Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils

Tsuboi et al. SpringerPlus (2015) 4:526 DOI 10.1186/s40064-015-1312-0 Open Access RESEARCH Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum‑contaminated soils Shun Tsuboi1,2*, Shigeki Yamamura1, Toshiaki Nakajima‑Kambe3 and Kazuhiro Iwasaki1 Abstract 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 appar‑ ently 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 impor‑ tant factors affecting petroleum hydrocarbon-degrading ability during phytoremediation. Keywords: Bacterial alkane hydroxylase genes, Grass roots, Petroleum contamination, Phytoremediation, Cultureindependent molecular approaches Background 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 *Correspondence: 1 National Institute for Environmental Studies (NIES), Center for Regional Environmental Research, 16‑2 Onogawa, Tsukuba 305‑8506, Japan Full list of author information is available at the end of the article 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 © 2015 Tsuboi et al. This 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. Tsuboi et al. SpringerPlus (2015) 4:526 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 petroleumdegrading 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. Methods Plant species 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 alkanedegrading 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) DNA of root-associated bacteria was extracted from about 0.2 g of each root sample of the carpeting grass Page 2 of 10 using the FastPrep® instrument and the FastDNA® spin kit for soil (Qbiogene, Carlsbad, CA, USA) according to the manufacturer’s protocol. The PCR reaction was performed with the PCR reaction mixture containing PCR buffer with MgCl2, 0.25 mM deoxynucleotide tr (...truncated)