Using Anaerobic Fungi to Hydrolyze Lignocellulosic Biomass in Anaerobic Digestion

American farms produce vast amounts of waste (leftover crop material and animal manure) that could become sources of clean energy and useful chemicals. A process called anaerobic digestion can convert this waste into energy, but it only works on large farms because plant fibers break down slowly and the equipment is costly. This project looks to nature for a faster path: cattle easily digest tough plants with the help of gut microbes called anaerobic fungi, which break down plant matter very quickly. These fungi could be powerful tools, but scientists have not learned how to keep them active inside human-built reactors. This project identifies conditions that enable the fungi to survive and stay productive in waste-treatment reactors. The results help engineers design less expensive, smaller systems that turn farm waste into valuable products. Project outcomes enable small farms to earn money from waste, reduce water pollution, and make the nation's food and energy supply more secure. The project also trains students and shares its findings with farmers and researchers worldwide. This project focuses on operational and ecological conditions that sustain anaerobic fungi (AF), the microbe with the largest known repertoire of carbohydrate-active enzymes, in mixed microbial communities. The research operates bench-scale, rumen-simulating, semi-continuous bioreactors that decouple hydraulic retention time from solids retention time to retain fungal biomass while preventing the inhibitory accumulation of product volatile fatty acids (VFAs). Three objectives are pursued: resolving whether and how simple sugars such as glucose repress fungal enzyme expression in mixed cultures, determining whether dormant AF in cow manure can be reactivated and sustained when supplied as co-substrate or inoculum under rumen-like conditions, and quantifying how elevated solids retention time affects lignocellulose hydrolysis yields and fungal stability. Mechanisms are investigated with molecular tools that include quantitative and reverse-transcription quantitative polymerase chain reaction targeting fungal 18S ribosomal RNA genes and GH5 endoglucanase transcripts, 16S and 28S ribosomal RNA sequencing, and 13C-glucose stable-isotope probing to track which populations consume sugars and help keeping AF active. Chemical, microbial, and multivariate statistical analyses are integrated to identify the drivers of community change. By establishing the design and operating parameters that keep AF hydrolytically active, the project advances the science of anaerobic microbial ecology and resource recovery and provides a foundation for scalable biomanufacturing that converts lignocellulosic agricultural wastes into biofuels and bioproducts. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.