Bioaugmentation of exogenous strain Rhodococcus sp. 2G can


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Agricultural and Environmental Chemistry

Bioaugmentation of exogenous strain Rhodococcus sp. 2G can efficiently mitigate DEHP contamination to vegetable cultivation Hai-Ming Zhao, Huan Du, Chun-Qing Huang, Sha Li, Xian-Hong Zeng, Xue-Jing Huang, Lei Xiang, Hui Li, Yan-Wen Li, Quanying Cai, Ce-Hui Mo, and Zhenli He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01875 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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Bioaugmentation of exogenous strain Rhodococcus sp. 2G can efficiently mitigate DEHP contamination to vegetable cultivation Hai-Ming Zhao†,‡,§, Huan Du†,§, Chun-Qing Huang†, Sha Li†, Xian-Hong Zeng†, Xue-Jing Huang†, Lei Xiang†, Hui Li†, Yan-Wen Li†, Quan-Ying Cai*,†, Ce-Hui Mo*,†, Zhenli He‡

† Guangdong

Provincial Research Center for Environment Pollution Control and Remediation Materials, College

of Life Science and Technology, Jinan University, Guangzhou 510632, China ‡ Indian

River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida,

Fort Pierce, FL 34945, USA

*Correspondence: [email protected] (Q. Cai); [email protected] (C. Mo); Tel: +86-20-8522-3405 (C. Mo)

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ABSTRACT: This work developed a bioaugmentation strategy that simultaneously reduced soil

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DEHP pollution and its bioaccumulation in Brassica parachinensis by inoculating the isolated

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strain Rhodococcus sp. 2G. This strain could efficiently degrade DEHP at a wide concentration

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range from 50~1600 mg/L and transformed DEHP through a unique biochemical degradation

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pathway that distinguished it from other Rhodococcus species. Besides, the strain 2G colonized well

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in the rhizosphere soil of the inoculated vegetable without competition with indigenous microbes,

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resulting in increased removal of DEHP from soil (~95%) and reduced DEHP bioaccumulation in

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vegetables (~75% in edible part) synchronously. Improved enzyme activities and DOC content in

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the rhizosphere of planting vegetable and inoculating strain 2G were responsible for the high

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efficiency in mitigating DEHP contamination to vegetable cultivation. This work demonstrated a

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great potential application to grow vegetable in contaminated soil for safe food production.

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KEYWORDS: phthalate; microbial degradation; bioaugmentation; soil bioremediation;

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vegetable cultivation

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1. INTRODUCTION

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Phthalic acid esters (PAEs) are widely used as plasticizers in plastic products to increase their

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flexibility and durability.1 They tend to be released into environment and may be ingested by human

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body through various approaches such as dietary and dermal contact.2,3 Di-(2-ethylhexyl) phthalate

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(DEHP), classified as the priority pollutant by many governments and regional organizations, one

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of the PAE compounds widely exists in the various environments.4 It is reported that the suspected

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endocrine-disrupting DEHP can cause hepatocellular tumors and developmental and reproductive

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toxicants.5,6 Because of widespread application of plastic film, wastewater irrigation, fertilizers, etc.

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that usually contain PAEs, elevated concentrations of DEHP were often found in agricultural

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fields.7-10 DEHP concentration in vegetable soil was reported with up to 57.4 mg/kg in Guangzhou,

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China.11 DEHP in soil can be taken up and accumulated by crops, especially for leaf

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vegetables, posing potential harms to the human health through diet.12,13 Our previous investigation

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suggests that Chinese flowering cabbage (Brassica parachinensis) accumulates the highest level of

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DEHP (up to 9.3 mg/kg, DW) in all the investigated crops collected from Pearl River Delta area,

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southern China.8 Therefore, it is of great urgency to eliminate PAEs from agricultural soils for safe

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agricultural product.

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Physico-chemical degradation of DEHP in the natural environment is very difficult.14

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Microbe-mediated biodegradation is the main degradation approach of DEHP, which is the most

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promising method to remediate DEHP-contaminated environments.15,16 To date, many

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DEHP-degrading bacteria have been isolated from soils, activated sludge, water, sediment, etc.,

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with degradation rates of 67-100% within 3-21 days,17-22 but further application potential analysis of

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these bacteria is poorly studied. Considering the poor nutrients, interspecific competition, and

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adverse survival conditions (such as unsuitable temperature and pH) in the actual environments, few 3

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of them showed good application potential.15 Moreover, the isolated strains that not only possess

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high efficiency of DEHP degradation but also ensure complete mineralization are still scarce in

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number, because DEHP has long side chain, high octanol–water partitioning coefficient

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(log Kow = 7.5), and low water solubility that make it recalcitrant to biodegrade.23 Therefore, it is

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urgent to isolate new DEHP-degrading bacteria for enriching strain resources. Besides, the

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application of the isolates in remediating environment contaminated by DEHP should be widely

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conducted for their real practice.

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Here, an effective DEHP degrading bacterium Rhodococcus sp. 2G was isolated and identified,

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and its biodegradation characteristics were investigated. Further, pot experiments were conducted to

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evaluate the interactions between the inoculated strain and the rhizosphere microorganism of B.

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parachinensis, and their effects on the DEHP elimination from contaminated soil and the growing

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vegetable. Meanwhile, the dynamic change of inoculated Rhodococcus sp. 2G in the rhizosphere

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soil was traced by PCR-DGGE analysis.

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2. MATERIALS AND METHODS

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2.1. Culture enrichment and isolation of the PAEs-degrading bacteria. The used

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chemicals and mediums were presented in Supporting Information. The enrichment-culture

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technique was used to isolate effective DEHP degrading bacteria from activated sludges, which

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were collected from a sewage treatment plant located at Guangzhou, southern China. The initial

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enrichment culture was started by inoculating 100 mL of sterile mineral salt medium (MSM) with 5

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mL activated sludge suspension and 50 mg/L DEHP in culture flasks (250 mL). These flasks were

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incubated for 7 days at 140 rpm and 30°C in an incubator shaker. Every 7 days the suspension (1

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mL) was transferred to new culture flasks containing fresh MSM (100 mL) supplemented with six 4

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increasing concentrations (100, 200, 300, 600, 1200, and 1500 mg/L) of DEHP, respectively. The

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enrichment medium was diluted serially after six rounds of transfers according to our previous

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method,19 and then was spread on MSM-agar plate containing DEHP (200 mg/L) as sole carbon

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source for isolating individual colonies. Finally, an isolated strain that could utilize DEHP for

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growth on MSM, designated as 2G (hereafter, strain 2G), was detailedly characterized in

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Supporting Information.

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2.2. Optimal conditions for DEHP biodegradation. Three main factors including medium

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temperature, pH, and inoculum size were chosen as independent variables based on preliminary

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single-factor experiment. Table S1 shows the range and center point values of three independent

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variables. The biodegradation of DEHP (200 mg/L) in MSM for 5 days was as the dependent

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variable. These factors and their interactive influences on DEHP biodegradation by the strain 2G

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were optimized using response surface methodology (RSM). Statistic Analysis System (SAS)

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software was used to generate a three-variable Box-Behnken design consisting of 15 experimental

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runs with three replicates at the midpoint (Table S2). The data were analyzed through response

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surface regression procedure to fit the following quadratic polynomial equation (Equation 1):

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𝑌𝑖 = 𝑏0 + ∑𝑏𝑖𝑋𝑖 + ∑𝑏𝑖𝑗𝑋𝑖𝑋𝑗 + ∑𝑏𝑖𝑖𝑋𝑖2

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Where Yi is the predicted response, Xi and Xj are variables, b0 is the constant, bi is the linear

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(1)

coefficient, bij is the interaction coefficient, and bii is the quadratic coefficient.

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The interaction between the variables and the responses was analyzed by analysis of variance

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(ANOVA) with a 95% confidence level. Three-dimensional response surface was drawn to

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demonstrate individual and interaction effects of the independent variables on the DEHP

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biodegradation rate.

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2.3. Preparation of the bacterial suspension. After centrifugation at 4600×g for 10 min, 5

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the degradation bacteria with late-exponential growth phase were collected. The bacteria were

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resuspended in the sterile saline (0.9%) after washing three times with the sterile saline. Unless

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otherwise stated, the densities of strain 2G were adjusted with the sterile saline to an OD600

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(optical density measurements at 600 nm) of 0.6. The dilution plate count technique was used to

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quantify colony forming units (CFU/mL) of the suspension.24 One percent of this suspension was

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used as the inocula for further study.

nm

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2.4. Biodegradation of DEHP by the strain 2G at different initial concentrations.

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Biodegradation experiments were performed in MSM containing DEHP at different initial

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concentrations (50-1600 mg/L). Under the optimum conditions, the culture mediums inoculated

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with bacterial suspensions were triplicately incubated for 5 days at 140 rpm, and then were

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collected daily to determine the residues of DEHP in MSM. The mediums without inoculation was

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kept as control. Besides, samples of cell-free filtrates at these different initial concentrations of

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DEHP were collected daily to measure cell growth (OD600) using a spectrophotometer, respectively.

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In the meantime, the intermediates of DEHP (200 mg/L) at days 1, 3, and 5 were identified by

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GC/MS (QP2010 Plus, Shimadzu, Japan).

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The first-order kinetic equation (2) was used to describe the effect of the initial concentrations of

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DEHP on biodegrading rate:

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(2)

𝑙𝑛𝐶 = ―𝑘𝑡 + 𝐴

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Where C is the DEHP concentration at time t; k and A are the slope and constant of first-order

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equation, respectively. The equation (3) was used to calculate the theoretical half-life (t1/2) of

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DEHP: 𝑙𝑛2

(3)

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𝑡1/2 =

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Where k is the rate constant (h-1).

𝑘

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The substrate inhibition model (Eq. (4)) was used to fit the specific degradation rate (q) at different

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initial concentrations:25

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𝑞𝑚𝑎𝑥𝑆

𝑞= (𝑆 +

(4)

( )+𝐾 ) 𝑆2 𝐾𝑖

𝑆

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Where S and qmax are the substrate concentration and maximum specific growth rate,

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respectively; KS and Ki are the constants of substrate affinity and inhibition, respectively. The model

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and kinetics parameters were analyzed using GraphPad Prism software.

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2.5. Pot experiments. To evaluate the effects of inoculated strain 2G on DEHP residues in soil

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and vegetable, soil pot experiments were conducted. The soil collected from an agricultural field

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was obtained by air drying and sieving (2 mm) . The soil physical properties were analyzed (DW)

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as follows: total organic C, 8.3 g/kg; total P, 1.42 g/kg; total N, 1.31 g/kg; a sandy loam texture

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including silt 53.5%, sand 34.8%, and clay 11.7%; and pH 6.9. Pot experiment was conducted in

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five replicates with four different designs: (i) soil (CK); (ii) soil planted with vegetable (Planting);

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(iii) soil inoculated with Rhodococcus sp. 2G (Inoculation); (iv) soil planted with vegetable and

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inoculated with Rhodococcus sp. 2G (Planting+Inoculation). All of the soils were spiked with 50

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mg/kg of DEHP based on the reported highest DEHP concentration in vegetable soil11 and aged for

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2 weeks prior to use. About 3.0 kg (DW) of the DEHP-spiked soil was loaded into a ceramic pot

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and mixed with chemical fertilizer (6 g; N:P:K=4:3:4). Five seedlings were transplanted into every

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pot after seed germination, and then the strain 2G was inoculated into the planted soil by drip

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irrigation. The sterile saline was kept as control and all treatments were performed in triplicate.

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These pots were kept for 35 days at natural light and moderate moisture conditions in the

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greenhouse. The rhizosphere soils were collected from the pots, refering to the soils adhering to

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roots after shaking. All the soil samples were collected at the 7th, 21st, 35th days after transplanting, 7

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respectively. The vegetables were collected at the 35th days and separated into shoots and roots. All

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the samples were stored at −70°C for further analysis.

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2.6. Instrumental analysis of DEHP and its degradation intermediates. The extraction,

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clean up, and GC/MS procedures for DEHP and its intermediate analyses were performed based on

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the methods reported in our previous papers.13,19 The limit of detection (LOD) of DEHP was 2.5

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μg/L according to three fold of the signal-to-noise ratio. The average procedural blank value (6.3

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μg/L) was subtracted from each sample. The recoveries of DEHP in soil/plant and filtrate samples

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ranged from 87.4% to 107.2% and from 97.7% to 104.7%, respectively.

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2.7. Analyses of enzyme activities and DOC in soils. Soil enzyme activities, including

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dehydrogenase, urease, protease, peroxidase, and polyphenol oxidase (PPO), were determined by a

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UV-2450 spectrophotometer (Shimadzu, Japan). Briefly, the supernatants for the enzymatic assays

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were extracted from 5 g of soil samples using 10 mL sterile water. Peroxidase and PPO were

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assayed based on the methods described in our previous report.20 The detailed description on the

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methods of dehydrogenase, urease, and protease activities were measured according to previous

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reports26-28 (see Supporting Information). The control for each enzyme test was carried out without

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the substrate addition. The concentrations of DOC in soil samples were measured by a TOC-VCSH

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analyzer (Shimadzu, Japan) according to previous study.20

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2.8. PCR-DGGE analysis. Total DNA was extracted from 0.5 g of frozen soil samples using

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the EZNA™ Soil DNA kit (Omega Bio-Tek, USA), and then was used to amplify the short and

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highly variable V3 region of the bacterial 16S rRNA gene by PCR for the following denaturing

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gradient gel electrophoresis (DGGE) analysis, using the universal primers 534R and 341F with GC

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clamp.19 For the DGGE assay, an amount of 25 μL of amplicons was loaded on 10% (w/v)

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polyacrylamide gels containing a denaturant gradient of 40-60% parallel to electrophoresis direction 8

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made of formamide and urea (100% denaturant contains 7 M urea and 40% formamide). Gels were

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electrophoresed at constant 60℃ and 80V in 1×TAE buffer (1 mM EDTA, 40 mM Tris-acetate, pH

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8.5) for 16 h, followed by colouration using 1× SybrGreen I nucleic acid gel stain for 30 min. Bands

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in digital images were detected by a UVP gel documentation system.

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2.9. Statistical analysis. The SPSS 17.0 for Windows statistical package was used for these data

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analyses including standard deviation, regression, and analysis of variance (ANOVA). The analyses

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were run at least in triplicate and the result is significant at P< 0.05.

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3. RESULTS AND DISCUSSION

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3.1. Identification of strain 2G and optimization of culture condition. The strain 2G

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was identified as Rhodococcus sp. and its detailed characterization was presented in Supporting

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Information. The effects of important variables containing pH (X1), temperature (X2), and inoculums

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size (X3) on DEHP biodegradation were determined by the Box-Behnken design according to

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previous single-factor experiments. Table S2 shows the related experimental design and response

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for DEHP biodegradation. The second order polynomial (regression) equation was used to represent

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the response surface Y as follows (5):

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𝑌2𝐺 = 98.3 ― 3𝑋1 +2.238𝑋2 ―10.252𝑋12 ―5.777𝑋22

(5)

Where Y2G is the predicted DEHP biodegradation (%) by strain 2G, and X1 and X2 are the coded values for the pH and temperature, respectively.

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As shown in Table 1, the determination coefficient R2 of the model is 0.9698, which is very

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close to 1. The result suggested that approximately 97% variation can be explained by this model,

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indication of good accuracy. The regression parameters in Table S4 indicated that the linear and

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square terms of pH (X1) and temperature (X2) values had significant effects (P<0.05) on the DEHP

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biodegradation by strain 2G, while the linear and square terms of inoculums size (X3) were 9

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insignificant (P>0.05). Hence, the three-dimensional (3D) plot displayed the effects of temperature

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and pH on DEHP biodegradation while the inoculum size was fixed at 0.6 (OD600nm) (Figure 1).

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The theoretical maximum value of DEHP biodegradation rate was 98.1%. At the theoretical

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maximum point, the optimal conditions for DEHP biodegradation by strain 2G were pH 7.1,

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temperature 29℃, and inoculum size 0.6 (OD600nm).

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3.2. Strain 2G growth and DEHP biodegradation in MSM. Strain 2G could utilize DEHP

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to grow rapidly without lag phases (Figure S3), reflecting rapid adaptation to DEHP at various

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initial concentrations. At low concentrations of DEHP (≤400 mg/L), strain 2G could grow rapidly

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and the cell densities increased to their maximum levels. While the initial concentrations increased

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from 800 to 1600 mg/L, the growing trends of strain 2G showed continuous rising within 5 days.

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Overall, strain 2G could degrade DEHP rapidly at the beginning of the incubation period without an

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adjustment process, further indicating its great application potential to remediate the

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DEHP-contaminated environment.

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As shown in Figure 2a, strain 2G could rapidly degrade up to 1600 mg/L of DEHP, with almost

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complete degradation at low concentrations (50-800 mg/L) within 5 days. The degradation rate kept

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higher when the DEHP concentration increased to 1600 mg/L, with 80% of the removal rate within

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5 days even initial DEHP concentration was up to 1600 mg/L. Inhibition was also found with

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increasing concentration of xenobiotics during the degradation by other bacteria.17,21

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Table 2 shows the kinetic parameters for different initial concentrations of DEHP. The R2 values

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were all higher than 0.9, indication of a good mathematical fit to the first-order kinetics model. It is

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concluded that the initial concentrations had critical impact on the biodegradation efficiency of

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DEHP due to the distinct difference in half-life (t1/2) of DEHP (varying from 0.86 to 2.53 days). To

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further clarify the impacts of initial concentrations of DEHP on the biodegradation, the specific 10

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degradation rate (q) was calculated based on the substrate inhibition model (Eq. (4) (Figure 2b). The

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coefficient of determination R2 (0.981) indicated that the experimental data agreed well with this

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model. The kinetic parameters of strain 2G determined from the non-linear regression analysis

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according to Chen et al.25 were qmax of 1.159 day-1, Ks of 213.3 mg/L, and Ki of 531.6 mg/L. The Ks

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and Ki numerically equal the lowest and the highest concentrations of substrate where the specific

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growth rates are equal to one-half the maximum specific growth rates in the absence of inhibition,

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respectively, which are important parameters in understanding the kinetics of the microorganism in

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the system.29,30 Both the two parameters were higher than those of the DEHP-degrading strain

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Microbacterium sp. J-1 (Ks of 180.2 mg/L and Ki of 332.8 mg/L),20 indicating a better substrate

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adaptability for strain 2G. Additionally, the maximum inhibitor concentration (Sm) was calculated to

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be 336.62 mg/L based on the square root of Ki×Ks,29 indicating that this concentration could

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partially inhibit the activity of strain 2G to degrade DEHP. However, it is hard to happen in the case

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of natural environments because the reported highest DEHP concentrations whether in water (≤

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0.36 mg/L), sewage (≤ 154 mg/kg) or in soil (≤ 149 mg/kg)11,31,32 are much less than this value of

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Sm. Therefore, it is expected that the strain 2G can work well in bioremediation of

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DEHP-contaminated environment.

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3.3. Biochemical degradation pathway of DEHP by strain 2G. To study the biochemical

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degradation pathway of DEHP by strain 2G, the degradation intermediates in culture filtrates were

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extracted and identified by GC/MS. After 3 days of culture, five distinct peaks were observed, and

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the corresponding retention times (RT), predicted chemical structures, characteristic ions of the

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mass spectra (m/z) are given in Table S5. At the 3rd day of the degradation experiment, five

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chromatographic peaks were found at the retention time of 10.05, 9.56, 6.89, 6.31, and 5.62 min,

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respectively (Figure 3), while only one peak at 10.05 min was found before the experiment. The 11

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five peaks were identified as DEHP, MEHP, PA, catechol and BA, respectively, based on their

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retention times and most typical fragment ions (Table S5). Afterwards, these peaks decreased

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gradually and disappeared finally with continuous culture. No persistent accumulative metabolites

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were detected in the final experiment. It was supported by our previous potential intermediate

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utilization tests that the strain 2G was able to live well on these intermediates as sole sources of

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carbon and energy.36

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It is reported that the primary metabolic pathways of PAEs include two steps: (i) transformation

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of PAEs to PA, and (ii) utilization of PA.15 In general, two kinds of reactions were involved in

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transforming PAEs to PA: the one is reduction of side chains’ length by β-oxidation or

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trans-esterification, and the other is hydrolyzing of ester bonds.16 In the present study, DEHP might

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be hydrolyzed by esterase firstly to MEHP and then further to PA based on the identified

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intermediates. PA is the central intermediate of reported PAEs biodegrading process, and its

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metabolic pathways are systematically reviewed elsewhere.15,16 Under aerobic condition, the key

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step in the biochemical degradation of PA is the hydroxylation of the aromatic ring by phthalate

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dioxygenase to form the common intermediate PCA.15,37 In this study, however, the

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chromatographic peak of PCA was not found in the identified DEHP degradation intermediates.

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Consistently, no gene involved in PA degrading to PCA was found based on the genome

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sequencing of strain 2G.36 Alternately, based on the detection of BA and catechol, it is supposed

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that Rhodococcus sp. 2G might metabolize PA to BA in priority through decarboxylation, and

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followed by ring hydroxylation and opening to form catechol, which was further utilized in

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tricarboxylic acid (TCA) cycle. Based on our previous results of functional genomic analysis, the

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identified genes, including transferase/decarboxylase genes, Xyl gene cluster, Pca-Cat gene cluster,

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Icl-Mhp gene cluster, etc., might be involved in the above pathway.36 Overall, a possible pathway 12

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for DEHP biochemical degradation by Rhodococcus sp. 2G was proposed based on the results and

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related chemical properties (Figure 3): DEHP was transformed to MEHP and PA by hydrolysis, and

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then BA was formed through the decarbxylation of PA; catechol was further produced through the

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corresponding hydroxylation and decarboxylation of BA; finally, the terminal degradation products

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of CO2 and H2O occurred after the generated catechol entering the TCA cycle. The detailed

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enzymatic steps and gene clusters involved in this biochemical degradation pathway were

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evidenced by functional genomic analysis of strain 2G previously.36 Taken together, this is the first

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time to comprehensively and systematically unravel such a unique biochemical degradation

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pathway of DEHP in a Rhodococcus species, which distinguishes strain 2G from other

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Rhodococcus species.

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3.4. Effect of the inoculated 2G on decontamination of DEHP-contaminated soil

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and vegetable. The microbial inoculation of strain 2G in DEHP-contaminated soils with or

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without planting vegetable was conducted to explore the impact of inoculated strain 2G on

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mitigating DEHP contamination to vegetable cultivation (Figure 4). The results showed that the

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introduction of strain 2G as a bioaugmentation strategy could efficiently enhance the degradation of

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DEHP in contaminated soil after 35 days, with removal rate (80%) significantly higher (P<0.05)

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than the control (20%) and comparable or higher than those made by the other isolations.18-20

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Notably, the dissipation rate of DEHP in rhizosphere soil of the planted B. parachinensis was up to

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95% when inoculated with strain 2G, while the planted B. parachinensis without inoculation

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removed only 59% DEHP from rhizosphere soil. This suggested an obvious synergistic effect

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between the inoculation of strain 2G and the planting of B. parachinensis on degrading DEHP,

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indicating a great potential of strain 2G to decontaminate DEHP-contaminated soil.

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More importantly, a significant decrease (~75%, P<0.05) of DEHP residue was found in the 13

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shoot of B. parachinensis inoculated with strain 2G (Figure 4b), while the vegetable growth was not

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affected obviously (data not shown). It was reported that numerous hydrophobic contaminants had

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faster rates of the surface accumulating than other biological processes.34 For this reason, DEHP is

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readily adsorbed onto the root surface of plants in priority before entering into plant tissues due to

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its marked hydrophobic character (logKOW ~7.5).33 Thus, the inoculated strain 2G could quickly and

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effectively degrade DEHP adsorbed onto root surface or in rhizosphere soil before its uptake by

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roots. That is, the vegetable cultivation with the inoculation of strain 2G not only greatly enhanced

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DEHP dissipation from soil but also reduced bioaccumulation of DEHP in the vegetable. Although

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there were many studies on accelerating removals of organic pollutants from contaminated soils by

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combining plants and microorganism,38-42 the vast majority of them focused on non-crops.

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Currently, a large scale of agricultural soils have been contaminated with organic pollutants,

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especially for PAEs,9,10 thus soil bioremediation by massively planting non-crop plants is

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impractical, especially in China for lack of cultivated land. Herein, the present work reflects the

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importance of synchronous soil remediation and agricultural production by constructing the

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partnerships between crops and bacterial inoculants. Overall, the strategy of bioaugmentation with

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strain 2G during vegetable cultivation could not only remediate contaminated soil efficiently, but

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also ensure safe vegetable production simultaneously. However, DEHP deposit from air was

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reported to contribute greatly to DEHP accumulation in crops.11 Hence, it is needed to develop new

293

technology for application of strain 2G, such as foliar spraying with strain 2G that can be expected

294

to remove adsorbed DEHP from the surfaces of crops.

295

3.5. Influence of the inoculated 2G on soil mesocosms. To reveal the mechanisms of

296

planting vegetable and inoculating strain 2G in efficiently removing DEHP from the soil, the DOC

297

content and enzyme activities in soils were examined. As sensors of soil organic matter 14

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decomposition, soil enzyme activities could reflect the comprehensive information of soil

299

physic-chemical conditions and microbial status.43 After 7 days, the inoculation of strain 2G in the

300

contaminated soils significantly (P<0.05) increased (approximately 53%~94%) soil enzyme

301

activities except for the urease (Figure 5), indirectly indicating that stain 2G was able to live in

302

harmony with indigenous microorganisms and adapt itself to the soil environment well. Among

303

these soil enzymes, the PPO showed twice higher activity (approximately 94%) when inoculated

304

with the strain 2G (Figure 5). This was conducive to enhancing DEHP removal efficiency in the soil

305

because PPO as an oxidoreductase plays an important role in catalyzing the degradation of aromatic

306

compounds in soils.20 In the presence of vegetable, all soil enzyme activities were significantly

307

(P<0.05) increased by approximately 8%~150% (Figure 5), suggesting positive effects of B.

308

parachinensis planting on indigenous microorganisms in the soil.44 Herein, the plant might

309

contribute to the degradation of DEHP by increasing microbial activity, modifying microbial

310

community, and improving desorption of DEHP from rhizosphere.45 Meanwhile, the elevated

311

activities of enzymes such as peroxidase secreted from root might also contribute to the dissipation

312

of DEHP (~59%, Figure 4a) in planted soil, because peroxidase activity has a direct contribution to

313

degrading aromatics in the natural environment.40 On the other hand, the inoculation of the strain

314

2G in the planted soils further increased (approximately 14%~96%, Figure 5) the enzyme activities

315

based on its role on increasing microbial respiration. The increase in activities of some enzymes,

316

such as dehydrogenase, protease, and urease might be due to the stimulation of respiration and

317

activities to inoculated 2G and indigenous microorganisms to take nutrients from the rhizosphere

318

soil.26 In return, more DOC that was released from the vegetable into rhizosphere was helpful to

319

desorb DEHP (Figure 4)46 and maintain a blooming bacterial population and longer time of enzyme

320

activities (Figure 5).40 Overall, the higher enzymes activities and more DOC content in the soil 15

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321

planting B. parachinensis and inoculating strain 2G simultaneously may be an important

322

mechanism accounting for efficient degradation of DEHP in soil.

323

3.6. Tracing the dynamic changes of inoculated 2G in soil by DGGE analysis.

324

Inoculation of soils with degrading bacteria may be one of important approaches to enhance the

325

bioremediation efficiency of contaminated soils. Nevertheless, a knotty problem is that the

326

degrading microbes are hard to survive for long time in the highly competitive environment because

327

inoculant strains are often out-competed rapidly by the natural microflora.47 In this study, the DNA

328

band of strain 2G (lanes 7 to 9) was found to be clear and bright at day 7 after inoculation (Figure 6),

329

illustrating that bioaugmentation was effective by inoculating strain 2G because it had the

330

advantage of competition growth to the indigenous microorganisms at an early stage. The additive

331

DEHP in soils might provide abundant energy sources for 2G growth and simultaneously

332

suppressed the growth of their competitors.48 Thus, the sharp decrease in DEHP in the first 7 days

333

(Figure 4a) was attributed to the sudden blooming of the introduced bacteria. Over time, however,

334

the DNA band blurred at day 21 and eventually disappeared at day 35, suggesting that strain 2G

335

could not survive in soil for nutrients shortage and/or failing to compete with indigenous

336

microbes.42 By contrast, the bands of strain 2G (lane 10 to 12) were clearly maintained in soils until

337

the vegetable were harvested at the 35th day. This indicated that the planting was conductive to

338

survival of the inoculated strain 2G in rhizosphere soil, reasonably explaining up to 95% dissipation

339

rate of DEHP in rhizosphere soil of the planted B. parachinensis when inoculated with stain 2G.

340

Moreover, some newly-emerging bands (a1-a8, Figure 6) suggested that the community

341

structure within the rhizosphere in the presence of 2G population was stable, and a mutually

342

beneficial relationship between microorganisms and plants in the rhizosphere was constructed.38,45

343

Here, B. parachinensis released DOC (Figure 5) into rhizosphere, which might be readily utilized 16

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by microorganisms and thereby facilitated a blooming bacterial population in the rhizophere soil. In

345

return, the rhizospheric microbes helped to decrease DEHP accumulation and toxicity in B.

346

parachinensis by degrading DEHP in rhizosphere soils. It was reported that the interactions

347

between Mycobacterium and root exudate could enhance PAH degradation in contaminated

348

agricultural soil by improving the diversity of soil microbial community structure.49 Furthermore,

349

the DOC released from vegetable roots might stimulate the population amount of DEHP-degrading

350

bacteria by increasing the bioavailability of DEHP in soil. Some studies demonstrated that DOC is

351

associated with increase in water solubility of the hydrophobic organic contaminants in soils,

352

leading to higher bioavailable for bacterial degradation.50,51 Overall, the planting vegetable B.

353

parachinensis with inoculation of Rhodococcus sp. 2G in the rhizosphere soil is a successful

354

strategy for mitigating DEHP contamination during agricultural production. Besides, some

355

innovative technologies should also be considered to use in combination. For instance, the

356

PAE-degrading strain Acinetobacter sp. LMB-5 could be immobilized by magnetic nanoparticles,

357

which provided many advantages such as improved catalytic efficiency, enhanced tolerance against

358

toxic compounds, recycled the catalytic cells, etc.52

359

In conclusion, an efficient DEHP-degrading bacterial strain Rhodococcus sp. 2G was identified

360

and its degradation parameters including the optimal condition, kinetics, and pathway of DEHP

361

biodegradation were characterized in detail. This strain was able to colonize the unplanted soil and

362

the rhizosphere soil of vegetable (B. parachinensis), with much higher survival and DEHP removal

363

in rhizosphere soil of B. parachinensis owing to improved DOC content and soil enzyme and

364

microbial activities. Especially, the inoculation could effectively reduce DEHP bioaccumulation in

365

vegetable to ensure food safety. Therefore, this strategy not only enhanced the decontamination of

366

agricultural soil, but also minimized the pollutant levels of crops, ensuring the simultaneous 17

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367

effective remediation of polluted soils and safe production of agricultural food. Further studies

368

should be conducted to (i) clarify the mechanism forming the stable community structure of the

369

inoculated strain in the rhizosphere soil; (ii) evaluate the viability of the inoculated strains on field

370

conditions; and (iii) investigate the applicability of strain 2G with other crop species.

371

ASSOCIATED CONTENT

372

Supporting information

373

The attached supporting information includes morphological characteristics of strain 2G (Figure

374

S1), phylogenetic tree analysis (Figure S2), bacteria growth (Figure S3), design matrix of

375

Box-Behnken (Table S1 and S2), physio-biochemical characteristics (Table S3), effect estimates for

376

RSM (Table S4), and chromatographic properties of DEHP intermediates (Table S5).

377

AUTHOR INFORMATION

378

Corresponding Author

379

*E-mail: [email protected] (Q. Cai); [email protected] (C. Mo); Tel: +86-20-8522-3405 (C. Mo)

380

Author Contributions

381

H.-M.Z., Q.-Y.C., and C.-H.M. designed the study and wrote this paper; H.-M.Z., H.D., C.-Q.H.,

382

X.-H.Z., X.-J.H. S.L., and H.L. conducted the experiments and analyzed the data; L.X., Y.-W.L.,

383

and Z.-L.H. revised this manuscript and provided technical and material support. §H.-M.Z. and H.D.

384

contributed equally to this work.

385

Funding

386

This work was funded by the Research Team Project of the Natural Science Foundation of

387

Guangdong Province (2016A030312009), the NSFC-Guangdong Joint Fund (U1501233), the

388

National Natural Science Foundation of China (41703085), the Natural Science Foundation of

389

Guangdong

Province

(2017A030313230),

the

China

Postdoctoral

18

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Science

Foundation

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Journal of Agricultural and Food Chemistry

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(2018T110924), the Program of the Guangdong Science and Technology Department

391

(2016B020242005), and the Project of the Guangzhou Science and Technology (201704020074).

392

Notes

393

The authors declare no competing financial interest.

394

REFERENCES

395

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396 397 398 399 400

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[52] Wang, J., Jiang, L. H., Zhou, Y., & Ye, B. C. Enhanced biodegradation of di-n-butyl phthalate by

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Acinetobacter species strain LMB-5 coated with magnetic nanoparticles. Int. Biodeter. Biodegr. 2017, 116,

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FIGURES Figure 1. Response surface for the effects of pH and temperature on DEHP biodegradation by Rhodococcus sp. 2G with a fixed inoculum size at 0.6 (OD600).

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Figure. 2. Biodegradation kinetics of various initial concentrations of DEHP by strain 2G (a) and the relationship between the initial DEHP concentration and specific degradation rate (b).

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Figure 3. Proposed degradation pathway of DEHP in Rhodococcus sp. 2G based on GC-MS chromatograms and spectra of detected DEHP intermediates at day 3 in mineral salt medium. DEHP: di-(2-ethylhexyl) phthalate, MEHP: mono-ethylhexyl phthalate, PA: phthalateacid, BA: benzoic acid.

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Figure 4. DEHP residues in soil (A) and vegetable (B) under different treatments. CK, non-treated control soil; Planting, soil planted with vegetable; Inoculation, soil inoculated with strain 2G; Planting+Inoculation, soil with planting and inoculation; Different lowercase letters indicate statistically significant differences at P≤0.05; Similarly hereinafter.

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Figure 5. Enzyme activities and DOC contents in different soil treatments at days 7, 21, and 35, respectively.

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Figure 6. DGGE-based bacterial profile of the total DNA present in DEHP contaminated-soils at days 7, 21, and 35. Lane 13 represents the DNA extracted from strain 2G, and lane M represents a DNA size marker. Triangles (a1-a8) indicate the newly-emerging bands, and arrows indicate the DNA bands of strain 2G.

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TABLES Table 1. ANOVA analysis for the fitted quadratic polynomial model in terms of coded variable for DEHP biodegradation. Source DF SS

MS

P-value*

F-value

X1

1

72

72

0.0078

18.4481

X2

1

40.0513

40.0513

0.0239

10.2621

X3

1

8.6113

8.6113

0.1976

2.2064

X1X1

1

398.7203

398.7203

0.0002

102.1617

X1X2

1

2.7225

2.7225

0.4417

0.6976

X1X3

1

5.0625

5.0625

0.3063

1.2971

X2X2

1

129.2564

129.2564

0.0022

33.1186

X2X3

1

1.44

1.44

0.5701

0.3690

X3X3

1

12.1856

12.1856

0.1375

3.1223

Model

9

627.4592

69.7177

0.0027

17.8634

Error

5

19.5142

3.9028

Total

14

646.9733

R2=0.9698 (adjusted R2=0.9155), coefficient of variation (CV)=2.30%. DF refers to the degrees of freedom, SS refers to the summation of squared deviation, MS refers to the mean of the summation of squared deviations where MS=SS/DF.*P-value indicates that the model term is significant when P<0.05.

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Table 2. Kinetic equations of DEHP biodegradation of different initial DEHP concentrations in mineral salt medium by strain 2G. DEHP concentrations (mg/L)

Regression equations

t1/2(d)

R2

50

ln C = -0.803t+3.912

0.86

0.976

100

ln C = -0.692t+4.605

1.00

0.976

200

ln C = -0.633t+5.298

1.10

0.974

400

ln C = -0.666t+5.991

1.04

0.944

800

ln C = -0.553t+6.685

1.25

0.918

1600

ln C = -0.274t+7.378

2.53

0.912

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Journal of Agricultural and Food Chemistry

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33

ACS Paragon Plus Environment