Ochrobactrum intermedium and saponin assisted phytoremediation of Cd and B[a]P co-contaminated soil by Cd-hyperaccumulator Sedum alfredii

Journal Pre-proof Ochrobactrum intermedium and saponin assisted phytoremediation of Cd and B[a] P co-contaminated soil by Cd-hyperaccumulator Sedum alfredii

Qi Tao, Jinxing Li, Yuankun Liu, Jipeng Luo, Qiang Xu, Bing Li, Qiquan Li, Tingqiang Li, Changquan Wang PII:

S0045-6535(19)32787-0

DOI:

https://doi.org/10.1016/j.chemosphere.2019.125547

Reference:

CHEM 125547

To appear in:

Chemosphere

Received Date:

15 September 2019

Accepted Date:

03 December 2019

Please cite this article as: Qi Tao, Jinxing Li, Yuankun Liu, Jipeng Luo, Qiang Xu, Bing Li, Qiquan Li, Tingqiang Li, Changquan Wang, Ochrobactrum intermedium and saponin assisted phytoremediation of Cd and B[a]P co-contaminated soil by Cd-hyperaccumulator Sedum alfredii, Chemosphere (2019), https://doi.org/10.1016/j.chemosphere.2019.125547

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Journal Pre-proof Title: Ochrobactrum intermedium and saponin assisted phytoremediation of Cd and B[a]P co-contaminated soil by Cd-hyperaccumulator Sedum alfredii

Authors: Qi Tao1, Jinxing Li2, Yuankun Liu2, Jipeng Luo2, Qiang Xu1, Bing Li1; Qiquan Li1; Tingqiang Li2#, Changquan Wang1#

The affiliation and address of the authors 1 College 2

of Resources, Sichuan Agricultural University, Chengdu 611130, China

Ministry of Education Key Laboratory of Environmental Remediation and Ecological

Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China

Corresponding author

#

Tingqiang Li, Ph.D and Professor, Ministry of Education Key Laboratory of Environmental Remediation and Ecological Health, College of Environmental and Resource Sciences, Zhejiang

University,

Hangzhou

310058,

China;

Tel:

+86-571-88982518,

Fax:

+86-571-88982907, E-mail: [email protected] Changquan Wang, Ph.D and Professor, College of Resources, Sichuan Agricultural University, Chengdu 611130, China, E-mail: [email protected]

Journal Pre-proof

B[a]P-16

Saponin

30 B[a]P

Cd

80

25

60 20

40

15

20

10

0 SC

Soil particle

SCB SCS SCBS Treatments

Degradation Cd2+ Cd2+

Cd

B[a]P

Cd-mineral

Cd2+

Uptake

?

B[a]P removal rate (%)

Cd Phytoextraction rate (%)

Graphic

Journal Pre-proof Abstract Pot-culture experiments were conducted to investigate the potential of microorganismsaponin assisted phytoremediation of cadmium (Cd) and benzo(a)pyrene (B[a]P) co-contaminated soil using Cd-hyperaccumulator Sedum alfredii. Results showed that B[a]P-degrading bacterium (Ochrobactrum intermedium B[a]P-16) inoculation significantly increased root (by 22.1-24.1%) and shoot (by 20.5-23.4%) biomass of S. alfredii, whereas the application of saponin had no effect on the growth of S. alfredii. The saponin solution at 2 g L-1 extracted more Cd and B[a]P than water, saponin enhanced Cd and B[a]P bioavailability in soil and thus promoted their uptake and accumulation in S. alfredii. The activity of B[a]P-16, dehydrogenase and polyphenol oxidase in co-contaminated soil was promoted by growing S. alfredii, and the application of B[a]P-16 and saponins caused a significant (P<0.05) increase in both enzyme activities. The maximum B[a]P removal rate (82.0%) and Cd phytoextraction rate (19.5%) were obtained by co-application of S. alfredii with B[a]P-16 and saponin. The B[a]P-16 and plant promoted biodegradation were the predominant contributors towards removal of B[a]P from soil. A significant (P<0.05) synergistic effect of B[a]P-16 and saponin on B[a]P and Cd removal efficiency was observed in this study. It is suggested that planting S. alfredii with application of B[a]P-16 and saponin would be an effective method for phytoremediation of soil co-contaminated with Cd and PAHs.

Keywords: Biodegradation; Co-contamination; PAHs; Phytoextraction; Saponin; S. alfredii

Journal Pre-proof 1. Introduction Heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) co-contamination in soil is a widespread environmental problem due to their toxic effects on both ecosystem and humans health (Ye et al., 2017; Sarma et al., 2019). Compared with single contamination, remediation of co-contaminated soil is more complicated and difficult, due to the different physical and chemical properties of diverse pollutants, the interactions among multiple pollutants, as well as the disparate remediation pathways for different pollutants (Chen et al., 2016; Yoo et al., 2017). Therefore, it is urgent to develop efficient and environmental-friendly methods to simultaneously remove HMs and PAHs from co-contaminated soils (Tang et al., 2018). However, most previous and current researches have paid their attention to the remediation of HMs or PAHs single contaminated soils (Kuppusamy et al., 2017). Phytoremediation has been recognized as a cost-efficient and environmental-friendly technology that uses plants to remove, degrade, assimilate, metabolize or detoxify metals and organic contaminants (Cristaldi et al., 2017; Sarwar et al., 2017). The feasibility of phytoremediation for soil contaminated by HMs or PAHs has extensively been investigated (Cristaldi et al., 2017). A lot of researches have addressed the uptake and accumulation of metal by both hyperaccumulating and non-hyperaccumulating plants, such as Thlaspi caerulescens (Lombi et al., 2001), Pteris vittata (Shelmerdine et al., 2009), Alyssum murale (Bani et al., 2007), Solanum nigrum (Sun et al., 2008), Nicotiana tabacum (Evangelou et al., 2006), Helianthus annuus L. (Hattab-Hambli et al., 2016) and hybrid poplar (Populus deltoids×Populus nigra) (Wu et al., 2010). On the other hand, the plant-enhanced dissipation

Journal Pre-proof of PAHs in PAH-contaminated soil was also reported by many researchers (Alagic et al., 2016; Afegbua and Batty, 2018). Furthermore, an accelerated reduction of PAHs was observed in the rhizosphere of remediation plants (Sun et al., 2010; Kotoky et al., 2018). However, few studies have focused on phytoremediation of the HMs and PAHs co-contaminated soils, especially for the effectiveness and processes of remediation using hyperaccumulators (Wang et al., 2012; Liu et al., 2017a). In natural environments, PAHs have a hydrophobic property, which leads to their decreased solubility in water (Bacosa and Inoue, 2015). In addition, both PAHs and HMs can be adsorbed strongly to the soil matrix, resulting in low bioaccessibility and bioavailability of the pollutants in soils, thus limiting the phytoremediation efficiency (Cristaldi et al., 2017; Lu et al., 2019). Surfactant can increase the solubilization of PAHs and HMs by partitioning them into the hydrophobic cores of micelles or chelating with metal by the oxygen-containing groups, thus enhancing the accessibility of pollutants in the soil to the plants (Almansoory et al., 2015; Liao et al., 2015; Liu et al., 2017a). However, synthetic chemical surfactants such as Tween80, sodium dodecyl sulfonate (SDS) and ethylene diamine tetraacetic acid (EDTA) had phytotoxicity and potential environmental risks (Gao et al., 2008; Cheng et al., 2018). Recently, biosurfactants (BS) with low toxicity, higher biodegradability, better environmental compatibility, have attracted more and more attention in phytoremediation. For example, Cay (2016) suggested that an increase in tea saponin concentration significantly increased Cd phytoextraction by Amaranthus caudatus. Liao et al. (2016) showed that biosurfactants (rhamnolipid and soybean lecithin) increased the removal of total petroleum hydrocarbons from the soil. On the other hand, plant-associated bacteria

Journal Pre-proof can enhance degradation of organic pollutants through direct metabolism or co-metabolism (Sun et al., 2017), contributing to improve organic pollutants and trace elements uptake by plants, and thus increase the efficiency of phytoremediation (Doty, 2008; Fan et al., 2018). To date, a variety of PAH-degrading microorganisms, as well as plant growth promoting bacteria have been isolated from contaminated soils (Hou et al., 2015; Saleem et al., 2018). The successful use of such microbial isolates, together with plants, in the remediation of PAHs or HMs-contaminated soils has been well documented (Sessitsch et al., 2013; dos Santos and Maranho, 2018). However, few studies have focused on synergistic effect of biosurfactants and microbes on the performance of phytoremediation, especially for the HMs and PAHs co-contaminated soils (Ye et al., 2017). Sedum alfredii (Crassulaceae) is a Cd hyperaccumulator with significant potential for Cd remediation (Li et al., 2012), which also showed high tolerance to PAHs and grew well in pyrene (184.9 mg kg-1) contaminated soil (Wang et al., 2012). Saponin is a representative plant-derived biosurfactant, which has the potential for simultaneous removal of both HMs and PAHs from the combined contaminated soils (Liu et al., 2017a). However, the use of S. alfredii to remediate Cd and PAH co-contaminated soils has not been well documented . It is unclear whether a combination of saponin and PAH-degrading bacterium might be advantageous for remediation of Cd and PAH co-contamination. Therefore, the aim of this study was to investigate the effectiveness of saponin-microbial assisted phytoremediation for soils co-contaminated with Cd and benzo(a)pyrene (B[a]P), using co-application of S. alfredii with B[a]P-degrading bacteria. B[a]P was chosen as the representative PAHs because it is classified as a priority contaminant due to its potential carcinogenicity,

Journal Pre-proof teratogenicity or acute toxicity. Results obtained from this study will provide practical insights on phytoremediation of Cd and B[a]P co-contaminated soils. 2. Materials and methods 2.1. Plant material and soil preparation The hyperaccumulator S. alfredii was collected from a Pb/Zn mining area in Quzhou (29°17′N, 118°56′E), China. Seedlings of S. alfredii were cultivated according to Li et al. (2013). Healthy and uniform seedlings were chosen and grown for two weeks in the basic nutrient solution. The paddy soil used in the pot experiment was collected from the surface layer (0–20 cm) from Fuyang County of Hangzhou, China. The soil had been contaminated with Cd and other metals due to mining activities and not suitable for crop growth. Soil samples were air-dried and sieved (< 2 mm). Soil properties were determined using standard analytical procedures (Lu, 1999). The pH of the soil was 6.72, the soil organic matter content was 20.3 g kg-1, the cation exchange capacity (CEC) was 9.2 cmol kg-1, and the composition of the soil was 37.0% sand, 40.4% silt, and 22.6% clay. The initial concentration of PAHs was undetectable. Benzo[a]pyrene was purchased from Sigma Aldrich Chemical (St. Louis, MO, USA). About 500 g soil was spiked with B[a]P dissolved in acetone and mixed thoroughly, after acetone was completely evaporated, the spiked soil was mixed evenly with approximately 4500 g and 45 kg of clean soil step by step. The contaminated soils were aged for 6 months. The total concentrations of Cd and B[a]P in the soils before the experiment were 10.5 mg kg-1 and 5.6 mg kg-1, respectively. The water extractable Cd and B[a]P in soil were 0.52 mg kg-1and 36 μg kg-1, respectively.

Journal Pre-proof 2.2. Bacterial incubation The B[a]P-degrading bacterial strain used in the experiment was isolated from PAH-contaminated soil, as described by Miller et al. (2004). The strain was isolated by enrichment culture using B[a]P as the sole source of carbon, and was identified as Ochrobactrum intermedium B[a]P-16. The dissipation rates of B[a]P in the medium by B[a]P-16 was 32.6% over a period of 15 days. The isolated B[a]P-degrading bacterium was then cultured in a liquid medium (Sun et al., 2017) in the presence of 10 mg L−1 B[a]P. The bacteria cells were harvested using a centrifuge at 6000 rpm and washed twice with decreasing concentrations of the medium solution, and re-suspended in 5 ml of sterilized deionized water. The strain showed the minimum inhibitory concentration of 100 mg L-1 for Cd. 2.3. Extraction experiments Extraction experiments were conducted in glass flasks. Saponin solutions at 0, 0.5, 1, 2 and 4 g L-1 were prepared in distilled water above the critical micellar concentration (CMC) (Davin et al., 2018). Each extraction was repeated five times. Briefly, 5 g of spiked dry soil was extracted using magnetic stirring with 10 mL of saponin solutions for 24 h in the dark. The aqueous phase was recovered by filtration. Cd concentration in the aqueous solution was determined by inductively coupled plasma optical emission spectrometer (ICP-OES, Thermo Scientific iCAP 6000). For B[a]P analysis, the aqueous phase was extracted twice with hexane for 1 h, and separated in a funnel. The organic phase was dried on anhydrous Na2SO4, eliminated with a rotative evaporation, and the residue was dissolved in hexane with a final volume of 1.0 ml for gas chromatography (GC, Agilent 6890N) analysis.

Journal Pre-proof 2.4. Pot experimental design Plastic pots were used for the present study and 2.5 kg of spiked soil was added in each pot. The experiment consisted of eight treatments: control (CK), bacterial inoculation (B), saponin (S), bacterial inoculation+saponin (BS), S. alfredii cultivation (SC), S. alfredii cultivation+bacteria (SCB), S. alfredii cultivation+saponin (SCS), and S. alfredii cultivation+bacteria+saponin (SCBS). After pre-culturing for three weeks in hydroponics solution, three S. alfredii plants were transplanted into each pot. The pots were placed randomly in a greenhouse where relative humidity was maintained at 75%, temperature at 26/20 ◦C (day/night). Soil moisture was maintained at 65% of field capacity by weight. Two days before planting, the inoculum suspension was transferred into the soil of corresponding pots and homogenized to obtain an inoculum density of about 1.5 × 107 colony-forming units (CFU) g-1 soil. After 30 days of growth, 200 mL saponin solution at 2 g L−1 was added to soil in the pots as settings before Soil solution samples to monitor Cd and B[a]P concentrations were collected 1 d before harvest using a soil moisture sampler installed in the pots. The plants were harvested after 90 d of growth and washed with deionized water, and separated into roots and shoots. 2.5. Bacterial quantification and enzyme activity The number of B[a]P-degrading bacteria in the soil was determined at the end of the experiment by conventional plate spread method, using the mineral medium described for bacterial culture, which contained 10 mg L-1 B[a]P. An aliquot of 0.1 mL diluted culture was spread over agar plates, and each dilution had three replicates. Colonies were counted after 5 days of incubation at 25 ℃, the number of bacteria was expressed as CFU g-1 dry soil.

Journal Pre-proof Dehydrogenase activity was measured as described previously (Davin et al., 2018). Briefly, moist soil (2 g) was treated with 2.5 mL of 1% triphenyl tetrazolium chloride (TTC)-Tris buffer (pH 7.6), and then incubated at 37 ℃ in dark for 24 h. Dehydrogenase activity was expressed in terms of triphenyl formazan (TPF) formed from TTC (μg TPF g−1 soil h−1). Soil polyphenol oxidase activity was determined by the colorimetric method described by Chen et al. (2016). The mixture of 1.0 g soil and 10 mL 1% pyrogallic acid was incubated at 30◦ C for 2 h, and 4 mL citric–phosphoric acid buffer (pH 4.5) was added to the mixture. The purpurigallin produced was extracted with ether, and then measured by a spectrophotometer at 430 nm. 2.6. Analysis of B[a]P and Cd in soils and plants Extraction of B[a]P in soils and plants was performed according to (Sun and Zhou, 2016). Briefly, 2.0 g of dry soil samples were extracted using 20 mL of dichloromethane for 2 h in an ultrasonic bath (the system was <40 oC). As for plant tissues, about 0.5 g of dried plant fragment was mixed into 10 ml solution of methanol, and extracted by ultrasonication for 30 min for 3 successive extractions. Then, 2ml of the supernatant was filtered through 2 g of silica gel column and was eluted consecutively with 10 ml of the mixture of 1:1 hexane and dichloromethane (v/v). The extract was concentrated by evaporation of the dichloromethane under a stream of nitrogen and the residue was dissolved in hexane with a final volume of 1.0 ml for GC analysis. B[a]P dissipation rate in soil was calculated as the B[a]P concentration difference before and after treatment versus the initial B[a]P concentration in soil. For B[a]P determination in soil solution, supelco-C18 solid phase extraction column was used. Methanol (10 mL) was added to 20 mL soil solution, after

Journal Pre-proof oscillating and mixing, the extraction was carried out on the solid phase extraction device at a rate of 2.0 mL/min, then the column was eluted consecutively with 5 ml of the mixture of 1:1 hexane and dichloromethane (v/v). B[a]P in the extract was determined using GC analysis as described above. Total Cd in the soil was analyzed after digestion with concentrated HNO3 (16 mol L-1), HClO4 (12 mol L-1) and HF (40%) (5:1:1 v/v/v). Plants were digested with a mixture of concentrated HNO3 (16 M) and HClO4 (12 M) (5:1 v/v) in PTFE vessels. The digest was transferred to a 50 ml volumetric flask, filled to 50 mL and filtered. Cd concentrations were determined using ICP-OES (Thermo Scientific iCAP 6000). Sample replicates, reagent blanks, and standard reference materials (GBW07440, the National Research Center for Certified Reference Materials of China) were included in each batch of analysis to ensure the quality of analysis. The recovery rate was 98% to 102.8%. The bioconcentration factor (BCF) was calculated as the Cd concentration in shoots versus the Cd concentration in soil. Phytoextraction rate was calculated as total Cd uptake by S. alfredii to the total Cd content in soil of each pot. Cd concentrations in soil solution were also determined as described above. 2.7. Statistical analysis All data were processed with Excel 2013 (Microsoft, Redmond, WA, USA). Data are means of three replicates, error bars indicate standard deviations. Statistical analysis was conducted using SPSS software Version 19.0 for Windows (SPSS, USA). Means of significant difference were separated by Duncan’s multiple range test at p < 0.05. 3. Results and discussions 3.1 Effects of saponin and B[a]P degrading bacterium on plant growth

Journal Pre-proof The accumulation and removal efficiency of pollutants mainly depend upon the biomass of plants (Li et al., 2012). Under different treatments, S. alfredii grew normally without showing any toxic symptoms, indicating that S. alfredii has high tolerance to Cd and B[a]P toxicity. This tolerance may be attributed to the low bioavailability of B[a]P due to its high octanole water partition coefficient (log Kow=6.04) (Gutierrez-Gines et al., 2014). In this study, B[a]P-degrading bacteria inoculation improved the growth of S. alfredii significantly (P < 0.05; Fig. 1). Compared with the SC treatment, bacterial inoculation showed a positive effect on the root and shoot biomass of S. alfredii (increased by 22.1% and 20.5% for SCB treatment and by 24.1% and 23.4% for SCBS treatment, respectively). These results suggest that B[a]P-16 could stimulate S. alfredii growth under metal as well as B[a]P stress. On the one hand, we speculate that B[a]P-16 accelerated the B[a]P degradation thus reduced the negative effects of B[a]P on plant growth (Bacosa and Inoue, 2015; Alagic et al., 2016). Furthermore, B[a]P-16 increased the nutrient uptake due to changes in rhizospheric environment (Gao and Zhu, 2004; Sun et al., 2016). For SCS treatment, however, no significant difference was observed as compared with SC treatment, indicating that the application of saponin, a biosurfactant, had no significant effect on the growth of S. alfredii. Previous studies showed that synthetic surfactants have certain toxicity and may affect plant growth (Bani et al., 2007; Cheng et al., 2018). For example, Triton x-100, an nonionic surfactant, has a toxic effect on the growth of Brassica juncea (Di Gregorio et al., 2006). Sudova et al. (2007) found that 2.5 mmol kg-1 EDDS significantly decreased plant biomass as well as reduced the development of AM fungi in the rhizosphere of tobacco. In this study, neither saponin nor its combined application with B[a]P-16 affected the growth of plants.

Journal Pre-proof This result indicated that saponin had no negative effects on S. alfredii and showed potential in the performance of phytoremediation. It should be pointed out that no significant difference between SCB and SCBS treatment was observed, which suggested that there was no interactive effect of bacterial inoculation and saponin on plant biomass (Fig. 1). 3.2. Accessibility of B[a]P and Cd in contaminated soils The extractions of Cd (a) and B[a]P by different saponin solutions are shown in Fig. 2a,b. Statistical analysis showed significant differences among different extraction solutions. For Cd (Fig. 2a), the concentration in solution increased significantly with increasing concentration of saponin from 0 to 4 g L-1. Compared with distilled water, the concentration of Cd in the 2 g L-1 and 4 g L-1 saponin solution was increased by 57.8% and 60.1%, respectively, however statistically non-significant difference was observed between these two treatments. Hong et al. (2002) found that saponin was effective in removing the exchangeable and carbonated fractions of heavy metals from soils in a washing process. Recently, Wang et al. (2016) demonstrated that saponin significantly enhanced the exchangeable fraction of Cd in soil. In accordance with previous study, our study showed that saponin could enhance solubilization of Cd in soil and thus increase its bioavailability. However, previous studies indicated that the efficiency of saponin in enhancing Cd desorption from soil was not ascribing to the ionic bond or surfactant micelle (Song et al., 2008). Therefore, the enhanced solubilization of Cd may be resulted from the carboxyl groups in saponin which could form complex with Cd in aqueous solution (Almansoory et al., 2015; Wang et al., 2016). Similarly, the saponin solutions provided a significantly better extraction of B[a]P than water (Fig. 2b). Compared with distilled water, 2 g L-1 saponin

Journal Pre-proof solution showed the highest concentration of B[a]P (increased by 87.8%). It is well known that surfactant can enhance the solubilization of hydrophobic organic compounds (HOCs) in the micelles when surfactant concentration above the critical micelle concentration (CMC) (Liu et al., 2017b). Zhou et al. (2011) have shown that the apparent solubilities of acenaphtene, phenanthrene and pyrene increased linearly with the saponin concentration above the CMC. Recently, Davin et al. (2018) found that 4 g L-1 saponin (130 fold of CMC) extracted significantly more B[a]P than water in aged-contaminated soils. However, our result suggested that in Cd and B[a]P co-contaminated soil, 2 g L-1 saponin showed greater solubilisation enhancements on B[a]P than 4 g L-1 saponin. This may be attributed to the fact that in some cases if the saponin concentration is too high, then micelles could raise the apparent sorption of the organic pollutants onto soil particles (Davin et al., 2018). In pot experiment, the concentrations of Cd and B[a]P in soil solution were determined after cultured for 90 d (Fig. 2c,d). Compared with no plant treatments, the Cd concentration decreased significantly after plant growth, which could be attributed to the tremendous Cd phytoextraction ability of S. alfredii (Li et al., 2012). For Cd concentration, no difference was observed between CK and B, as well as SC and SCB (Fig. 2c), suggesting that B[a]P-16 inoculation had no effect on Cd availability. However, in pots without plants, the application of saponin (S and BS treatments) showed a significant increase in Cd concentration, this result was consistent with extraction experiment and further confirmed that saponin could enhance solubilization of Cd in soil (Almansoory et al., 2015; Wang et al., 2016). For SCS and SCBS treatments, Cd concentration in soil solution decreased significantly (by 40.9% and 41.8%, respectively) as compared with single S. alfredii treatment (SC), indicating that

Journal Pre-proof saponin promoted the uptake of Cd in S. alfredii and resulted in enhanced phytoextraction efficiency. As for B[a]P, in pots without plants, B[a]P-16 inoculation resulted in a significant decrease in B[a]P concentration (P<0.05), whereas saponin addition showed an opposite effect on B[a]P availability. After plants growth, the addition of B[a]P-16 resulted in a greater decrease, implying that B[a]P-16 could effectively degrade B[a]P. For SCBS treatment, the concentration of B[a]P was the lowest, this phenomenon indicated that there was synergistic effect of B[a]P-16 and saponin on B[a]P dissipation. 3.3 Microbial population and activities of enzymes in co-contaminated soils The activity of microorganisms in contaminated soils is very important for PAHs dissipation, which were considered as biological indicators for soil remediation efficiency (Ramadass et al., 2018). Almost no B[a]P-16 were detected in the soil without bacterial inoculation (Fig. 3). B[a]P-16 was more in the soil planted with S. alfredii than in the un-planted soil (Fig. 3a). The population sizes of B[a]P-16 in the SCB and SCBS treatments were 1.32×10-7 and 1.22×10-7 CFU g-1 dry soil, respectively, which were significantly (P<0.05) higher than that of the un-planted soil (1.04×10-7 and 0.99×10-7 CFU g-1 dry soil for B and BS treatments, respectively). This result was in agreement with previous studies on Lolium multiflorum (Wang et al., 2016) and Pteris vittata (Sun et al., 2017), and indicated that the activity of PAH-degrading bacterium was promoted by plants due to the enhanced release of plant-derived carbon and nitrogen sources through root exudation, which provided numerous organic substrates to B[a]P-16 (Sun et al., 2016). Our result also suggested that S. alfredii combined with B[a]P-16 is an effective method for the phytoremediation of B[a]P-Cd co-contaminated soils.

Journal Pre-proof Soil microbial activities and community diversities are critical indicators of the ecological health of soils, thus it is important to evaluate the dynamics of the whole microbial community rather than just PAHs degrading microbes during remediation (dos Santos and Maranho, 2018). Dehydrogenase activity in soil is an estimation of total oxidative potential of the microbes which has been used as an index for monitoring microbial activity (Ramadass et al., 2018). The dehydrogenase activity was higher in soil planted with S. alfredii than in the un-planted soil (Fig. 3b), which showed a contrary tendency with the level of B[a]P and Cd in soil solution (Fig. 2c,d). The lowest dehydrogenase activity appeared in saponins-treated soils without plant (Fig. 3b), where the B[a]P and Cd concentration in soil solution was the highest. Clearly, dehydrogenase activity correlated well with the bioavailability of pollutants in soil (Chen et al., 2016). The dehydrogenase activity in SCBS treatments increased by 42% compared with SC treatment (Fig. 3b), indicating that the application of B[a]P-16 and saponins caused a significant (P<0.05) increase in dehydrogenase activity. Furthermore, a significant synergistic effect of B[a]P-16 and saponin on dehydrogenase activity was observed (P<0.05). The results on dehydrogenase activity clearly supported the data on B[a]P and Cd removal in different soil treatments (Table 2, Fig.4), and suggested that the dehydrogenase could be considered as an indicator for evaluating bioremediation potential of a soil contaminated with B[a]P and Cd. The polyphenol oxidase (PPO) is one of the most important oxidoreductases in soil involved in the conversion of PAHs in humus. The change in the PPO activity is depicted in Fig. 3c. As shown, the changing trend of PPO activity was similar to dehydrogenase, which showed that the PPO activity was higher in soil planted with S. alfredii than in the

Journal Pre-proof un-planted soil. This result was consistent with that of Liu et al.( 2013) and Chen et al. (2016), indicating that S. alfredii growth enhanced PPO activity and increased the degradation rate of B[a]P in the soil. As expected, the above results confirmed that S. alfredii clearly enhanced the total number of B[a]P-16 (Fig. 3a) due to root exduates secretion which stimulate the growth of specific microbial community in soil (Liu et al., 2013; Ye et al., 2017; Sarma et al., 2019). The PPO activity in SCBS treatment was also the highest among planted treatments (P < 0.05), which was attributed to the synergistic effect of B[a]P-16 and saponin, as well as the rhizospheric effect of S. alfredii. 3.4 Cd phytoextraction in co-contaminated soils Considering the rare change on Cd concentration in soil without plants, we only showed Cd concentrations in S. alfredii after 90 days of transplantation (Table 1). The concentrations of Cd in shoot were much greater than in the root, which was supported by our previous study that S. alfredii showed great Cd uptake and transport ability (Li et al., 2012). B[a]P degradation bacteria had no significant effect on the Cd concentration in shoot. However, compared with the SC treatment, the addition of saponin significantly increased Cd concentrations in both shoot and root (by 11.9% and 13.9% for SCS treatment and by 18.2% and 26.1% for SCBS treatment, respectively). This increase was mainly attributed to the enhanced solubilization of Cd in soil by saponin (Fig. 2c). Previous studies on Saccharum officinarum (Xia et al., 2009) and Lolium multiflorum (Liu et al., 2017a) also found that tea saponin could promote the uptake of Cd under PAHs and Cd combined pollution. However, another study found that surfactant SDS inhibit Cd uptake in soybean plants (Zhang et al., 2008). These contrasting results suggest that the effects of surfactants on the uptake and

Journal Pre-proof accumulation of Cd depend on plant or surfactant type. It should be pointed out that the interaction between B[a]P-16 and saponins had no significant effect on the Cd concentration in the shoot (Table 1). The high concentrations of high heavy metals accumulated in the aerial parts of plants are ideal for the removal metals from soils by simply harvesting aboveground parts (Sun et al., 2008; Cay, 2016). After 90 days, the accumulation of Cd in the shoot of S. alfredii was extraordinarily higher (45-50 folds) than that in the root (Table 1). B[a]P-16 inoculation significantly enhanced Cd accumulation in the shoot due to the increased shoot biomass (Fig. 1b). At the same time, saponin application also increased Cd accumulation in the shoot by increasing Cd concentrations in the shoot. Interestingly, a significant (P<0.05) synergistic effect of B[a]P-16 and saponin on Cd accumulation in the shoot of S. alfredii was observed, implying that a combination of these two applications showed great potential in increasing phytoextraction of Cd from soil. Bioaccumulation factor (BF) is an important parameter to assess the effectiveness of a plant to concentrate pollutant into aerial part, and translocation factor (TF) is used to measure the effectiveness of a plant in transferring a pollutant from roots to shoots (Sun et al., 2011). As shown in Table 2, for all treatments, the BF and TF were 3.64-3.84 and 47.0-53.7, indicating that S. alfredii was efficient in taking up Cd from soil and transfer them to the aboveground parts. B[a]P-16 and saponin had no effect on TF, however, saponin addition increased BF significantly (P < 0.05). The phytoextraction rate (PR) can reflect the amount of a pollutant extracted by a plant from the soil, thus indicating the phytoextraction efficiency. As listed in Table 2, the PR values of Cd were 13.3-19.5%, which was much

Journal Pre-proof greater than other plants, such as Tagetes patula (Sun et al., 2011), ryegrass (Chen et al., 2016). Therefore, S. alfredii would be more promising to phytoextract Cd from contaminated soils. Furthermore, combination of B[a]P-16 and saponin increased PR values significantly (P < 0.05), as compared with B[a]P-16 and saponin single treatments. 3.5 B[a]P degradation in Cd co-contaminated soils Table 3 exhibits B[a]P uptake and partitioning in different tissues of S. alfredii. Concentrations of B[a]P in the root were much greater than those in the shoot, which was in accordance with previous study and confirmed that S. alfredii showed weak ability of B[a]P uptake and translocation as compared with Cd (Chen et al., 2016). B[a]P-16 had no effect on B[a]P concentration in both shoot and root, on the contrary, the addition of saponin significantly increased B[a]P concentrations in both shoot and root (P < 0.05). After 90 days, the accumulation of B[a]P in the shoot and root of S. alfredii were 15.7-33.8 μg pot-1 and 2.19-4.44 μg pot-1, respectively. The accumulation of B[a]P in S. alfredii was relatively small, which accounted for 0.13–0.28% of total B[a]P in soil. Other studies on Juncus subsecundus (Zhang et al., 2012), Tagetespatula and Mirabilisjalapa (Sun and Zhou, 2016) and Lolium multiflorum (Liu et al., 2017a) also found that the accumulation of PAHs in shoots was relatively small. Our results suggested that the uptake of B[a]P by plants was not a major pathway for B[a]P removal compared with biodegradation of B[a]P (Fig. 4). Since the extraction of B[a]P by plant failed to explain the dissipation of B[a]P, we calculated the B[a]P removal rate based on the residual concentration of B[a]P in the soils at the end of the experiment (Fig. 4). Compared with CK, B[a]P-16 inoculation significantly decreased (P < 0.05) B[a]P concentrations in soil (by 45.1%, 56.1%, 68.2 and 81.4% for B,

Journal Pre-proof BS, SCB and SCBS treatments, respectively). The dissipation of B[a]P from soils was significantly influenced by B[a]P-16 inoculation after 90 days of plant growth. On average, 47.0-82.0% of B[a]P was removed in B[a]P-16 inoculation treatments, whereas only 2.7-31.9% of B[a]P was removed in treatments without B[a]P-16. This significant difference further confirmed the efficient degradation ability of B[a]P-16. At the same time, due to the enhanced solubilization of B[a]P (Fig. 2), saponin application significantly promoted B[a]P dissipation, as also found by other studies (Wang et al., 2016; Davin et al., 2018). The B[a]P removal rate in SCBS treatment (82.0%) was higher (P < 0.05) than in SCB (69.3%) and SCS (31.9%) treatments, this phenomenon also confirmed that there was synergistic effect of B[a]P-16 and saponin on B[a]P dissipation. In addition, plant-enhanced B[a]P dissipation was observed in our study, for example, the B[a]P removal rate in SCB treatment was increased by 47.5%, as compared with single B[a]P-16 inoculation, indicating that S. alfredii play an important role in removing B[a]P from soils. The release of plant-derived carbon and nitrogen sources through root exudation provides a heterogeneous rhizosphere environment, such as increased microbial activity and functional diversity (Liu et al., 2014), catabolic enzymes activity (Chen et al., 2016; Davin et al., 2018), as well as bioavailability of pollutants (Sun et al., 2010), which contributed to the biodegradation of B[a]P. The dissipation mechanisms of PAHs in soils are volatilization, leaching, plant uptake and metabolism, biodegradation and incorporation into soil organic material (Zhang et al., 2012). For 4–5 rings of PAHs, the leaching and volatilization was negligible due to low solubility (Reilley et al., 1996). Therefore the dissipation of PAHs mainly derive of plant accumulation and promoted biodegradation. In present study, the promoted biodegradation of B[a]P may

Journal Pre-proof be attributed to both B[a]P-16 inoculation and plant growth. Thus the loss of B[a]P could be calculated as follows: L= Lbb + Lpb + Lpac where Lbb and Lpb were the loss of B[a]P by B[a]P-16 and plant-promoted biodegradation, Lpac refers B[a]P uptake and accumulation in plants. As listed in Table 4, in different treatments, the dissipation of B[a]P was totally different, which was mainly contributed to B[a]P-16 and plant-enhanced biodegradation (Sun and Zhou, 2016), accounting for 61.0-66.3% and 29.7–87.1%, respectively. However, the amount of Lpac was 0.21-0.72% of dissipation of B[a]P, suggesting that plant uptake and metabolism in the present study was not significant. 4. Conclusions In this study, a novel B[a]P-degrading bacterium Ochrobactrum intermedium B[a]P-16 was identified which showed a positive effect on the plant growth of S. alfredii. On the other hand, saponin could enhance Cd and B[a]P solubilization and bioaccessibility in soil and thus promoted their uptake and accumulation in S. alfredii. The maximum B[a]P removal rate (82.0%) and Cd phytoextraction rate (19.5%) were obtained by co-application of S. alfredii with B[a]P-16 and saponin. The B[a]P-16 and plant promoted biodegradation were the predominant contributors towards removal of B[a]P in the co-contaminated soil. The results indicate that combined use of S. alfredii, Ochrobactrum intermedium B[a]P-16 and saponin would be an effective method for phytoremediation of soils cocontaminated with Cd and PAHs. Acknowledgments This research was supported by the National Natural Science Foundation of China

Journal Pre-proof (Grant Nos. 41807126, 41671315 and 41977107), Natural Key Research and Development Projects of China (2016YFD0800802, 2018YFD0800600), the Applied Basic Research Programs of Sichuan Science and Technology Department (2018JY0002) and Zhejiang Provincial Natural Science Foundation of China (No. LZ18D010001). References Afegbua, S.L., Batty, L.C., 2018. Effect of single and mixed polycyclic aromatic hydrocarbon contamination on plant biomass yield and PAH dissipation during phytoremediation. Environ. Sci. Pollut. Res. 25, 18596-18603. Alagic, S.C., Jovanovic, V.P.S., Mitic, V.D., Cvetkovic, J.S., Petrovic, G.M., Stojanovic, G.S., 2016. Bioaccumulation of HMW PAHs in the roots of wild blackberry from the Borregion (Serbia): Phytoremediation and biomonitoring aspects. Sci. Total Environ. 562, 561-570. Almansoory, A.F., Abu Hasan, H., Idris, M., Abdullah, S.R.S., Anuar, N., 2015. Potential application of a biosurfactant in phytoremediation technology for treatment of gasoline-contaminated soil. Ecol. Eng. 84, 113-120. Bacosa, H.P., Inoue, C., 2015. Polycyclic aromatic hydrocarbons (PAHs) biodegradation potential and diversity of microbial consortia enriched from tsunami sediments in Miyagi, Japan. J. Hazard. Mater. 283, 689-697. Bani, A., Echevarria, G., Sulce, S., Morel, J.L., Mullai, A., 2007. In-situ phytoextraction of Ni by a native population of Alyssum murale on an ultramafic site (Albania). Plant Soil 293, 79-89. Cay, S., 2016. Enhancement of cadmium uptake by Amaranthus caudatus, an ornamental plant, using tea saponin. Environ. Monit. Assess. 188. Chen, F., Tan, M., Ma, J., Zhang, S.L., Li, G., Qu, J.F., 2016. Efficient remediation of PAH-metal co-contaminated soil using microbial-plant combination: A greenhouse study. J. Hazard. Mater. 302, 250-261. Cheng, M., Zeng, G.M., Huang, D.L., Yang, C.P., Lai, C., Zhang, C., Liu, Y., 2018. Tween 80 surfactant-enhanced bioremediation: toward a solution to the soil contamination by hydrophobic organic compounds. Crit. Rev. Biotechnol. 38, 17-30.

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Journal Pre-proof and polynuclear aromatic hydrocarbons (PAHs). Geoderma 175, 1-8. Zhou, W.J., Yang, J.J., Lou, L.J., Zhu, L.Z., 2011. Solubilization properties of polycyclic aromatic hydrocarbons by saponin, a plant-derived biosurfactant. Environ. Pollut. 159, 1198-1204.

Journal Pre-proof Table 1 Cd concentration and accumulation in S. alfredii shoot and root after 90 days of transplantation. Cd concentration （mg kg-1 DW）

Cd accumulation（mg pot-1）

shoot

root

shoot

root

SC

493.7 ± 29.6 b

132.1 ± 12.8 b

3.31 ± 0.26 c

0.068 ± 0.013 c

SCB

495.0 ± 16.7 b

125.1 ± 17.3 b

4.14 ± 0.41 b

0.079 ± 0.011 b

SCS

552.3 ± 45.3 a

150.5 ± 12.3 a

3.84 ± 0.48 b

0.078 ± 0.006 b

SCBS

583.5 ± 15.2 a

166.6 ± 6.3 a

5.00 ± 0.43 a

0.107 ± 0.011 a

Treatments

Values are means ± standard deviations (n = 3). Means with different letters in the same column are significantly different from each other (P < 0.05).

Journal Pre-proof Table 2 Translocation factor, bioaccumulation factor and phytoextraction rate of Cd in different treatments. Treatments

Translocation factor

Bioaccumulation factor

Phytoextraction rate（%）

SC

3.67 ± 0.27 a

47.0 ± 2.82 b

13.3 ± 1.06 c

SCB

3.84 ± 0.43 a

47.1 ± 1.59 b

16.1 ± 1.61 b

SCS

3.64 ± 0.15 a

52.6 ± 4.43 a

14.9 ± 1.86 b

SCBS

3.45 ± 0.13 a

55.7 ± 1.45 a

19.5 ± 1.64 a

Values are means ± standard deviations (n = 3). Means with different letters in the same column are significantly different from each other (P < 0.05).

Journal Pre-proof Table 3 B[a]P concentration and accumulation in S. alfredii shoot and root after 90 days of transplantation. B[a]P concentration （mg kg-1 DW）

B[a]P accumulation（μg pot-1）

shoot

root

shoot

root

SC

2.26 ± 0.31 b

4.24 ± 0.39 b

15.7 ± 2.61 c

2.19 ± 0.29 c

SCB

2.17 ± 0.20 b

3.98 ± 0.36 b

18.2 ± 0.95 c

2.50 ± 0.17 c

SCS

4.14 ± 0.28 a

6.71 ± 0.51 a

28.7 ± 2.45 b

3.50 ± 0.37 b

SCBS

3.94 ± 0.40 a

6.92 ± 0.47 a

33.8 ± 4.84 a

4.44 ± 0.48 a

Treatments

Values are means ± standard deviations (n = 3). Means with different letters in the same column are significantly different from each other (P < 0.05).

Journal Pre-proof Table 4 Contribution of Ochrobactrum intermedium B[a]P-16 and S. alfredii to the removal of B[a]P in soils after 90 days of transplantation. L

Lbb

Lpb

Lpac

(mg pot-1)

(mg pot-1)

(mg pot-1)

(μg pot-1)

SC

3.02

-

2.63

17.89

SCB

9.71

6.44

3.13

SCS

4.48

-

SCBS

11.49

7.01

Treatments

Lbb/L (%)

Lpb/L (%)

Lpac/L (%)

-

87.09

0.55

20.70

66.32

32.23

0.21

2.90

32.20

-

64.73

0.72

3.41

38.24

61.01

29.68

0.33

Lbb were calculated by subtracting the corresponding data without B[a]P-16 from L. Lpb were calculated by subtracting the corresponding data without plant from L.

Journal Pre-proof Figure legends Fig. 1. Root (a) and shoot (b) dry weight of S. alfredii grown in Cd and B[a]P co-contaminated soil with different treatments: S. alfredii cultivation (SC), S. alfredii cultivation+bacteria (SCB), S. alfredii cultivation+saponin (SCS), and S. alfredii cultivation+bacteria+saponin (SCBS). Means with different letters indicate significant differences among treatments at P < 0.05. Error bars represent ± SD (n = 3). Fig. 2. Accessibility of Cd and B[a]P in contaminated soils. Cd (a) and B[a]P (b) extractions by different saponins solutions. Cd (c) and B[a]P (d) in soil solution after plant growth. Means with different letters indicate significant differences among treatments at P < 0.05. Error bars represent ± SD (n = 3). Fig 3. B[a]P-16 number (a), dehydrogenase activity (b) and polyphenol oxidase activity (c) in Cd and B[a]P co-contaminated soil at the end of the experiment with different treatment: control (CK), bacterial inoculation (B), saponin (S), bacterial inoculation+saponin (BS), S. alfredii

cultivation

(SC),

S.

alfredii

cultivation+bacteria

(SCB),

S.

alfredii

cultivation+saponin (SCS), and S. alfredii cultivation+bacteria+saponin (SCBS). Means with different letters indicate significant differences among treatments at P < 0.05. Error bars represent ± SD (n = 3). Fig. 4 The residual B[a]P in the soils (a) and B[a]P removal rate (b) at the end of the experiment with different treatment: control (CK), bacterial inoculation (B), saponin (S), bacterial inoculation+saponin (BS), S. alfredii cultivation (SC), S. alfredii cultivation +bacteria

(SCB),

S.

alfredii

cultivation+saponin

(SCS),

and

S.

alfredii

cultivation+bacteria+saponin (SCBS). Means with different letters indicate significant differences among treatments at P < 0.05. Error bars represent ± SD (n = 3).

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Dry biomass (g pot-1)

0.8 0.6

a

a

b

b

0.4 0.2 0.0

10.0 Dry biomass (g pot-1)

(a)

8.0

c

SC

(b)

c

SCB SCS Treatment a b

b

SCBS a a

6.0 4.0 2.0 0.0

c

SC

Fig. 1

c

SCB SCS Treatments

SCBS

0.8

b

c 0.6

90

a

a

(a)

B[a]P concentration (μg kg-1)

Cd concentration (mg kg-1)

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d

0.4 0.2 0.0

Water

0.5

1

2

4

80

a

(b)

ab

b

70 60

c

50

d

40 30 20 10 0

Water

Saponins concentrations (g L-1)

0.5

1

2

4

Saponins concentrations (g L-1)

0.8

(c)

0.6

b

a

a

b c

0.4

c d

d

0.2 0.0

c CK

B

S

BS SC SCB SCS SCBS

Treatments

Fig. 2

B[a]P concentration (μg L-1)

Cd concentration (mg L-1)

70 60 50 40

(d)

a

b

b c

c

30

d

20

f

10 0

e

c CK

B

S

BS SC SCB SCS SCBS

Treatments

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CFU g-1 dry soil ×107

(a) a

1.5 b

a

b

1.0

0.5

0.0

Dehydrogenase activity (μg TPF g-1 dry soil))

300

c

c

c

a

(b) b

250

c

c

200 150

c

d

e f g

100 f 50

Polyphenol oxidase activity (μg g-1 dry soil h-1)

0 160

a

(c) b

120 c

c d

80

f

CK

B

S

BS SC SCB SCS SCBS

Treatments

Fig. 3

b

c

40 0

b

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B[a]P concentration (mg kg-1)

7.0 6.0

a b

5.0 4.0

c

c

d e

3.0

f

2.0

g

1.0 0.0 100 90

B[a]P removal rate (%)

(a)

c

(b)

a

80

b

70

c

60

d

50

e

40 f

30 20 10 0

g

h

c CK

B

S

BS SC SCB SCS SCBS

Treatments

Fig. 4

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Author Contributions Section Qi Tao: Investigation, Writing-Original draft preparation; Jinxing Li: Investigation, Visualization; Yuankun Liu: Investigation, Data Curation; Jipeng Luo: Visualization, Formal analysis; Qiang Xu: Validation; Bing Li: Writing-Reviewing and Editing; Qiquan Li: Data curation; Tingqiang Li: Conceptualization, Writing-Reviewing and Editing, Supervision; Changquan Wang: Project administration,Writing-Reviewing and Editing.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Journal Pre-proof Highlights 

A novel B[a]P-degrading bacterium Ochrobactrum intermedium B[a]P-16 was identified



Inoculation with B[a]P-16 promoted the gorwth of S. alfredii



2g L-1 saponin increased Cd and B[a]P solubilization significantly



Combined use of B[a]P-16 and saponin significantly increased B[a]P and Cd phytoremediation efficiency by S. alfredii



B[a]P-16 and plant promoted biodegradation played predominant role in removal of B[a]P