In Vitro Bioremediation: A Development Process of Cadmium and Mercury Removal by Environmental Biotechnologies of UV-Mutated Escherichia coli K12 and Bacillus subtilis 168

E. coli K12 and B. subtilis 168 were investigated for their cadmium and mercury tolerance abilities. They were developed by UV mutagenesis technique to increase their tolerances either to cadmium or mercury, and their names then were designated depend on the name and concentration of metals. E. coli K12 Cd3 R exhibited bioremediation amount of 6.5 mg Cd/g dry biomass cell. At the same time, its wild-type (E. coli K12 Cd3) was able to remove 5.2 mg Cd/g dry biomass cell in treatment of 17 mg Cd /L within 72 hours of incubation at 37 °C (pH=7) in vitro assays. The results show that E.coli K12 Hg 20 was able to remove 0.050 μg Hg/g dry biomass cell and more removal by its mutant E.coli K12 Hg 20 R to 0.060 μg Hg/ g dry biomass cell in the treatment of 0.15 μg Hg /L. On the other hand, B. subtilis168 Cd2 was able to remove the least amount of cadmium (5 mg Cd/ g dry biomass cell) and of mercury (0.045 μg Hg/ g dry biomass cell) under the same conditions were used for E. coli K12. Also, the complete removal of both metals was confirmed by scanning electron microscopy (SEM) showing that the effect of cadmium and mercury on the bacterial mass. Also, the SEM images showed that the removal amounts had relationships in changing the morphology of cells under in vitro assays.

These mechanisms are caused by biotic or abiotic activities of cells that lead to the absorption of cadmium ions in the cell surface or accumulation inside or outside the cell (4). Intracellular accumulation is a more complicated process, and the collection could be involved within specific enzymatic detoxification and organelles systems. However, there are differences in the bioremediation for mercury as the main mechanisms include bioaccumulation and volatilisation rather than biosorption or bioaccumulation mechanisms as for cadmium. Bacteria have a detoxification system of transport Hg (II) into cellular space to reduce it into Hg (0) by volatilisation (5). All these mechanisms, either for cadmium or mercury bioremediation, depend on the resistance ability of bacteria as higher resistance to metal leading to increase the process and the efficiency of removal (6). Thus, the resistance ability of bacteria is one of the most biotechnological approaches in the bioremediation process. One of the simple methods for improving resistance ability of bacteria is using ultraviolet radiation (UV). UV in wavelength above 260 nm can enter into DNA bases, producing pyrimidine dimers that can cause an error in replication of DNA and growing within media that had specific metal ions which could improve the gens response of resistant to that metal ions during the duplication of the wild-type sequence (7). It is challenging to find bacteria which can remove cadmium or mercury efficiency. As, few researchers have studied the mechanisms of cadmium or mercury removal by either E. coli ATCC25922 (8) or B. subtilis BNi11 (9), so in this study, we evaluate to use E. coli K12 and B.subtilis168 as biosorbents for cadmium or mercury.
Four objectives of this study were carried out, firstly determine the minimum inhibitory concentration (MIC) of E. coli K12 and B.subtilis168 in the presence of Cd and Hg, separately, secondly develop their MIC ability by UV mutagen, thirdly evaluate their abilities to remove their concentrations in vitro assay and finally investigate the effect of cadmium or mercury onto bacterial surface by SEM.

Materials and Methods:
Bacteria, cultural medium, and cadmium or mercury stock solutions E. coli K12 and B. subtilis168 used in this study were acquired from Sigma-Aldrich Co and chemicals also were purchased from Sigma-Aldrich Co. The stock culture was grown in 50 mL of a nutrient medium using 250 mL Erlenmeyer flasks. Stock solutions of cadmium and mercury (1 M) were prepared in distilled water and sterilised by filtration using paper size pore 0.22 μm.

Determination of MIC
The MIC values of E. coli K12 and B. subtilis168 against cadmium and mercury ions were determined separately in E-Basel Salts (EBS) media (10). 1 mL of cell inoculum was inoculated into 50 mL of EBS broth medium supplemented with fructose (10 mM) as a source of carbon-containing cadmium or mercury and incubated at 37 °C for 48 hours. Starting concentration of cadmium or mercury was started from 5 µM until the growth of cell inhibited. The MIC was forward growth where there was no growth at a known concentration.

Effect of cadmium and mercury on microbial growth and substrate utilisation
After determining the MIC of each strain, the effect of either cadmium and mercury on the growth of E. coli K12 and B. subtilis168 were evaluated by growing the strains in the presence of cadmium and mercury at concentrations 0.1 X MIC, 0.5 X MIC, and 1 X MIC. 1 mL of cell inoculum (OD 440 = 0.1) was inoculated into 50 mL EBS both medium supplemented with fructose (10 mM) as carbon source with the presence of cadmium or mercury in 250 mL Erlenmeyer flasks in triplicate and incubated shaken at 150 rpm at 37 °C. The cell growth was determined from the measuring of optical density (OD 440 ) by spectrophotometry at interval times. The fructose utilisation by bacterial cells was determined by collecting samples at interval times through cultural growth. Collected samples were centrifuged, and the supernets were kept in a freezer to determine the amount of fructose by using fructose kit assay (Sigma, kit number FA-20).
The growth was observed by measuring the optical density (OD 440 ) as 0.1 of OD assumed to equal to 23 mg/l of cells. Monod`s equation (1947) was used to determine the growth rate (µ): And the dry biomass formed was measured from the differences between the final concentration (X f ) and the initial concentration of biomass (X 0 ) to the differences between the final concentration (S f ) and the initial concentration of substrate (S 0 ) (11).

Mutagenesis
The mutagenesis was done for E.coli K12 and B. subtilis168 in aiming for getting mutants with higher resistance either to cadmium or mercury. Cells at concentrations 6 x10 6 were collected from early stationary phase prior mutation assay. Firstly, to optimise the best time of exposure that kill 90% of cells, cells ( 50 CFU/mL) were irradiated by ultraviolet light distance from light 50 cm for different times of vulnerability and colonyforming units were counted after growing in nutrient agar medium. Then the mutation assay was done at the best times for exposures. The irradiated cells finally were grown in EBS liquid media supplemented with 0.5 X MIC, 1 X MIC, 1.5 X MIC, and 2 X MIC concentrations of either cadmium or mercury of both wild-types in triplicate and incubated at 37 °C for 72 hours. The screening of mutant was done in EBS agar medium at the cadmium or mercury concentrations of cultures that the mutants were obtained from it. One mutated colony appeared on an EBS agar plate having the highest concentration of Cd or Hg was selected and purified for use in in vitro bioremediation of either of cadmium or mercury. In vitro bioremediation of cadmium and mercury For the preparation of absorbent, cells from early stationary phase of growth cultures grown in nutrient broth medium were harvested by centrifugation at 40000g for 15 minutes. Cells were washed twice with buffer solution (pH 7) and kept at -8°C prior using for the bioremediation of cadmium and mercury. For bioremediation experiments, cells in concentration 2.5 g/L were inoculated into 50 mL of cadmium at concentration was 17 mg/L and of mercury solution at concentration was 0.15 µg/L in 250 ml flasks in triplicate and incubated at 37 °C shaken at 150 rpm. Samples were taken from the batch flasks at interval times and centrifugated. The pellets were washed with a buffer solution to observe on the cell surface under SEM. The supernets were used to determine the concentrations of either cadmium or mercury by ICP-MS. The amounts of determined Cd or Hg (concentration in mg/L) were normalised into mg Cd or Hg depending on the molecular weight of each metal and the cell mass as follows: where M i and M f are the initial and the final amounts of metal (mg); m is the dried biomass of cell (g).

SEM
SEM was used to observe the cadmium and mercury that binding on cell mass and how the cellsurface changed morphologically. Pellets of cells loaded with cadmium/mercury were treated with 5% glutaraldehyde (12). The fixed pellets were viewed under SEM (JEM-6610LV).

Determination of the concentrations of cadmium and mercury after bioremediation
The concentrations of cadmium and mercury in the batch experiment solutions that bioremediated by E. coli K12 and B. subtilis168 or their mutants were determined using ICP-MS. The samples of 20 mL were collected from each bioremediation flask, and 1 % of nitric acid was added to acidifications the samples. An internal standard solution, indium at 50 mg/l concentration and gold at 1 %( v/v) were added to the acidic samples for precise the measurement of Hg to avoid the volatilisation of it.  The growth curves of both strains shown in Fig.1 and the growth rate for E. coli K12 was 0.14 and for B. subtilis168 was 0.16 h -1 .

Effect of cadmium and mercury on growth activity of bacterial cells
The effects were estimated by measuring of fructose utilisation by E. coli K12 and B. subtilis168. Based on the measurements, the results show that 100% (10 mM) of fructose was consumed by E. coli K12 and 93.7% (9.6 mM) was consumed by B. subtilis168 grown in EBS with 10 mM fructose for 72 hours at 37 °C and shaken at 100 rpm (Fig. 2). However, the growth rates of both strains were slightly affected by 1 and 1.5 mM Cd compared to the control cultures grown without cadmium (Fig. 3) leading to decrease in growth rate that was resulting in increasing the biomasses.
However, at increasing Hg concentrations, the growth rates were considerably reduced of growing strains at 10 µM Hg showed the growth rate of E. coli K12 and B. subtilis168 were 0.10 and 0.12 h. -1 , respectively (Fig. 4). K12 and (b) B. subtilis168. Strains were exposed to (•) 0 (▲) 1 and (■) 1.5 mM Cd. Cells were grown in EBS liquid medium containing 10 mM fructose and incubated at 37 °C for 72 hours and shaken at 150 rpm.

Mutagenesis
The killer curves of E. coli K12 and B. subtilis168, as presented in Fig. 5 a,b show that 20 minutes kill 90% of cells in exposure to UV light, and this time was the best time to generate a mutant. K12 and (b) B. subtilis168. Strains in concentration (50 CFU/plate) were exposed to UV light. Total viable count (CFU/mL) was accounted, and error bars represent the standard error of the mean (n = 3).

Mutant characteristics
One mutant E. coli K12 Cd3 R was obtained from the UV mutagen of E. coli K12 Cd3 in that media had cadmium (9 mM) and no mutant of B. subtilis 168 Cd2 was obtained. Also, one mutant E.coli K12 Hg20 R was obtained in grown in the media that had mercury from E.coli K12 Hg20 and no mutant obtained from B. subtilis 168 Hg20. The MIC of developed mutant, E. coli K12 Cd3 R was 9 mM Cd much higher than the MIC of its wild-type, E. coli K12 Cd3 (3 mM Cd). And the MIC of developed mutant, E.coli K12 Hg20 R was 30 µM Hg bit higher than the MIC of its wild-type, E.coli K12 Hg20 (20 µM Hg).

In vitro bioremediation of cadmium and mercury
E. coli K12 Cd3, mutant E. coli K12 Cd3 R and B. subtilis 168 Cd2 were used as biosorbents for cadmium removals in batch flasks containing 2 mM[Cd]. While, E.coli K12 Hg20, mutant E.coli K12 Hg20 R and B. subtilis 168 Hg20 was used as biosorbents for mercury removals in batch flasks containing 20 µM Hg. The removal amounts were compared between wild-types and mutants to evaluate the improvement of their MIC in the upgrades of the bioremediation process. The precipitation of cadmium was observed in the EBS broth medium after 72 hours in flasks incubated at 37 °C, as shown in Fig. 6. The results, as shown in Fig.7b showed the mutant E. coli K12 Cd3 R removed 6.5 mg Cd/ g dry biomass cell from solution. In comparison, its wild-type E. coli K12 Cd3 was able to remove less amount to 5.2 mg Cd/ g dry biomass cell within 72 hours of incubation at 37 °C (pH 7) in vitro assays (Fig. 7a). But B. subtilis 168 Cd2 was able to remove a similar amount of E. coli K12 Cd3 to 5 mg Cd/ g dry biomass cell (Fig 7c). The removal by E.coli K12 Hg20 and B. subtilis168 Hg20 were to 0.050, and 0.045 µg Hg/g biomass cell, respectively (Fig 8a and  c) and more removal by mutant E.coli K12 Hg20 R to 0.060 µg Hg/ g dry biomass cell (Fig. 8c).

SEM analysis
The SEM images are shown in Fig. 9a, b, c and the interpretations of these images are presented in Tables 3 and 4. SEM showed that the change of the morphology of bacterial surface to irregularity shape due to extracellular biosorption of cadmium responded for cadmium removal (28). It showed that the cell-surface had the original size, rod-shape and clear surface in E. coli K12 Cd3 (Fig. 9a) and B. subtilis 168 Cd2 (Fig. 9c). In comparison, in mutant E. coli K12 Cd3 R (Fig. 9b), the cell surface was bigger and shiny due to the presence of cadmium ions that appeared by SEM as metallic colour. In addition, it can be seen in Table 3 that there was variance in cell length and width between E. coli K12 Cd3 and mutant E. coli K12 Cd3 R despite they belong to one genus and are because of their differences in removal abilities. While the SEM images of cells from batch flasks in media supplemented with mercury are shown in Fig. 9d, e, f and their line analyses data of their images are shown in Table 4. The comparison between E.coli K12 Hg20, mutant E.coli K12 Hg20 R and B. subtilis168 Hg20 in SEM images and line analyses of these images showed nearly small differences in cell-shapes.   Overall, E. coli K12 and B.subtilis168 were used to remove more than 97 % of Cd and Hg ions in the treatment of the solution 17 mg/L Cd and 0.15 µg/L Hg within 72 hours of incubation at 37 °C (pH=7) in vitro assays. The development process of cadmium and mercury removal was carried out using a biological system in E. coli K12, environmental biotechnology. The mutant E. coli K12 Cd3 R and mutant E. coli K12 Hg20 R were also effectively developed the process in cadmium and mercury removal because of their high MIC. These strains were considered as a potential agent for cadmium and mercury bioremediation as increasing the MIC of bacteria led to improve the bioremediation in terms of removal amounts. This process was lower cost and higher efficiency, which depends on different factors including bacterial species, strains, resistant abilities and environmental conditions. It is recommended that further studies could be conducted using the mutants, E. coli K12 Cd3 R and E.coli K12 Hg20 R in laboratory and pilotscale-reactors to confirm the development of bioremediation processes of cadmium and mercury by environmental biotechnologies of this work. It is possible to study the surface complexation of Cd or Hg in strains for determining the model adsorption reaction (27). Also, more analysis, such as transmission electron microscopy (TEM) could be used to identify whether the removals were by adsorption or bioaccumulation mechanisms. In addition, as shown in Fig. 5 that the cadmium was removed by bacterial cultures as precipitation of CdS as shown the yellow colour so, it is important to investigate the forms of precipitated cadmium.