M1. Supplementary methods
Enrichment and isolation of bacterial strain
Five gram of fresh soil was collected as inoculum from a landfill in Guangzhou, China. The mineral salt medium (MSM) containing (g L-1) K2HPO4 (5.8), KH2PO4 (4.5), (NH4)2SO4 (2.0), MgCl2 (0.16), CaCl2 (0.02), Na2MoO4·2H2O (0.0024), FeCl3 (0.0018), and MnCl2·2H2O (0.0015) was used in this study. The final pH was adjusted to 7.0. The initial enrichment culture was established in a 250-mL Erlenmeyer flask containing 100 mL MSM supplemented with DEHP (25 mg L−1). The flasks were incubated in the dark at 30°C under shaking for a week. Then, 1.0-mL aliquots of the active culture were transferred to new Erlenmeyer flasks containing 100 mL of freshly made MSM with gradually increasing concentrations of DEHP (25-2000 mg L−1). The DEHP-degrading enrichment cultures were transferred more than ten times serially into fresh medium before the isolation of DEHP-degrading strains.
Identification of the DEHP-degrading bacteria
The isolate was characterized and identified by the morphological, physio-biochemical characteristics, and 16S rRNA gene analysis. Bacterial morphology was observed under a scanning electron microscope (PhilipsXL30, Netherlands). Physio-biochemical tests were examined with reference to Bergey’s Manual of Determinative Bacteriology. Genomic DNA was isolated and subjected to PCR amplification of the 16S rRNA gene using the universal primers F27 (5′-AGAGTTTGATCCTGGCTCAG-3′) and R1492 (5′-ACGGHTACCTTGTTTACGACTT-3′). The service of purifying and sequencing of amplification products were provided bySangonCorporation (Shanghai, China). The resulting sequences of the bacteria were aligned and compared with those in the GenBank. Sequence data of the closest relatives were retrieved from GenBank and aligned using Clustal W with all parameters set at their default values. A phylogenetic tree was then constructed using the neighbor-joining method with MEGA 5.05 software.
Inoculum preparation
To prepare the inoculum, strain J-1 growing in LB medium was harvested by centrifugation at 2500×g for 5 min and washed three times with 0.9% sterile saline. The washed bacteria were then resuspended in the saline. After the cell density had been adjusted to OD600 = 0.8, one percent of this suspension (1.0 ×108 CFU·mL−1) was used as inoculum, unless otherwise mentioned.
Theaccession numbersof the phthalate 3,4-dioxygenasesequencesin GenBank
The accession numbers of ten conserved sequences of the phthalate 3,4-dioxygenase reported by NCBIare as follow: DQ007994, EF494237, AB154537,AB048709,AY502076,AB084235, FJ528989, FJ528991, AF33104, and CP000433.
M2. Supplementary Tables
Table S1 The symbols and levels of three independent variables used in Box-Behnken design
Independent variables / Symbol / Code levels of variables-1 / 0 / 1
Temperature (℃) / X1 / 20 / 30 / 40
pH / X2 / 6.0 / 8.0 / 10.0
Inoculum size (OD600 nm) / X3 / 0.2 / 0.6 / 1.0
Table S2 Physio-biochemical characteristics of strain J-1
Physiological and biochemical test / Results / Physiological and biochemical test / ResultsMethyl red / － / Starch hydrolysis / －
Voges-Proskauer (V-P) / － / Urease production / －
Catalase test / ＋ / Gelatin liquefaction (20℃) / －
Citrateutilization / － / Nitrate reduction / －
D-fructose product acid / ＋ / Oxidase test / －
D-glucose product acid / ＋ / Gas from glucose / －
Oxidasetest / ＋ / Indol reaction / －
＋: Positive; －: Negative
Table S3Box-Behnken design matrix and the response of dependent variable for DEHP degradation.
Run / Code levels of independent variables / ResponseX1 / X2 / X3 / DEHP degradation Y1 (%)
1 / -1 / -1 / 0 / 56.7±3.4e
2 / -1 / 1 / 0 / 64.9±2.1d
3 / 1 / -1 / 0 / 65.2±1.8d
4 / 1 / 1 / 0 / 66.6±2.6d
5 / 0 / -1 / -1 / 57.7±2.3e
6 / 0 / -1 / 1 / 74.7±4.8c
7 / 0 / 1 / -1 / 63.4±4.5d
8 / 0 / 1 / 1 / 79.9±5.6bc
9 / -1 / 0 / -1 / 65.1±1.5d
10 / 1 / 0 / -1 / 67.4±2.4d
11 / -1 / 0 / 1 / 83.7±4.1b
12 / 1 / 0 / 1 / 86.2±4.4b
13 / 0 / 0 / 0 / 93.7±3.6a
14 / 0 / 0 / 0 / 94.4±1.9a
15 / 0 / 0 / 0 / 93.5±2.4a
X1 refers to temperature; X2 refers to pH; X3 refers to inoculum size. The data are means of three replicates with standard deviation. Data followed by different letters in the same column indicate significant differences at p<0.05
Table S4 Analysis of variance (ANOVA) for the fitted quadratic polynomial model for DEHP degradation.
Source / DF / SS / MS / F-Value / P-Value*X1 / 1 / 494614.6 / 494614.6 / 12.46775 / 0.0167
X2 / 1 / 895008.3 / 895008.3 / 22.56048 / 0.0051
X3 / 1 / 13185679 / 13185679 / 332.3715 / < 0.0001
X1 X1 / 1 / 13376539 / 13376539 / 337.1825 / < 0.0001
X1 X2 / 1 / 165079.7 / 165079.7 / 4.161165 / 0.0969
X1 X3 / 1 / 3600 / 3600 / 0.090745 / 0.7754
X2 X2 / 1 / 30611787 / 30611787 / 771.6316 / < 0.0001
X2 X3 / 1 / 3229.081 / 3229.081 / 0.081395 / 0.7869
X3 X3 / 1 / 4496219 / 4496219 / 113.3362 / 0.0001
Model / 9 / 58032743 / 6448083 / 162.5369 / < 0.0001
Error / 5 / 198357.5 / 39671.5
Total / 14 / 58231101
R2 = 0.9936 (adjusted R2 = 0.9889), coefficient of variation (CV) = 3.5%. DF refers to degrees of freedom, SS refers to sum of sequences, MS refers to mean square. *p<0.05 indicates the model terms are significant
Table S5Effect estimates for the fitted quadratic polynomial model for DEHP degradation
Term / Estimate / Standard Error / t-Value / P-Value*X1 / 248.65 / 70.41973 / 3.530971 / 0.0167
X2 / 334.47875 / 70.41973 / 4.749788 / 0.0051
X3 / 1283.8263 / 70.41973 / 18.23106 / < 0.0001
X1 X1 / -1903.369 / 103.655 / -18.3625 / < 0.0001
X1 X2 / -203.15 / 99.58853 / -2.03989 / 0.0969
X1 X3 / 30 / 99.58853 / 0.301239 / 0.7754
X2 X2 / -2879.356 / 103.655 / -27.7783 / < 0.0001
X2 X3 / 28.4125 / 99.58853 / 0.285299 / 0.7869
X3 X3 / -1103.506 / 103.655 / -10.6459 / 0.0001
*p<0.05 indicate the model terms are significant
Table S6Kinetic parameters of DEHP degradation in MSM with different initial concentrations
Initial concentration (mg L−1) / Kinetic equations / t1/2 (d) / R250 / ln C = －0.5330t＋3.9204 / 1.30 / 0.9611
200 / ln C = －0.5192t＋5.2956 / 1.34 / 0.9859
500 / ln C = －0.3782t＋6.2170 / 1.83 / 0.9658
800 / ln C = －0.3381t＋6.6831 / 2.05 / 0.9478
1200 / ln C = －0.2494t＋7.1001 / 2.78 / 0.9453
1600 / ln C = －0.1687t＋7.3848 / 4.11 / 0.9238
2000 / ln C = －0.1078t＋7.6053 / 6.43 / 0.9257
Table S7 Degradation kinetics of other PAEs by strain J-1 with the initial concentration of 200 mg L−1.
PAEs / Kinetic equations / t1/2 (d) / R2DMP / ln C = －1.0232t＋5.2995 / 0.68 / 0.9570
DEP / ln C = －0.9917t＋5.2971 / 0.70 / 0.9564
DBP / ln C = －0.8896t＋5.2952 / 0.78 / 0.9556
BBP / ln C = －0.4483t＋5.3043 / 1.55 / 0.9486
DnOP / ln C = －0.6487t＋5.2955 / 1.07 / 0.9537
DINP / ln C = －0.1300t＋5.2994 / 5.33 / 0.9272
M3. Supplementary Figures
Fig. S1 Morphological characteristics of Microbacterium sp. J-1 under scanning electron microscopy (SEM, × 20000)
Fig. S2Response surface plots showing the effects of three variables on DEHP degradation by strain J-1. a: the effects of temperature (X1) and pH (X2) on DEHP degradation rate; b: the effects of temperature (X1) and inoculum (X3) on DEHP degradation rate; c: the effects of pH (X2) and inoculum (X3) on DEHP degradation rate.
Fig. S3Bacterial growth of strain J-1 during biodegradation of DEHP at different initial concentrations. CK: non-inoculated control containing 2000 mg L-1 DEHP
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