This document discusses green genes and microalgae as promising sources for biofuel production. It notes that microalgae have advantages over plants for biofuel production, including higher oil yields while using less land area. The document also summarizes research on genetic manipulation of plants and microalgae to improve traits related to biofuel production, such as reducing lignin in plants to improve saccharification or modifying lipid synthesis pathways in microalgae.

1. Welcome

2. 2
• Fossil fuels- primary and non renewable source of energy
• Contributes 86.4 % - global fuel consumption
• Major cause - global warming and environmental pollution
• High cost
(Kumar and Sharma, 2014)

3. 182
643772.05
3509
0
500
1000
1500
2000
2500
3000
3500
4000
production consumption
1980 2013
India’s crude oil production and consumption
Productio
n
Consumptio
n
Oilinthousandbarrelsperday
4000
3500
3000
2500
2000
1500
1000
50
0
0
1980 2013
3(Kumar and Sharma, 2014)

4. Centre for Plant Biotechnology and Molecular Biology
College of Horticulture, Vellanikkara
Kerala Agricultural University
Green genes: Promising fuel source for future
4
Narasimha Reddy P. K.
2014-11-104

5. 5
 Introduction
 Evolution of biofuels
 Biofuel production methods
 Target areas for biotechnological interventions
 Current research and developments
 Success stories
 Applications
 Future line
 Summary
 Conclusion
Outline

6. Green genes
6
 Green genes- plants and algae
 Hydrocarbons, polysaccharides and triacylglycerides -
precursors for biofuel
(Reijnders et al., 2014)
Introduction

7. Biofuel
7
 From renewable biological processes
 Forms of biofuel:
1. Biodiesel
2. Bioethanol
3. Biomethane
4. Biohydrogen
 Biodegradable and ecofriendly
 Major sources- plants and algae
(Kumar and Sharma, 2014)

8. Evolution of biofuel
8

9. Biofuel
First
generation
Sucrose-
Containing
feedstock
Sugar beet
Sugar cane
Sweet
sorghum
Starchy
material
Corn
Potato
Oil
seeds
Second
generation
Lignocellulosic
biomass
Wood
Jatropha
Switch
grass
Third
generation
Algal
biomass
(Halim et al., 2012)
9
…evolution of biofuel

10. 10
 Food crops such as sugarcane, wheat, corn, barley, potato, etc.
 Sugar and starch biomass
 Bioethanol - fermentation
 Fuel vs food - global increase in food prices
 Require high agricultural inputs such as labour and fertilizers
First generation biofuels
(Wong and Sanggari, 2014)

11. 11
Second generation biofuel
 Non-food crops such as jatropha, wood, etc.
 Lignocellulosic biomass and triacylglycerols
 Biodiesel and bioethanol
 Feedstock extraction from woody or fibrous biomass
 Depletion of biodiversity
(Halim et al., 2012)

12. 12
Third generation biofuels
 Algal biomass - triacylglycerols
 Overcome the problems
 Transesterification
 Biodiesel, bioethanol, biomethane and biohydrogen
( Kumar and Sharma, 2014)

13. 13
Biodiesel - transesterification
Biohydrogen - photobiological process
Bioethanol - fermentation
(Nagle and Lemke, 1990)
(Fedorov et al., 2005)
(Dexter et al., 2009)
Biomethane - anaerobic digestion (Spolaore et al., 2006)
Biofuel Production methods

14. (Metting and Pyne, 1986)
Biodiesel production
14
Carbon dioxide
and sunlight
Oxygen
Fats(oil) grown inside
algae and plant
Oil is extracted Oil converted to
biodiesel
Biodiesel placed in
the market

15. 15
Photosynthesis
6CO2 + 6 H20 + Light C6H12O6 + 6CO2
(Carbon dioxide) (Water) (Glucose)
Fermentation
C6H12O6 C2H5OH + 2CO2 + Heat
(Glucose) (Ethanol) (Carbon dioxide)
Reactions during bioethanol production
(Wong and Sanggari, 2014)

16. 16
Preparation of
sugarcane
bagasse
Liquefaction of
sugarcane
Saccharification
of sugarcane
bagasse
Fermentation
of sugarcane
Distillation of
ethanol
Production of bioethanol from sugarcane
(Wong and Sanggari, 2014)

17. Biomethane
17
 Agricultural waste, manure, plant
material, green waste, etc.
 Anaerobic digestion
 Cooking
 Compressed biomethane - vehicle
Biomethane bus, Sweden
Biomethane train, Sweden
(Barakat et al., 2012)

18. Biohydrogen
18
 Source - algal biomass
 Biological process – fermentation
 Organic acid as substrate – higher fermentation rate
 Fuel for vehicles
(Kruse and Hankamer, 2010)
Biohydrogen car
Biohydrogen bus

19. 19
1. Improve – photosynthetic efficiency
2. External input reduction
3. Improve penetration of light - dense cell cultures
4. Metabolic pathway modifications
5. Improve the lipid synthesis
(Chisti, 2010)
Target areas for biotechnological interventions

20. Bioethanol from lignocellulose biomass
20
 Presence of lignin in vascular tissue - barrier
 Enzymatic digestion of lignin - improve plant carbohydrate
production
 Genes encoding enzymes hydroxyphyl (H), guaiacyl (G) and
syringyl (S) - building blocks of lignin
 Antisense constructs to knock out genes encoding enzymes
(Chen et al., 2007)

21. …bioethanol from lignocellulose biomass
21
Mature stem harvested - late flowering stage
Plants with least lignin have high carbohydrate level
Hydroxycinnamoyl - highly contributes for lignin blocking
than enzymes like C 3-H and C 4-H
(Chen et al., 2007)

22. 22
 C 4H : Cinnamate 4-hydroxylase
 HCT : Shikimate hydroxycinnamoyl transferase
 C 3-H : Coumaroyl shikimate 3-hydroxylase
 CCoAOMT : Caffeoyl CoA 3-O-methyltransferase
 F 5-H: Ferulate 5-hydroxylase
 COMT: Caffeic acid 3-O-methyltransferase
Saccharification efficiencies for biomass
(Chen et al., 2007)

23. 23
 Higher saccharification efficiency - transgenic lines
 Pathway - conserved across plant kingdom
 Targeted genes - candidate genes for improving saccharification
in bioenergy crops like jatropha, switchgrass etc.
…bioethanol from lignocellulose biomass
(Chen et al., 2007)

24. Biodiesel from algal biomass
24
 Photosynthetic, heterotrophic organisms
 Potential for cultivation as energy crops
MacroalgaeMicroalgae
(Parker et al., 2008)

25. Microalgal species with oil content
25
Microalgae Oil content (% dry wt.)
Botryococcus braunii 25 - 75
Chlorella sp. 28 - 32
Crypthecodinium cohnii 20
Cylindrotheca sp. 16 - 37
Isochrysis sp. 25 - 33
Nannochloris sp. 20 - 35
Nannochloropsis sp. 31 - 68
Neochloris oleoabundans 35 - 54
Nitzschia sp. 45 - 47
Phaeodactylum tricornutum 20 - 30
Schizochytrium sp. 50 - 77
Tetraselmis suecica 15 - 23
(Chisti, 2007)

26. 1
10
100
1000
10000
100000
1000000
Corn Soyabean Canola Jatropa Coconut Oilpalm Microalgae Microalgae
Land(M ha)
Oil yield(
1540
172
594 446
1190
1892 2689
5950
136,000
58,700
223
140 99
45
2
4.5
L/ha)
Landareaneeded(mha)
Oilyield(L/ha)
( Kumar and Sharma, 2014)
100000
0
100000
10000
1000
100
10
0 Corn Coconut Oil palm Microalgae̽Soybean canola Microalgae̽̽̽ ̽Jatropha
Comparison of some sources of biodiesel
100000
0
100000
̽ - 70 %
Oil
̽̽ ̽- 30 % 26
Sources
Land (mha)
Oil yield (L/ ha)

27. Why microalgae than plants?
27
 More oil yield
 Small area of land
 Lesser need of labour, nutrients and water
 Grow rapidly with high solar energy conversion efficiency
 Wider adaptability
(Chisti, 2007)

28. 28
Downstream processing for biodiesel
(Scott et al., 2010)

29. 29
CH₂-OCOR₁ R₁-COOCH₃ CH₂-OH
I KOH I I
CH₂-OCOR₂ + 3HOKH₃ R₂-COOCH₃ + CH-OH
I I I
CH₂-OCOR₃ R₃-COOCH₃ CH₂-OH
Triacylglycerol Methanol Fatty acid methyl esters Glycerol
Reaction during transesterification
(Scott et al., 2010)

30. 30
Draft genome sequence of Nannochloropsis
gaditana
(Radakovits et al., 2012)
• Lack of a genetically tractable model alga capable of industrial
biofuels production
• Nannochloropsis gaditana - high photoautotrophic
- rapid lipid accumulation
- grow on waste water

31. 31
Pathway Genes
• TAG biosynthesis • PAP (Nga21116), PDAT(Nga02737)
• Gluconeogenesis • TAG lipases (Nga30958, Nga30749)
• Acyl-coA oxidases (Nga03053,Nga04370.1,
Nga30819)
• Carbon assimilation • Carbonic anhydrases (Nga01240, Nga01717,
Nga03728, Nga30848,Nga10007, Nga21222)
• Putative bicarbonate transporters
(Nga00165.01, Nga06584)
Identified genes in N.gaditana
(Radakovits et al., 2012)

32. 32
Genome sequencing and annotation
Nuclear genome assembly
Estimated genome size 29 Mb
Genomic G+C content 54.2 %
number of assembled scaffolds 2,087
Number of contigs 5,823
Gene statistics
Predicted number of genes 8,892
Chloroplast genes 124
Mitochondrial genes 36
Total number of genes 9,052
Average exon length 1,069 bp
Average intron length 220 bp
(Radakovits et al., 2012)

33. Current research and
developments
33

34. Sl. no. Institution/Organization Microalgae Reference
1 Central Food Technological
Research Institute (CFTRI),
Mysore
Botryococcus
braunii
(Dayananda
et al., 2006)
2 University of Madras, Chennai Seaweeds (Rengasamy, 2009)
3 Vivekananda Institute of Algal
Technology (VIAT), Chennai
Microalgae (Ramachandra et
al., 2009)
4 University of Madras, Chennai Sargassum sp. (Rengasamy, 2011)
5 Alternate Hydro Energy
Centre, Indian Institute of
Technology, Roorkee
Microalgae (Rajvanshi, 2011)
Researches in India
34

35. 35
Biodiesel from jatropha and pongamia
Ethanol production from cashew fruit
Ethanol 7.7% mixed diesel in the transport buses
Ethanol from arecanut peels, coffee seed peels and powder waste
Biodiesel production from rubber seeds
…researches in India
Biofuel park, Hassan

36. AlgaePARC
36
Research and development in Netherlands
Research:
 Cellular processes
 Strain improvement
 Cultivation optimization
 Scale up
 Biorefinery and product development
 Chain analysis and design

37. 37
Offshore Membrane Enclosure for Growing Algae (OMEGA) system
NASA
Research and development in United States

38. 38
 Algenol, USA and Reliance, India - algae fuel demonstration
project in India on 21st January 2015
• Patented for gasoline, biodiesel and jet fuels production by CO₂
• Environmental protection Agency(EPA), USA approved Algenol’s
ethanol
 Algenol ,USA and Zhongyuan New Energy Company ltd. (ZYNE)-
Algal biofuel production projects in China, 23rd September 2015
…research and development in United States
Algenol, USA

39. Success stories
39

40. 40
Sapphire ,USA
 Two‐engine Boeing 737‐800 by
Continental Airlines, January 2009
 Blend of 50% conventional and 50%
synthetic jet fuel (blend of algae and
jatropha jet fuel)
 Toyota Prius, September 2009
 Blend of 5% algae derived
gasoline
Success stories of algal biodiesel

41. 41
Solazyme ,USA
US Navy’s riverine command boat,
Oct 2010
US Navy’s MH-605 sea hawk, June 2011
…success stories of algal biodiesel

42. 42
 Central Salt Marine and Chemical Research Institute (CSMCRI),
Bhavnagar
 B20 biodiesel Chevrolet tavera
 Marine algae
 Council of Scientific and Industrial Research (CSIR) ,Ministry of
Earth Science (MoES) and nine institutes
CSMCRI, India
…success stories of algal biodiesel
(Vijyanish, P. 2012)

43. Applications
43

44.  UAS and IISc, Bengaluru
 Oct 2nd 2015
 B20
Biodiesel – Saves Rs. 5 per litre
Biofuel application in India
44
 Indian railway, 2002
 Southern online biotechnologies ltd.
Hyderabad
 Tiruchirapalli to Lalgudi, Tamil Nadu
(Gadekal, 2015)

45. 45
• World's 2nd biggest producer of ethanol (5.9 billion gallons)
• Sugarcane
• About 6 million flex-fuel vehicles and 3 million motorbikes - E100
• Bioethanol - 48% of light vehicle fuel consumption
• Gasoline blend with ethanol - 20 to 25 percent in 2013
Biofuel in Brazil
(Smith, 2013)

46. 46
…biofuel in Brazil
0
5
10
15
20
25
30
35
40
45
50
2004 2005 2006 2007 2008 2009 2010 2011
Production Consumption
Biofuel production and consumption in Brazil
(Smith, 2013)

47. Biodiesel production and consumption
0
50
100
150
200
250
Production Consumption Production Consumption Production Consumption Production Consumption
Argentina Europe Germany India
Thousandbarrelsperday
2005 2007 2009 2011 47(Smith, 2013)

48. 48
Company Country
Algenol Biofuels USA
Aquaflow New Zealand
Aurora Algae USA
Bioalgene USA
Bodega Algae USA
Joule Unlimited USA
LiveFuels USA
OriginOil USA
Solix Biofuels USA
Sapphire Energy USA
Seambiotic Ltd. USA
Solazyme USA
(Chisti and Yan, 2011)
Startup companies for commercialization of
algal biofuels

49. Identification of new sources of biofuel
Appropriate genetic transformation methods and insilico
approaches
49
Future line

50. 50
Summary
• Green genes for biofuel production
• Advantage of microalgae over plants
• Genetic manipulation in plant and microalgae
• Research and developments
• Application in transportation and defence

51. Green biofuel - promising alternate to the fossil fuels
Genetic engineering and insilico methods of green genes - a
revolution
Issues related to environment can be reduced
51
Conclusion

52. Thank you