Sample collection and preparation
10 samples of effluents were randomly collected from different car washes in Tehran (light-heavy machine) and transferred to the laboratory under sterile conditions for microbial analysis. Then, to prepare the dilution, 1 mL of the sample was transferred to 9 mL of physiological saline, and completely mixed, and this operation was performed up to 10− 6 dilution and from the prepared dilutions, the media were inoculated.
Isolation of aerobic mesophilic bacteria
From the prepared dilutions, surface culture was performed directly on the surface of nutrient agar media (direct culture) and all inoculated media were incubated for 30–72 hours at 30 ° C. Then, based on the appearance characteristics of growth bacteria were selected by neddle, and cultured on the surface of TSA (Tryptic Soy Agar), TSB (Tryptic Soy Broth), and Sabouraud dextrose agar (SDA), containing chloramphenicol.
Determination of biodegradation of alkyl benzene sulfonates in a minimal salt medium (MSM) containing LABS
This test method is a criterion for measuring the environmental compatibility of sulfonates used as surfactants by measuring the biodegradability of LABS. In this test, the microorganisms are inoculated into a container containing the MSM and the surfactant being tested. After two stages of compatibility, biodegradability is determined by measuring the reduction of surfactant during the experiment. If the reduction of surfactant during the test period is more than 80%, it indicates the degradation of surfactant by bacteria. Moreover, the reference surfactant material was Dodecyl hydrogen sulfate sodium ((MERCK) NO.2969). To this aim, 500 mL of the MSM was poured, and one of them was identified as a control stock containing 10 mL of LABS surfactant from LABS storage solution and was free of bacteria. In other stock, surfactant was added to the MSM in addition to each of the bacteria in a ratio of 1 mL to 100 mL. Thus, stocks containing MSM medium, surfactant and inoculated bacteria were prepared for degradability test.
Before starting the biodegradability test, two initial compatibility steps must be performed for 72 hours in the mentioned stock. After the initial adjustment step, 500 mL of these stocks were added to the stocks containing fresh medium plus LABS, at a rate of 10 mL of storage solution and kept for 72 hours, for secondary compatibility. At the beginning of the degradability test (immediately after inoculation and mixing the contents of the stock) and also on the eighth day, samples were taken and the amount of surfactant in these samples was measured by MBAS method and the results were recorded.
Surfactant measurement test by MBAS method
The basis of this test is to measure the amount of surfactant by complexion with methylene blue in the chloroform phase. This method is able to measure the amount of LABS from 0.05 to 1.5 mg / l per 100 mL of sample. To measure the amount of LABS in the sample, certain amounts of LABS storage solution of 0, 0.1, 0.2, 0.3 and 0.5 ml per sample were taken first, and a few drops of phenolphthalein and a sufficient amount of NaOH were taken. Added to create a pink color. Then, dilute sulfuric acid was added drop wise to color the solution. Then, 25 mL of methylene blue was added and after mixing, 25 mL of chloroform was added to the separating funnel. After 30 seconds, when the two phases were separated, the chloroform layer was transferred to another 250 ml separating funnel and 25 ml of chloroform was added again. In the second step, 50 ml of phosphate washing solution was added, and after 1 minute of settling, the chloroform layer was extracted. Washing with chloroform was continued until the sample volume reached 100 mL.
Then the prepared samples were stored and a calibration diagram for different values was drawn with a spectrophotometer at 650 nm.
Using a pH meter, the solution containing (LABS) and bacteria was adjusted to pHs of 5, 7 and 9 using 1 Normal hydrochloric acid (HCl), and Sodium Hydroxide (NaOH).
Measurement of LABS concentration based on spectrophotometry analysis
To measure the optical absorption to obtain the concentration of LABS by MBAS method on day zero, the device with a wavelength of 650 nm was set to 0 absorption. Then some of the solutions containing chloroform and methylene blue together with concentrations of LABS were transferred into the cell of the spectrophotometer and with a wavelength of 650 nm the absorption rate of each of them was read by the device. The results of light absorption and calculation of LABS concentration for samples containing bacteria in mg / L on day 0 were recorded.
To measure the amount of light absorption to calculate the concentration of LABS, after passing the biodegradation period by MBAS method on the eighth day with standard solution No. 1, the concentration of LABS was 0 mg / l, the device with a wavelength of 650 nm was set to 0 absorption. Then, some of the contents of LABS-containing samples were poured into the cells of the spectrophotometer for bacteria, respectively, and the absorption of each of them was read with a wavelength of 650 nm. Then, from the calibration curve, the concentration of LABS in mg / l was calculated from the light absorption.
The percentage of LABS decomposed by Bacillus licheniformis bacterium was calculated by reducing the concentration of surfactant tested by MBAS method on day 0 and after day 8 using the following formula:
LABS concentration (%)=((S_0-B_0 )-(S_x-B_x))/(S_0-B_0 )×100
Where S_0 and S_x are the amount of surfactant on day 0 (start day) and day x, and B_0 and B_x are the amount of surfactant on day 0 and day x, expressed in mg/L. The test results were calculated as the mean of the decomposed percentage on the eighth day, and then displayed as the mean.
Macroscopic, microscopic and biochemical analyses
Regarding the identification of the main bacterial isolate in LABS degradation, macroscopic (colony characteristics), microscopy (gram staining) and biochemical (lecithinase, citrate, and growth in anaerobic conditions) tests were done.
In order to confirm the isolates, DNA extraction was performed by CTAB method, as described previously(Cota-Sánchez et al. 2006). Then, the quantity and quality of the isolated DNA was determined using spectrophotometry and gel electrophoresis. After that, 16s rRNA gene was amplified by Polymerase Chain Reaction (PCR) using Master Mix (Sinaclone, Iran), in a BioRad thermal cycler system according to the following program: One step Initial at 5 ° C for 5 minutes, 35 cycles at 95 ° C for 1 minute, 57 ° C for 1 minute, 75 ° C for 75 seconds and a final expansion step at 10 ° C for 10 minutes. In this study, universal 27F (forward primer) and 1492R (reverse primer) were used, which were purchased from Pishgam company (Tehran, Iran). After confirming the single band, the PCR products were sequenced by Bioneer company (Tehran, Iran). The obtained sequences were analyzed through BLASTN online program. Finally, the phylogenetic tree was draw by MEGA7 software to determine the genetic similarity of the obtained isolates.