Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in the environment and are of concern because of their toxic and carcinogenic potential (Menzie et al. 1992). PAHs persist within the ecosystem for years due to poor solubility in water and their adsorption to solid particles. Microbial degradation is the major route through which PAHs are removed from the environment (Habe and Omori 2003), and biological treatment of PAHs should be efficient, economical, and versatile compared to physicochemical treatment. Obtaining bacterial isolates capable of degrading PAHs is a first step towards understanding the microbiology and fate of PAHs in the environment. Enumeration is essential to describe a PAH-degrading microbial community. However, it is difficult to screen and enumerate the bacteria on agar plates because PAHs cannot be incorporated into agar. Traditionally, the three methods used for the detection of PAH-degrading bacteria are the spraying-plate, imbedding-plate, and sublimating-plate methods in which PAH-degradation-positive colonies are surrounded by a clear zone in PAH haze (Kiyohara et al. 1982; Bogardt and Hemmingsen 1992; Shuttleworth and Cerniglia 1997; Alley and Brown 2000), and these methods have been used by many researchers (Heitkamp et al. 1988; Kästner et al. 1994; Goyal and Zylstra 1996; Bogan et al. 2003; Edlund and Jansson 2006). In fact, all these procedures are labour intensive and have other disadvantages such as the frequent contamination of PAH on the surface of the plate and the laboratory fume hood during spraying or sublimating, and the degree of homogenization and quantity of PAH deposited on the agar plate which is difficult to control.
In this paper, we describe a method that can solve some of these problems. The method is based on adsorption of PAH by cellulose acetate/nitrate filters lying on the surface of a mineral salt agarose solidified media. This procedure is a rapid and simple method to screen and/or count PAH-degrading bacteria.
Soil samples from 5 m from the mouth of Number 77 well, which is located in saline soil of the Shengli Oilfield of Shangdong Province, China, were collected in sterile 250-ml bottles and stored at 4°C without loss of humidity until samples of bacteria were investigated in the laboratory. Dry weight of soil samples were determined by drying known amounts in duplicate at 65°C to a constant weight.
For the screening or cultivation of PAH-degrading bacteria, a 3·5% (w/v) mineral salts medium (MSM) was prepared (g l−1): (NH4)2SO4 (4·0), K2HPO4 (0·5), NaCl (23·84), MgCl2·6H2O (2·07), MgSO4·7 H2O (3·14), CaCl2·7H2O (0·22), KCl (0·63), NaHCO3 (0·03) and NaBr (0·07). The medium was supplemented with 1 ml of trace lement solution (mg l−1): Ca(NO3)2·4H2O (800), FeSO4·7H2O (400), CuSO4·5H2O (80), ZnSO4·7H2O (40), MnSO4·4H2O (40), NaMO4·2H2O (8) and H3BO3 (6). The pH values were adjusted to 7·2 with 5 mol l−1 KOH. When agarose plates were prepared, 1·5% agarose was added to MSM prior to autoclaving. The phosphate solution was autoclaved separately and added to each previously sterilized salt solution before pouring into the plates.
The procedures were performed in a fume hood with the appropriate personal safety equipment. First, the edge of a piece of cellulose acetate/nitrate filter (0·45 μm; Millipore; disk diameter, 90 mm; Beijing Dingguo Biotechnology Co., Ltd, North Qijia Town, Beijing, China) was held by using sterilized antimagnetic tweezers, placed onto the surface of a predried mineral salts agarose medium in a standard glass Petri dish (100 by 15 mm). Then, any air between the cellulose acetate/nitrate filter and the agarose surface was expelled using a glass spreader. The PAH substrates (98% purity; Bailingwei, Beijing, China) were phenanthrene, anthracene, pyrene and fluoranthene. The PAHs were dissolved in ethyl ether (99% purity; Dingguo) at an optimal concentration of 4 mg ml−1 (anthracene, 2 mg ml−1). Then, 1 ml of these solutions was added to the centre of the filter disk and was gently, rapidly and evenly spread with a glass spreader on the surface of the filter. Subsequently, about 90% of the Petri dish base was covered with the lid so that ethyl ether could evaporate under sterile conditions in the fume hood. After about 5 min, the Petri dish was covered completely with the lid. In this manner, the mineral salt solidified medium was coated with a single PAH compound such as phenanthrene that was absorbed by the cellulose acetate/nitrate filter. The soil sample was then serially diluted from 10−3 to 10−8 in mineral salt liquid media and spread on the surface of the cellulose acetate/nitrate filter on agarose plates. These plates were incubated at 25°C and examined regularly for growth and colony formation. To determine whether these colonies can utilize PAHs as the sole carbon and energy source, some control plates without PAH were prepared and examined for growth at intervals for 30 days. The colony-forming units (CFUs) on plates with PAH were counted as the number of potential degrading bacteria per gram of soil dry weight (gdw) by comparison with the control plates. All tests were carried out in triplicate.
In order to validate this method, the diluted oil-polluted saline soil samples were spread on the surface of cellulose acetate/nitrate filter loaded with phenanthrene. After incubation for 96 h, a number of colonies could be observed. More colonies appeared later, and the diameter of the colonies extended with the prolonged incubation while no growth took place on the control plates. Figure 1 shows that many colonies were able to produce a diffusible, intensely colored pigment. The colonies could be tooth picked from the plates without disrupting the layer of phenanthrene.
In order to evaluate the number of PAH-degrading bacteria in the oil-polluted soil of Shengli, a series of diluted soil samples were spread onto the cellulose acetate/nitrate filter, which adsorbed a single PAH compound on 3·5% mineral salt agarose plates. The phenanthrene-degrading bacteria were counted after 5 days, and the bacteria degrading anthracene, fluoranthene or pyrene were counted after 9 days. Direct counts indicated that the number of degraders of phenanthrene, anthracene, fluoranthene or pyrene in the soil example was (5·6 ± 0·7) × 107, (2·1 ± 0·1) × 106, (1·2 ± 0·1) × 106 and (1·4 ± 0·2) × 106 CFU per gdw respectively. These colonies could not grow on the control plate, which indicates that these bacteria can utilize PAH as a single carbon and energy source. It should be pointed out that the plate count allows detection and isolation of a subset of total bacteria. Therefore, the oil-contaminated soil of Shengli may contain more PAH-degrading bacteria than the number counted with this method.
Screening and enumeration of PAH-degrading bacteria from the environment has been quite difficult on agar plates because of the aqueous insolubility of PAH. In this study, a rapid procedure was developed for screening bacterial isolates for the ability to grow with PAH adsorbed on a cellulose acetate/nitrate filter on agarose plates. The cellulose acetate/nitrate filter method has several obvious advantages over techniques previously published. First, this method is safer, faster and less expensive. Secondly, the homogenization and quantity of deposited PAH can be controlled. Thirdly, because the samples are spread after the evaporation of ethyl ether, enumeration of PAH-degrading bacteria is more accurate by avoiding the toxic effects of organic solvents such as ether or acetone on the bacteria. Fourth, the compound layer of the filter surface cannot be disrupted by streaking, which occurs in the previous published methods. Finally, the method can also be used for screening of mutants, which makes it useful in genetic screenings.