Effect of cellulose reducing ends and primary hydroxyl groups modifications on cellulose-cellulase interactions and cellulose hydrolysis.

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents. The effects of cellulose reducing ends modification on cellulose reducing ends, cellulose structure, and cellulose accessibility to cellulase were evaluated along with the impact on cellulose hydrolysis with complete as well purified cellulase components. Sodium borohydride (NaBH4) reduction and sodium chlorite-acetic acid (SC/AA) oxidation were able to modify more than 90% and 60% of the reducing ends, respectively, while the bicinchoninic acid (BCA) reagent applied for various cycles oxidized cellulose reducing ends to various extents. X-ray diffractograms of the treated solids showed that these treatments did not change the cellulose crystalline structure and the change in crystallinity index was insignificant. Surprisingly, it was found that the cellulose reducing ends modification, either through selective NaBH4 reduction or BCA oxidation, had a negligible impact on cellulose accessibility as well on cellulose hydrolysis rates or final conversions with complete cellulase at loadings as low as 0.5 mg protein/g cellulose. In fact, in contrast to what is traditionally believed, modifications of cellulose reducing ends by these two methods had no apparent impact on cellulose conversion with purified cellulase components and their synergy. However, SC/AA oxidation resulted in significant drop in cellulose conversion (10%-50%) with complete as well purified cellulase components. Nonetheless, further research revealed that the cause for drop in cellulose conversion for the SC/AA oxidation case was due to primary hydroxyl groups (PHGs) oxidation and not the oxidation of reducing ends. Furthermore, it was found that the PHGs modification affects cellulose accessibility and slows the cellulase uptake as well resulting in significant drop in cellulose conversions.

Technoeconomic and life-cycle analysis of single-step catalytic conversion of wet ethanol into fungible fuel blendstocks.

Technoeconomic and life-cycle analyses are presented for catalytic conversion of ethanol to fungible hydrocarbon fuel blendstocks, informed by advances in catalyst and process development. Whereas prior work toward this end focused on 3-step processes featuring dehydration, oligomerization, and hydrogenation, the consolidated alcohol dehydration and oligomerization (CADO) approach described here results in 1-step conversion of wet ethanol vapor (40 wt% in water) to hydrocarbons and water over a metal-modified zeolite catalyst. A development project increased liquid hydrocarbon yields from 36% of theoretical to >80%, reduced catalyst cost by an order of magnitude, scaled up the process by 300-fold, and reduced projected costs of ethanol conversion 12-fold. Current CADO products conform most closely to gasoline blendstocks, but can be blended with jet fuel at low levels today, and could potentially be blended at higher levels in the future. Operating plus annualized capital costs for conversion of wet ethanol to fungible blendstocks are estimated at $2.00/GJ for CADO today and $1.44/GJ in the future, similar to the unit energy cost of producing anhydrous ethanol from wet ethanol ($1.46/GJ). Including the cost of ethanol from either corn or future cellulosic biomass but not production incentives, projected minimum selling prices for fungible blendstocks produced via CADO are competitive with conventional jet fuel when oil is $100 per barrel but not at $60 per barrel. However, with existing production incentives, the projected minimum blendstock selling price is competitive with oil at $60 per barrel. Life-cycle greenhouse gas emission reductions for CADO-derived hydrocarbon blendstocks closely follow those for the ethanol feedstock.

Polyurethanes Based on Unmodified and Refined Technical Lignins: Correlation between Molecular Structure and Material Properties.

The structural complexity and robust intermolecular interactions have challenged the incorporation of technical lignin into value-added polymeric materials for decades. To study the correlation between lignin molecular structure and material properties of lignin-based polyurethanes, we applied co-solvent enhanced lignocellulosic fractionation pretreatment followed by sequential precipitation to produce three distinct lignin preparations with narrowly distributed (molecular weight dispersity <2) and comparatively low molecular weight (<1500 g/mol) from poplar biomass. Structural characterization indicated that these lignin preparations differed in average molecular chain length and stiffness as well as hydroxyl group distribution. Secondary hydroxyl group providers such as aliphatic diols and polyethers were incorporated as building blocks into the lignin-based polyurethanes to provide additional hydrogen capacity to improve the dispersion of lignin in the polyurethane network. The selected aliphatic diols and polyethers interacted with lignin molecules at different levels of strength depending on their molecular structure, and their impacts were ultimately reflected in the mechanical and thermal properties of the resulting lignin-based polyurethanes. The copolymerization of technical lignin with tailored structure and secondary hydroxyl providers could provide new strategies in formulating lignin-based/containing polyurethanes for various functional applications.

Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol.

Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a long-standing limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery. Here, we illustrate how competing factors that limit ethanol yields during high-solids fermentations are overcome by integrating newly developed cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment with SSF. First, fed-batch glucose fermentations by Saccharomyces cerevisiae D5A revealed that this strain, which has been favored for SSF, can produce ethanol at titers of up to 86 g⋅L-1 Then, optimizing SSF of CELF-pretreated corn stover achieved unprecedented ethanol titers of 79.2, 81.3, and 85.6 g⋅L-1 in batch shake flask, corresponding to ethanol yields of 90.5%, 86.1%, and 80.8% at solids loadings of 20.0 wt %, 21.5 wt %, and 23.0 wt %, respectively. Ethanol yields remained at over 90% despite reducing enzyme loading to only 10 mg protein⋅g glucan-1 [∼6.5 filter paper units (FPU)], revealing that the enduring factors limiting further ethanol production were reduced cell viability and glucose uptake by D5A and not loss of enzyme activity or mixing issues, thereby demonstrating an SSF-based process that was limited by a strain's metabolic capabilities and tolerance to ethanol.

A Multifunctional Cosolvent Pair Reveals Molecular Principles of Biomass Deconstruction.

The complex structure of plant cell walls resists chemical or biological degradation, challenging the breakdown of lignocellulosic biomass into renewable chemical precursors that could form the basis of future production of green chemicals and transportation fuels. Here, experimental and computational results reveal that the effect of the tetrahydrofuran (THF)-water cosolvents on the structure of lignin and on its interactions with cellulose in the cell wall drives multiple synergistic mechanisms leading to the efficient breakdown and fractionation of biomass into valuable chemical precursors. Molecular simulations show that THF-water is an excellent "theta" solvent, such that lignin dissociates from itself and from cellulose and expands to form a random coil. The expansion of the lignin molecules exposes interunit linkages, rendering them more susceptible to depolymerization by acid-catalyzed cleavage of aryl-ether bonds. Nanoscale infrared sensors confirm cosolvent-mediated molecular rearrangement of lignin in the cell wall of micrometer-thick hardwood slices and track the disappearance of lignin. At bulk scale, adding dilute acid to the cosolvent mixture liberates the majority of the hemicellulose and lignin from biomass, allowing unfettered access of cellulolytic enzymes to the remaining cellulose-rich material, allowing them to sustain high rates of hydrolysis to glucose without enzyme deactivation. Through this multiscale analysis, synergistic mechanisms for biomass deconstruction are identified, portending a paradigm shift toward first-principles design and evaluation of other cosolvent methods to realize low cost fuels and bioproducts.

Sugar yield and composition of tubers from Jerusalem Artichoke (Helianthus tuberosus) irrigated with saline waters.

Currently, major biofuel crops are also food crops that demand fertile soils and good-quality water. Jerusalem artichoke (Helianthus tuberosus, Asteraceae) produces high tonnage of tubers that are rich in sugars, mainly in the form of inulin. In this study, plants of the cultivar "White Fuseau" grown under five salinity levels were evaluated for tuber yield. Results indicated that this cultivar is moderately salt-tolerant if the goal is tuber production. Hydraulic pressings of the tubers produced juice that contained 15% (wet weight) or 55% (dry weight) free sugars, with 70% of these in the form of inulin and the rest as fructose, sucrose, and glucose. Importantly, salinity did not affect the total free sugar or inulin content of the tubers. Tubers were composed of about 12% dry washed bagasse (wet weight) or 44% (dry matter basis) and bagasse retained such high quantities of free sugars after pressing that washing was required for complete sugar recovery. Chemical composition analysis of tuber bagasse suggested that it had low lignin content (11-13 wt%), and its structural sugar composition was similar to chicory root bagasse. Because of the high hemicellulose and pectin content of the bagasse, adding xylanase and pectinase to cellulase substantially improved sugar yields from enzymatic hydrolysis compared to at the same protein loading as cellulase alone. In addition to the high total sugar yield of tuber, these first findings on the sugar and lignin content and enzymatic hydrolysis of tuber bagasse can lead to low-cost production of ethanol for transportation fuels.

Dynamic changes of substrate reactivity and enzyme adsorption on partially hydrolyzed cellulose.

The enzymatic hydrolysis of cellulose is a thermodynamically challenging catalytic process that is influenced by both substrate-related and enzyme-related factors. In this study, a proteolysis approach was applied to recover and clean the partially converted cellulose at the different stages of enzymatic hydrolysis to monitor the hydrolysis rate as a function of substrate reactivity/accessibility and investigate surface characteristics of the partially converted cellulose. Enzyme-substrate interactions between individual key cellulase components from wild-type Trichoderma reesei and partially converted cellulose were followed and correlated to the enzyme adsorption capacity and dynamic sugar release. Results suggest that cellobiohydrolase CBH1 (Cel7A) and endoglucanases EG2 (Cel5A) adsorption capacities decreased as cellulose was progressively hydrolyzed, likely due to the "depletion" of binding sites. Furthermore, the degree of synergism between CBH1 and EG2 varied depending on the enzyme loading and the substrates. The results provide a better understanding of the relationship between dynamic change of substrate features and the functionality of various cellulase components during enzymatic hydrolysis. Biotechnol. Bioeng. 2017;114: 503-515. © 2016 Wiley Periodicals, Inc.

Local Phase Separation of Co-solvents Enhances Pretreatment of Biomass for Bioenergy Applications.

Pretreatment facilitates more complete deconstruction of plant biomass to enable more economic production of lignocellulosic biofuels and byproducts. Various co-solvent pretreatments have demonstrated advantages relative to aqueous-only methods by enhancing lignin removal to allow unfettered access to cellulose. However, there is a limited mechanistic understanding of the interactions between the co-solvents and cellulose that impedes further improvement of such pretreatment methods. Recently, tetrahydrofuran (THF) has been identified as a highly effective co-solvent for the pretreatment and fractionation of biomass. To elucidate the mechanism of the THF-water interactions with cellulose, we pair simulation and experimental data demonstrating that enhanced solubilization of cellulose can be achieved by the THF-water co-solvent system at equivolume mixtures and moderate temperatures (≤445 K). The simulations show that THF and water spontaneously phase separate on the local surface of a cellulose fiber, owing to hydrogen bonding of water molecules with the hydrophilic cellulose faces and stacking of THF molecules on the hydrophobic faces. Furthermore, a single fully solvated cellulose chain is shown to be preferentially bound by water molecules in the THF-water mixture. In light of these findings, co-solvent reactions were performed on microcrystalline cellulose and maple wood to show that THF significantly enhanced cellulose deconstruction and lignocellulose solubilization at simulation conditions, enabling a highly versatile and efficient biomass pretreatment and fractionation method.

4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein.

The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-L-methionine to O-4 of α-D-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co(2+) for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.

Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars.

Cellulase enzymes contribute a major fraction of the total cost for biological conversion of lignocellulosic biomass to fuels and chemicals. Although a several fold reduction in cellulase production costs and enhancement of cellulase activity and stability have been reported in recent years, sugar yields are still lower at low enzyme doses than desired commercially. We recently reported that hemicellulose xylan and its oligomers strongly inhibit cellulase and that supplementation of cellulase with xylanase and ß-xylosidase would significantly reduce such inhibition. In this study, mannan polysaccharides and their enzymatically prepared hydrolyzates were discovered to be strongly inhibitory to fungal cellulase in cellulose conversion (>50% drop in % relative conversion), even at a small concentration of 0.1 g/L, and inhibition was much greater than experienced by other known inhibitors such as cellobiose, xylooligomers, and furfural. Furthermore, cellulase inhibition dramatically increased with heteromannan loading and mannan substitution with galactose side units. In general, enzymatically prepared hydrolyzates were less inhibitory than their respective mannan polysaccharides except highly substituted ones. Supplementation of cellulase with commercial accessory enzymes such as xylanase, pectinase, and ß-glucosidase was effective in greatly relieving inhibition but only for less substituted heteromannans. However, cellulase supplementation with purified heteromannan specific enzymes relieved inhibition by these more substituted heteromannans as well, suggesting that commercial preparations need to have higher amounts of such activities to realize high sugar yields at the low enzyme protein loadings needed for low cost fuels production.

Fast hemicellulose quantification via a simple one-step acid hydrolysis.

As the second most common polysaccharides in nature, hemicellulose has received much attention in recent years for its importance in biomass conversion in terms of producing high yields of fermentable sugars and value-added products, as well as its role in reducing biomass recalcitrance. Therefore, a time and labor efficient method that specifically analyzes hemicellulose content would be valuable to facilitate the screening of biomass feedstocks. In this study, a one-step acid hydrolysis method was developed, which applied 4 wt% sulfuric acid at 121°C for 1 h to rapidly quantify XGM (xylan + galactan + mannan) contents in various types of lignocellulosic biomass and model hemicelluloses. This method gave statistically identical results in XGM contents compared to results from conventional two-step acid hydrolysis while significantly shortening analysis time.

Investigation of lignin deposition on cellulose during hydrothermal pretreatment, its effect on cellulose hydrolysis, and underlying mechanisms.

In dilute acid pretreatment of lignocellulosic biomass, lignin has been shown to form droplets that deposit on the cellulose surface and retard enzymatic digestion of cellulose (Donohoe et al., 2008; Selig et al., 2007). However, studies of this nature are limited for hydrothermal pretreatment, with the result that the corresponding mechanisms that inhibit cellulosic enzymes are not well understood. In this study, scanning electron microscope (SEM) and wet chemical analysis of solids formed by hydrothermal pretreatment of a mixture of Avicel cellulose and poplar wood showed that lignin droplets from poplar wood relocated onto the Avicel surface. In addition, nuclear magnetic resonance (NMR) showed higher S/G ratios in deposited lignin than the initial lignin in poplar wood. Furthermore, the lignin droplets deposited on Avicel significantly impeded cellulose hydrolysis. A series of tests confirmed that blockage of the cellulose surface by lignin droplets was the main cause of cellulase inhibition. The results give new insights into the fate of lignin in hydrothermal pretreatment and its effects on enzymatic hydrolysis.

Lignin content in natural Populus variants affects sugar release.

The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plant's recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production.

Application of high throughput pretreatment and co-hydrolysis system to thermochemical pretreatment. Part 1: dilute acid.

Because conventional approaches for evaluating sugar release from the coupled operations of pretreatment and enzymatic hydrolysis are extremely time and material intensive, high throughput (HT) pretreatment and enzymatic hydrolysis systems have become vital for screening large numbers of lignocellulosic biomass samples to identify feedstocks and/or processing conditions that significantly improve performance and lower costs. Because dilute acid pretreatment offers many important advantages in rendering biomass highly susceptible to subsequent enzymatic hydrolysis, a high throughput pretreatment and co-hydrolysis (HTPH) approach was extended to employ dilute acid as a tool to screen for enhanced performance. First, a single-step neutralization and buffering method was developed to allow effective enzymatic hydrolysis of the whole pretreated slurry. Switchgrass and poplar were then pretreated with 0.5% and 1% acid loadings at a 5% solids concentration, the resulting slurry conditioned with the buffering approach, and the entire mixture enzymatically hydrolyzed. The resulting sugar yields demonstrated that single-step neutralizing and buffering was capable of adjusting the pH as needed for enzymatic saccharification, as well as overcoming enzyme inhibition by compounds released in pretreatment. In addition, the effects of pretreatment conditions and biomass types on susceptibility of pretreated substrates to enzymatic conversion were clearly discernible, demonstrating the method to be a useful extension of HTPH systems.

Application of high throughput pretreatment and co-hydrolysis system to thermochemical pretreatment. Part 2: Dilute alkali.

High throughput pretreatment (HTPH) and enzymatic hydrolysis systems are now vital for screening large numbers of biomass samples to investigate biomass recalcitrance over various pretreatment and enzymatic hydrolysis conditions. Although hydrothermal pretreatment is currently being employed in most high throughput applications, thermochemical pretreatment at low and high pH conditions can offer additional insights to better understand the roles of hemicellulose and lignin, respectively, in defining biomass recalcitrance. Thus, after successfully applying the HTPH approach to dilute acid pretreatment [Gao et al. (2012) Biotechnol. Bioeng. 110(3): 754-762], extension to dilute alkali pretreatment was also achieved using a similar single-step neutralization and buffering concept. In the latter approach, poplar and switchgrass were pretreated with 1 wt% sodium hydroxide at 120°C for different reaction times. Following pretreatment, an H2Cit⁻/HCit²â» buffer with a pH of 4.5 was used to condition the pretreatment slurry to a pH range of 4.69-4.89, followed by enzymatic hydrolysis for 72 h of the entire mixture. Sugar yields showed different trends for poplar and switchgrass with increases in pretreatment times, demonstrating the method provided a clearly discernible screening tool at alkali conditions. This method was then applied to selected Populus tremuloides samples to follow ring-by-ring sugar release patterns. Observed variations were compared to results from hydrothermal pretreatments, providing new insights in understanding the influence of biomass structural differences on recalcitrance.

Carbohydrate derived-pseudo-lignin can retard cellulose biological conversion.

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo-lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo-lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT-IR, and SEM imaging. It was found that hemicelluloses (xylan) derived-pseudo-lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ~ 1.94) dilute acid pretreatment of a xylan-Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo-lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions.

Chromatographic determination of 1, 4-ß-xylooligosaccharides of different chain lengths to follow xylan deconstruction in biomass conversion.

Xylooligosaccharides released in hydrothermal pretreatment of lignocellulosic biomass can be purified for high-value products or further hydrolyzed into sugars for fermentation or chemical conversion. In addition, characterization of xylooligosaccharides is vital to understand hemicellulose structure and removal mechanisms in pretreatment of cellulosic biomass. In this study, gel permeation chromatography was applied to fractionate xylooligosaccharides produced from birchwood xylan according to their specific degree of polymerization (DP). Then, each fraction was identified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS); and their concentrations were determined by a downscaled post-hydrolysis method. Based on PAD responses and sugar concentrations for each fraction, a series of response factors were developed that can be used to quantify xylooligosaccharides of DP from 2 to 14 without standards. The resulting approach can profile xylooligosaccharides and help gain new insights into biomass deconstruction.

Rewiring metabolism of Clostridium thermocellum for consolidated bioprocessing of lignocellulosic biomass poplar to produce short-chain esters.

Consolidated bioprocessing (CBP) of lignocellulosic biomass uses cellulolytic microorganisms to enable enzyme production, saccharification, and fermentation to produce biofuels, biochemicals, and biomaterials in a single step. However, understanding and redirecting metabolisms of these microorganisms compatible with CBP are limited. Here, a cellulolytic thermophile Clostridium thermocellum was engineered and demonstrated to be compatible with CBP integrated with a Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters with industrial use as solvents, flavors, fragrances, and biofuels. The recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of alcohol acetyltransferases improved ester production without compromised deacetylation activities. These esterases were discovered to exhibit promiscuous thioesterase activities and their deletion enhanced ester production by rerouting the electron and carbon metabolism. Ester production was further improved up to 80-fold and ester composition could be modulated by deleting lactate biosynthesis and using poplar with different pretreatment severity.

Application of a reaction model to improve calculation of the sugar recovery standard for sugar analysis.

A kinetic model was applied to improve determination of the sugar recovery standard (SRS) for biomass analysis. Three sets of xylose (0.10-1.00 g/L and 0.999-19.995 g/L) and glucose (0.206-1.602 g/L) concentrations were measured by HPLC following reaction of each for 1 h. Then, parameters in a kinetic model were fit to the resulting sugar concentration data, and the model was applied to predict the initial sugar concentrations and the best SRS value (SRS(p)). The initial sugar concentrations predicted by the model agreed with the actual initial sugar concentrations. Although the SRS(e) calculated directly from experimental data oscillated considerably with sugar concentration, the SRS(p) trend was smooth. Statistical analysis of errors and application of the F-test confirmed that application of the model reduced experimental errors in SRS(e). Reference SRS(e) values are reported for the three series of concentrations.

Prospects of thermotolerant Kluyveromyces marxianus for high solids ethanol fermentation of lignocellulosic biomass.

Simultaneous saccharification and fermentation (SSF) is effective for minimizing sugar inhibition during high solids fermentation of biomass solids to ethanol. However, fungal enzymes used during SSF are optimal between 50 and 60 °C, whereas most fermentative yeast, such as Saccharomyces cerevisiae, do not tolerate temperatures above 37 °C. Kluyveromyces marxianus variant CBS 6556 is a thermotolerant eukaryote that thrives at 43 °C, thus potentially serving as a promising new host for SSF operation in biorefineries. Here, we attempt to leverage the thermotolerance of the strain to demonstrate the application of CBS 6556 in a high solids (up to 20 wt% insoluble solid loading) SSF configuration to understand its capabilities and limitations as compared to a proven SSF strain, S. cerevisiae D5A. For this study, we first pretreated hardwood poplar chips using Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) to remove lignin and hemicellulose and to produce cellulose-enriched pretreated solids for SSF. Our results demonstrate that although CBS 6556 could not directly outperform D5A, it demonstrated similar tolerance to high gravity sugar solutions, superior growth rates at higher temperatures and higher early stage ethanol productivity. We discovered that CBS 6556's membrane was particularly sensitive to higher ethanol concentrations causing it to suffer earlier fermentation arrest than D5A. Cross-examination of metabolite data between CBS 6556 and D5A and cell surface imaging suggests that the combined stresses of high ethanol concentrations and temperature to CBS 6556's cell membrane was a primary factor limiting its ethanol productivity. Hence, we believe K. marxianus to be an excellent host for future genetic engineering efforts to improve membrane robustness especially at high temperatures in order to achieve higher ethanol productivity and titers, serving as a viable alternative to D5A.