Synergistic Inhibition of Xylose Fermentation by AFEX-CS Hydrolysate

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.Table 4

Plant cell wall-derived decomposition products and water-soluble extractives present in AFEX-CS hydrolysate (ACH)

Category	Compound	Concentration (mg/L)
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.

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.