# Lignocellulosic Biomass as a Substrate for Oleaginous Microorganisms: A Review
## 5 Pretreatment By-Products Affecting Enzymatic Hydrolysis and Fermentation  
By-products generated during the pretreatment of lignocellulosic biomass are detrimental to sugar yield and also in certain concentrations may reduce the efficiency of enzymatic hydrolysis, as well as the growth and metabolism of most microorganisms used in fermentations. Specifically, several compounds derived from lignocellulosic pretreatment, even at low concentrations, can inhibit growth and lipid accumulation in oleaginous microorganisms [15; 73; 191]. Therefore, it is necessary to study the inhibitory effects, in order to adopt appropriate strategies for the production of SCO from lignocellulosic hydrolysates. The type and concentrations of each compound generated in principal pretreatments depend on the operating conditions and the composition of the biomass used (Table 1). Among the inhibitory compounds derived from sugars are furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF), generated in acidic media from the dehydration of pentoses and hexoses, respectively. Both compounds have a significant effect on enzymatic hydrolysis at concentrations of 2.0 g/L for furfural and 4.0 g/L for HMF [203]. In addition, research has shown that furfural and 5-HMF inhibit the metabolism of alcohol-producing yeasts, prolonging the lag phase, inhibiting cell growth and decreasing ethanol productivity. For instance, furfural is found to directly inhibit alcohol dehydrogenase, aldehyde dehydrogenase, pyruvate dehydrogenase and some enzymes involved in glycolysis [228; 229]. Additionally, it is known to induce DNA damage or breakdown, inhibit protein and RNA synthesis [172; 230; 231; 232], and affect the production of NADH and NADPH, decreasing the uptake rates of specific sugar [232]. Other inhibitory compounds derived from sugars are aliphatic carboxylic acids such as acetic acid, formic acid and levulinic acid. Acetic acid is formed by acid hydrolysis of hemicellulose acetyl groups, levulinic acid is the terminal product of hexose oxidation and formic acid has two pathways of formation, one by the oxidation of pentoses and the other by the oxidation of hexoses [15; 233; 234]. These acids cause a pH decrease which negatively affects the microbial metabolism, e.g., acetic acid can penetrate the cell membrane in its undissociated form inhibiting product formation, causing pH imbalances at high concentrations and eventually cell growth inhibition or death [172]. These compounds are mainly generated during acid, alkaline and organosolv pretreatment [50; 108; 235], and are commonly produced within a range of 0.1-5 g/L (Table 1), while being especially toxic to microorganisms at concentrations ranging between 0.3 and 5 g/L (Table 3). Regarding acetic acid, due to the low content of acetyl groups present in softwoods, pretreatments generate lower concentrations of acetic acid, while in hardwoods and agricultural residues with high acetyl group content, the concentration of acetic acid is higher resulting in an important inhibition effect [15]. In contrast, the low concentrations of acetic acid in pretreated softwood stimulate ethanol production due to the lower yeast biomass formation. This is due to the increased ATP demand and/or inefficient ATP production due to uncoupling of the respiratory chain and the oxidative phosphorylation of ADP, which leads to increased ATP-generating glycolytic activity at the expense of cell proliferation [15].