Role of PGPR and Chemical Fertilizers on Oil Yield and Biodiesel Production of Canola ( Brassica napus L.)

1. 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

2. 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

3. 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).

4. 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).

5. 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.

6. 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.

7. 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)

8. 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.

9. 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).

10. 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.

11. 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.

12. 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).

13. 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

31. 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.

32. 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.

33. 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.

34. 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

35. 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.

36. 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.

37. Thanks for Attention