Chemicals and culture medium
DMP, DEP, DBP, BBP, DEHP, DOP, monoethyl phthalate (MEP), monomethyl phthalate (MMP), monobutyl phthalate (MBP), phthalic acid (PA), protocatechuic acid (PCA) and butanol (analytical grade, above 98% of purity) used for biodegradation experiments was purchased from Aladdin Chemistry Co. (Shanghai, China). Standard stock solution of PAEs in methanol was prepared. All organic solvents used were of HPLC grade, while other reagents were of analytical grade.
Improved Luria-Bertani (LB) medium contained (g/L) NaCl (10), beef peptone (10), yeast extract (5), glucose (5) and its final pH was adjusted to 7.5.
Mineral salt medium (MSM) contained (g/L) NH4Cl (0.5), Na2HPO4 (0.3), KH2PO4 (0.05), MgSO4 (0.1), CaCl2 (0.01), ammonium ferric citrate (0.01), 0.5 mL trace elements (Karn et al., 2010), and its final pH was adjusted to 7.5. The media and vessels (e.g., Erlenmeyer flask) were sterilized in an autoclave at 121°C for 20min.
Enrichment and isolation of DBP biodegrading bacteria
Activated sludge samples were collected from Weifang Kangda sewage treatment factory of Shandong in China. Standard stock solution of DBP was added to a 250-mL triangular flask and the solvent methanol was allowed to evaporate overnight before adding 50 mL MSM and 10 grams of activated sludge. These flasks were incubated at 30°C and 200 rpm. An aliquot of 5 ml was subcultured to fresh medium every week, the concentration of DBP increasing from 50 to 1000 mg/L (50, 100, 200, 400, 600, 800, and 1000 mg/L). After 7 weeks, some bacteria could grow in the medium with 1000 mg/L DBP; then serial dilutions of the final bacterial culture were conducted and inoculated with spread method onto the LB agar plates and cultured for 48 h at 30°C. Different isolates were screened by biodegradation ability of DBP, and a promising bacterial strain USTB-Y was isolated and used.
Identification and characterization, whole genome sequencing of strain USTB-Y
The morphology of strain USTB-Y was observed by a microscope (OLYMPUS DP72, Japan). Total DNA was extracted from the purified bacterium using the Bacterial Genome Extraction kit (Tiangen, Beijing) following the manufacturer’s instructions. Following centrifugation, the supernatant was used as template for polymerase chain reaction (PCR) with the primer pair of 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (48). The PCR products were sequenced by Shenggong Biotechnology Co., Ltd. (Shanghai, China) and deposited in the GenBank database under the accession number MW828316. The resulting sequence of strain USTB-Y were aligned and compared with the known gene sequences in EzBioCloud database. The nearest neighbor sequences were aligned using Clustal W, and a phylogenetic tree based on the 16S rDNA sequence data was constructed by the neighbor-joining method with MEGA 7 software. To define the phylogenetic relationship, the genome sequences of available Microbacterium sp. strains in EzBioCloud database were downloaded from NCBI and whole genome average nucleotide identity (ANI) values were obtained from pairwise comparison online (EzBioCloud database).
The draft genome was sequenced by Illumina NovaSeq platform of Beijing Fixgene Co., Ltd. Low reads were trimmed by fastp software. High quality reads were then assembled by Spades software. The genome sequence was annotated by the PGAP on NCBI (October, 2020). The sequence data were submitted to NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/ ) with SRA number SRR14179763.
Inoculum preparation
Strain USTB-Y was pre-cultured in LB liquid medium at 30°C and 200 rpm for 18 h (at logarithmic phase), and cell pellets were harvested by centrifugation (6000 rpm for 10 min), washed three times with sterile physiological saline water and re-suspended in MSM to obtain the inoculum suspension (OD600 = 1.8, tested by SHIMADZU UV-2450 spectrophotometer, approximately 1.2×109 CFU/mL determined by colony counting method), and 2% of inoculation proportion (volume ratio) was used in all the biodegradation experiments.
Optimization of DBP degradation conditions
The biodegradation experiments were performed in 20 mL MSM supplementing with 50 mg/L DBP in triplicates and cultured at 200 rpm and 30℃ for 24 h. In this experiment, three single factors including pH, temperature and initial concentrations were used as independent variables to investigate their effects on the biodegradation ratios of DBP by USTB-Y. The pH of culture medium was set at 5.5, 6.5, 7.5, 8.5 and 9.0 (30°C, initial concentration of DBP at 50 mg/L); temperature at 15, 25, 30, 37 and 40°C (pH at 7.5, initial concentration of DBP at 50 mg/L); and initial concentrations of DBP at 50, 100, 500 and 1000 mg/L (pH at 7.5, 30°C).
Substrate utilization tests
Liquid MSM of 20 mL was supplemented with each compound of PA, PCA, MMP, MEP, MBP, DMP, DEP, DBP, DOP, DEHP and BBP (50 mg/L) as the sole carbon source to test the substrate utilization of strain USTB-Y. After culturing at optimal condition for 72 h, the residual of these compounds in the culture was analyzed by HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific Inc., USA) after 72 h, respectively.
Biodegradation Kinetic of DBP by Microbacterium sp. USTB-Y
To investigate biodegradation kinetic of DBP by whole cells of strain USTB-Y, 20 mL MSM with initial concentration of 50, 100, 500 and 1000 mg/L were prepared in triplicates, respectively. DBP biodegradation experiments were carried out under the optimum conditions in dark for 5 d, and samples of culture solution were collected at regular intervals. Non-inoculated medium served as a control.
Biodegradation of DBP by cell-free extracts solution
For measurement of enzymatic degrading activity, supernatant of cell-free extracts was prepared. After cultured in 1000 mg/L DBP-MSM for 96 h, cells were sedimented by centrifugation (6000 rpm, 15 min). The biomass (about 0.7 g of wet weight) was used to prepared 7 mL cell-free extracts using M5 Bacterial Protein Extraction Kit purchased from Mei5 Biotechnology, Co., Ltd (China). Cell debris was removed by centrifugation (4℃, 12000 rpm and 15 min). After filtered with 0.22 µm organic nylon micropore membrane, 200 µL supernate of cell-free extracts was added into 10 mL of 100 mg/L DBP-PBS (phosphate buffer saline, 0.05 M, pH = 7.4) and cultured at 200 rpm and 30℃, and PBS without cell-free extracts supernate was as a control. Samples at 0, 2, 4, 8 and 12 h were collected and treated for HPLC analysis.
Analysis of residual substrates by HPLC
HPLC was used to determine the concentrations of DBP and their metabolites followed an improved method described by Lu et al. (2020). Twice the volume of methanol was added to the culture or the enzyme assay solution, and the mixture was vortexed for 5 min before it was centrifuged at 10000 rpm for 5 min, then the supernatant was filtered through a 0.22 µm organic nylon micropore membrane and analyzed by an Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific Inc., USA) which was equipped with a C18 reverse phase column (250×4.6 mm2, 5 µm particle size, Dikema Technology Co. Ltd., China), and a UV-detector at 228 nm. For DBP analysis, methanol and an aqueous solution of 0.5% acetic acid (80:20, v/v) were used as the mobile phase with a flow rate of 1.0 mL/min. The temperature of column oven was 30℃. For quantification, an external standard method was applied and the standard curve was built following a six-point calibration curve (ranged from 25 to 500 mg/L, and the R2 was above 0.99). All experiments were conducted in triplicates and were subjected to statistical analysis.
Identification of biodegradation products by GC-MS
Liquid MSM with 100 mg/L of DBP was inoculated with strain USTB-Y and cultured at 200 rpm under the optimal conditions. The culture samples were collected at 0, 4, 10 and 12 h, respectively. All treatments were in triplicates and non-inoculation served as the control. HPLC analysis followed the method described above. For GC-MS analysis, cultured solution was extracted twice with equal volume of dichloromethane, and the combined organic phase was concentrated to near dryness by a rotary evaporator, and then the residue was redissolved in acetone for GC-MS (QP2010 Plus, Shimadzu, Japan) analysis. A capillary column (HP-5MS, 0.25 µm× 0.25 mm× 30m) was used for separation. Helium (> 99.99% of purity) was employed as carrier gas at a flow rate of 1.0 mL/min. The temperatures of injection and ion source were set at 250°C and 220°C, respectively. The GC oven temperature was programmed as follows: 100°C held for 2 min, raised at 15°C/min to 129°C, then at 40°C/min to 280°C (held for 5min). The extract (1 µL) was injected in the splitless mode. Mass spectra were acquired in the electron ionization (EI) mode using an electron impact ionization of 70eV and scanning at 45–500 amu.
Bioremediation of DBP artificially contaminated soil by strain USTB-Y
Soil samples were taken from top 20 cm of soil profile on a botanical garden in Weifang and contained no detectable residual PAEs. The soil was mixed thoroughly, sieved through 2-mm mesh to remove stones and debris, and stored at room temperature overnight. Soil properties were measured as follows: pH 6.83, organic carbon 15.4 g/kg, total nitrogen 1.63 g/k, total phosphorus 0.79 g/kg, total potassium 13.75 g/kg. Soil samples were autoclaved and treated with DBP thoroughly to get a concentration of 50 mg/kg dry soil, and followed by methanol evaporation for 12 h at room temperature.
Prepared soil of 50 g was put into 150 mL glass bottles sealed with a gas-permeable membrane, and inoculated with the prepared inoculum to give an initial bacterial population of 6×107 CFU/g as the biodegradation treatment. All soil samples were cultured at 30℃ in the dark for 12 h. DBP-contaminated soil without inoculation served as a blank control and all treatments were prepared in triplicate. Soil samples of 2 g were collected and soaked with 20 mL of a mixture of acetone and hexane (1:1, v/v) overnight in a glass centrifuge bottle, and then extracted ultrasonically three times to collect organic phase. After centrifugation at 6000 rpm for 10 min, the supernate solution was concentrated to near dryness with a rotary evaporator. The residue was redissolved in 5 mL methanol, which was diluted to a suitable concentration for HPLC analysis.
Statistics analyses
The data were calculated, analyzed and plotted by Microsoft Excel 2010 (Microsoft Co., USA).The degradation rate of DBP was calculated by Eq. (1):
Degradation rate (%) = (C0 − Ct )/C0 ×100% (1)
Where C0 is the initial concentration of DBP in culture (or soil), and Ct is the residual concentration of DBP after biodegradation experiment.
The biodegradation process of DBP was confirmed by first-order kinetic model as Eq. (2), the half-life of first-order reaction could be calculated by Eq. (3) (Suzuki et al. 1998; Heo et al. 2020):
lnC = − kt + A (2)
t1/2 = ln2/k (3)
Where C is the DBP concentration (mg/L) at time t (h), k is the first-order kinetic constant (h− 1), and A is a constant. t1/2 is the half-life corresponding to the time interval of the DBP concentration to decrease to half of its initial value.