Sampling and isolation
Form July 2015 to August 2017, a total number of 90 environmental samples were collected from sea and river sediments, drinking and non-drinking water, industrial, agricultural and hospital soil and wastewaters in different parts of Iran (Fig 1). The samples were collected aseptically in sterile bottles and processed based on standard procedures. In brief, the water samples were decontaminated with cetylpyridinium chloride (CPC) 0.005%, for 15 minutes and were filtered by vacuum through cellulose nitrate filters (0.45 µm, Sartorius AG, Gottingen, Germany). The filters were rinsed and macerated in tubes containing 15 ml of distilled water. Almost 100 µL aliquots of dissolved samples were transferred into Sauton’s medium, and incubated at 25°, 30° and 35°C in a 5% CO2 atmosphere for 3 weeks.[10].
For soil samples, 15-30 g soil were taken from 3-5 cm depth of a sampling point at the polluted site and transferred directly to the laboratory. Five grams of the soil sample was transferred to 50ml sterile centrifuge tube. 20 ml sterile distilled water was added to the tube and vortexed for 5 min, and centrifuged at 4300 ×g at room temperature for 20 min. The pellet and supernatant were decontaminated in separate tubes by 3% sodium lauryl sulfate and 1% NaOH. 100 µL of the decontaminated sample was used to inoculate into the Sauton’s media which was supplemented with antifungal and antibacterial antibiotics including kanamycin, nystatin and nalidixic acid (each at 50µg/ml) and were incubated as described earlier [3].
For sediment samples, up to 3 grams of sediment samples stirred for 30 minutes in 100 ml of sterile Ringer’s solution (5% v/v). Tenfold serial dilutions of each homogenized suspension were prepared and 200 μl of each of the pretreated 10−2, 10−3, and 10−4 dilutions were inoculated into the Sauton’s media supplemented with antifungal and antibacterial antibiotics including nystatin, kanamycin and nalidixic acid (each at 50µg/ml). The samples were incubated for 3 weeks as described earlier [29]. The details of environmental samples are given in Table 1.
2.2. Conventional identification
The isolates were primarily identified by conventional phenotypic tests including partial acid-fast staining, growth at 25°C, 37°C and 42°C, pigment production and standard biochemical assays, including resistance to hydrolysis of tyrosine, lysozyme, xanthine, and hypoxanthine tests [10]. The identification was further pursued by molecular testing as follows:
2.3. Molecular identification
Chromosomal DNA from Nocardia isolates was extracted using a simple boiling method. In short, a few colonies of the isolates were added into 200 mL of TE buffer (Tris EDTA), boiled for 30 minutes and centrifuged at 10000 rpm for 10 minutes. The supernatant was transferred to another sterile microtube and centrifuged at 11000 ×g for 10 minutes. The Precipitated DNA was re-suspended in 50 µl of Milli-Q water and stored at -20° C .
The identity of the Nocardia isolates was verified to the genus level using a specific PCR protocol based on a 596-bp region of the 16S rRNA as recommended by Laurent [7]. For species identification, amplification and direct sequence analysis of 16S rRNA gene was carried out as described by Roth [2]. Sequencing was performed by ABI 3100 genetic analyzer in Bioneer Company (South Korea). The obtained sequences were aligned manually with all existing sequences of the closely related Nocardia species retrieved from GenBankTM database, compared with the relevant sequences and analyzed using the jPhydit program [14].
2.4. Nucleotide sequence accession numbers
The GenBank accession number for the 16S rRNA sequencing of isolated Nocardia in this study are listed below., N.cyriacigeorgica ( KX685347), N. coublieae like (KX685350), N. asteroides (KX685345), N. fluminea like ( KX685346), N. otitidiscavarum ( KX685341), N. kroppenstedtii ( KX685348), N. cashijiensis (KT372140.2), N. sungurluensis like (KT372141.1)
2.5. Bioremediation analysis
The sample collection locations have been polluted with crude oil and its derivatives such as PAHs (Polycyclic Aromatic Hydrocarbon), Phenol and sodium sulfate. With regard to widespread existence of petroleum resource all around Iran, petrochemical and its derivatives pollutions are the most common environmental pollutants in different region of Iran and have been found in agricultural soil and water, hospital and industrial wastewater and sewages[2; 7; 12]. The bioremediation capacity of isolates for these pollutants was evaluated according to kanali et al.[8] as follow:
2.5.1. Media preparation
To evaluate the bacterial growth in presence of PAH, Phenol and sodium sulfate, 100 mL aliquots of Mineral Salt Medium (MSM) were prepared in 250 mL flask as the base media for this experiment. The MSM medium containing (7H20. 0.25-MgSO4, 0.5-KH2PO4, 0.5-K2HPO4, 1-NaCl, 0.009-CaCl2.2H2O, 0.5-KNO3, 0.1-Mn Cl2.4H2O, 0.07-ZnCl2, 0.015-CuCl2.2H2O, 0.025-Ni Cl2.6H2O, 0.12- COCl2.6H2O, 0.025-Na2MO4.2H2O (g/l). The MSM media then mixed with different substrates as follow.
The media supplemented with 1% PAH mix solution (1-1) purchased from AccuStandard. The PAH mix solution content the following ingredients each at a concentration of 0.2 mg/mL dissolved in dichloromethane and methanol: Acenaphthene, Acenaphthylene, Anthracene, Benzo(b)fluoranthene, 1,2-Benzanthracene, Benzo(a)pyrene, Benzo(k)fluoranthene, Benzo(g,h,i)perylene, Chrysene, Dibenz(a,h)anthracene, Fluoranthene, Fluorene, Indeno(1,2,3-cd)pyrene, Phenanthrene, Naphthalene, Pyrene.
Another set of MSM media were supplemented with 1% phenol (Merck, Germany) dissolved in deionized water, and the third MSM media set were supplemented 1% sodium sulfate (Merck, Germany) dissolved in deionized water.
An amount of 1mL of 0.5 McFarland turbidity (1 × 108 CFU ml-1) of the selected isolates were prepared in normal saline, and inoculated the respected media, incubated for 144 h at 30C in an orbital shaker (3 RCF). To evaluate the bacterial growth in presence of PAH, Phenol or sodium sulfate, samples were collected at 24-hour intervals and the absorbance at 560 nm was measured by Spectrophotometer.
Showing sign of growth in the media indicates decomposition and/or consumption of target material by studied isolates. For final confirmation of degradation of the studied material, the amount of 5 mL of medium were removed from the flask and examined for the PAH, phenol and sodium sulfate degradation yield according to standard procedure [C; 2; 3-4] explained in the following method.
2.5.2. Determination of PAHs phenol and sodium sulfate degradation
5 ml of the MSM medium containing PAH that shown bacterial growth was transferred to a screw cap glass tube and supplemented with 0.6 ml of a mixture of tetrachlorethylene and methanol (1:100) as the extraction solvent, vortexed for 10 seconds, then centrifuged at 3000 ×g for 10 minutes. The organic phase then collected and transferred to a clean tube to be further analyzed by HPLC. The amount of PAH was measured by injecting 100µl of the collected organic phase into the HPLC device (Manager 5000, Knauer, Germany), equipped with C18 ultra-sep ES PAH QC specia 6o×2mm ID, with water and acetonitrile as mobile phase at a ratio of 5:95, and 0.3 ml min-1 flow rate. The adsorption at 254 nm was measured and the PAH content of the sample was calculated using the standard curve and function previously calculated using sterile PAH standards [3-4] .
Gibbs method was used for determination of phenolic compound[2]. In brief, 5 ml of MSM medium contain phenol which showed bacterial growth was transferred to a sterile tube and the pH was adjusted at 8.0, then subjected to centrifugation at 2700 g for 20 minutes. 150 µl of the collected supernatant was mixed with 30 µl NaHCO3, then 20 µl of Gibbs reagent (2, 6-Dichloroquinone 4- chloroimide) was added to the mixture and shacked for 30 minutes at 25 0C. The absorbance at 620 nm was recorded and the phenol content determined using the standard curve previously calculated using sterile standards.
From the MSM media supplemented with sodium sulfate that showed bacterial growth an amount of 5 ml sample was taken and transferred to a sterile tube. One ml acetic acid (1%) and 1 ml Acetate buffer was added and mixed for 3 minutes. Afterwards, 1 mL of barium chloride was added and mixed for another 3 minutes. The turbidity was calculated using Spectrophotometry at a wavelength of 420 nm. The amount of consumed sodium sulfate was measured by using the standard curve previously calculated using sterile sodium sulfate standards.