Processing Lignocellulosic Biomass into Ethanol

Fuel ethanol from lignocellulosic biomass has the potential to provide a sustainable replacement for traditional oil-based fuels. This dissertation assesses the processing of three different lignocellulosic materials – spruce, wheat straw and giant reed – at industrially relevant solid loadings. The work is divided into two main parts. The first part deals with the degradation of biomass to sugars, focusing on the complex rheological behavior of biomass slurries and the connection to mixing during high solids hydrolysis. The second part deals with the process design of combined hydrolysis and fermentation processes, focusing on efficient xylose co-consumption at high solids loadings. Rheological characterization of steam pretreated spruce revealed strong nonNewtonian flow behavior with rapidly increasing viscosities and yield stress at high solid loadings, for instance the yield stress more than doubled when increasing the WIS content from 10 to 12 % (from 10 Pa to 24.5 Pa). Moreover, a strong effect of particle size distribution was found on both the viscosity and the yield stress. High viscosities create a need for efficient mixing during enzymatic hydrolysis of pretreated spruce. The hydrolysis rate was significantly influenced by both the amount and type of agitation. For pretreated spruce, for example, an increased agitation rate from 75 rpm to 500 rpm doubled the hydrolysis yield after 96 hours (an increase in yield from 35 to 72 %). The positive effect remained during scale-up to cubic meter scale and could be correlated to the flow conditions in the reactor. However, large discrepancies were found between different pretreated materials, and it became evident that the hydrolysis rate of giant reed was not affected by mixing. This was likely due to the much more rapid liquefaction achieved during the hydrolysis of giant reed. In addition to glucose, many potential raw materials contain considerable amounts of the pentose sugar xylose. Xylose metabolism has today been successfully implemented in Saccharomyces cerevisiae through genetic engineering, although glucose is still the preferred substrate. In this work, xylose co-consumption was significantly enhanced by applying different process design strategies. By using a dual feed strategy, xylose consumption could be increased by 25 %, which resulted in a 10 % increase in final ethanol titer. It was also found that, in the presence of high acetic acid concentrations, xylose uptake could be significantly enhanced by increasing the pH. Whether or not this was beneficial for ethanol production, however, was found to be dependent on the specific process design. Popularvetenskaplig sammanfattning Anstrangningen att framstalla nya fornyelsebara drivmedel har under de senaste artionden okat markant. Anledningarna har varit flera, dels oron for klimatpaverkan vid anvandningen av fossila branslen, dels fran ett nationellt energisakerhetsperspektiv da lokal produktion skulle kunna stabilisera tillgangen. Bioetanol ar ett av de storsta biobranslena i varlden och produceras idag framforallt fran sockerror och majsstarkelse. Nar man betraktar totala vaxthusgasutslapp ar i synnerhet majsstarkelse i langden nodvandigtvis inte hallbart. Analyser visar att ett mer hallbart alternativ ar att anvanda hela vaxtmaterial och inte bara fro-delen som det gors idag. Omvandlingen av vaxtmaterial ar dock en mycket mer komplicerad process dar materialet forst maste brytas ned enzymatiskt till fria sockermolekyler som sedan jases till etanol. Vidare bestar vaxtmaterial generellt av flera olika sockerarter. Den mest forekommande sockerarten ar glukos, som kan jasas till etanol naturlig av vanlig bagerijast, Saccharomyces cerevisiae, vilken traditionellt anvands inom etanolindustrin. For att jasa xylos, den nast mest forekommande sockerarten i manga vaxtyper, kravs daremot genmodifiering av jasten. Forskningen har nu kommit sa langt att de forsta kommersiella anlaggningarna har borjat tas i drift. For dessa anlaggningar ar vikten av att kunna arbeta vid hoga torrhalter stor, da detta forutspas ge stora ekonomiska fordelar i form av lagre investeringsoch driftskostnader. Att arbeta vid hoga torrhalter ar dock en stor processteknisk utmaning, bland annat pa grund av hog viskositet samt hoga koncentrationer av fermentationsinhibitorer. Arbetet i denna avhandling har syftat till att forsta den komplexa reologin, dvs. flodesegenskaperna, hos forbehandlat granmaterial. Dessutom kopplas dessa till hur omblandning paverkar den enzymatiska nedbrytningen av materialet till socker. Flodesegenskaperna visade sig vara starkt kopplade till mangden torrmaterial samt till fordelningen av fiberstorlek i materialet. Vidare pavisades en stark inverkan av omrorning pa den enzymatiska hydrolysen av forbehandlad gran, vilket kunde kopplas till flodesbetingelserna i reaktorn. For mer graslika material kunde dock ingen paverkan av omrorning pavisas. Detta var sakerligen kopplat till den mycket snabbare forvatskning som skedde under den enzymatiska hydrolysen. Nya processtrategier utvecklades dessutom for att med hjalp av genmodifierade jaststammar effektivare omvandla bade glukos och xylos till etanol. Genom att mata reaktorn med bade material och enzymer sa kunde xylosupptaget i processen okas med 25 %, vilket gav en 10 %-tig okning av den slutliga etanolkoncentrationen. Vid hoga koncentrationer av attiksyra kunde dessutom xylosupptaget okas markant genom en okning av pH. List of publications This dissertation is based on the following publications, which will be referred to in the text by their Roman numeral: I. Wiman M, Palmqvist B, Tornberg E, Liden G. (2011) Rheological characterization of dilute acid pretreated softwood. Biotechnol Bioeng. 108:1031 – 41 II. Palmqvist B, Wiman M, Liden G. (2011) Effect of mixing on enzymatic hydrolysis of steam-pretreated spruce: a quantitative analysis of conversion and power consumption. Biotechnol Biofuels 4:10. III. Palmqvist B, Liden G. (2012) Torque measurements reveal large process differences between materials during high solid enzymatic hydrolysis of pretreated lignocellulose. Biotechnol Biofuels 5:57 IV. Kadic A, Palmqvist B, Liden G. Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed (Submitted) V. Palmqvist B, Kadic A, Hagglund K, Petersson A, Liden G. Mixing considerations when scaling up enzymatic hydrolysis of pretreated spruce. (Submitted). VI. Olofsson K, Palmqvist B, Liden G. (2010) Improving simultaneous saccharification and co-fermentation of pretreated wheat straw using both enzyme and substrate feeding. Biotechnol Biofuels 3:17 VII. Palmqvist B, Liden G. Combining the effects of process design and pH for improved xylose conversion in high solids ethanol production from Arundo donax. (AMB Express – Accepted) I have also contributed to the following book chapter (not part of this dissertation): Mutturi S, Palmqvist B, Liden G. Developments in bioethanol fuel-focused biorefineries, In Advances in biorefineries, Ed. K. Waldron, Woodhead Publishing, Cambridge, in print, (2014). (ISBN 9780857095213) My contributions to the publications I. I participated in the design of the study, the experimental work and the discussion about the results. I was involved in the preparation of the manuscript. II. I participated in the design of the study, performed the experimental work and wrote the manuscript. III. I designed the study, performed the experimental work and wrote the manuscript. IV. I participated in the design of the study, the experimental work and the discussion about the results. I was involved in the preparation of the manuscript. V. I participated in the design of the study, performed the lab-scale part of the experimental work and wrote the manuscript. VI. I participated in the design of the study, the experimental work and the discussion about the results. I was involved in the preparation of the manuscript. VII. I designed the study, performed the experimental work and wrote the manuscript.