Ovvero: come aumentare (per ora) il prezzo del cibo

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1 Ovvero: come aumentare (per ora) il prezzo del cibo
Biofuel crops Ovvero: come aumentare (per ora) il prezzo del cibo

2 Qualche numero * Consumo combustibili liquidi: benzine (12,7 Mt), gasolio autotrazione (25,4 Mt) olio combustibile (8,3 Mt) la domanda complessiva di gasoli (30,6 Mt) (prodotti petroliferi totali, pari a 84,7 Mtep) * Consumo di energia totale in Italia nel 2006 pari a 195,6 Mtep * Nel 2006 l'Italia ha importato 87 milioni di tonnellate di greggio * Consumo di gas naturale: 69,7 Mtep Consumo italiano circa 709 M barili /anno 1 barile sono 159 litri, 1 TEP sono 7.5 barili Consumo italiano petrolio: 94,3 M TEP

3 Un poco di storia Il biofuel storicamente più usato: il legno
Comporta una serie di vantaggi (rinnovabile, spesso accessibile facilmente, a basso costo) e di svantaggi (combustione spesso poco efficace con rilascio di polveri sottili, poco pratico,...) …‘‘traditional’’ biofuels are not new, have not always been good for health or for the environment and have competed with food production. Will the next generations be better?

4 Biofuels Ethanol (sugar fermentation) Butanol (fermentation)
Diesel (vegetable oil trans-esterification) Methane/H2 (waste fermentation) Microbial Fuel Cells

5 Sugar soln. Corn Ethanol + CO2 Ethanol saccharification
α-amylase, gluco-amylase Fermentation Cooking Sugar soln. Corn A bushel of corn (56 lbs.) used in ethanol manufacture yields the following products and co-products:  17.6 lbs Ethanol,  17 lbs. Dry distiller’s grains,  18.4 lbs. Carbon dioxide Ethanol + CO2 Distillation Ethanol 1 ac = 0.4 ha; 1 gal = 4.54 l; 1 lb = 0.45 kg; 1 bu = 36.3 l Yield: lbs/bu = 100 gal/ton (Theoretical 3.1 gal/bu) Yield: bu/ac = 430 gal/ac; Yield: 4824 l/ha Yield 12.7 l/25.4 kg l/t

6 Ethanol Production in the U.S.
Renewable Fuels Standard, RFS = amount of ethanol to be blended 12.0 billion gal/year by end of 2006 One new dry grind ethanol plant every 30 days Over 100 dry mill plants in operation or under construction

7 Where biotech can help? Energy consuming
α-Amylase Breaks starch into dextrins or short chains in Liquefaction. Reduces viscosity for pumping. Bacillus licheniformis Glucoamylase Releases sugars from dextrins or starch chains in Saccharification. Aspergillus niger, Trichoderma reesei Pullulanase Breaks branched amylopectin starch molecules into straight chains. Protease Breaks starch-protein matrix for yeast nutrition. Trichoderma reesei Phytase Converts phytic acid to Pi. Improves Liquefaction, Fermentation and animal nutrition of DDGS

8 Corn wet vs. dry milling Products: Ethanol Corn Syrup Corn Gluten Meal
Corn Oil CO2

9 Prodotti ad alto valore
Co-Products: DDGS (Distiller’s dried grain Fiber Steep Water (acqua di macerazione?) Corn Gluten Feed Senza questi co-prodotti la produzione di Etanolo NON sarebbe economicamente conveniente

10 Microorganisms capable of producing n-butanol by fermentation are Clostridium acetobutylicum, C. beijerinckii, and C. tetanomorphum. Fermentazione butanolica: Glucosio  2 Acetil CoA  butanolo

11 Butanol plant - Commercial Solvents Corp. Terra Haute, Indiana
Commercial Solvents Corporation was established at the end of World War I and earned distinction as the pioneer producer of acetone and butanol by fermentation processes developed and patented by Dr. Chaim Weizmann

12 Caratteristiche carburanti
Il butanolo contiene più energia a parità di peso e non corrode

13 Butanol as a transportation fuel
Butanol (4-carbons) is more like petrol (4-12 carbons) Higher energy density than ethanol (88% vs. 66%) Less corrosive and less water soluble than ethanol 85% Butanol/gasoline blends used in unmodified engines Can use same pipelines as petrol Easier to integrate into existing transportation infrastructure The n-butanol biosynthetic pathway. The enzymes in green are from Clostridium beijerinckii. Enzymes in black are from other organisms: AtoB, Escherichia coli; Erg10, S. cerevisiae; PhaA, Ralstonia eutropha; PhaB, Ralstonia eutropha; Ccr, Streptomyces collinus. Each enzyme candidate was evaluated in the pathway for n-butanol production (except thl, which is native to Clostridia). Slide from: “Metabolic Engineering of New Routes to Biofuels” by Steve Wilkinson

14 Partendo dal metabolismo centrale (glicolisi, ciclo di krebs e sintesi di aminoacidi) è possibile sintetizzare diversi composti utilizzabili come carburanti o comunque precursori per l’industria chimica

15 Higher alcohols through synthetic non-fermentative pathways
Atsumi et al (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86-89

16 Biodiesel Estrazione di olio da colture oleaginose (colza, girasole, soia, palma da olio, ricino...) Trasesterificazione per ottenere l’estere (metilico in genere) dell’acido grasso Separazione degli esteri metilici dal glicerolo Gli esteri sono utilizzati come carburanti Il glicerolo è sottoprodotto ad alto valore aggiunto

17 Trasesterification FAME
Sostituzione della funzione alcolica del glicerolo con la funzione di un alcool primario a catena corta (il più usato è il Metanolo) La reazione può avvenire per catalisi acida, basica o con diversi altri modi (sonicazione, lipasi, controlled substrate feeding...) Transesterification typically takes place in batch and takes one or more hours to reach high levels of conversion. It is a typical two-phase reaction due to the immiscibility of oil and alcohol, therefore, the rate of transesterification is primarily controlled by the rate of mass transfer between the methanol and oil phases.

18 Nuovi reattori e processi per aumentare le rese e diminuire i tempi (costi)

19 Economy (settimanale) 09/04/2008

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21 The second generation biodiesel Special oilseeds
Ricinus communis (castor bean) Pongamia pinnata Calophyllum inophyllum Jatropha curcas (black vomit nut) Jatropha curcas is the most highly promoted oilseed crop: * can grow in the desert without water and fertilizer, but without commercial yield * fuel properties of Jatropha biodiesel are comparable to commercial diesel None of the websites promoting its use mention its common name: black vomit nut, purge nut, physic nut, nor the names of its oil: hell oil, oil infernale. The fruits contain irritants and the seeds contain alkaloids as well as curcin, a toxalbumin similar in structure and effect to ricin. The seed oil is irritant/cancer potentiator/synergist and contains curcusones, diterpenoids of the tiglian (phorbol) type with levels between 0.03 and 3.4% of phorbol esters

22 Raccolta e estrazione Raccolta a mano quando i frutti sono ancora verdi. I semi vengono spremuti con sistemi artigianali per estrarre l’olio: Price for farmers: $0.14/kg seed

23 Ingesting 4 seeds can be toxic to a child, with symptoms resembling organophosphate insecticide intoxication, yet with no known antidote for the lethal mixture. “Oh, did I mention that Japtropha has no need for pesticides and deters pests from entering the field? Yeah, jatropha is naturally disease- and pest-resistant. And the matter that is left over after the seeds have been pressed for oil is naturally high in nitrogen, phosphorus and potassium, which are the big three nutrients used to fertilize other crops. “ Problem: what do you do with the protein residue after crushing? The amino acid content of Jatropha meal is exceptional, except for low lysine. If the seed cake were rendered non-toxic and could be used as animal feed, the profitability of cultivating Jatropha, which was more expensive than diesel in India in 2005, would be ‘‘dramatically increased’’. Unesterified farmgate price of oil would be near $0.50/l, close to the retail USA pump price for processed diesel. Price of soybeans, where most of the value is in the meal, is $0.37/kg.

24 Domesticazione accelerata
Dwarf the stalks for easier harvesting as well as to increase the harvest index (seed yield divided by biomass). Suppress branching, rendering the plants to have single or less branched stalks can facilitate mechanical harvesting. Anti-shattering genes. The fruits are picked green, because if they were allowed to dry on the crop, the fruits would open, and drop the seeds to the ground. Supress curcin expression. This toxalbumin has been purified, sequenced and the gene cloned. Suppress phorbol ester production.  la pianta diventerebbe più sensibile a insetti e malattie…

25 Ricino Ricin, a toxalbumin is the major toxic protein of the seeds (0.2–3% ricin). The estimated oral lethal dose of ricin to man is 1 mg/kg. Two to four seeds may cause severe poisoning in an adult, and eight are generally fatal. The amount of ricin in the residue from manufacturing 50 l of biodiesel has sufficient ricin to kill about two average size people at the lowest ricin levels, and 30 at the highest levels. Imperative to suppress Ricin production by transgenesis

26 researchers, led by Federico García Maroto, have genetically altered the castor-oil plant so as to use it as a factory to produce bio lubricants.

27 Lignocellulosic materials for biofuels
Wheat straw & corn stover are by-products with negative value Cellulose  30-50% plant matter, produces C6 sugars (glucose) Hemicellulose  20-30% plant matter, produces C5 sugars (xylose) Lignin  15-30% biomass, “glue” holds plant together, no conversion Biofuels production from lignocellulosic biomass needs to overcome the cell-wall plant fibers recalcitrance to enzymatic hydrolysis. Bioethanol from straw achieve only a 20% efficiency of conversion due to hinderance by lignin. Pre-treatment needed to expose the crystalline cellulose core to hydrolisis by cellulase (acid, alkali, ammonia, steam explosion…)

28 Energy consuming Where biotech can help?

29  es. Energypoplar: progetto per aumentare la resa nel pioppo
Reduced lignin content can increase yields of fermentable sugars after pretreatment of plant biomass with hot acid and can also reduce or eliminate the need for this step.  es. Energypoplar: progetto per aumentare la resa nel pioppo

30 Genetically engineering plants to produce cellulases and hemicellulases, and to reduce the need for pretreatment processes through lignin modification, are promising paths to solving this problem, together with other strategies, such as increasing plant polysaccharide content and overall biomass.  ad es. nel pioppo si ottiene un aumento di resa di EtOH del 50% in seguito alla riduzione di pochi % di lignina * cultivating perennial grasses as crops such as switchgrass (Panicum virgatum) to produce biofuel is nonsense * yields can vary more than fourfold from less than two to more than nine tonnes per hectare Alternative più sensate: canne come Arundo donax e Miscantus con una produttività maggiore e che richiedono poco fertilizzante

31 2nd problem: carbohydrate recovery
I pentosi sono il principale componente delle emicellulose, ma il lievito comune non riesce a fermentarli. Altri organismi riescono a metabolizzarli e quindi si possono usare i loro geni.

32 Pentose metabolism • Introduce the following genes (enzymes) from Pichia stipitis: – D-xylose reductase (XYL1) – xylitol dehydrogenase (XYL2) – D-xylulokinase (XYL3) • Growth of this engineered yeast strain is very slow because… • …Reductive step and oxidative step both require co-factors: – NADPH and NAD+ – producing NADP+ and NADH respectively Excess accumulation of NADH under oxygen limitation

33 Terzo problema: la lignina
La lignina è un polimero molto complesso difficile da degradare Le zone più lignificate (xilema) sono le parti più difficili da biodegradare anche da parte dei microorganismi del terreno. Il reticolo del sistema vascolare rimane intatto mentre tutta la parte del mesofillo è stata distrutta dopo pochi mesi di permanenza nel residuo umido del sottobosco.

34 Third generation technologies: algae and cyanobacteria for biofuel production
Organism survival: contaminated and taken over by indigenous local organisms Growth and lipid content: Most algae either grow or alternatively they produce lipid (fat) bodies, but not both simultaneously. Carbon dioxide enrichment Light penetration Forme di domesticazione che indeboliscono la capacità di competere: occorre fornire un vantaggio o fare colture pure

35 Quattro conti sulle superfici
Di quanta terra avremmo bisogno per produrre biocarburanti come * Ethanolo e butanolo da zuccheri e amidi * diesel da oleaginose Quattro conti sulle superfici Cropping area needed to replace 15% of transport fuels in the USA Attenzione che se applichiamo questi conti all’Italia...

36 * Resa media per ettaro: Mais: 10 t,
* La superficie agricola utile (SAU) in Italia è di circa 13 milioni di ettari Nel 2006 la produzione di etanolo da canna da zucchero in Brasile è quasi di 6 mila litri per ettaro coltivato 34 milioni di veicoli italiani consumano circa mille litri ciascuno di carburante all'anno  equivalente a 5.7 milioni di ettari di suolo brasiliano coltivato a canna da zucchero. Siccome la canna da zucchero non cresce in Italia, potremmo forse usare mais o barbabietola…. Quanta energia spendiamo per produrre un litro di biodiesel o bioetanolo? (quanto carburante si consuma per produrne un litro?)  Indice ‘EROEI’ (rapporto tra energia ottenuta (ricavata) e energia spesa (investita))

37 In Italia, produzioni medie di biodiesel da colza e girasole pari a 966 litri per ettaro (850 Kg/ha). In USA di 1029 litri per ettaro. Il bioetanolo da cellulosa è molto più costoso di quello ottenuto dalla canna da zucchero e solo importanti progressi scientifici possono renderlo conveniente. Il costo non è dovuto alla materia prima (cellulosa) ma alla sua trasformazione in bioetanolo. I processi industriali attuali fanno costare il bioetanolo da cellulosa tre volte quello ottenuto da canna da zucchero. La media degli agricoltori statunitensi consuma 82 litri di carburante per ettaro di terreno per la produzione di un raccolto (senza contare l'energia per fare il concime, gli antiparassitari, i diserbanti, per fabbricare i trattori, ecc.)

38 EROEI Energy Return On Energy Investment
Per il biodiesel è un valore pari a circa 3, un impianto eolico (20-30); per l’estrazione e raffinazione del petrolio (10-100). Per il bioetanolo il valore del EROEI è intorno a 7-8 nel caso migliore. Per bioetanolo da barbabietola o da mais è vicino a 1. Se non addirittura meno, il chè significa che abbiamo bisogno di combustibili fossili per produrlo oltre che di sussidi economici (es. riduzione della tassa sul carburante) IN SOLDONI: la produzione di biodiesel da soia e girasole, sia quella dell'etanolo da mais, legno ed erba, consumano allo stato attuale tanta o più energia di quanta se ne possa ricavare, non tenendo conto né delle tasse, né dei danni ambientali.

39 First generation (conventional) biofuels
Biofuel type Specific name Biomass feedstock Production process Bioethanol Conventional bioeth. Sugar beets, grains Hydrolysis & fermentation Vegetable oil Pure plant oil (PPO) Oil crops (e.g. rape seed) Cold pressing/extraction Biodiesel fatty acid methyl/ethyl ester & transesterification Biodiesel from waste FAME/FAEE Waste/cooking/frying oil Transesterification Biogas Upgraded biogas (Wet) biomass Digestion Bio-ETBE ethyl tertiary butyl ether bioethanol Chemical synthesis Biofuels in the European Union A VISION FOR 2030 AND BEYOND Final draft report of the Biofuels Research Advisory Council ftp://ftp.cordis.europa.eu/pub/fp7/energy/docs/biofuels_vision_2030_en.pdf

40 Second generation biofuels
Biofuel type Specific name Biomass feedstock Production process Bioethanol Cellulosic bioethanol Lignocellulosic material Advanced hydrolysis & fermentation Synthetic biofuels Biomass-to-liquids (BTL) Fischer-Tropsch (FT) diesel Synthetic (bio)diesel Biomethanol Heavier (mixed) alcohols Biodimethylether (Bio-DME) Gasification & synthesis Biodiesel hybrid 1st-2nd gener. NExBTL Vegetable oils and animal fat Hydrogenation (refining) Biogas SNG (Synthetic Natural Gas) Biohydrogen Upgraded biogas Gasification & synthesis or Biological process

41 Bibliografia * Gressel (2008) Transgenics are imperative for Biofuel crops. Plant Science, 174: * Chisti Y. (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol Jan 21; * Schmer MR, Vogel KP, Mitchell RB, Perrin RK. (2008) Net energy of cellulosic ethanol from switchgrass. Proc Natl Acad Sci U S A. 105:464-9. * Searchinger et al., (2008) Use of U.S. Croplands for Biofuels Increases Greenhouse Gases Through Emissions from Land Use Change. Science (Feb. 2008)

42 Butanol Steen et al (2008) Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact. 7: 36. Atsumi et al (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86-89. Keasling & Chou (2008) Metabolic engineering delivers next-generation biofuels. Nature Biotechnology 26:298 – 299.

43 Lignocellulosic Mielenz JR. (2001) Ethanol production from biomass: technology and commercialization status. Curr. Op. Microbiol. 4: Himmel, M.E. et al., (2007) Science, 315, Hal A, Joel M, Elke N, et al. (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314: Mariam B. Sticklen (2008) Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nature Reviews Genetics 9: | doi: /nrg2336. Lionetti et al., (2010) Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion. PNAS 107: Edward M. Rubin (2008) Genomics of cellulosic biofuels Nature 454: (EROEI ethanol)

44 Hydrogen Rühle T, Hemschemeier A, Melis A, Happe T. (2008) A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtii strains. BMC Plant Biol. 8:107. Melis A, Seibert M, Ghirardi ML. (2007) Hydrogen fuel production by transgenic microalgae. Adv Exp Med Biol. 2007;616: Review. Generali Alper & Stephanopoulos (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nature Reviews Microbiology 7,