J Soils Sediments DOI 10.1007/s11368-015-1217-0
SEDIMENTS, SEC 4 • SEDIMENT-ECOLOGY INTERACTIONS • RESEARCH ARTICLE
Responses of endophytic and rhizospheric bacterial communities of salt marsh plant (Spartina alterniflora) to polycyclic aromatic hydrocarbons contamination Jianqiang Su 1 & Weiying Ouyang 1,5 & Youwei Hong 1,2,3 & Dan Liao 4 & Sardar Khan 6 & Hu Li 1,5
Received: 27 February 2015 / Accepted: 22 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose Plants and their root-associated microbes play important roles in the remediation of polycyclic aromatic hydrocarbons (PAHs) present in the sediments of contaminated coastal wetlands. The detailed information about the effects of PAHs on root-associated bacterial communities could help to isolate PAH-degrading bacteria and optimize the process of phytoremediation. Materials and methods The community structures of rhizospheric (RB) and endophytic bacteria (EB) of salt marsh plant (Spartina alterniflora) grown in phenanthrene (PHE)and pyrene (PYR)-contaminated sediments (for 70 days) were investigated using the barcoded Illumina paired-end sequencing technique. Results and discussion The diversity and community structure of EB and RB were more sensitive to PHE and PYR Responsible editor: John R. Lawrence Electronic supplementary material The online version of this article (doi:10.1007/s11368-015-1217-0) contains supplementary material, which is available to authorized users. * Youwei Hong
[email protected] 1
Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
2
Department of Environmental Sciences, University of California, Riverside, CA 92521, USA
3
Southern California Coast Water Research Project, Costa Mesa, CA 92626, USA
4
Xiamen Huaxia University, Xiamen 361024, China
5
University of Chinese Academy of Sciences, Beijing 100049, China
6
Department of Environmental Sciences, University of Peshawar, Peshawar 25120, Pakistan
contamination as compared to those in the sediments. All samples were strongly clustered according to EB, RB, and sediments, suggesting the niche-specific influence of PAHs on bacterial communities. Potential PAH-degrading bacteria (Pseudomonas sp., Paenibacillus sp., and Flavobacterium sp.) in EB and RB were stimulated by PAH contamination. The results revealed an increase prevalence of specific bacteria which may be responsible for PAH degradation. PHE contamination increased the abundance of Proteobacteria in EB but decreased the number of Firmicutes, Bacteroidetes, and Chloroflexi. The numbers of Proteobacteria and Firmicutes in EB were enhanced by PYR treatment. However, both PHE and PYR treatment showed similar effects on the bacterial communities in RB. Conclusions The results suggested that PAH pollution could alter root-associated bacterial communities of S. alterniflora, whose EB might play an important role in the phytoremediation of PAH-contaminated sediments. Keywords 16S rRNA gene . Endophytic bacteria . Illumina sequencing . PAHs . Rhizospheric bacteria
1 Introduction Polycyclic aromatic hydrocarbons (PAHs), a group of highly toxic and recalcitrant organic contaminants, have been widely studied during the past several decades, due to their serious threats to human health and ecosystems (Haritash and Kaushik 2009; Yergeau et al. 2014; Johnston and Leff 2015). Coastal wetland is considered an important intertidal and endangered ecosystem, suffering from severe PAH contamination because of numerous human activities such as oil spills, ship traffic, wastewater, and industrial discharges into the coastal zone (Qiu 2011; Oliveira et al. 2014). Therefore,
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the potential effects of PAHs on the functions of coastal ecosystems have attracted both the public and scientific attentions. Plant-microbe interactions play key roles in various aspects of PAH biogeochemistry at the salt marsh ecosystem, including plant detoxification and in the biodegradation of PAHs (Khan et al. 2013; Oliveira et al. 2014). Large amounts of PAHs could be taken up and translocated into plant tissues, while root exudates might trigger root-associated microorganisms to participate in PAH-degrading processes (Khan et al. 2009; Sun et al. 2011; Khan et al. 2013). Recently, several authors have demonstrated that endophytic bacteria (EB) have distinct degrading potentials for PAHs (Germaine et al. 2009; Weyens et al. 2011; Andreolli et al. 2013; Khan et al. 2014). The diversity of EB has been previously reported in association with many plants including poplar, willow, and prairie grown in PAH-contaminated soils (Phillips et al. 2008; Peng et al. 2013; Yergeau et al. 2014). However, little work has been done about the variations of EB diversity and its composition responses to PAH contamination in coastal wetland ecosystems. The spatial variation of microbes provides clues for the investigation of underlying mechanisms that help in structuring the ecological communities and isolation of PAHdegrading bacteria. Jiang et al. (2013) reported that mangrove plants have significant rhizospheric effects on diversity and composition of the bacterial community present in mudflat, edge, bulk, and rhizosphere sediments. Yergeau et al. (2014) found that genes related to hydrocarbon degradation were generally more expressed in the PAH-contaminated rhizosphere than bulk soil, which could help to optimize microorganisms associated with phytoremediation systems. Therefore, knowledge regarding the success of plantassociated bacterial communities in specific niche will help to explore the plant-endophytic symbiosis and its uses for improvement of phytoremediation of PAH-contaminated environment. Spartina alterniflora is a perennial salt marsh grass, widely distributed in nine coastal provinces present in East and South of China (Wan et al. 2009). The displacement of the native plant species has caused a number of ecological impacts on these ecosystems (Wan et al. 2009; Zhang et al. 2012). In our previous studies, we have only compared the plant uptake of PAHs from sediment and microbial activity in roots of S. alterniflora (Hong et al. 2015). Abundance of PAH-ring hydroxylating dioxygenase genes for gram-negative bacteria in the rhizoplane and endophyte was enhanced, indicating the phytoremediation potential of root-associated bacteria on PAH-contaminated sediments. However, the impacts of PAHs on the bacterial diversity and composition of rhizophere (RB) and EB of S. alterniflora remained unclear. The main objectives of this study were to investigate the responses of the root-associated bacterial community to PAH contamination
and the changes in bacterial diversity and composition occurring in the sediment, rhizoplane, and endophytic environment. This study will help us to understand the potential PAH phytoremediation using endophytes and guiding the isolation of PAH-degraded bacteria.
2 Material and methods 2.1 Pot experiment setup S. alterniflora seedlings and surface sediments were collected from Jiulong River Estuary Mangrove Nature Reserve (24° 24′ N, 117° 55′ E), China, in July 2012. The surface layer (10 cm) of the sediment from the bare mudflat, which was adjacent to the S. alterniflora-invaded zone in the Jiulong River Estuary, was collected using a stainless steel spoon. A total of 150-kg surface sediment was sampled from three sites of bare mudflat and placed into a bucket and then transported to the lab for preparation and analyses. Aliquots of the sediments (150 g) were air-dried and spiked with phenanthrene (PHE, purity 98 %, Sigma-Aldrich Co. USA) and pyrene (PYR, purity 98 %) dissolved in acetone. The spiked sample was left for 12 h to evaporate acetone, and then, a portion of spiked sediment was first mixed with 50 kg of sediment using mechanical mixing technique. The final concentrations of PHE and PYR in treated sediments were 100 mg kg−1. After aging for 2 weeks, the sediments were used for the culture experiments. The uniform and healthy seedlings of S. alterniflora were cultured (three plants) in each pot (25 cm×12 cm), according to the method mentioned in our previous paper (Hong et al. 2015). Each seedling was planted in nylon rhizobag (25-μm pore size) to separate the rhizosphere sediment (RS) from the non-rhizosphere sediment (NRS). Treatments included (a) planted control pots with unspiked sediments (CK), (b) planted pots with spiked 100 mg kg−1 PHE, and (c) planted pots with spiked 100 mg kg−1 PYR. Each treatment was performed in three replicates. Sediment moisture content was adjusted to 100 % of the water holding capacity by irrigating with deionized water with 7-day interval to simulate the anoxic conditions. The plants were grown under greenhouse conditions with natural illumination and the temperature (from 26 to 32 °C) for 70 days. During growing period, the humidity varied between 45 and 60 %. After harvesting, the roots of S. alterniflora were carefully washed with Milli-Q water, while the sediments from RS and NRS were collected and homogenized then kept at −20 °C for further analyses. 2.2 PAH measurements Sediments were freeze-dried, grounded into a fine powder, and then filtered through 0.250-mm mesh. PAH extraction
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and cleanup methods were adopted as mentioned by Hong et al. (2015). Briefly, sediments (5 g) were extracted with a mix (60 mL) of acetone/hexane (1:1 v/v) using accelerated solvent extraction system (Dionex ASE™ 350, Thermo Scientific Co., USA). The extracts were purified with a silica/alumina column. These cleanup samples were concentrated and the volume was adjusted to 1 mL and analyzed using GC/MS (7890-5975C, Agilent Co., USA). Pore water was obtained by centrifugation of 100 g wet sediment and analyzed using F-4600 fluorescence spectrophotometer (Hitachi, Japan). The recoveries of PAH surrogate standards were satisfactory and observed as 81.8±12.0 % for PHE-d10 and 87.6±11.9 % for chrysene-d12. 2.3 DNA extraction Roots of S. alterniflora were thoroughly washed with deionized water, and RB were isolated by vigorously shaking the root segments (2 g) in 200 mL of PBS buffer for 1 h (Mendes et al. 2007). For EB samples, washed root segments (5 g) were surface sterilized by sequential washing in 70 % ethanol for 1 min, sodium hypochlorite (2 %, v/v) for 3 min, and 70 % ethanol for 30 s and five rinses with ample sterilized distilled water (Hong et al. 2015). Coated onto tryptic soy agar plates, the final rinse was used to verify surface sterilization. Surfacesterilized root segments were ground in a mortar with liquid nitrogen. Total DNA from sediment and strains was extracted and purified according to the manufacturer’s instructions, using the Fast DNA kit (MP Biomedicals, California, USA) and the DNA purification kit (Tiangen, China).
of combined DNA was collected by centrifugation, washed with 75 % ethanol, eluted in 50 μL water, and submitted to Beijing Genomics Institute (BGI), Shenzhen, China, for sequencing (pair-end) on Illumina HiSeq 2000 platform. 2.5 Data analysis and statistical methods Raw pair-end reads were assembled after filtering adaptor, low-quality reads, ambiguous N, and barcode to generate clean joined reads capturing the complete V3 region of the 16S rRNA gene by BGI (Hamady et al. 2010). All the sequences were analyzed using Quantitative Insights Into Microbial Ecology (QIIME, version 1.6) (Caporaso et al. 2010). According to the previous studies (Xie et al. 2015), the operational taxonomic units (OTUs) were picked at 97 % sequence similarity, and their representative sequences were chosen for alignment and taxonomic assignment with Ribosomal Database Project (RDP) classifier with a cutoff value of 0.8. Chimeric sequences, mitochondrial, chloroplast, and singleton OTUs were removed. In this study, the barcodes used for each sample are given in Table S1. Rarefaction analysis, α-diversity, and β-diversity were conducted according to OTU table with minimal sequencing depth (1378) of the samples (Table S2, Electronic Supplementary Material). For βdiversity analysis, dissimilarity of bacterial communities was determined using principal coordinate analysis (PCoA) on unweighted and weighted UniFrac distances among all samples. All sequences were deposited in the National Center for Biotechnology Information Sequence Read Archive under the accession number SRP052212.
2.4 16S rRNA gene amplification and sequencing
3 Results The V3 region of bacterial 16S ribosomal RNA (rRNA) gene was amplified using 338F (5′-ACTCCTACGGGAGGCA GCAG-3′ and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) with identified barcodes (Huse et al. 2008), and barcodes used for each sample are listed in Table S1 of the Electronic Supplementary Material. PCR amplifications were performed using a thermocycler (Eppendorf, Hamburg, Germany) in 50-μL reaction volumes containing 25 μL Dream Taq Green PCR Master Mix (2×) (Thermo Scientific Co., USA), 0.5 μL 1 % bovine serum albumin (BSA), 0.2 μmol L−1 of each primer, 40–50 ng of template DNA, and 20.5 μL sterile water. PCR was carried out with the following temperature profiles: initial denaturation at 94 °C (3 min), 35 cycles including 30 s of denaturation at 94 °C, 30 s at the primer annealing temperature (55 °C), 30 s of elongation at 72 °C, followed by a final extension at 72 °C for 5 min. PCR products were purified with Universal DNA Purification kit (Tiangen, China) following the instructions of the manufacturer. The PCR products from all samples were combined and kept in 0.1 vol 3 mol L−1 sodium acetate and 3 vol 100 % ethanol overnight. The pellet
3.1 Diversity of bacterial communities A total of 225,172 assembled high-quality sequences were obtained from 60 samples (average 38,155 per sample) and were clustered into 73,206 OTUs. Alpha diversity using phylogeny-based metrics (phylogenetic diversity (PD)) showed a significant difference between sediment (RS and NRS) and root-associated samples (EB and RB), while significantly higher diversity (P
