Biodegradation of dimethyl phthalate, diethyl phthalate and di-n-butyl phthalate by Rhodococcus sp. L4 isolated from activated sludge

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Abstract

In this study, an aerobic bacterial strain capable of utilizing dimethyl phthalate (DMP), diethyl phthalate (DEP) and di-n-butyl phthalate (DBP) as sole carbon source and energy was isolated from activated sludge collected from a dyeing plant. According to its morphology, physiochemical characteristics and 16S rDNA sequence, the strain was identified as Rhodococcus ruber. The biodegradation batch tests of DMP, DEP and DBP by the Rhodococcus sp. L4 showed the optimal pH value, temperature and substrate concentration: pH 7.0–8.0, 30–37 °C and PAEs concentration ≤450 mg/L. Kinetics of degradation have also been performed at different initial concentrations. The results show that the degradation can be described with exponential model. The half-life of degradation was about 1.30 days when the concentration of PAEs mixture was lower than 300 mg/L. PAEs contaminated water samples (300 mg/L) with non-emulsification and completed emulsification were prepared to investigate the effect on PAEs degradation rate. Little difference between the above two sample preparations was observed in terms of ultimate degradation rate. Rhodococcus sp. L4 can also grow on phenol, sodium benzoate or naphthalene solution as sole carbon source and energy which suggests its ability in resisting environmental toxicants. This work provides some new evidence for the possibility of applying Rhodococcus for contaminated water remediation in the area of industry.

Introduction

Phthalic acid esters (PAEs) which are widely used in plastic, coatings and cosmetics industries are a class of refractory organic compounds. Some of PAEs have received more and more attention in recent years since they are considered as estrogenic disrupting compounds (EDCs) and may have reproductive development toxicity [1], [2], [3], [4]. Animal studies have proved PAEs can cause repetitive abortions [5], [6] and male sterility [7]. In addition, some PAEs are suspected to be mutagens and carcinogens [8], [9], [10]. The United States Environmental Protection Agency and China National Environmental Monitoring Center have classified most of the PAEs, such as diethyl phthalate (DEP), benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP) and di-(2-ethyl hexyl) phthalate (DEHP) as priority pollutants [11], [12].

There are more than 60 kinds of PAEs produced and consumed for diverse purposes, mainly as plasticizers. In particular, soft polyvinyl chloride (PVC) has been manufactured with more than 60% by weight of plasticizers [13]. As it bound covalently to the plastic resin, PAEs are able to migrate into the environment during use or disposal. Because of their low water solubility and high octanol-water partition coefficients, they tend to accumulate in the soil or sediment and in the biota living in the PAEs containing waters [14]. Presently, pollution of PAEs is ubiquitous in variable water environment including river water, ground water, drinking water, open ocean and lake [15], [16], [17], [18], which may do harm to aquatic organisms and human health through food chain transmission and bioamplification.

Unfortunately, due to its chemical structure, PAEs can not be removed well by natural processes such as hydrolyzation and photodecomposition. However, metabolic breakdown of PAEs by microorganisms is considered as one of the major routes of environmental degradation for this widespread pollutant [19], [20]. Many bacterial strains with the ability of degrading PAEs and their isomers have been isolated from different sources such as activated sludge, mangrove sediment and wastewater [21], [22].

In the present study, an aerobic bacterial strain was isolated from PAEs acclimated activated sludge of a dyeing plant. This strain was recognized to have the ability to utilize DMP, DEP and DBP individually and all together. Besides, some physiochemical characteristics, the environmental factors and the kinetics of biodegradation were also investigated.

Section snippets

Chemicals

DMP, DEP and DBP (A.P.) were purchased from Beijing Chemical Reagent Factory, China. Dichloromethane (DCM, C.P.) and acetone (A.P.) were obtained from Shanghai Chemical Reagent Factory, China. Other chemicals used in this study were all of analytical grade and commercially available.

Preparation of medium and PAEs wastewater

The inorganic salt solution used for acclimation of the activated sludge contained NaCl 1.0 g/L, NH4NO3 0.5 g/L, FeCl3 0.01 g/L, K2HPO4·3H2O 1.0 g/L, MgSO4·7H2O 0.4 g/L, CaCl2·2H2O 0.1 g/L.

The Luria-Bertani (LB) broth for

Identification and characterization of the bacterial strain L4

Viable bacteria were isolated from activated sludge of a dyeing plant after 8 weeks PAEs acclimation in an aerated basin. One pure bacterial strain named strain L4 was obtained as dominant PAEs-degrading strain through screening test. The strain with size of 1.1–1.9 μm × 4.0–5.4 μm was gram-positive, non-flagellated and short rod shape. The morphology of strain L4 was shown in Fig. 1. The colonies of this strain with diameter of 0.2–0.5 mm on LB agar cultured in 18–24 h showed ivory, opaque and round

Conclusions

A strain able to degrade PAEs, isolated from activated sludge of a dyeing plant was identified as R. ruber through analysis of morphology, physiochemical properties and 16S rDNA sequence. This strain can survive by using DMP, DEP and DBP mixture as carbon source and energy. The article showed the optimal pH value, temperature and substrate's concentration that influenced the degradation rate in water. Kinetics of degradation can be described with exponential model. Little difference was

Acknowledgements

This study was supported by grants from the National Science Council, Republic of China (No. 30400346 and No. 30771776). The authors wish to express their appreciation to Dr. D. Zheng, who has given technical assistance in GC analysis.

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