Reagents, samples and cultivation medium
DBP, DEP, DOP, dimethyl phthalate (DMP), diisooctyl phthalate (DIOP) and diisononyl phthalate (DINP) were purchased from Sigma-Aldrich (USA). The methanol was HPLC grade from Merck. The rest of chemical reagents were analytical grade, obtained from China National Medical Medicine Group. The soil samples of peanut field were collected from Laixi Experimental Station of Shandong Peanut Research Institute in Qingdao, Shandong Province, China. All samples were stored at 4 oC before analysis. The mineral salt medium (MSM) was prepared as previously described (Wu et al. 2011). DBP was added, and the solution was sterilized at 120 oC for 15 min.
Enrichment, screening and identification of DBP-degrading bacteria
DBP-degrading bacteria were screened from peanut field soil by continuous enrichment cultivation. 1 gram soil sample was inoculated to 200 ml of solution with 200 mg/L DBP. After 7 days, 1000 μl of the culture added to 200 ml solution with 1000 mg /L DBP. After 7 days, 100 μL samples were spread onto MSM agar with DBP under sterile conditions, and then the plates were incubated at 28 oC for 5-10 days until colonies appeared. Single colony was isolated and subsequently transferred to MSM agar for three times, and the pure culture was named J2.
The morphology of strain J2 was observed on LB agar plates after 16 h at 28 oC. The hydrolysis of casein, starch, gelatin, Tween 40, Tween 80 and gelatin was studied as described in previous reports (Romanenko et al. 2013). The strain J2 was identified by 16S rRNA gene sequencing. 27F and 1492R primers were used for PCR amplification (Frank et al. 2008). PCR amplification products were analyzed using 0.8% agarose gel, and were then purified. The fragments were linked into pMD19-T, and then were transformed into competent cells. The DNA sequencing was performed using an DNA sequence analyzer ABI 3720 (Applied Biosystems, United States) by Shanghai Personalbio. The 16S rRNA gene sequence was analyzed in Ezbiocloud database. The phylogenetic tree was then constructed using MEGA X (Kumar et al. 2018).
Substrate utilization experiments
In order to study strain J2 to degrade PAEs, the bacterium was inoculated into MSM containing 0.3 g/L of one of PAEs: DBP, DMP, DEP, DOP, DIOP, DINP. Uninoculated media supplemented with each substrate were used negative controls. The suspension was cultured on a rotating shaker at 28 oC. 5 days later, substrate utilization was measured using optical density method.
Biodegradation of DBP by strain J2
Strain J2 was first cultured in LB medium at 28 oC for 24 h. Strain J2 was harvested and washed three times with phosphate buffer, and then was suspended in the phosphate buffer. Inoculated 1% bacterial solution into 50 mL MSM with 1000 mg/L DBP. In order to study the optical conditions of degradation, the degradation of 1000 mg/L DBP by strain J2 was investigated under pH 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, and different temperature (18, 28, 35, 40, 45 oC). After 5 days, the residue remaining DBP was investigated using HPLC.
Analysis of DBP biodegradation using HPLC
The samples were mixed with ethyl acetate at a 1:1 vol ratio. The aqueous phase was separated using centrifugation at 8,000 g. The aqueous solution samples were extracted twice. The ethyl acetate was evaporated by a rotary evaporator (Buchi, Switzerland). The residue was dissolved in 10 ml methanol, and then filtered. Finally, HPLC was used to analyze the residual concentration of DBP with C18 column. The experiment was carried out at a column temperature of 30 ℃, with a mixture of methanol and water at a ratio of 90:10 (v/v). The concentrations of residual DBP were quantified by comparing the integral values of the peak area with the calibration standard curve. DBP degradation efficiency was evaluated, based on the initial and final concentrations of DBP in the test solution.
The whole genome of strain J2 was sequenced by using PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platform, provided by Shanghai Majorbio Biopharmaceutical Technology Co., LTD. For PacBio sequencing, 20 μg DNA of strain J2 was spun in a Covaris G-tube. The DNA fragments were then purified, repaired at the end, and linked with SMRTbell sequencing adapters. The sequence library was constructed by 0.45 x volumes of Agencourt AMPure XP beads. An insertion library of about 10 kb was sequenced on one SMRT cell. For Illumina sequencing, more than 1 μg DNA samples were used for sequencing. According to the manufacturer's protocol, genomic DNA samples were cut into 400-500 bp fragments. The libraries were sequenced on Illumina HiSeq X Ten machine with 2 × 150 bp.
Annotation of Genomic Information
CDS, transfer RNA (tRNA) genes and ribosomal RNA (rRNA) genes were predicted by Glimmer (Delcher et al. 2007), Trnascan-SE (Lowe et al. 1997) and rRNAmmer (Lagesen et al. 2007), respectively. The CDSs were annotated based on six databases, including NR, Swiss-Prot, Pfam, GO, COG and KEGG, using several sequence alignment tools (Besemer et al. 2001; Kanehisa et al. 2004; Tatusov et al. 2003). DNAMAN software was used for multiple sequence alignment of esterase.