Role of PGPR and Chemical Fertilizers on
Oil Yield and Biodiesel Production of Canola
(Brassica napus L.)
Asia Nosheen, Asghari Bano and Faizanullah
Dept. of Plant Sciences
Quaid-i-Azam University, Islamabad
Introduction
Due to wide spread exhaustion of the world
petroleum reserves and increased environmental
concerns have stimulated recent interest in
alternative resources for petroleum based fuels.
In such a situation, biodiesel has arisen as a
potential candidate to substitute petroleum
based diesel (Mehar et al., 2006). It does not
contribute to increase the level of carbon dioxide
in the atmosphere and consequently, to the
Green House Effect.
• In
recent years, biofertilizers have
emerged as a promising component of
integrating nutrient supply system in
agriculture.
•Microbial fertilizers are an important
part of environment friendly sustainable
agricultural practices (Ramazan 1999).
•Biofertilizer mainly include the nitrogen
fixing, phosphate solubilizing and plant
growth promoting microorganisms (Goel
1999).
•Biofertlizers are supplemented with
chemical fertilizers to minimize the
pollution problems and to cut down the cost
of chemical fertilizers.
•The efficiency of nitrogen fertilizer can be
improved by the application of plant growth
promoting bacteria (Choudhury 2004).
Aims and Ojectives
The present investigation was aimed to
compare the effects of Azospirillum, Azotobacter
and chemical fertilizers on seed yield and oil
quality of canola (Brassica campestris L.)
pertaining to biodiesel production.
Materials and methods


A field experiment was conducted in the
Department of Plant Sciences, Quaid-e-Azam
University Islamabad. A plot size of 3x3m2 was
used with randomized complete block design
(RCBD) with 4 replica.
Seeds of canola (Brassica napus L.) cv. Pakola,
obtained from National Agricultural Research
Center (NARC), Islamabad, were surface
sterilized prior to sowing with 0.1% mercuric
chloride (HgCl2) and subsequently washed
with sterile water.
Treatments





Azospirillum and Azotobacter were used as a
source of biofertilizers whereas Urea and
DAP were utilized as a source of chemical
fertilizers. The treatments includes
T0 control (without inoculation and without
urea and Diamonium phosphate)
T1 (Azospirillum)
T2 (Chemical fertilizers (urea 160 kg/hec +
Diamonium Phosphate 185 kg/hec) )
T3 (Azotobacter)
•The Azospirillum and Azotobacter were applied as
seed inoculation @106cells/ml.
•First dose of chemical fertilizers was applied at the
time of sowing while other 3 doses were applied at
40 days interval.
•Soil samples collected prior to cultivation and
analyzed for phosphorus content according to
Soltanpur and Workman (1979).
•The Exchangeable K+1 and Mg+2 were extracted
according to Lanyon and Heald (1982), while Mn+2,
Zn+2, Cu+3, Ni, Co+3 and Fe+2 were extracted with a
solution of DTPA (Lindsay and Norvell 1978) and
then determined by means of the atomic absorption
spectrophotometer.

Seed oil content was estimated by NMR (Nuclear
Magnetic Resonance) test (Robertson and
Morrison 1979).

For estimation of seed protein content, 100 mg
seeds were grounded and digested in Kjeldhal
digestion flask. The total seed protein was then
determined following the method as described by
AOAC (1982).

Glucosinolate content of the oil was determined
following the method of Smith et al. (1985).




The oil was extracted in Soxhlet extractor
(AOAC 1960).
The oil acid value was determined
according to AOAC (1997).
The specific gravity was determined by the
method of Pearson (1976) using density
bottle.
For quantification of fatty acids, oil sample
(50-100 mg) was converted into its fatty acid
methyl esters.
• The methyl esters of the fatty acids (0.5 µl)
were analyzed in a gas chromatograph
(Shimadzu QP 5050) equipped with a flame
ionizing detector (FID) and a fused silica
capillary column (MN FFAP (50 m x 0.32 mm
i.d.; film thickness 0.25 µm).
•Helium was utilized as carrier gas. The
column temperature was kept at 110 °C for
0.5 min, heated to 200 °C at 10 °C/min, and
then maintained for 10 min.
•The temperatures of the injector and detector
were set at 220 °C and 250 °C respectively.
Biodiesel Preparation
The Biodiesel was prepared via base
catalyzed transesterification (Freedman et
al. 1986) and biodiesel yield was determined
as % conversion of vegetable oil to biodiesel
(w/w) (Rashid and Anwar 2008).
Statistical analysis
The data was analyzed statistically by
Analysis of Variance (Steel and Torrie 1980)
technique and comparison among mean
values of treatments was made by Duncan’s
Multiple Range Test (DMRT) (Duncan
1955).
Table 1 Macro and micronutrients (µg/g) analysis
of soil used for cultivation of Canola crop
Ca2+
K1+
Mg2+
Na1+ Cu3
Fe2+ Mn+2
+
23
11.6 0.53
5.3
0.08 0.64 6.2
Ni+2 Co+3 Zn+2 Cr+3 P
0.05
0.15
0.59
0.02
189
Results
8
a
ab
No.of Branches/Plant
7
6
5
b
c
4
3
2
1
te
r
P
zo
to
ba
c
A
D
+
a
A
re
U
zo
sp
ir
ill
um
A
C
on
tr
o
l
0
Tr e atm e nts
Fig.1. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Number of Branches/Plant
No.of Siliqua/Branch
60
a
50
b
b
40
b
30
20
10
A
zo
to
ba
c
te
r
P
A
D
+
a
re
U
zo
sp
ir
ill
um
A
C
on
tr
o
l
0
Treatments
Fig.2. Effect of Azospirillum, Azotobacter and chemical
fertilizers on Number of Siliqua/Branch
35
a
No.of Seed/Siliqua
30
b
25
b
b
20
15
10
5
te
r
P
to
ba
c
A
zo
+
re
a
U
A
zo
C
sp
iri
lu
D
A
m
on
tro
l
0
Tre atm e nts
Fig.3. Effect of Azospirillum, Azotobacter and chemical
fertilizers on Number of Seed/Siliqua
1400
a
Seed Yield (Kg/ha)
1200
1000
800
b
600
b
b
400
200
ac
te
r
A
P
A
zo
to
b
D
+
re
a
U
A
zo
s
C
pi
ril
on
tro
lu
m
l
0
Tre atm e nts
Fig.4. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Seed yield (Kg/ha)
3.1
a
3.05
a
1000 Seed Weight (g)
3
2.95
2.9
ab
2.85
2.8
b
2.75
2.7
2.65
2.6
Control
Azospirillum Urea + DAP Azotobacter
Tre atm e nts
Fig.5. Effect of Azospirillum, Azotobacter and
chemical fertilizers on 1000 Seed Weight (g)
49
a
Seed oil contents (%)
48
47
b
46
45
c
c
44
43
42
Control
Azospirillum
Tre atm e nts
Urea + DAP Azotobacter
Fig.6. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Seed oil contents (%)
3
a
a
Acid value
2.5
2
b
b
1.5
1
0.5
+
re
a
U
A
zo
to
ba
ct
er
P
D
A
lu
m
iri
l
A
zo
sp
C
on
tr
ol
0
Tre atme nts
Fig.7. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Oil Acid value (mgKOH/g)
1.4
a
a
Free Fatty Acids
1.2
b
1
b
0.8
0.6
0.4
0.2
+
re
a
U
A
zo
to
ba
ct
er
P
D
A
lu
m
iri
l
A
zo
sp
C
on
tr
ol
0
Tre atm e nts
Fig.8. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Free Fatty Acid (%)
0.912
Specific Gravity (g/cm3)
a
a
0.91
a
0.908
0.906
0.904
b
0.902
0.9
0.898
te
r
zo
to
b
A
Treatments
ac
A
P
U
re
a
+D
llu
zo
sp
iri
A
co
n
tr
ol
m
0.896
Fig.9. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Oil Specific gravity (g/cm3)
42
a
41
a
Glucosinolate contents
40
a
39
38
37
36
b
35
34
33
32
Control
Azospirillum Urea + DAP Azotobacter
Tre atm e nts
Fig.10. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Seed Glucosinolate content
Moisture contents
6
a
b
b
c
5
4
3
2
1
zo
to
ba
ct
er
A
U
re
a
+
D
A
P
llu
m
zo
sp
iri
A
C
on
tro
l
0
Treatments
Fig.11. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Seed Moisture content (%)
23
22.5
Protein contents
a
a
22
21.5
21
b
20.5
b
20
19.5
+
re
a
U
A
zo
to
ba
ct
er
P
D
A
lu
m
iri
l
A
zo
sp
C
on
tr
ol
19
Tre atm e nts
Fig.12. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Seed Protein content (%)
57.5
a
Oleic Acid (C18:1)
57
56.5
a
56
55.5
b
55
54.5
b
54
53.5
53
+
re
a
U
A
zo
to
ba
ct
er
P
D
A
lu
m
iri
l
A
zo
sp
C
on
tr
ol
52.5
Tre atm e nts
Fig.13. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Oleic Acid (%)
Linolenic Acid (C18:3)
12
b
c
a
d
10
8
6
4
2
zo
to
ba
ct
er
A
U
re
a
+
D
A
P
llu
m
zo
sp
iri
A
C
on
tro
l
0
Treatments
Fig.14. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Linolenic Acid (%)
Erucic Acid (C22:1)
30
a
29
a
28
27
b
26
c
25
24
te
r
P
to
ba
c
D
A
A
zo
+
re
a
U
A
zo
C
sp
i ri
lu
on
tro
m
l
23
Treatments
Fig.15. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Erucic Acid (%)
Biodiesel Yield (% w/w)
94
a
93
a
92
91
b
90
b
89
88
Treatments
zo
to
ba
ct
er
A
P
A
re
a+
D
U
zo
sp
iri
l
A
co
nt
ro
l
lu
m
87
Fig.16. Effect of Azospirillum, Azotobacter and
chemical fertilizers on Biodiesel Yield (% w/w)
Significant Findings




The application of Biofertilizers influenced the
growth and yield of canola.
Biofertilizers improved the seed proteins which
will lead to the production of good quality oil
seed cake as food for cattle
Biofertilizers decreased the glucosinolate
content of canola, glucosinolate content contain
sulpher that reduces the quality of biodiesel and
cause environmental pollution.
Biofertilizers decreased the oil acid value and
free fatty acid content.
Cont……

Biofertilizers enhanced the oil oleic acid
content which leads to the production of
good quality biodiesel with better cold flow
properties.

Biofertilizers improved the biodiesel yield in
transesterification reaction.
Conclusion
It is inferred from the present investigation that
Azospirillum and Azotobacter could be highly
effective in improving yield and oil quality of
canola for biodiesel production and could be
helpful in reducing the cost of chemical fertilizers
as biofertilizers have been reported to replace
50% of chemical fertilizers.
FUTURE PERSPECTIVES



Determination of the mechanism of PGPR
on oil yield and quality of canola at
molecular level.
Determination of the impact of PGPR on
the activities of enzymes like desaturase and
lipase.
Extension of this work to non-edible oil
yielding plants



Extension of this work to farmers level and
determination of the possible role of PGPR
on growth and yield of oil yielding crops
under biotic and abiotic stresses and soil
fertility.
Further investigation for the impact of
PGPR on soil nutrients status
Investigations on the impact of biofertilizers
in combination with chemical fertilizers on
biodiesel yield of oil seed crops.
Acknowledgement

This work was a part of the Research
Project funded by HEC and authors are
highly grateful for financial support
extended to accomplish this piece of work.
Thanks
for
Attention