Abstract
The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.
Highlights
Lignin is a heterogeneous aromatic polymer and a major component of the plant cell wall
Vanillin, and acetovanillone are the most abundant monoaromatic compounds in LignoforceTM softwood black liquor (BL). These are the same major monoaromatic compounds that have been reported in other depolymerized softwood lignin samples, including an oxidative depolymerized Lignoboost softwood kraft lignin (Abdelaziz et al, 2019), an alkaline depolymerized pine kraft lignin (Indulin AT; Prothmann et al, 2017; Ravi et al, 2019), and OCF pine lignin (Zhu et al, 2020)
This suggests that these compounds are characteristic monoaromatic compounds generated by oxidative depolymerization of softwood lignin. 4-Propanol guaiacol (4PG) and 4-ethanol guaiacol (4EG) constituted a relatively minor proportion of the BL monoaromatic compounds reported here (~5% total), but have not been reported in similar softwood lignin samples
Summary
Lignin is a heterogeneous aromatic polymer and a major component of the plant cell wall. It can comprise over 30% of lignocellulosic biomass, it is under-utilized in biorefineries, typically being used to power the extraction of carbohydrates (Ragauskas et al, 2014; Becker and Wittmann, 2019). Considerable effort has been invested in developing processes to depolymerize lignin and convert it to value-added bioproducts (Ragauskas et al, 2014; Schutyser et al, 2018; Sun et al, 2018; Becker and Wittmann, 2019). BL contains lower molecular weight compounds, including monoaromatics, sugars, and small organic acids that represent unmined value
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