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MOESM1 of Conversion of lignocellulosic agave residues into liquid biofuels using an AFEX™-based biorefinery

Additional file 1: Figure S1. Box and whisker plot for sugar yields at varying AFEX pretreatment conditions on Agave biomass. Here, % sugar yields (glucose and xylose) from the whole set of experiments (DoE) for each biomass, minimum and maximum values, as well as the interquartile range, are shown. Enzyme hydrolysis was carried at 1% glucan loading, using Cellic® CTec2 (9 mg protein/g glucan) and Cellic® HTec2 (6 mg protein/g glucan), pH 4.8, 250 rpm and 50 °C. Figure S2. SEM Images for untreated and AFEX-pretreated A. tequilana bagasse. Here, untreated (left), AFEX treated (right). Figure S3. Effects of AFEX parameters on monomeric sugar conversions from A. salmiana bagasse. Here, glucan conversion A to C (green) and xylan conversion D to F (blue). Figure S4. Effects of AFEX parameters on monomeric sugar conversions from fibers of two agave species leaves. Here, glucan conversion A to C (green) and xylan conversion D to F (blue). Figure S5. Ternary contour plots showing effects of varying the ratio of commercial enzymes on sugar conversion. Glucan conversion (left column) and xylan conversion (right column) to monomeric sugars from the four AFEX-pretreated agave feedstocks. Here, (a) A. tequilana bagasse, (b) A. tequilana leaf fibers, (c) A. salmiana bagasse and (d) A. salmiana leaf fibers. Enzymatic hydrolysis was conducted at different enzyme mixtures with CTec3, HTec3 and Multifect Pectinase at 6% glucan loading, at total enzyme loading of 20 mg of protein/g glucan, pH 5.0, 250 rpm and 72 h. Figure S6. Monomeric glucose release during high solids loading EH of pretreated A. tequilana leaf fiber. As a function of enzyme loading at 20% total solids. Figure S7. Different untreated and pretreated agave residues. Here, A. tequilana bagasse (untreated) (A), A. tequilana bagasse (AFEX treated) (B), A. salmiana bagasse (untreated) (C), A. salmiana bagasse (AFEX treated) (D), A. tequilana leaf fibers (untreated) (E), A. tequilana leaf fibers (AFEX treated) (F), A. salmiana leaf fibers (untreated) (G) and A. salmiana leaf fibers (AFEX treated) (H). Table S1. AFEX conditions tested in the statistical design of experiments performed on each agave biomass. Table S2. Regression Coefficients of Mixture Design Model from the Enzyme ratio optimization of pretreated Agave residues

MOESM4 of Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries

Additional file 4: Fig. S2. Profiling the effect of ammonia loading and temperature on the combined glucose and xylose yields for AFEX™-treated bagasse and cane leaf matter after 1% glucan loading enzymatic hydrolysis with 15 mg protein g−1 glucan

MOESM6 of Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries

Additional file 6: Fig. S4. Material balances during pretreatment, washing, hydrolysis and fermentation for Processes I–IV

MOESM2 of Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries

Additional file 2: Table S2. AFEX™ -bagasse pretreatment conditions used for evaluating the effect of pretreatment conditions on the monomeric glucose, xylose and combined sugar yield using a central composite design of experiments (DOE)

MOESM5 of Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries

Additional file 5: Fig. S3. Statistical optimization of Cellic® CTec3, Cellic® HTec3, Pectinex Ultra-SP combinations for maximizing combined glucose and xylose yields from AFEXTM and Steam exploded sugarcane bagasse and CLM

Water-soluble phenolic compounds produced from extractive ammonia pretreatment exerted binary inhibitory effects on yeast fermentation using synthetic hydrolysate

<div><p>Biochemical conversion of lignocellulosic biomass to liquid fuels requires pretreatment and enzymatic hydrolysis of the biomass to produce fermentable sugars. Degradation products produced during thermochemical pretreatment, however, inhibit the microbes with regard to both ethanol yield and cell growth. In this work, we used synthetic hydrolysates (SynH) to study the inhibition of yeast fermentation by water-soluble components (WSC) isolated from lignin streams obtained after extractive ammonia pretreatment (EA). We found that SynH with 20g/L WSC mimics real hydrolysate in cell growth, sugar consumption and ethanol production. However, a long lag phase was observed in the first 48 h of fermentation of SynH, which is not observed during fermentation with the crude extraction mixture. Ethyl acetate extraction was conducted to separate phenolic compounds from other water-soluble components. These phenolic compounds play a key inhibitory role during ethanol fermentation. The most abundant compounds were identified by Liquid Chromatography followed by Mass Spectrometry (LC-MS) and Gas Chromatography followed by Mass Spectrometry (GC-MS), including coumaroyl amide, feruloyl amide and coumaroyl glycerol. Chemical genomics profiling was employed to fingerprint the gene deletion response of yeast to different groups of inhibitors in WSC and AFEX-Pretreated Corn Stover Hydrolysate (ACSH). The sensitive/resistant genes cluster patterns for different fermentation media revealed their similarities and differences with regard to degradation compounds.</p></div

Fermentation performance of Y128 using different WSC fractions.

<p><b>Here,</b> (A) Glucose consumption; (B) Xylose consumption; (C) Ethanol production and (D) Cell growth OD₆₀₀. ACSH: AFEX corn stover hydrolysate; SynH-W: SynH with 20 g/L water phase extract after ethyl acetate-water partitioning; SynH-P: SynH with 20 g/L ethyl acetate phase extract after ethyl acetate-water partitioning; SynH-WSC: SynH with 20 g/L WSC; SynH-Control: SynH-base media with no inhibitors added. Both phenolic compounds and nutrient components were re-dissolved in SynH-base media at 20 g/L. Fermentations were conducted in Erlenmeyer flasks (50 mL at pH 4.8, 30 °C and 150 RPM with inoculum at 0.8 OD₆₀₀.</p

Methodology and mass balance of water extraction and ethyl acetate extraction of crude lignin.

<p>Methodology and mass balance of water extraction and ethyl acetate extraction of crude lignin.</p

Correlation between chemical genomic profiles of SynH control media, ACSH and SynH+WSC.

<p>Chemical genomics is the study of chemical compound interactions with specific genes within an organism. This approach determined whether hydrolysate variability existed using a biological ‘‘sensor” (individual gene mutants) to create a genome-wide, biological ‘‘fingerprint” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194012#pone.0194012.ref014" target="_blank">14</a>]. In this study, we combined chemical genomics profiling with SynH, therefore determined both hydrolysate variability and gene fingerprints. This is a high-throughput method to test different compounds for their inhibitory effects, which can be widely applied in fermentation study and media development.</p