Fungal pretreatment is a biological process that uses rotting fungi to reduce the recalcitrance and enhance the enzymatic digestibility of lignocellulosic feedstocks at low temperature, without added chemicals and wastewater generation. Thus, it has been presumed to be low cost. However, fungal pretreatment requires longer incubation times and generates lower yields than traditional pretreatments. Thus, this study assesses the techno-economic feasibility of a fungal pretreatment facility for the production of fermentable sugars for a 75,700 m 3 (20 million gallons) per year cellulosic bioethanol plant. Four feedstocks were evaluated: perennial grasses, corn stover, agricultural residues other than corn stover, and hardwood. The lowest estimated sugars production cost ($1.6/kg) was obtained from corn stover, and was 4-15 times as much as previous estimates for conventional pretreatment technologies. The facility-related cost was the major contributor (46-51%) to the sugar production cost, mainly because of the requirement of large equipment in high quantities, due to process bottlenecks such as low sugar yields, low feedstock bulk density, long fungal pretreatment times, and sterilization requirements. At the current state of the technology, fungal pretreatment at biorefinery scale does not appear to be economically feasible, and considerable process improvements are still required to achieve product cost targets.Fermentation 2019, 5, 30 2 of 23 of water [5]. Additionally, most commercially available pretreatments, such as dilute acid and liquid hot water pretreatment, require an additional detoxification step, because their severe conditions promote the generation of compounds derived from lignocellulose, such as furans, weak acids and aromatic compounds, which could inhibit fermentative microorganisms, such as yeast and bacteria [6].A series of alternative methods have been proposed to reduce the severity of the conditions required for biomass fractionation and pretreatment. Use of extrusion, microwaves, ultrasound, or pulse electric field has shown to improve the biomass pretreatment process and allowed for the use of milder pretreatment conditions [7]. Biomass pretreatment with aqueous organic solvents (organosolv) [8,9], ozone (ozonolysis) [10], ionic liquids [11,12], or deep eutectic solvents [13] has shown promising results in biomass fractionation [14,15]. However, concerns about the costs, handling, and recovery of the chemicals present limitations in their large-scale applicability [14,16].Fungal pretreatment is as an alternative to conventional pretreatments that is performed at 25-30 • C, atmospheric pressure, in solid-state with low water use, and without added chemicals that can be corrosive and would need disposal or recovery [17]. Fungal pretreatment is generally performed using wood-rotting fungi, such as white-, brown-, or soft-rot fungi, due to their ability to modify the components of the lignocellulosic biomass. Brown-rot fungi mainly degrade cellulose and hemicellulose, with little modificati...