2.1. Cultivation, Isolation of bacteria
The bacterium sample was taken from Babol-Rood River, Babolsar, Mazandaran, Iran and diluted to 10 by 9% NaCl, and then cultured in nutrient agar. After 48 hours, the colonies were cultured in nutrient agar again (Merck KGaA 54271 Darmstadt, Germany) for many times because of purification. Babol-Rood River is an important river in the north of Iran. After that, the colonies cultured in chromium medium nutrient agar with different concentrations of chromium (50 to 1000mg/L), in 37°C, in the pH of 7, and with shaking at 200rpm to isolate more chromium resistant bacteria [22].
2.2. Preparation of Chromium Stock Solution
Chromium stock solution was prepared by means of dissolving K₂Cr₂O7 (Potassium dichromate Merck KGaA 64271 Darmstadt Germany) in deionized distilled water. The concentration of K₂Cr₂O7 ranged from 50 to 1000mg/L in order to have different experiments and was measured by atomic adsorption spectrometer [23].
2.3. Bacterial identification tests, preparation of living and dead bacterial biomass, and metal solutions preparation
Biochemical tests such as gram staining, TSI test, catalase test, and oxidase test have been done for selected bacteria. For obtaining a living bacterial biomass, bacteria were cultured in nutrient broth (Merck KGaA 105443 Darmstadt Germany) and incubated at 37°C, in the pH of 7, and in shake culture (200rpm) for 24 hours; Then they were centrifuged at 7000rpm in 25°C for 10 minutes. To gain a dead bacterial biomass, living biomass should be put in the oven at 55°C for 72hours after they obtained. Thereafter this biomass can be used in the experiments, which need dead bacterial biomass [24].
After each experiment, the amount of total chromium was measured by atomic adsorption spectrometer. The amount of Cr (VI) was computed spectrophotometrically by measuring pink complex of Cr (VI) with 0.2g of 1, 5-diphenyl carbazide, in 100mL of acetone, at 540nm after 10 minutes. The total chromium and Cr (VI) uptake was calculated using the mass balance equation:
Where CI and Cf are the initial and the final total chromium concentration (mg/L), V is the solution volume, and M is the mass of biomass used in experiments [25].
2.4. Biosorption test of Cr (VI)
The effects of many items were determined as follow: pH (different pH experiment that varied from 1 to 13); isotherm (from 0 to 1000mg/L); temperature (0 to 50°C); initial biomass concentration (from 0 to 1g); contact time for defining desorption kinetic that had been done with living and dead bacteria to have a comparison between them (0 to 120min); age of bacteria (24,48, and 72h); the effect of shaker (50 to 200 rpm); the effect of desorption agents (KCl, HCl, CaCl2); isotherm data for binary absorption of (K2CrO4 and CdCl2 + H2O); (K2CrO4 and Pb(NO3)2 + H2O) and for ternary absorption of (K2CrO4, CdCl2, and Pb(NO3)2 + H2O) with equal concentration of each metal that had been used in the experiments
The different way of killing bacteria such as autoclave, incubator, Na3N2 solute in Tris buffer (pH: 10.7), had been applied for the adsorption process. Batch biosorption studies were performed by using 20mL of chromium solution, 1g/L of bacterial biomass concentration, and incubated at 37°C with 200rpm of shaking for 60minutes in different 250mL Erlenmeyer flasks. To set pH, 1M HCL and 0.1M NaOH had been used.
To measure recycling ability, chromium was washed from bacterial biomass after 60minutes of absorption. It was obtained by centrifuge with desorption agents that were named above and after 3times washing with deionized-double distilled water. The optimal result that had been gained after each experiment had been performed for all the other experiments. The experiments such as pH, isotherm, and concentration of bacterial biomass had been done with dead bacteria and other experiments had been done with living bacteria [26].
2.5. Analysis of SEM-EDX, and FTIR of Lelliottia amnigena
In order to study the external appearances, scanning electron microscopy with energy dispersive X-ray (SEM-EDX) had been done before and after chromium biosorption. The bacteria were coated with gold in the presence of argon, an inert gas, and then analyzing was started [27].
FTIR analysis was required to detect the bacterial functional groups in absorption. Dead Lelliottia amnigena infrared spectroscopies with and without metal ion were acquired. The dead and dried samples which were blended with 2% KBr, analyzed in the range 400 to 4000 cm-1 and with the resolution of 8 cm-1 [28].
2.6. Genotypic identification and 16S rRNA gene sequencing
To extract the genomic DNA of isolate, Cinna Pure DNA kit ex6021 was applied. The isolate 16S rRNA gene was amplified using the universal primers of 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1488R (5’-CGGTTACCTTGTTACGACTTCACC-3’). The amplification was done by initial denaturation at 95°C for 15minutes followed by 40 cycles of 95°C for 30seconds, 58°C for 30seconds, 72°C for 90seconds, and final extension at 72°C for 5minutes. The purified PCR product was sequenced using ABI Genetic Analyzer 3500 in both directions.
Isolated bacterial was kept in a dark place in a refrigerator to have a longer and better durability to protect them from different environmental factors such as fungal, other bacteria, and viruses. After each experiment, sample container was acid washed. The specimen stored in 80% glycerol solution and freezed in −80°C for longer maintenance.
All of the experiment had been repeated 3times and average results was calculated. The experiment conditions were the same unless the items required to be experimented in different conditions.