Fig 1.
Methodology and mass balance of water extraction and ethyl acetate extraction of crude lignin.
Fig 2.
Size exclusion chromatography (SEC) fractionation and liquid chromatography-mass spectrometry (LC-MS) screening of WSC.
Here, (A) SEC of WSC using P6 gel (Polyacrylamide, 1–6 kDa, water as mobile phase) with UV-Vis detector at 280nm. 96 SEC fractions from A1-H12 were collected in a 96-well microtiter plate and (B) LC-MS screening of the fractions collected in SEC were performed in the same order as they were collected (A1-H12). Peak 1 to peak 5: Measured signals from LC-MS extracted ion chromatograms for abundant components profiled by LC-MS and assigned based on SEC retention times, aligned from peak No. 1 to peak No. 5 in the SEC chromatogram.
Fig 3.
Water soluble aromatic compounds isolated from EA crude lignin stream using ethyl acetate.
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.
Table 1.
The major compositions in SynH.
Detailed recipe with concentrations can be found in S2 Table.
Fig 4.
Fermentation performance of Y128 under varying concentrations of WSC.
(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).
Fig 5.
Fermentation performance of Y128 using different WSC fractions.
Here, (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.
Fig 6.
Chemical genomic profiling of WSC on using a yeast deletion strain library.
(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 ERJ1 (involved in ER protein folding), PDX1 (a subunit of the mitochondrial dehydrogenase complex), and GOS1 (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 (Fig 7). The strong correlation suggests that the degradation compounds in WSC can represent the real inhibitors in ACSH to a large extent.
Fig 7.
Correlation between chemical genomic profiles of SynH control media, ACSH and SynH+WSC.
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” [14]. 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.