Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils
© Tsuboi et al. 2015
Received: 3 February 2015
Accepted: 7 September 2015
Published: 18 September 2015
The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with the unplanted system. Among the representative alkane hydroxylase genes alkB, CYP153, almA and ladA, the first two were detected in this study, and the genotypes of both genes were apparently different among the systems studied. Their diversity was also higher on the rhizoplanes of the grasses than in unplanted oil-contaminated soils. Actinobacteria-related genes in particular were among the most diverse alkane hydroxylase genes on the rhizoplane in this study, indicating that they are one of the main contributors to degrading alkanes in oil-contaminated soils during phytoremediation. Actinobacteria-related alkB genes and CYP153 genes close to the genera Parvibaculum and Aeromicrobium were found in significant numbers on the rhizoplanes of grasses. These results suggest that the increase in diversity and genotype differences of the alkB and CYP153 genes are important factors affecting petroleum hydrocarbon-degrading ability during phytoremediation.
The exploration, extraction, refining, transport, and use of petroleum and derivative products has resulted in soil pollution with petroleum hydrocarbons, which is of critical environmental concern worldwide (Khan et al. 2013). Techniques for cleaning these soils include physicochemical/chemical treatments such as chemical oxidation using ferrous compounds and soil thermal desorption (Langbehn and Steinhart 1995; Ferguson et al. 2004), but these are expensive and environmentally invasive (Pandey et al. 2009; Segura et al. 2009). Biological remediation methods using plants (that is, “phytoremediation”, a green technology) have been recognized as excellent alternatives (Khan et al. 2004; Jain et al. 2011).
Grasses and legumes have been selected and used for phytoremediation of petroleum-polluted soils because of their tolerance to petroleum pollution. Grasses in particular are regarded as candidate plants for efficient phytoremediation because they have fibrous roots (Kaimi et al. 2007) that can loosen soil aggregates and effectively introduce oxygen, which is needed to activate alkanes by terminal oxidation by alkane hydroxylases (van Beilen et al. 2003), along root channels from the atmosphere (Adam and Duncan 1999; Merkl et al. 2005).
A primary concept of phytoremediation is that the petroleum-degrading microorganisms in the rhizosphere, which consists of rhizoplanes (the external surface of roots) and soil close to roots, have their degradation activity enhanced by exudates from the plant roots (Kuiper et al. 2001) and by molecular oxygen introduced from the atmosphere (Adam and Duncan 1999; Merkl et al. 2005). Although previous studies reported that these plants effectively reduced petroleum concentration in the contaminated soils, presumably via stimulation of petroleum-degrading bacteria, the bacterial communities involved in the remediation remain largely unknown. Thus, characterization of the petroleum hydrocarbon-degrading bacteria on the rhizoplanes is indispensable to understanding the phytoremediation mechanisms and improving the efficiency of remediation. This study aims to acquire novel insights into the community structures and diversity of alkane-degrading bacteria on the rhizoplanes of grasses, based on culture-independent molecular approaches.
Four types of grass were used in this study: two Japanese lawngrasses [Zoysia japonica Steud. and drought-resistant Z. japonica Steud. (described as “dr-Z. japonica” in this paper)], Manilagrass (Z. matrella Merr.), and Tifton Bermuda grass (Cynodon dactylon Pers.) were used in this study. The carpeting grasses were obtained from commercial gardening stores.
Soil preparation, plant experiment and sampling
To compare the diversity and phylogeny of alkane-degrading bacteria among the rhizoplane samples of the four grasses planted under the same experimental conditions, petroleum-contaminated soils (10,000 mg/kg) were prepared by mixed commercial river sands and oil obtained from an actual petroleum-polluted site in Yamaguchi, Japan, in experimental containers (height, 500 mm; width, 600 mm; depth, 800 mm; and volume, 240 L). To increase the water- and nutrient-holding capacity of the soils, they were covered by a 50-mm layer of commercial Akadama soil (small: 2–6 mm diameter, Makino, Tochigi, Japan). Sections measuring 100 mm × 100 mm (length × width) were periodically cut from the 400 mm × 600 mm carpeting grasses for sampling, and the roots sampled were stored at −20 °C for molecular analysis after removing the petroleum-contaminated sands. The contaminated soils were collected to measure total petroleum hydrocarbon (TPH) concentration. Total petroleum hydrocarbon from the polluted soils was extracted and measured as soon as possible (see below). Samples collected at 856 or 891 and 494 days into the study were used to analyze alkB genes and CYP153 genes, respectively.
DNA extraction from roots and detection of four alkane degradation genes (alkB, almA, CYP153 and ladA)
PCR primers used in this study
Paisse et al. (2011)
Wang and Shao (2012)
Wang et al. (2011)
Lo Piccolo et al. (2011)
M13 primer M4
M13 primer RV
Clone library constructions, sequencing and phylogenetic analysis of alkB and CYP153 genes
Successfully amplified target genes were cloned into the pMD20-T vector with Mighty TA-cloning Kit (Takara Bio) according to the manufacturer’s protocol. The constructed vectors were transformed into Escherichia coli JM109 competent cells (Takara Bio). The selected colonies were checked by direct PCR using the vector primers M13 primer M4, and M13 primer RV (Table 1) and Quick Taq™ HS DyeMix (Toyobo, Osaka, Japan) if they had an insert fragment of the correct size. From each sample, about 50 E. coli JM109 colonies with the PCR fragment of the correct size were selected randomly and used in further sequencing analysis. The positive fragments were sequenced using the BigDye® Terminator kit v.3.1 (Applied Biosystems, Carlsbad, CA, USA) and the vector primers as above, and the sequences were obtained on an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems). We used BLASTx to perform a homology search of the cloned alkB and CYP153 gene sequences against the GenPept database at the National Center for Biotechnology Information (NCBI). Distance matrices were calculated based on DNADIST of PHYLIP (PHYLogeny Inference Package) 3.695 (http://evolution.genetics.washington.edu/phylip.html) and were used to group the obtained sequences into operational taxonomic units (OTUs) with a distance cut-off of 0.15 (alkB) and 0.07 (CYP153) using Mothur (Schloss et al. 2009). Rarefaction curves were calculated using “R” statistics software (R Development Core Team, version 2.15.2). Evolutionary distance dendrograms were constructed by the maximum likelihood method with the Molecular Evolutionary Genetics Analysis (MEGA) 6 software package (Tamura et al. 2013). Confidence of the dendrogram topology was evaluated using bootstrap analysis with 100 resamplings.
Real-time quantitative PCR (qPCR) assay
The standard samples of the target gene quantification were constructed from retrieved and cloned DNAs from the petroleum-contaminated soil and its PCR products. The qPCR was carried out using a Takara Thermal Cycler Dice® Real Time System Single (Takara Bio) and KOD SYBR® qPCR Mix (Toyobo) according to the manufacturers’ protocols. The thermal conditions for alkB qPCR were as follows: initial denaturation at 98 °C for 2 min, followed by 40 cycles of 98 °C for 10 s, 55 °C for 15 s and 68 °C for 30 s. For CYP153, the same thermal conditions were used with the difference that the final phase at 68 °C was extended to 1 min. All analyses were carried out in triplicate on each extracted DNA sample.
Total petroleum hydrocarbon was extracted from 0.2 g of sampled soil (wet weight) with 10 mL of polychlorotrifluoroethylene (H-997; Horiba, Kyoto, Japan) as the extraction solvent. Total petroleum hydrocarbon in the solvent was quantified with an oil-measurement instrument OCMA-350 (Horiba, Japan) based on infrared absorption analysis. A standard mixture of OCB (Horiba, Japan) consisting of 2,2,4-trimethylpentane, hexadecane and benzene was used as standard. Total petroleum hydrocarbon concentration per dry weight soil (g) was calculated and converted to mg/kg units from the actual TPH concentration data and moisture ratios of soil samples.
Nucleotide sequence accession numbers
The nucleotide sequences of the partial alkB and CYP153 genes obtained in this study have been deposited into the DDBJ/EMBL/GenBank databases under the following accession numbers: LC019409 through LC019680 for the alkB genes, and LC019154 through LC019408 for the CYP153 genes.
Decrease in total petroleum hydrocarbon (TPH) concentration
Genotypic diversity of cloned alkB and CYP153 genes
Phylogenetic analyses of the alkB and CYP153 genes
Sequence similarities of the alkB and CYP153 genes at amino acid levels to NCBI database
Distribution of representative AlkB and CYP153 sequences in each system
Closest BLAST match
Range of % ID
b, c, d, e
Uncultured soil bacterium
Pristine and hydrocarbon-contaminated soil
Caulobacter sp. K31
Pseudoxanthomonas spadix BD-a59
b, d, e
Nocardia sp. NRRL WC-3656
b, c, d, e
Granular activated carbon
Mycobacterium chubuense NBB4
Polluted farmland soil
Hexane degrading biofilter
a, c, d
Barley field soil
a, b, d
Crude oil-contaminated soil
a, b, c, d, e
Parvibaculum lavamentivorans DS-1
Parvibaculum lavamentivorans DS-1
a, b, c, d, e
Uncultured Rhizobiales bacterium HF4000_48A13
a, b, c, d, e
b, c, d, e
Alpha proteobacterium MA2
As CYP153, OTU C1, which was the most similar to the uncultured bacterium clone (accession no. BAE47472), indicated a considerable large proportion (76.9 %; 40/52) in the unplanted system. OTU C5 (88–90 % similarity to Aeromicrobium marinum, accession no. WP007077898) was found in the Z. matrella and dr-Z. japonica systems. OTU C4 (83–85 % similarity to the uncultured bacterium clone, accession no. CCO96723) and OTU C6 (97–100 % similarity to Parvibaculum lavamentivorans DS-1, accession no. WP012110693) were more abundant in dr-Z. japonica and Z. japonica systems, respectively. Origins of database sequences with the highest similarity were associated with a range of environments such as oil-contaminated and uncontaminated soils, estuarine and marine sediments and seawater.
Quantification of two alkane hydroxylase genes
Various bacterial phylogenies possess alkB and CYP153 genes, such as Alpha-, Beta-, Gamma- and Deltaproteobacteria; Actinobacteria; Bacteroides; Firmicutes; Spirochetes and Planctomycetes (Wang et al. 2010a, b; Nie et al. 2014a). In the present study, these two genes were also detected in abundance on the rhizoplanes of grasses (Fig. 5). In phylogenetic analyses of these two genes, Alpha-, Beta-, Gammaproteobacteria and Actinobacteria-related alkB and CYP153 genes were detected on the rhizoplane of grasses. These results suggest that these phylogenies play an important role in degrading the oil in the contaminated soils on the rhizoplane of grasses during phytoremediation. Culture-dependent methods show that the genera Bacillus, Ochrobactrum, Enterobacter, Pontola, Arthrobacter, Rhodococcus, Nocardia and Pseudoxanthomonas have been observed on the rhizoplanes of petroleum-contaminated soils, as the alkanes-degrading bacteria (Al-Awadhi et al. 2009). Alkane hydroxylase genes close to those of phylogenies other than those described above (such as the genera Mycobacterium, Nocardia, Aeromicrobium, Parvibaculum and Caulobacter) were also detected in abundance on the rhizoplanes in this study. Most of the retrieved sequences were also similar to clones derived from other environments such as oil-contaminated soils and estuarine sediments (Table 2). These results show that genotypes of the alkane hydroxylase genes on the rhizoplanes of grasses are more diverse than previously supposed, and the alkane-degrading rhizobacteria do not consist of rhizosphere-specific bacterial assemblages.
Both alkane hydroxylase genes show higher diversity on the rhizoplanes than in unplanted oil-contaminated soils (Fig. 2). Actinobacteria-related alkB and CYP153 genes in particular were more diverse on the rhizoplanes than in the unplanted oil-contaminated soils (Figs. 3, 4; Additional file 1: Figures S1 and S2). The Actinobacteria-related alkane hydroxylase genes on the rhizoplanes contained the genes phylogenetically close to those of the genera Pseudonocardia, Marmoricola, Aeromicrobium, Actinomycetospora, Mycobacterium, Rhodococcus, Gordonia and Nocardia. To the best of our knowledge, this is the first study to report that alkB and CYP153, phylogenetically close to the first four of these genera, have been detected from the rhizoplanes. Whereas it has been previously discussed (Singh et al. 2007) that Actinobacteria are not seemed to be dominant in environments with continuous carbon substrate supply such as the rhizospheres of grasses, Smalla et al. (2001) found the abundance of Actinobacteria in the rhizosphere. Our results also imply that Actinobacteria were among the most diverse phyla on the rhizoplanes of grasses in oil-contaminated soils. Thus, various actinobacterial species might be some of the main contributors in degrading alkanes in contaminated soils during phytoremediation using grasses.
The root-associated bacteria were different from bacterial communities in bulk soils (Grayston et al. 1998), although the reason(s) why diversity of hydrocarbon-degrading genes on the rhizoplane increased are unclear. The genera Parvibaculum, Caulobacter and Mycobacterium, which were likely to be abundant from the detected genotypes of alkB and CYP153 genes on the rhizoplane in this study, can produce biofilms (Schleheck et al. 2000; Smit et al. 2000; Carter et al. 2003; Ojha and Hatfull 2007). Regonne et al. (2013) proposed that formation of bacterial biofilms might be associated with an increase in diversities of the polycyclic aromatic hydrocarbons (PAHs)-specific ring-hydroxylating dioxygenase alpha subunit gene responsible for phenanthrene degradation on a hydrophobic membrane laid in contaminated soils. Biofilms are considered to enhance PAH availability by increasing contact surface areas between bacteria and hydrophobic hydrocarbons (Eriksson et al. 2002). Bacterial communities in the biofilms physically and physiologically benefit each other (Stach and Burns 2002), and our results suggest that formation of biofilms is likely to help to increase genotypic diversity of alkane hydroxylase genes on the rhizoplanes.
The TPH effectively decreased in all grass-planted systems (Fig. 1), in which alkB and CYP153 genes were more diverse than in the unplanted system. However, copy numbers of both genes were not correlated with degradation efficiencies (Fig. 5). Thus, the diversity of alkane hydroxylase genes may enhance phytoremediation efficiency. It was reported that inoculation of elite alkane degraders increased degradation efficiency during phytoremediation (Soleimani et al. 2010; Afzal et al. 2011). The diversification of alkane hydroxylase genes probably increases the probability of elite alkane degraders appearing in the bacterial community on the rhizoplane. Furthermore, the co-existence of alkB and CYP153 genes in a bacterial cell enlarges the range of alkane degradation (Schneiker et al. 2006; Nie et al. 2014b). Nie et al. (2013) reported that homologues of alkane hydroxylase gene in a bacterial cell expressed at different range of alkanes. Our results suggest that the diversity and genotypes of alkane hydroxylase genes on the rhizoplane is significant in influencing alkane degradation efficiency during phytoremediation. However, further studies regarding the gene expression and activity of both alkane hydroxylases and the link between chain length of degradable alkanes and genotypic patterns of both genes is necessary to test this hypothesis.
Study conception and design: ST, TNK and KI. Acquisition of data: ST. Analysis and interpretation of data: ST and SY. Drafting of manuscript: ST and SY. All authors read and approved the final manuscript.
The authors thank Ms. Megumi Okawa, Ms. Mahoro Tajima and Mr. Akira Tsurukoya for their assistance with collection of soils and root samples and cultivation of grasses on the oil-contaminated soils.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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