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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Journal of Agricultural and Food 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†, 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 (P0.05). 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