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Welcome
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)
production consumption0
500
1000
1500
2000
2500
3000
3500
4000
182
643772.05
3509
1980 2013
India’s crude oil production and consumption
Production
Consumption
O
il in
thou
sand
bar
rels
per
day
40003500300025002000
15001000500
0
1980 20133(Kumar and Sharma, 2014)
Centre for Plant Biotechnology and Molecular BiologyCollege of Horticulture, Vellanikkara
Kerala Agricultural University
Green genes: Promising fuel source for future
4
Narasimha Reddy P. K. 2014-11-104
5
IntroductionEvolution of biofuelsBiofuel production methodsTarget areas for biotechnological interventionsCurrent research and developmentsSuccess storiesApplicationsFuture lineSummary Conclusion
Outline
Green genes
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Green genes- plants and algae
Hydrocarbons, polysaccharides and triacylglycerides -precursors for biofuel
(Reijnders et al., 2014)
Introduction
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)
Evolution of biofuel
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Biofuel
First generati
onSucrose-
Containing feedstock
Sugar beet
Sugar cane
Sweet sorghum
Starchy materia
l
Corn
Potato
Oil seeds
Second generati
on
Lignocellulosic biomass
Wood
Jatropha
Switch grass
Third generatio
n Algal bioma
ss
(Halim et al., 2012) 9
…evolution of biofuel
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
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
Third generation biofuels
Algal biomass - triacylglycerols
Overcome the problems
Transesterification
Biodiesel, bioethanol, biomethane and biohydrogen
( Kumar and Sharma, 2014)
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
(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
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)
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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)
Biomethane
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Agricultural waste, manure, plant
material, green waste, etc.
Anaerobic digestion
Cooking
Compressed biomethane - vehicle
Biomethane bus, Sweden
Biomethane train, Sweden
(Barakat et al., 2012)
Biohydrogen
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Source - algal biomass
Biological process – fermentation
Organic acid as substrate – higher fermentation rate
Fuel for vehicles
(Kruse and Hankamer, 2010)
Biohydrogen car
Biohydrogen bus
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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
Bioethanol from lignocellulose biomass
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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)
…bioethanol from lignocellulose biomass
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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)
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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
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)
Biodiesel from algal biomass
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Photosynthetic, heterotrophic organisms
Potential for cultivation as energy crops
MacroalgaeMicroalgae
(Parker et al., 2008)
Microalgal species with oil content
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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)
Corn Soyabean Canola Jatropa Coconut Oilpalm Microalgae Microalgae1
10
100
1000
10000
100000
1000000Land(M ha)Oil yield(
1540
172594 446
11901892 268
9
5950
136,000 58,700
223140 99
45
2
4.5
L/ha)
Land
are
a ne
eded
(m
ha)
Oil
yiel
d (L
/ha)
( Kumar and Sharma, 2014)
1000000 100000 10000 1000
100 10 0 Corn Coconut Oil palm MicroalgaeSoybean canola Microalgae Jatropha
Comparison of some sources of biodiesel1000000 100000
- 70 % Oil - 30 % Oil
26
Sources
Land (mha)Oil yield (L/ ha)
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
Downstream processing for biodiesel
(Scott et al., 2010)
29
CH₂-OCOR R -COOCH ₁ ₁ ₃ CH₂-OH I KOH I I CH₂-OCOR₂ + 3HOKH R₃ ₂-COOCH + ₃ CH-OHI I I CH₂-OCOR₃ R -COOCH₃ ₃ CH₂-OH
Triacylglycerol Methanol Fatty acid methyl esters Glycerol
Reaction during transesterification
(Scott et al., 2010)
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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
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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
Genome sequencing and annotationNuclear genome assembly
Estimated genome size 29 MbGenomic G+C content 54.2 %number of assembled scaffolds 2,087Number of contigs 5,823
Gene statisticsPredicted number of genes 8,892Chloroplast genes 124Mitochondrial genes 36Total number of genes 9,052Average exon length 1,069 bpAverage intron length 220 bp
(Radakovits et al., 2012)
Current research and developments
33
Sl. no. Institution/Organization Microalgae Reference
1 Central Food Technological Research Institute (CFTRI), Mysore
Botryococcus braunii
(Dayanandaet 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
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 IndiaBiofuel park, Hassan
AlgaePARC
36
Research and development in Netherlands
Research:
Cellular processes
Strain improvement
Cultivation optimization
Scale up
Biorefinery and product development
Chain analysis and design
37Offshore Membrane Enclosure for Growing Algae (OMEGA) system
NASAResearch and development in United States
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
Success stories
39
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
Solazyme ,USA
US Navy’s riverine command boat, Oct 2010
US Navy’s MH-605 sea hawk, June 2011
…success stories of algal biodiesel
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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)
Applications
43
UAS and IISc, Bengaluru
Oct 2nd 2015
B20
Biodiesel – Saves Rs. 5 per litre
Biofuel application in India
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Indian railway, 2002
Southern online biotechnologies ltd.
Hyderabad
Tiruchirapalli to Lalgudi, Tamil Nadu
(Gadekal, 2015)
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
…biofuel in Brazil
2004 2005 2006 2007 2008 2009 2010 20110
5
10
15
20
25
30
35
40
45
50
Production Consumption
Biofuel production and consumption in Brazil(Smith, 2013)
Biodiesel production and consumption
Production Consumption Production Consumption Production Consumption Production ConsumptionArgentina Europe Germany India
0
50
100
150
200
250
2005 2007 2009 2011
Thou
sand
bar
rels
per d
ay
47(Smith, 2013)
48
Company Country Algenol Biofuels USA
Aquaflow New ZealandAurora Algae USA
Bioalgene USABodega Algae USA
Joule Unlimited USALiveFuels USAOriginOil USA
Solix Biofuels USASapphire Energy USASeambiotic Ltd. USA
Solazyme USA
(Chisti and Yan, 2011)
Startup companies for commercialization of algal biofuels
Identification of new sources of biofuel
Appropriate genetic transformation methods and insilico
approaches
49
Future line
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
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
Thank you