J. Cent. South Univ. Technol. (2009) 16: 0948-0953

DOI: 10.1007/s11771-009-0158-4

Aerobic biodegradation of di-n-butyl phthalate by

Xiangjiang River sediment and microflora analysis

ZHOU Hong-bo(周洪波)¹, LIN Feng(林 峰)¹, HU Pei-lei(胡培磊)¹, JING De-cai(金德才)¹,

REN Hong-qiang(任洪强)², ZHAO Jing(赵 晶)³, QIU Guan-zhou(邱冠周)¹

(1. School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China;

2. State Key Laboratory of Pollution Control and Resources Reuse, Nanjing University, Nanjing 210008, China;

3. School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China)

Abstract: Di-n-butyl phthalate (DBP), one of phthalate acid esters (PAEs), was investigated to determine its biodegradation rate using Xiangjiang River sediment and find potential DBP degraders in the enrichment culture of the sediment. The sediment sample was incubated with an initial concentration of DBP of 100 mg/L for 5 d. The biodegradation rate of DBP was detected using HPLC and the degraded products were analyzed by GC/MS. Subsequently, the microbial diversity of the enrichment culture was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results reveal that almost 100% of DBP is degraded after merely 3 d, generating two main degraded products: mono-butyl phthalate (MBP) and 9-octadecenoic acid. After a six-month enrichment period under the pressure of DBP, the dominant family in the final enrichment culture is clustered with the Comamonas sp., the remaining are affiliated with Sphingomonas sp., Hydrogenophaga sp., Rhizobium sp., and Acidovorax sp. The results show the potential of these bacteria to be used in the bioremediation of DBP in the environment.

Key words: bioremediation; di-n-butyl phthalate; sediment; polymerase chain reaction-restriction fragment length polymorphism; microbial diversity

1 Introduction

Phthalate acid esters (PAEs) have enjoyed a wide application in the industrial production as well as people’s daily life since the middle of the 20th century. They belong to a class of refractory organic compounds extensively used in plastic industry, occupying 80%-85% of the global plasticizer market[1]. As PAEs can enter environment directly, they are widely distributed in different environments at concentrations ranging from 0.3 to 98 μg/L in surface water, 0.2 to   8.4 mg/kg in sediment, and 28 to 154 mg/kg in sewage sludge[2]. What’s worse, some of them are toxic to mammals and aquatic organisms[3], interfering with their reproductive systems and endocrine function[4]. Furthermore, certain PAEs are suspected of exerting adverse effects on human health[5].

Di-n-butyl phthalate (DBP) that belongs to the family of PAEs is used in the preparation of insect repellents and paper coatings, and as an agent for textile lubrication, with developmental and reproductive toxicity on animals[6]. As one of the most frequently identified PAEs in diverse environmental samples, DBP is listed as a top-priority environmental pollutant by several regulatory bodies, such as U.S. Environment Protection Agency, European Union and China National Environmental Monitoring Center[7].

Unfortunately, DBP is relatively stable in the environment. The hydrolysis half-life of DBP was estimated to be about 20 years. Besides, its photolysis half-life is also quiet long, which is from several months to a few years[8]. Apart from physical and chemical ways, biological pathway is indisputably an effective and efficient route to eliminate this widespread pollutant. LI et al[9] studied the kinetics of DBP biodegradation in soil. When the initial concentration of DBP is below   50 mg/kg, the half life of DBP is only 5.20 d. YUAN   et al[10] reported that the biodegradation of PAEs is more effective under aerobic than anaerobic conditions.

Xiangjiang River is the longest river in Hunan Province, China. It is also seriously polluted by pollutants including PAEs since the biggest cities in Hunan are all located along this river. In this work, the DBP biodegradation ability of Xiangjiang River sediment was tested under aerobic condition and the functional microorganisms in the enrichment culture were then explored.

2 Experimental

2.1 Chemicals

DBP with 99.5% purity was purchased from Hunan Huihong Chemicals Corporation China. Chemicalsused for diluting and extracting DBP were analytical-reagent grade and were redistilled. Methanol used was HPLC grade (SK Chemicals, Korea). Other chemicals and solvents were analytical-reagent grade.

2.2 Sediment sample and culture conditions

Sediment sample was taken from Xiangjiang River in Changsha City, Hunan Province, China. Enrichment culture was obtained from the sediment sample using DBP as the sole carbon source. The culture medium contained the following salts (g/L): (NH₄)₂SO₄ (3.00), KCl (0.10), KH₂PO₄ (3.00), MgSO₄·7H₂O (0.50), CaCl₂·2H₂O (0.25), Na₂S₂O₃ (10.00). The enrichment was performed for 180 d.

2.3 Biodegradation trails

1 g of sediment was added to a 50 mL flask with 20 mL medium. The initial concentration of DBP in the system was 100 mg/L. The flask was incubated at 30 ℃ for 5 d in a rotary shaker operated at 150 r/min. Sampling was performed every 24 h.

2.4 HPLC and GC/MS analysis

DBP concentrations in the samples were analyzed using a reverse-phase HPLC system. In order to maintain a homogenous system for accurate quantification of the residual DBP, 1.0 mL tween 80 stock solution (10 g/L) was added to each flask as a solubilizing agent. The flasks were then autoclaved at 115 ℃ for 30 min to ensure a strictly aseptic homogenous environment before they were put in a rotary shaker at 150 r/min for 24 h. After equilibration, samples were centrifuged at  12 000 r/min for 30 min, and then 1.0 mL of each sample was removed to a clean tube to which 1.0 mL of dichloromethane was added. The aqueous and organic phases were formed in the tube after vortexing for 1 min and centrifugation at 12 000 r/min for 3 min. After extracting from the aqueous phase two times, the dichloromethane was evaporated to dryness and the residue was redissolved in 1.0 mL of methanol. Approximately 0.5 mL of the DBP-containing methanol passed through a 0.22 μm membrane filter before a portion (20 μL) was injected into HPLC.

HPLC analysis was carried out using Elite series HPLC (Elite, China) consisting of an online DG 230-2 degasser, a P230/P230p pump, and a UV230⁺ UV-Vis detector set at 228 nm. A Hypersil BDS-C18 column (200 mm×4.6 mm, 5 μm) was used for the separation. The mobile phase consisted of a methanol-water solution (90?10, volume ratio), and the flow rate was 0.5 mL/min.

GC/MS analysis was performed on Shimadzu GCMS-QP2010 analyzer. An OV-5 capillary column (30.0 m×0.32 mm (i.d.), 0.32 μm film thickness) was used. The injection temperature was 310 ℃ and the sample was injected in the split mode for 1 min. The split ratio was 10?1. The oven temperature was programmed from 100 to 280 ℃. For MS detection, standard EI conditions (70 eV) were used with a source temperature of 200 ℃. Helium was used as carrier gas with     0.91 mL/min of flow rate. The collected scans of metabolites were identified by comparing with the published mass spectrum at NIST (National Institute of Standards and Technology) Database.

Each sampling was run in triplicate. Besides, additional sediment samples without adding DBP were used to eliminate the effect of the compounds in the sediment to the HPLC and GC/MS analysis.

2.5 DNA extraction and 16S rDNA gene amplification

Bacterial DNA was extracted from the enrichment sample using E.Z.N.A.^TM Bacteria DNA Kit (OMEGA, USA). 16S rDNA gene was then amplified by PCR from the extracted DNA. PCR mixture consisted of 5 μL of PCR buffer (Mg²⁺ plus), 1 μL of dNTP (10 mmol/L),   1 μL of each primer: forward primer (27f: 5′-AGAGTTTGATCCTGGCTCAG-3′, 5 μmol/L) and reverse primer (1492r: 5′-GGTTACCTTGTTACGA- CTT-3′, 5 μmol/L), 0.25 μL of Taq polymerase and 2 μL of DNA extraction products. Double-distilled water was added until its final volume reached 50 μL. The PCR conditions were an initial denaturation step at 95 ℃ for 6 min, followed by 36 cycles at 94 ℃ for 1 min, 56 ℃ for 1 min and 72 ℃ for 2 min, and a final step at 72 ℃ for 10 min. PCR products were purified by E.Z.N.A.^TM Gel Extraction Kit (OMEGA, USA).

2.6 Cloning and restriction fragment length poly- morphism (RFLP) analysis

The purified PCR products were cloned into pGEM-T vectors that were used to transform DH5α Escherichia coli competent cells. After blue-white screening, 176 white colonies were randomly selected and re-amplified by PCR. 72 of the positive reamplified products were digested by the restriction endonuclease Hae Ⅲ and Msp I (New England Biolabs) at 37 ℃ overnight. The system for the reaction of Msp Ⅰ and Hae Ⅲ digestion consisted of 1 μL buffer, 0.25 μL of each enzyme, and 5 μL of purified clone PCR products. Double-distilled water was added until its final volume reached 10 μL. The digestion products were analyzed on a 3% agarose gel prestained with ethidium bromide by electrophoresis (3 h, 80 V). Restriction profiles can be divided into 7 different groups. Representatives of each group were selected for sequencing.

2.7 Sequencing and phylogenetic analysis

Seven representative clones were sequenced (completed by Beijing Sun biotech Co, Ltd) and these sequences were compared with the existing GenBank 16S rDNA gene sequences. The closest 16S rDNA gene sequences were aligned with CLUSTALX 1.83. Phylogenetic tree was constructed by the neighbor joining method, using MEGA 3.1[11].

3 Results and discussion

3.1 DBP biodegradation

In order to make a preliminary evaluation of the biodegradation ability of the sediment, its original potential to degrade DBP was tested under aerobic condition. The biodegradation of DBP by the sediment sample was monitored by detecting the concentration of this substrate in the flask system through HPLC. The degradation pattern of DBP is shown in Fig.1.

Fig.1 Degradation pattern of DBP in flask system

In the first 24 h, the degradation rate is relatively slow. Subsequently, the degradation rate becomes quite rapid and almost 100% of DBP is degraded in 72 h. During this period, the microflora in the sediment probably has acclimatized to the new substrate (DBP), resulting in the rapid degradation of DBP. Low substrate degradation rate during the initial stage can be attributed to the acclimatization phase, which is a prerequisite for bacteria to be acclimated to the new environment.

GC/MS was used to identify the metabolites and intermediates of DBP during the biodegradation process performed by the microflora in the sediment sample. Three main compounds are identified as mono-butyl phthalate (MBP), DBP and 9-octadecenoic acid, respectively (Fig.2) by comparing each mass spectrum with the published mass spectrum in the National Institute of Standards and Technology (NIST) Database.

Based on the intermediate compounds detected by GC/MS, a preliminary pathway for metabolism of DBP by the river sediment is proposed and illustrated as follows:

(1)

One biochemical degradation pathway of DBP proposed by XU et al[12] is that the hydrolysis of the esters linkage of DBP would form MBP and subsequent phthalic acid (PA) and protocatechuic acid (PCA), and then PA can be further metabolized to produce carbon dioxide and water. The detection of MBP as intermediate of DBP degradation in this work is consistent with previous reports. However, the most frequently reported products such as PA and PCA[13] are not detected. CHANG et al[14] pointed out that the high degradation rate of DBP might be the reason for that PA was not detected by GC/MS. Besides, a kind of fatty acid named 9-octadecenoic acid was detected. Actually, it was reported that various fatty acids such as tetradecanoic and hexadecanoic were detected during metabolization of DBP by fungi[15]. Nevertheless, it is still not clear what role the fatty acids might play in the pathway of DBP biodegradation. As stated above, the former proposed biochemical degradation pathway for DBP by isolated bacteria is simple but uncompleted. According to this work, the degradation pathway of DBP by the microflora in sediment is probably more complicated. Thus, further investigation is necessary to reveal more information about the pathway by the microbial community in the sediment.

3.2 Phylogenetic analysis

The relative rapid DBP biodegradation rates by the sediment reveal that there are microorganisms that probably possess strong capability to degrade the pollutant. It is, however, not easy to find those real functional microorganisms directly from the sediment, considering its complicated microbial community composition. The enrichment process can serve as a good method to reduce this complicity and help us to focus on the right microorganisms.

72 positive clones from the enrichment sample were analyzed by PCR-RFLP, presenting seven operational taxonomic units (OTUs). Rarefaction curve (data are not shown) evaluation of the clones reveals that 72 clones are sufficient to represent the microflora diversity in the final enrichment system. Representative clones of the seven restriction profiles were selected respectively for sequencing. The sequence information of the tested clones is listed in Table 1. The library which consists of five genera is dominated by clones related to Comamonas (XJ-L67, XJ-L79, and XJ-L144). Fig.3 shows the detailed information of the abundance of each genus in the enrichment system. Based on all the available sequences, phylogenetic tree of the microflora in the enrichment system was constructed, as shown in Fig.4.

The original enrichment sample was tested by PCR-RFLP, presenting more than 40 OTUs (data are not

Fig.2 Mass spectra of MBP (a), DBP (b) and 9-octadecenoic acid (c) with retention time of 17.091, 24.487 and 27.018 min, respectively

Table 1 Sequence information of tested clones

shown). After the six-month enrichment, the biodiversity of the enrichment sample was dramatically diminished to merely 7 OTUs. However, studies of certain scholars have already demonstrated that the microbial community in the contaminated ecosystems tends to be dominated by organisms that are capable of utilizing the toxic contaminants or resisting to their toxicity[16]. Therefore, all of the five genera in this work should possess the capability to utilize DBP or at least resist its toxicity. Actually, various species and strains in all these five genera have already been reported to engage in the biodegradation of considerable pollutants in the environment[17-18]. The dominant genus Comamonas in the enrichment system is a group of species that are able to degrade a wide range of aromatic compounds[19]. Particularly, many species of Sphingomonas have also

Fig.3 Distribution of genera in clone library of enrichment sample

been identified as PAEs degraders by some scholars. CHANG et al[20] used Sphingomonas sp. strain O18 to degrade DBP and found that it merely took 3 d for the strain to completely remove DBP out of the culture system when the starting concentration of DBP was 30 mg/L. Nevertheless, when the initial concentration of DBP was increased to 100 mg/L. Unsatisfactorily, there was still approximately one fourth of the DBP remaining in the culture system after 7 d incubation by strain O18. Compared with their researches, the microflora in the sediment of Xiangjiang River definitely possesses a strong capability to degrade DBP since it merely takes 3 d to decompose all the DBP with an initial concentration of 100 mg/L. Moreover, the organic pollutants tend to attach to sediment particles, which may reduce the degrading effectiveness of microorganisms, thus retarding the biodegradation process[20]. That is to say,

Fig.4 Phylogenetic tree based on 16S rDNA sequences from enrichment sample

our sediment-free enrichment system may possess an even stronger capability to degrade DBP than its original sediment. Although few reports have been found to demonstrate that Comamonas sp., Hydrogenophaga sp., Rhizobium sp. and Acidovorax sp. are the PAEs degraders, seven species that are able to survive in the enrichment system with DBP as the sole carbon source after the six-month enrichment period have unquestionably established a harmonious cooperation to degrade DBP.

4 Conclusions

(1) The microflora in the sediment of Xiangjiang River possesses a strong capability, which can degrade DBP (100 mg/L) in merely 3 d.

(2) Some of the intermediate of DBP during the biodegradation process are detected by GC/MS, namely MBP and 9-octadecenoic acid. But further work is still needed to find the exact DBP biodegradation pathway by the mircroflora in the sediment.

(3) PCR-RFLP analysis of the final enrichment culture further demonstrates that there are seven species (belong to five genera) possessing strong potential capability to degrade DBP rapidly. Except Sphingomonas sp., Comamonas sp., Hydrogenophaga sp., Rhizobium sp. and Acidovorax sp. were seldom reported as PAEs degraders previously.

(4) Based on these findings, future work will be focused on this novel DBP-degrading community, and the exact DBP metabolic relationships among the seven species will be revealed. Additionally, strong DBP degraders will be isolated from the enrichment system to seek more efficient approach to degrade DBP and other PAEs.

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(Edited by YANG You-ping)

Foundation item: Project(50621063) supported by the National Nature Science Foundation of China; Project(NCET-06-0691) supported by the Program for New Century Excellent Talents in University

Received date: 2009-01-12; Accepted date: 2009-05-20

Corresponding author: ZHOU Hong-bo, Professor; Tel: +86-731-88877216; E-mail: zhouhb@mail.csu.edu.cn