Chemosphere 234 (2019) 789e795 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Enhanced degradation of polycyclic aromatic hydrocarbons (PAHs) in the rhizosphere of sudangrass (Sorghum  drummondii) * John Jewish A. Dominguez a, , Hernando P. Bacosa a, b, Mei-Fang Chien a, Chihiro Inoue a a Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai, 980-8579, Japan b Department of Marine Sciences, Texas A&M University at Galveston, Galveston, TX, United States highlights graphical abstract Sudangrass emerged superior in removing PAHs in soil than other previously reported grasses. Sudangrass specially enriched Sphingomonadales, a potent PAHs degrader, in its rhizosphere. The abundance of PAHs degradation biomarker genes, PAH-RHDa and nidA, was highest in soil planted with sudangrass. article info abstract Article history: Grasses are advantageous in the removal of polycyclic aromatic hydrocarbons (PAHs) in soil because of Received 30 January 2019 their fibrous root, high tolerance to environmental stress, and low nutritional requirements. In this study, Received in revised form a pot experiment was conducted to test the ability of four grasses to remove PAHs in the soil, and to 21 May 2019 investigate the corresponding bacterial community shift in the rhizosphere of each. Sudangrass achieved Accepted 30 May 2019 the maximum removal of PAHs at 98% dissipation rate after 20 days. Polymerase chain reaction- Available online 3 June 2019 denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing revealed that su- Handling Editor: T. Cutright dangrass specially enriched the growth of a known PAHs degrader, Sphingomonadales, regardless of the presence or absence of PAHs in the soil. Moreover, the gene copy numbers of PAHs catabolic genes, PAH- Keywords: RHDa and nidA, as measured by real time-PCR (RT-PCR) were highest in the soil planted with sudangrass. Polycyclic aromatic hydrocarbons Overall, this study suggested that sudangrass further enhanced the dissipation of PAHs by enriching Phytoremediation Sphingomonadales in its rhizosphere. Rhizodegradation © 2019 Elsevier Ltd. All rights reserved. Sphingomonadales Rhizosphere effect 1. Introduction carcinogenic, teratogenic, mutagenic, and eco-toxic (Abdel-shafy and Mansour, 2016; Brown et al., 2017; Samanta et al., 2002; Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants Wang et al., 2018). Once released into the environment, PAHs tend known to pose risks to both ecological and human health. It is to adsorb in soil humic contents in the topsoil where they tend to concentrate (Kanu and Anyanwu, 2005; Okere and Semple, 2012). In order to remove PAHs with low cost and environmental burden, one method is to use living organisms such as plant, bacteria or * Corresponding author. Inoue Laboratory, Graduate School of Environmental both (Abdel Ghany et al., 2015; Gan et al., 2009; Shukla et al., 2013). Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai, 980-8579, In particular, rhizoremediation is proposed to be the approach with Japan. E-mail address: [email protected] (J.J.A. Dominguez). most potential in remediating PAHs in soil (Shukla et al., 2013). https://doi.org/10.1016/j.chemosphere.2019.05.290 0045-6535/© 2019 Elsevier Ltd. All rights reserved. 790 J.J.A. Dominguez et al. / Chemosphere 234 (2019) 789e795 Rhizoremediation is the process in which plants indirectly 30 seeds of Italian ryegrass, tall fescue, and perennial ryegrass were degrade PAHs by stimulating the microbial community in its root accordingly dispersed in each pot and covered with 3 cm of soil. (Dzantor, 2007). This can be achieved in multiple ways, namely: 1) Sudangrass seeds are bigger than the other grasses. The average plant provides aeration to the soil and microbes thereby enhancing germination rate for sudangrass was 86%, 85% for tall fescue, 88% aerobic reactions (Anderson et al., 1993; Bisht et al., 2015); 2) plants for perennial rye grass, and 90% for Italian ryegrass. The soil secrete organic compounds (i.e. sugar, organic acids, secondary moisture content was maintained to 20% throughout the plant metabolites) that can stimulate microbial growth, select experiment. PAHs-degrading bacteria, and induce PAHs catabolic reactions Depending on the plant used, five experimental treatments (Anderson et al., 1993; Balasubramaniyam, 2015; Rohrbacher and were prepared with (1) or without (0) the addition of PAHs solution St-Arnaud, 2016); and finally, plants can increase the bioavail- (Table 1). The treatments include the following: unplanted control ability of PAHs through physical and chemical means (Lefevre et al., (H1, H0); tall fescue (T1, T0); sudangrass (S1, S0); perennial ryegrass 2013; Rohrbacher and St-Arnaud, 2016; Zhu et al., 2009). (P1, P0); lastly, Italian ryegrass (I1, I0). Each treatment was prepared Plants under the family Poaceae, collectively known as grasses, in triplicate. After 20 days of cultivation at room temperature, soil are often employed in rhizoremediation studies due to their fast samples were stored in 4 C until extraction. growth, tolerance to PAHs, deep and fibrous roots, resistance to environmental stress, and low nutritional requirements 2.3. Analytical procedure (Balasubramaniyam, 2015; Sivaram et al., 2018a). Furthermore, in a study conducted by Olson et al. (2007), grasses emerged as the The remaining PAHs in the soil were extracted using methods most effective in dissipating PAHs in soil among the eight families from Lee et al. (2008). Briefly, 10 g fresh weight of soil were ho- tested. Thus, it is not surprising that numerous PAHs rhizor- mogenized and transferred to 50 mL centrifuge tube and shaken emediation studies have been conducted using grasses (Fu et al., vertically with 25 mL of dichloromethane at 300 rpm for 1 h. The 2012; Gaskin and Bentham, 2010; Khan et al., 2009; Kuiper et al., tubes were then centrifuged at 5000 rpm and the supernatant was 2001). decanted and stored in 4 C until analysis. PAHs were analyzed Of particular interest in this study is sudangrass (Sorghum x using GC-4000 gas chromatography (GL Sciences Inc., Tokyo, Japan) drummondii). Despite reports of superior PAHs dissipation equipped with flame ionization detector (FID) and an InertCap 17 enhancing ability (Reilley et al., 1996; Sivaram et al., 2018b; Su et al., MS column. The column temperature was held at 80 C for 2 min, 2008), sudangrass remained relatively not studied and the infor- then increased to a maximum temperature of 300 C at the rate of mation about how the plant influences its rhizosphere during rhi- 10 C/min. The injector and detector temperatures were main- zoremediation is lacking. Among the four plants tested in a study tained at 350 C. Helium was used as a carrier gas. The concentra- conducted by Su et al. (2008), sudangrass dissipated most of pyrene tion of PAHs was calculated based on a five-point standard curve. but enriched the soil microbial population the least. Hence, it is worth investigating how sudangrass possibly achieve dissipation of 2.4. Microbial community analyses PAHs with respect to its rhizosphere microbial community. fl In this study, the ability of sudangrass to dissipate PAHs ( uo- Total DNA was extracted from the soil using Powersoil DNA fl rene, phenanthrene, uoranthene, and anthracene) in soil and the extraction Kit (Qiagen, Inc., Hilde, Germany) based on the manu- corresponding changes in the bacterial community of the rhizo- facturer's protocol. The extracted DNA was used as template for sphere soil was investigated. Furthermore, known PAHs degrada- polymerase chain reaction-denaturing gel gradient electrophoresis a tion biomarkers such as PAH-RHD and nidA genes were analyzed (PCR-DGGE). Briefly, the V3 region of 16s rRNA gene was amplified in parallel. These were conducted together with other grasses by PCR using universal primers 341F-GC (50-CGC CCG CCG CGC GCG previously reported to enhance PAHs degradation, namely: tall GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG fescue (Festuca arundinacea), perennial ryegrass (Lolium perenne) CAG-30) and 518R (50-ATT ACC GCG GCT GCT GG-30). PCR was per- fl and Italian ryegrass (Lolium multi orum). formed in reaction mixtures composed of 25 ml of Promega Master Mix (Promega Corp., WI, USA), 2 mL(10mM) each of forward and 2. Materials and methods reverse primers, approximately 10 ng of DNA extract and filled with DNAse free water to a volume of 50 mL. The amplification conditions 2.1. Chemicals and plant samples in the 2720 Thermal Cycler (Applied Biosystems, Foster, CA, USA) were as follows: initial denaturation at 94 C for 5 min, followed by fl fl Analytical grade uorene, phenanthrene, uoranthene, and 30 cycles of 94 C for 30 s, 55 C for 30s and 72 C for 30s, and a final pyrene were procured from Wako Chemicals, Ltd. (Osaka, Japan). extension step at 72 C for 7 min. The presence of PCR products was Four grasses were used in the study, namely: tall fescue (Festuca confirmed by gel electrophoresis. arundinacea), sudangrass (Sorghum x drummondii), perennial DGGE was performed using the DCode Universal Mutation fl ryegrass (Lolium perenne), and Italian ryegrass (Lolium multi orum). Detection System (Bio-Rad, California, USA). An 8% polyacrylamide & The seeds were purchased from Takii Co., Ltd (Miyagi, Japan). gel with a 30e70% gradient was prepared using urea and form- amide as denaturants. The samples were then loaded and run at a 2.2. Biodegradation experiment constant voltage of 70V and constant temperature of 60 C for 16 h. For the soil, four bags of lawn soil with unknown composition (Daisin Co., Ltd., Miyagi, Japan) were thoroughly mixed to achieve a Table 1 homogenous soil composition. The PAHs in the lawn soil were Experimental set-up employed in the biodegradation experiment. below detection limit. Four hundred grams of the soil was weighed, Soil with 400 ppm PAHs No PAHs added fl sprayed with PAHs solution (1 mg/mL each of uorene, phenan- Unplanted control H1 H0 threne, fluoranthene, and pyrene) to a final concentration of Tall fescue T1 T0 400 mg/kg, and mixed thoroughly using a hand mixer.