With the aim of increasing the sugars concentration in dilute-acid ligno-cellulosic hydrolyzate to more than 100 g/l for industrial applications, the hydrolyzate from spruce was concentrated about threefold by high-pressure or vacuum evaporations. It was then fermented by repeated fed-batch cultivation using flocculating Saccharomyces cerevisiae with no prior detoxification. The sugars and inhibitors concentrations in the hydrolyzates were compared after the evaporations and also fermenta-tion. The evaporations were carried out either under vacuum (VEH) at 0.5 bar and 80°C or with 1.3 bar pressure (HPEH) at 107.5°C, which resulted in 153.3 and 164.6 g/l total sugars, respectively. No sugar decomposition occurred during either of the evaporations, while more than 96% of furfural and to a lesser extent formic and acetic acids disappeared from the hydrolyzates. However, HMF and levulinic acid remained in the hydrolyzates and were concentrated proportionally. The concentrated hydrolyzates were then fermented in a 4 l bioreactor with 12-22 g/l yeast and 0.14-0.22 h-1 initial dilute rates (ID). More than 84% of the fermentable sugars present in the VEH were fermented by fed-batch cultivation using 12 g/l yeast and initial dilution rate (ID) of 0.22 h-1, and resulted in 0.40±0.01 g/g ethanol from the fermentable sugars in one cycle of fermentation. Fermentation of HPEH was as successful as VEH and resulted in more than 86% of the sugar consumption under the corresponding conditions. By lowering the initial dilution rate to 0.14 h-1, more than 97% of the total fermentable sugars were consumed, and ethanol yield was 0.44±0.01 g/g in one cycle of fermentation. The yeast was able to convert or assimilate HMF, levulinic, acetic, and formic acids by 96, 30, 43, and 74%, respectively
In order to obtain a sugar concentration of more than 100g.l-1of fermentable sugars, aspruce wood hydrolysate was subjected to high pressure and vacuum concentration andthe fermentability of each hydrolysate was assessed by fermentation experiments withflocculating S. cerevisiae. The hypothesis that high pressure evaporated hydrolysate(evaporation carried out at 108°C and 1.3 bar) would be more difficult to ferment thanvacuum evaporated hydrolysate (evaporation carried out at 80°C and 0.5 bar) was notconfirmed by the results. Minor amount of cells lost their flocculating ability afterfermentation which their ratio and their viability and vitality was assessed.By vacuum and high pressure concentration, the fermentable sugars (defined as theconcentration of glucose, mannose and galactose) in the hydrolysates reached to 120g.l-1and 129g.l-1 respectively. Compared to the initial hydrolysate the concentration factorrepresented a 3-fold increase of fermentable sugars. Furfural was evaporated in both trialsand its concentration reached to 0.03g.l-1 and 0.1g.l-1 after vacuum and high pressureevaporation respectively. Fermentation with both 0.14h-1and 0.22h-1 initial dilution rateswas possible, while more than 96% of furfural and to less extent formic and acetic acidsdisappeared from the hydrolyzates. However, HMF and levulinic acid remained in thehydrolyzates and concentrated proportionally. More than 84% of the fermentable sugarspresent in VEH were fermented by fed-batch cultivation using 12g.l-1 yeast and initialdilution rate (ID) of 0.22h-1, and resulted into 0.40±0.01g.g-1 ethanol in 21h.Fermentation of HPEH was as successful as VEH and resulted into more than 86% of thesugar consumption at the corresponding conditions. With an ID of 0.14h-1, more than97% of the total fermentable sugars were consumed, and ethanol yielded 0.44±0.01g.g-1.A viability and vitality determination from the supernatant of fermentation liquorrepresented that about 76% of the cells which lost their flocculating ability kept theirvitality. Cultivation of yeast with beet molasses was tricky in both batch and fed-batchcultivation as the concentration more than 50g.l-1 in batch cultivation prevent from yeastgrowing.Uppsatsnivå:
In order to obtain a sugar concentration of more than 100g.l-1of fermentable sugars, aspruce wood hydrolysate was subjected to high pressure and vacuum concentration andthe fermentability of each hydrolysate was assessed by fermentation experiments withflocculating S. cerevisiae. The hypothesis that high pressure evaporated hydrolysate(evaporation carried out at 108°C and 1.3 bar) would be more difficult to ferment thanvacuum evaporated hydrolysate (evaporation carried out at 80°C and 0.5 bar) was notconfirmed by the results. Minor amount of cells lost their flocculating ability afterfermentation which their ratio and their viability and vitality was assessed.By vacuum and high pressure concentration, the fermentable sugars (defined as theconcentration of glucose, mannose and galactose) in the hydrolysates reached to 120g.l-1and 129g.l-1 respectively. Compared to the initial hydrolysate the concentration factorrepresented a 3-fold increase of fermentable sugars. Furfural was evaporated in both trialsand its concentration reached to 0.03g.l-1 and 0.1g.l-1 after vacuum and high pressureevaporation respectively. Fermentation with both 0.14h-1and 0.22h-1 initial dilution rateswas possible, while more than 96% of furfural and to less extent formic and acetic acidsdisappeared from the hydrolyzates. However, HMF and levulinic acid remained in thehydrolyzates and concentrated proportionally. More than 84% of the fermentable sugarspresent in VEH were fermented by fed-batch cultivation using 12g.l-1 yeast and initialdilution rate (ID) of 0.22h-1, and resulted into 0.40±0.01g.g-1 ethanol in 21h.Fermentation of HPEH was as successful as VEH and resulted into more than 86% of thesugar consumption at the corresponding conditions. With an ID of 0.14h-1, more than97% of the total fermentable sugars were consumed, and ethanol yielded 0.44±0.01g.g-1.A viability and vitality determination from the supernatant of fermentation liquorrepresented that about 76% of the cells which lost their flocculating ability kept theirvitality. Cultivation of yeast with beet molasses was tricky in both batch and fed-batchcultivation as the concentration more than 50g.l-1 in batch cultivation prevent from yeastgrowing.Uppsatsnivå:
Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange
Publication date
01/01/2013
Field of study
The current raw materials for the production of bioethanol are starch-based materials as well as sugar cane and molasses. However lignocellulosic biomass (e.g. agricultural residues and herbaceous grasses) hold a great potential to be used as carbohydrtae source for fermentation to ethanol. Between the two routes developed for hydrolysis of biomass to carbohydrate monomers, acid and enzymatic hydrolysis, the latter is favored. However, a foremost concern for economically efficient ethanol production from biomass through enzymatic hydrolysis pathway is the large volume and high cost of the Cellulolytic enzymes used in this process. Due to the recalcitrant nature of biomass a typical dose of 15 FPU/g glucan is used for enzymatic hydrolysis. This level of enzyme is equivalent to ~30 g enzyme/l ethanol produced, a costly dose (at an enzyme price of 10/kg)thatcausethecellulolyticenzymetoaccountfor27−400.94/l. Thus, enzyme costs must either be reduced to lower than 2/kgproteinorstrategiesdevelopedtosubstantiallyreduceloadings(3˘c5FPU/gglucan)sothatcellulolyticenzymeswouldbecomparablewithenzymescurrentlyusedincorntoethanolindustry.Ithasbeenshownthatcellulolyticenzymesaredeactivatedduetoavarietyofreasonssuchasdeformationduetothermal,sheareffect,highsurfacetensionofsolutionthatwouldexposetheenzymetoair−liquidinterfaceandirreversibleadsorptiontoligninandcrystallinecellulosethatinhibittheircatalyticaction.Inthisdissertation,thepotentialroleofnovelprotein−basedmicellesandsurfactantorprotein−basedpolymericmicelles(PMs)wastestedonenzymeactivity,hydrolysis,fermentationandenzymerecycling.Andthepotentialmechanismsofactionoftheseamphiphilesandtheireconomicalviabilitywereinspected.Formationofirreversible−boundmonolayersofcasein,onhydrophobicsurfaceswasshowntoalleviatethede−activationofproteinofinterest.ApplicationofcolloidalproteinsofcaseinonthesurfaceofSiO2hasbeenusedtopreventfromdeactivationofkinesin.Alsoavarietyofblockingbuffersrangingfromcasein,milkornormalserumtohighlypurifiedproteinsiscommonlyusedtoblockthefreesitesonamicrocellulosemembraneonwhichtheantibodies(proteins)hasbeentransferredfromagel,topreventnonspecificbindingofthedetectionantigens(protein)duringthesubsequentsteps.Casein,aphospho−protein,thatmakesthe804-6/kg could not justify the economically use of Tween at 0.47 g/g and Casein at 2.5 g/g glucan during SHF of corn stover. Even reduction of the Tween and casein utilization to 0.1 g/g glucan would potentially yield in a permissible cost of ¢16.0/kg and ¢78.0/kg, respectively. In order for the casein application to be justifiable as additives, a recycling strategy or addition of 0.06 g/g glucan PEG would be necessary to increase the permissible cost by up to 2.5/kg.However,evenwiththesestrategies,thepermissiblecostofTween20wasonly1.3/kg which wouldn’t permit the application of this additive as enzyme stabilizer. As a result of the sensitivity analysis it was found that, the permissible cost of the additive is increased with the increase in cost of enzyme. The extent of improvement in ethanol yield obtained and the level of additives used, along with the price of enzyme were the determinative factors in permissible cost of additive
One of the concerns for economical production of ethanol from biomass is the large volume and high cost of the cellulolytic enzymes used to convert biomass into fermentable sugars. The presence of acetyl groups in hemicellulose and lignin in plant cell walls reduces accessibility of biomass to the enzymes and makes conversion a slow process. In addition to low enzyme accessibility, a rapid deactivation of cellulases during biomass hydrolysis can be another factor contributing to the low sugar recovery. As of now, the economical reduction in lignin content of the biomass is considered a bottleneck, and raises issues for several reasons. The presence of lignin in biomass reduces the swelling of cellulose fibrils and accessibility of enzyme to carbohydrate polymers. It also causes an irreversible adsorption of the cellulolytic enzymes that prevents effective enzyme activity and recycling. Amphiphiles, such as surfactants and proteins have been found to improve enzyme activity by several mechanisms of action that are not yet fully understood. Reduction in irreversible adsorption of enzyme to non-specific sites, reduction in viscosity of liquid and surface tension and consequently reduced contact of enzyme with air-liquid interface, and modifications in biomass chemical structure are some of the benefits derived from surface active molecules. Application of some of these amphiphiles could potentially reduce the capital and operating costs of bioethanol production by reducing fermentation time and the amount of enzyme used for saccharification of biomass. In this review article, the benefit of applying amphiphiles at various stages of ethanol production (i.e., pretreatment, hydrolysis and hydrolysis-fermentation) is reviewed and the proposed mechanisms of actions are described
Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively