Abstract
BackgroundLignocellulosic 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.ResultsThe 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.ConclusionsWe 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.
Highlights
Lignocellulosic biomass represents an underutilized renewable resource readily available from forestry and agricultural residues
Selective consumption of aromatic compounds Acinetobacter baylyi ADP1 consumes benzoate and 4HB by converting them to central metabolites via the catechol and protocatechuate branches, respectively, of the β-ketoadipate pathway (Fig. 1). When both compounds are present, ADP1 metabolizes them sequentially: 4HB consumption commences only when most of the benzoate is depleted [4]. We hypothesized that both benzoate and 4HB could be simultaneously consumed by a consortium of two different ADP1-derived mutants, one of which would consume only benzoate and another that would consume only 4HB
We described approaches to remove two aromatic hydrocarbons simultaneously from a simulated lignocellulosic hydrolysate
Summary
Lignocellulosic biomass represents an underutilized renewable resource readily available from forestry and agricultural residues. Corn is the most widely planted agricultural crop in the U.S, and estimated 40 million tons of corn stover are sustainably available for bio-based products [20]. Estimated 400–600 billion liters of ethanol could be generated from these lignocellulosic crops, residues, and waste [23, 36, 41]. 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 used a thermotolerant yeast, Kluyveromyces marxianus, to convert the glucose remaining after detoxification to ethanol
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