Distribution of Culturable Endophytic Bacteria in Aquatic Plants and Their Potential for Bioremediation in Polluted Waters
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Vol. 15: 99–110, 2012 AQUATIC BIOLOGY Published May 15 doi: 10.3354/ab00422 Aquat Biol OPENPEN ACCESSCCESS FEATURE ARTICLE Distribution of culturable endophytic bacteria in aquatic plants and their potential for bioremediation in polluted waters Wei-Min Chen1, Yue-Qin Tang1, Kazuhiro Mori2, Xiao-Lei Wu1,* 1Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100871, PR China 2Department of Civil and Environmental Engineering, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 400-8511, Japan ABSTRACT: Plant−bacterial asscociations can improve the degradation of organic pollutants in soil. How- ever, little is known about the diversity and distribu- tion of endophytic bacteria associated with aquatic plants and their potential to enhance phytoremedia- tion of aquatic environments. In the present study, endophytic bacteria were isolated from 4 species of aquatic plants: Phragmites communis, Potamogeton crispus, Nymphaea tetragona and Najas marina. The isolated bacteria were classified into 12 genera in the Gammaproteobacteria, Bacilli, Alphaproteobacteria, Flavobacteria and Actinobacteria. In addition, differ- ent strains and/or different concentrations of the bac- teria were isolated from different parts of the 4 plants, suggesting the different parts of the 4 plants harbored different endophytic bacteria. Some of the isolates de - Guanting Reservoir shoreline where endophytic bacterial graded naphthalene and pesticides and some showed strains were isolated from aquatic plants. potential to dissolve insoluble phosphate. This is one Photo: Yue-Qin Tang of the first studies to isolate and compare culturable endophytic bacteria among different aquatic plants. This research indicates that culturable endophytes in aquatic plants are very diverse, but are dominated by Gammaproteobacteria, and have the potential to KEY WORDS: Aquatic plants · Endophytic bacteria · enhance in situ phytoremediation. Phytoremediation · Organic pollutants · Phosphorus Resale or republication not permitted without written consent of the publisher INTRODUCTION to Alpha proteo bac teria, Betaproteobacteria and Gam - maproteobacteria. They can supply nutrients to Plant endophytic bacteria have been studied since plants by fixing atmospheric nitrogen and solubiliz- the 1940s. They have been classified into 82 gen- ing iron (Marx 2004, Porras-Soriano et al. 2009) and era within Alpha proteobacteria, Betaproteobacteria, can protect the host plants from infection by plant Gam ma pro teo bacteria, Firmicutes, Actinobacteria pathogens through competition for space and nutri- and Bactero idetes (Lodewyckx et al. 2002, Rosen- ents, the production of hydrolytic enzymes and anti- blueth & Martínez-Romero 2006), and most belong biosis, and by inducing plant defense mechanisms *Corresponding author. Email: [email protected] © Inter-Research 2012 · www.int-res.com 100 Aquat Biol 15: 99–110, 2012 (Ryan et al. 2008). In addition, recent studies have MATERIALS AND METHODS shown that endophytic bacteria have the potential to enhance the removal of soil contaminants by Sample collection and isolation of endophytic bacteria phytoremediation (Barac et al. 2004, Compant et al. 2005, Germaine et al. 2006, Doty et al. 2009, Tag- Guanting Reservoir (from 40° 25’ 96’’ N, 115° 59’ 74’’ E havi et al. 2009, Janda & Abbott 2010). For example, to 40° 45’ 64’ N, 115° 96’ 53’’ E) is located in the north- a genetically modified endo phytic strain, Burkhol - west of Beijing city. It was once Beijing’s second deria cepacia, together with yellow lupine Lupinus largest water source for drinking, agricultural and luteus, could improve degradation of to luene (Barac industrial purposes. Since the 1970s, Guanting Re- et al. 2004). Addition of petroleum to a sediment servoir has been heavily polluted owing to rapid doubled the prevalence of naphthalene dioxygenase industrialization and intensive application of chemi- (ndoB)-positive endophytes in Scirpus pungens (Sici - cal fertilizers and pesticides to arable fields in up - liano et al. 2001). Some endophytic bacteria from stream regions (T. Wang et al. 2007, Hu et al. 2009). poplar trees exhibited the ability to degrade ben- Pollutants detected in Guanting Reservoir include zene, toluene, ethylbenzene and xylene (BTEX) pesticides (organochlorines, OCPs) and persistent compounds at one contaminated site (Moore et al. organic pollutants (POPs) at concentrations of 16.7 to 2006). However, reports about the biodegradational 791 ng l−1 (mean, 234 ng l−1), 275 to 1600 ng l−1 (mean, abilities of endophytic bacteria were mostly focused 644 ng l−1) and 5250 to 33 400 ng kg−1 (mean, 13 000 ng on rhizospheric bacteria in terrestrial plants. Apart kg−1) in the surface water, pore water and sediment from Toyama et al. (2009), who showed that an (dry weight), respectively. An analysis of the sedi- aquatic plant−bacterial collaboration could acceler- ment yielded the following: chemical oxygen de- ate contaminant degradation in the aquatic environ- mand, 49.7 mg l−1; total phosphorus, 53.5 mg l−1; ment, there has been little research into endophytic total nitrogen, 62.4 mg l−1 and chlorophyll a (chl a), bacteria associated with aquatic plants. 51.6 mg m−3 (Xue et al. 2005, 2006). Environmental water pollution is a serious problem Along the shoreline of the reservoir, 4 species of in China and other developing countries because of healthy plants, Phragmites communis, Potamogeton the discharge of organic pollutants such as pesticides crispus, Nymphaea tetragona and Najas marina, which and nutrients including phosphorus (Zhao et al. 2006, are dominant aquatic plants in this region, were H. Wang et al. 2007, Perelo 2010). Because organic sampled from Daying (40° 44’ 30’’ N, 115° 90’71’’ E), pollutants in aquatic environments are generally pre- GuiS lake (40° 45’ 16’’ N, 115° 96’ 08’’ E) and KZ sent in low concentrations, bioremediation and/or bridge (40° 45’ 00’’ N, 115° 88’ 02’’ E) where one of phytoremediation may be the most economic and the rivers flows into the reservoir. At each sampling reliable approach to address the problem. In addi- site, 4 plants from each species were sampled and tion, phosphorus, which can stimulate eutro phica - cleaned with sterilized water to get rid of sediment tion, usually precipitates in sediments. This makes and dust. The roots, stems and leaves of the entire phosphorus largely unavailable to plants. Further- plant were sliced into 1 to 2 cm × 1 to 2 cm pieces. more, the phosphorus precipitate can persist in a lake The roots, stems and leaves were taken separately for a considerable length of time and continue to from different plants within each species and mixed periodically cause algal blooms. The release of in - completely for that species as root, stem and leaf soluble phosphorus and its removal from the en- samples, respectively. The mixed root, stem and leaf vironment using aquatic plants is a so-called eco- samples from each plant species were then divided engineering approach (van Bohemen 2005), in which into 3 subsamples (each 6 g), washed in sterilized released phosphorus is taken up by plants and then water for 5 min, surface-sterilized with a solution removed from the aquatic environment by harvest- containing 5% active chloride (w/v, added as a ing the plants. However, little research has been NaOCl solution) for 3 min and 70% ethanol for 1 min undertaken to understand more about endophytic and then rinsed 6 to 8 times in sterile distilled water. bacteria in aquatic plants, in particular their diver- They were then ground separately with scissors and sity, distribution within the plant, bioremediation a glass rod to make plant slurries. The root, stem and abilities and their association with the plants. leaf subsample slurries were mixed again to make The present study was therefore undertaken to one integrated root sample, stem sample and leaf investigate endophytes found in 4 species of aquatic sample, respectively, for each of the original species plants growing in a reservoir in Beijing, China, by of plant. The slurry samples were then diluted with using the culture-dependent method. sterilized double-distilled H2O. The final diluted slur- Chen et al.: Endophytic bacteria in aquatic plants 101 ries (100 µl each) from the integrated root, stem and maximum-likelihood and maximum-parsimony algo- leaf samples of each plant species were respectively rithms. spread onto Luria-Bertani (LB) agar plates (12 agar plates in total), which is often used to isolate endo- phytic bacteria from plants (Bhore et al. 2010, Guo et Functional analysis of the isolates al. 2010, Luo et al. 2011), and incubated at 28°C for 5 to 7 d. In addition, 100 µl of the last rinse and dilution Representative isolates were selected from each water were also plated on LB medium as a sterility genotype in combination with their location in the check. Then 100 morphologically different bacterial 4 aquatic plants. After being precultured in LB colonies from the 12 LB agar plates were selected medium for 5 d at 28°C, cells of the representative and subcultured for purification. isolates were harvested by centrifugation and washed 3 times with sterilized water. They were then inocu- lated onto the minimal salt agar plates containing −1 −1 −1 Sequencing and analyses of DNA 1.5 g l K2HPO4, 0.5 g l KH2PO4, 1.0 g l NaCl, 1.0 −1 −1 g l NH4NO3 and 0.20 g l MgSO4 at pH 6.8 to 7.0 Genomic DNA was prepared from the pure indi - and supplemented with 20 mg l−1 chlorpyrifos, fen- vidual isolates using FastDNA Spin Kit for soils (MP propathrin and bifenthrinas and naphthalene, respec- Biomedicals) and PCR was performed with the uni- tively, which have been detected as serious pollu- versal 16S rRNA gene primers: 8f: 5’-AGA GTT TGA tants in Guanting Reservoir (Xue et al. 2006). The TCC TGG CTC AG-3’ and 1492r: 5’-GGT TAC CTT final cell concentration was 108 cells ml−1 in each cul- GTT ACG ACTT-3’.