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Soil is a three dimensional, dynamic, natural body occurring on the surface of earth and it is a medium for plant growth. It plays a fundamental role in the regulation of pollutants in the ecosystem (Ranjan, 2005). Pollution due to industrial activities has created a serious problem for the safe use and rational utilization of soils (Ashraf et al., 2007). Emissions of liquid effluents due to industrial activities are capable of causing soil and groundwater pollution (Surita et al., 2007). When the effluent comes in contact with land it affects the soil and leads to its degradation in varying degrees depending upon its physicochemical characteristics (Dwivedi et al., 2006).
Tanning is one of the oldest industries in Pakistan and is the major foreign exchange earners for the country. Tanning industrial wastes are of serious consequences from the pollution point of view. The wastes from this industry rank among the most polluting of all industrial wastes. Non degradable persistent trace metals present in tanneries are the most pressing problem these days. Soils act as the sinks for these metals. Tanneries discharge effluents containing toxic heavy metals like copper, nickel, zinc, lead, arsenic, cadmium, and chromium (Sharma et al., 2009). Heavy metal pollution of soil deteriorates environmental quality by influencing the yield and quality of crops, atmosphere and quality of water environment. Human health is also affected via food chains (Huamain et al., 1999).
Soil contains wide variety of microbial populations and these microorganisms significantly contribute to the maintenance of the matter and energy turnover in land environment (Paul, 2007). Microbes such as fungi and bacteria play essential role in nutrient transformations (Critter et al., 2002). They affect the soil erosion and metal remediation efforts and also play important role in plant interaction with soil environment. These microbes may be essential for the revegetation efforts by aiding in removal of excessive soil metals (Khan, 2003). Microorganisms mineralize, oxidize, reduce and immobilize mineral and organic materials in soils. These compounds may alter the number or activity of the microorganisms and can affect soil biochemical process and ultimately influence the soil fertility and plant growth (Araujo et al., 2006). As tanneries are major source of heavy metal pollution, they may have damaging effects on the decomposition and nutrient mineralization in soil near pollution sources. This can be the result of harmful effect of metals on microbes (Komulainen, 1992).
The objectives of this study were to isolate, purify and characterize bacteria from the soil affected with tannery effluents and also to check their heavy metal resistance, which may have a potential role in biodegradation/ bioremediation of these metals from the polluted environment.
Materials and Methods:
Sample collection and bacterial isolation:
Tannery effluents affected soil sample was collected in airtight sterilized plastic bags from the vicinity of Leather Field Industry, Sambarial where these effluents were thrown. Bacterial population were determined by serial dilution and plating of soil suspension on nutrient agar medium. After incubation at 37°C for 24-48 hrs plates were observed for the presence of bacterial colonies. Pure cultures of distinct colonies were obtained by single colony streaking. After many rounds of streaking purified bacterial cultures were obtained. Morphological and biochemical characterization of pure cultures was done after their isolation.
Antibiotic Resistance of Bacterial Isolates:
Bacterial strains may be resistant or susceptible for a particular type of antibiotics. Resistant bacteria have the ability to withstand the effects of antibiotics. The disc diffusion method was used for determining antibiotic resistance spectra. Efficacy of nine antibiotics including Vancomycin (VA30), Erythromycin (E15), Chloramphenicol (C30), Streptomycin (S10), Ampicllin (AMP25), Ciprofloxacin (CIP5), Kanamycin (K30), Rifampicin (RD5), and Tetracycline (TE30) was checked for each strain.
Heavy metal resistance of Bacterial Strains:
To check heavy metals resistance bacterial strains were grown in the presence of salts of different heavy metals. Sterilized autoclaved molten nutrient agar was supplemented with known concentrations of salts of various heavy metals. Bacterial strains were grown in the presence of salts of heavy metals of K2CrO4 (Cr), ZnSO4 (Zn), CdCl2 (Cd), CoCl2 (Co), Fe2(SO4)3 (Fe), HgCl2 (Hg), NiSO4 (Ni), CuSO4 (Cu), (CH3COO)2Pb (Pb), MnSO4 (Mn). Growth of bacterial strains was examined after incubation at 37°C for 24 to 48 hrs.
Effect of pH on Growth of Bacterial Strains:
Effect of pH on growth of bacterial strains was observed through spectrophotometric analysis. Nutrient broth was prepared and adjusted at different pH from 5-11.Test tubes containing 5ml of nutrient broth of each pH was autoclaved and inoculated using micropippeter and incubated at 37°C for 24 to 48 hrs. A spectrophotometer was used to measure the optical density at 600nm to determine the bacterial biomass.
Results and Discussions:
The effects of sludge and liquid wastes produced from leather tanneries on soil biodiversity can be amplified through the food chain and also threaten sustainability of natural ecosystems (Iram et al., 2009).
In present study sixteen bacterial strains were isolated and their morphological and biochemical characterization was done from tannery effluent affected soil. 69% of the total isolates under study were gram-negative, while remaining were gram- positive and majority of them were rods (Table 1).
All the strains in the present research were facultative anaerobes and showed negative nitrate reduction but exhibited positive catalase test. Srinath et al. (2001) reported five facultative anaerobes from tannery effluents in Kanpur, India which showed negative nitrate reduction and catalase test.
In present study 6% bacterial isolates showed positive methyl red, 69% bacterial isolates showed positive Voges Proskuer, 19% showed positive urease test, 38% were motile and all were unable to hydrolyze starch. Whereas, Pal et al. (2004) reported an isolate AND303 from serpentine polluted soil in India that exhibited negative methyl Red, voges proskuer, urease and starch hydrolysis test. Yazdi et al. (1999) reported 20 bacterial isolates from industrial wastewater treatment unit. These bacterial strains gave positive catalase test and negative voges proskuer test. Twelve of them showed positive nitrate reduction test, 4 strains showed positive urease test and nine were motile.
Haq et al. (1999) reported three bacterial isolates from industrial effluents of chemical and textile mill industries. These isolates exhibited pink growth on MacConkey agar. Four strains documented by Faisal et al. (2004) from tannery effluent showed positive growth on MacConkey agar and no growth on Brilliant Green Bile agar while three of them exhibited growth on EMB agar as well. However, in present study only TS-11 isolate showed pink growth on EMB agar. Two isolates TS-2 and TS-11 exhibited growth on Simmons Citrate agar. Growth of TS-7 and TS-11 isolates was observed on Brilliant Green Bile agar and MacConkey's agar (Table 2).
Shafiani et al. (2003) documented 35% chloramphenicol and 20% tetracycline antibiotic resistant strains from wastewater irrigated soil. Altaf et al. (2008) reported 15 bacterial isolates from agricultural soil as well as from Trifolium alexandrinum plant nodule. They were 26.6% resistant against ampicllin and 20% against kanamycin. Verma et al. (2001) documented streptomycin resistant but chloramphenicol sensitive bacteria from tannery effluents. In the present study all the bacterial isolates were sensitive to tetracycline and 13% of bacterial isolates were resistant to chloramphenicol. Only one bacterial isolate TS-4 was resistant to ampicllin, one isolate TS-14 was resistant to streptomycin and no resistance was observed by any bacterial isolate to kanamycin (Table 3).
Ashraf et al., (2007) isolated stress tolerant bacteria from the soil amended with varying concentrations of heavy metal salts including silver (Ag), zinc (Zn) and lead (Pb). In present study about 75% of bacterial isolates were resistant to cadmium up to 1000μgml-1, 50% were resistant to chromium up to 1000μgml-1 and isolate TS-11 was sensitive to chromium and exhibited no growth at any concentration. Majority of the isolates were resistant to lead at maximum concentration of 1000μgml-1. Six strains showed growth against zinc at 100μgml-1 and two isolates TS-2 and TS-5 exhibited growth at 200μgml-1 as well, while remaining were sensitive to zinc and no growth was documented. 31% bacterial isolates were resistant against copper up to 500μgml-1, 44% were resistant up to 400μgml-1 and 13% exhibited no growth. Majority of bacterial isolates (75%) were resistant to nickel (up to 500μgml-1). Only three isolates TS-3, TS-5, and TS-6 were resistant to mercury and could tolerate 200μgml-1 of mercury in medium, while all others were sensitive (Table 4).
Haq et al. (1999) reported three bacterial isolates from industrial effluents. Out of these three, two isolates showed maximum resistance up to 110μgml-1 against cadmium and third one showed up to 220μgml-1. One strain showed resistance against chromium and lead up to a maximum concentration of 800μgml-1 and 1400μgml-1, respectively. Second strain showed resistance against chromium up to 600μgml-1 and lead up to 1200μgml-1, whereas third strain showed resistance against chromium at maximum concentration of 500μgml-1 and lead at 900μgml-1. Zahoor et al. (2009) reported two bacterial isolates from industrial effluents of Sheikhupura. One strain was able to resist cadmium (50μgml-1), copper (200μgml-1), lead (800μgml-1), mercury (50μgml-1), nickel (4000μgml-1), and zinc (50μgml-1). Second strain resisted cadmium (50μgm-1l), copper (200μgml-1), lead (800μgml-1), mercury (50μgml-1), nickel (4000μgml-1), and zinc (50μgml-1).
Haq et al. (1999) reported optimal growth of bacterial isolates at different pH values from 5 to 9. Two isolates showed optimal growth at pH 7. One bacterial strain studied by Zahoor et al. (2009) showed maximum growth at pH 6 and other showed maximum growth at pH 7. All the bacterial isolates in present study were able to grow at different pH values ranged from 5 to 11. Optimal growth showed by TS-4 isolate from pH 6 to 9. Isolates TS-7 and TS-12 showed approximately same growth at 5 and 6 pH. TS-11 isolate was neutrophile and showed maximum growth at pH 7. Optimum growth of TS-15 isolate was observed at 6 and 7 pH. Most of the isolates were slight acidophilic or acidophilic in nature.
From the present study it is concluded that the soils affected with tannery effluents are inhabited by a number of bacteria having diverse characters. Although they shared few characteristic like all of them were facultative anaerobes and they were highly tolerant to different heavy metals but the resistance pattern was different for different isolates. Majority of them were sensitive to various antibiotics. Further studies are still required to find out their role in bioremediation.