Isolation and Identification of Nickel Resistant Bacteria in Khor Mousa Sediments and Study of Bacterial Capability in Nickel Biosorption

Introduction
Because of population growth, human needs to produce raw materials have increased. Different industries have developed that discharge pollutants into the environment. One type of the contaminants is heavy metals that are abundant in municipal and industrial waste waters. These compounds aren’t capable of biological degradation and in certain quantities are considered toxic to many aquatic animals. Heavy metals may be accumulated in the various parts of the ecosystem and organism tissues along the food chain. Transfer of these pollutants may eventually lead to human health threat.
Petroleum consists of considerable amounts of heavy metals especially lead, nickel and vanadium. While loading and mining, amounts of petroleum intentionally and unintentionally enter the marine environment and besides pollution, the concentration of heavy metals like nickel increases. Most of the petroleum constituents which enter the marine environment are either volatile or dissolvable by the microorganisms, while the metals cannot be disintegrated and last more. Methods should be considered to eliminate these metals from the environment in order to prevent pollution.
Nickel due to its wide application in metal planting industry is concerned. These are various methods for nickel metal removal from aqueous environment including oxidation and reduction, ion exchange and metal deposits. However these methods may also impose high costs and low metal concentrations have little efficiency. Biosorption is a relatively newer method in which the absorption ability of microorganisms to remove pollutants from the environment is used. Fungi, algae and bacteria are among the microorganisms which are capable of biological degradation and are used frequently in studies of biosorption.
Various species such as Aspergillus, Pseudomonas, Sporpophyticu, Bacillus and Phanercheate are reported as nickel biosorbents. Bacteria are preferred due to high adaptation and quick growth. The cell wall of these microorganisms has lipo-polysaccharide which contains active groups such as carboxyl, hydroxyl and amine that enabled bacteria to adsorb metal cations. The objective of this study was to isolate and identify nickel-resistant bacteria from contaminated sediments in the Khor Mousa and determine its ability to remove nickel from the environment.
Materials and methods
Sediment samples from the surface layer of Khor Mousa contaminated sediment were taken using a grab. For isolation of bacteria, initially one gram of each sample was added to 10 ml of a %0.85 salt solution and was diluted to 10 ^-3. 0.1 ml on nutrient agar medium containing nickel concentrations of 50 and 100 mg/l were cultured. 
Thereafter, the prepared mediums were incubated at 30° C for 4 days. After colonies growth on medium, the largest colony was selected and cultured frequently on solid media to get a purified bacterium. Bacterium identification was carried out through biochemical tests for example oxidase, catalase, TSI, citrate, VP, phenylalanine, macConkey, indole, MR, lactose, motility and decarboxylase.
To measure the bacterial growth, first LB broth culture was used to prepare suspensions. The isolated colony was cultured into 50 ml of LB broth and incubated for 24 hours at 150 rpm. Afterwards, 10 ml of the solution was extracted and centrifuged at 7000 rpm for 20 minutes. The upper phase was extracted, washed and centrifuged and eventually the bacterial suspension was prepared. Three ml of the suspension medium was added to LB broth containing concentrations of 50, 100 and 200 mg/l of nickel and the assay was performed with a spectrophotometer for 12 days. The primary step is to use 3 ml LB broth as a blank sample. After that 0.6 ml of medium containing bacteria were mixed with 2.4 ml of fresh LB broth to measure the optical density at 600 nm wave length.
 The nickel solutions at concentration of 50, 100 and 200 mg/l were used for evaluating the bacterial potential for biosorption of nickel. Thereafter, 1 ml of the bacterium suspension was added to metal samples and incubated at 30˚C for 2 hours. Moreover, solutions without bacteria were considered for comparison with other samples. Five ml of each solution was centrifuged at 4000 rpm in 0, 30, 60, 90, 120 and 150 minutes. Finally the amount of nickel remained into the solutions was measured by Atomic Absorption Savant AAS in concentrations of 50, 100 and 200 mg/l at 30 minutes intervals for 150 minutes.
Results and discussion
Three colonies of bacteria were observed on nutrient agar medium containing 100 mg/l nickel and the largest colony was selected for further experiments. The isolated bacterium was a kind of gram-positive rod bacterium that was identified as Bacillus anthracis using biochemical tests. Growth curve in the first 24 h after inoculation in LB broth, in all three concentrations showed a similar trend, but in the days after, the bacteria at concentrations of 50 mg/l had a better performance compared to other concentrations. The maximum growth rate at concentrations of 50, 100 and 200 mg/l nickel was 0.331, 0.199 and 0.161, respectively. The sample without nickel showed the maximum optical density (0.633). Also study the maximum growth of B. anthracis depicted that bacterial growth was reduced along with increase of metal concentration. There was a significant different between the highest optical density of the bacterium in the media without nickel and the maximum bacterial growth in the presence of nickel. Moreover, a significant different was not observed between the maximum growth of B. anthracis in concentrations of 100 and 200 mg/l (P>0.05).
The isolated bacterium in the duration of 150 minutes reduced the amount of nickel from 50 mg/l to 15.7 ± 0.99 mg/l. The reduction of nickel metal at concentrations of 100 and 200 mg/l was respectively 52.8 ± 0.99 and 118.2 ± 0.71 mg/l. Meanwhile % 68.6 nickel metal concentration of 50 mg/l of solution was absorbed by the bacterium B. anthracis which was compared to other concentrations won the highest percentage of absorption. The ANOVA test also revealed the significant difference in nickel percentage at concentrations of 50, 100 and 200 mg/l. Furthermore, the percentage of nickel absorption is reduced with increasing Ni concentration.
Many studies have been carried out on Bacillus that proved the bacterial potential in heavy metal absorption. Bacillus due to a high stability in the terrestrial and marine ecosystems can be distributed in various environments. The purpose of this study is to isolate the native bacterial species which is nickel resistant and is able to eliminate nickel from the environment.
 B. anthracis isolated from Khor Mousa sediments and it's capability to eliminate nickel from the environment was studied. In the same field strain Bacillus sp. SJ-101 and Bacillus cereus were respectively isolated from contaminated soil and Anzali wetland sediments. Another study reported the isolation of 4 kinds of marine bacillus which had a great ability in absorbing nickel, from the polluted waters in Egypt. The results of isolation step showed that the isolate could grow in nickel concentration of 100 mg/l. Thus, a range of nickel concentrations (50, 100 and 200 mg/l) was considered in growth and absorption steps.
Evaluating the growth of B. anthracis in different concentrations of nickel metal showed that this bacterium has the maximum growth in a concentration of 50 mg/l and with increasing the concentrations of the metal in the culture, the growth decreased and this is probably due to increased toxicity of metal and its inhibition of bacterial growth. There is also a 24 hour delay in bacterial growth in each of three concentrations of bacteria due to capability with the new medium. In 2007, scientists reported the isolation and identification of the species Bacillus Sp. CPB4 from the pollutant sediments of heavy metals in Korea. The ability of the bacterium in the presence of lead, cadmium, copper, nickel, cobalt, manganese, chromium and zinc was studied in 20-40 ˚C and 40-400 mg/l concentration after 24 hours.
More than 90% of absorption was done by the outer membrane of the bacterium cell because metal ions attach to the outer membrane proteins of cell. The results showed high capability of this species in biological absorption of heavy metals. Comparison of nickel absorption by B. anthracis displayed that it is able to remove % 68.6 of the metal in a concentration of 50 mg/l and % 40.9 in a concentration of 100 mg/l. The ability of nickel absorption in the presence of two bacterial species Bacillus Subtilis 117S and Pseudomonas Cepacia showed that the Bacillus strain could absorb 351/1 mµ/ml nickel from the medium. The nickel removal showed a remarkable increase during 1 to 8 hours after the bacterium inoculation and it reached to a balance after 24 hours. As the concentration of biomass increased, the nickel's adsorption increased too.
In the field of pH effect on metal absorption we can say that the biological absorption of nickel increases by the increase in the amount of pH from 2 to 7. The report in 2010 also showed that Bacillus sp. is able to remove % 50.5 of nickel in a concentration of 200 mg/l. Another study was carried out on the ability of Bacillus sp. MG-75 in nickel absorption.  The mentioned strain in a concentration of 140 mg/l was able to absorb % 70 of the nickel metal.
Most nickel removal occurred in the early 30 minutes of the measurement. The amount of nickel at concentrations of 50, 100 and 200 decreased to 31.65±0.63, 80.50±0.282 and 182.25±0.494 mg/l in the first 30 minutes after the inoculation, respectively. In the primary hour of measurement the bacterium had more active sites to absorb the metal but as the time passes and these sites are occupied so the amount of absorption remains constant.
With increasing metal concentrations in the media, the percentage of absorption will decrease so the maximum percent of biosorption (68.8%) was observed in a concentration of 50 mg/l and the minimum percent (40.9%) was in a nickel concentration of 200 mg/l. Other scientists argued that in lower concentrations, there are fewer amounts of metal ions in comparison with the active sites on the cell wall of bacteria. Therefore, more percentage of the metal will be up taken by non-occupied sites, while with increasing the concentration of the metal in the media, the percent of absorption will decrease.
Conclusions
B. anthracis isolated from Khor Mous sediments and its growth was studied in various nickel concentrations. Although the bacterium had the highest growth in 50 mg/l, it could survive in 200 mg/l concentration.  B. anthracis removed 68/8% nickel from the culture. Thus, with regard to the ability of bacterium to grow in medium containing nickel and the biological uptake of this heavy metal, it can be recommended as an appropriate organism to reduce the nickel-metal pollution.