Abstract
Biorefinery processes converting C-rich feedstocks such as lignocellulosic biomass and starch residues often generate side-streams with HMF and furfural [1]. These compounds are formed when high temperatures and strong acids are used. Furthermore, they can be washed from the biomass and found in the washing filtrates. The presence of inhibitors in the washing filtrates makes their reuse more complex and energy-intensive. HMF and furfural are known inhibitors of microbial growth and can significantly reduce the efficiency of metabolic pathways [2], thereby limiting the feasibility of microbial fermentation using such side-streams. Oleaginous yeasts are able to produce single-cell oils from various side streams [3]. In this study, we screened a set of oleaginous yeasts to assess their tolerance, growth, and lipid accumulation using HMF- and furfural-containing side streams. The goal of this study was to identify strains suitable for converting HMF-furfural-rich side streams into microbial oils.
In this study, 8 oleaginous yeast strains from the genera Yarrowia, Cystofilobasidium, Rhodotorula, Sporidiobolus, Phaffia, and Debaryomyces were used. The steam-exploded biomass from Arbaflame was sent to RISE PFI for the washing trials [4]. 4 HMF-furfural containing side streams with different concentrations of HMF and furfural were prepared and used in cultivation trials. Cultivations were performed on three media: control media without inhibitors, Arbaflame washing filtrates media containing carbohydrates and different loadings of HMF and furfural, and synthetic media with added HMF and furfural at different concentrations. Cultivations were done using high-throughput screening systems such as the Duetz-MTPs (Enzyscreen, Netherlands) and BioLector I (Beckman Coulter, USA). The following analysis was performed after the cultivation: estimation of biomass production, total lipid content, fatty acid profile, substrate consumption, and HMF and furfural bioconversion.
Strains of Y. lipolytica, C. infirmominiatum, D. hansenii, and R. graminis can tolerate various loading of HMF and furfural without a significant decrease in lipid-accumulation. HPLC results showed that all sugar and acid components of Arbaflame side streams were consumed, and HMF and furfural were transformed into less toxic derivatives. These strains were selected for further screening on 11 synthetic media with different concentrations of HMF and furfural and combinations. It was observed that C. infirmominiatum and D. hansenii can tolerate HMF (up to 3 g/L) and combinations. Y. lipolytica and R. graminis can tolerate up to 3 g/L of HMF, while could not tolerate furfural and combinations of both. Total lipid content of R. graminis grown in media with 1-2 g/L of HMF was higher compared to the control. Total lipid content of C. infirmominiatum grown in the presence of 1-2 g/L HMF and furfural was similar to that of control media.
C. infirmominiatum and D. hansenii showed to be tolerant to HMF, furfural, and combinations and were able to transform them into less toxic derivatives and produce lipid-rich biomass. Total lipid content of R. graminis was higher in Arbaflame media than in control. This strain was less tolerant to furfural and combinations of HMF and furfural.