Bioremediation of heavy metals by employing resistant microbial isolates from agricultural soil irrigated with Industrial Waste water

Introduction

The presence of heavy metals and pesticides in the environment has been a subject of great concern due to their toxicity, non-biodegradable nature and the long biological half-lives for their elimination from biological tissues^1-5. Pollutants deteriorates the quality of soil and crops produced. Excessive metal concentrations and pesticides in contaminated soils can result in decreased soil microbial activity and soil fertility, and yield losses^2-3. Agricultural irrigation with wastewater is common in arid areas but has possible public health and environmental side effects, as effluent may contain pathogens, high level of salts, detergents and toxic metals^2-5.

There is a need for monitoring of toxic effects of wastewaters and such irrigation practices should be carried out only after treatment of wastewater. Numerous methods have been proposed to remove heavy metals from sewage sludge, including chlorination, use of chelating agents and acid treatments at high temperatures ^6-7. However, those methods are generally ineffective in practical applications due to high cost, operational difficulties and low metal leaching efficiency. An alternative way to replace chemical methods in removing heavy metals is bioremediation through microbial isolates.

The bioremediation techniques are effective and efficient for remediation of pollutants so as the bioremediation technology from laboratory to field to clean up the environment can be taken up⁸. For bioremediation to be effective, microorganisms must enzymatic ally attack the pollutants and convert them to harmless products⁹. As bioremediation can be effective only where environmental conditions permit microbial growth and activity, its application often involves the manipulation of environmental parameters to allow microbial growth and degradation to proceed at a faster rate. These factors include the existence of a microbial population capable of degrading the pollutants; the availability of contaminants to the microbial population; the environment factors¹⁰.Therefore, this study was designed with the objective to isolate and characterize metal (Zinc and lead) resistant bacteria from heavy metal contaminated soil to determine their feasibility on the removal of metals through bio-accumulation.

Methodology

Collection and Physicochemical Analysis of Soil Samples

Soil samples were collected from the top 15 cm from industrial effluents of the Ludhiana (Punjab) region (30. 91^o N 75. 85^o E). These samples were collected in sterile zip lock bags with the help of sterilized spatula, properly sealed and labeled, and sent to the laboratory within 24 hours. Heavy metals in soil were analyzed by inductively coupled plasma-atomic emission spectrometry at P.A.U., Ludhiana (ICP-AES).

Isolation & Identification of the Micro organism

Isolation was done by the method as described by Jyothi¹¹. Isolated microbes were identified through morphological, biochemical& molecular characterization¹².

Determination of Comparative Growth and Growth Pattern

Extent of heavy metal resistance of selected microbial isolates was evaluated in bacillus cereus broth (bacteria) containing 25, 100, 250 and 300 ppm of Lead nitrate Pb(NO₃)₂, Copper sulphate CuSO₄, Zinc nitrate Zn(NO₃)₂ and Ferrous sulphate FeSO₄ and growth is determined by measuring Optical density (O.D.)at 540 nm with un-inoculated broth as control. To check the growth pattern of the isolates, cultures were inoculated into broth, treated with 0 (control), 25, 100, 250 and 300 ppm of Pb(NO₃)₂, CuSO₄, Zn(NO₃)₂ and FeSO₄, and incubated at 37^oC^13.

Molecular Analysis

Isolates showing high resistant against heavy metals were sent for 16 S r RNA analysis to Ahmedabad, Gujarat for molecular analysis^14-15.

Minimum Inhibitory Concentration (MIC) Determination of the Isolated Strains

Bacillus cereus agar plates and yeast pep tone dextrose agar plates were inoculated with 100µl aliquots of 24 hr culture with all isolates with different concentrations of Pb(NO₃)₂, CuSO₄, Zn(NO₃)₂ and FeSO₄ (250, 500,750,1000,1250 and 1500 mg/l).Diameter of inhibition zones were measured (in mm) in order to determine the MIC¹⁶.

Efficacy of Isolated Strains on Bioaccumulation of Heavy metals

80µl of the bacterial inoculum was inoculated into test tubes containing 8.0 mL of broth supplemented with 100 ppm of zinc & lead. The control treatment was prepared by mixing 9.0 ml of broth with 1.0 ml of bacterial suspension. All the tubes were sealed with para film and kept at 27 ± 2 °C for 7 days. To determine degradation efficacy, the heavy metal was first extracted with sequential extraction method¹⁷ diluted 10^-4 times and the absorbance was recorded at 225 nm¹⁸. The biodegradation efficacy (BE) was calculated using the following formula BE (%) = 100 – (As/Aac × 100)^19,20.

Results & Discussion

Soil Analysis Report and Enumeration of Bacteria

Soil analysis report shows the presence of various toxic heavy metals and sample contains Arsenic(0.756 mg/kg), Calcium (327.9 mg/kg), Cadmium (0.09 mg/kg), Cobalt (0.02 mg/kg), Chromium (0.053 mg/kg), Copper (9.4 mg/kg), Iron (53.71 mg/kg), Potassium (118.6 mg/kg), Magnesium (202.7 mg/kg), Manganese (4.539 mg/kg), Sodium (59.91 mg/kg), Nickel (0.723 mg/kg), Phosphorous (65.02 mg/kg), Lead (8.387 mg/kg), and Zinc (10.8 mg/kg). A total of fourteen different isolates (IS1,IS2, IS3, IS4, IS5, IS6, IS7, IS8, IS9, IS10, IS11, IS12, IS13, and IS14) were isolated and are biochemically and molecularly characterized and best two having high resistant against metals were further selected for bio accumulation & heavy metal testi.e. IS1 and IS 14. The biochemical and isolated results were shown in fig. 1 and table 1.

Figure1: Isolation of the microorganisms Click here to View figure

 

Test	IS1	IS14
Indole test	–	–
Methyl red	+	+
VogesProskauer	–	+
Citrate	+	–
Catalase	+	+
Coagulase	–	+
Motility	+	–
Nitrate reduction	+	+

Table no1: Biochemical tests:

Relative growth determination of isolates and their growth pattern

It was observed that the growth of the isolate decreases with the increase in metal concentration.IS1 Bacillus thuringiensis strain Simi and IS14 was found to have maximum relative growth against Lead and Zinc solution. The relative growth rate was observed at different concentration of heavy metals. The results are consisted with Ahemad and Malik¹⁵. The growth pattern of the microbial isolate sat different concentration(25,100,250&300 µgml^-1)are shown below:

Figure2: Isolates amended with different concentrations of metals & Graph showing relative growth of isolates at different concentrations (Control, 25 ppm, 100 ppm 200 ppm, 300 ppm of metal solution) of Zinc and Lead. Click here to View figure

 

Heavy metal tolerance test

All the isolates were tested for heavy metal tolerance test against Pb(NO3)2, and Zn (NO3)2. The IS1 exhibited maximum tolerance for zinc and IS14 exhibited maximum tolerance for lead and the results are shown in figure 3.

Heavy metal tolerance test for IS1 & IS14

Figure 3Click here to View figure

 

16 sr RNA Sequence Analysis

The isolates IS1 and IS14 were found similar to Bacillus thuringiensis strainsimi & Bacillus subtilis under accession number KF 916618.1 & KJ 489411.1, when submitted in NCBI. The phylogenetic tree shows relationship of isolated strains with other species.

Figure 4Click here to View figure

 

Determination of Minimum Inhibitory Concentration (MIC)

A great deal of variation among the isolates against the different heavy metals was observed.The results in Table 8 reveal that MIC of Zinc for Bacillus thuringiensis strain Simi was at 1000 µg/ml where as Bacillus subtiliss train PSB had maximum MIC for lead.

Metals	IS1	IS14
Zinc	960±10	100±10
Copper	100±10	500±10
Lead	250±10	1000±10
Iron	250±10	500±10

Table 2 Minimum Inhibitory Concentration (µgmL-1).

Biodegradation Efficacyon Heavy Metals

The degradation efficacy of Bacillus thuringiensis strain Simionzinc showed rapid degradation in the first three days, with mean of 54% biodegradation efficacy after fourth day the degradation efficacy decreased and reached to the point of 31% and Bacillus subtilis strain PSB  showed less degradation activity but the decrease in degradation efficacy started after fifth day. Similar work has already been reported against the hydrocarbon degradation by the isolate P. lundensis UTAR FPE2^18.

Figure 5Click here to View figure

 

Efficacy of IS1 and IS14 on Biodegradation of lead and Zinc solution.

Conclusion

The removal of heavy metals or break down into harmless state has become necessary. Thus bioremediation can be employed for the removal of such contaminants. This study of Zinc and lead accumulation by the isolates from heavy metal contaminated soils revealed a good and positive sign for its further use in bioremediation of zinc and lead in contaminated sites. The current study has illustrated some basic considerations that are important for the use of metal accumulating bacteria for bioremediation under field conditions.

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