Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover

Abstract Background Inefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans. Results In this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme cocktails used in this work are unable to effectively target those substituted polysaccharide residues. Conclusion To our knowledge, this is the first report using glycome profiling as a tool to dynamically monitor recalcitrant cell wall carbohydrates during the course of enzymatic hydrolysis. Glycome profiling of UHS and liquid hydrolysates unveiled some of the glycans that are not cleaved and enriched after enzyme hydrolysis. The major polysaccharides include 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan, suggesting that enzymes with glucuronidase and arabinofuranosidase activities are required to maximize monomeric sugar yields. This methodology provides a rapid tool to assist in developing new enzyme cocktails, by supplementing the existing cocktails with the required enzyme activities for achieving complete deconstruction of pretreated biomass in the future

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 under varying concentrations of WSC.

<p>(A) Glucose consumption; (B), Xylose consumption; (C), Ethanol production and (D), Cell growth OD₆₀₀. Syn-4WSC: SynH with 40 g/L WSC added; SynH-3WSC: SynH with 30 g/L WSC added; SynH 2WSC: SynH with 20 g/L WSC added; SynH-1WSC: SynH with 10 g/L WSC added; SynH-Control: SynH-base media with no inhibitors added. Fermentation was conducted in Erlenmeyer flasks (50 mL) at pH 4.8, 30 °C and 150 RPM with inoculum at 2 (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

Chemical genomic profiling of WSC on using a yeast deletion strain library.

<p>(A). Chemical genomic profile of SynH+WSC, with the top sensitive deletion mutants (red) and top resistant mutants (green) highlighted (mean profile n = 3); (B). Gene clusters correlation between SynH, ACSH and SynH+WSC (n = 3). Mutants in <i>ERJ1</i> (involved in ER protein folding), <i>PDX1</i> (a subunit of the mitochondrial dehydrogenase complex), and <i>GOS1</i> (involved Golgi transport) were especially sensitive to WSC. The sensitive genes gave insight into the mechanism of toxicity, confirming that cell membranes were the likely target of WSC toxicity; and overexpression of the sensitive genes could be used to confer resistance. Comparing the chemical genetic profiles between different hydrolysates and fermentation media, we found that the profile of SynH + WSC exhibited a high correlation with that of ACSH (Fig 6B, R = 0.69), and showed greater similarity to ACSH compared to SynH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194012#pone.0194012.g007" target="_blank">Fig 7</a>). The strong correlation suggests that the degradation compounds in WSC can represent the real inhibitors in ACSH to a large extent.</p

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

Water soluble aromatic compounds isolated from EA crude lignin stream using ethyl acetate.

<p>Here, (A) Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and (B) gas chromatography-mass spectrometry (GC-MS) chromatograms were used to identify phenolic compounds.</p