INTRODUCTION:

Raw waste water from various industries contain huge amount of heavy metals that are generally not degraded by the conventional process of wastewater treatment and removal of these heavy metals from waste water needs advance chemical technology, which is relatively very expensive and may generate further toxic waste in the form of sludge, that may pose a serious disposal problem. Presence of these higher concentrations of toxic heavy metals in wastewater leads to contamination of receiving water bodies and has a significant negative impact on aquatic life¹. Of the major industries, mining industry generates waste residues, with high levels of heavy metals (Ni, Cd, Pb, Mn, Cu, and Zn) that are deposited in open areas creating ecological disaster and leading to human health risk. One such heavy metal Lead (Pb) a common occurring heavy metal we encounter in our daily life via paints, batteries, toys, drinking water, according to LEAD is highly toxic to human, animals, plants and microbes at very low concentrations ².

The microorganisms respond to these heavy metals by several inbuilt mechanisms and processes; biosorption to the cell walls and or entrapment in extracellular capsules, precipitation of metals inside the cell, complexation and oxidation-reduction reactions via suitable enzymes ^3,4,5. Naturally occurring bacteria that are capable of metal accumulation, tolerance and biotransformation capabilities have been extensively studied. It is difficult to assume that a single species of bacterium could be capable of removing series of heavy metals from its polluted site ⁶, so it is very much interesting to explore consortium of bacteria and or unique bacterium capable of exhibiting tolerance towards a broad spectrum of heavy metals. The objective of this study is to explore and find heavy metals resistance of bacteria in a broader spectrum, biochemical characteristics of the versatile bacterium and its growth studies for further application of these isolates for clean-up of tannery effluent.

MATERIALS AND METHODS:

Isolation of screening of bacterial isolates from tannery effluent:

The sewage water samples were collected in and around Ezar Tanneries, Vaniyambadi, Vellore district, South India. The samples were collected in sterile plastic container and transport to laboratory for bacteriological analysis. The bacterial isolates were screened on Luria Bertani (LB) agar plates supplemented with 5 mg/l concentration of Cr, Cu, Ni, Pb and Zn metals by the standard pour plate method. Plates were incubated at 37°C for 48 hours and colonies differing in morphological characteristics were selected and used for further studies.

Identification and biochemical characterization of screened bacterial isolates:

The shape and color of the colonies were examined under the microscope after Gram staining and were also checked for motility. Isolates were biochemically analyzed for the activities of catalase, starch hydrolysis and gelatin hydrolysis, mannitol test, indole production and citrate utilization. The tests were used to identify the isolates according to Bergey’s Manual of Systematic Bacteriology⁷.

Growth studies of screened bacterial isolates:

Growth studies of sewage bacterial isolates were studied in 250 ml flasks containing 50 ml LB medium supplemented with 0.1 mM concentration lead. Flasks were inoculated with 0.5 ml of overnight culture and incubated 37°C at 100 rpm. Growth was monitored as a function of biomass by measuring the absorbance at 620 nm using spectrophotometer and the optimal growth conditions with reference to pH and temperature were determined. The effect of pH on the growth of bacteria in the presence of lead was analyzed at five pH values 4.5, 5.5, 6.5, 7.5 and 8.5 and different temperatures 30ºC, 37ºC and 45ºC was studied.

RESULTS AND DISCUSSION:

Isolation of screening of bacterial isolates from tannery effluent:

The microbial colonies were isolated by pour plate method. Pure colonies of these eleven bacterial isolates were obtained using streak plate method. The bacterial isolates were named based on the source from which it was isolated. The isolated bacterial colonies were screened with the metal concentration of 100 mg/l and the metal resistant isolates were isolated based on their cell density values measured at 620 nm by using UV-visible spectrophotometer (ELICO, India).

Identification and biochemical characterization of screened bacterial isolates:

From the gram-staining procedure it was found that the microbial strain was a gram positive spherical bacterium that occurred in microscopic clusters formed yellow colonies in nutrient medium. Further biochemical tests resulted positive for catalase, mannitol sugar fermentation and coagulase test confirmed that the isolated microbial strain VLB-9 was identified as Staphylococcus aureus.

Growth studies of Staphylococcus aureus:

Effect of temperature:

The measurements from the cultures incubated for 24 hr were in good agreement according to bacterial resistance. Growth was monitored as a function of biomass by measuring the absorbance at 620 nm using spectrophotometer and the optimal growth conditions with reference to different temperature such as 30ºC, 37ºC and 45ºC. Fig. 1 shows maximum removal of the metal which was found to be at 30ºC. Thus the optimum temperature was found as 30ºC.

Fig 1.Effect of Temperature on biosorption of lead by Staphylococcus aureus

Table.1 Screening of metal resistant bacterial isolates:

S.No

METALS

(5 mg/lit)

DAYS

MICROBIAL STRAINS

VLB-

VLB-2

VLB-3

VLB-4

VLB-5

VLB-

VLB-7

VLB-8

VLB-9

VLB-10

VLB-11

+++

++++

Note:- no growth; + poor growth; ++ moderate; +++ growth

*VLB- Vaniyambadi Leather effluent Bacteria

Table. 2 The morphological and biochemical characterization of bacterial isolates (VLB-9)

Sl. No.	Characteristics	*Staphylococcus aureus* (VLB-9)
1.	Colony diameter	1-3 mm
2.	Colony color	yellow
3.	Colony morphology	Slightly elevated, circular
4.	Gram staining	positive
5.	Turbidity in broth	Uniform dispersed growth
6.	Motility	Non-motile
7.	Oxidase	negative
8	Catalase	positive
9.	Mannitol sugar fermentation	negative
10.	Coagulase	positive
11.	Temperatures (ºC)
	30	+++
	37	++
	45	_
12.	pH
	4.5	+
	5.5	++
	6.5	+++
	7.5	++
	8.5	_

Note: - no growth; + poor growth; ++ moderate growth; +++ growth

Effect of pH:

The effect of pH on the growth of bacteria in the presence of lead was analyzed at five pH values such as 4.5, 5.5, 6.5, 7.5 and 8.5.Fig. 2 shows maximum removal of the metal was found to be at 6.5 pH. Thus the optimum pH was found as 6.5.

Fig. 2 Effect of pH on biosorption of lead by Staphylococcus aureus

CONCLUSION:

The heavy metal resistant organism could be a potential agent for bioremediation of heavy metals pollution. The microbial resistance to heavy metal is attributed to a variety of detoxifying mechanism developed by resistant microorganisms such as complexation by xopolysaccharides, binding with bacterial cell envelopes, metal reduction, metal efflux etc. These mechanisms are sometime encoded in plasmid genes facilitating the transfer of toxic metal resistance from one cell to another⁸. Wastewater bacteria isolate Psuedomonas aeroginosa, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus resistant to heavy metals and antibiotics⁹. In the present study high degree of heavy metals resistance associated with multiple antibiotic resistances was detected in Staphylococcus aureus. Staphylococcus aureus exposed to high levels of heavy metals in their environment have adapted to this stress by developing various resistance mechanism. These mechanisms could be utilized for detoxification and removal of heavy metals from polluted environment¹⁰. According to these results, the present study evaluates that Staphylococcus aureus were used to remediate heavy metal contaminated tannery effluent waste.

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Received on 25.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 239-241