Traditionally, furfuryl alcohol (FOL) is produced from biomass-derived furfural (FAL) by hydrogenation using metal-based chemocatalysts. It is challenging due to high metal toxicity, high hydrogen partial pressure, and the need for organic solvents. NAD­(P)­H-dependent yeast alcohol dehydrogenase I (YADH), which offers high atom economy and up to 100% product selectivity at room temperature in an aqueous medium, is used in this study for the biocatalytic production of FOL from FAL using ethanol (EtOH) as the terminal reductant for in situ regeneration of NAD­(P)­H. Up to 74% FAL conversion was observed at pH 8 with 40 and 160 mM initial FAL and EtOH concentrations, respectively. The conversion was determined to be equilibrium-limited. Circular dichroism spectroscopy and differential scanning fluorimetry studies of YADH show a significant change in the secondary structure upon treatment with increasing concentrations of aldehydes resulting in loss of catalytic activity. Benign reaction conditions support efforts toward sustainable processing, but opportunities for further improvement by increasing product titer and catalyst stability have been identified. This study lays the framework for developing the science and process for alternatives to biomass-derived ethanol.

Over the past decade, market saturation has drastically reduced the value of ethanol, which threatens the viability of industrial bioethanol facilities throughout the Midwest. In response, we are exploring enzymatic methods to convert ethanol and other biomass substrates into higher value chemical derivatives, such as acetaldehyde, to enhance the economic sustainability of bioethanol production. Acetaldehyde has a market price 2.5 times greater than ethanol and the volatility of acetaldehyde offers the potential for ready industrial separation. We are currently evaluating a coupled enzyme reaction in which horse liver alcohol dehydrogenase produces acetaldehyde from ethanol while an N191D mutant of Pseudomonas fluorescens mannitol‐2‐dehydrogenase (PfM2DH) is used to produce mannitol from fructose and to oxidize NADH regenerating NAD+. We have chosen the N191D mutant based on work by Klimacek and Nidetsky demonstrating that this mutation converts wild type PfM2DH, which is reversible, into an enzyme selective for fructose reduction (1). In previous work, we have shown that PfM2DH is active under a wide range of pH and temperature conditions making it a promising enzyme for industrial production. We are developing a GC‐MS solid phase microextraction (SPME) assay to evaluate accumulation of acetaldehyde in the head space of our reaction vessel. This assay will allow us to optimize conditions for the industrial stripping of acetaldehyde from a reactor. We are also optimizing a HILIC‐LC‐MS assay to quantify mannitol production in solution. Combining these assays allows the determination of total acetaldehyde production and the acetaldehyde fraction entering the gas phase. In addition to evaluating the viability of our horse liver ADH and PfM2DH_N191D coupled reaction, these assays will allow the characterization of future enzyme pairs in our efforts to valorize bioethanol using an industrial enzymatic process.

Bioethanol is predominantly produced by fermentation of corn feedstocks in the United States. In recent years, market saturation has reduced the value of ethanol, threatening the viability of bioethanol plants throughout the Midwest. The overall goal of this project is to develop enzymatic and catalytic methods for converting ethanol into higher value chemical derivatives that enhance the economic sustainability of bioethanol plants. Acetaldehyde has a market price 2.5 times greater than ethanol. Alcohol dehydrogenases may be used to oxidize ethanol to acetaldehyde, but they require the cofactor NAD+, which must be regenerated in a recycling reaction. In this study, we examined a mannitol‐2‐dehydrogenase from Pseudomonas fluorescens (PfM2DH) as a candidate recycling enzyme to regenerate NAD+. PfM2DH catalyzes the reversible, NADH‐dependent reduction of fructose to mannitol. Fructose is a readily available substrate at bioethanol plants as it is derived from corn starches. The efficacy of PfM2DH in an industrial setting will depend on the effect temperature, time (enzyme durability), pH and potential inhibitors (high ethanol or acetaldehyde concentration) have on PfM2DH activity. We have purified PfM2DH using published methods and have investigated the factors above using continuous assays measuring NADH oxidation by UV‐Vis spectrophotometry. We are currently developing methods to assay the coupled production of acetaldehyde by gas chromatography. Our results demonstrate that PfM2DH retains activity from pH 7.5 up to pH 9.0, which is valuable for coupling with alcohol dehydrogenases that have pH optimums between 8.0 and 9.0. PfM2DH shows modest inhibition by acetaldehyde and ethanol at high concentrations and retains activity at room temperature for several days. These data are guiding our design of coupled enzyme reactions to efficiently generate acetaldehyde as a higher value derivative of ethanol. The success of this project will lay the groundwork for the future industrial valorization of bioethanol.