Flowthrough pretreatment with very dilute acid provides insights into high lignin contribution to biomass recalcitrance.

BACKGROUND: Flowthrough pretreatment is capable of removing much higher quantities of hemicellulose and lignin from lignocellulosic biomass than batch pretreatment performed at otherwise similar conditions. Comparison of these two pretreatment configurations for sugar yields and lignin removal can provide insights into lignocellulosic biomass deconstruction. Therefore, we applied liquid hot water (LHW) and extremely dilute acid (EDA, 0.05%) flowthrough and batch pretreatments of poplar at two temperatures and the same pretreatment severity for the solids. Composition of solids, sugar mass distribution with pretreatment, sugar yields, and lignin removal from pretreatment and enzymatic hydrolysis were measured. RESULTS: Flowthrough aqueous pretreatment of poplar showed between 63 and 69% lignin removal at both 140 and 180 °C, while batch pretreatments showed about 20 to 33% lignin removal at similar conditions. Extremely dilute acid slightly enhanced lignin removal from solids with flowthrough pretreatment at both pretreatment temperatures. However, extremely dilute acid batch pretreatment did realize greater than 70% xylan yields largely in the form of monomeric xylose. Close to 100% total sugar yields were measured from LHW and EDA flowthrough pretreatments and one batch EDA pretreatment at 180 °C. The high lignin removal by flowthrough pretreatment enhanced cellulose digestibility compared to batch pretreatment, consistent with lignin being a key contributor to biomass recalcitrance. Furthermore, solids from 180 °C flowthrough pretreatment were much more digestible than solids pretreated at 140 °C despite similar lignin and extensive hemicellulose removal. CONCLUSIONS: Results with flowthrough pretreatment show that about 65-70% of the lignin is solubilized and removed before it can react further to form low solubility lignin rich fragments that deposit on the biomass surface in batch operations and hinder enzyme action. The leftover 30-35% lignin in poplar was a key player in biomass recalcitrance to enzymatic deconstruction and it might be more difficult to dislodge from biomass with lower temperature of pretreatment. These results also point to the possibility that hemicellulose removal is more important as an indicator of lignin disruption than in playing a direct role in reducing biomass recalcitrance.

Comparison of enzymatic reactivity of corn stover solids prepared by dilute acid, AFEX™, and ionic liquid pretreatments.

BACKGROUND: Pretreatment is essential to realize high product yields from biological conversion of naturally recalcitrant cellulosic biomass, with thermochemical pretreatments often favored for cost and performance. In this study, enzymatic digestion of solids from dilute sulfuric acid (DA), ammonia fiber expansion (AFEX™), and ionic liquid (IL) thermochemical pretreatments of corn stover were followed over time for the same range of total enzyme protein loadings to provide comparative data on glucose and xylose yields of monomers and oligomers from the pretreated solids. The composition of pretreated solids and enzyme adsorption on each substrate were also measured to determine. The extent glucose release could be related to these features. RESULTS: Corn stover solids from pretreatment by DA, AFEX, and IL were enzymatically digested over a range of low to moderate loadings of commercial cellulase, xylanase, and pectinase enzyme mixtures, the proportions of which had been previously optimized for each pretreatment. Avicel® cellulose, regenerated amorphous cellulose (RAC), and beechwood xylan were also subjected to enzymatic hydrolysis as controls. Yields of glucose and xylose and their oligomers were followed for times up to 120 hours, and enzyme adsorption was measured. IL pretreated corn stover displayed the highest initial glucose yields at all enzyme loadings and the highest final yield for a low enzyme loading of 3 mg protein/g glucan in the raw material. However, increasing the enzyme loading to 12 mg/g glucan or more resulted in DA pretreated corn stover attaining the highest longer-term glucose yields. Hydrolyzate from AFEX pretreated corn stover had the highest proportion of xylooligomers, while IL produced the most glucooligomers. However, the amounts of both oligomers dropped with increasing enzyme loadings and hydrolysis times. IL pretreated corn stover had the highest enzyme adsorption capacity. CONCLUSIONS: Initial hydrolysis yields were highest for substrates with greater lignin removal, a greater degree of change in cellulose crystallinity, and high enzyme accessibility. Final glucose yields could not be clearly related to concentrations of xylooligomers released from xylan during hydrolysis. Overall, none of these factors could completely account for differences in enzymatic digestion performance of solids produced by AFEX, DA, and IL pretreatments.

A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover.

BACKGROUND: In a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production. RESULTS: An on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEX(TM)), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation. CONCLUSIONS: Compositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.

Agave proves to be a low recalcitrant lignocellulosic feedstock for biofuels production on semi-arid lands.

BACKGROUND: Agave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants. RESULTS: Here, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon's Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass. CONCLUSIONS: This study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave's low recalcitrance for developments in improving cellulosic energy crops.

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.

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.

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.

Spiropyran-based fluorescent anion probe and its application for urinary pyrophosphate detection.

In recent decades, numerous spiropyran derivatives have been designed and utilized for optical sensing of metal ions. However, there is still less research on spiropyran-based anion sensors. In this work, a new spiropyran compound (L) appended with a pendant bis(2-pyridylmethyl)amine was synthesized and used in fluorescent sensing of pyrophosphate ion (PP(i)) in aqueous solution. The molecular recognition and signal transduction are based on the cooperative ligation interactions and the ligation-induced structural conversion of the spiropyran, which leads to a significant change in the photophysical property of the spiropyran. In an ethanol/water solution (30:70, v/v) at pH 7.4, ligation of L with Zn(2+) causes an intense fluorescence emission at 620 nm at the expense of the original fluorescence at 560 nm. Once PP(i) was introduced, interaction between PP(i) and the L-Zn(2+) complex leads to full quenching of the 620 nm band emission which was concomitant with recovery of the 560 nm band emission, and the fluorescence intensity ratio, F(560)/F(620), is proportional to the PP(i) concentration. Under the optimum condition, the L-Zn(2+) complex responds to PP(i) over a dynamic range of 1.0 x 10(-6) to 5.0 x 10(-4) M, with a detection limit of 4.0 x 10(-7) M. The fluorescence response is highly selective for PP(i) over other biologically related substrates, especially the structurally similar anions, such as phosphate and adenosine triphosphate. The mechanism of interaction among L, Zn(2+), and PP(i) was primarily studied by (1)H NMR, (31)P NMR, and HRMS. To demonstrate the analytical application of this approach, the PP(i) concentration in human urine was determined. It was on the order of 3.18 x 10(-5) M, and the mean value for urinary PP(i) excretion by three healthy subjects was 62.4 micromol/24 h.

Spiropyran-based optical approaches for mercury ion sensing: improving sensitivity and selectivity via cooperative ligation interactions using cysteine.

Spiropyrans are an attractive starting point in design of optical approaches for metal ions sensing. However, the high background in aqueous solution and non-specific chelation of the spiropyran with heavy metal ions has hindered their application as reliable sensors for environmental and biological species. Here, we report on a new spiropyran-based approach for sensitive and selective sensing of Hg(2+) in aqueous solution, based on cooperative ligation interactions among the spiropyran probe, an intermediate, cysteine, and the metal ion. To test the feasibility of this design, three spiropyran scaffolds, L1-L3, with different ligation functions at the 8'-position were examined as model systems. The results demonstrate that by using cysteine, a potential ligand of Hg(2+), the spiropyran could detect 1.0x10(-7) M Hg(2+) in aqueous solution. Due to the specific metal-amino acid interaction, the approach exhibits selective response toward Hg(2+) over other metal ions and anions, although possible interference from Cu(2+) has to be considered at the high level of the metal ion. This approach has been used for the determination of Hg(2+) in water samples containing potential interferents with satisfactory recovery.