Journal of the Faculty of Environmental Science and Technology.

Okayama University
Vol.13 No1, pp.97-101, March 2008

Enumeration, Isolation and Identification of Nitrogen-Fixing Bacterial Strains

at Seedling Stage in Rhizosphere of Rice Grown in Non-Calcareous Grey Flood
Plain Soil of Bangladesh

Md. Harunor Rashid KHAN*, Md. MOHIUDDIN** and M. RAHMAN*

Non-symbiotic diazotrophic systems for biological nitrogen fixation (BNF) in agriculture are most
promising but the possibility for the extension of nitrogen fixation by rice is still speculative. Accordingly, the
present study was conducted for the Enumeration, isolation and identification of nitrogen fixing bacterial
strains at seedling stage (30 days after seed sowing) in rhizosphere of rice (BR 10, Oryza sativa L.) grown in
Non-Calcareous Grey Flood Plain soil of Bangladesh. The soil is classified as ‘Inceptisol’ order and ‘Aquept’
suborder. It was identified as ‘Dhamrai series’, had ‘silt’ texture, pH 7.1 and 5.5 C/N ratio. The present results
of the microbial tests on the rice rhizosphere soil evinced that out of 263 isolates, only 91 were branded as
nitrogen fixing organisms per gram of soil, which was about 34.6 % of the total isolates. As per selection
criteria, four individual strains were considered for identification. Biochemical tests were conducted for
proper identification and the selected strains were identified as Enterobacter spp., Klebsiella spp., Bacillus
spp. and Azospirllum spp.

Keywords: Azospirllum spp., Bacillus spp., diazotrophs, Enterobacter Spp., Klebsiella spp. and Oryza sativa.

1 INTRODUCTION

The source of soil nitrogen is the atmosphere where as the primary food grain, making it the most important
nitrogen gas occupies about 79% of the total atmospheric food crop currently produced (Cottyn, et al. 2001; Klipp,
gases. Living organisms that are present in the soil have et al. 2004). Hence for the higher yield of rice for over
profound effect on transformation, which provides food population, people of the world use expensive
and fiber for an expanding world population. Although nitrogenous fertilizers. These are used to fulfill the
nitrogen is very abundant in nature, it often limits plant nitrogen demand of rice that can be overcome partially by
productivity because atmospheric nitrogen is only using biofertilizers when they are scientifically applied.
available to a very range of organisms symbiotically Biofertilizer is important in crop farming systems
associates with higher plants and non-symbiotically. because it is an inexpensive source of nitrogen for the
About 386 x 1016 kg nitrogen contains in the Earth’s higher yields of crops. This process diminishes the need
atmosphere (Stevensen, 1986). He stated that nitrogen for expensive chemical fertilizer. Thus the extensive use
returned to the earth every year through of biofertilizers would provide economic benefits to
microbiologically is of the order of 139 x 109 kg of which farmers, improve the socio-economic condition of people
about 65% (89 x 109 kg) contributed by nodulated and preserve natural resources.
legumes. Biological fixation of the atmospheric nitrogen Now days, scientists of the world in the field of
can be estimated at about 175 million metric tons per agriculture are very much concerned about the fixation of
year or about 70% of all nitrogen fixed on the Earth per atmospheric nitrogen associated with rice. In Bangladesh
year, the remaining is by some micro-organisms, rice is the main staple cereal crop. It is the basic nutrition
autotrophs or heterotrophs ‘free’ fixers. According to of the inhabitants of this country. Rice covers 80% of the
Ishizuka (1992), total world biological nitrogen fixation total cropped areas and is the main source of cash income
is 17.2 x 107 tons per year. This figure is three times of for farmers.
those of industrially or other ways of nitrogen fixation. Sen (1992) suggested that the heterotrophic bacteria
The world population increased day by day but the associated to the root system of rice could contribute
expansion of land is limited. Moreover, almost half of the efficiently to the nitrogen fixation. Youshida (1972) in
world’s population is consuming rice (Oryza sativa L.) the Philippines and Balandreau and Dommerques (1978)
in the Ivory Coast demonstrated that BNF supplies a part
of the nitrogen necessary for the growth of rice.
*Department of Soil, Water and Environment, University of Dhaka, Statistically significant grain yield increases due to
Dhaka 1000, Bangladesh. Present address: Department of
Environmental Management Engineering, Faculty of Environmental
Azospirillum inoculation have been reported from India
Science and Technology, Okayama University. and Israel (Dobereiner, 1981 and 1988). But most of the
**Department of Soil Science, Govt. Barisal College, Barisal, cases, these trials have been done using bacterial strains
Bangladesh. from international collections. Using an axenic rice

97
98 J. Fac. Environ. Sci and Tech., Okayama Univ.13 (1) 2008

plant lets as a selective medium, a collection of 23 N2 represents the growth of a single bacterial species. The
fixing bacterial strains were isolated from the rhizosphere colonies, which are different in appearances and
of rice cultivated in the Brahmaputra alluviam soil tract characters were picked and purified.
of Bangladesh. The aim of the present research was to
characterize and identify nitrogen-fixing organisms at the 2.5 Preparation for microscopic examination
seedling stage of rice rhizosphere soil in order to reduce The strains were studied following the methods of
the use of chemical nitrogen fertilizers by the production Cerney (1993) including morphological and
of biofertilizer. physiological features. Gram staining was used for the
study of the bacterial morphology. Bacteria were grown
2 MATERIALS AND METHODS on nutrient slants for overnight incubation at 30±0.5˚C. A
portion of the bacterial culture was taken out by a sterile
2.1 Soil Sampling loop and was suspended in sterile normal saline. The
Soil at a depth of 0 to 15 cm was sampled during suspension was sufficiently diluted. A drop of suspension
October from Dautia Bil at Dhamrai under the district of was taken on the slide and was spread evenly covering an
Dhaka at the seedling stage (30 days after seed sowing) area of about 15-20 mm diameter. The slide was then
of rice (BR 10, Oryza sativa L.). Six samples of kept in a safe place to air-dry. The smear was fixed by
rhizosphere soil were collected from an area of 100 m2. rapidly passing the dry slide; smear uppermost three
According to 1981 reviewed Reconnaissance Soil Survey times through the flame of a Bunsen burner. After
report of Dhaka district, the studied site is identified as passing the slide through the flame, it should be possible
Non-Calcareous Grey Flood Plain soil as per the general to lay the slide on the back of the hand without the hand
soil type of Bangladesh and the collected soil is feeling uncomfortably hot. The slide was allowed to cool
representing Dhamrai series. The soil responded well before staining.
when Aus and Boro-aman were cultivated. The field was
used to receive N-fertilizers at the rate of 60 kg ha-1yr.-1. 2.6 Identification of gram stain
In the winter season Rabi crops are cultivated. The soil A dried fixed smear was covered with oxalate crystal
was deeply flooded (about1.5 to 2.5 m) during rainy violet reagent for sixty seconds. The strain was then
season. Selected morphological and physico-chemical rapidly washed off with clean water. All the water was
properties of the studied soil are presented in Tables 1 then tipped off and the smear was covered with Lugol’s
and 2, respectively. iodine for sixty seconds. The iodine was then washed off
with clean water. The smear was then decolorized rapidly
2.2 Dilution of Soil Samples for 10 seconds with acetone alcohol and was washed
The sampled rhizosphere soil was mixed thoroughly to immediately with clear water. Finally, the smear was
make a composite soil. Then 10 gm of sub-soil sample covered with safranine for 30 seconds. The slide was then
diluted to 100 ml that considered being 10-1 dilution washed thoroughly in water and blotted dry. The smear
factor. Transferring of 1 ml of 10-1 dilution to 9 ml was examined under microscope by using high power (5
sterilized water with the help of a sterilized pipettes x 1000) immersion oil objective. The gram-negative
yielded 10-2 dilution. In this way, a series of up to 10-8 organisms were stained pink and the gram positives were
dilution was prepared under aseptic condition. Screw cap dark violet in color.
test tubes and glass petridishes were used to culture
microorganisms. Sterility is the hallmark for successful 2.7 Physiological studies of the selected strains
works in the microbiological studies were also kept in The physiological activities of the selected strains
mind throughout the study. were tested through oxidase, catalase motility indole
urease (MIU), methyl red (MR), aceton production
2.3 Bacterial counts (Voges-Proskauer), nitrate reduction, citrate utilization,
The calculation for the total numbers of bacteria was hydrogen sulfide (H2S) production, gelation liquification
done by plating soil dilutions on nutrient agar and total and carbohydrate fermentation methods as demonstrated
numbers of N2 fixing bacteria were counted by plating by Collee and Miles (1989).
soil dilutions on nitrogen free medium –RCV media. One
ml of the suspension from each (10-1, 10-2, 10-3, 10-4, 10-5, 2.8 Maintenance of culture
10-6, 10-7, 10-8) was taken and poured into the nutrient Stock cultures were maintained in soft agar (0.7% agar
agar media and the nitrogen free media on petridish in nutrient broth, Difco Lab, Detroit) stab on one-dram
separately. Then incubated the plates at 30˚C for 48 hours. airtight screw capped tubes, stored at 4 to 8˚C. The
The total count of the microorganisms was obtained by working cultures were maintained on Trypticase Soyagar
multiplying the number of cells per plate by the dilution (TSA) slant in one dram airtight screw capped (150 x 16
factor, which was the reciprocal of the dilution. mm) test tubes and stored at 4 to 8˚C. Isolated colonies
were then streaked on TSA slant in 5 mm screw capped
2.4 Isolation of pure culture test tubes and incubated overnight at 37±0.5˚C. The TSA
Discrete well-developed and separated colonies on the slant cultures were stored at 4 to 8˚C and were used as
surface of a nutrient medium plate culture were each working culture. For routine culture or routine use,
picked up with a sterile niddle and transferred separately culture was transferred from TSA slant on TSA plate.
in RCV medium slant. Each of these new slant cultures
 Md. Harunor Rashid KHAN et al / Enumeration, Isolation and Identification of Nitrogen Fixing Bacteria in Rhizosphere of Rice 99

Table 1 Selected morphological characteristics of the soil used in the experiment.

Parameters Description
1. Location 1. Mouga and Vill.: Dautia Bil; Union and P.S.: Dhamrai; Dist.: Dhaka.
2. Soil Series 2. Dhamrai series
3. General Soil Type 3. Non-Calcareous Grey Flood Plain Soil
4. FAO-UNESCO System 4. Areni Euteric Gleysols
5. USDA Siol Taxonomy 5. Subgroup: Typic Halaquepts; Greatgroug: Halaquepts;
Suborder: Aquept; Order: Inceptisol.
6. Topography 6. Low land
7. Present land use 7. Mainly mixed Aus, Aman, Jute and Rabi crops
8. Soil Color 8. Grey

Table 2 Selected physical and chemical properties of the soil used in the experiment.
Physical properties Values Chemical properties Values
Soil sampling depth 0-15 cm pH 7.1
Maximum water holding capacity 49 % EC (1 : 5) 0.02 mS cm-1
Particle sizes: Organic carbon 10.5 g kg-1
Sand 15 % Organic matter 18.1 g kg-1
Silt 59 % C/N ratio 5.5
Clay 26% Total Nitrogen 1.9 g kg-1
Textural Class Silt Available Nitrogen 25 mg kg-1

The plates were incubated overnights at 37±0.5˚C to 3 RESULTS AND DISCUSSION

yield 10 g phase culture.
The studied soil was classified as ‘Inceptisol’ order
and ‘Aquept’ suborder on the basis of the USAD Soil
2.9 Media and Reagent
Taxonomy, 1999. The general soil type of the studied
The RCV and complete nutrient media were used for
area was Non-Calcareous Grey Flood Plain Soil. It was
the present study in order to enumeration, isolation and
identified as ‘Dhamrai series’, had ‘silt’ texture, pH 7.1
identification of bacterial strains.
and 5.5 C/N ratio (Tables 1 and 2). The enumeration of
The RCV media: A carbon and nitrogen free media,
the existing total biomass and only nitrogen-fixing
which was adjusted for the culture of Rhodopseudomonas
bacteria were done through serial dilution agar plating
capsulate. The composition of this media is Elements
technique. Molten agar at 45˚C was poured into a
solution (ZnSO4 7H2O 430 mg, MnSO4 H2O 1.30 g,
petridish. One ml of the suspension was putted onto a
Na2MoO4 2H2O 750 mg, H3BO3 2.80 g, CuSO4 26 mg,
petridish from the each fold of dilution. Then the plate
CoSO4 2H2O 70 mg and distilled water 1 L), Super salt
was gently rotated in a circular motion to achieve
solution (Elements solution 20 ml, EDTA 400 mg,
uniform distribution. This procedure was repeated for all
MgSO4 7H2O 2.00 g, FeSO4 7H2O 440 mg, CaCl2 2H2O
dilutions to be plated. Dilutions were plated in duplicate
200 mg and distilled water 1 L), and Phosphate buffer
for greater accuracy, incubated overnight before colonies
(KH2PO4 40 mg, K2HPO4 60 mg and distilled water 1 L).
develop and counted by naked eye. The total counts of
The super salt solution and phosphate buffer were
the suspension are obtained by multiplying the number of
sterilized separately and then they were carefully mixed
cells per plate by the dilution factor, which is the
after cooling in order to avoid flocculation. Carbon
reciprocal of the dilution. It was observed that 263
source (yeast extract 0.10 g L-1, Malic acid 3.5 g L-1) was
microorganisms was randomly counted from incubated
added in the super salt solution and adjusted to pH 6.8
plated of solid complete nutrient agar media and 91
with 1N KOH. The final solution media was a mixture of
organisms were picked up in the same manner from the
super salt solution 50 ml, phosphate buffer 15 ml and
N-free medium (RCV) plates and the results are
distilled water 1 L. The complete media for the
presented in Table 3.
development of bacterial strain were Luria Bertani Broth
The present results have close conformity with
(LB), Nutrient agar (NA), Nutrient Broth (NB), Gelation
findings of Torres, et al. (2000). They obtained
agar (AG), MR-VP medium, Nitrate reduction medium
Azotobacter Chroococcum, Azotobacter vinelandii,
(NR), Simmons citrate medium, TSA+0.6% yeast extract,
Pseudomonas aeruginosa and Klebsiella pneumoniae
starch agar, Tributyrin agar, milk agar and TSA agar.
strains from rhizosphere of rice cultivated in the Tolima
The chemicals were Kovac’s reagent, mercuric
region, Colombia S.A. Thomas-Bauzon and Balandreau
chloride solution, methyl red indicator, NR test reagent,
(1982) isolated N-fixing bacteria with a frequency of
Phosphate buffer saline, VP reagent, ammonium oxalate
65%, 32 of the many isolates were Klebsiella spp.,
crystal violet solution, Lugol’s iodine solution and
safranine solution. Enterobacter spp., Pseudomonas spp. and Azospirillum.
100 J. Fac. Environ. Sci and Tech., Okayama Univ.13 (1) 2008

Table 3 Enumeration of total biomass and N-Fixing bacteria. The appearances of the colonies of strains (strain-1,
strain-2, strain-3 and strain-4) on the nutrient agar (NA)
Dilution Total No. of % of plate were circular, flat, raised and convex in elevation;
Factor Number N-Fixer N-Fixer small and pinpoint in size; non-pigment, yellow, grey, off
10-1 120 30 white in color, respectively. On the other hand, the
10-2 58 21 colonies appearances on RCV petridish were white, off
10-3 42 18 white, grey and grey to white in color; circular, flat,
10-4 29 14 raised, serrate in elevation; small and pinpoint in size,
10-5 08 06 34.6 % which are presented in Table 4.
10-6 03 02 All the selected strains were identified as gram-
10-7 02 0 negative rods and mostly true motile. All the strains were
10-8 01 0 oxidase positive, indole positive, starch and lipid
Grant Total: 263 91 34.6 % hydrolysis positive, but did not produce H2S in KIA
media, catalase positive except strain-2, methyl red
Rennic and Vose (1983) used single nitrogen free positive (strain-2, strain-3) and methyl red negative
medium for isolating nitrogen-fixing bacteria and showed (strain-1, Strain-4), Voges-Proskauer (acetone
that at the higher dilutions 75% of the isolates exhibited production) positive (strain-1, strain-4) and negative
acetylene reduction. They also showed that Erwinia (strain-2, strain-3), only strain-2 was found to be reduced
herbicola comprised 50% of the total population, which nitrite to nitrate, did not utilize citrate or liquefication.
13 Polymixa and K. Pneumoniae accounted for 33% and The results obtained from the biochemical and
17% exist in the rhizosphere of rice, respectively. But the carbohydrate fermentation tests are stated in Tables 5
present results showed that only 91 microorganisms of and 6, respectively. According to Bargey’s Manual of
the total of 263 were nitrogen fixing organisms which Systemic Bacteriology, the above (Tables 5 and 6)
was about 34.6% of the total isolates per gram of the biochemical and carbohydrate fermentation tests
rhizosphere soil (Table 3). Watanabe and Baraqui (1979) indicated that the characters represented by the strain-1 is
revealed that nitrogen-fixing bacteria are present in similar to Enterobacter spp., strain-2 is as Klebsiella spp.,
greater number in the root of wetland rice. Azospirillum strain-3 is as Bacillus spp. and strain-4 is as Azospirillum
spp. were found in large numbers were associated with spp.
rice. Yoshida (1972) also reported that biological
nitrogen fixation in Philippine rice fields were ranged 4 CONCLUSIONS
from 2.30 to 33.3 kg ha-1. Sanoria and Maurya (1982)
also reported that significantly much higher yields of The 34.6% of the total isolates was identified as
grain and straw of rice by inoculation Azospirillum nitrogen fixing microorganisms during the seedling (30
compared with control. The present results have proved days after seed sowing) stage of rice (BR 10) rhizosphere
the above-mentioned facts and also have similarities with soil. Based on the selection criteria, the four individual
the ways of investigation and findings as reported by strains were microbiologically identified. Their
Mukhopadhyay, et al. (1996) and Stoltzfus, et al. (1997). biochemical tests were strictly similar to Enterobacter
Among the 91 nitrogen fixers (34.6%), only 4 types of spp., for strain-1, Klebsiella spp. for strain-2, Bacillus spp.
strains (strain-1, strain-2, strain-3 and strain-4) were for strain-3 and Azospirillum spp. for strain-4. They were
selected for the identification on the basis of their anaerobic in nature.
colonies appearances on NA media and on RCV media.

Table 4 Colony appearance on NA media and RCV media.

# *
NA media RCV media
Features Appearances of the strains Appearances of the strains
Strain-1 Strain-2 Strain-3 Strain-4 Strain-1 Strain-2 Strain-3 Strain-4
Size Small Pinpoint Small Pinpoint Small Pinpoint Small Pinpoint
Elevation Circular Flat Raised Convex Circular Flat Raised Serrate
Pigmentation Non. pig. Yellow Grey Off-white White Off-White Grey White-Grey
# *
NA = Nutrient agar; RCV = A carbon and nitrogen free media which was adjusted for the culture of
Rhodopseudomonous capsulate.

REFERENCES Cerney, G. (1993): Method for the distinction of gram negative

Balandreau, J. and Dommerques, Y. (1978): Limitations and and gram positive bacteria. EU J. Appl Micro. 3, pp. 223-
potentials for biological nitrogen fixation in the tropics. 225.
Pleurum Publ. Crop. pp. 275-302.
 Md. Harunor Rashid KHAN et al / Enumeration, Isolation and Identification of Nitrogen Fixing Bacteria in Rhizosphere of Rice 101

Table 5 Selected biochemical tests of unknown strains after 48 Klipp, W., Masepohl, B., Gallon, J.R. and Newton, W.E.
hours of incubation at 37±0.5ºC. (2004): Genetics and Regulation of Nitrogen fixation in free-
living bacteria. Nitrogen fixation: origins, application and
Name of the Response of strain research progress, Vol. 2: Kluwer academic publishers,
Tests Boston.
Strain-1 Strain-2 Strain-3 Strain-4 Mukhopadhyay, K., Garrison, N.K. Hinton, D.M., Bacon, C.W.,
Gram strain - - - - Khush, G.S., Peck, H.D. and Datta, N. (1996): Identification
and characterization of bacterial endophytes of rice.
H2S production - - - - Mycopathologia, 134, pp. 151-159.
NO3 production Neyra, C.A. and Dobereiner, J. (1977): Nitrogen fixation in rice.
- + - - Adv. Agronomy, 29, pp. 1-38.
Indole production + + + + Rennic, R.J. and Vose, P.V. (1983): 15N-isotope dilution of
quantity dinitrogen fixation associated with Canadian and
Methyl Red - + + - Brazillian wheat. Can. J. Bot., 61, pp. 967-971.
reaction Riando, G. and Dommerques, Y. (1971): Validity of estimating
Voges-Proskauer + - - + biological nitrogen fixation in the rhizosphere by acetylene
reaction reduction method. Ann. Inst. Pasteur (Paris), 121, pp. 993-
999.
Citrate Utilization - - - - Sanoria, C.L. and Maurya, B.R. (1982): Field trials with
Urease activity Azospirillum brasilense in an Indo-Gangetic Alluvium. J.
+ + + - Indian Soc. Soil Sci., 30, pp. 208-209.
Catalase + - + + Sen, M.A. (1992): In bacterial association a factor in nitrogen
assimilation by rice plant. Agri. J. India, 24, pp. 229-231.
Oxidase + + + + Stevenson, F.J. (1986): Cycles of soil. John Willey & Sons Inc.
NY, pp. 116.
Gelatin - - - - Thomas-Bauzon, D. and Balandreau, J. (1982): The
liquefication Supermosphere model on its use in growing, counting and
Starch hydrolysis + + + + isolating N-fixing bacteria from the rhizosphere of rice. Can.
J. Micro., 28, pp. 922-928.
Liquid hydrolysis + + + + Watanabe, I. and Baraqui, W.L. (1979): Low levels of fixed
nitrogen required for isolation of free living nitrogen fixing
Motility ++ ++ ++ ++ organisms from rice roots. Appl. Env. Micro., 7, pp. 566-569.
Yoshida, T. (1972): Soil Microbiology. Ann. Report. The
International Rice Research Institute, Manila. pp. 35-42.
Stoltzfus, J.R., So, R., Malarvizhi, P.P., Ladha, J.K. and de
Table 6 Selected carbohydrate fermentation tests for the
studied strains. Bruijn, F.J. (1997): Isolation of endophytic bacteria from
rice and assessment of their potential for supplying rice with
Carbohydrate Response of strain biologically fixed nitrogen. Plant & Soil, 194, pp. 25-36.
tests Strain-1 Strain-2 Strain-3 Strain-4 Torres-Rubio, M.G., Valencia-Plata, S.A., Bernal-Castillo J.
and Martienez-Nieto, P. (2000): Isolation of Enterobacteria,
Glucose + + + + Azotobacter sp. And Pseudomonas sp., Producers of Indole-
Lactose + + + + 3-Acetic Acid and Siderophores, from Colombian Rice
Sucrose + + + - Rhizosphere. R. L. De Microbiologiea, 42, pp. 171-176
Maltose + - + -
Mannose + + - +
Mannitol - - + -
Inositol - + - +

Collee, J.G. and Miles, R.S. (1989): Tests for identification of

bacteria in Mackie and Maccartney practical medical
microbiology. Churchill Livingstone, London. pp. 141-160.
Cottyn, B., Regalado, E., Lanoot, B., De Cleene, M., Mew T.W.
and Swings, J. (2001): Bacterial Populations Associated
with Rice Seed in the Tropical Environment.
Phytopathology, 91, pp. 282-292.
Dobereiner, J. (1981): Emerging technology based on biological
nitrogen fixation by associative nitrogen fixing organisms.
Paper presented in a workshop held at CIAT (Centro
International de Agriculture Tropica), March 9-13, Cali,
Colombia.
Dobereiner, J. (1988): Isolation and identification of root
associated diazotrophs. Plant & Soil., 110, pp. 207-212.
Ishizuka, J. (1992): Trends in biological nitrogen fixation
research and application. Plant & Soil, 141, pp. 197-209.