“BIOETHANOL FROM NON-CONVENTIONAL SOURCES”

1. “BIOETHANOL FROM NON-CONVENTIONAL SOURCES” José A. Teixeira IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering University of Minho, PORTUGAL e-mail: jateixeira@deb.uminho.pt

2. Raw materials and processes currently used for bioethanol production Simple Sugars Starches Sugarcane Brazil, India Corn United States, China, Canada Sugar beet Wheat China, Canada, Europe Europe (France) India Sorghum Cassava Thailand Cheese Whey New Zealand Milling for sugars extraction, and fermentation of sugars Milling, liquefaction, saccharification, and fermentation of sugars

3. 89% Bioethanol production worldwide

4. Brazil: main exporter United States, Japan, Europe: main importers Bioethanol consumption worldwide

5. Rice straw Wheat straw Sugarcane bagasse LIGNIN CELLULOSE HEMICELLULOSE Bioethanol production from non-conventional sources Bioethanol from lignocellulosic biomass Technology under development

6. Pre-treatment: diluted acid hydrolysis Bioethanol production from non-conventional sources Hemicellulose Cellulose Cellulose hydrolysis:concentrated acid hydrolysis and enzymatic hydrolysis

7. LIGNOCELLULOSIC BIOMASS (Xylose) Fermentation Milling Distillation (Glucose) Fermentation Obtainment of a fermentable sugars solution Fermentation of sugars Ethanol separation and purification Pre-treatment Ethanol Cellulose conversion (hydrolysis) Bioethanol production from non-conventional sources Bioethanol from lignocellulosic biomass – Iogen (straw – Canada); Abengoa (straw – Spain, US); Etek (softwood – Sweden); Elsam (straw –Denmark); TMO (straw etc. –UK); Tavda (wood – Russia); NEDO (rice straw – Japan)

8. (1) (3) (2) Challenges to be overcome for an efficient bioethanol production from lignocellulosic biomass 1) High energy consumption for biomass pretreatment Cellulose fibers

9. Challenges to be overcome for an efficient bioethanol production from lignocellulosic biomass 2) Development of a suitable and economically viable hydrolysis process step Specific enzymes Glucose CELLULOSE

10. Challenges to be overcome for an efficient bioethanol production from lignocellulosic biomass 3) Improvement in the conversion rate and yield of hemicellulose sugars fermentation HEMICELLULOSE SUGARS Ethanol Pentoses (Xylose and Arabinose) and Hexoses Toxic compounds: low sugars conversion yield

11. Challenges to be overcome for an efficient bioethanol production from lignocellulosic biomass 3) Other important considerations for the process implementation • To develop microorganisms able to metabolize pentose and hexose sugars simultaneously withstanding the stress imposed by the process inhibitors • To evaluate the process scalability • To perform an analysis of the costs involved for commercial production • To establish alternatives for the recovery of pretreatment chemicals and wastewater treatment

12. Other energy crops Sweet potato Sweet corn Jerusalem artichoke Sweet sorghum

13. Carbohydrate and expected ethanol yields for sweet corn, Jerusalem artichoke, sweet potato ad sweet sorghum Current ethanol yield from grain corn in the US and sugarcane in Brazil is approximately 3,500 and 6,000 L/ha, respectively.1

14. SweetSorghum as anenergycrop • Ithas a photosyntheticefficiency (~ 4 g biomass/MJ of solar radiation) twotimesor more thatofC3 crops (forest) • Itisable to growanywhereindryclimateswithhighyieldsoffermentablesugars, grainsandlignocellulosics. • In some regionsitispossible to obtaintwoplantationsperyearreachingfullmaturityand a largeproduction. • Ithaslowwaterrequirements 1/3 of sugar cane, 1/2 ofcorn, 1/4 ofShortRotationForestry. • Sweet sorghum (Sorghum bicolor) is frequently called as smart crop for its ability of not only produce food but fuel as well

15. Agave vs sucarcane needs

16. Agave and sugar cane bioethanol production

17. Miscanthus x giganteusproperties • relatively high yields — 8-15 t/ha (3-6 t/acre) dry weight, • low moisture content (as little as 15-20% if harvested in late winter or spring), • annual harvests, providing a regular yearly income for the grower, • good energy balance and output/input ratio compared with some other biomass options, • low mineral content, especially with late winter or spring harvest, which improves fuel quality. • can be grown in a cool climate like that of northern Europe

18. Miscanthus x giganteus ethanol production yield • a typical acre of corn yields around 7.6 tons of input per acre and 756 gallons of ethanol.. • switchgrass, which yields around 3-6 tons of biomass and 400-900 gallons of ethanol fuel • giant Miscanthus is capable of producing up to 20 tons of biomass and 3,250 gallons of ethanol fuel

19. Represents 85-95% of the milk volume and its world production is estimated to be over 108 tons/ year Lactose (5-6% w/v) is assumed to be responsible for 90% of the whey’s BOD and COD. Liquid remaining after the precipitation and removal of milk casein during cheese-making CHEESE WHEY Biological treatment by conventional aerobic process is very expensive Bioethanol production from non-conventional sources Bioethanol from cheese whey Bioconversion of lactose to ethanol represents a process which can provide a value-added product from cheese manufacturing, allied with efficient bioremediation of plant effluent.

20. Obtainment of a fermentable sugars solution Fermentation of sugars Ethanol separation and purification CHEESE WHEY Concentrated cheese whey or cheese whey powder solution (Lactose) Fermentation Distillation Ethanol Bioethanol production from non-conventional sources Bioethanol from cheese whey • Direct fermentation of whey or whey permeate to ethanol is generally not economically feasible because the low lactose content results in low ethanol titre (2–3% v/v), making the distillation process too expensive.

21. Bioethanol production from non-conventional sources Bioethanol from cheese whey Important process considerations: • Cheese whey concentration: by ultrafiltration and/or reverse osmosis processes. • Yeasts that ferment lactose: Kluyveromyces lactis, K. marxianus, Candida pseudotropicalis, genetically modified Saccharomyces cerevisiae.

22. Whey to bioethanol… • 8 million tons oflactose (worldwideannualwheyproduction) • ~50% not transformed into added-value sub-products • ~2.3 millionm3ethanolconsidering a 85% conversionyield • Worldwideproductionofbioethanol for fuel in 2008: ~65 million m3

23. Whey to Ethanol Industrial Plants • IrelandCarberyMilkProducts • since 1978, potableethanol & ethanol for fuel (since 2005) • 11 000 tons ethanol /year • New Zealand Fonterra • AnchorEthanol (Fonterrasubsidiary) • potableethanol & ethanol for fuel (since 2007) • 17 millionlitersethanol /year • UnitedStatesGoldenCheeseLandO’Lakes • Germany Müllermilch • near Dresden; 10 million litres ethanol /year • from dairy by-products

24. Flocculent Saccharomyces cerevisiae strains able to metabolize lactose Continuous high cell density systems Whey permeate biotechnological treatment • S. cerevisiae traditionally used in industry • Molecular biology techniques well developed • Good fermentative capacity • Higher volumetric productivity • Improvement of separation processes • Higher stability

25. A Saccharomyces cerevisiae strain that efficiently metabolizes lactose was developed, with a lactose metabolization capacity comparable to the one presented by other natural lactose users • A high ethanol productivity (10 gl-1h-1) system using cheese whey as a substrate was developed • The developed fermentation system proved its long term stability • Pilot scale experiments validated the developed fermentation process

26. Fermentation of Cheese Whey powder solutions by T1-E 110–150 gL-1 Lactose + Corn Steep Liquor (10 gL-1) Repeated-batch operation with biomass recycling by flocculation 6-L Air-lift Bioreactor Aerated at 0.1 vvm pH 4.2 ± 0.2 Temperature: 30 ± 1 ºC • 8% (v/v) ethanol (max.) – Ethanol productivity 0.7 gL-1 h-1

27. Sun light O2 O2 CO2 CO2 ETHANOL CO2 nutrients Water nutrients Water Bioethanol production from non-conventional sources Microalgae as a feedstock for bioethanol production The microalgae Chlorella vulgaris, particularly, has been considered as a promising feedstock for bio-ethanol production

28. Bioethanol production from non-conventional sources Microalgae as a feedstock for bioethanol production Technology under development sugars Fermentation Enzymatic Starch hydrolysis Microalgae cultivation Cell rupture ETHANOL Some algal species are able to conduct self-fermentation

29. Bioethanol production from non-conventional sources Microalgae as a feedstock for bioethanol production Advantages of this process: • microalgae can be harvested batch-wise nearly all-year-round • they grow in aqueous media, but need less water than terrestrial crops, therefore reducing the load on freshwater sources • the ability of microalgae to fix CO2

30. Bioethanol production from non-conventional sources Bioethanol yield from different sources:

31. Conclusions • World ethanol production and consumption will continue to grow strongly • Corn is the main raw material used today, but in the future..... Don’t affect the food provision Microalgae Cheese whey Lignocellulose Agave • Development of ethanol production systems all over the world

32. Thank you for your attention!