GREEN GENES- A PROMISING FUEL SOURCE FOR FUTURE Narasimha Reddy Palicherlu

Welcome

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• 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

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Narasimha Reddy P. K. 2014-11-104

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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

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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

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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)

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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)

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Third generation biofuels

Algal biomass - triacylglycerols

Overcome the problems

Transesterification

Biodiesel, bioethanol, biomethane and biohydrogen

( Kumar and Sharma, 2014)

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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

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Carbon dioxide and sunlight Oxygen

Fats(oil) grown inside algae and plant

Oil is extracted Oil converted to biodiesel

Biodiesel placed in the market

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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)

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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

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Sources

Land (mha)Oil yield (L/ ha)

Why microalgae than plants?

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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)

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Downstream processing for biodiesel

(Scott et al., 2010)

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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)

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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

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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

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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

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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

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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

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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

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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

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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)

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• 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)

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…biofuel in Brazil

2004 2005 2006 2007 2008 2009 2010 20110

5

10

15

20

25

30

35

40

45

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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)

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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

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Future line

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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

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Conclusion

Thank you