ISOLATION, IDENTIFICATION AND SCREENING OF ENDOPHYTIC NITROGEN FIXING BACTERIA FROM SUGARCANE AND SELECTION OF EFFICIENT STRAINS FOR THEIR MASS PRODUCTION AS LIQUID STATE BIOINOCULANT WITH FORMULATIONS BY FERMENTATION BASED BIOTECHNOLOGY. Project Report Submitted to the Jai Dhaneshwari Education Society College OF Agriculture Biotechnology Raipur-492006 (C.G) INDIA. By Laxman A. Savalkar Roll No. 4537 ID No. UG/BIO/03/18A-37
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Agrobacterium diazotrophicus as a Liquid biofertilizer.
ISOLATION, IDENTIFICATION AND SCREENING OF ENDOPHYTIC NITROGEN FIXING BACTERIA FROM SUGARCANE AND SELECTION OF EFFICIENT STRAINS FOR THEIR MASS PRODUCTION AS LIQUID STATE BIOINOCULANT WITH FORMULATIONS BY FERMENTATION BASED BIOTECHNOLOGY.
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ISOLATION, IDENTIFICATION AND SCREENING OF ENDOPHYTIC NITROGEN FIXING BACTERIA FROM SUGARCANE AND SELECTION OF EFFICIENT STRAINS FOR THEIR MASS PRODUCTION AS LIQUID STATE BIOINOCULANT WITH FORMULATIONS BY FERMENTATION BASED BIOTECHNOLOGY.
Project Report
Submitted to the
Jai Dhaneshwari Education SocietyCollege OF Agriculture Biotechnology
Raipur-492006 (C.G) INDIA.
By
Laxman A. Savalkar
Roll No. 4537 ID No. UG/BIO/03/18A-37
JUNE 2007
CERTIFICATE
This is to certify that Project entitled “ Isolation, Identification and Screening
of Endophytic nitrogen fixing bacteria from sugarcane and selection of
efficient strains for their mass production as liquid state bioinoculant with
formulations by Fermentation based biotechnology.” Submitted by Mr.
Laxman A. Savalkar to the Jai Dhaneshwari Education Society, Collage of
Agricultural Biotechnology, Raipur ( C.G.) has been approved by the students
advisory committee after on oral examination in collaboration with the external
examiner.
Major Advisor: Mrs. Chaitali Niratker
Members of advisory committee
1. Mrs. Archana Prasad
2. Mrs. Kiran Kumari
“Dedicated to
My beloved
ParentsWho gave me a
solid foundation in life.”
Acknowledgement
I take this golden opportunity to express heartfelt and deepest sense of gratitude to those who have helped me to complete this thesis. My debts to many individuals can warmly be acknowledged but never fully recompensed. Any project report is the culmination of any course of study for undergraduate degree. As such it forms the crown piece; the crown piece of my B.Sc. (Ag) Biotechnology degree would take its shape due to the assiduous efforts of my major advisor, Mrs. R. R. More, Scientific officer, Plant pathology and Agril. Microbiology,& Mr. D. B. Phonde Scientist, soil Science Department, Vasantdada Sugar Institute(VSI), Manjari(Bk), Pune, Completion of my thesis is the result of his cooperative labour and intellect of honorable guidance.
Most humbly and respectfully I wish to express my profound sense of gratitude to Dr.A.S. Patil, Directorate Of Research, V.S.I. Pune.
I wish to express my profound sense of gratitude to Mrs. Chaitali Niratker, for her guidance, patience and encouragement towards my work and in my studies.
I am indebted to all my teachers for having shared their wisdom, especially Mr. Chavan, Mrs. Archana Prasad, Miss. Aditi Sharma, Mr. Suhas kadam, Mr. Niraj kumar, Mr.Rupesh Deshmuk, Mr. Krishna, Mr. Amit Deokar and other staff members of the College of Ag-Biotechnology for their kind help and co-operation during my study period. Working under a single roof, it was a good company of Mr.V.C.Vasekar. Scientist, P.R.surve. Senier boiler, S.D. Ghule assistant of plant pathology & Agril. Microbiology laboratory, who have always helped me and made me enthusiastic to come up with good results in one way or the other. It will be a sin if I forget love affection and cooperation of my beloved one Miss. Sayali Pungaonkar who care, support, guided whenever me needed. I take this opportunity to offer my emotional thanks in works to Mrs. Reshu and Mr. Alok Shrivastava, Miss Ashu, Mr.
Alok Verma, Mr. Sanjay Dvivedi, Mr. & Mrs. Mishra, Shanu & Golu for their encouraging words and their cooperation throughout my work.
It is indeed a great pleasure to acknowledge the love, affection, cooperation and inspiration rendered by my batch mates and friends Vishvajeet, Pravin, Anurag, Amol, Harish, Ram, Sarjerao, Vikrant, Vivek, Vinod, Ankur, Ajay, Tripti, Mohan, Pavan2, Ashish, for their continued affection and unending encouragement during the course of this research work.
Diction is not enough to express my gratitude to my beloved parents Shri. Ashok Savalkar and Smt. Sagarbai A. Savalkar and brother Lakhan Savalkar. Whose selfless love, constant encouragement, obstinate sacrifices, sincere prayers, expectations and blessings has always been the most vital source of inspiration and motivation in my life. I am highly indebted to my beloved Parents whose affection has been the source of inspiration and encouragement throughout my career.
I would like to thank all those who helped me directly or indirectly to fulfill this huge task.
Indeed the words at my command are not adequate, either in form of spirit, to express the depth of my humility and humbleness before Almighty God without whose endless benevolence and blessings this tedious task could not have been accomplished.
Place:
Date: Laxman Savalkar
Content
ChapterNo
Topics Page
No. 01 Introduction
02 Review of literature
03 Material and Method
04 Result & Discussion
05 Summery & Conclusion
Abstract
Bibliography
List of Figures/Graphs
Figure No.
Particulars Page No.
4.1 Isolated Strain Of Agrobacterium
Diazptrophicus
4.2 Isolated Strain Of Azosperrillum
4.3 Isolated Strain Of Azoarcus
4.4 Pure Strain of Endophytes Given by
Institute
45 Dilution scheme for reducing sugar by
DNSA method.
4.6 Dilution scheme for sucrose by phenol
sulphuric acid method.
4.7 Dilution scheme for protein by Folin-
Lowery method.
List of tables
Table Particulars Page No.
No.
3.1 Dilution scheme for reducing sugar by
DNSA method.
3.2 Dilution scheme for sucrose by phenol
sulphuric acid method.
3.3 Dilution scheme for protein by Folin-
Lowery method.
4.3 Biochemical characteristics of
endophytes
4.4 Utilization of different carbon source by
endophytes.
4.5 Screening of Endophytes for N2 fixation
in vitro.
4.6 Temperature range for growth of
Endophytic bacteria.
4.7 pH range for growth of Endophytic
bacteria.
4.8 Response of Endophytes to various
sucrose concentrations.
4.9 Dilution scheme for reducing sugar by
DNSA method.
4.10 Dilution scheme for sucrose by phenol
sulphuric acid method.
4.11 Dilution scheme for protein by Folin-
Lowery method.
4.12 Chemical analysis
4.13 Microbial analysis
ABREVATIONS
Agr. - Agro bacterium Diazotrophicus
Azr. - Azoarcus
Act - Acetobacter Diazotrophicus.
i.e. – That is.
ha-1. - Per hectare
BNF - Biological Nitrogen Fixation.
D/W - Distilled Water
BTB - Bromothymol Blue Indicator
Fig. - Figure
Chapter 1
Introduction
Introduction
Endophytic bacterial Nitrogen fixing liquid Bioinoculant is a
unique agro-based product in liquid state, formulated with
growth boosters and cell protectants and it is a consortium of
group of efficient Endophytic Nitrogen fixing bacteria in live
form. Endophytic bacterial Nitrogen fixing Bioinoculant is
special product with newly developed A4H medium with high
cell count, zero contamination, longer shelf life, greater
protection against environment stresses, increased field
efficiency with respect to spreading and penetration and
convenience of handling are main features of the this product.
(Baldani et al., 1978 ).
Endophytic bacteria are those bacteria that fix nitrogen
internally in plant tissues; mostly they are present in the
apoplast i.e. intercellular spaces and xylem vessels. Hence they
are called as endophytic bacteria. Endophytic bacteria such as
Acetobacter, Azoarcus, Herbaspirillum, Azosperrillum, and
Agrobacterium are present in all parts of sugarcane plant
including leaf, stem, roots and juice. These endophytic bacteria
actively participate in biological nitrogen fixation and fix more
nitrogen as compare to ectophytic bacteria. (Dobereiner, J.
1998).
“Biofertilizers” are products consisting of selected, efficient
and beneficial live or latent (resting stage- spores)
microorganisms, which help to improve plant growth and
productivity mainly through supply of plant nutrients.
Biofertilizers are also known as microbial inoculants or bio-
inoculants. Biofertilizers have been introduced in Indian
agriculture since last three decades in view of their cost
effectiveness, contribution to crop productivity, soil
sustainability, and eco-friendly characteristics. Biofertilizers
form an integral part of integrated plant nutrient supply system
(IPNS or INM) and organic farming which constitutes the present
as well as the future mandate of Indian agriculture. (Bellone,et
al., 1989)
Nitrogen is the most essential nutrient required in fairly
large amount for increased productivity of sugarcane and other
cereal crops. It is universal fact that atmosphere is highly rich in
nitrogen (78% N) but without an aid of microorganisms not a
single molecule of atmospheric nitrogen can be utilized by the
plant can utilize the plant. Biological nitrogen fixation (BNF)
is a process either carried out symbiotically or non-symbiotically
by ectophytic and endophytic bacteria which converts
atmospheric nitrogen into “ammonia” and further converted
into “nitrate” readily available form of nitrogen through agency
of nitrifying bacteria or taken up by the plants for their growth
and development. Unless atmospheric nitrogen is fixed it is not
available to plant. So, Biological Nitrogen Fixation through
agency of microbes plays important role in agriculture from
economic point of view. Use of BNF bacteria along with organic
matter and reducing dose of inorganic fertilizer is best source
for maintaining soil fertility as well as achieving the potential
crop yield. . (Bellone,et al., 1989).
Introduction of Endophytic nitrogen fixing
bacterial Bioinoculant in Indian agriculture for monocotyledons
will completely change the concept of symbiotic nitrogen
fixation restricted to dicotylydons like legumes with root nodule
formation. These five major groups of nitrogen-fixing bacteria
and their interaction with the host plants are compared and
many scientists have reported the potential of their use in
agriculture. Hence mass production of these endophytic
nitrogen-fixing bacteria as liquid bioinoculants will be a mile
stone in field of Agriculture with respect to biological nitrogen
fixation and will be a road map for organic farming for all crops.
(More et al., 2007)
The proposed investigation was carried out with following
objectives:
1) Isolation, Identification and screening of efficient strains of
Endophytes liquid Bioinoculant production and for Biological
Nitrogen Fixation.
2) Formulation of liquid endophytic Bioinoculant with cell protects
ants.
3) Efficiency test for Liquid Bioinoculant.
4) Growth and Chemical analysis.
Review of Literature
Chapter 2
REVIEW OF LITERATURE:
Biological Nitrogen Fixation (BNF) is a vital component of
agricultural sustainability. ‘Sustainability’ is defined as ‘
successful management of resources for agriculture to satisfy
human needs while maintaining or enhancing the quality of the
environment and conserving resources’ (TAC, CGIAR, 1988).
Economists measure sustainability as the ratio of output to
input taking into account stock depletion. Stocks in agriculture
include soil, water, non-renewable energy resources and
environmental quality.
Modern agriculture is based on maximum output in the
short term with inadequate concern for input efficiency or stock
maintenance (Odum, 1989). Nitrogen fertilizer ranks first
among the external inputs to maximize output in agriculture.
Input efficiency of Nitrogen fertilizer is one of the lowest among
the plant nutrients and in turn contributes sustainability to
environment pollution. The continued and unabated use of N
fertilizer would further accelerate depletion of stocks of non-
renewable energy resources used in fertilizer production. The
removal of large quantities of crop produce from the land
additionally depletes soil of its native N reserves. On the other
hand, BNF, a microbiological process in the biosphere, converts
atmospheric dinitrogen into a plant usable form through the
microbial enzyme nitrogenase.
This chapter gives the comprehensive review of literature
of the project work & review is summarized under following
headings.
2.1 N2 fixing Endophytic bacteria.
2.1.1 Acetobacter Diazotrophicus
2.1.2 Agrobacterium Diazotrophicus
2.1.3 Azoarcus
2.1.4 Azospirrillum
2.1.5 Herbaspirrillum
2.2 Importance of N2 fixing endophytic bacteria.
2.3 Endophytes as a Biofertilizer.
2.1 N2 fixing Endophytic bacteria.
Nitrogen input through BNF can help maintain soil
N reserves as well as substitute for N fertilizer to attain large
crop yields. (Peoples and Croswell, 1992). Biological Nitrogen
Fixation (BNF) can therefore be a major source of N in
agriculture when symbiotic N fixing systems are used. The
amount of N input reported to be as high as 360 kg N ha-1. On
the other hand, a contribution for non-symbiotic (associative
and free-living) N2-fixation in upland agriculture is generally not
Substantial, although N2-fixation to the order of 160kg N ha-1
has been reported for sugarcane (Ladha et al., 1992). Brazilian
cultivars of sugarcane rarely respond to N fertilizer applications
during the plant crop. Among 135 NPK fertilizer trials all over
the country, only 19% showed significant increase in cane yield
due to N application. This indicates that some endophytic
bacteria may contribute for Biological Nitrogen fixation. Initially
reported by Azeredo et al., (1986). Tremendous progress in
BNF has been made during the last more than 30 years and yet
we are still hoping for breakthrough during the years to come
and as such abundant literature is already available especially
on the BNF except BNF in sugarcane associations and therefore
the review of literature is especially is confined to the
rhizospheric associative diazotrophs in sugarcane. Johanna
Döbereiner initiated research on BNF with grasses in Brazil
when she joined the research team at the National Center of
Education and Agricultural Research of the Ministry of
Agriculture, in the fifties. The first studies were Dedicated to the
memory of Dr. Johanna Döbereiner by two of her disciples who
learned through working with her that research could be done
with simplicity, perseverance, honesty, ethics and sagacity.
2.1.1 Acetobacter:
Acetobacter is Gram negative, Micro aerophilic
bacteria motile with 1-3 lateral flagella present in high number
in roots and stems showing optimum growth with 19% sugar
and pH around 5.5 precisely this condition prevailing in
sugarcane. First isolation of Acetobacter diazotrophicus strains
from roots and stems of sugarcane and classified them under
the genera, viz., Gluconobacter and Acetobacter (De Ley et al.,
1984) or to the genus Frateuria (Swings et al., 1984) on
observing that this organism has capacity to grow at low pH
values and their ability to form acetic acid from ethanol by De
Ley and Swings (1984) from Brazil. A new N2 fixing bacterium
Acetobacter diazotrophicus found in high numbers in roots and
stem of Sugarcane samples from all over the Brazil and also in
Australia and Mexico,It was reported by Cavalcante and
Dobereiner, 1988 and Gills et al. Dobereiner et al., (1988) and
Paula et al., (1989) observed that these bacteria were not
however found in soil between rows of sugarcane plants or
roots of 12 different weed species, which grew in sugarcane
fields. It was also not found in grain of sugar sorghum but was
isolated from few samples of washed roots and aerial parts of
Pennisetum purpureum CV Cameroon and from sweet potatoes.
Gillis et al., (1988, 1989) reported that this nitrogen fixing
bacterium that seems to be specific for sugarcane (Dobereiner
et al., 1988) has been identified through DNA-rRNA
hybridizations and DNA-DNA homology values, as a new species
of Acetobacter diazotrophicus. Reis et al., (1988) also observed
that A. diazotrophicus occur only in plants propagated
vegetatively but not in plants grown from seeds. Dobereiner et
al., (1988) reported the endophytic occurrences of Acetobacter
diazotrophicus in sugarcane, sweet potatoes and Cameroon
grass; all plants that are propagated vegetatively and that
contain high sugar concentrations which was later confirmed by
Li and Mac Rae (1992). Boddey et al., (1991) from the above
observations concluded that this Acetobacter diazotrophicus
must be considered as an endophytic in nature, which
propagated within stem cuttings. Mahesh Kumar-KS; Krishnaraj
Dharwad, India (1999) carried out mineral phosphate
solubilizing activity of Acetobacter diazotrophicus a bacterium
associated with sugarcane, Li and Mac Rae (1992) reported the
presence of A. diazotrophicus in soil samples collected between
cane rows. This was later confirmed by Reis et al., (1993). Paula
et al., (1992); Reis and Dobereiner (1991) reported the
presence of A. diazotrophicus in stems, tubers and roots of
sweet potato collected from various regions in Brazil.Dobereiner
et al., (1993) further reviewed the work on endophytic
diazotroph in sugarcane, cereals and tuber plants.
2.1.2 Agrobacterium Diazotrophicus:
Agrobacterium diazotrophicus are rod in shape &
motile by 1-6 peritrichous flagella. They are gram negative,
microaerophillic, showing optimum growth with 10-20% sugar,
pH around 5.5-6.0, temperature 250C & highly obtained from
internode region of a sugarcane plant. They cause proliferation
in many plants. Study on influence of nitrogen fertilization on
the population of diazotrophic bacteria A. diazotrophicus in
sugar cane (Saccharum spp.) by Reis-Junior-FB-dos; Reis-VM;
Urquiaga-S; Dobereiner-Brazil (2000). Stephen et al., (1991)
studied the physiology and dinitrogen fixation of Acetobacter
diazotrophicus. Reis et al.,(1994) have therefore reexamined
several alternatives and gave the most successful methods and
some results on the specific occurrence of this diazotroph in
By the help of colony counter, the viable count of an isolates were calculated, & these can be compared with before data. i.e., Growth rate of Nitrogen Fixation
COMPOSITION OF MEDIA
1. Azosperillum medium (for 1 liter.) (pH- 6.6-7)
(For Azosperillum)
Malic acid - 5gm
K2HPO4 -4gm
FeSo4 x 7 H20 – 0.05gm
Na2 Mo04 x 2 H20 – 0.002gm
MnSo4 x H20 - 0.01gm
MgS04 x 7 H20 -0.10gm
Nacl - o.o2gm
CaCl2 x 2 H20 - 0.01gm
Distil water - 1000ml
Agar-Agar - 30gm
2. Azoarcus medium (for 1 liter)(pH- 6.6-7)
(For Azoarcus)
Malic acid - 2-5gm
KOH - 2-5gm
KH2PO4 - 1.5gm
MgS04 x 7 H20 -1gm
Nacl -1gm.
Sodium Molybdate – 2mg.
CaCl2 -1gm
MnSo4 x H20 - 10mg
Fe EDTA - 66mg
Biotin - 1 mg
NH4Cl - 2mg
Beef Extract - 3gm
Yeast extract - 1gm
Agar-Agar - 15gm
Distil water - 1000ml
3. Agrobacterium Diazotrophicus medium (for 1 liter) (pH-5.5-
6.0) For (Agrobacterium Diazotrophicus)
Sucrose - 100gm
Nacl - 0.2 gm
MgS04 - 0.02 gm
CaCo3 - 1gm
Na MoO4 - 0.005gm
Agar - 15gm
4. LGI Medium (for 1 liter) (pH-5.5-6.0)
for Acetobacter Diazotrophicus.
Cane Sugar – 100 gm
KH2PO4 - 0.6 gm
K2HpO4 - 0.2 gm
MgS04 - 0.02 gm
Sodium molybdate – 0.002gm
Ferric Chloride - 0.01gm
CaCl2 - O.O2 gm
BTB - 5ml
Yeast Extract - 0.5 gm
Agar agar - 30gm
D/W - 1000ml.
5. Herbasperrillum medium (for 1 liter) (pH-7)
For HerbasperrillumKH2PO4 - 0.400 gm
K2HpO4 - 0.100 gm
MgS04 X 7 H20 - 0. 200 gm
Nacl - 0.100gm
Cacl2 - 0.020 gm
Fecl2 X 6 H20 - 0.010 gm
Sodium Molybdate - 0.002 gm
Yeast Extract - 0.025gm
D/W - 950 ml.
Agar Agar - 15 gm
Autoclave at 1200c for 15 min after sterilization add filter
sterilized solution A
Solution A = Sodium Malate 5.0 gm
Water 50ml (pH 7.0)
Chapter 4
RESULT AND DISCUSSION
Endophytic bacteria are those bacteria that fix nitrogen
internally in plant tissues; Endophytic bacterial Nitrogen fixing
liquid Bioinoculant is a unique agro-based product in liquid
state, formulated with growth boosters and cell protectants and
it is a consortium of group of efficient Endophytic Nitrogen
fixing bacteria in live form.
This chapter gives the result and discussion of project work
under following headings.
Results&
Discussion
4.1.Result
4.1.1 Isolation:
The three different isolates of an endophytes were obtained
the help of respective selective media. Shows in Fig 4.1, 4.2 and
4.3
4.1.2 Identification and Characterization: - It was carried
out by morphological studies - Colony Characteristics and
microscopic studies.
The isolates are identified with Bergyess Manual.
4.1.2.1 Colony Characteristics of Endophytes: -
Table 4.1: Colony Characteristics of different isolates of Endophytes (Azospirillum, Agrobacterium diazotrophicus, Azoarcus) grown respective solid media at 30° c for 120 hrs
M.Org/ Medium with temp. & time
Size (mm)
Shape Colour Margin Elevation
Consistency
Opacity
Azosprillium 300C for 120 hrs
0.4 Circular Greenish with white
metallic shine
Entire Convex Smooth Opaque
Azoarcus 300C for 120 hrs
0.3 Circular Insipid(Creamish white)
Entire Flat Smooth Opaque
Agrobacter diazotrophicus 300C for 120 hrs
1.0-2.0
Circular Dull white Entire Flat Moist Opaque
4.1.2 Microscopic Observations: -
Microscopic observation showed that the Endophytic bacteria
are Gram negative, short rods, motile with 2–3 lateral flagella.
Table 4.2 – Staining and Motility test of different isolates
Graph. 4.5. Standard Graph for Reducing Sugar Estimation:
Graph 4.6 Standard Graph for Sucrose Estimation:
Graph 4.7 Standard Graph For Protein Estimation:
Table 4.13 Chemical Analysis:
Observations
Reducing Sugar content
Sucrose content
Protein content
Acidity (pH)
(g/100 ml)
(OD at 550n)
(g/100ml)
(OD at 550nm)
(g/100ml)
(OD at 550nm)
Initial A4H BrothAfter sterilization
2.5 x104
1.0345
9.6 x104
3.730 9.6 x 103
0.252 6.5
A4H culture + G. booster/cell protectant
8.8 x104
3.600 4.6 x104
3.008 1.1 x 104
0.290 7.00
After 7 days of adding cell protectants
4.3 x105
17.590
4.9 x104
3.067 2.0 x104
0.458 5.35
After 15 days of adding cell protectants
5.1 x105
20.75 3.6 x104
2.900 1.4 x104
0.367 4.84
4.4.3. Microbial Analysis:
Microbial analysis of culture prior to addition of cell protectant 2
showed Acetobacter count in range of 53 x 10 –12 at pH 3.65.
After addition of cell protectant 2 after 7 days microbial count
was found to be more than 300 for 10 –12 dilution with decrease
in pH to 4.84 from 7. Further 15 days count in range of 19.2 x
10 –12 with pH 4.65.
After 21 days analysis it was found to be 154 x 10 –12 with pH
4.3-6. Microbial count and pH studies indicates that after
formulation with cell protectant 2 there was sudden increase in
microbial count but gradual decrease after 15 days to 21 days
with respect to pH there was sudden decrease in pH during first
7 days after formulation with cell protectant 2 and further
gradual decrease in pH was observed up to 21 days.
Comparative Analysis Studies of chemical and microbial
parameter shows that initial sucrose content of A4H broth was
reduced during sterilization due to inversion of sucrose resulting
in formation of reducing sugar. Endophytes utilize both during
their growth period of 5 days. Further formulations of
Endophytic culture with cell protectant 2 to pH 7 increase the
sucrose content, which was further utilize by bacteria with
increase in reducing sugar. During this period it has been
observed that both reducing sugar and sucrose was utilized
simultaneously with minute fluctuation in pH. Further it was
observed that cell metabolism is leading to decrease in protein
content in minute quantity.
Microbial count of initial broth (53 x 10-12) has been boosted to
more than 300 x 10-12 it indicates that bacteria are utilizing
sucrose provided by cell protectant 2 and also reducing sugar
during metabolism and showing steady decrease in their count
up to 21 days.
Further studies will be carried out for 6 months of period for
sucrose, reducing sugar, protein, pH and microbial count in
order to estimate this contents and shelf life of product. Prior to
packing of the product depending on the final pH adjustment
will be carried out with some weak bases and antitox after
addition of 10 ml of cell protectant 1 per liter cell protectants.
Table.4.14- Microbial Analysis
Dilution no. TVC of mother culture
TVC of cell protectant added culture After 7 days
TVC of cell protectant added culture After 15 days
10-1 >300 >300 >300
10-2 >300 >300 >300
10-3 >300 >300 >300
10-4 >300 >300 >300
10-5 >300 >300 >300
10-6 245 >300 >300
10-7 232 >300 >300
10-8 192 >300 >300
10-9 178 >300 >300
10-10 135 >300 >300
10-11 96 >300 >300
10-12 53 >300 192
DISCUSSUION
From the observations recorded (Table 3) shows that the
bacteria did not hydrolyze the starch, which is in agreement
worth report presented by Gills et al., (1989) and Bhowmik
(1995) who showed negative response of Endophytes
(Azospirillum, Ag. diazotrophicus, Azoarcus) to hydrolysis of
starch. The investigations show (Table 4.3) that Endophytes
(Azospirillum, Ag. diazotrophicus, Azoarcus) isolates were
catalase positive further it was confirmed by the reports
mentioned by Dobereiner (1988); Stephan et al., (1991); L.E.
Fuentes – Ramirez et al., (1997). The observation recorded
(Table 4.3) shows that Endophytes (Azospirillum, Ag.
diazotrophicus, Azoarcus) is weakly gelatin liquefier. It showed
a zone of clearance. Bhowmik, 1995 and Gillis et al., (1989)
showed that Acetobacter diazotrophicus was unable to liquefy
the gelatin. Calvalcate and Dobereiner (1988) reported that
besides 30% of sucrose, which was proved to be best carbon
source for growth of A.diazotrophicus, they also observed good
response for glucose, fructose, ethanol (1%), mannitol, and
maltose. They also found bet growth at high sucrose or glucose
concentration (10%) and strong acid production led to a final
pH of 3.0 or below. Bhowmik (1995) reported that glucose and
sucrose are best carbon source for growth of A.diazotrophicus.
The optimum temperature was observed at 30˚C (Bhowmik,
1995). There was a report that optimum temperature for
growth of A.diazotrophicus about 30˚C (Cavcalcante and
Dobereiner, 1988 and Gillis et al., 1989).Cavalcant and
Dobereiner (1988) reported that the suitability for the growth
of A.diazotrophicus at pH 4.5. The faster growth was obtained
at more acid i.e. pH 3.9. Stephan et al., (1988) reported that
pH 3.0 or below were suitable for growth and N2 fixation.
Further Stephan et al., (1991) revealed from their studies that
pH range was from 2.5 to 7.5, optimum pH of 5.5. Gillis et al.
(1989) found the excellent growth at pH 5.5 but no growth
occurs at pH 7.0.Therefore, the present investigation for
response to pH was in conformity with the above-mentioned
reports. (Cavalcante and Dobereiner 1988, Gillis et al., 1989;
Stephan et al., 1991) The same can be confirmed from report
of Bhowmik, (1995) that the optimum range of pH was between
5.6 to 6.6.There was report that the best growth occurred at
high sucrose concentration (10%) and even up to 30%
(Cavalcante and Dobereiner, 1988; Boddy et al., 1991).
Bhowmik, (1995) also reported that Nitrogen dependent growth
occurred between 1% to 30% cane sugar concentration with an
optimum between 10 and 15%.
Summary, Conclusion
Chapter v
SUMMARY AND CONCLUSION:
Isolation, Identification & Screening of different endophytic N2
fixing bacteria from sugarcane. Selection of efficient strains of
these endophytic N2 fixing bacteria was carried out for mass
production of endophytic N2 fixing bacterial bioinoculant through
fermentation based biotechnologies, by designing a new
common media and formulation of the same with cell
protectants & cell growth boosters. This unique product of
consortium of efficient endophytic N2 fixing bacteria.
A quality product formulated with Cell Growth Booster & Cell
Protectant with neutral pH, higher shelf life, easy in handling
storage and application with benefit ratio & ideal cost has been
developed. Such type of liquid formulation with CGB & CP will
also increase utilization efficiency of Liquid Bioinoculant by
stem leaves & plantlets, over average standard set or seed
treatment, foliar application & deeping of plantlets.
This endophytic N2 fixing Bioinoculant for different crops having
sucrose, including sugarcane for increasing yield and quality of
crop.
Considering the importance of endophytic bacteria in sugarcane
and other crops, with respect to biological nitrogen fixation,
present studies of isolation, screening and selection of efficient
strain of endophytic bacterial isolates and their mass production
as liquid bioinocolants with fermentation based biotechnology
has been undertaken.
In nonlegumes such as sugarcane from gramenecios family
endophytic diazotrophs such as Acetobacter, Azoarcus,
Herbaspirillum, Agrobacterium diazotrophicus and
Azosperrillum are presents in all parts of plant including leaf,
stem, roots and juice. The recent discovery of the endophytic
diazotrophs bacteria such as Acetobacter diazotrophicus,
Herbaspirillum spp. and Azoarcus spp. colonizing the interior of
sugarcane, rice and Kallar grass (Leptochloa fusca [Diplachne
fusca]), respectively, and other species of grasses as well as
cereals, has led to a considerable interest in exploring these
novel associations. There is a general consensus that plant
genotype is a key factor to higher contributions of BNF together
with the selection of more efficient bacterial strains. Nitrogen-
fixing bacteria are important in modern agriculture - exploiting
these bacteria would decrease the present dependency on
nitrogen fertilizers, which would have positive results for the
ecosystem and the health of humans and other animals.
CONCLUSION:
Endophytic bacterial Nitrogen fixing Bioinoculant is special
product with newly developed A4H medium with high cell
count, zero contamination, longer shelf life, greater
protection against environment stresses, increased field
efficiency with respect to spreading and penetration and
convenience of handling are main features of the this
product.
In sugarcane endophytic diazotrophic bacteria like
Azospirillum, Azorcus, Agrobacterium diazotrophicus are
present in all parts of plant including left, stem, roots and
juice. These endophytic diazotrophs actively participates in
biological nitrogen fixation and fixes more Nitrogen as
compare to ectophytic bacteria.
These bacteria’s were isolated successfully and they were
screened and compared with Bergyess manual.
Biological nitrogen fixing system offers an economically attractive and
ecologically sounds means, of externally reducing external inputs and
improving internal resources. Hence Biological Nitrogen fixation has been
an interesting area of research over several decades.
“Isolation, Identification and Screening of Endophytic NITROGEN FIXING bacteria from sugarcane and selection of efficient strains for their mass production as liquid state Bioinoculant with Formulations by fermentation based biotechnology.”
By Laxman Savalkar
ABSTRACT
Endophytic bacterial Nitrogen fixing Bioinoculant is special product with newly developed A4H medium with high cell count, zero contamination, longer shelf life, greater protection against environment stresses, increased field efficiency with respect to spreading and penetration and convenience of handling are main features of the this product. The proposed investigation was carried out with following objectives: Isolation, Identification and screening of efficient strains of Endophytes liquid Bioinoculant production and for Biological Nitrogen Fixation., Formulation of liquid entophytic Bioinoculant with cell protects ants., Efficiency test for Liquid Bioinoculant.,Growth and Chemical analysis.
Entophytic bacteria were isolated from different parts of various sugarcane Varieties, they are screened, and used for mass production as an liquid biofertiliser. These bacteria fix nitrogen internally on utilizing starch (byproduct of sugarcane and many cereal crops), as well as some strains fixes atmospheric nitrogen also. Formulation of Endophytic liquid Bioinoculant with cell growth booster and cell protectant may result into development of quality product with neutral pH, higher shelf life, ease in handling, storage and application with benefit ratio ideal cost help in application and its utilization by plant. It will increase utilization efficiency of liquid Bioinoculant by stem; leaves and plantlets average standard treatment, foliar application and dipping in plantlets.
Date: Mrs.Chaitali Niratker
(Major Advisor)
Bibliography
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