (Beijing Center for Physical and Chemical Analysis, China) for providing facilities and helping with GC and HPLC analysis.

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