Author ORCID Identifier

Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation


Agriculture; Engineering


Biosystems and Agricultural Engineering

First Advisor

Dr. Jian Shi


Due to the overuse of antibiotics in our society, there has been a steady rise in highly antimicrobial-resistant bacteria in the last decade. This has created a renewed interest in natural phenolic compounds for antimicrobial discovery amongst the scientific community. To this end, lignin is the most abundant naturally occurring phenolic polymer on earth and has already been known to have antimicrobial properties due to its polyphenolic structure. In addition, lignin is considered a major waste product for lignocellulosic biorefineries, and its valorization into value-added products will generate extra profit for a biorefinery, making biofuels less expensive, increasing their marketability as an alternative to fossil fuels. However, the retention of lignin’s antimicrobial properties in different materials, as depolymerized products, or even the prediction of their antimicrobial properties is not well understood in the literature.

Much work has utilized lignin as a functional polymer in a variety of composites and materials, but their antimicrobial properties have not been as widely explored. Therefore, ionic liquids were used in the facile preparation of cellulose-based hydrogels, and the addition of different lignocellulosic components (lignin and xylan) or the use of whole biomass (poplar and sorghum) were evaluated for their effects on hydrogel properties (mechanical and antimicrobial). The addition of both lignin and xylan improved hydrogel mechanical strength/stiffness, and lignin-containing hydrogels showed retained antimicrobial properties when screened against the target organism (Escherichia coli). Utilizing raw biomass provided increased mechanical strength (poplar), similar water retention abilities (poplar and sorghum), and retained antimicrobial properties (poplar). These results indicate that the different components of lignocellulose can be used to fine tune the properties of cellulose-based hydrogels and that lignin can confer its antimicrobial properties when incorporated into hydrogels.

The antimicrobial properties of different lignin depolymerization products were explored using a reductive and oxidative depolymerization method to produce phenolic rich lignin-based bio-oils. Purified alkali-enzymatic corn stover lignin (AEL) was depolymerized by catalytic transfer hydrogenolysis using supercritical ethanol and a Ru/C catalyst, generating a bio-oil stream at high yields. Sequential extraction was used to fractionate the bio-oil into five fractions with different phenolic compositions using hexane, petroleum ether, chloroform, and ethyl acetate. Antimicrobial properties of the bio-oils were screened against Gram-positive/negative bacteria and yeast by examining microbial growth inhibition. The monomers in the bio-oil fractions contained primarily alkylated phenols, hydrogenated hydroxycinnamic acid derivatives, syringol and guaiacol-type lignins created from reductive cleavages of ether linkages. After sequential extraction, the lignin derived compounds were fractionated into groups depending on solvent polarity. Results suggest that the total monomer concentration and the presence of specific monomers (i.e., syringyl propane) may correlate to the antimicrobial activity of lignin depolymerization products, but the exact mode of action or antimicrobial activity caused by the complex mixtures of monomers and unidentified oligomers remains unclear.

The same AEL lignin was depolymerized through oxidative procedures using peracetic acid, and its applications as an antibiotic replacement in the fuel ethanol industry were explored. The resulting bio-oil had a low degree of depolymerization that mostly produced unidentifiable lignin oligomers. Nonetheless, this bio-oil displayed highly selective antimicrobial properties, with up to 90% inhibition of commercially sampled lactic acid bacteria (LAB) at 4 mg/ml and no inhibition of yeast. Using the bio-oil (4 mg/ml) as an alternative antibiotic treatment during simultaneous-saccharification and fermentation of raw corn starch showed an 8% increase in ethanol production at a yeast to LAB ratio of 1:100, compared to untreated contaminated controls. The ability of the bio-oil to improve ethanol yields clearly shows its efficacy as an alternative antibiotic and that depending on depolymerization method lignin derivates can display a variety of useful antimicrobial properties/applications.

The final study was the first attempt in the literature to predict the antimicrobial properties of lignin derivatives using quantitative structure−activity relationship (QSAR) models. First, the open-access database ChEMBL, with non-lignin specific compounds, was used to create datasets of compounds with MIC activity measurements against both B. subtilis and E. coli. Machine learning algorithms were used to develop the QSARs for the large ChEMBL datasets and were found to underpredict the antimicrobial activity of actual lignin compounds. Conversely, as metanalysis of the literature containing MIC data of lignin derivatives were used to build QSAR models with ordinary least square regressions (OLS). An accurate QSAR model for E. coli was not found, but a satisfactory model was obtained for the B. subtilis metanalysis dataset. Molecular Operation Environment (MOE)-type descriptors and the number of aliphatic carboxylic acid groups showed strong correlations to the MIC values (R2 of 0.759). Comparatively, an additional dataset was experimentally derived by screening 25 lignin monomers and three dimers against B. subtilis by measuring bacterial load difference (BLD). This datasets QSAR, using OLS, found that MOE-type descriptors and the number of aromatic hydroxyl groups were better predictors of BLD (R2 of 0.831). Thus, the smaller datasets highlighted how the variability in antimicrobial measurements and the specific compounds used will impact the predictive nature of the resulting QSARs. Overall, this entire work provides critical knowledge and guidance on using lignin as an antimicrobial source in different industrial processes/products and the identification of lignin derivatives with enhanced activity.

Digital Object Identifier (DOI)

Funding Information

This study was supported by the National Science Foundation under Cooperative Agreement No. 1355438 and 1632854 (2016-2020). This work was also supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch-Multistate project under accession numbers 1018315/1003563 (2018 - 2023).