Coupled enzymatic hydrolysis and ethanol fermentation: ionic liquid pretreatment for enhanced yields.

BACKGROUND: Pretreatment is a vital step upon biochemical conversion of lignocellulose materials into biofuels. An acid catalyzed thermochemical treatment is the most commonly employed method for this purpose. Alternatively, ionic liquids (ILs), a class of neoteric solvents, provide unique opportunities as solvents for the pretreatment of a wide range of lignocellulose materials. In the present study, four ionic liquid solvents (ILs), two switchable ILs (SILs) DBU-MEA-SO2 and DBU-MEA-CO2, as well as two 'classical' ILs [Amim][HCO2] and [AMMorp][OAc], were applied in the pretreatment of five different lignocellulosic materials: Spruce (Picea abies) wood, Pine (Pinus sylvestris) stem wood, Birch (Betula pendula) wood, Reed canary grass (RCG, Phalaris arundinacea), and Pine bark. Pure cellulosic substrate, Avicel, was also included in the study. The investigations were carried out in comparison to acid pretreatments. The efficiency of different pretreatments was then evaluated in terms of sugar release and ethanol fermentation. RESULTS: Excellent glucan-to-glucose conversion levels (between 75 and 97 %, depending on the biomass and pretreatment process applied) were obtained after the enzymatic hydrolysis of IL-treated substrates. This corresponded between 13 and 77 % for the combined acid treatment and enzymatic hydrolysis. With the exception of 77 % for pine bark, the glucan conversions for the non-treated lignocelluloses were much lower. Upon enzymatic hydrolysis of IL-treated lignocelluloses, a maximum of 92 % hemicelluloses were also released. As expected, the ethanol production upon fermentation of hydrolysates reflected their sugar concentrations, respectively. CONCLUSIONS: Utilization of various ILs as pretreatment solvents for different lignocelluloses was explored. SIL DBU-MEA-SO2 was found to be superior solvent for the pretreatment of lignocelluloses, especially in case of softwood substrates (i.e., spruce and pine). In case of birch and RCG, the hydrolysis efficiency of the SIL DBU-MEA-CO2 was similar or even better than that of DBU-MEA-SO2. Further, the IL [AMMorp][OAc] was found as comparably efficient as DBU-MEA-CO2. Pine bark was highly amorphous and none of the pretreatments applied resulted in clear benefits to improve the product yields.

Detoxification of acid pretreated spruce hydrolysates with ferrous sulfate and hydrogen peroxide improves enzymatic hydrolysis and fermentation.

The aim of the present work was to investigate whether a detoxification method already in use during waste water treatment could be functional also for ethanol production based on lignocellulosic substrates. Chemical conditioning of spruce hydrolysate with hydrogen peroxide (H2O2) and ferrous sulfate (FeSO4) was shown to be an efficient strategy to remove significant amounts of inhibitory compounds and, simultaneously, to enhance the enzymatic hydrolysis and fermentability of the substrates. Without treatment, the hydrolysates were hardly fermentable with maximum ethanol concentration below 0.4 g/l. In contrast, treatment by 2.5 mM FeSO4 and 150 mM H2O2 yielded a maximum ethanol concentration of 8.3 g/l.

Simultaneous saccharification, filtration and fermentation (SSFF): a novel method for bioethanol production from lignocellulosic biomass.

Simultaneous saccharification, filtration and fermentation (SSFF) was developed for lignocellulosic ethanol production. In SSFF, pretreated lignocellulosic material is enzymatically hydrolyzed in a reactor, while the suspension is continuously pumped through a cross-flow membrane. The retentate goes back to the hydrolysis vessel, while a clear sugar-rich filtrate continuously perfuses through the fermentation vessel before it is pumped back to the hydrolysis vessel. The capacity and life span of the cross-flow filter module was examined for 4 weeks using enzymatically hydrolyzed slurry, initially with 14.4% suspended solids, without clogging or fouling. An ethanol yield of 85.0% of the theoretical yield was obtained in SSFF and a flocculating strain of Saccharomyces cerevisiae was successfully reused for five cultivations of SSFF.

Influence of cultivation procedure for Saccharomyces cerevisiae used as pitching agent in industrial spent sulphite liquor fermentations.

The cell viability and fermentation performance often deteriorate in fermentations of spent sulphite liquor (SSL). This investigation therefore addresses the question of how different cultivation conditions for yeast cells influence their ability to survive and boost the ethanol production capacity in an SSL-based fermentation process. The strains used as pitching agents were an industrially harvested Saccharomyces cerevisiae and commercial dry baker's yeast. This study therefore suggests that exposure to SSL in combination with nutrients, prior to the fermentation step, is crucial for the performance of the yeast. Supplying 0.5 g/l fresh yeast cultivated under appropriate cultivation conditions may increase ethanol concentration more than 200%.

Continuous fermentation of wheat-supplemented lignocellulose hydrolysate with different types of cell retention.

Medium supplementation and process alternatives for fuel ethanol production from dilute acid lignocellulose hydrolysate were investigated. Dilute acid lignocellulose hydrolysate supplemented with enzymatically hydrolysed wheat flour could sustain continuous anaerobic cultivation of Saccharomyces cerevisiae ATCC 96581 if further supplemented with ammonium sulphate and biotin. This medium composition allowed for a hexose utilisation of 73% and an ethanol production of 36 mmol l(-1) h(-1) in chemostat cultivation at dilution rate 0.10 h(-1). Three different methods for cell retention were compared for improved fermentation of supplemented lignocellulose hydrolysate: cell recirculation by filtration, cell recirculation by sedimentation and cell immobilisation in calcium alginate. All three cell retention methods improved the hexose conversion and increased the volumetric ethanol production rate. Recirculation of 75% of the bioreactor outlet flow by filtration improved the hexose utilisation from 76% to 94%. Sedimentation turned out to be an efficient method for cell separation; the cell concentration in the reactor was 32 times higher than in the outflow after 60 h of substrate feeding. However, chemostat and continuous cell recirculation cultures became severely inhibited when the dilution rate was increased to 0.20 h(-1). In contrast, an immobilised system kept producing ethanol at a stable level also at dilution rate 0.30 h(-1).

Fed-batch cultivation of Mucor indicus in dilute-acid lignocellulosic hydrolyzate for ethanol production.

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.

Continuous fermentation of undetoxified dilute acid lignocellulose hydrolysate by Saccharomyces cerevisiae ATCC 96581 using cell recirculation.

Saccharomyces cerevisiae ATCC 96581 was cultivated in a chemostat reactor with undetoxified dilute acid softwood hydrolysate as the only carbon and energy source. The effects of nutrient addition, dilution rate, cell recirculation, and microaerobicity were investigated. Fermentation of unsupplemented dilute acid lignocellulose hydrolysate at D = 0.10 h(-1) in an anaerobic continuous reactor led to washout. Addition of ammonium sulfate or yeast extract was insufficient for obtaining steady state. In contrast, dilute acid lignocellulose hydrolysate supplemented with complete mineral medium, except for the carbon and energy source, was fermentable under anaerobic steady-state conditions at dilution rates up to 0.14 h(-1). Under these conditions, washout occurred at D = 0.15 h(-1). This was preceded by a drop in fermentative capacity and a very high specific ethanol production rate. Growth at all different dilution rates tested resulted in residual sugar in the chemostat. Cell recirculation (90%), achieved by cross-flow filtration, increased the sugar conversion rate from 92% to 99% at D = 0.10 h(-1). Nutrient addition clearly improved the long-term ethanol productivity in the recirculation cultures. Application of microaerobic conditions on the nutrient-supplemented recirculation cultures resulted in a higher production of biomass, a higher cellular protein content, and improved fermentative capacity, which further improves the robustness of fermentation of undetoxified lignocellulose hydrolysate.

The fermentation performance of nine strains of Saccharomyces cerevisiae in batch and fed-batch cultures in dilute-acid wood hydrolysate.

Large differences in colony forming capacity, ethanol production and inhibitor conversion were noted between nine different strains of Saccharomyces cerevisiae in anaerobic batch and fed-batch cultures on dilute acid wood hydrolysate. S. cerevisiae ATCC 96581 was able to metabolize all added glucose and mannose in fed-batch experiments. The choice of production strain will have a significant effect on the performance of a hydrolysate-based ethanol production plant.