Bioremediation of Historically Chlorimuron-Ethyl-Contaminated Soil by Co-Culture Chlorimuron-Ethyl-Degrading Bacteria Combined with the Spent Mushroom Substrate

In this study, a novel chlorimuron-ethyl-degrading Pleurotus eryngiu-SMS-CB was successfully constructed for remediation of soil historically contaminated with chlorimuron-ethyl. The P. eryngiu-SMS-CB was prepared using efficient chlorimuron-ethyl-degrading cocultured bacteria, Rhodococcus sp. D310-1 and Enterobacter sp. D310-5, with spent mushroom substrate (SMS, a type of agricultural waste containing laccase) of Pleurotus eryngiu as a carrier. The chlorimuron-ethyl degradation efficiency in historically chlorimuron-ethyl-contaminated soil reached 93.1% at the end of 80 days of treatment with the P. eryngiu-SMS-CB. Although the P. eryngiu-SMS-CB altered the microbial community structure at the beginning of the 80 days, the bacterial population slowly recovered after 180 days; thus, the P. eryngiu-SMS-CB does not have an excessive effect on the long-term microbial community structure of the soil. Pot experiments indicated that contaminated soil remediation with P. eryngiu-SMS-CB reduced the toxic effects of chlorimuron-ethyl on wheat. This paper is the first to attempt to use chlorimuron-ethyl-degrading bacterial strains adhering to P. eryngiu-SMS to remediate historically chlorimuron-ethyl-contaminated soil, and the microbial community structure and P. eryngiu-SMS-CB activity in chlorimuron-ethyl-contaminated soil were traced in situ to evaluate the long-term effects of this remediation.


The chlorimuron-ethyl extraction assay
For the extraction of chlorimuron-ethyl from the medium, ten milliliters of the culture medium was centrifuged at 12,000 rpm for 10 min, and then 5 mL of the supernatant was transferred to a 250 mL glass separation funnel and extracted with 10 mL of dichloromethane. The supernatants were extracted three times each with 10 mL of dichloromethane, and then the organic phases were combined.
For the extraction of chlorimuron-ethyl from the soil samples, 5 g of soil sample was weighed into a 50 mL polystyrene tube, soaked overnight in 20 mL of dichloromethane and then centrifuged at 12,000 rpm for 10 min. The supernatant was extracted with 10 mL of dichloromethane. Each sample was extracted three times with dichloromethane, and the organic phases were combined.
After dehydration with anhydrous sodium sulfate, the organic phases were collected in a 100 mL flat bottom flask and concentrated to almost dryness with a rotary evaporator (RE52C, Shanghai, China). Acetonitrile was added to dissolve chlorimuron-ethyl, the volume was increased to 1.5 mL, and the samples from all of the biodegradation experiments were filtered through 0.22-μm Millex-GP PES filter for HPLC analysis [1].

HPLC instrumentation and conditions
The chlorimuron-ethyl concentration was determined using a Waters 600 reverse-phase HPLC (Waters, MA, USA) with a Waters 2487 dual wavelength detector and an autosampler with a C18 column (250×4.6 mm i.d., 5 µm particle size).
The column temperature was 25℃. The applied mobile phase was methanol/water/glacial acetic acid (70/30/0.5, v/v/v) at a flow rate of 1.0 mL min -1 .
The injection volume was 20 µL. Ultraviolet absorption was monitored at 254 nm [1,2]. The peak areas were recorded and calculated using Empower software. The degradation efficiency was calculated with the following formula: where A 0 is the average concentration of the control group, and A is the average concentration of the experimental group.

Extraction of laccase crude extract
A 20 g sample of SMS was placed in an Erlenmeyer flask (250 mL) with 100 mL of sodium citrate buffer (50 mM, pH4.8) and agitated at 150 rpm for 1 h. The crude enzymatic extract was obtained after separation of the residues using filter paper [2]. The crude enzymatic extract was stored in a refrigerator until use.

Assay of Enzymes Activity
Laccase activity was determined by registering the oxidation of 2,2-azo-bis-(ethylbenzothiazoline-6-sulfonic acid) (ABTS) every 20 s during 2 min (λ=420 nm). The reaction mixture included 0.1 mL of crude enzymatic extract, 2.5 mL of sodium acetate buffer (pH 4.5), and 1 mL of ABTS solution (1 mM). The temperature was adjusted to 30°C. One activity unit was defined as the amount of 3 enzyme needed to oxidize 1 M ABTS per minute.

Effect of pH-values on chlorimuron-ethyl degradation
pH4.0, 5.5, 7.0, 8.5 and 10.0 phosphate buffers was added to MSM supplemented with 20 mg L -1 chlorimuron-ethyl, respectively, and then incubated at 27°C for 7 d, and the degradation efficiency of chlorimuron-ethyl was determined.
A 4% (v/v) bacterial suspension was inoculated into MSM supplemented with 20 mg L -1 chlorimuron-ethyl, cultured at 27℃ for 7 d. The pH value was measured continuously for 7 d.
A 4% (v/v) bacterial suspension was inoculated into MSM supplemented with 20 mg L -1 chlorimuron-ethyl, and the pH of the culture medium was 6.0, 6.5 and 7.0 respectively, cultured at 27℃ for 7 d. Non-inoculated medium was as control group.
The degradation efficiency was determined.

Co-culture bacteria
Under aseptic conditions, the chlorimuron-ethyl degrading bacteria Rhodococcus sp. D310-1 and Enterobacter sp. D310-5 were streaked on LB solid plates and cultured at 30℃ for 48 h, respectively, picking a well-growing single colony on the LB plate, inoculating into LB liquid medium (100 mL, pH 6.5), and incubated at 27°C for about 18 h. After cultured to the logarithmic phase, the culture solution was centrifuged at 5000 rpm for 3 min, the supernatant discarded, and the solution was washed with a phosphate buffer (0.2 mol L -1 ) for 3 times. The bacteria were collected, and then the concentration of the bacteria was adjusted with a phosphate buffer to OD600nm = 2.0 ± 0.1. Finally, D310-1 and D310-5 were inoculated into the fermentation medium with 2% inoculum, respectively, and the co-culture bacteria were cultured to logarithmic phase (OD=1.92) for subsequent experiments.

Optimization of the conditions for preparing the chlorimuron-ethyl-degrading Pleurotus eryngiu-SMS-CB
The results indicated that chlorimuron-ethyl was degraded by laccase crude extract from SMS, and the SMS of P. eryngiu was chosen as a carrier for the 4 preparation of chlorimuron-ethyl-degrading P. eryngiu-SMS-CB.
The P. eryngiu-SMS was inoculated with the co-culture bacteria to prepare the P. Based on preliminary experiments, a Box-Behnken Design (BBD) was used to optimize P. eryngiu-SMS-CB preparation conditions. A second-order polynomial equation was a typical model that could describe the response surface:

eryngiu-SMS-CB
where Y is the predicted response; A is a constant and A j , A jj and A ij are coefficients of the linear, square and interaction terms, respectively.
To validate the predicted results with the optimized model, the degradation efficiency was measured in triplicate under optimum conditions.

Effect of pH-values on chlorimuron-ethyl degradation
In general, sulfonylurea herbicides exist as a mixture of ions and molecules in the environment. Under acidic conditions, sulfonylurea herbicides are easily hydrolyzed. Under alkali conditions, the negative charge is distributed on the sulfonylurea bridge, which reduces the nucleophilic reactivity of the carbonyl carbon atoms, and these herbicides are not easily hydrolyzed [4]. Therefore, the hydrolysis of sulfonylurea herbicides is affected by pH. In this study, the effects of pH on chlorimuron-ethyl hydrolysis were examined, and the results are shown in Fig. S1a.  Fig. S1b. The medium pH increased from 6.5 to 7.0 after strains D310-1 and D310-5 were cultured continuously for 7 d, and the pH of the control group remained at the initial pH of 6.5. At different pH values (6.0, 6.5 and 7.0) of the culture medium, the chlorimuron-ethyl degradation efficiencies by D310-1 and D310-5 reached 80.7%, 84.5%, 81.5% after 7 d, respectively, and the hydrolysis efficiencies were only 11.3%, 10.7%, and 10.1%, respectively (Fig. S1c).
During the degradation of chlorimuron-ethyl, the pH of the culture medium did not change significantly, so the degradation of chlorimuron-ethyl was mainly based on microbial degradation, not an acidolysis. where Y is the predicted degradation efficiency of chlorimuron-ethyl, A is the inoculum quantity, B is the culture time and C is the culture temperature.

6
The ANOVA for the second-order polynomial model is shown in Table S1. The regression model, with an R 2 =0.9981, was considered to exhibit very high correlation.
The closer the value of the correlation coefficient is to 1, the better the correlation is