LV Hui-ying, LI Gao-yang, FANG Zhi-hui, ZHANG Fan, LIANG Zeng-enni, SHAN Yang*

1. Hunan Academy of Agricultural Sciences, Agricultural Product Processing Institute, Changsha 410125, PRC;

2. Changsha Environmental Protection College, Changsha 410004, PRC

Abstract In this research, soil samples were first polluted with 6 PAEs (i.e. DMP,DEP, DIBP, DBP, DEHP, and BBP) at 3 different concentration gradients (0, 10, and 20 mg/kg) and then treated with a highly-efficient degradation fungus, Fusarium oxysporum (PO-Yi), to investigate the biodegradation of PAEs in pepper and eggplant soil. The findings revealed that PO-Yi can accelerate the degradation of PAEs in vegetable soils including pepper and eggplant soil to varying degrees. The highest absolute degradation rate (up to 39.5%) was observed in DEP-treated soils at the high pollution level (20 mg/kg), which was 14.2% higher than that at the middle pollution level (10 mg/kg). The degradation effect in the pepper soil was superior to that in the eggplant soil. Vegetable soil, either pepper soil or eggplant soil, which was polluted by various PAEs at different pollution gradients and then treated with PO-Yi fungus presented good bioremediation results. In the pepper and eggplant soil, 76.8%and 63.1% of the PAEs with a total volume of 60 mg/kg were degraded within 30 d respectively. PO-Yi, indigenous microorganisms, and the vegetables, i.e. pepper and eggplant had good synergistic effects on the degradation of compound PAEs in PAE-polluted pot soil.

Key words Fusarium oxysporum (PO-Yi); Phthalates; Soil; Biodegradation; Pot experiment

1. Introduction

Phthalate esters (PAEs) are synthetic organic compounds that have been widely produced and applied to a large variety of products worldwide[1]. They are mainly used as plasticizers to increase the flexibility of paint solvents, as plastic modifiers, coatingsetc[2-3]. Globally, approximately 80% of the total plasticizers are PAEs. Plasticizers can migrate because of exposure to organic solvents and fats[4-7]. They can also evaporate and change into gases in the air under high temperature[8-10],causing considerable concern about health risks[11-12]. Currently, about 5.2 million t/a of PAEs are produced globally[13-14], among which 0.87million t/a are produced by China, making PAEs one of the most common pollutants in the world. In China,DEP (Diethyl phthalate), DMP (Dimethyl phthalate),and DNOP (Di-n-octyl phthalate) are classified as the three “priority pollutants”[15].

China consumes the largest amount of plastic mulch in the world and has the biggest mulch-covering area. As a result, plastic mulching is seen as a main cause of plasticizer pollution in soil. PAE pollutants could affect the soil quality as well as the quantity and diversity of edaphon[16-18]. Substantial concerns have been expressed over the potential hazardous effects of PAEs on human health due to their endocrine disrupting toxicity and bioaccumulation[19-20]. PAEs are very stable and can hardly be broken down by light or natural chemistry. In contrast, biodegradation is considered the most promising technology for PAE control. In recent years, extensive research has been done on the degradation of PAEs in soil by bacteria, providing a large quantity of highlyefficient degradation strains[21]. But there are very few reports concerning the degradation of PAEs by fungi. Eupenicillum javanicum has been proven to be capable of degrading the compound PAEs in soil.

PO-Yi (Fusarium oxysporum) is a highlyefficient degradation fungus screened out by our research group. It can be used to break down DMP,DEP and DEHP. In this research, a pot experiment was designed to investigate the degradation of PAEs in vegetable soil, such as pepper and eggplant soil,by PO-Yi, which can provide a theoretical basis and scientific grounds for the microbe-plant combined bioremediation of PAE polluted soil.

2. Materials and Methods

2.1. Materials

2.1.1. Soil

The vegetable soil used for testing was collected from a vegetable base in Changsha County. The pH value of the soil was 6.5. The organic matter was 25.20 g/kg. The cation exchange capacity was 7.97 cmol/kg. The content of total nitrogen, total phosphorus,and total potassium was 1.45 g/kg, 1.35 g/kg, and 16.30 g/kg, respectively. The relative proportion of sand, silt and clay was 9 ∶7∶1. The soil was placed in the room and dried by air naturally before being grounded and screened using 10-mesh and 20-mesh sieves for the pot experiment. In this soil, the background values of 6 PAEs (namely, DMP, DEP, DIBP, DBP, DEHP, and BBP) were 0.019, 0.050, 0.040, 0.032, 0.028, and 0 mg/kg, respectively.

2.1.2. Plants

Peppers (Xinshu 215) and eggplants (Xiangzaoqie 2), two predominant plant sources in Hunan Province,were selected as the testing plants.

2.1.3. Fungal strain

PO-Yi (Fusarium oxysporum) was screened out by our research team and preserved in the China Center for Type Culture Collection (Wuhan) under Item No. CCTCC M 2018571.

2.1.4. Culture medium

The formula for the liquid fermentative degradation medium is: KH2PO41.5 g/L, (NH4)2SO42.0 g/L, MgSO4·7H2O 0.2 g/L, CaCl20.1 g/L, a little bit of microelements (e.g. Fe, Co, Mn, Mo, and Zn),and double distilled water 1.0 L.

2.1.5. Main reagents and instrument

DMP, DEHP, DEP, DBP, DIBP, and BBP were purchased from Sinopharm Chemical Reagent Co.,Ltd (Shanghai, China). The contents of these 6 PAEs were ≥99.5%. Methyl alcohol (chromatographically pure) was bought from MERCK & Co., Inc. (USA).Methyl alcohol (analytically pure), n-hexane(analytically pure), and dichloromethane (also analytically pure) were purchased from China National Medicines Corp. The Gas Chromatography-Mass Spectrometer (GC-MS) was bought from Shimadu(Japan) (Model No.: QP 2010 Plus).

2.2. Methods

2.2.1. Preparation of PO-Yi fungal suspension

First, individual colonies of PO-Yi were introduced into PDA seed culture solution and incubated at 28±0.5℃ and 150 r/min for 72 h. Then, the mixture was centrifuged at 5 000 r/min for 15 min, after which the supernatant was discarded. The mycelia was washed with sterile water to remove the non-PAE carbon-source matter (e.g. saccharides). Then an equal quantity of sterile water was added to the seed culture solution and the mixture was thoroughly blended.

2.2.2. Pot experiment

The experiment was conducted from July 12th,2017 to March 20th, 2018. The plants were cultivated in pottery pots (40 cm in diameter and 60 cm in height). Each pot was filled with 30 kg testing soil.The seedlings were kept in nutrient soil for 36 d. Then,the two groups of seedlings (pepper and eggplant)were transplanted into pottery pots to grow for 60 d with a compound base fertilizer (content: nitrogen:phosphorus: potassium=1∶1∶1). Next, we prepared DMP, DEP, DIBP, DBP, BBP, and DEHP with 3 concentration gradients (0, 10, and 20 mg/kg) and added them into the pots.

The two groups of pot plants processed with 6 PAE pollutants at 3 different concentration gradients were subdivided into two categories: the group treated with PO-Yi and the group containing no POYi. The group containing no PO-Yi was used as the control. Each treatment was repeated 3 times. PO-Yi suspension was prepared according to the method mentioned in 2.2.1. Tap water was added into 100 mL POYi suspension to make 1 000 mL solution. After the plants had grown in the pots for 90 d (namely, 30 d after the addition of the PAE pollutants), we applied the above 1 000 mL solution to the pots and further watered the soil until its water content reached 20%.

2.2.3. Pretreatment and determination of samples

Fresh soil samples were collected from the plough layer (0～20 cm) of the pottery pots using columnar samplers; each pot contained 4 symmetrical sampling points. The soil samples were dried in the room by natural air and grounded into particles. Next,the soil was screened using a 60-mesh sieve. The soil samples were gathered 30 d after the PO-Yi treatment.

1.00 g of PO-Yi treated soil sample was weighed and poured into a screwed glass tube. 1 mL of water was added into the tube and the mixture was blended thoroughly. Then the mixture was diluted with 2 mL of n-hexane and oscillated on the oscillator for 3 min.After this, the mixture was centrifuged for 5 min at 7 000 r/min and the supernatant was set aside for further purification. Then, the high purity singlewalled carbon nanotube (SWNT) glass solid phase extraction (GSPE) column was activated using 3 mL of acetone solvent and 3 mL of n-hexane solvent. The supernatant was added into the activated high purity SWNT GSPE column at a flow velocity of 1 mL/min and the effluent was discarded. 5 mL of n-hexane was used to further leach away the impurities, then the leaching liquid was discarded. The plasticizers would then be bound to the high purity SWNT, thus realizing purification. Using 3 mL of methylbenzene solvent as the eluent for elution, all the eluate was collected.The eluate was concentrated on the Termovap Sample Concentrator at 40℃ and its volume was reduced to 1 mL for the GC-MS analysis. The contents of the 6 PAE plasticizers (i.e. DMP, DEP, DIBP, DBP, BBP,and DEHP) were measured.

2.2.4. Data processing

The data were processed with Microsoft Excel 2016. All the data were averages. The degradation rate=100%×(Residual amount of the control sample–Residual amount of the treated sample)/Original value of the soil.

3. Results and Analysis

3.1. Degradation of PAEs in pepper soil by PO-Yi

In this research, we polluted the soil samples with PAEs at three gradients: low (L), middle (M), and high (H) level. More specifically, PAEs were added to the soil samples at a rate of 0, 10, and 20 mg/kg,respectively. Low level PAE pollution corresponded to the original soil without the addition of PAEs. As the background concentration of PAE pollutants in the original soil samples was lower than 0.05 mg/kg,and no PAEs were detected, the preexisting content of PAEs in the soil samples was seen as negligible.New soil samples were collected from the pots 30 d after PO-Yi treatment to test the PAE residue (Table 1). The results showed that PO-Yi, to some extent,can accelerate the degradation of PAEs in the soil. For the PO-Yi treated samples, the total PAE degradation rate reached 76.8% and 57.8% in pepper soil with middle and high level PAE pollution, respectively.These degradation rates showed a significant increase in comparison with the PO-Yi-free soil samples.Particularly, the total degradation rate of soil with middle and high level PAE pollution had increased by 16.7% and 25.3%, respectively. The data proved that PO-Yi not only has a competitive growth advantage,but also presents good degradation performance when combining with other microorganisms in the soil. This collaborative advantage is quite evident in highlypolluted soil. The highest absolute degradation rate was found in DEP-polluted soil; in highly-polluted soil, this value reached up to 39.5%, which was 14.2%higher than that in soil with middle level DEP pollution(Fig. 1). For the removal rate of various PAEs (Fig.2), we had DEHP＞DMP＞DEP＞BBP＞DBP＞DIBP in middle-level polluted soil and DEHP＞DMP＞BBP＞DEP＞DIBP＞DBP in highly-polluted soil, the highest removal rate occurred in DEHP-polluted soil samples,which was 95%. To conclude, microorganisms present certain selection priority when degrading compound PAEs. There was also some decrease of PAE contents in the PO-Yi-free soil samples, indicating the potential existence of microorganisms capable of degrading PAE pollutants. These microorganisms can form a specific PAE degradation product food chain with PO-Yi to stimulate the effective degradation of PAEs.Besides, the plants themselves could also absorb and degrade some PAEs.

Table 1 PAE residue in pepper pot soil 30 d after treatment mg/kg

Fig. 1 Absolute degradation rate of PAEs in pepper pot soil 30 d after PO-Yi treatment

Fig. 2 Effects of PO-Yi on the contents of PAEs in pepper soil

3.2. Degradation of PAEs in eggplant soil by PO-Yi

According to the PAE residue in eggplant soil 30 d after PO-Yi treatment (Table 2 and Fig. 3), it is clear that PO-Yi can facilitate the degradation of various PAEs in the soil. For the PO-Yi treated samples, the total PAE degradation rate reached 63.1% and 47.0%in soil with middle and high level PAE pollution,respectively, representing a significant increase in comparison with that of the PO-Yi-free samples; the total PAE degradation rate of soil with middle and high level PAE pollution had increased by 20.4% and 23%, respectively.

The data indicated that PO-Yi not only has a competitive growth advantage, but also presents good degradation performance combining with other microorganisms in the soil. This collaborative advantage is quite obvious in highly-polluted soil as well. The highest absolute degradation rate was found in DEP-polluted soil; in highly-polluted soil, its value reached up to 37.7%, which was 11.0% higher than that in soil with middle level DEP pollution (Fig. 3).But the overall degradation effect of PO-Yi in eggplant soil was inferior to that in pepper soil. One possible cause might be that eggplant and pepper plants have different microbial environments and varied PAE absorbing capacities. For PO-Yi-treated soil samples at the same PAE pollution level, the removal rate of each PAE pollutant in eggplant soil ranked in the same order as that in pepper soil (Fig. 4). But the degradation effect was not as good as that in the pepper pot experiment, indicating that microorganisms have selection priority when degrading compound PAEs.

Table 2 PAE residue in eggplant pot soil 30 d after the treatment mg/kg

Fig. 3 Absolute degradation rate of PAEs in eggplant pot soil 30 d after PO-Yi treatment

Fig. 4 Effects of PO-Yi on the contents of PAEs in eggplant soil

4. Conclusion

(1) PO-Yi can facilitate the degradation of PAEs(i.e. DMP, DEP, DIBP, DBP, BBP and DEHP) in vegetable soil including pepper and eggplant soil, to varying degrees. The highest absolute degradation rate (up to 39.5%) was observed in DEP-treated soil at the high pollution level (20 mg/kg), which was 14.2%higher than that at the middle pollution level (10 mg/kg).The degradation effect in pepper soil was superior to that in eggplant soil.

(2) Vegetable soil either pepper soil or eggplant soil, which were polluted by various PAEs at different pollution gradients and then treated with PO-Yi fungus have presented good bioremediation results. In pepper and eggplant soil, 76.8% and 63.1% of the PAEs with a total volume of 60 mg/kg were degraded within 30 d respectively, the 6 individual PAEs were 10 mg/kg each. PO-Yi, indigenous microorganisms, and the vegetables including pepper and eggplant had good synergistic effects on the degradation of compound PAEs in PAE-polluted pot soil.