Demonstrating a separation-free process coupling ionic liquid pretreatment, saccharification, and fermentation with Rhodosporidium toruloides to produce advanced biofuels

Abstract

Achieving low cost and high efficiency lignocellulose deconstruction is a critical step towards widespread adoption of lignocellulosic biofuels. Certain ionic liquid (IL)-based pretreatment processes effectively reduce recalcitrance of lignocellulose to enzymatic degradation but require either costly separations following pretreatment or novel IL compatible processes to mitigate downstream toxicity. Here we demonstrate at benchtop and pilot bioreactor scales a separation-free, intensified process for IL pretreatment, saccharification, and fermentation of sorghum biomass to produce the sesquiterpene bisabolene, a precursor to the renewable diesel and jet fuel bisabolane. The deconstruction process employs the IL cholinium lysinate ([Ch][Lys]), followed by enzymatic saccharification with the commercial enzyme cocktails Cellic CTec2 and HTec2. Glucose yields above 80% and xylose yields above 60% are observed at all scales tested. Unfiltered hydrolysate is fermented directly by Rhodosporidium toruloides -with glucose, xylose, acetate and lactate fully consumed during fermentation at all scales tested. Bisabolene titers improved with scale from 1.3 g L −1 in 30 mL shake flasks to 2.2 g L −1 in 20 L fermentation. The combined process enables conversion of saccharified IL-pretreated biomass directly to advanced biofuels with no separations or washing, minimal additions to facilitate fermentation, no loss of performance due to IL toxicity, and simplified fuel recovery via phase separation. This study is the first to demonstrate a separation-free IL based process for conversion of biomass to an advanced biofuel and is the first to demonstrate full consumption of glucose, xylose, acetate, and lactic acid in the presence of [Ch][Lys].

Engineering Robust Production Microbes for Large-Scale Cultivation

“…Miniaturizing commercial-scale culture conditions and the associated cost-effective, highthroughput capabilities can significantly increase the quantity of parameters tested at laboratory-scale. This has allowed testing new hosts for performance at different scales [87] (Table 1). However, the traditional statistical design-of-experiments (DOE) approach to process development is often limited by researchers' understanding of commercial-scale conditions.…”

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides

“…One of the advantages of R. toruloides as a host is its ability to grow on lignocellulosic hydrolysates and to perform well when cultivated in bioreactors [26,35]. To demonstrate production of ent-kaurene from lignocellulose, DMR-EH hydrolysate was prepared from corn stover as described previously [21].…”

Strategies for the production of biochemicals in bioenergy crops

“…The development of solvents and extraction methods compatible with existing biorefineries should enable the integration of novel streams that generate valuable coproducts while reducing recovery costs [321,322]. Furthermore, in a concept of one-pot biomass conversion process, the release from engineered lignocellulosic feedstocks of either target bioproducts or their immediate metabolic precursors during biomass pretreatment and saccharification offers a potential for increasing final bioproduct yields, but this approach will necessitate the development of microbial strains that are tolerant to inhibitors found in lignocellulosic hydrolysates [323,324]. For our future bioenergy crops, exploiting diverse metabolic pathways inherent to plants such as the shikimate and isoprenoid pathways will certainly contribute to the supply of several valuable biochemicals that find multiple industrial applications.…”