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A Comparative Study of Azotobacter Spp. from Different Soil Samples

M. Z. Islam
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh

D. I. Sharif
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh

M. A. Hossain
Fisheries and Marine Research Technology Discipline, Khulna University, Khulna-9208, Bangladesh

Abstract/ Executive Summary:

Azotobacter spp. were isolated from different soil samples and analyzed for number of Azotobacter per gram of soil, saline tolerant and heat resistant. The maximum number (CFU) of Azotobacter in legume crop field soil (sample No. 1) was 11 x 105 / gm of soil. The lowest number in grain crop field soil (Sample no. 3) was 3 x 105 / gm of soil. There was not any Azotobacter observed in river water sediment. To determine the prominence of Azotobacter in the soil sample other determinants such as pH and moisture. All isolate show maximum growth at 0% NaCl, only two isolate 1 and 4 show growth at 0.8 % NaCl. All isolate content was studied which showed maximum growth at 30ºC temperature but isolate 2, 5 and 6 showed optimum growth 20 ºC whereas isolate 1, 3 and 4 showed optimum growth at 40 ºC and no isolate survive at 50 ºC. The result revealed that legume crop field soil contained the high varieties of Azotobacter spp. in terms of temperature and salt tolerance, which became a promising source for further study.

Keywords: Azotobacter, water sediment, legume crops

Location: Microbilology laboratory of Biotechnology and Genetic Engineering Discipline of Knulna University

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Bio fertilizer

Objectives:

To isolate effective strains of Azotobacter from the different soil could be used as bio-fertilizer in future.

Methodology:

Collection of soil samples and preservation: In this study, isolation of Azotobacter from different soil samples was investigated in the Microbilology laboratory of Biotechnology and Genetic Engineering Discipline of Knulna University. Soil samples were collected from seven different places, which are as follows: Legumes crop field, Non legume (Vegetable) crop field, Grain (rice) crop field, Grass land, Forest and wood land, Unused land, River water sediment. Soil samplers were collected from seven different places. For the purpose of soil sample collection several plastic bags, a marking pen, spatula, alcohol and knives were taken. At first we selected a field for sampling, then selected four or five points in that field and collected soil and mixed the four or five point soil samples. Sufficient amount of soil was collected from each site; kept in a polythene bag and tagged. Soil sample were collected from top 4 cm of the soil profile, as this is where most of microbial activities takes place, and thus where most of the bacterial population is concentrated. Determination of field soil moisture: 50 gm fresh samples were taken separately in a clean 150 ml beaker, weight of the beaker was taken before pouring soil sample. It was then kept in the over at 105ºC ± 3ºC for 24 hours, and then again weight of soil sample and beaker was taken combinedly. Difference of moisture content of the soil was recorded and calculated for the moisture content. Determination of field soil pH: 25 gm (field moist) was taken in a clean dry 150 ml beaker and 50 ml distilled water was added. The contents were thoroughly stirred with vortex machine. pH of the suspension was measured with a digital pH meter. Media Preparation: To prepare of one liter of culture media Ashby’s media or Nutrient agar media or Jensen’s media) , the following steps were followed. According to media composition, the reagents were weighted by electronic balance. 1000 ml distilled water was measuring by volumetric flask and taken in a conical flask. The reagents (except agar) were mixed with the distilled water. After mixing reagents, pH was adjusted by adding HCl or NaoH solution if required. After pH adjustment, agar was mixed into the solution. After mixing the agar, the media was autoclaved by autoclave machine. Finally the media was poured in sterile Petri Plates.Isolation: 10 gm collected sample was added to 90 ml of sterile distilled water in a sterile conical flask (250 ml), shaken well by vortex machine than allowed to stand for 30 minutes. 1 ml of sample suspension was then transferred to sterile 9 ml distilled water containing McCarty bottle and shaken well by hand and again allowed to stand for 30 minutes. In this way, samples were diluted up to 105 dilution fraction. One ml of sample suspension (from 101 to 105 fraction) were taken in a sterilized Petri Plates contain approximately 15 ml melted (45ºC), Ashby’s medium, and then incubated at 28±2ºC temperature for about 2 – 3 days. After incubation the individual colony was appeared on the medium. The number of Azotobacter per gram of soil was then calculated. Purification: The isolated organisms were purified through repeated plating. Media used for the purpose were Ashby’s media, Jensen’s media and nutrient agar media. Preservation: The purified isolated was then transferred to the slants of Nutrient agar media. The 1 dram vial was kept in the polyethylene bags, properly tied and preserved as stock culture. Physiological studies of selected Azotobacter The following studied were made on the physiological activities of the organisms. a. Salt tolerance: Nutrient agar slant containing different concentration of Sodium chloride (viz. 0%, 0.2%, 0.4%, 0.6% and 0.8%) were inoculated and incubated at 28ºC for 48 hours. The growth of different concentrations of NaCl was then compared with the control. b. Temperature tolerance: To find out the optimum temperature for growth, Nutrient agar slants were inoculated and were allowed to grow at different temperature (Viz. 10ºC, 20ºC, 30ºC, 40ºC, 50ºC).

Source of Information: J. Soil. Nature. 2 (3):16-19, 2008

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Azotobacter contained in different soil sample was varied. The legume crop field soil sample contains highest number of Azotobacter than other soil sample and it’s more potential than other isolate. This isolate can be used as suitable substrate for production of bio-fertilizer. Bangladesh has cheap manpower and other facilities to production of Azotobacter bio-fertilizer at low cost from this isolate.

Knowledge Management: Journal