Integrated Process for the Enzymatic Production of Fatty Acid Sugar Esters Completely Based on Lignocellulosic Substrates

Abstract

Lignocellulose can be converted sustainably to fuels, power and value-added chemicals like fatty acid esters. This study presents a concept for the first eco-friendly enzymatic synthesis of economically important fatty acid sugar esters based on lignocellulosic biomass. To achieve this, beech wood cellulose fiber hydrolysate was applied in three manners: as sugar component, as part of the deep eutectic solvent (DES) reaction system and as carbon source for the microbial production of the fatty acid component. These fatty acids were gained from single cell oil produced by the oleaginous yeast Cryptococcus curvatus cultivated with cellulose fiber hydrolysate as carbon source. Afterwards, an immobilized Candida antarctica lipase B was used as the biocatalyst in DES to esterify sugars with fatty acids. Properties of the DES were determined and synthesized sugar mono- and di-esters were identified and characterized using TLC, MS, and NMR. Using this approach, sugar esters were successfully synthesized which are 100% based on lignocellulosic biomass.

Keywords: Cryptococcus curvatus; biorefinery; deep eutectic solvents; lignocellulose; single cell oil; sugar esters; synthesis.

Figure 1

Flowchart of the study. Carbohydrates from beech wood cellulose fiber hydrolysates were used to produce fatty acids in a yeast bioprocess, to form the solvent system and as substrate for the enzymatic reaction to produce fatty acid sugar esters (a.k.a glycolipids). Analytical methods are indicated at each work package.

Figure 2

Production of single cell oil with the oleaginous yeast Cryptococcus curvatus ATCC 20509 in a 2.5 L bioreactor using cellulose fiber hydrolysate from beech wood. All concentrations are given as averages of two independent bioreactor cultivations. The individual values can be found in Supplement 1.

Figure 3

Lipase catalyzed transesterification reaction in the DES with sugar (e.g., glucose) and a methylated fatty acid ester produced from SCO (methyl myristate, methyl palmitate, methyl stearate, methyl oleate and methyl linoleate). The reaction leads to a sugar ester and methanol as side product. Methanol can enter the gas phase and push the reaction forwards or inhibit the enzyme.

Figure 4

Thin layer chromatography of synthesis products from the enzymatic reaction of microbial fatty acid methyl esters and beech wood cellulose fiber hydrolysate sugars catalyzed by iCalB in a DES reaction system. The crude synthesis extract, the purified and unified sample 1 and 2 were applied; sample 3, as middle fraction of sample 2, was not spotted and direct analyzed by NMR. After the run, the plate was dyed with anise aldehyde solution. Spots with RF 0 represents glucose (dark gray) and xylose (yellow) as leftovers from the DES. The light gray-yellowish spots between RF 0.05 and 0.42 indicate the formation of various fatty acid glucose- and xylose-esters. The high of the spots depends on the sugar, fatty acid chain length and saturation state. The two dark purple spots (RF 0.75 and 0.92) represents fatty acid methyl esters which were not consumed during the synthesis reaction.