Role of PGPR and Chemical Fertilizers on Role of PGPR and Chemical Fertilizers on Oil Yield and Biodiesel Production of Canola Oil Yield and Biodiesel Production of Canola

((Brassica napusBrassica napus L.) L.)

Asia Nosheen, Asghari Bano and Faizanullah Dept.

of Plant SciencesQuaid-i-Azam University,

Islamabad

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.

Introduction

• In recent years, biofertilizers have In recent years, biofertilizers have emerged as a promising component of emerged as a promising component of integrating nutrient supply system in integrating nutrient supply system in agriculture. agriculture. •Microbial fertilizers are an important Microbial fertilizers are an important part of environment friendly sustainable part of environment friendly sustainable agricultural practices (Ramazan 1999).agricultural practices (Ramazan 1999).•Biofertilizer mainly include the nitrogen Biofertilizer mainly include the nitrogen fixing, phosphate solubilizing and plant fixing, phosphate solubilizing and plant growth promoting microorganisms (Goel growth promoting microorganisms (Goel 1999).1999).

•Biofertlizers are supplemented with Biofertlizers are supplemented with chemical fertilizers to minimize the chemical fertilizers to minimize the pollution problems and to cut down the cost pollution problems and to cut down the cost of chemical fertilizers. of chemical fertilizers.

•The efficiency of nitrogen fertilizer can be The efficiency of nitrogen fertilizer can be improved by the application of plant growth improved by the application of plant growth promoting bacteria (Choudhury 2004).promoting bacteria (Choudhury 2004).

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.

Aims and OjectivesAims and Ojectives

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.

TreatmentsTreatments AzospirillumAzospirillum and and AzotobacterAzotobacter were used as a were used as a

source of biofertilizers whereas Urea and source of biofertilizers whereas Urea and DAP were utilized as a source of chemical DAP were utilized as a source of chemical fertilizers. The treatments includesfertilizers. The treatments includes

T0 control (without inoculation and without T0 control (without inoculation and without urea and Diamonium phosphate)urea and Diamonium phosphate)

T1 (T1 (AzospirillumAzospirillum) ) T2 T2 (Chemical fertilizers (urea 160 kg/hec + (Chemical fertilizers (urea 160 kg/hec +

Diamonium Phosphate 185 kg/hec)Diamonium Phosphate 185 kg/hec) )) T3 (T3 (AzotobacterAzotobacter))

•The The AzospirillumAzospirillum and and AzotobacterAzotobacter were applied as were applied as seed inoculation @10seed inoculation @1066cells/ml. cells/ml. •First dose of chemical fertilizers was applied at the First dose of chemical fertilizers was applied at the time of sowing while other 3 doses were applied at time of sowing while other 3 doses were applied at 40 days interval. 40 days interval. •Soil samples collected prior to cultivation and Soil samples collected prior to cultivation and analyzed for phosphorus content according to analyzed for phosphorus content according to Soltanpur and Workman (1979). Soltanpur and Workman (1979). •The Exchangeable KThe Exchangeable K+1+1 and Mg and Mg+2 +2 were extracted were extracted according to Lanyon and Heald (1982), while Mnaccording to Lanyon and Heald (1982), while Mn+2+2, , ZnZn+2+2, Cu, Cu+3+3, Ni, Co, Ni, Co+3+3 and Fe and Fe+2+2 were extracted with a were extracted with a solution of DTPA (Lindsay and Norvell 1978) and solution of DTPA (Lindsay and Norvell 1978) and then determined by means of the atomic absorption then determined by means of the atomic absorption spectrophotometer.spectrophotometer.

Seed oil content was estimated by NMR (Nuclear Seed oil content was estimated by NMR (Nuclear Magnetic Resonance) test (Robertson and Magnetic Resonance) test (Robertson and Morrison 1979). Morrison 1979).

For estimation of seed protein content, 100 mg For estimation of seed protein content, 100 mg seeds were grounded and digested in Kjeldhal seeds were grounded and digested in Kjeldhal digestion flask. The total seed protein was then digestion flask. The total seed protein was then determined following the method as described by determined following the method as described by AOAC (1982). AOAC (1982).

Glucosinolate content of the oil was determined Glucosinolate content of the oil was determined following the method of Smith et al. (1985).following the method of Smith et al. (1985).

The oil was extracted in Soxhlet extractor The oil was extracted in Soxhlet extractor (AOAC 1960).(AOAC 1960).

The oil acid value was determined The oil acid value was determined according to AOAC (1997). according to AOAC (1997).

The specific gravity was determined by the The specific gravity was determined by the method of Pearson (1976) using density method of Pearson (1976) using density bottle. bottle.

For quantification of fatty acids, oil sample For quantification of fatty acids, oil sample (50-100 mg) was converted into its fatty acid (50-100 mg) was converted into its fatty acid methyl esters. methyl esters.

• The methyl esters of the fatty acids (0.5 µl) The methyl esters of the fatty acids (0.5 µl) were analyzed in a gas chromatograph were analyzed in a gas chromatograph (Shimadzu QP 5050) equipped with a flame (Shimadzu QP 5050) equipped with a flame ionizing detector (FID) and a fused silica ionizing detector (FID) and a fused silica capillary column (MN FFAP (50 m x 0.32 mm capillary column (MN FFAP (50 m x 0.32 mm i.d.; film thickness 0.25 µm).i.d.; film thickness 0.25 µm). •Helium was utilized as carrier gas. The Helium was utilized as carrier gas. The column temperature was kept at 110 °C for column temperature was kept at 110 °C for 0.5 min, heated to 200 °C at 10 °C/min, and 0.5 min, heated to 200 °C at 10 °C/min, and then maintained for 10 min. then maintained for 10 min. •The temperatures of the injector and detector The temperatures of the injector and detector were set at 220 °C and 250 °C respectively.were set at 220 °C and 250 °C respectively.

Biodiesel Preparation The Biodiesel was prepared via base The Biodiesel was prepared via base

catalyzed transesterification (Freedman et catalyzed transesterification (Freedman et al. 1986) and biodiesel yield was determined al. 1986) and biodiesel yield was determined as % conversion of vegetable oil to biodiesel as % conversion of vegetable oil to biodiesel (w/w) (Rashid and Anwar 2008).(w/w) (Rashid and Anwar 2008).

The data was analyzed statistically by The data was analyzed statistically by Analysis of Variance (Steel and Torrie 1980) Analysis of Variance (Steel and Torrie 1980) technique and comparison among mean technique and comparison among mean values of treatments was made by Duncan’s values of treatments was made by Duncan’s Multiple Range Test (DMRT) (Duncan Multiple Range Test (DMRT) (Duncan 1955).1955).

Statistical analysisStatistical analysis

Table 1 Macro and micronutrients (µg/g) analysis Table 1 Macro and micronutrients (µg/g) analysis of soil used for cultivation of Canola cropof soil used for cultivation of Canola crop

Ca2+ K1+ Mg2+ Na1+ Cu3+ Fe2+ Mn+2 Ni+2 Co+3 Zn+2 Cr+3 P

23 11.6 0.53 5.3 0.08 0.64 6.2 0.05 0.15 0.59 0.02 189

ResultsResults

c

aba

b

0

1

2

3

4

5

6

7

8

Treatments

No

.of B

ran

ch

es/P

lan

t

Fig.1. Effect of Azospirillum, Azotobacter and chemical fertilizers on Number of Branches/Plant

Fig.2. Effect of Azospirillum, Azotobacter and chemical fertilizers on Number of Siliqua/Branch

b

a

bb

0

10

20

30

40

50

60

Treatments

No

.of

Siliq

ua/B

ran

ch

bb

a

b

0

5

10

15

20

25

30

35

Treatments

No

.of

Seed

/Siliq

ua

Fig.3. Effect of Azospirillum, Azotobacter and chemical fertilizers on Number of Seed/Siliqua

b

b

a

b

0

200

400

600

800

1000

1200

1400

Treatments

Seed

Yie

ld (

Kg

/ha)

Fig.4. Effect of Azospirillum, Azotobacter and chemical fertilizers on Seed yield (Kg/ha)

b

a

a

ab

2.6

2.65

2.7

2.75

2.8

2.85

2.9

2.95

3

3.05

3.1

Control Azospirillum Urea + DAP AzotobacterTreatments

1000 S

eed

Weig

ht

(g)

Fig.5. Effect of Azospirillum, Azotobacter and chemical fertilizers on 1000 Seed Weight (g)

c

c

b

a

42

43

44

45

46

47

48

49

Control Azospirillum Urea + DAP AzotobacterTreatments

Seed

oil c

on

ten

ts (

%)

Fig.6. Effect of Azospirillum, Azotobacter and chemical fertilizers on Seed oil contents (%)

a

b b

a

0

0.5

1

1.5

2

2.5

3

Treatments

Ac

id v

alu

e

Fig.7. Effect of Azospirillum, Azotobacter and chemical fertilizers on Oil Acid value (mgKOH/g)

a

bb

a

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Treatments

Fre

e F

att

y A

cid

s

Fig.8. Effect of Azospirillum, Azotobacter and chemical fertilizers on Free Fatty Acid (%)

Fig.9. Effect of Azospirillum, Azotobacter and chemical fertilizers on Oil Specific gravity (g/cm3)

a

a

b

a

0.896

0.898

0.9

0.902

0.904

0.906

0.908

0.91

0.912

Treatments

Sp

ec

ific

Gra

vit

y (

g/c

m3

)

a

a a

b

32

33

34

35

36

37

38

39

40

41

42

Control Azospirillum Urea + DAP AzotobacterTreatments

Glu

co

sin

ola

te c

on

ten

ts

Fig.10. Effect of Azospirillum, Azotobacter and chemical fertilizers on Seed Glucosinolate content

b ab

c

0

1

2

3

4

5

6

Treatments

Mo

istu

re c

on

ten

ts

Fig.11. Effect of Azospirillum, Azotobacter and chemical fertilizers on Seed Moisture content (%)

b

aa

b

19

19.5

20

20.5

21

21.5

22

22.5

23

Treatments

Pro

tein

co

nte

nts

Fig.12. Effect of Azospirillum, Azotobacter and chemical fertilizers on Seed Protein content (%)

b

a

a

b

52.5

53

53.5

54

54.5

55

55.5

56

56.5

57

57.5

Treatments

Ole

ic A

cid

(C

18:1

)

Fig.13. Effect of Azospirillum, Azotobacter and chemical fertilizers on Oleic Acid (%)

b

dc

a

0

2

4

6

8

10

12

Treatments

Lin

ole

nic

Acid

(C

18:3

)

Fig.14. Effect of Azospirillum, Azotobacter and chemical fertilizers on Linolenic Acid (%)

a

c

b

a

23

24

25

26

27

28

29

30

Treatments

Eru

cic

Ac

id (

C2

2:1

)

Fig.15. Effect of Azospirillum, Azotobacter and chemical fertilizers on Erucic Acid (%)

b

a

a

b

87

88

89

90

91

92

93

94

Treatments

Bio

die

sel Y

ield

(%

w/w

)

Fig.16. Effect of Azospirillum, Azotobacter and chemical fertilizers on Biodiesel Yield (% w/w)

Significant Findings Significant Findings The application of Biofertilizers influenced the The application of Biofertilizers influenced the

growth and yield of canola.growth and yield of canola. Biofertilizers improved the seed proteins which will Biofertilizers improved the seed proteins which will

lead to the production of good quality oil seed cake as lead to the production of good quality oil seed cake as food for cattlefood for cattle

Biofertilizers decreased the glucosinolate content of Biofertilizers decreased the glucosinolate content of canola, glucosinolate content contain sulpher that canola, glucosinolate content contain sulpher that reduces the quality of biodiesel and cause reduces the quality of biodiesel and cause environmental pollution.environmental pollution.

Biofertilizers decreased the oil acid value and free Biofertilizers decreased the oil acid value and free fatty acid content.fatty acid content.

Cont……Cont……

Biofertilizers enhanced the oil oleic acid Biofertilizers enhanced the oil oleic acid content which leads to the production of content which leads to the production of good quality biodiesel with better cold flow good quality biodiesel with better cold flow properties.properties.

Biofertilizers improved the biodiesel yield in Biofertilizers improved the biodiesel yield in transesterification reaction.transesterification reaction.

ConclusionIt 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 PERSPECTIVESFUTURE PERSPECTIVES

Determination of the mechanism of PGPR Determination of the mechanism of PGPR on oil yield and quality of canola at on oil yield and quality of canola at molecular level.molecular level.

Determination of the impact of PGPR on Determination of the impact of PGPR on the activities of enzymes like desaturase and the activities of enzymes like desaturase and lipase.lipase.

Extension of this work to non-edible oil Extension of this work to non-edible oil yielding plantsyielding plants

Extension of this work to farmers level and Extension of this work to farmers level and determination of the possible role of PGPR determination of the possible role of PGPR on growth and yield of oil yielding crops on growth and yield of oil yielding crops under biotic and abiotic stresses and soil under biotic and abiotic stresses and soil fertility. fertility.

Further investigation for the impact of Further investigation for the impact of PGPR on soil nutrients statusPGPR on soil nutrients status

Investigations on the impact of biofertilizers Investigations on the impact of biofertilizers in combination with chemical fertilizers on in combination with chemical fertilizers on biodiesel yield of oil seed crops.biodiesel yield of oil seed crops.

AcknowledgementAcknowledgement This work was a part of the Research This work was a part of the Research

Project funded by HEC and authors are Project funded by HEC and authors are highly grateful for financial support highly grateful for financial support extended to accomplish this piece of work.extended to accomplish this piece of work.

Thanks Thanks for for AttentionAttention