Identification of Small Aliphatic Aldehydes in Pretreated

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Identification of Small Aliphatic Aldehydes in Pretreated Lignocellulosic Feedstocks and Evaluation of Their Inhibitory Effects on Yeast Adnan Cavka, Stefan Stagge, and Leif J. Jönsson* Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden ABSTRACT: Six lignocellulosic hydrolysates produced through acid pretreatment were analyzed for the occurrence of formaldehyde, acetaldehyde, and glycolaldehyde. Acetaldehyde was found in all six (0.3−1.6 mM) and formaldehyde in four (≤4.4 mM), whereas glycolaldehyde was not detected. To assess the relevance of these findings, fermentations with yeast and formaldehyde or acetaldehyde were performed in the concentration interval 0.5−10 mM. Formaldehyde already inhibited at 1.0 mM, whereas 5.0 mM acetaldehyde was needed to obtain a clear inhibitory effect. After 24 h of fermentation, 1.5 mM formaldehyde reduced the glucose consumption by 85%, the balanced ethanol yield by 92%, and the volumetric productivity by 91%. The results show that formaldehyde and acetaldehyde are prevalent in pretreated lignocellulose and that formaldehyde in some cases could explain a large part of the inhibitory effects on yeast by lignocellulosic hydrolysates, as three of six hydrolysates contained ≥1.9 mM formaldehyde, which was shown to be strongly inhibitory. KEYWORDS: formaldehyde, acetaldehyde, glycolaldehyde, mass spectrometry, lignocellulose, pretreatment, fermentation inhibition

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INTRODUCTION

been identified, and it is also possible to quantitate the total phenolic content.4,5,7,10 Apart from furan aldehydes, such as furfural and HMF, it is conceivable that small aliphatic aldehydes, such as formaldehyde, 1, and acetaldehyde, 2, are formed during pretreatment. The role of small aliphatic aldehydes as inhibitors may have been overlooked considering that much of the interest has been focused on furan aldehydes.5−9 Recently, glycolaldehyde, 3, another small aliphatic aldehyde, received attention as a possible inhibitor for production of biofuels with yeast.11 As pretreatment is often performed at temperatures of around 200 °C, small volatile substances may evaporate during the process, so even if they would form during pretreatment, their presence in hydrolysates is not self-evident. Besides the handling of the pretreated material, the nature of the feedstock and the pretreatment conditions could potentially influence the levels of small aliphatic aldehydes. To investigate this further and to obtain a comprehensive view of the presence of small aliphatic aldehydes in acid-pretreated lignocellulose, we analyzed six pretreatment liquids (PLs) produced from different lignocellulosic feedstocks using different acidic pretreatment procedures with regard to their content of 1, 2, and 3. Fermentations with S. cerevisiae were carried out to determine relevant concentrations of small aldehydes with regard to their inhibitory effects. Problems with inhibitors can be alleviated through detoxification, and even strongly inhibitory lignocellulose hydrolysates can exhibit good fermentability after detoxification.5 A recent development is chemical in situ detoxification with reducing agents, for example, sulfur oxyanions such as

Liquid biofuels and other commodities can be produced from lignocellulosic feedstocks through biochemical conversion processes in which lignocellulose is pretreated to make the cellulose more susceptible to enzymatic hydrolysis.1 The resulting sugar is used in microbial fermentation processes based on bacteria or fungi. Conversion of lignocellulosic hydrolysates to ethanol by the yeast Saccharomyces cerevisiae is a basic example of such a process. Pretreatment using acid catalysts, such as sulfuric acid or sulfur dioxide, is a well-established pretreatment method that is also suitable for more recalcitrant types of lignocellulose.1−3 Acid pretreatment solubilizes and hydrolyzes hemicellulose to sugars, which are desired as substrate for the microbial fermentation process, but there are also reactions that give rise to byproducts such as aliphatic carboxylic acids (including acetic acid, formic acid, and levulinic acid), phenolic compounds, and furanic compounds.4−7 Acetic acid is formed through hydrolysis of acetylated xylan, whereas formic acid and levulinic acid are formed as degradation products under harsh pretreatment conditions.5,7 Apart from these three commonly occurring aliphatic carboxylic acids, Du et al.4 identified and quantitated 13 less abundant aliphatic acids in acid-pretreated biomass. Acid pretreatment of lignocellulosic biomass gives rise to a wide variety of phenolic compounds, which are mainly formed through unintentional partial degradation of lignin.5,7 Furan aldehydes, such as furfural and 5-hydroxymethylfurfural (HMF), are formed through dehydration of sugars under harsh pretreatment conditions.5,7 The presence in lignocellulosic hydrolysates and the effects of carboxylic acids and furan aldehydes on fermenting microorganisms have been studied extensively.5−9 The large number of phenols in lignocellulosic hydrolysates makes them more challenging to characterize than the carbohydrate degradation products, but many phenolic compounds have © 2015 American Chemical Society

Received: Revised: Accepted: Published: 9747

July 7, 2015 October 23, 2015 October 24, 2015 November 3, 2015 DOI: 10.1021/acs.jafc.5b04803 J. Agric. Food Chem. 2015, 63, 9747−9754

Article

Journal of Agricultural and Food Chemistry Table 1. Pretreatment Liquids and Aldehyde Concentrations feedstock and pretreatment conditions

formaldehyde (mM)

PL1 aspen, H2SO4, 165 °C, 10 min PL2 sugar cane bagasse, SO2, 188 °C, 10 min PL3 birch wood, SO2, 190 °C, 7 min PL4 cassava stems, H2SO4, 170 °C, 20 min PL5 corn cobs, H2SO4, 186 °C, 6.4 min PL6 spruce wood, SO2, 204 °C, 7−8 min

0.651