Biofuels and industrial chemicals from microbes

Fossil fuels

Limited supplyPollutingUsed also for chemicals for synthesis of e.g. plasticsEventually oil and coal will be too precious to burn anyway

Biofuels and Industrial chemicals from microbesCan be liquid or gasFrom renewable biomass, frequently waste from other processes, no dwindling stocksLess pollutingMicrobes can also produce chemical feedstocks for syntheses

Alkanes: methaneNatural gas from gas/oil fields or town gas produced from coalProduced by anaerobic bacteria (methanogens) found in swamps, organic sediments and rumen of ruminantsExploited locally as methane digesters where economic animal faeces converted to usable fuel

Methane production by microbesBy a mixed population of bacteria First population hydrolyse fats, proteins and carbohydrates to fatty acids, amino acids, and sugars Second population metabolize these to organic acids (acetic [mainly] propionic and butyric)Third population oxidize acids to corresponding alkanes and carbon dioxideCarbon dioxide, acetic acid converted to methane

ButanolAcetone, butanol, butyric acid and isopropanol (plus higher homologues) obtained from Clostridium spp. fermentations Fermentable base usually starch, mollasses, or hydrolysed cellulose Amount of each product depends on strain and fermentation conditions

Clostridial fermentationsAcetone-butanol (C. acetobutylicum) yields also butyric, acetic acids, acetoin, ethanol CO2 & H2Butanol-isopropanol (C. butylicum) yields also butyric, acetic acids, CO2 & H2Butyric-acetic acid (C. butyricum) yields also CO2 & H2

Industrial acetone-butanol fermentationWeizmann - basic research on Clostridial fermentation Acetone produced used to make cordite (explosive) during W W I Produced riboflavin as a byproduct Butanol feedstock for lacquers, rayon, plasticizers, coatings, detergents, brake fluids, butadiene (synthetic rubber)solvent for fats, waxes, resins, shellac, varnishextractant/solvents in food industry

Butanol todayLargely from petroleum in the West Beer molasses in USSR Still performed in China As the price of oil escalates fermented butanol may become important again

Butanol as a fuelFuel properties Octane enhancing properties when mixed with gasoline High heat of combustion -- butanol: water 4:1 equivalent to anhydrous ethanol More volatile than methanol or ethanol Highly miscible with gasoline and diesel

Butanol productionBatch processUnstirred -- gaseous by-products mix fermentationsStarch or molasses fermentable base (corn and by products in US)

Butanol fermentationanaerobiosis achieved with carbon dioxide purge low level of inoculum pH drops as organic acids formed pH rises as acids metabolized to neutral solvents final concentration 2 %acetone/butanol/ethanol 6/3/1 final yield 37%carbon dioxide by-product sold products recovered by distillation

Problems with butanol fermentationLactobacillus contamination Phage infection Product inhibition Cost of distillation Multiple products Commercial viability depends on oil price

Industrial ethanol30 billion litres globally per annum 70 % by fermentation 30 % by catalytic hydrogenation of ethyleneFermentation ethanol 12% for beverages20% solvent/feedstock68% fuel

Ethanol as a fuelCan be used alone on as a mixture "Gasohol" ethanol mixed with gasoline Brazil makes 46 % global ethanol -- offsets oil imports -- but now has to import ethanol! Brazilian ethanol from cane sugar (sucrose) fermented by yeast can use fermentable bases from plant and dairy wastecomplex polysaccharides require pre-processing

*The world is going towards biofuels

Chart1

1.1

1.2

1.9

2.3

3.1

3.8

3.5

3.75

3.95

4

4

4.15

4.1

4.15

4.5

4.8

4.9

5.45

5.1

5

4.85

5

6

7

10.77

12

Year

Billions of gallons

World Ethanol Production 1980-2005Source: Worldwatch/International Energy Agency and F.O. Licht

2004 prod

Ethanol Production and Capacity USA and Brazil

Existing plantsActual prod (2004)Actual Existing CapacityPlants under ConstructionPlanned Capacity AdditionsInstalled Capacity (end 2006)

USA893,4104,075.402311475222.4

Note: All #'s in millions of gallons

2004 prod

1100

1200

1900

2300

3100

3800

3500

3750

3950

4000

4000

4150

4100

4150

4500

4800

4900

5450

5100

5000

4850

5000

6000

7000

10770

11500

Year

Millions of gallons

World Ethanol Production 1980-2004Source: Worldwatch/International Energy Agency and F.O. Licht

US Brazil 82-04

YearUnited StatesBrazil

1982350977

19833751,374

19844301,744

19856102,113

19867102,800

19878302,827

19888453,104

19898703,329

19909003,042

19919503,359

19921,1003,090

19931,2002,981

19941,3503,354

19951,4003,324

19961,1003,799

19971,3004,070

19981,4003,675

19991,4703,435

20001,6302,798

20011,7703,047

20022,1303,335

20032,8103,912

20043,4103,989

Source: Compiled by Earth Policy Institute from U.S. Energy Information Administration, cited in Renewable Fuels Association, Homegrown for the Homeland: Ethanol Industry Outlook 2005, Washington, DC: 2005, pp. 2, 14-15; So Paulo Sugarcane Agroindustry U

US Brazil 82-04

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00

00

00

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00

00

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USA

Brazil

Year

Millions of gallons

Ethanol Production in Brazil and USA, 1982-2004

By Country

CountryProduction2004 World Ethanol Production

Brazil3989(All grades, in millions of gallons)

China964Brazil3989Italy40

India462U.S.3535Australia33

France219China964Japan31

Russia198India462Pakistan26

South Africa110France219Sweden26

U.K.106Russia198Philippines22

Saudi Arabia79South Africa110South Korea22

Spain79U.K.106Guatemala17

Thailand74Saudi Arabia79Cuba16

Germany71Spain79Ecuador12

Ukraine66Thailand74Mexico9

Canada61Germany71Nicaragua8

Poland53Ukraine66Mauritius6

Indonesia44Canada61Zimbabwe6

Argentina42Poland53Kenya3

Italy40Indonesia44Swaziland3

Australia33Argentina42Others338

Japan31Total10770

Pakistan26Source: F.O. Licht

Sweden26

Philippines22

South Korea22

Guatemala17

Cuba16

Ecuador12

Mexico9

Nicaragua8

Mauritius6

Zimbabwe6

Kenya3

Swaziland3

Others338

Total10770

Sheet2

YearBillions of Gallons

19801.1

19811.2

19821.9

19832.3

19843.1

19853.8

19863.5

19873.75

19883.95

19894

19904

19914.15

19924.1

19934.15

19944.5

19954.8

19964.9

19975.45

19985.1

19995

20004.85

20015

20026

20037

200410.77

200512

Sheet2

Year

Billions of gallons

World Ethanol Production 1980-2005Source: Worldwatch/International Energy Agency and F.O. Licht

*The world is going towards biofuels

*

Sugar/Starch feedstockSugar cane - sucrose

Corn - starchNot a preferred option for NZBrazil

United States

Fuel alcohol in BrazilSugar cane: 53% converted into alcohol 47% converted into sugar

Sugar: 50% domestic marketEthanol production capacity: 16.0 billion litres

Ethanol production: 12.5 billion litres or 205,000 barrels of oil equivalent per day

Total production of sugar & alcohol: 40 million tons of sugar equivalent.

Ethanol from maizeDry milling separates oil from starch -- generates animal feed Starch gelatinized and digested to hexoses (mostly glucose) using thermostable amylasesGlucose base fermented by S. cerevisiae In continuous or batch mode at 32-38 oC and pH 4.5 -5.0Generate "beer" of about 10% alcohol Yeast recovered for reuse Beer distilled, azeotrope distilled (half the cost of whole procedure)

Improvements to ethanol fermentationsUse oleyl alcohol as continuous extractant passed through fermenter Top layer removed and centrifuged - fermentation mix returned to fermenter Ethanol recovered by flash vaporization - less volatile solvent recycled

Alternatives to yeastZymomonas spp. afford higher alcohol yield but have low alcohol tolerance and ferment only glucose, fructose, sucrose genetic engineering to improve yield and range of fermentable substrates E coli engineered with Zymomonas alcohol dehydrogenase and pyuvate decarboxylase produce ethanol under aerobic and anaerobic conditions

Ethanol from lignocellulosic materialBillions of tonnes of waste products produced per year - under -utilized resource Composition Lignin (10-35 %) polymer of phenolic alcohols - not degraded by microbes Cellulose (15-55 %) polymer of glucose - not readily degraded by microbes Hemicellulose (25-85 %) - hexose (glc, galactose, mannose/pentose (xylose, arabinose) heteropolymer - few microbes ferment pentoses to alcohol but this is required for commercial viability

Lignocellulosic feedstock Plant residues, agricultural wastes, forestry biomass Large, cheap resource

Lignocellulose pretreatmentPretreatment bymilling chemical pulpingacid/alkali treatment solvent extractionsteam treatmentsUsually require acid (0.5 - 5% hot sulphuric acid under pressure or conc hydrochloric acid at ambient temperature) or amylase treatment for saccharification

Lignocellulose pretreatment (2)Acids corrosiveNeed acid recycling to make process commercially viableAcid hydrolysis hard to control hemicellulose preferentially degraded and resulting sugars degraded by the acidSugars generated: glc, cellobiose (disaccharide), xyloseXylose not fermented by yeast Organisms that ferment xylose produce poor yields of ethanol, and are ethanol intolerant

LignocelluloseCrystalline cellulose ~ 44%Hemicellulosic polysaccharides ~30%Lignin ~ 26%

Lignocellulose Pre-treatment

160-260C, steam, acid/alkaline treatment

Cooling required prior to fermentation Pre-treatment - 160-260C Saccharification - 45-50C Fermentation - 25-30C Contributes to high operating costs of bioethanol production

*Starch To Ethanol -Reaction Mechanism- Enzyme+EnergySUBSTRATE + H2O ---------------------------> PRODUCT (Starch) Alpha Amylase (SPEZYME Fred, SPEZYME Xtra) (Dextrins)

EnzymeSUBSTRATE + H2O ---------------------------> PRODUCT (Dextrins) Glucoamylase (Optidex L400, Fermenzyme) (Glucose)

YeastSUBSTRATE ----------------------------> PRODUCT (Glucose) (EtOH,CO2,Biomass)

*Conventional Ethanol Production ProcessGRINDINGSLURRY TANKWaterLiquefactionThermo-StableAlpha AmylaseGlucoamylaseYeastAlcoholRecoverySaccharificationFermentationDistillation &DehydrationSTORAGETANKDDGS*JET COOKER>100 C58 MINSECONDARYLIQUEFACTION95 C~90 MIN*60 C810 HRS(optional)MiloCornWheatRyeBarleyTapioca

Biofuel Technology InnovationPreparation/glycationFermentationEnrichment/dehydrationDrainingMaterial productionDevelopment of energy conservation technology for separation through membranes instead of distillationSelection of high-yield plants growing in non-cultivatable land and development of technologies for growing such plantsDevelopment of enzymes featuring higher ethanol yields and lower costs

Fermentation of xyloseUse bacterial xylose isomerase to convert xylose to xylulose (pentose phosphate phosphate pathway intermediate) Genetic engineering make yeast produce bacterial xylose isomerase themselves introduce genes from yeasts (Candida, Pichia spp) able to ferment pentoses (xylose reductase/xylitol dehydrogenase) into S cerevisiae Genetically engineered bacteria (Zymomonas, E. coli) utilize xylose

Fuel ethanolMain characteristics are in line with Sustainable Development:High performance & versatile energy source Environmentally friendly & safe Renewable Energy efficient

SummaryPossible to produce range of chemicals by microbial actionMost important is industrial ethanolButanol, acetone and organic acids can be produced by anaerobic fermentationsCommercial viability depends on oil price

*Brazil has a well established industry that uses sugar cane as a feedstock for yeast fermentation, however NZ does not have the right climate to grow this crop.In the US, the preferred feedstock for bioethanol production is cornHowever, if we were to use the starch from corn alone, it would require a tremendous amount of land area to produce enough corn that could be converted into sufficient volumes of bioethanol to meet our needs. *A more sustainable and effective option is for yeast to ferment feedstock that comes from lignocellulose. And as Donna Giltrap discussed before me, this could come from plant residues like leftover stalks and leaves from corn, waste plants from agricultural production and forestry biomass. Because lignocellulose is already present in large amounts in the form of waste, it would be a cheap resource to utilise. *Unfortunately, the composition of Pinus radiata, and other forms of lignocellulose makes it challenging to be used in bioethanol industries.. Lignocellulose comes from plant cell walls and is composed mostly of crystalline cellulose, tightly packed into microfibrils, which are partially coated in an interconnecting network of polysaccharides known as hemicelluloses. This tight network of polysaccharides is embedded and linked together by lignin, which in pine, is difficult to break down as it is present in large amounts. *Because of this, lignocellulose requires significant pre-treatment prior to it been used as a feedstock for yeast fermentation.Various pre-treatments, like very high temperature, steam explosion, acid or alkaline treatments can be employed to remove the lignin,and break down the hemicelluloseAnd by the end of the process, the cellulose microfibrils are loosened

*The optimal temperature for yeast fermentation is 25-30C. Therefore, after pretreatment and saccharification, the feedstock must be cooled extensively. This contributes to the high operating costs required to run a bioethanol production facility, and such costs would make it difficult to establish and run a local bioethanol industry in New Zealand based on lignocellulose from forestry waste. *