I= ISOLATION AND CHARACTERIZATION OF HEAVY METAL TOLERANT BIOTA FROM INDUSTRIALLY POLLUTED SOILS AND THEIR ROLE IN BIOREMEDIATION
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Title of Thesis
ISOLATION AND CHARACTERIZATION OF HEAVY METAL TOLERANT BIOTA FROM INDUSTRIALLY POLLUTED SOILS AND THEIR ROLE IN BIOREMEDIATION

Author(s)
Ali Abbas Qazilbash
Institute/University/Department Details
Department of Biological Sciences / Quaid-i-Azam University, Islamabad
Session
2004
Subject
Microbiology
Number of Pages
192
Keywords (Extracted from title, table of contents and abstract of thesis)
heavy metal tolerant biota, bioremediation, heavy metals, pollutant, biotreatment regimens, fungal biomass, bacillus strains, bacterial strains

Abstract
Over the years, with the active spread and development of the industries, heavy metals, which are either used, or produced, as by-products, by numerous manufacturing, industrial, refining and mining processes, have become ubiquitous, persistent environmental pollutants. Pakistan, too, is not exempted from such devastating environmental degradation caused by these pollutants. With its rapidly growing population, the demand of major economic activity in the agricultural sector has been phenomenal. Associated with this, has been the rapid development of the manufacturing industry to cater for the growing demand of mechanization, technology and the drive for an enhanced standard of living. Simultaneously, the processing of and disposal of waste has been given less priority, as a result, over the past 20 years there has been a gradual be marked increase in soil, air and water pollutant levels. Researchers have developed a method to accelerate the process of removal by encouraging the microbial and associated biota to degrade and/or remove the pollutants from the identified site. This process, aptly known as bioremediation, has been labeled as a cost-effective technology for the treatment of a variety of pollutants and contaminated sites. This study was designed to identify and characterize microbial flora present in industrially polluted areas and to determine their role in bioremediation.

The area under study for this research work was a 170 km stretch of highway linking Rawalpindi /Islamabad to Peshawar, peppered with a variety of industries from textiles, steel and paper mills, to cement and marble factories and leather tanneries, and pharmaceuticals. Three sites were identified along the GT road, based on the extent of pollutants being discharged and the land and waterways being polluted by the effluents and waste being dumped by the numerous industrial units. Samples were collected for microbiological and physio-chemical analysis in sterilized plastic bags (soil) polypropylene bottles (effluent) and maintained a temperature of 4°C, or less to ensure minimal biological activity. Processing of the samples for the isolation of bacteria was carried out within 24 hrs of sample collection. The samples were then split into two portions; one was portion was stored at 4°C in the refrigerator till its processing for isolation of fungi, while the other portion was preserved for the analysis of heavy metals using the atomic absorption spectrophotometer.

HM tolerant microbial biota were isolated from contaminated soils and industrial effluent, and subjected to biochemical tests and microscopy to help in their identification. The initial tests included heavy metal and physio-chemical analyses of both soil and effluent samples, followed by the isolation, identification and characterization of HM tolerant bacteria and fungi from the identified samples. Optimization experiments were carried out, through a series of experiments, to enhance the removal of the toxic heavy metals. The effluent from the first site was acidic in nature (pH 3.3) with a stale, fishy smell and bluish green color. The temperature, of this running stream of effluent, was, at midstream, 30°C. The electrical conductivity of the effluent was found to be 3.14 µΩ/cm, total dissolved solids (TDS) at 322,100 mg/l and total suspected solids (TSS) at 7,643.3 mg/1. The effluent from the second site was alkaline in nature (pH 11.47), whereas the pH of the soil was lower, towards the neutrality (pH 8.24). The effluent had a dark brown color with a pungent fishy smell, whereas the soil was very much, €˜soil colored€™; light brown with an earthy smell. The effluent temperature was recorded at 31°C and the EC of both soil and effluent samples were 3.83 µΩ/cm and 2.86 µΩ/cm, respectively.

Bacterial strains were isolated from soil contaminated by industrial effluent, consisting mainly of tannery wastewater. Isolation was achieved by the serial dilution method and cultured on nutrient agar plate. Morphological and biochemical studies of the isolates showed that all the 6 bacterial strains were Gram-positive rods and belonged to the Bacillus species. All the isolates were found to be Catalase-positive. For the TSI agar test, strains QIP 1, 2, 4 showed alkaline slant and butt with no gas production, while strains QIP 3 and 5, showed alkaline slants only with no gas production. For the nitrate reduction test, strains QIP 1,2, 3 and 5 were negative after 24 hrs, but after 48 hrs strains QIP 1 and 5 were found to be positive for nitrate reduction, yet strains QIP 4 and 6 were positive within 24 hrs. All the strains were Indole-negative and citrate negative, as derived from the appropriate tests. The strains were also incubated at higher temperatures, e.g. 42°C for 24 hrs. and the results indicated that Bacillus species QIP 1 and 4 grew better at this higher temperature, as compared to the other strains, while all the strains showed minimal growth on NA media with 6% NaC1 .

This series of experiments were designed to determine the tolerance/resistance levels of the Bacillus strains and removal of chromium from the minimal salt media, using the shake flask incubator. Bacillus spp. QIP 1 and 5 showed maximum resistance to chromium, as they sowed moderate growth at Cr concentrations as high as 900 ppm, with good growth at 700 - 800 ppm. All strains displayed maximum growth on media with a Cr concentration of 400 ppm. Bacillus strains QIP 1 and QIP 5 showed maximum chromium removal in media containing 400 ppm metal salt solution over the stipulated time. Strain QIP 1 removed 37.2% of the Cr6+, whereas QIP 5 went better removing 44.12% of the chromium at the same concentration over the same period of time. Strain QIP 1 removed the highest percentage ofCr6+ ions (53.01%) from the media at pH 5.5, with QIP 5 removing 38.85% Cr at the same pH, within 24 hours of incubation. Strains QIP 1 and 5 removed maximum Cr6+ ions within 24 hours of incubation at 28°C, 42.6% and 38.85%, respectively. For the optimum glucose concentration, maximum Cr6+ ions were removed from media containing 400 mM of glucose, for both strains, after 24 hours of incubation, QIP 1 removed 40.10% and QIP 5 removed 32% of the Cr6+ ions from the media. Under the optimized conditions, a significant positive correlation between the Bacillus strains was recorded, as strain QIP 1 adsorbed 46.81% of the Cr6+ ions in 60 minutes, whereas QIP 5 removed 42.5% of the Cr6+ ions over the same incubation period.

Fungal isolates showed a greater degree of variation for resistance against the chosen heavy metals, namely: Pb, Cr, Zn, Mo, and Ni. For Pb, the strains of Aspergillus species showed the highest degree of tolerance, specifically those of Aspergillus niger which should moderate to minimal growth in media with Pb concentrations ranging from 10000 ppm to 18,000 ppm. Two strains of Aspergillus niger, NP 17 and 18 the highest tolerance at 15,000 ppm and 18,000 ppm of Pb. These two strains differed only in that A. niger NP 18 had a higher minimal growth concentration limit (18,000 ppm) than strain NP 17, whose minimal growth observed on 7th day of incubation between concentration range of 15,000 ppm, above which range no growth was recorded. Based on these experiments, strains NP 17 and NP 18 of Aspergillus niger were chosen, for subsequent experiments on removing Pb from the media.

Strains NP 17 & 18, showed maximum growth at pH 4, after 7 days of incubation, with NP17 dry weight of 0.59 g/100 ml and strain NP 18 dry weight of 0.88 g/ml. These strains also showed good growth rates at higher pH values, from 7 to 9, with strain NP 17 dry weight readings of 0.56, 0.57 and 0.63 g/100 ml at pH 7, 8 and 9, respectively, and strain NP with dry weight readings of 0.57, 0.51 and 0.62 g/100 ml at the respective pH values, all after 7 days of incubation. Both the fungal strains showed maximum growth at 40°C, with A. niger NP 17 attaining a dry weight of 0.94 g/100 ml and A. niger NP 18 a dry weight of 0.92 g/100 ml, following 7 days of incubation.

Following optimization of the growth conditions, both Aspergillus strain were tested for their adsorption of Pb from SDB in oven-dried (dead) and pelleted non-growing (living) forms, using the shake-flask incubation method. Both strains of Aspergillus niger showed very high percentage of Pb adsorption in the oven-dried, as compared to the non-growing state.

Furthermore, the adsorption by oven-dried strains was also high at higher concentrations of the heavy metal, whereas adsorption of Pb effectively took place at the low concentration levels for the non-growing strains. This was exemplified in the correlation matrices, where the comparison showed significant positive correlation between strains at high Pb concentrations under the oven-dried condition and significant positive correlation at low Pb concentrations under the non-growing conditions. The percentage Pb adsorption of both strains under oven-dried conditions was over 90% for the concentration range of 100-500 ppm, with the highest being 99.95% at 500 ppm for strain NP 18 after 80 minutes of incubation. Furthermore, for the non-growing strains this maximum adsorption was achieved for the concentration range of 100 - 300 ppm and that too was 96.37% achieved by strain NP 17 at 300 ppm concentration after 30 minutes, after which the toxic effects of the HM on the live fungal strains diminished their capacity to adsorb the metal.

Bioremediation may be considered a panacea that occurs naturally in the polluted soils and effluents and in all probability involves a consortium of microorganisms working together to ensure an equally good job of removing the toxin from the contaminated sites, as would a particular isolate. Of course, they required the necessary time to ensure proper removal and may require some augmentation, on a case to case basis, depending on the physio-chemical nature of the contaminated sites. When comparing the two biotreatment regimens of oven-dried fungal biomass, verses the use of non-growing pellets of fungi, there is overwhelming evidence to support the use of the former as a method to removal HM contamination from polluted sites, be it effluents or soil.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
550.27 KB
2 1 Introduction 1
544.19 KB
3 2 Review Of Literature 18
813.77 KB
4 3 Materials And Methods 41
664.69 KB
5 4 Results 67
1723.33 KB
6 5 Discussion 142
1586.92 KB
  5.1 Conclusions 165
  5.2 Recommendation 166
  5.3 References 167