To simplify this study, all characterized AFEX pretreatment-derived biomass decomposition products were divided into five groups (Table 4 ): 1) nitrogenous compounds, 2) furans, 3) aliphatic acids, 4) aromatic compounds, and 5) carbohydrates.
The effect of these five groups of compounds on xylose fermentation was tested individually and in combination (five groups in combination) in order to investigate their synergistic inhibitory effect. The fermentations were conducted in SM supplemented with 60 g/L glucose and 26 g/L xylose. The decomposition products in each group and their concentrations are given in Table 2 , and matched their absolute abundance as found in 6% glucan loading-based ACHs. To make stock solutions of decomposition products, all compounds were dissolved in water according to the categories of aliphatic acids, aromatic acids, aromatic aldehyde/ketones, furans, imidazoles, and pyrazines at 50-fold higher concentrations and the stock solutions were sterile filtered prior to their addition into the SM. Ferulic acid, p-coumaric acid, amides, and carbohydrates were directly added to the fermentation media at the desired concentrations (Table 2 ) due to their lower solubility in water. Fermentations of SM without any decomposition products (blank) and ACHs were used as negative and positive controls, respectively. The ACH was adjusted to pH 5.5 before inoculum addition.
Nitrogenous compounds¶ | Feruloyl amide | 1065 |
p-Coumaroyl amide | 886 | |
Acetamide | 5674 | |
2-Methylpyrazine | 10 | |
2,5-Dimethylpyrazine | 1 | |
2,6-Dimethylpyrazine | 4 | |
2,4-Dimethyl-1 H-imidazole | 24 | |
4-Methyl-1 H-imidazole | 95 | |
Furan¶ | 5-Hydroxymethyl furfural | 145 |
Aliphatic acids | Malonic acid | 33 |
Lactic acid | 181 | |
cis-Aconitic acid | 111 | |
Succinic acid | 60 | |
Fumaric acid | 30 | |
trans-Aconitic acid | 329 | |
Levulinic acid | 2.5 | |
Itaconic acid | 8.2 | |
Acetic acid | 1958 | |
Formic acid | 517 | |
Aromatic compounds | Vanillic acid | 15 |
Syringic acid | 15 | |
Benzoic acid | 59 | |
p-Coumaric acid | 345 | |
Ferulic acid | 137 | |
Cinnamic acid | 14 | |
Caffeic acid | 2 | |
Vanillin | 20 | |
Syringaldehyde | 29.5 | |
4-Hydroxybenzaldehyde | 24 | |
4-Hydroxyacetophenone | 3.4 | |
Carbohydrates | Glucose | 60 g/L |
Xylose | 26 g/L | |
Arabinose | 5 g/L | |
Gluco-oligomers | 12 g/L | |
Xylo-oligomers | 18 g/L |
¶The concentration of nitrogenous compounds and furan were calculated from the content of the analyte in dry pretreated biomass [15 (link)] based on 18% solids loading (w/v) assuming 100% solubilization into the liquid phase.
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