Keywords

Azotobacter, Vermicast, Charcoal and Biofertilizer

Introduction

Application of biofertilizer for crop production is environmental friendly and sustainable for ecological system. Several types of biofertilizer have been developed from bacteria, particularly Rhizobium species, Azospirillum species, Azotobacter species and used in the production of various plants [6]. Biofertilizers are defined as preparations containing living cells or latent cells of efficient strains of microorganisms that help crop plants uptake of nutrients by their interactions in the rhizosphere when applied through seed or to soil. Biofertilizer from nitrogen fixing bacteria come in three forms: liquid, solid and lyophilized. For liquid and lyophilized ones, only solution medium is used, but for solid form, carriers such as peat, activated charcoal and chicken dung are needed. Azotobacter is a free living nitrogen fixing bacteria is a beneficial biofertilizer which has profitable effects on plants and soil fertility. Azotobacter is gram-negative, motile, pleomorphic aerobic bacterium which produces catalase, oval or spherical that form thick walled cysts and may produce large quantities of capsular slime[3, 4].

Presently lignite powder is being used as carrier material by most of the bioinoculant producing units in India. Often it has also been found that its availability is also made difficult, as it is being used as fuel by thermal power stations, etc. Availability of quality lignite powder is also in doubt because of adulteration by agents and improper mesh size in the pulverizing unit. Several scientists have suggested compost as carrier material for biofertilizers. But the role of good compost in maintaining microbial population has not been studied much. The existing studies exhibit that the earthworm casts is ideal material for carrying microbial culture from the agriculture point of view[2]. In the present study, the effect of charcoal and vermicast as carrier in maintaining the shelf life of Azotobacter in powder inoculum at room temperature.

Materials and Methods

Preparation of carriers

Charcoal and vermicast were used in this study. The raw material were ground, sieved with 0.5 cm mesh screen and dried in a hot air oven at 60oC for two days. The materials were autoclave at 121oC at a pressure of 15 lb for 30 minutes.

Analysis of physical and chemical properties

The carriers were analyzed for physical and chemical properties including pH, temperature, moisture, total nitrogen (Ntot), total phosphorus (Ptot) and total potassium (Ktot).

Collection of soil samples

Soil samples were collected from Semmedu-Kolli hill forest, Namakkal, Tamil Nadu.

Isolation of Azotobacter from soil sample

Azotobacter was isolated from the soil sample by serial dilution as 1.0g of air dried samples was dissolved in 99ml of distilled water. The soil suspension was further diluted up to 10-6 level. The diluted soil suspension (0.1ml) was spread on the surface of Jensen agar medium which is a selective medium for isolating Azotobacter. The pH of the medium was adjusted to 7.0 with the help of 1N HCl/ 1N NaOH. The plates were incubated at 28oC for 5-7 days and the colonies were observed. Strains of Azotobacter were picked out and purified by repeated streaking on Jensen medium and were preserved as slant culture for further usage.

Identification of isolates

The isolate was identified by Gram staining and the biochemical characterization of the isolate was carried out by using standard method.

Cultivation of Azotobacter powder inoculum and packaging

A loopfull of Azotobacter pure culture was transferred into 250ml Erlenmeyer flask containing 100ml of Ashby’s broth and incubated at 28oC on 120 rpm rotary shaker for 72 hours. After incubation, 10ml of the inoculums was transferred to 1000ml of respective broth and kept in shaking incubator for mass multiplication. 750ml was mixed thoroughly with 1000g of each sterile carrier, adjusted the moisture content to 75% water holding capacity, packed in polyethylene bags, sealed and incubated under room temperature. The inoculums were repacked in sterile polyethylene bags and stored at room temperature for further usage.

Evaluation for survival of the Azotobacter during storage at room temperature

The survival of Azotobacter was determined after the inoculum was subjected to different carriers at room temperature. Ten grams of each sample was taken for estimating viable cells at the initial day, 15, 30, 45 and 60 days after storage using dilution plating method on Ashby’s agar and incubated at 28oC for 5-7 days. The number of apparent Azotobacter colonies after incubation from both carriers was counted and calculated into viable cells.

Results

Physical and chemical properties of carriers

Some physical and chemical properties of the carriers are shown in (Table 1). The pH of vermicast was nearly neutral compared to charcoal. The highest moisture content was found in vermicast, while that of charcoal was the lowest. There was a significantly difference among the physical and chemical properties of the carriers.

Isolation of Azotobacter

Azotobacter was isolated from rhizosphere soil by serial dilution method. Azotobacter strain was recovered from the soil samples collected using Jensen agar medium. This media is very specific for the isolation of Azotobacter.

Colony morphology

Large mucoid opaque colonies were observed.

Gram staining

The smear was examined under the microscope, the isolates does not retain violet colour and it was to be confirmed as Gram-negative and it showed rod or spherical shape thick walled cyst in the cell morphology.

Characterization of Azotobacter on various biochemical tests

The confirmation of Azotobacter was done through various biochemical tests. Isolates was showed positive results to MR, Citrate, Urease, Oxidase, Catalase and Nitrate where they expressed negative result to Indole and VP. The isolates were efficient in hydrolyzing Starch (Table 2).

Survival of Azotobacter in the carriers at room temperature

After 60 days of inoculants maintenance, the population of the bacteria was determined through Colony Forming Unit method. At initial days the bacterial population was higher in charcoal. After 15 days of incubation, the bacterial population in the charcoal was intensively declined (Figure 1). While the population of bacteria in the vermicast increased after 15 days (Figure 2). As listed in Table.3, the best carrier was treatment of vermicast. The weakest carrier was charcoal.

Table 1

Physical and chemical properties of carriers

Parameter Carrier materials
Vermicast Charcoal
pH 7.15 7.45
Moisture (%) 18.18 15
Water uptake (mins) 18(mins) 28(mins)
Ntot (%) 8.5 0.83
Ptot (%) 6 0.39
Ktot (%) 5 0.24

Table 2

Characteristics of Azotobacter

Characteristics Observations
Shape Rod or spherical
Gram’s reaction Negative
Indole production
Methyl red +
Voges proskauer
Citrate utilisation +
Nitrate reduction +
Urease +
Catalase +
Oxidase +
Starch hydrolysis +
TSI Alkaline slant, Acid butt

Table 3

Population of inoculants during storage at room temperature

Days Carrier Colony forming unit/gram
10-4 10-5
0 Charcoal 350 197
Vermicast 132 108
15 Charcoal 243 129
Vermicast 142 117
30 Charcoal 158 109
Vermicast 159 128
45 Charcoal 117 87
Vermicast 179 140
60 Charcoal 62 20
Vermicast 192 165

Figure 1: Survival of Azotobacter inoculants in charcoal carrier on different days of incubation (0, 15, 30, 45, and 60 days)

Figure 2: Survival of Azotobacter inoculants in vermicast carrier on different days of incubation (0, 15, 30, 45, and 60 days)

Discussion

Previous studies have suggested that high temperature could cause growth and increase of the bacterial population, it produced wastes that were not only toxic for bacteria, but also changed the pH of medium that could be the reason of population reduction and death of bacteria[1]. Mendez and Videira (2005) stated that bacterial maintenance at 28oC for 41 days caused an increase in number of viable bacterial cells on all carriers so that the population reached nearly 109 bacteria per gram of carrier[7].

The study by Kalra et al. (2008) that the granular vermicompost as a carrier is capable of holding 108 viable bacteria after 180 days[5].Sekar and Karmegam (2010) reported that vermicasts from E. euginae as a carrier material which supports the survival of more than 1×107 g-1 viable cells of A. chroococcum, B. megaterium and R. leguminosarum till the end of 10th month which is longer than observed in lignite (a commercial carrier material)[9]. Saleh et al. (2001) reported that the population of Azotobacter vinelandii A1 in rice husk carrier rise up to 128% from the initial population after storing at 30oC. At 30 days after storage, the bacterial population increased slightly in Pt and ptLC stored at 16oC, and Pt, PtCC, PtMC and PtLC at 5oC[8]. The vermicasts when used as carrier material for biofertilizers supported the survival rate for more than one year[9]. The findings of the present study also showed similar results when the vermicast used as carrier for the survival and viability of the biofertilizer Azotobacter inoculant for long period during storage than the charcoal as a carrier.

Conclusion

Use of organic sources of fertilizers improved the soil chemical properties through increasing the content of macro and micronutrients and organic carbon in the subtropical climate of India. The biological property of soil with regard to microbial count was strengthened when Azotobacter was applied along with vermicast. The vermicast prepared from the earthworm Perionyx excavatus is the most suitable inoculum carrier after storage for 60 days. Vermicast inoculum can be stored at room temperature for a period 60 days and still maintained the bacterial population when compared to the Azotobacter inoculated in charcoal.

Acknowledgements

Authors are thankful to the Tamil Nadu government state council for science and technology to the supported this research work through scholarship sanctioned.