The Pichia stipitis genome

History and economics of cellulosic ethanolThomas Jeffries

Specialized Library AssociationChicago, IllinoisJuly 17, 2012

If we are to survive as a society we must find a way to convert our fossil energy capital into the means for renewable energy income.R. Buckminster FullerBiofuels have been under development for 200 yearsEthanol production from wood is much older than many think.The chemistry has not changed.Biotechnology has provided new impetus.Emphasis increased during wars and times of fuel shortage.Earliest attempts - 1819Henri Braconnot (1819) treated wood with cold 91.5% sulfuric acid, and fermented the sugarFrench chemist elected member of the Paris Acadmie des Sciences in 1823Braconnot, H. 1819. Gilbert's Annalen der Physik, 63:348-371

Commercialization in Europe - 1855Arnould (1854) used 110 parts of concentrated sulfuric acid per 100 parts of wood and obtained 80 to 90% of wood in solutionMelsens (1855) used 3 to 5% sulfuric acid under pressure at 180CPelouze (1855) erected a factory for the recovery of ethanol from wood in ParisVon Demuth, R. 1913. Zeitschr. F. angew Chemie Aufsatzteil 1913, 26:786-792 The Simonsen process (1894-1898)First comprehensive examination of engineering parametersUsed dilute acid under high pressure 15 minute hydrolysis, 0.5% sulfuric acid, 9 atm steam (180C). Yielded 26.5% sugar on a dry wood basisProduced 7.6 L ethanol/100 kg woodKressmann studied dilute acid hydrolysis at FPL from 1910 to 1922Need for ethanol in synthetic rubber synthesisExcess wood residues accumulated at sawmills ($0.50 per ton)Raw material cost was 2 cents per gallon of ethanol.Sugars from softwoods were about 70 percent fermentable while those from hardwoods were 30 percent fermentable by yeast.First US commercialization in 1910 by the Standard Alcohol Company Built a cellulosic ethanol plant in Georgetown, South Carolina to process waste wood from a lumber mill Later built a second plant in Fullteron, LouisianaEach produced 5,000 to 7,000 gal ethanol per day from wood wasteBoth were in production for several yearsRobert Rapier Sep 10, 2009Sherrad EC & Kressman FW (1945) Review of Processes in the United States Prior to World War II. Industrial and Engineering Chemistry 37(1):5-8Problems with pentoses and sugar degradationFoth noted in 1913 that the unfermentable sugars in hydrolysates mainly come from pentosansThe pentosans are completely converted to pentoses in the first cookGlucose was degraded by acid at high temperaturesFoth, G. 1913. The recovery of alcohol from wood. Chemiker Zeitung 37(120), p. 1221From 1916 to 1922, FPL took acid hydrolysis to the pilot scale

Settling tank, Single effect evaporator, HydroextractorHydrolyzerEvaporator, CondenserThese findings led to the percolation processHemicellulosic sugars (xylose , arabinose) are hydrolyzed rapidly but then break down in the acidCellulosic sugars (glucose) are hydrolyzed more slowly and are more stableUse a percolation process with rising acidity and temperature to extractMadison Wood Sugar process - 1943

Developed in response to need for ethanol for the synthesis of synthetic rubber.

Based on the Scholler process in which dilute acid is percolated over a bed of wood chips.

Differs in that dilute acid is percolated initially at a lower temperature then at progressively higher temperatures until only lignin remains.

Sugars are collected in a series of tanks, neutralized with CaO and fermented. Development of the Scholler processFollowing World War II, scientists modified the German Sholler process for use in the United StatesJ.A. Hall directed pilot plant studies at the Dow Chemical Company plant in Marquette, Michigan and Vulcan Copper and Supply Co. at Cincinnati Ohio.Designed a pilot plant to produce 11,500 gal of ethanol/day (4 million gallons/year)Based on Douglas fir (lowest xylan)0.4 to 0.85% sulfuric acid6 hour hydrolysis; 8:1 L:S ratio50 to 150 psig; 298-366FYield of 52 gallons per ton (2% beer)Vulcan Wood to ethanol plant, Springfield Oregon, 1945Designed by Ray Katzen Operated by Jerry SaemanThe plant did run and made ethanol but had lots of problems. Low concentration of sugar; lots of organic matter ran down the river; no alternative to thatJerome Saeman, May 1, 2003Tars, calcium sulfate made a hard scale and lining in pumps and valves requiring cleaning and maintenanceRay Katzen, May 6, 2003

Constructed in 1944 operated until 1946: met target of 15,000 gal/day, 50 gal per tonWood to ethanol plant, Springfield Oregon, 1945

Arial viewInteriorHistory doesnt repeat itselfTo render automotive transportation independent of fuel imports and to produce domestically this fuel in the desired quantities, are the questions to be faced from the national point of view -- Meunier 19221 bushel of corn yielded 2.4 gal EtOH in 1922Cost about $0.27/gal prior to WWIToday one bushel of corn yields 2.75 gal EtOHCosts about But it rhymesOne ton of sawdust yielded about 12 to 20 gal EtOH/ton in 1922If the manufacturing cost of producing ethyl alcohol from wood can be reduced to the same figure or nearly the same figure as that for making it from grain or molasses, there will be a large margin in favor of producing the alcohol from wood waste. -- F.W. Kressman, USDA Bulletin No. 983, 1922, p. 2.Today, one ton of sawdust could yield 70-90 gal of ethanolThe maximum theoretical yield is 110-140 galWe have made much progress with cellulosicsTwo paths to cellulose saccharification

Jerry Saeman 80th birthday1996Elwin Reese at age 62 1973

18Elwin Reese was born in Scranton, Pennsylvania On January 16, 1912; died on December 17, 1993. Worked in the laboratory for approximately 20 years after his initial retirement in 1971

Jerry Saeman was born in Cross Plains, Wisconsin in 1916Enzymatic saccharification of celluloseReese, Siu and Levinson - 1950Cellulase is not a single enzyme but a complexC1, Cx hypothesis (later replaced with endo/exo)Reese organized and chaired an ACS symposium in Washington, DC on cellulase in 1962Katz and Reese produced 30% glucose from 50% cellulose in 1968Second ACS symposium on cellulase in Atlantic City 1969Natick symposium on Enzymatic Conversion of cellulose 1975Early contributors to cellulose enzymatic saccharificationKendall KingVirginia PolytechnicGeoffery HalliwellRowett Res. InstituteKazutosi NisizawaTokyo UniversityKarl Erick ErickssonSwedish Forest Products LaboratoryKeith SelbyShirley InstituteEllis CowlingYale School of ForestryNobuo ToyamaMiyazaki UniversityTarun K. GhoseIndian Institute, New DelhiMary MandelsNatick Lab20Cowling was studying the structural features of cellulose that contributed to its susceptibility to enzymatic hydrolysis.Toyama was investigating the uses of cellulases in extraction of cell contentsNotes from Enzymatic hydrolysis of cellulose to glucose A report on the Natick Program, September 1981. Development of Trichoderma reeseiQM6a first isolated from deteriorated shelter from Bougaineville Island at the end of WW2Originally identified as T. viride; in 1977 recognized as T. longibrachiatum named T. reesei by Simmons in 1977Produces a complete extracellular cellulase complexScheduled for complete genome sequencing by DOE in 2003QM6AQM91231969QM94141971TK0411977MCG771977Linear acceleratorLinear acceleratorUV -KabicidinUVM7NG14C-30MCG801976197719781980UVnitrosoguanidineUVUV -Kabicidin21Reese, E.T. 1976. History of the cellulase program at the U.S. Army Natick Development Center. Biotechnol. Bioengineer. Symp. No. 6 91-93.

Simmons, E.G. 1977. Classification of some cellulase producing Trichoderma species. Abst. 2nd Int. Mycological Congress. Tampa Florida p. 618Development of hyper secreting strainsBland Montenecourt and Doug Eveleigh developed RutC30Looking for carbon catabolite resistance - discovered hyper-secreting strainUsed oxgall extract and phosphon D as colony restriction agentsBlocked phospholipid production

Discovery of pentose fermenting yeastsWang and Schneider - NRC, CanadaFermentation of D-xylulose (1980)Clete Kurtzman - USDA, NRRLFermentation by P. tannophilus (1981)C.S. Gong - Purdue UniversityCandida sp. Mutant (1981)Tom Jeffries - FPLAerobic conversion by C. tropicalis (1981)23Michael Flickinger emphasized the importance of xylose fermentation at the FCBM meeting in 1980The virtual community -1981-1982First international computer conference on biotechnology for fuels and chemicals; Organized through IEAOne of the very first computer conferences.Initiated by Swedish innovator; coordinated by John Black, University of Western OntarioBrought together researchers from around the world to exchange information on bioconversion for renewable fuels and chemicalsSweden, Canada, Japan, United States, Soviet Union, India, France, Mexico, Brazil (et al.)Metabolic engineering - 1984Lonnie IngramMetabolic engineering of Escherichia coliPET operon -- from Zymomonas mobilisMin Zhang, Steve PicataggioMetabolic engineering of Z. mobilis Pentose metabolic genes from E. coliAccelerating forcesEnzymes from uncultured organismsIn-vitro recombinationDirected evolutionPathway optimizationGenome-wide expression analysisMetabolic modelingPetroleum pricesSource: U.S. Energy Information Administration Annual Energy Review, Table 5.21. Composite of domestic and imported crude oil. In chained (2005) dollars, calculated by using gross domestic product implicit price deflators. See "Chained Dollars" in Glossary.Average in 2011 - $111Arab-IsraeliConflict 1973Iranian hostageCrisis 11/79-1/81Peak oil 2005?Collapse of oilcartel 1980-86US Production has passed its peak

Ethanol production has tracked with petroleum price

Global warmingWhat are the drivers?30The greenhouse effect has been recognized for 185 yearsJoseph Fourier discovered greenhouse effect in 1827John Tyndall discovered in 1861 that H2O and CO2 were largely responsibleSvante Arrhenius showed the role of CO2 in 1896 and he and Chamberlin recognized the feedback effect with water by 1905Jean Baptiste Joseph Fourier (21 March 1768 16 May 1830) was a French mathematician and physicist best known for initiating the investigation of Fourier series and their applications to problems of heat transfer and vibrations. The Fourier transform and Fourier's Law are also named in his honour. Fourier is also generally credited with the discovery of the greenhouse effect.[1]

John Tyndall FRS (2 August 1820 4 December 1893) was a prominent 19th century physicist. His initial scientific fame arose in the 1850s from his study of diamagnetism. Later he studied thermal radiation, and produced a number of discoveries about processes in the atmosphere.

Svante August Arrhenius (19 February 1859 2 October 1927) was a Swedish scientist, originally a physicist, but often referred to as a chemist, and one of the founders of the science of physical chemistry. He received the Nobel Prize for Chemistry in 1903. The Arrhenius equation, lunar crater Arrhenius and the Arrhenius Labs at Stockholm University are named after him.31Projected surface temperature of the globe in 150 yearsNine of the world's 10 warmest years since records began were in the 1990s, including.Temperatures in the 1990s were 0.33 C higher than in 1961-90 and 0.7 C higher than those at the turn of the century

32If we heed only our own needs, we endanger the capacities of the ecosystem to sustain itself. In the past 100 years the average temperature of our planet has increased by about 0.6C (1F). Even though this rise might not sound significant, it has caused the vast majority of plant and animal species to respond to spring about 5 days earlier each decade , and each decade animal and plant ranges have moved about 6 km farther north . These effects are most dramatic in the arctic. For example, the icepack in Greenland has thinned at a rate of 10 to 14 cm/yr over the past 40 years .We are already seeing the effects of global changeEach decadeSpring comes 5 days earlier Animal and plant ranges move 6 km further northIce thinning in arctic and alpine glaciersVegetation changes in arctic

Temperature correlates closely with CO2 levels395 ppm34Ice core records of gases trapped in the ice from the Russian Vostok station in East Antarctica show a remarkable correlation between atmospheric CO2 and methane concentrations and surface temperature over a 450,000 year period in which our planet has experienced four inter-glacial periods.

Supplemental text:How have global surface temperatures and concentrations of greenhouse gases such as carbon dioxide (CO2) and methane (CH4) varied over the last 420,000 years, relative to the last 150-200 years? How did "ice ages" and intervening warm periods come and go during this period of time? Is there evidence that changes in the concentration of CO2 and other greenhouse gases bring about changes in the surface temperature of the Earth? By comparison, how do the recent (the last 150-200 years) global trends in CO2, CH4, and surface temperature compare to these historic trends? From an historical perspective, how large an impact, if any, is society having on the Earth's climate system?

INTRODUCTION: Dr. Julie Palais Program Manager for Antarctic Glaciology, National Science Foundation, Arlington, VA

Climate over the Last 420,000 Years: Results and Implications The extended Vostok record of climate now enables scientists and others to compare the climatic evolution of the present-day interglacial period (often referred to as the Holocene warm period) in which we are living, with previous periods of global climate warming. As judged from this detailed record which encapsulates the main trends in global climate change, the long, stable Holocene period (the last 10,000 years) appears to be a unique feature of the Earth's climate during the last 420,000 years. The Vostok results show an overall remarkable correlation between greenhouse gases and climate over the four glacial-interglacial cycles (naturally recurring at intervals of approximately 100,000 years) which, in particular, confirm that the periods of CO2 build-up have most likely contributed to the major global warming transitions at the Earth's surface. The results further indicate that the succession of changes in climate parameters such as temperature, greenhouse gases and continental ice volume during each of the major warming periods corresponding to four glacial-interglacial (cold-warm) transitions, are similar. This suggests that the same sequence of climate triggers and feedbacks occurred in each instance as follows changes in solar insolation (as a result of changes in the Earth's orbit) is typically followed by two strong amplifiers of climate warming, with greenhouse gases acting first, followed by deglaciation (melting) due to feedbacks related to changes in the reflectivity of glacial ice (Snow and ice typically reflect sunlight, but as ice melts the amount of solar radiation absorbed at the Earth's surface increases, resulting in further warming). During these major transitions in the Earth's climate, Antarctic warming appears to precede warming in the Northern Hemisphere. The lessons from the Vostok ice core can be summarized as follows. Past changes in greenhouse gases have been initially triggered by climatically induced changes in the oceanic and terrestrial pools or reservoirs of carbon Changes in these pools of carbon resulted in the amplification of the original weak, orbitally-driven changes in the amount of solar radiation reaching the Earth's surface. Once in the atmosphere, greenhouse gases then played an important role as amplifiers of climate change, accounting for about half of the global warming observed in the Vostok ice core, corresponding to the four glacial-interglacial (cold-warm) climate transitions. The main difference between the Vostok record of climate change and the present climate situation is that today the sharp increase in greenhouse gases (i.e., approaching unique levels of greenhouse gas concentrations relative to the last 420,000 years of climate change) is being triggered by human activities at an unprecedented rate. In addition, ice-core records (and other records) of past climates indicate that changes in the concentration of greenhouse gases, whatever the causes, induce important global climatic changes. By comparison, society's impact on the concentration of greenhouse gases during the last 150 years has already enhanced the CO2 concentration of the atmosphere by an amount equivalent to the glacial-interglacial CO2 increases documented in the Vostok ice-core records described above.

Regional emissions commitment from existing energy and transportation infrastructureRegional emissions normalized by regional populationRegional emissions normalized by regional GDPFuture CO2 Emissions and Climate Change from Existing Energy Infrastructure Steven J. Davis, et al. Science 329, 1330 (2010)

Future CO2 Emissions and Climate Change from Existing Energy Infrastructure Steven J. Davis, et al. Science 329, 1330 (2010)36Global emissions of CO2 have an intergenerational effectThe last and the current generation contributed approximately two thirds of the present day CO2-induced warming.

Global mean temperatures would increase by several tenths of a degree for at least the next 20 years even if CO2 emissions were immediately cut to zero.

Friedlingstein and Solomon, 2005 PNAS 102(31):1083210836

Solid line shows contribution to CO2 by each generation continuing at same rateDotted line shows contribution if CO2 emissions were immediately stopped

CO2 is rising at a faster rate than seen in 400,000 years

Domestication Of first plants38Fluctuations in temperature and in the atmospheric concentration of carbon dioxide over the past 400,000 years as inferred from Antarctic ice-core records (45). The vertical red bar is the increase in atmospheric carbon dioxide levels over the past two centuries and before 2006.Biofuels can reduce CO2 productionEthanol, methane and biodiesel are the most immediate bioenergy sourcesEthanol and biodiesel recovered in processingMethane recovered from feedlot operationsGreatly reduces CO2 emissions Summary of energy efficienciesFuel Energy yield Net Energy (loss) or gain Gasoline 0.805 (19.5 %) Diesel 0.843 (15.7 %) Ethanol 1.34 34 % Biodiesel 3.20 220 %Source: Minnesota Department of AgricultureBiofuels account for 7% of the US automotive and light truck fuel supply >14 billion gallons of ethanol/yrVirtually all derived from grainEthanol can be blended at up to 10% by vol.Has only 2/3 the energy content of gasolineProduction of ethanol from corn is reaching unsustainable levelsCTL = coal to liquids; GE = grain ethanol; CE = cellulosic ethanol; BTL = biomass to liquids; Gas = gasolineCellulose to ethanol reduces CO2 emissions

National Academy of Sciences, National Academy of Engineering, National Research Council. (2009.) Americas Energy Future Panel on Alternative Liquid Transportation Fuels, p.250. Accessed at: http://www.nap.edu/openbook.php?record_id=12620&page=25043Isobutanol could provide 12% of US automotive and light truck fuels> 14 billion gallons of ethanol annuallyVirtually all derived from grain (corn)Ethanol can be blended at up to 10% by vol.Has only 2/3 the energy content of gasolineEquivalent to 7%Isobutanol can be blended at 16% by volHas the energy content of gasolineProduction from cellulosics is essential for market expansionDomestic biomass resource is sufficientWheat straw and forest residues are potentially the most economical feedstocksFeedstockWTAWTPPrice gap ($/dry ton)Price gap ($/gal)Corn stover9225670.96Alfalfa11826921.31Switchgrass11726901.29Miscanthus11027841.20Wheat straw7527490.70SR woody crops8924650.93Forest Residues7824540.77Source: National Research Council, 2011 Renewable Fuel Standard (prepublication)WTA = willing to accept; WTP willing to payImplicit subsidy required for cellulosic ethanol at $111/bbl oilThe US produces large amounts of biomass annuallyBasic advances are needed in cellulase saccharification and biocatalyst researchMore funding for basic energy research is desperately neededCompetitive funding for basic research in plant biology by all federal agencies totals only about 1% of the National Institutes of Healths budgetChris Somerville Science 312:1277 (2 JUNE 2006)46R. D. Perlack et al., Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-TonAnnual Supply (DOE/GO-102005-2135, Oak Ridge National Laboratory, Oak Ridge, TN, 2005).Barriers to commercializationCellulose is recalcitrant and requires large amounts of enzymes to produce sugarLignin occludes polysaccharides and inhibits enzymatic hydrolysis of carbohydratesEnergetically expensive and corrosive chemical pretreatments are required. Yeast currently used in large-scale ethanol production cannot efficiently ferment sugars other than glucose. Why are we doing this work?Ethanol fuels can help alleviate global warmingWood and agricultural residues are availableMetabolic engineering can increase ethanol production