Identification of Small Aliphatic Aldehydes in Pretreated Lignocellulosic Feedstocks and Evaluation of Their Inhibitory Effects on Yeast

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.

Factors Affecting Detoxification of Softwood Enzymatic Hydrolysates Using Sodium Dithionite

“…Thus, the formaldehyde concentration found in the hydrolysate would be expected to be strongly inhibitory, while the acetaldehyde concentration would not be inhibitory. The observed concentrations are in line with previous studies of pretreated spruce showing higher concentrations of formaldehyde than of acetaldehyde [15,17]. It is probable that formaldehyde primarily comes from lignin, and that acetaldehyde primarily comes from acetyl groups in hemicelluloses.…”

“…With regard to aliphatic aldehydes, the hydrolysate contained a high concentration of formaldehyde (6.1 mM) whereas the concentration of acetaldehyde was rather low (1.7 mM). Cavka et al [15] and Martin et al [17] found that 1.0 mM of formaldehyde and 5.0 mM of acetaldehyde was sufficient to inhibit S. cerevisae. Thus, the formaldehyde concentration found in the hydrolysate would be expected to be strongly inhibitory, while the acetaldehyde concentration would not be inhibitory.…”

“…Ultra-high performance liquid chromatography-electrospray ionization-triple quadrupole-mass spectrometry (UHPLC-ESI-QqQ-MS) was used for determination of formaldehyde, acetaldehyde, p-benzoquinone, vanillin, coniferyl aldehyde, syringaldehyde, p-hydroxybenzaldehyde, p-coumaraldehyde, and acetovanillone. Derivatization with 2,4-dinitrophenylhydrazine (DNPH) and calibration was performed as previously described [15,16]. Briefly, UHPLC-ESI-QqQ-MS analysis was carried out using an Agilent 1290 Infinity system coupled to an Agilent 6490 TripleQuad mass spectrometer (QqQ-MS) used in negative electrospray ionization (ESI) mode and equipped with Agilent Jetstream technology.…”

“…Certain groups of by-products that inhibit microorganisms have been extensively studied, and these include aliphatic carboxylic acids (such as acetic acid, formic acid, and levulinic acid), furan aldehydes (such as furfural and 5-hydroxymethylfurfural [HMF]), and phenolic and other phenylic substances [10,13,14]. More recently, small aliphatic aldehydes (such as formaldehyde and acetaldehyde) [15] and benzoquinones (such as p-benzoquinone) [16] were also found to be commonly occurring in pretreated biomass. Martín et al [17] reported that formaldehyde was the most important inhibitor of Saccharomyces cerevisiae yeast in a set of slurries of pretreated Norway spruce.…”

Cross-Feeding of a Toxic Metabolite in a Synthetic Lignocellulose-Degrading Microbial Community

“…Formaldehyde is a small aliphatic aldehyde that is often overlooked in bioprocessing. Yet it is inhibitory to ethanol-generating yeast at concentrations as low as 1.0 mM, lower than is found in many chemically pretreated lignocellulosic feedstocks, resulting in a reduction in product generation of up to tenfold [18]; formaldehyde accumulation is often a challenge overcome via engineered resistance [19]. It can be inhibitory even to the organisms that generate it: formaldehyde detoxification can prove a rate-limiting step in the microbial degradation of methoxylated aromatic compounds [8,9,20].…”