Green Oil as the High-energy Multipurpose Biofuel of Choice

Katayoon DeheshUC Davis

Genetic and genomic approaches to enhance production of plant oil

The fossil oil is soon to be depleted and causes huge geopolitical problems and carbon dioxide emissions that cause a global warming that are threatening the future of human civilization.

Plant are the perfect chemical factory!

Petrochemical industry cracks carbon chainsand use advanced organic chemistry to builddesired products with these building blocks. Usually, this require more energy than the product contain.Plants design the complex lipid productsdirect in the seeds without extra cost and energy, thereby minimizing downstreamprocessing costs..

In many industrial applications, plant oilshave successfully competeted with fossil oildespite that their price have been over fivetimes higher.

Courtesy of Sten Stymne

Everything you can do with mineral oil, you canalso do with plant oil

It is just a matter of costs!!

$146 USD $130 USD$17 USD $88 USD

1997 2008Courtesy of Sten Stymne

We must find sustainable and environmental neutral alternatives to

fossil oil very soon.

How can plant lipid contribute?

Mission I:Mission I:

Replacement of fossil oil with Replacement of fossil oil with plant oils in the chemical industryplant oils in the chemical industry

Mission II:Mission II:

Replacement of fossil oil with plant oil Replacement of fossil oil with plant oil as biofuelas biofuel

Plant oils are the major source of highly-reduced carbon available from Nature. The abi l i ty to control the chemical structure of this stored carbon represents a vital step towards producing renewable biomaterials that can r e p l a c e p e t r o - c h e m i c a l s .

050

100150200250300350400450

Mill

ion

met

ric to

nFossil oilused in chemicalindustry

2007

Courtesy of Sten Stymne

125 million tones of vegetableoil are produced per year

About 20% (25 million tonnes) are used in industrialapplications today.

Soap, detergentsPaints, inksPlasticLubricantsCosmeticsetc.

Courtesy of Sten Stymne

What has to be done to change chemical industry from fossil to fresh oil based

1.Optimization of plant oil chemical structure(lowering downstream processing costs)

2. Increase the total production of plant oils

It was 19 years since the first report of modification of plant oil quality by genetic engineering.

Since then probably thousands of patent applications in the plant lipid area have been filed around the world to the cost of hundreds of millions of $.

A number of wild plants have very high amount of industrial valuable fatty acids in their seed oils

90% Ricinoleic acid95% Capric acid (10:0)73% Caprylic acid (8:0)

Cuphea painteri, Cuphea koehneana

OH OH

Castor (Ricinus communis)

88% α−Eleostearic acid 78% Sterculic acid

Aleurites fordii Sterculia foetida

+ several hundreds of other fatty acid structures….

8

Cuphea species Castor Aleurites fordii Sterculia foetidaC t f St St

Transferring the biosyntheticmachinery for the production of

unsusal fatty acids to high yielding oil crops by genetic

engineering could yieldproduction at a low price and

with stable supply.

Transgenic canola seeds with medium chain thioesterasesVoelker et al. (1992) Science, 257, 72–74.Knutzon et al. (1999) Plant Physiol. 120, 739–746.

% 12:0 in oil

Native seed oil GM-seed12:0 -TE from California bay tree 60% 67%

Courtesy of Sten Stymne

Fatty Acid Composition8:0 50%

10:0 25%8:0 95%

10:0 2.5%

Cuphea pulcherrimaCuphea hookeriana

Medium Chain Fatty Acidsacetyl-CoA

C4:0-ACP

C6:0-ACP

C8:00-ACP

C10:0-ACP

C12:0-ACP

C14:0-ACP

C16:0-ACP

C18:0-ACP

C18:1-ACPchloroplast/proplastid

C8:0C10:0

Free fatty acids

acyl-CoAs

Structurallipids

Storagelipids

{further desaturation}

endoplasmic reticilum

Electron Micrograph of WT Brassica Seed (30 DPA)

Electron Micrograph of WT Brassica Seed (30 DPA)

Electron Micrograph of High MCFA Transgenic Brassica Seed (30 DPA)

Electron Micrograph of High MCFA Transgenic BrassicaSeed (30 DPA)

Other Unusual Fatty Acids

Cyclopropane Fatty Acids (CPE-FAs)

CPE-FAs are prized for oxidative stability while retaining low viscosity, thus they have wide applications as: lubricants and potentially in biodiesel.

Cyclopropene fatty acids (CPE-FAs)The biosynthesis of the cyclopropane ring occurs when a methyl group is transferred from S-adenosylmethionine to the double bond of oleic acid.

The enzyme cyclopropane synthase has been identified from Sterculia foetida. Expression of thisgene in tobacco led to production of ~ 6% cyclopropanefatty acids in callus.

Bao X, Katz S, Pollard M, Ohlrogge J (2002) Carbocyclic Fatty Acids in Plants: Biochemical and Molecular Genetic Characterization of Cyclopropane Fatty Acid Synthes is o f Stercul ia foet ida. Proc Nat l Acad Sci USA 99: 7172-7.

Bao X, Thelen JJ, Bonaventure G, Ohlrogge JB (2003) Characterization of Cyclopropane Fatty Acid Synthase From Sterculia foetida. J Biol Chem 278:12846-53.

Cyclopropane synthase was identified in 2003:

Cyclopropene fatty acids (CPE-FAs)

Cyclopropane synthase in cotton

The cyclopropane synthase found in cotton and other plants adds a methyl group across the double bond of 18:1 to synthesize cyclopropaneFA.

Conjugated Fatty AcidsThe double bonds of conjugated fatty acids are arranged at adjacent carbon atoms and ARE NOT separated by a methylenegroup (-CH2-) as is the case in linoleic and α-linolenic acids.

The conjugated unsaturation makes these fatty acids more prone to oxidation. This is a desired property of vegetable oils that are used as solvents in inks, paints, and varnishes, as the polymerization associated with oxidation of the double bonds results in faster drying rates.

There are at least seven types of conjugated fatty acids that can occur in seed oils of plants

α-Eleostearic acidα-Eleostearic acid is one of type of conjugated fatty acids

CH3(CH2)3CH=CHCH=CHCH=CH(CH2)7COOH

The primary source of α-Eleostearic acid is Tung oil

Other unsaturated plant oils, such as castor oil and linseed oil, take longer to dryand leave an oily residue until they soak into the wood surface.

BUT:Tung oil 's ability to dry quickly and polymerize into a tough, glossy, waterproof coating has made it especially valuable in paints, varnishes, linoleum, oilcloth and printing inks.

WHY Tung Oil?

Fruit and seeds of the Tung oil tree (Aleurites fordii)

Tung Tree: belongs to the Euphorbia Family (Euphorbiaceae)

The oil-rich Tung seeds are the source of the oil used on fine furniture. The lower left fruit has completely dried out.

Candlenuts And Kukui NutsThe seed of candlenut (Aleurites molucanna) contains about 50 percent oil

A closely related candlenut tree (Aleurites molucanna): a native to Asia that has been spread by people throughout the tropical Pacific, because its seeds are rich in oil. The valuable oil expressed from seeds is used as a light source.

The candlenut tree (Aleurites molucanna)In the Hawaiian Islands candlenuts are known as "kukui nuts" and are polished and made into shiny dark brown or black bracelets and leis.

Colorful Hawaiian necklaces, made from paintedand polished kukui "nuts."

Why not Tung seeds?Like many other Euphorbiaceae, Tung seeds contain toxins. Other potential sources of α-eleostearic acid-enriched oils include seeds of the Cucurbitaceae Momordica charantia (bitter melon).

Bitter melon is grown as a vegetable, but its viny growth morphology and low production of seeds limit its potential as an oilseed crop.

The FA conjugase from Momordica charantia

Conjugated fatty acids are used as drying oils and can serve as replacements to petroleum additives to reduce VOC emissions from paints and other coating materials.Cahoon EB, Carlson TJ, Ripp KG, Schweiger BJ, Cook GA, Hall SE, Kinney AJ (1999) Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos. Proc. Natl. Acad. Sci. USA 96: 12395-12940.

Cahoon EB, Dietrich CR, Meyer K, Damude HG, Dyer JM, Kinney AJ (2006) Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds. Phytochemistry 67: 1166-1176.

Fatty acid conjugase

The bitter melon fatty acid conjugase has been expressed in seeds of Arabidopsis and in seeds and somatic embryos of soybean to produce α-eleostearic acid to levels of nearly 20% of the total seed oil. Seeds from these plants, however, typically have a wrinkled morphology and strongly impaired germination. Inaddition, α-eleostearic acid not only accumulates in the storage triacylglycerols of the engineered seeds, but is also present inelevated levels in PC and other membrane phospholipids. By contrast, seeds from plants that naturally produce conjugated fatty acids (e.g., bitter melon) have apparently evolved enzyme(s) that efficiently remove these fatty acids from PC and other phospholipids and prevent their accumulation in membranes

Ricinoleate (12-hydroxy-octadeca-cis-9-enoic acid: 18:1-OH)Other Unusual Fatty Acids

Ricinoleate: an important natural raw material with great value as a petrochemical replacement in products such as:

lubricants, nylon, dyes, soaps, inks, adhesives, and biodiesel.

RicinoleateThe Major source: seeds of castor plant (Ricinus communis L.) ( 90% of the total fatty acids ) .

Availability of 18:1-OH form castor is limited because:

1- Oilseed castor cultivation is limited to tropical and sub-tropical regions, and seeds are laboriously harvested by methods that are difficult to adapt to large-scale production.

2- Castor seeds contain the poisonous ricin as well as strongly allergenic 2S albumins, which pose health threats for workers during planting, harvesting and processing.

Castor plant (Ricinus communis L.)

RicinoleateRicinoleate biosynthesis in castor seeds is catalyzed by an oleate ∆12-hydroxylase (FAH12) [1].

Expression of FAH12 in transgenic tobacco and Arabidopsis caused the accumulation of hydroxyfatty acids, but only to about 17% of total seed oil, far less than that in the native castor seeds [2].

1-van de Loo FJ, Broun P, Turner S, Somerville C, 1995: An oleate 12-hydroxylase from Ricinus communis L is a fatty acyl desaturase homolog. Proceedings of the National Academy of Sciences of the United States of America 92, 6743-6747.

2-Chaofu Lu, Martin Fulda, Jim Wallis, John Browse, 2006. A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. The Plant Journal 45, 847-856.

A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seedsDurrett, TP. et. al., 2010, PNAS, 107: 9464-9469.

Euonymus alatus (Burning Bush) accumulatehigh levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs)

in its embryo and endosperm.

Developing fruit

Long chain TAGsAcetyl diacylglycerol

3-acetyl-1,2-diacyl-sn-glycerols: a low viscosity oil

Measured viscosity values for acTAGs and lcTAGs

Sample Intrinsic Density, Kinematicviscosity, mPa s g/cm3 viscosity,

mm2·s−1

Soybean oil 49.2 ± 2.4 0.921 53.5Trioctanoin 23.3 ± 3.5 0.948 24.5LcTAG 42.6 ± 2.90 0.916 46.5AcTAG 26.3 ± 0.9 0.924 28.5

Data were obtained at ambient temperature (24 °C).

Frédéric Beisson et al 2003. Arabidopsis Genes Involved in Acyl Lipid Metabolism. A 2003 Census of the Candidates, a Study of the Distribution of Expressed Sequence Tags in Organs, and a Web-Based Database. Plant physioloy 132: 681-697.

Y. Niu, G.-Z. Wu, R. Ye, W.-H. Lin, Q.-M. Shi, L.-J. Xue, X.-D. Xu, Y. Li, Y.-G. Du, and H.-W. Xue. 2009.

Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana.Mol Plant, 2(5): 1107 - 1122.

Oat as a model plant for converting cereals into oil crops

Oat is unique among cereals having the major part of the oil in the endosperm.

Oil content of oat grain can vary from 2-10% andThe differences is due to the oil content of the endosperm

Oil rotein

tarch

Endosperm cellsof high-oil oat

Identifying the genetic switch between oil and starch synthesis in oat endosperm cells open the possibility to create high oil cereals

Starch OilPlastid

Oil i trees

Pine 1,5% Spruce 0,5% Birch1.3%

OljaStarchOil Mongolian oil firewood (5%)

(Tetraena mongolica Maxim)

Courtesy of Sten Stymne

In Sweden, more fatty acids are extracted frompine and spruce (tall oil) in the pulping industry than produced by oilseed crop canola,

That is:

350,000 ton of fatty acids/year

Compare with canola =100,000 ton oil/year

Courtesy of Sten Stymne

Nut sedge (Cyperus esculentus) tubers as a model for oil accumulation in roots and tubers

Courtesy of Sten Stymne

Increase the production of plant oils

New oil crops adapted to marginal land.

Jathropha curcas

Courtesy of Sten Stymne

BIODIESELRudolf Diesel used peanut oil as liquid fuels in internal combustion engines (1900)

Because of its low cost and easy availability, petroleum became the dominant energysource and petroleum diesel was then developed as the primary fuel for diesel engines

Petroleum and its derivatives fuels were in short supply in 1930 and hence in the 1930s and 1940s, neat vegetable oils were used in diesel engines under an emergency situation (Ma and M.A. Hanna, Biodiesel production: a review, Bioresource Technology 70 (1999), pp. 1–15. ) Two approaches were used:

1- Hydrocarbons were produced in China by a tung oil pyrolysis batch system and used as liquid fuels

2- Fatty acids’ ethyl or methyl esters, obtained by transesterification oralcoholysis of vegetable oils

alcoholysis /al·co·hol·y·sis/ (al″kah-hol´ĭ-sis) decomposition of acompound due to the incorporation and splitting of alcohol

WHY BIODIESEL?1- Biodiesel is biodegradable and harmless

2- Can be produced from renewable materials

3- Ethyl or methyl fatty acid esters contain no sulfur

4- Biodiesel decreases soot emission considerably (up to 50%)

5- Biodiesel emits about the same amount of CO2 that is absorbed during cultivation of the oilseed

9- Has numerous social and economic advantages, particularly in countries such as Brazil.

6- Does not contain any of the carcinogens found in diesel oil7- Biodiesel is not considered a hazardous material8- Increases engine lifetime owing to a superior lubrication capability

10- Biodiesel represents a suitable outlet for vegetable oil industry, serving as an important tool for market regulation.

1- The first governmental program concerning the use ofbiofuel was during the Second World War using cottonseed oilas the main vegetable oil produced in Brazil at that time,as fuel in trains Export of this oil was forbidden in order to force a drop inits price!!2- PROALCOOL : In 1980 Brazil's Federal Government created this program which implemented and regulated the use of hydrated ethanol as fuel (engine's adaptationswere needed to use this fuel) and anhydrous ethanol that could be blended with petroleum gasoline Since 1980 no pure petroleum gasoline is used in Brazil, only the ethanol/gasoline. The ethanol content in those blends started as 5% and has been increased duringthe three decades of PROALCOOL and actually varies from 20% to 25%.

Biofuel Program in Brazil

PRO-ÓLEO: Program created by National Energy Commission, through Resolution No. 007 dated October 22, 1980. It was expected a 30% mixtureof vegetable oils or derivatives in diesel and a full substitution at long term. But after thedrop of petroleum prices in the international market, this program was abandoned in 1986.

Biofuel Program in Brazil1- The first governmental program concerning the use of biofuel was during the SecondWorld War using cottonseed oil, as the main vegetable oil produced in Brazil at that time,as fuel in trains Export of this oil was forbidden in order to force a drop in its price

2- PROALCOOL : In 1980 Brazil's Federal Government created this program which implemented and regulated the use of hydrated ethanol as fuel (engine's adaptations were needed to use this fuel) and anhydrous ethanol that could be blended with petroleum gasoline

The ethanol content in those blends started as 5% and has been increased during the three decades of PROALCOOL and actually varies from 20% to 25%.

PRO-ÓLEO: Program created by National Energy Commission, through Resolution No. 007 dated October 22, 1980. It was expected a 30% mixtureof vegetable oils or derivatives in diesel and a full substitution at long term. But after thedrop of petroleum prices in the international market, this program was abandoned in 1986.

Biofuel Program in Brazil1- The first governmental program concerning the use of biofuel was during the SecondWorld War using cottonseed oil, as the main vegetable oil produced in Brazil at that time,as fuel in trains Export of this oil was forbidden in order to force a drop in its price

2- PROALCOOL : In 1980 Brazil's Federal Government created this program which implemented and regulated the use of hydrated ethanol as fuel (engine's adaptationswere needed to use this fuel) and anhydrous ethanol that could be blended with petroleum gasoline

The ethanol content in those blends started as 5% and has been increased duringthe three decades of PROALCOOL and actually varies from 20% to 25%.

PRO-ÓLEO: Program created by National Energy Commission, through Resolution No. 007 dated October 22, 1980. It was expected a 30% mixture of vegetable oils or derivatives in diesel and a full substitution at long term.

But After the drop of petroleum prices in the international market, this program was abandoned in 1986.

Launch of: National Program of Production and Use of Biodiesel (PNPB)

Launched in the Palácio do Planalto (Government seat) on December 4, 2004: Main objective is to guarantee the economically viable production of biodiesel .

Law No. 11097 dated January 13, 2005. Between 2008 and 2013, it will be possible to use blends up to 5% of biodiesel, and after this period B5 will be mandatory.

Eight parameters are established to control the quality of diesel and diesel/biodiesel blend:oxidation stability, composition, volatility, viscosity, combustion, copper corrosion, contaminants, and lubricity.

History and policy of biodiesel in Brazil Gabriella P.A.G. Pousa, André L.F. Santosa and Paulo A.Z. Suarez

Energy Policy, 35: 5393- 5398

Brazil’s biodiesel production target for 2020 with different feedstocks

Soybean Sunflower/rapeseed Jatropha curcas Oil Palm

(red) Direct land-use changes; (blue) indirect land-use changes

Estimated biodiesel production costs by region, in US$/L

History and policy of biodiesel in Brazil Gabriella P.A.G. Pousa, André L.F. Santosa and Paulo A.Z. Suarez

Energy Policy, 35: 5393- 5398

Lapola et al., PNAS 2010 Indirect land-use changes can overcome carbon savings from biofuels in Brazil

Modeled direct (A) and indirect (B) LUC caused by the fulfillment of Brazil’s biofuel (sugarcane ethanol and soybean biodiesel) production targets for 2020.

Genetic and genomic approaches to enhance production of plant oil