Journal of Applied Sciences & Environmental Management, Vol. 9, No. 1, 2005, pp. 115-121

Therefore, the present study was undertaken, with the soil collected from the agricultural field and amended with a graded, known concentration of metal ions and its combinations in the lab conditions. The survival pattern of the common major groups of indigenous bacterial population (aerobic heterotrophs, asymbiotic diazotrophs and actinomycetes) was studied.

MATERIALS AND METHODS

Collection and preparation of soil sample:  Soil samples, during the months of September-October 2001 were collected from agricultural farm of Mathura Oil Refinery, Mathura, U.P. India. This field is under cultivation and has received oil refinery treated wastewater for the last 15years. Before use, soil was sieved through a 1mm-pore size screen. Soil was mixed with chloride salts of heavy metals (Cu, Cd, Cr, Hg, Mn, Ni, Pb and Zn) at the concentrations of 50, 100, 150 and 200 μg/g of soil. Soil samples (100 g each) were then packed in sterile polythene bags and maintained at 50% water holding capacity and stored at 28±2°C for 35 days.

Physico-chemical characteristics of soil under study:  The physico-chemical properties of soil were analysed as per method of Ghosh et al. (1983) and APHA, AWWA-WPCF (1985).

Heavy metal analysis: Well mixed, acid preserved soil sample was transferred to a beaker containing 5 ml of concentrated nitric acid and a few boiling chips. The sample was heated on a hot plate for complete digestion, as shown by light coloured, clear solution and filtered through whatman filter paper (No.42) (Vanloon and Lichwa, 1973). The filtrate was used for the determination of various metals, using a GBC 902 double beam atomic absorption spectrophotometer.

Enumeration of microbial population in soil: Astandard spread plate method with modification was used (Cuppucino and Sherman, 1983). Total viable count of heterotrophic aerobic bacteria, actinomycetes and asymbiotic nitrogen fixers were evaluated on appropriate selective media (Herbort, 1990), as adopted earlier (Hayat et al., 2002). The composition (g/l) of media was as follows: Nutrient agar: (peptic digest of animal tissue 5 g; sodium chloride 5 g; beef extract 1.5 g; yeast extract 1.5 g; agar 15 g); Kennight medium (dextrose 1 g; potassium dihydrogen orthophosphate 0.1 g; sodium nitrate 0.1 g; potassium chloride 0.1 g; magnesium sulphate 0.1 g; agar 15 g); Jensen’s medium (sucrose 20 g; dipotassium phosphate1 g; magnesium sulphate 0.5 g; sodium chloride 0.5g; ferrous sulphate 0.1 g; sodium molybdate 0.005 g; calcium carbonate 2g; agar 15 g). All the above microbiological media are sterilized by autoclaving at 121 °C for 15 min. The pH of each medium was adjusted according to bacterial groups.

Determination of survival of indigenous soil bacteria from soil amended with heavy metal: One gram of the soil samples amended with different concentrations of heavy metals was serially diluted in sterile normal saline solution. 0.1 ml of diluted sample was spread over the surface of plate count agar of the respective medium at 0, 7, 14, 21, and 28 days after incubation. The total viable count of each group of population was noted. Decrease in viable count in the heavy metal amended soil over control sample indicates the effect of heavy metals on metal sensitive population and survival of indigenous soil bacteria.

RESULTS AND DISCUSSION

Physicochemical and heavy metal characteristics of soil before amendments: The physico-chemical and heavy metal characteristics of the soil are presented in Table 1. All the parameters are below the permissible limit, recommended for soil as described by Krishna Murti and Vishwanathan (1991).       

Aerobic heterotrophic bacteria: The plate viable count in control soil (without heavy metal) was in the range of 7.2 x 10⁷ to 1.1x10⁸ CFU/ g of soil. There was no significant inhibition in the viable count of aerobic heterotrophs for any metals at 50 and 100 μg/g of concentration up to 28 day of incubation (Table 2 ). On the other hand, treatment of  Cu, Cd, Hg, Mn and Ni at 150 and 200 μg/g concentration led to significant decline in viable count or complete inhibition of growth after 21 day of incubation. This behaviour of indigenous soil bacteria was similar to our previous study where indigenous metal resistant aerobic- heterotrophs were directly isolated in significant number on nutrient agar plates supplemented with 200 and 400 μg/ml of metal concentration (Hayat et al. 2002). Because of the great diversity within this   group of bacteria , plate viable count may not be affected at lower concentration of heavy metal but the diversity  of the population is  reduced.. Random selection of 50 colonies from different plates indicated that almost 75% of the strains were gram negative at higher metal concentration and remaining were gram positive or gram variable. Common gram positive bacteria at higher concentration was belonged to genus Bacillus ( data not shown). At lower concentration morphological diversity was more as compared to at higher metal concentration. The predominance of gram-negative bacteria at higher concentration of metal is probably due to their higher level of intrinsic metal resistance than majority of the gram-positive bacteria. The basis of this difference might be due to the differences in the chemical composition of cell wall of gram-negative bacteria and gram- positive bacteria (Babich and Stotzky,1977).

Potential aerobic asymbiotic nitrogen fixers Most of the free nitrogen fixing bacteria are capable of growing on nitrogen free solid medium belong to genera,Azotobacter, Azomonas, Derxia, some species of Pseudomonas, and Klebsiella  (Alexander, 1984). All the above bacteria are primarily gram-ve, therefore, may have certain common characteristics of metal tolerance due to their similar cell wall composition, also the majority of the nitrogen fixers are good extra-cellular polysaccharide producers which may further protect the cell from the toxic effect of heavy metal (Geesey and Jang, 1990). In contrast to aerobic heterotrophs, this group of bacteria could not survive for 28 days even at 100 μg/g of heavy metals like Cd, Hg & Pb, while other heavy metal (Cu, Cr, Mn and Ni) inhibited growth after 21 or 28 days of incubation at 150 μg/g of concentration. Similarly, Pb, at 200 μg/g inhibited microbial growth after 14 day of incubation followed by Ni, Hg, Mn, Cr, Cd and Cu and Zn (Tables 3 ). Higher sensitivity to toxic metals may be due to lower diversity among asymbiotic nitrogen fixers compared to heterotrophic groups. Morphological diversity of asymbiotic nitrogen fixers revealed the presence of three (I, II &III) major groups of bacteria. At elevated metal concentration in soil, the group-I  bacterial population was found predominant. Morphological, cultural, and certain biochemical characteristics of these bacteria are given in table 4.

Actinomycetes: Soil actinomycetes are the filamentous gram +ve bacteria representing several morphological types, which includes isolated genera like Streptomyces, Nocardia, Pseudonocardia, Micromonospora and some unidentified isolates.. This group of soil bacteria are considered to play very important role in maintaining soil properties although ,they are poor competitor than other soil bacteria. (Alexander, 1984). The data presented in table 5  and 6 revealed inhibition of growth  even at 50 μg/g concentration of Mn, Ni and Pb after 28 day of incubation. However, Cd showed toxicity at100 μg/g of soil, Cr, Zn and Hg were effective at 150 μg/g and Cu was effective only at 200 μg/g of soil. In general it can be stated that actinomycetes were  less tolerant than gram negative heterotrophic and nitrogen fixing bacterial population . These observations are comparable with our previous findings (Hayat et al., 2002). 

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