WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 7.632

Volume 11, Issue 5, 1519-1526 Research Article ISSN 2278 – 4357

ISOLATION AND PRODUCTION OF LIQUID NITROGEN FIXER

BIOFERTILIZER AND GROWTH EFFECTS ON CHICKPEA (CICER
ARIETINUM)

Somnath De1*, Koustuv Maity1, Souvik Santra1, Manoj Kumar Jana1, Sirsendu Bikash
Maiti2 and Tanusri Mandal2

1
Department of Biotechnology, Panskura Banamali College (Autonomous) West Bengal,
India.
2
MSV Laboratories (Pvt.) Ltd., Kanakpur, Panskura R.S., Purba Medinipur, West Bengal,
India.

Article Received on
21 Feb. 2022,
Revised on 13 March 2022,
Accepted on 03 April 2022
DOI: 10.20959/wjpps20225-21847
ABSTRACT
The aim of present study was the production of liquid nitrogen fixer
biofertilizer using selective and optimized media. Nitrogen containing
products which helps to increase germination rate, increase production
rate with growth of plants. Field inoculation of plants with Azotobacter

*Corresponding Author
and Rhizobium after cultivation of experimental plants it is noted that
Dr. Somnath De 20-40 kg nitrogen remain back which benefits for further plantation
Department of and reduce use of 25 % chemical fertilizers. Due to the function of
Biotechnology, Panskura
Azotobacter and Rhizobium in nitrogen fixation and to their probable
Banamali College
biotechnological applications, there are few challenges in developing
(Autonomous) West Bengal,
India.
an effectual approach for the selective isolation of these micro-
organisms from soil. During this study experimental plants of chickpea
Cicer arietinum shows that the growth of plant is better than normal growth and it increase
yields.

KEYWORDS: Liquid Biofertilizer, Plant Growth, Cicer arietinum.

INTRODUCTION
Nitrogen fixer biofertilizer are very important strains of microorganism which can uptake the
N2 from air and soil to the plant. Nitrogen containing biofertilizer exists in three forms: solid,
lyophilized and liquid. For solid form needed saw dust, activated charcoal and chicken dunk

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1519
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

but for liquid and lyophilized one only solution medium is used.[1,2] Numbers of bacterial spp.
(Rhizobium, Azospirillum, Azotobacter), Fungi (Mycorrhizae like glomus), Blue – green
algae or Cyanobacteria (Anabena, Nostoc) and Azolla (a fern containing symbiotic anabena
azollae) are acts as a biofertilizer. It’s a live microbial inoculant which have enormous
significance to improve plant growth and productivity.[3,4]

Azotobacter is gram-negative, motile, pleomorphic aerobic bacterium which establish

symbiotic relationships with different parts of plants and may develop special structures as
the site of nitrogen fixation.[5,6] Nearly all of the bacteria included in biofertilizer include a
close association through plant roots. Rhizobium has symbiotic interaction by legume roots
and rhizobacteria in habit on roots surface or in rhizosphere soil.[7]

Azotobacter, a free-living microbe, acts as plant growth promoting rhizobacteria (PGPR) in

the rhizosphere of almost all crops. A group of beneficial plant bacteria, as potentially useful
for stimulating plant growth and increasing crop yields has evolved over the past several
years to where today researchers are able to repeatedly use them successfully in field
experiments Such PGPRs also fix nitrogen for non-legume crops like wheat, rice, sunflower,
sugarcane, cauliflower, cotton, maize and sorghum which helps in saving 20-40kg chemical
nitrogen i.e., 45-90 kg urea per hectare. Yield of several non-legume was increased by
PGPRs symbionts through plant growth promoting substances, it helps in root expansion,
improve uptake of plant nutrients protects plants from root diseases and most important
improves biomass production of fast growing at wasteland.[8]

MATERIALS AND METHODS

Isolation of Azotobacter species from soil sample
Rhizospheric microbes were isolated from soil that were composed from MSV Laboratories
(Pvt.) Ltd. and inoculate in Ashby’s agar medium and was incubate at 24 hrs. Colony trait of
arbitrarily isolated colonies was recorded. Gram staining was performed. For mass
production of Azotobacter bacterial strain isolated from various regions and grown on slants
for preservation as per need culture from slant were transferred to liquid broth of selective as
well as optimized medium in the rotary shaker for 4 days to prepare starter culture. Later the
starter cultures are incubated at 300C and continuous agitation for 4-9 days. when cell count
reached to 108- 109 cells/ml, the broth used as inoculant. After proper mixing carrier
containing inoculant was left for 7 days and above formulated microbial inoculants used as
biofertilizer.

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1520
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

Isolation of Rhizobium species from soil sample

Rhizospheric microbes were isolated from soil that were composed from MSV Laboratories
(Pvt.) Ltd. and inoculate in YEMA agar medium and was incubate at 24 hrs. Colony trait of
arbitrarily isolated colonies was recorded. Gram staining was performed.

Biochemical characterization
Chosen colonies were more characterize for a variety of biochemical and morphological test.
The staining is followed by use of various biochemical reagents and tests to get closer to the
identification of bacteria. There are many biochemical tests available for bacterial
identification. Few of them are required to be carried out depending upon the bacteria. The
commonly used biochemical tests are as mentioned below (a) Catalase test (b) Coagulase test
(c) Oxidase test (d) Sugar fermentation test (e) Indole test (f) Citrate test (g) Urease test etc.

Pot experiment for Chickpea growth effects

For mass production of Azotobacter and Rhizobium bacterial strain isolated from various
regions and grown on slants for preservation as per need culture from slant were transferred
to liquid broth of selective as well as optimized medium in the rotary shaker for 3 days to
prepare starter culture. In present study, different concentration of broth culture prepared i.e.,
20%, 40%, 70% and 100% broth of Azotobacter and Rhizobium bacterial strain. Three sets of
sterile soil were prepared for the experiment. Then broth is mixed with sterile soil. After
proper mixing, Chickpea was planted in each set of sterile soil.

RESULTS AND DISCUSSION

Azotobacter forms large, flat, soft, milky, mucoid and gummy colonies (Fig 1). Biofertilizer
applied as seed treatment and through pouring of slurry i.e., soil application where it
functions to mobilize the availability of nutrients especially NPK by their biological activity
in particular area and help to enhance soil health by building up microflora to supply different
kind of nutrients in the soil. This study clearly indicates that the effect of biofertilizer in Cicer
arietinum was enhanced, when compare with pea plants were inoculated with carrier material
compared to uninoculated control. All parameter were significantly showed favourability
more for seed treatment with biofertilizer than pouring of slurry in soil over control. Plant
height was one of the parameters used to assess performance for crops showed that treatments
involving Azotobacter with crude coal powder were more pronounced growth throughout the
growing period than activated charcoal where nutrient contents studied showed good
nutritional value with crude powder and enhance N-fixation thus disease free high growth of

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1521
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

plants was resulted and economically it is better to use biofertilizer of Azotobacter alone for
non-leguminous plants by using mass inoculums of optimized as well as selective media.
However, pea plants grown in control pots were showing less growth. Although, the NPK of
soil was measured, it appears that high percent of NPK in pots inoculated with biofertilizer
than uninoculated pots. This study not only showing improved growth potentiality with
optimized media also considering seed germination found early almost 100% when compared
with control. At maturity data regarding plants height, leaf area, root weight, shoots weight
and grain yield were recorded using standard procedures. During study N influences
vegetative and reproductive phase of plant growth by biofertilizer containing crude coal
powder with optimized media were showed enhancement in uptake of NO3, NH4, H2PO4,
K, P and improved water status of plants, nitrate reductase activity and antifungal compounds
whenever compare with control. For one litter of bacterial culture 5 kg of carrier material is
required for biofertilizer preparation.

Fig 1: Rhizobium spp. Fig 2: Azotobacter spp.

Table 1: Biochemical Characteristics of Azotobacter spp.

Character tics Observations
Shape Oval
Texture Mucoid
Surface Flat
Gram`s reaction Negative
Motility Motile
Indole production +
Methyl red +
Voges prokauer -
Citrate utilization +
Urease +
Starch hydrolysis +
Sugar fermentation glucose +
Mannitol +

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1522
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

Table 2: Biochemical Characteristics of Rhizobium spp.

Character tics Observations
Shape Rod
Texture ---
Surface ---
Gram`s reaction Negative
Motility Motile
Indole production +
Methyl red +
Voges proskauer +
Citrate utilization -
Urease +
Starch hydrolysis +
Sugar fermentation Glucose +
Mannitol +

Table 3: Effect of liquid Azotobacter inoculant on chickpea from day 1 to 15 of plant

growth.
AZOTOBACTOR
DAY (GROWTH IN CM)
TEST
1 2 3 4 5 6 7 10 15 30
K1 (CONTROL) 0.0 0.7 1.7 2.7 4.1 7.8 8.5 12.3 19.3 29.8
K2 (20%) 0.0 4.2 8.1 10.6 12.2 18.3 21.3 24.7 31.2 48.7
K3 (40%) 0.0 3.7 6.5 8.0 11.2 16.8 19.7 22.8 29.4 40.5
K4 (70%) 0.0 2.5 4.5 6.4 8.3 13.2 15.4 19.5 26.1 38.6
K5(100%) 0.0 0.7 2.5 3.5 5.7 9.3 11.8 15.3 23.4 35.4

Fig. 3: Effect of liquid Azotobacter inoculant on chickpea from day 1 to 15 of plant

growth.

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1523
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

Table 4: Effect of liquid Rhizobium inoculant on chickpea from day 1 to 15 of plant

growth.
RHIZOBIUM
DAY (GROWTH IN CM )
TEST
1 2 3 4 5 6 7 10 15 30
K1 (CONTROL) 0.0 0.7 1.7 2.7 4.1 7.8 8.5 12.3 19.3 29.8
K6(20%) 0.0 4.0 8.3 11.2 14.5 20.7 23.1 29.3 36.4 49.3
K7(40%) 0.0 2.9 5.3 7.2 8.4 14.6 17.8 22.5 31.8 44.3
K8(70%) 0.0 0.0 0.0 0.1 0.3 2.1 2.8 4.5 11.2 21.3
K9(100%) 0.0 1.5 2.0 3.5 4.6 6.3 7.6 12.3 16.5 27.2

Fig. 4: Effect of liquid Rhizobium inoculant on chickpea from day 1 to 15 of plant

growth.

Table 5: Effect of liquid Azotobacter and Rhizobium inoculant on chickpea from day 1
to 15 of plant growth.
AZOTOBACTOR + RHIZOBIUM
DAY (GROWTH IN CM )
TEST
1 2 3 4 5 6 7 10 15 30
K1 (CONTROL) 0.0 0.7 1.7 2.7 4.1 7.8 8.5 12.3 19.3 29.8
K10(20%) 0.0 3.0 5.5 8.7 10.2 16.9 19.8 26.8 37.6 47.3
K11(40%) 0.0 3.3 5.7 8.7 10.0 15.6 17.2 22.8 31.2 41.3
K12(70%) 0.0 3.5 7.3 10.1 12.2 18.4 20.4 25.3 31.8 39.5
K13(100%) 0.0 2.5 4.2 5.2 6.4 10.9 12.3 15.8 21.6 29.2

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1524
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

Fig. 5: Effect of liquid Azotobacter and Rhizobium inoculant on chickpea from day 1 to
15 of plant growth.

Fig. 6: Effect of Azotobacter inoculant on the growth of chickpea.

Fig. 7: Effect of Rhizobium inoculant on the growth of chickpea.

Fig. 8: Effect of Azotobacter and Rhizobium inoculant on the growth of chickpea.

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1525
Somnath et al. World Journal of Pharmacy and Pharmaceutical Sciences

CONCLUSION
The experimental plants of chickpea (Cicer arietinum) shows that the growth of plant is
better than normal growth and it increase yields. Therefore, use of biofertilizer may be a good
option. This study has demonstrated that productivity can potentially be improved through
the use of appropriate biofertilizer it resulted in the most of inoculated legumes plants colour
found to be greenish with proper growth as compare to control. In further study media
optimization for growth of new improved inoculant may contain Rhizobia and PGP
organisms for design of biofertilizer.

ACKNOWLEDGEMENT
This project work done in the laboratory of MSV Laboratories Pvt. Ltd. Recognized by
DSIR, Ministry of Science and Technology, Government of India.

REFERENCES
1. Gandora V., Gupta R. D., Bhardwaj K. K. R."Abundance of Azotobacter in great soil
groups of North-West Himalayas". Journal of the Indian Society of Soil Science, 1998;
46(3): 379–383.
2. Martyniuk S., Martyniuk M. "Occurrence of Azotobacter Spp. in Some Polish Soils".
Polish Journal of Environmental Studies, 2003; 12(3): 371–374.
3. Chen M., Alexander M. “Survival of soil bacteria during prolonged desiccation”. Soil
Biol. Biochem, 1973; 5: 213-221.
4. Moffett M.L., Giles JE., Wood BA. “Survival of Pseudomonas solanacearum biovars 2
and 3 in soil: effect of moisture and soil type”. Soil Biol. Biochem, 1983; 15: 587-591.
5. Stephens J. H.G., Rask H. M. “Inoculant production and formulation”. Fields Crops Res.,
2000; 65: 249-258.
6. Aneja K.R. fourth edition Experiments in “Microbiology Plant Pathology and
Biotechnology”. New age international (P) Ltd, publishers Hyderabad.
7. Gupta A.K. The complete technology book on biofertilizers and organic farming.
National Institute of Industrial Research Press. India, 2004.
8. Thomas J. Burr, Anthony Caesar &M. N. Schrolh. Beneficial plant bacteria, 02 Dec 2008;
1-20.

www.wjpps.com │ Vol 11, Issue 5, 2022. │ ISO 9001:2015 Certified Journal │ 1526