Green pretreatment strategies for enhanced microbial lipid fermentation and synergistic high-quality lignin recovery for next-generation integrated biorefineries

Authors: RAJ, TIRATH - University Of Illinois; SHARMA, POONAM - University Of Illinois; Thompson, Stephanie; Dien, Bruce; SINGH, VIJAY - University Of Illinois

Submitted to: Chemical Engineering Journal Advances
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2025
Publication Date: 1/1/2026
Citation: Raj, T., Sharma, P., Thompson, S., Dien, B.S., Singh, V. 2026. Green pretreatment strategies for enhanced microbial lipid fermentation and synergistic high-quality lignin recovery for next-generation integrated biorefineries. Chemical Engineering Journal Advances. https://doi.org/10.1016/j.ceja.2025.101031.
DOI: https://doi.org/10.1016/j.ceja.2025.101031

Interpretive Summary: Miscanthus is a newly emerging crop grown for biomass production in temperate regions. It is a warm season perennial grass that is prized for its productivity and genetic diversity.  However, production is limited by the absence of sufficient value-added markets.  ARS and University Illinois researchers have developed a highly integrated bioprocess that converts the biomass into two high-value products: oil (produced using yeast) and extracted lignin.  The oil can be used for making renewable fuels, food ingredients, and biobased consumer care products. The lignin can be further broken down to yield high-value chemicals or be converted to high quality carbon-based products, such as carbon fibers. Development of new markets for miscanthus allows farmers to repurpose non-commercial farmland and creates an additional revenue stream for agricultural crop processors.

Technical Abstract: Miscanthus is a warm season perennial grass that is being commercialized for biomass production for temperate climate farms. Three process options are compared for processing Mxg biomass into single-cell oil and extracted lignin as intermediates for biofuel and chemical production, respectively. The three processes tested were chemical-free hydrothermal processing and natural deep eutectic solvents (NADES). Two NADES formulations tested were ChCl:LA (choline chloride: lactic acid) and ChCl:Gly (choline chloride: glycerol). Pretreatments were performed at 10% and 50% solids loading under the following conditions: NADES (140 °C, 2 h) and hydrothermal (190 °C, 10 min). Various enzymatic hydrolysates were prepared by hydrolyzing at 10% solids loading using washed and unwashed biomass to investigate the lipid production. Overall, washed biomass yielded maximum glucose conversions of 83.5%, 52.7%, and 74.0% for ChCl:LA, ChCl:Gly, and HT pretreatments, respectively. Notably, NADES-derived hydrolysates can effectively replace refined sugars for microbial lipid production using the oleaginous yeast Rhodotorula toruloides, achieving ~51% higher biomass (OD 90.7) and lipid titers (19.36 g/L) within 45 h compared to refined sugars, using a two-stage fermentation strategy. Lipid content ranged from 34.5–44.5% % dry weight lipid following a state fermentation approach, reducing reliance on purified sugars. Furthermore, ChCl:LA pretreatment enabled high-purity lignin recovery (>89%) following a lignin-first approach. Structural characterization by 2D-HSQC and ³¹P NMR revealed lignin compositions of S (78.15%), G (15.15%), and H (6.41%) units in ChCl:LA lignin, with higher phenolic hydroxyl content (0.91 mmol g'¹) and a lower S/G ratio (0.19) compared to hydrothermal lignin (0.87 mmol g'¹ phenolic OH, S/G 0.22). These structural features may facilitate downstream lignin depolymerization into low-molecular-weight aromatics. Overall, NADES pretreatment offers a dual advantage by enabling enhanced production of microbial lipid production and recovery of high-quality lignin, thereby improving the economic feasibility of producing renewable diesel and SAFs from bioenergy crops within an integrated biorefinery framework.