Pilot-scale fermentation to produce trichodiene, a fungal volatile that suppresses trichothecene production in mycotoxigenic fungi

Trichodiene is a sesquiterpene hydrocarbon and the precursor of trichothecene mycotoxins produced by <i>Fusarium</i> and other fungi. Interestingly, utilizing trichodiene as a volatile treatment has been shown to reduce mycotoxin production in <i>Fusarium graminearum</i>-infected wheat heads. This research developed a pilot-scale fungal fermentation method to produce trichodiene as a biofumigant to mitigate mycotoxin contamination. A <i>TRI4</i> mutant strain of <i>Fusarium sporotrichioides</i> was used to mass-produce trichodiene. Xanthotoxin, a furanocoumarin produced by parsnips, was used to enhance trichodiene production in this mutant. Xanthotoxin treatments significantly increased trichodiene yield and were found to promote lipid droplet release from the fungal germlings. Benchtop scale evaluations were conducted to determine the impacts of xanthotoxin concentration, fermentation time, extraction solvent, and filtration on overall yield. After optimal conditions were identified, the fungal cultures treated with xanthotoxin were fermented for 1 week in a 30 L bioreactor. After an organic extraction of the fungal culture and concentration of the extract, trichodiene was isolated using silica gel column chromatography. Purified trichodiene reduced mycotoxin production in <i>F. graminearum</i> in a dose-dependent manner. This research will allow the production of trichodiene in sufficient quantities to further evaluate its efficacy as a biofumigant to suppress mycotoxin production in <i>F. graminearum</i>.IMPORTANCEFood contamination from microbial toxins is a threat to human and animal health. Globally, the pathogen <i>Fusarium graminearum</i> causes annual losses in billions of dollars for cereal farmers and producers. Previous studies have shown that the fungal terpene trichodiene can suppress the production of vomitoxin (deoxynivalenol) by <i>F. graminearum</i>. We developed a way to scale production of trichodiene and use it to inhibit <i>F. graminearum</i> toxin contamination. A mutant strain of <i>Fusarium sporotrichioides</i> that produces trichodiene was grown in large 30 L fermenters and treated with xanthotoxin, a natural compound made by parsnips. Xanthotoxin caused lipid droplet release from the fungus and increased trichodiene yield. The purified trichodiene effectively reduced toxin production by <i>F. graminearum</i> by direct contact or as a volatile. Based on these research findings, trichodiene can be produced using common large-scale fermentation methods. Field formulations can now be developed to suppress mycotoxin contamination in food and feed.