Improved Methodologies for Biomass Wet Chemical Analysis

The purpose of this thesis study was to further the development of lignocellulosic biomass as a potential renewable energy source by investigating new wet chemical compositional analysis techniques to be used to monitor changes in biomass composition resulting from size reduction and separation processes such grinding and sieving. Numerous disadvantages to the standard wet chemical analysis procedure as developed by US Dept of Energy and the National Renewable Energy Laboratory (NREL) were identified as targets for possible improvements. The overall objective was utilization of ionic liquids as a “green” alternative to the use of aqueous acidic solvents employed in the NREL protocol. These experiments included direct spectral analyses to quantify the lignin constituent, and successive enzymatic hydrolysis for quantification of the cellulose constituent. Results contained herein revealed that solubilization of biomass occurred in ionic liquids, which allowed for rapid spectroscopic determination of its lignin composition. The enzymatic hydrolysis of cellulose occurred in an ionic liquid-rich solvent system, and quantification of the cellulolytic monosaccharide products was achieved using high performance liquid chromatography. Motivated by the disadvantages associated with the NREL biomass compositional analysis procedure, a new analysis procedure utilizing ionic liquids was proposed and developed as an approach aimed towards improving laboratory safety and analysis time. The study was approached by first quantifying the solubility of biomass in ionic liquids. Direct quantification of the lignin content was conducted by two methods, UV-visible spectrophotometric analyses after the addition of a dilution agent, acetonitrile, and Fourier Transform Infrared Spectroscopy. The cellulose component of yellow poplar was then completely hydrolyzed using a cellulolytic enzyme in the ionic liquid-rich reaction media, and the hydrolysate was then analyzed by high performance liquid chromatography for the quantification of glucose monomeric units. Success was achieved in the design of the analysis procedure, and it was employed for the quantification of lignin and cellulose in yellow poplar. There was also a highly predictable conversion of cellulose to glucose and cellobiose by the cellulase in the ionic liquid-rich reaction media. A biomass compositional analysis procedure for the quantification of lignin and cellulose was created and was observed to be consistent in comparison with the results from the NREL protocol. The total lignin content as a percent of dry mass in yellow poplar was found to be 25.1% ± 0.8 using the NREL protocol, and 21.5% ± 0.4 and 25.6% ± 0.1 by the UV-visible and Fourier Transform Infrared Spectroscopy approaches, respectively, in the methods described herein. The glucan component was quantified as 43.5% ± 0.5 utilizing the NREL protocol and 43.6% ± 0.3 through analysis of the enzymatic hydrolysate as part of these methodologies

SIMPLIFIED DETERMINATION OF LIGNIN CONTENT IN HARD AND SOFT WOODS VIA UV-SPECTROPHOTOMETRIC ANALYSIS OF BIOMASS DISSOLVED IN IONIC LIQUID

A new simple and safe method for quantifying lignin content in lignocellulosic biomass is described. The approach consists of measuring the absorbance of a solution of whole biomass dissolved in the ionic liquid 1-n-butyl-3-methyl imidazolium chloride, [Bmim][Cl], at 440 nm via ultraviolet- (UV-) visible spectrophotometry. An extinction coefficient for a lignin standard, highly pure lignin isolated from biomass through an organosolv process, is used in conjunction with the Beer-Lambert Law to calculate the lignin concentration. Principal component analysis (PCA) of Fourier Transform-Infrared (FTIR) spectra collected for several different lignin standards was performed to understand the differences in their chemical structure and composition (e.g., the relative amounts of syringyl and guaiacyl units). A rapid FTIR analysis of the whole biomass sample with unknown lignin content is required to assist in the proper selection of the lignin standard for the subsequent spectrophotometric analysis. The proposed method was tested and validated on two biomass types: Yellow poplar and Southern pine. The spectrophotometric approach yielded lignin contents for Yellow poplar and Southern pine of 25.7 ± 1.1% and 26.7 ± 0.7%, respectively, which are comparable to the values obtained by a standard wet chemical protocol, 25.1% ± 0.7 and 26.6 ± 0.4%, respectively