Nitrogen fixing efficiency and physiological characteristics of Azospirillum isolates from the paddy fields of North Bengal

The microbial activity of ten selected Azospirillum isolates was measured in terms of the amount of CO2 evolved by the isolates after incubation for 5 days which ranged from 6.88 to19.25 mg. Nitrogen fixing efficiency all of the isolates was determined by microkjeldhal method and the nitrogen fixing efficiency ranged from 10.03 to 13. 1 mg N/g substrate. The growth of Azospirillum isolates was significant affected by different phy siological factors such as pH, temperature and salinity. Most of the isolates show ed optimum growth at pH 6.8, temperature 37°C and in the absence of NaCl. Azospirillum has the potential to be used as a substitute and o r supplement of N-fertilizers. Further research is ne eded to estimate N-supplement potentials of biological nitrogen fixation (BNF) systems at the fa rm level.


INTRODUCTION
Biological approaches are usually less expensive, harmless and in the reach of all the countries.The utilization of biological nitrogen fixation (BNF) technology can also decrease the use of urea-N, prevent the depletion of soil organic matter and reduce environmental pollution to a considerable extent.Azospirillum a plant growth prtomoting bacteria is being used as biofertilizer in several countries of the world.It is a soil bacterium capable of producing associative symbiosis in the roots of various plants including grain crops including rice.Azospirillum promotes plant growth by fixing atmospheric nitrogen and by some other ways like production of growth promoting substances and influencing root development, causing increased uptake of nutrients from the land, and inhibiting pathogenic fungi and bacteria in the rhizosphere.Azospirillum inoculation increases percentage of rice seed germination (treated 50% : untreated 20%) (Kannan & Ponmurugan, 2010;Ravikumar et al., 2002).Inoculation of plants with Azospirillum has been found to cause significant increases in growth and yield which is equivalent to that is attainable by application of 15-20 kg N/ha.A yield increase in rice due to inoculation of Azospirillum is reported to be in the 5-60% range (Kumar & Balasubramanian, 1986).The aims and objectives of the present study were to determine microbial activity, nitrogen fixing efficiency and the effect of different physiological factors such as pH, temperature, salinity on the growth of different Azospirillum isolates.

MATERIALS AND METHODS
Isolates collection: Ten Azospirillum isolates (Table 1), isolated from rhizosphere soils (M-6 and M-9) and roots of rice(M-1, M-2, M-3, M-4, M-5, M-7, M-8 and M-10) (Oryza sativa) growing on rice fields of particular locations of three different districts of North Bengal-Bogra, Naowgoan and Dinajpur were selected for inoculation.These isolates were identified by different biochemical tests.Determination of microbial activity: Microbial activity of the selected isolates was determined by measuring the amount of CO 2 evolved (mg) by the culture.CO 2 evolution was estimated according to the method described by Pramer & Schmidt (1964).

Determination of nitrogen-fixing efficiency of the Azospirillum isolates:
Nitrogen fixation was determined in terms of the quantity of nitrogen (mg N/g substrate) gained in the 72 hours old cultures of each isolates developed in 25 ml semi-solid nitrogen free malate medium (without bromothymol rblue).Total nitrogen content in culture was estimated by microkjeldahl method.
Effect of pH on the activity of the Azospirillum isolates: Semi-solid Nfb medium was prepared and pH of the medium was adjusted to 5.0, 6.0, 6.8, 8.0 and 9.0 respectively just prior to adding agar.Effect of different pH on the activity of selected isolates was determined by CO 2 evolution method (Pramer & Schmidt, 1964).

Effect of temperature on the activity of the Azospirillum isolates:
To find out the maximum, minimum and optimum temperature, each isolate was allowed to grow in Nfb semi-solid medium at temperature 30°C, 37°C, 40°C, 45°C respectively for 5 days.Then the activity of each isolate was estimated by CO 2 evolution method (Pramer & Schmidt, 1964).

Effect of salinity on the growth of the Azospirillum isolates:
To determine the effect of salinity on the growth of the Azospirillum isolates tubes of liquid malate medium with no Bromothymol blue but containing 1.0% NH 4 Cl was used.Tubes of the medium containing various concentrations of NaCl (1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, and 4.0%) were inoculated with the selected isolates and incubated for 48 hours.The growth level was measured by spectromertically at 620 nm (Usha & Kanimozhi, 2011).

RESULTS AND DISCUSSION
The activities of the selected isolates of Azospirillum under N 2 -fixing condition have been studied.Carbon dioxide is one of the principle metabolic products of microorganisms and CO 2 evolution during microbial growth has frequently been used as a measure of microbial activity (Waksman & Starkey, 1957).Activity of selected isolates of Azospirillum measured in terms of quantity of CO 2 (mg) evolved after 5 days varied significantly (Table 2).Amounts of CO 2 evolved by the selected isolates ranged from 6.88 to 19.25 mg.M-6 and M-8 evolved more CO 2 than the other selected isolates.On the basis of activities the selected isolates could be arranged as M-6, M-8>M-1>M-9>M-7>M-4>M-10>M-2>M-5>M-3.Azospirillum readily utilize organic acids like malate, succinate, pyruvate, and lactate for its growth (Tarrand el al., 1978).In this study malate was used as sole carbon source for the determination of nitrogen fixing potential of the selected isolates.Values equivalent to the highest efficiencies of nitrogen fixation first reported by Dobereiner and Day (1976).115 mg N per g lactate has not been reported in other studies.About in vitro nitrogen fixation, Okon et al., (1977) reported values of 20 to 24, Nelson & Knowles (1978) 4.7 to 28 and Lakshmi el al., (1977) 12 to 36 mg N per g substrate.Lakshmi & Dhala (1984) reported that some aquatic isolates of Azospirillum has nitrogen fixing potential ranging from 3.08 to 11.9 mg N/100 ml culture.In the present investigation, the selected isolates were found to fix nitrogen ranging from 10.03 to 13.11 mg N per g substrate (malate) (Table 3).As per their nitrogen fixing capability the selected isolates could be arranged as M-10>M-7>M-6>M-1>M-4>M-9>M-8>M-3>M-2>M-5.Khan & Hossain (1990) found that nitrogen fixation by ten isolates of Azospirillum ranged from 2.9 to 7.3 mg N/50 ml culture.Khan & Akond (1996) however reported lower values, the amount of nitrogen fixed by their isolates ranged from 448 to 658 µg N/25 ml culture.Khan et al. (2001) reported that the N 2 -fixing potentials of Azospirillum isolated from wheat fields of Dhaka ranged from 15.12 to 22.16 mg N/g substrate.Khan et al. (2001) also reported that some thermophilic isolates of Azospirillum isolated from Bangladesh could fix nitrogen well at 55°C, and the values ranged from 10.08 to 28.00 mg N/g substrate.In this study, activities of all the selected isolates were observed optimum at pH 6.8 but least at pH 5.0 and 9.0 (Table 4).At pH 6.8 both M-6 and M-8 showed the highest activities evolving 19.25 mg CO 2 , whereas M-3 demonstrated its lowest activity evolving 6.88 mg CO 2 after 5 days incubation.Tilak et al. (1988) found that N 2 -fixation for 3 isolates of Azospirillum ranged for 6.5 to 8.5 mg at their optimum pH values.In this study, the activity of the selected isolates was found to be optimum at 37°C, except M-9 the optimum temperature of which was 40°C (Table 5).At 37°C the values of activity ranged from 19.25 mg CO 2 in M-6 and M-8 to 6.88 mg CO 2 in M-3 after incubation for 5 days.The activities were low at 30°C and 45°C.The results indicated that activity decreases with the increase of temperature above 40°C.Khan et al. (2001) found that some thermophilic isolates of Azospirillum exhibited higher growth and N 2 -fixation at 55°C than at 35°C.The high temperature requirements of these organisms are of great ecological importance as in temperate regions soil temperatures seldom reach 28°C for any significant period.In the tropics, however, optimal temperatures for nitrogenase activity of this system occur during the main growing season almost daily for most of the days.Nitrogen fixation by a tropical Azospirillum isolate was highly reduced when the isolate was transferred from 36°C to 17°C (Day & Dobereiner, 1976).It shows that Azospirillum are highly adaptive to their native environment.In this study, growth of the selected isolates of Azospirillum was highly affected by NaCl, and growth in all isolates, except isolate M-8 and M-9 gradually declined with increasing concentrations of NaCl in the medium (Table 6).Growth of isolate M-1, M-2 and M-5 was absent at all concentration of NaCl.Growth was almost completely inhibited at 4% NaCl in all isolates except M-8 and M-9 at which little growth was observed for M-8 and highest growth was observed for M-9.The growth of M-9 gradually increased with the increasing of NaCl concentration.Low concentrations of NaCl produce an accelerating effect on the growth of bacteria (Salle, 1967).High concentrations of NaCl are generally inhibitory.Maximum count of E. coli was found at a concentration of 0.2 M (1.17%) (Salle, 1967).In addition to affecting osmotic pressure, high salt concentrations tend to denature proteins and obligate halophiles possess specialized enzymes that are in their active configuration only at high salt concentrations (Atlas & Bartha, 1981).In the present investigation none of the isolates, except M-9 preferred saline condition for proper growth.Similar results were reported by Khan & Akond (1996).They found that N 2 -fixation by 5 isolates of A. brasilense gradually decreased with the increase of the concentrations of NaCl in the medium.Only isolate M-9 preferred saline condition as reported by Rahman et al. (2007).