Removal of aromatic inhibitors produced from lignocellulosic hydrolysates by Acinetobacter baylyi ADP1 with formation of ethanol by Kluyveromyces marxianus

Abstract

Background Lignocellulosic biomass is an attractive, inexpensive source of potentially fermentable sugars. However, hydrolysis of lignocellulose results in a complex mixture containing microbial inhibitors at variable composition. A single microbial species is unable to detoxify or even tolerate these non-sugar components while converting the sugar mixtures effectively to a product of interest. Often multiple substrates are metabolized sequentially because of microbial regulatory mechanisms. To overcome these problems, we engineered strains of Acinetobacter baylyi ADP1 to comprise a consortium able to degrade benzoate and 4-hydroxybenzoate simultaneously under batch and continuous conditions in the presence of sugars. We furthermore used a thermotolerant yeast, Kluyveromyces marxianus , to convert the glucose remaining after detoxification to ethanol. Results The two engineered strains, one unable to metabolize benzoate and another unable to metabolize 4-hydroxybenzoate, when grown together removed these two inhibitors simultaneously under batch conditions. Under continuous conditions, a single strain with a deletion in the gcd gene metabolized both inhibitors in the presence of sugars. After this batch detoxification using ADP1-derived mutants, K. marxianus generated 36.6 g/L ethanol. Conclusions We demonstrated approaches for the simultaneous removal of two aromatic inhibitors from a simulated lignocellulosic hydrolysate. A two-stage batch process converted the residual sugar into a non-growth-associated product, ethanol. Such a two-stage process with bacteria ( A. baylyi) and yeast ( K. marxianus ) is advantageous, because the yeast fermentation occurs at a higher temperature which prevents growth and ethanol consumption of A. baylyi. Conceptually, the process can be extended to other inhibitors or sugars found in real hydrolysates. That is, additional strains which degrade components of lignocellulosic hydrolysates could be made substrate-selective and targeted for use with specific complex mixtures found in a hydrolysate. Electronic supplementary material The online version of this article (10.1186/s13068-019-1434-7) contains supplementary material, which is available to authorized users.

Engineering CatM, a LysR-Type Transcriptional Regulator, to Respond Synergistically to Two Effectors

“…Bacteria were grown on Luria-Bertani (LB) medium at 37 °C [13]. In some cases, A. baylyi strains were grown in minimal medium [14,15] with succinate (10 mM), pyruvate (20 mM), benzoate (2 mM), muconate, (2.5 mM), or anthranilate (1.5 mM) as the carbon source. Antibiotics were added when needed at the following final concentrations: ampicillin, 150 μg/mL, kanamycin, 25 μg/mL, spectinomycin, 13 μg/mL, and streptomycin, 13 μg/mL.…”

Formate Dehydrogenase Improves the Resistance to Formic Acid and Acetic Acid Simultaneously in Saccharomyces cerevisiae

“…Despite the many merits of lignocellulosic feedstocks, many challenges still exist in the conversion of lignocellulosic feedstocks into fuels and chemicals by microorganisms, especially multiple toxic compounds released by pretreatment [ 6 ]. Lignocellulosic feedstocks release toxic compounds such as acetic acid, formic acid, furfural, and 5-hydroxymethylfurfural (5-HMF), which have severe inhibition on cell growth and fermentation performance at a large scale [ 7 , 8 , 9 ].…”

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“…Currently, a single natural microbial that can detoxify or tolerate the non-sugar ingredients of pretreated lignocellulose hydrolysate while validly transforming sugar mixtures into valuable products is less well reported [ 8 ]. Adaptive evolution is considered a powerful tool to improve substrate utilization and tolerance to toxic degradation products of biochemical-producing bacteria [ 9 ].…”

“…Furthermore, microbial consortia synergies exist that can result in more efficient substrate utilization and increased product yield through functional modularity [ 18 ]. The design and establishment of microbial consortia possessing the capability of performing detoxification and biochemical production from pretreated lignocellulosic feedstock hold many appealing properties in practical lignocellulose biorefining [ 8 ]. Zhu et al developed a consortium composed of two Saccharomyces cerevisiae strains with specific functions for bioethanol production from pretreated biomass.…”