ISOLATION, SCREENING AND CHARACTERIZATION OF HEAVY METAL RESISTANCE BACTERIA FOR THEIR POTENTIAL USE IN BIOREMEDIATION OF POLLUTED WATER

Snehal Kale 1 , Narsingrao Bandela 1 and * Sayaji Mehetre 2 . 1. Dept of Environmental Science, Dr Babasaheb Ambedkar Marathwada University, Aurangabad. 2. Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Collection of sample:
The effluent water samples were collected from Common Effluent Treatment Plant (CETP), Navi Mumbai. The samples (in duplicate) were collected in a sterile plastic container (5 liter capacity) which was sealed subsequently with paraffin and brought in the laboratory.

Isolation of heavy metal resistance bacteria:
For isolation and purification, 1 ml of effluent water was inoculated in nutrient broth medium (Himedia, India) and incubated at 37 0 C. The stock solution of different metals salts was prepared as Arsenic Sulfate (AsSO 4 ), Cadmium Chloride (CdCl 2 ), Chromium (K 2 Cr 2 O 7 ), Cobalt Sulfate ( CoSO 4 ), Copper Sulfate (CuSO 4 ), Lead Sulfate (PbSO 4 ), Mercury Sulfate (HgSO 4 ) , Nickel chloride (NiCl 2 ) and Zinc Sulfate (ZnSO 4 ) in distilled water. Subsequently desired metal concentration was added in a petri plates containing Nutrient agar medium (Himedia, India) and incubated at 37 0 C (Joshi and Modi 2013). After complete growth single colony was isolated and transferred to another plate containing the respective metal.

Characterization of bacteria.
All the strains were characterized for the growth parameters mentioned as follows.

Optimum pH:
The optimal growth conditions with reference to pH was determined by growing the strains in Nutrient broth with different pH (viz. 4,6,7,9,10,11) and incubated at 37 0 C for 24 hr. Growth was quantified in terms of OD measured by UV-Spectrophotometer (Model V 530, Jasco, Japan) at 600 nm.

Optimum temperature:
The optimal growth conditions with reference to temperature were studied by growing each strain in a Nutrient medium and incubated at 25 0 C, 30 0 C, 35 0 C, 37 0 C, 40 0 C for 24 hr. Growth was quantified in terms of OD measured as mentioned earlier.

Salt tolerance:
Test tubes containing nutrient broth medium with different concentration of sodium chloride (1 to 10%) were inoculated with each strain and incubated at 37 0 C for 24 hr and the growth was measured as mentioned earlier (Zhang 2008).

Enzyme potential of bacteria:-Starch Hydrolysis:
Potato starch (10%) was added to nutrient agar medium and inoculated with all the cultures and incubated at 37°C.

Cellulose:
Medium containing 1% cellulose was inoculated with with culture and incubated at 37°C for 2 days the plates were flooded by iodine to detect the zone of clearance due to cellulose production. (Coleman et.al. 2009)

Carbon assimilation capacity of bacteria:
The ability of the isolates to utilize different carbon substrate was tested by adding different carbon sources (Lactose, Dextrose, Sorbitol, Mannitol, Arabinose, and xylose) in minimal medium at 1% concentration. Individual strains were streaked on the plates and incubated at 37 0 C for 24 hr .(Denef et al. 2009)

Study of growth of bacteria:
Each isolate was studied for its growth pattern after inoculating overnight grown culture and incubating at 37 0 C. Aliquots of samples were drawn at every 2 hr interval and growth were measured as optimal density (OD) using UV spectrophotometer (Model V 530, Jasco, Japan) @ 600 nm.

(Wingender et al. 2001)
Bioremediation of effluent waste water using isolated strains. All the selected strains were gown in nutrient broth and incubated at 37 0 C for 24hr and centrifuged @ 10000 rpm to get the desired pellet and 0.1% (w/v) inoculum was used per 100ml of waste water (Garecha et al 2016). The flasks were incubated for 3 to 4 days at room temperature with a control flask without culture .Observation like change in colour, pH, and COD reduction were done at the end of incubation (Bayoumi et al. 2014).

Result and Discussion:-Isolation and selection of heavy metal resistance bacteria:
Based on the maximum threshold limit 9 strains were isolated on the basis of colony characterization and growth behavior and designated as WAs1 , WCd2, WCr3, WCo4, WCu5, WPb6, WHg7, WNi8, WZn9. It was found that Arsenic, lead and Zinc tolerance was observed up to 1000 ppm to the respective strains and WCr4, WHg6 isolate showed tolerance below 50ppm for Chromium and Mercury. The maximum threshold limits for respective strain are shown in Table.

Optimization of growth parameters:-pH
Each strain was studied for its optimum pH requirement based on maximum OD observed for each strain. It was found that strain WAs1 showed optimum pH at 11 and all other strains showed optimum grow at pH 7 (Fig. 1).

Fig 1:-pH requirement of different strains
Temperature:-Based on the maximum growth observed by the bacteria it was found that all the strains grew at 37 0 C temperature (Fig. 2).

Enzyme potential of bacteria:
The details of the enzyme activity of isolated strains are given in Table 2. Strain resistant to cadmium (WCd2) was found to be cellulose positive and lead resistant strain (WPb8) was citrate positive and other strain showed mixed response among the studied enzymes (Sidkey et al. 2010).

Isolates
Enzymes potential of bacteria

Growth Curve.
Growth curve of all 9 strains was studied in order to understand the growing nature of the isolates to get the maximum growth in minimum time. Growth curve of each isolates are given in  Morphological and biochemical study:-Bacterial strains were characterized on the basis of Gram nature and shape. It was found that the strains WAs1, WCd2, WCo3, WCr4, WCu5, WHg6, WNi7 and WPb8 were gram negative and strain WZn9 was gram positive in nature. Strains WAs1, WCd2, WCo3, WCr4, WCu5, WHg5 and WZn9 were cocci in shape and strain WNi7 and WPb8 were rod shape. Colony characteristics of each strain are given in Table 4.

Conclusion:-
Different bacterial strains were isolated from effluent water and used for their ability to reduce the COD being major parameter of water quality. It was observed that there was 80-90% COD reduction by these isolates. These isolates were also tested for their resistance to heavy metals based on the maximum threshold limits. Strains WAs1, WCd2, WCo3, WCr4, WCu5, WHg6, WNi7, WPb8 and WZn9 showed tolerance to Arsenic (1000 ppm), Cadmium (200 ppm), Cobalt (400 ppm) Chromium (20 ppm), Copper (100 ppm) Mercury (20 ppm), Nickel (50 ppm), lead (1000 ppm) and Zinc (1000 ppm). Morphological, biochemical and molecular characterization of all these strains was completed. This study revealed the potential of heavy metal resistant isolates for bioremediation of waste water for its application to different purpose.