Beruflich Dokumente
Kultur Dokumente
Project Report
Submitted to the
Jai Dhaneshwari Education Society
College OF Agriculture Biotechnology
Raipur-492006 (C.G) INDIA.
By
Laxman A. Savalkar
JUNE 2007
CERTIFICATE
This is to certify that Project entitled “ Isolation, Identification and Screening
efficient strains for their mass production as liquid state bioinoculant with
examiner.
Place:
Date: Laxman Savalkar
Content
List of Figures/Graphs
Institute
45 Dilution scheme for reducing sugar by
DNSA method.
4.6 Dilution scheme for sucrose by phenol
Lowery method.
List of tables
DNSA method.
3.2 Dilution scheme for sucrose by phenol
Lowery method.
4.3 Biochemical characteristics of
endophytes
endophytes.
4.5 Screening of Endophytes for N2 fixation
in vitro.
4.6 Temperature range for growth of
Endophytic bacteria.
bacteria.
sucrose concentrations.
4.9 Dilution scheme for reducing sugar by
DNSA method.
4.10 Dilution scheme for sucrose by phenol
Lowery method.
4.12 Chemical analysis
Azr. - Azoarcus
Fig. - Figure
Introduction
Chapter 1
Introduction
1998).
al., 1989)
and their interaction with the host plants are compared and
fixation and will be a road map for organic farming for all crops.
objectives:
Nitrogen Fixation.
protects ants.
Chapter 2
REVIEW OF LITERATURE:
environmental quality.
headings.
2.1 N2 fixing Endophytic bacteria.
2.1.3 Azoarcus
2.1.4 Azospirrillum
2.1.5 Herbaspirrillum
during the plant crop. Among 135 NPK fertilizer trials all over
the country, only 19% showed significant increase in cane yield
BNF has been made during the last more than 30 years and yet
2.1.1 Acetobacter:
stem of Sugarcane samples from all over the Brazil and also in
fields. It was also not found in grain of sugar sorghum but was
Li and Mac Rae (1992). Boddey et al., (1991) from the above
cane rows. This was later confirmed by Reis et al., (1993). Paula
2.1.3 Azoarcus:
They were firstly isolated from salt tolerant plant. They are
2.1.4 Azosperrillum:
bacteria, motile with flagella & highly present in roots & leaves
2.1.5 Herbaspirillum:
reported that this crop was able to obtain more than 60% of its
Nitrogen needs from BNF, which was later reexamined by
I.R., and Y.T. Tchan. 1992. Recent advances in BNF with non-
Chapter 3
3.1 Materials:
3.1.2 Microorganism:
3.2 Methods:
three sets.
each set.
3. 10 ml sample from bottle first and transferred it to next
Pour plating was done for the selection for the isolates in respective
media.
dilution’s petriplate.
media.
Colony Characters: -
1) Shape
The culture growth of 48 hours of all eight isolates along with
2) Size
3) Motility
objective.
3.2.2.2. Staining
Procedure:
1. Smear of sample were prepared, air-dried and heat
fixed.
water
reaction.
immersion lens.
1. Hydrolysis of starch
The type strain of Endophytes (Azospirillum,
plates were flooded with weak logust iodine solution after 3 days
2. Catalase test
3. Liquefaction of gelatin
30°C for 3 days. After 2 days, the plates were flooded with 10
ml, solution of HgCl2 in 100 ml, distilled water and 200 ml conc.
colony of endophytes.
KJELDAHL method)
Procedure
30°C at 110 rpm, the contents of flask were checked for purity
bath (50 to 60°C) to dryness. The dried culture was washed and
taken as a sample. The contents of the flask in inoculated
3.2.4. Purification:
in Zigzag manner.
analysis.
3.2.6. Formulation:
concentrations
control.
Mass production was done with three isolates and two pure
Broth:
and the dilution system is given in table 3.1 as the dilutions were
method
acid method and the dilution system is given in table 3.2 as the
Lowry method and the dilution system is given in table 3.3 as the
(For Azosperillum)
K2HPO4 -4gm
Nacl - o.o2gm
Agar-Agar - 30gm
(For Azoarcus)
KOH - 2-5gm
KH2PO4 - 1.5gm
CaCl2 - 1gm
Fe EDTA - 66mg
Biotin - 1 mg
NH4Cl - 2mg
Agar-Agar - 15gm
Sucrose - 100gm
Nacl - 0.2 gm
MgS04 - 0.02 gm
CaCo3 - 1gm
Na MoO4 - 0.005gm
Agar - 15gm
K2HpO4 - 0.2 gm
MgS04 - 0.02 gm
CaCl2 - O.O2 gm
BTB - 5ml
D/W - 1000ml.
For Herbasperrillum
KH2PO4 - 0.400 gm
K2HpO4 - 0.100 gm
Nacl - 0.100gm
Cacl2 - 0.020 gm
Agar Agar - 15 gm
Autoclave at 1200c for 15 min after sterilization add filter
sterilized solution A
Results
&
Discussion
Chapter 4
4.1.Result
4.1.1 Isolation:
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
microscopic studies.
are Gram negative, short rods, motile with 2–3 lateral flagella.
of Endophytes.
Organism Gram’s staining Motility
Azosperrillum Gram Negative Rods Sluggishly
motile
Ag.diazotropicus Gram Negative Rods Sluggishly
motile
Azoarcus Gram Negative Rods Sluggishly
motile
starch.
Azoarcus
-Ve +Ve +Ve
Azoarcus + + + + -
Growth Analysis:
phytes
>
10-1 >30 >30 >300 >30 >30 >300 >30 >30 >300
0 0 0 0 0 0
10 -2 >30 >30 >300 >30 >30 >300 >30 >30 >300
0 0 0 0 0 0
10 -3 >30 >30 >300 >30 >30 >300 >30 >30 >300
0 0 0 0 0 0
10-4 >30 >30 >300 >30 >30 >300 >30 >30 >300
0 0 0 0 0 0
10 -5 >30 >30 >300 >30 >30 >300 >30 >30 >300
0 0 0 0 0 0
10-6 234 265 188 >30 >30 >300 >30 >30 >300
0 0 0 0
10 -7
232 238 154 >30 >30 >300 >30 287 >300
0 0 0
10-8 189 176 100 >30 >30 >300 >30 254 >300
0 0 0
10 -9
176 166 98 >30 >30 >300 >30 232 214
0 0 0
10-10 123 122 76 >30 >30 209 234 212 209
0 0
10 -11
100 98 65 >30 >30 167 212 198 189
0 0
10-12 65 53 34 193 >30 123 178 167 187
0
consumed.
N2 fixed in mg/gm
of sucrose consumed
Sr.No Name of Dry weight basis Liquid weight
. Endophyte 50 ml medium basis
broth 50 ml medium
broth
01. Agr. Diazotrophicus 38.46 11.36
03 Azoarcus 46.66 10
Screening was further carried out for its efficiency of
4.3. Formulations:
with control.
inert oil base cell protectant with pH around 6.8 to 7.0. Hence it
helps to increase pH of culture, which are around 3.5 to 4.5
after growth.
protectant 1 and
4.3.1. Optimization:
Endophytes.
bacteria
Endo. Bacteria
Azospirilum ++ +++ ++ ++ + - -
+ +
Ag.diazotrophicus ++ +++ ++ ++ ++ + ++
+ + + +
Azoarcus ++ +++ ++ ++ + + -
+ +
of microbial population.
concentrations
were taken. (Table 4.7) The observations showed that there was
hampered.
concentrations.
growth
to 10 2
and reducing sugar to 10 4. It suggest that bacteria
1.1 x 10 4
at this stage increase in both sucrose as well as
counts during this stage suggest the same trend. After growth
be decreased to 10 2
. It indicates sterilization denaturates the
DNSA Graph
y = 0.0002x - 0.0219
0.2
Abs at 550 nm
0.15
0.1
0.05
0
100 300 500 700 900 1100
ug Glucose
Abs at 480 nm
0.5
0.4
0.3
0.2
0.1
0
5 25 45 65 85 105
ug conc. in Sucrose
1.2
Abs at 750 nm
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100
ug Conc. in protein
10 –12
with pH 4.65.
up to 21 days.
will be carried out with some weak bases and antitox after
DISCUSSUION
Chapter v
storage and application with benefit ratio & ideal cost has been
crop.
CONCLUSION:
product with newly developed A4H medium with high cell count, zero
all parts of plant including left, stem, roots and juice. These
internal resources. Hence Biological Nitrogen fixation has been an interesting area of
By
Laxman Savalkar
ABSTRACT
(Major Advisor)
Bibliography
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