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 Sugarcane Sugar beet Sorghum Cheese Whey Brazil, India Europe (France) India New Zealand Milling for sugars extraction, and fermentation of sugars United States, China, Canada Corn China, Canada, Europe Wheat Thailand Cassava Milling, liquefaction, saccharification, and fermentation of sugars

3. Bioethanol production worldwide 89%

4. Brazil: main exporter United States, Japan, Europe: main importers BrazilVehicles use ethanol in the pure form or in mixture with the gasoline, where ethanol corresponds up to 25% of the mixture United StatesEthanol is used in two forms: mixed with gasoline in the maximum proportion of 10%, or in mixtures containing 85% ethanol and 15% gasoline, as an alternative fuel BoliviaCurrent blend levels are 10%, but efforts are being directed to expand ethanol blends to 25% ThailandGasoline must be blended with 10% ethanol ColombiaRequires 10% ethanol blends in cities with populations over 500,000 ChinaSome regions of China use mixtures containing up to 10% ethanol in gasoline IndiaAddition of 5% ethanol to gasoline is mandatory. Efforts will be directed to increase the ethanol percentage in the mixture to 10% Canada5% of all motor vehicle must be ethanol or biodiesel SwedenMixtures containing 5% ethanol in gasoline are used JapanThe replacement of 3% of gasoline by ethanol is authorized, but efforts will be done to increase this value to 10% Bioethanol consumption worldwide

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

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

7. Obtainment of a fermentable sugars solution Fermentation of sugarsEthanol separation and purification LIGNOCELLULOSIC BIOMASS Distillation Ethanol Milling Cellulose conversion (hydrolysis) Pre-treatment Fermentation (Xylose) (Glucose) Bioethanol production from non-conventional sources – 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. Challenges to be overcome for an efficient bioethanol production from lignocellulosic biomass 1) High energy consumption for biomass pretreatment Cellulose fibers (1) (2) (3)

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

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

11. 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 Challenges to be overcome for an efficient bioethanol production from lignocellulosic biomass

12. Sweet cornJerusalem artichoke Sweet potato Sweet sorghum Other energy crops

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 1

14. Sweet Sorghum as an energy crop It has a photosynthetic efficiency (~ 4 g biomass/MJ of solar radiation) two times or more that of C3 crops (forest) It is able to grow anywhere in dry climates with high yields of fermentable sugars, grains and lignocellulosics. In some regions it is possible to obtain two plantations per year reaching full maturity and a large production. It has low water requirements 1/3 of sugar cane, 1/2 of corn, 1/4 of Short Rotation Forestry. 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 giganteus properties 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. 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 Miscanthus x giganteus ethanol production yield

19. Bioethanol production from non-conventional sources CHEESE WHEY Liquid remaining after the precipitation and removal of milk casein during cheese-making Represents 85- 95% of the milk volume and its world production is estimated to be over 10 8 tons/ year Lactose (5-6% w/v) is assumed to be responsible for 90% of the whey’s BOD and COD. Biological treatment by conventional aerobic process is very expensive 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. Bioethanol production from non-conventional sources Obtainment of a fermentable sugars solution Fermentation of sugarsEthanol separation and purification CHEESE WHEY Distillation Ethanol Fermentation (Lactose) Concentrated cheese whey or cheese whey powder solution  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 Cheese whey concentration: by ultrafiltration and/or reverse osmosis processes. Yeasts that ferment lactose: Kluyveromyces lactis, K. marxianus, Candida pseudotropicalis, genetically modified Saccharomyces cerevisiae. Important process considerations:

22.  8 million tons of lactose (worldwide annual whey production) ~50% not transformed into added-value sub-products  ~2.3 million m 3 ethanol considering a 85% conversion yield Worldwide production of bioethanol for fuel in 2008: ~65 million m 3 Whey to bioethanol…

23. Whey to Ethanol Industrial Plants Carbery Milk Products  IrelandCarbery Milk Products since 1978, potable ethanol & ethanol for fuel (since 2005) 11 000 tons ethanol /year Fonterra  New ZealandFonterra Anchor Ethanol (Fonterra subsidiary) potable ethanol & ethanol for fuel (since 2007) 17 million liters ethanol /year Golden Cheese Land O’Lakes  United StatesGolden Cheese Land O’Lakes Müllermilch  GermanyMüllermilch near Dresden; 10 million litres ethanol /year from dairy by-products

24. Whey permeate biotechnological treatment Flocculent Saccharomyces cerevisiae strains able to metabolize lactose Continuous high cell density systems  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 -1 h -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. 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 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

27. Sun light Water CO 2 nutrients ETHANOL O2O2 O2O2 The microalgae Chlorella vulgaris, particularly, has been considered as a promising feedstock for bio- ethanol production Bioethanol production from non-conventional sources

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

29. 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 CO 2 Advantages of this process: Bioethanol production from non-conventional sources

30. SourceEthanol yield (gallons/acre) Ethanol yield (L/ha) Corn stover112-1501,050-1,400 Wheat2772,590 Cassava3543,310 Sweet sorghum326-4353,050-4,070 Corn370-4303,460-4,020 Sugar beet536-7145,010-6,680 Sugarcane662-8026,190-7,500 Switch grass1,15010,760 Microalgae5,000-15,00046,760-140,290 Bioethanol yield from different sources: Bioethanol production from non-conventional 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..... Lignocellulose Microalgae Don’t affect the food provision  Development of ethanol production systems all over the world Cheese whey Agave