We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover.

Background Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions.ResultsCarbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 % for Caldicellulosiruptor bescii to 65 % for Clostridium thermocellum, with intermediate values for a thermophilic horse manure enrichment, Clostridium clariflavum, Clostridium cellulolyticum, and simultaneous saccharification and fermentation (SSF) featuring a fungal cellulase cocktail and yeast. Under a variety of conditions, solubilization yields were about twice as high for C. thermocellum compared to fungal cellulase. Solubilization of mid-season harvested switchgrass was about twice that of senescent switchgrass. Lower yields and greater dependence on particle size were observed for Populus as compared to switchgrass. Trends observed from data drawn from six conversion systems and three substrates, including both time course and end-point data, were (1) equal fractional solubilization of glucan and xylan, (2) no biological solubilization of the non-carbohydrate fraction of biomass, and (3) higher solubilization for three of the four bacterial cultures tested as compared to the fungal cellulase system. Brief (5 min) ball milling of solids remaining after fermentation of senescent switchgrass by C. thermocellum nearly doubled carbohydrate solubilization upon reinnoculation as compared to a control without milling. Greater particle size reduction and solubilization were observed for milling of partially fermented solids than for unfermented solids. Physical disruption of cellulosic feedstocks after initiation of fermentation, termed cotreatment, warrants further study.ConclusionsWhile the ability to achieve significant solubilization of minimally pretreated switchgrass is widespread, a fivefold difference between the most and least effective biocatalyst—feedstock combinations was observed. Starting with nature’s best biomass-solubilizing systems may enable a reduction in the amount of non-biological processing required, and in particular substitution of cotreatment for pretreatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0412-y) contains supplementary material, which is available to authorized users.

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 DA revealed that this strain, which has been favored for SSF, can produce ethanol at titers of up to 86 g⋅L Then, optimizing SSF of CELF-pretreated corn stover achieved unprecedented ethanol titers of 79.2, 81.3, and 85.6 g⋅L 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 [∼6.5 filter paper units (FPU)], revealing that the enduring factors limiting further ethanol production were reduced cell viability and glucose uptake by DA 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.

† The authors declare no conflict of interest. Electronic Supplementary Information (ESI) available: Ethanol yields from the SSF of CELF pretreated corn stover at enzyme loading of 5 mg-protein g-glucan-in-RCS -1 , component mass balance from the SSF of DA pretreated corn stover at enzyme loadings of 15 mg-protein gglucan-in-RCS -1 , cell viability of SSF flasks with CELF pretreated corn stover at an enzyme loading of 2 mg-protein g-glucan-in-RCS -1 . See A major challenge to economically produce ethanol from lignocellulosic biomass is to achieve industrially relevant ethanol titers (> 50 g L -1 ) to control operating and capital costs for downstream ethanol operations while maintaining high ethanol yields. However, due to reduced fermentation effectiveness at high biomass solids loadings, excessive amounts of enzymes are typically required to obtain reasonable ethanol titers, thereby trading off reduced operating and capital costs with high enzyme costs. In this study, we applied our newly developed Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment to produce highly digestible glucan-rich solids from corn stover. Simultaneous saccharification and fermentation (SSF) was then applied to pretreated solids from CELF at 15.5 wt% solids loadings (corresponding to 11 wt% glucan loadings) in modified shake flasks to achieve an ethanol titer of 58.8 g L -1 at 89.2% yield with an enzyme loading of 15 mg-protein g-glucan-in-raw-corn-stover -1 (-RCS) -1 in only 5 days. By comparison, SSF of corn stover solids from dilute acid pretreatment at 18.3 wt% solids loading (or 10 wt% glucan loadings) only achieved an ethanol titer and yield of 47.8 g L -1 and 73.0%, respectively, despite needing longer fermentation times (~20 days) and an additional 18 h of prehydrolysis at 50°C. Remarkably, although longer fermentation times were required at more economical enzyme loadings of 5 and 2 mg-protein g-glucan-in-RCS -1 , high solids SSF of CELF pretreated corn stover realized final ethanol titers consistently above 50 g L -1 and yields over 80%.