Toward a sustainable biorefinery using high-gravity technology

The realization of process solutions for a sustainable bioeconomy depends on the efficient processing of biomass. High-gravity technology is one important alternative to realizing such solutions. The aims of this work were to expand the knowledge-base on lignocellulosic bioconversion processes at high solids content, to advance the current technologies for production of second-generation liquid biofuels, to evaluate the environmental impact of the proposed process by using life cycle assessment (LCA), and to develop and present a technically, economically, and environmentally sound process at high gravity, i.e., a process operating at the highest possible concentrations of raw material. The results and opinions presented here are the result of a Nordic collaborative study within the framework of the HG Biofuels project. Processes with bioethanol or biobutanol as target products were studied using wheat straw and spruce as interesting Nordic raw materials. During the project, the main scientific, economic, and technical challenges of such a process were identified. Integrated solutions to these challenges were proposed and tested experimentally, using wheat straw and spruce wood at a dry matter content of 30% (w/w) as model substrates. The LCA performed revealed the environmental impact of each of the process steps, highlighting the importance of the enzyme dose used for the hydrolysis of the plant biomass, as well as the importance of the fermentation yield

Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii

Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition, and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingolipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid, and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid, was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stres

Co-production of biofuels and value-added compounds from industrial Eucalyptus globulus bark residues using hydrothermal treatment

In this work, hydrothermal treatment was assessed for the fractionation of industrial Eucalyptus globulus bark residue (EBR) to obtain biofuels and value-added compounds (such as oligosaccharides and phenolic compounds) in separated streams. Hydrothermal treatment was evaluated under non-isothermal regimen in the range of maximum temperature (Tmax) of 177228 °C or severities (S0) between 2.76 and 4.25. The highest oligosaccharides concentration (17.5 g/L) was achieved at S0 of 3.69, corresponding to hemicellulose recovery of 77.30%. Under all severities evaluated in this work, over 90.94% and 84.17% of cellulose and lignin remained in the solid phase, respectively. The increase of S0 improved 4.38-fold the enzymatic saccharification of cellulose, the highest glucose yield (84%) being achieved at S0 of 4.04. Considering the maximal recovery of polysaccharides as glucose and oligosaccharides from the liquid and solid phases, S0 of 4.04 was selected for bioethanol production using high solid loadings and following different strategies (simultaneous saccharification and fermentation SSF and pre-saccharification and simultaneous saccharification and fermentation PSSF). The utilization of 15% hydrothermally pretreated EBR without nutrient supplementation resulted in 26 g/L of ethanol, independently of the strategy used. An increase up to 17.5% solids and employing nutrient supplementation enabled the production of 38 g/L (or 4.8% v/v) of ethanol by PSSF.This work has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI) – LISBOA-01-0145-FEDER-022059, supported by Operational Programme for Competitiveness and Internationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Programme (Lisboa 2020) and by North Portugal Regional Operational Program (Norte 2020) under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and has been supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/ 04469/2020 and through Project EcoTech (POCI-01-0145-FEDER032206) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

A process for energy-efficient high-solids fed-batch enzymatic liquefaction of cellulosic biomass

The enzymatic hydrolysis of cellulosic biomass is a key step in the biochemical production of fuels and chemicals. Economically feasible large-scale implementation of the process requires operation at high solids loadings, i.e., biomass concentrations >15% (w/w). At increasing solids loadings, however, biomass forms a high viscosity slurry that becomes increasingly challenging to mix and severely mass transfer limited, which limits further addition of solids. To overcome these limitations, we developed a fed-batch process controlled by the yield stress and its changes during liquefaction of the reaction mixture. The process control relies on an in-line, non-invasive magnetic resonance imaging (MRI) rheometer to monitor real-time evolution of yield stress during liquefaction. Additionally, we demonstrate that timing of enzyme addition relative to biomass addition influences process efficiency, and the upper limit of solids loading is ultimately limited by end-product inhibition as soluble glucose and cellobiose accumulate in the liquid phase

A novel hybrid organosolv: steam explosion method for the efficient fractionation and pretreatment of birch biomass

Background: The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance sac- charification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractiona- tion into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction. Results: In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pre - treatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/w biomass H 2 SO 4 was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum. Conclusions: The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive dis- charge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass

Evolutionary engineering of xylose utilizing S. cerevisiae to improve the tolerance against inhibitors present in the pretreated lignocellulosic raw materials

The development of inhibitor tolerant Saccharomyces cerevisiae is needed for the efficient fermentation of lignocellulosic hydrolysates. In this work, evolutionary engineering was carried out on a xylose utilizing recombinant S. cerevisiae to generate strains tolerant to inhibitors in spruce hydrolysate. A broad range of inhibitors generated in pretreated spruce hydrolysate were chosen from available literature information. Potent inhibitors were selected to represent three inhibitor categories: furans, weak acids and phenolic compounds to make an inhibitor cocktail. Repetitive shake flask experiments were carried out by increasing the concentration of inhibitor cocktail in defined media containing glucose and xylose as carbon sources. The strains were screened for increase in specific growth rate and decrease in length of lag phase. Adaptive evolution was also accompanied by UV induced mutations. Strains developed during the course of adaptive evolution showed 89% increase in specific growth rate and length of lag phase decreased from 48 h to 24 h. The strains were screened on YPD plates containing inhibitor cocktail, YP-hydrolysate and YPX plates for inhibitor tolerance and improved xylose utilization. The two strains RK60-5 and RKU90- 3 were selected and anaerobic fermentations in defined media and spruce hydrolysate were carried out. Both strains displayed higher specific growth rates and glucose consumption rates. In spruce hydrolysate, the maximum ethanol productivity of RK60-5 and RKU90-3 were 0.017 and 0.018 g g-1 h-1 respectively compared to 0.012 g g-1 h-1 of parental strain. However, RK60-5 showed low glycerol yield and no traces of xylitol formation indicating absence of xylose utilization, whereas, RKU90-3 produced considerable levels of xylitol in spruce hydrolysate. Nevertheless, both strains showed high tolerance to inhibitors and the strategy of adaptive evolution proved to be promising. Keywords: Evolutionary engineering, adaptive evolution, inhibitors, spruce hydrolysate, specific growth rate and lag phase