The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 ºC to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2 3 full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo), Brazil. Novozymes ( FAPESP )Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Influence of enzyme loading and physical parameters on the enzymatic hydrolysis of steam-pretreated softwood

Softwood is an interesting raw material for the production of fuel ethanol as a result of its high content of hexoses, and it has attracted attention especially in the Northern hemisphere. However, the enzymatic hydrolysis of softwood is not sufficiently efficient for the complete conversion of cellulose to glucose. Since an improvement in the glucose yield is of great importance for the overall economy of the process, the influence of various parameters on the cellulose conversion of steam-pretreated spruce has been investigated. The addition of beta -glucosidase up to 50 IU g(-1) cellulose to the enzymatic hydrolysis process resulted in increased cellulose conversion at a cellulase loading up to 48 FPU g(-1) cellulose. Despite very high enzyme loading (120 FPU g(-1) cellulose) only about 50% of the cellulose in steam-pretreated spruce was converted to glucose when all of the material following pretreatment was used in the hydrolysis step. The influence of temperature, residence time, and pH were investigated for washed pretreated spruce at a dry matter (DM) content of 5% and a cellulase activity of 18.5 FPU g(-1) cellulose. The optimal temperature was found to be dependent on both residence time and pH, and the maximum degree of cellulose conversion, 69.2%, was obtained at 38 degreesC and pH 4.9 for a residence time of 144 h. However, when the substrate concentration was changed from 5% to 2% DM, the cellulose conversion increased to 79.7%. An increase from 5% to 10% DM resulted, however, in a similar degree of cellulose conversion, despite a significant increase in the glucose concentration from 23 g L-1 to 45 g L-1. The deactivation of beta -glucosidase increased with increasing residence time and was more pronounced with vigorous agitation

Reduced inhibition of enzymatic hydrolysis of steam-pretreated softwood

Softwood constitutes the main source of lignocellulosic material in Sweden which can be used for ethanol production from renewable resources. To make the biomass-to-ethanol process more economically feasible, it is preferable to include the sugar-rich prehydrolysate, i.e. the liquid obtained after the pretreatment step, in the enzymatic hydrolysis of the solid fraction. This study shows that the prehydrolysate inhibits cellulose conversion in the enzymatic hydrolysis step. When the prehydrolysate was included in the enzymatic hydrolysis, the cellulose conversion was reduced by up to 36%. However, this inhibition can be overcome by fermentation of the prehydrolysate prior to enzymatic hydrolysis. (C) 2001 Elsevier Science Inc. All rights reserved

Recycling of process streams in ethanol production from softwoods based on enzymatic hydrolysis

In ethanol production from lignocellulose by enzymatic hydrolysis and fermentation, it is desirable to minimize addition of fresh-water and waste-water streams, which leads to an accumulation of substances in the process. This study shows that the amount of fresh water used and the amount of waste water thereby produced in the production of fuel ethanol from softwood, can be reduced to a large extent by recycling of either the stillage stream or part of the liquid stream from the fermenter. A reduction in fresh-water demand of more than 50%, from 3 kg/kg dry raw material to 1.5 kg/kg dry raw material was obtained without any negative effects on either hydrolysis or fermentation. A further decrease in the amount of fresh water, to one-fourth of what was used without recycling of process streams, resulted in a considerable decrease in the ethanol productivity and a slight decrease in the ethanol yield

The generation of fermentation inhibitors during dilute acid hydrolysis of softwood

The influence of the severity of dilute sulfuric acid hydrolysis of spruce (softwood) on sugar yield and on the fermentability of the hydrolysate by Saccharomyces cerevisiae (Baker's yeast) was investigated. Fermentability was assessed as the ethanol yield on fermentable sugars (mannose and glucose) and the mean volumetric productivity (4 h). The hydrolysis conditions, residence time, temperature, and sulfuric acid concentration were treated as a single parameter, combined severity (CS). When the CS of the hydrolysis conditions increased, the yield of fermentable sugars increased to a maximum between CS 2.0-2.7 for mannose, and 3.0-3.4 for glucose above which it decreased. The decrease in the yield of monosaccharides coincided with the maximum concentrations of furfural and 5-hydroxymethylfurfural (5HMF). With the further increase in CS, the concentrations of furfural and 5-HMF decreased while the formation of formic acid and levulinic acid increased The yield of ethanol decreased at approximately CS 3; however, the volumetric productivity decreased at lower CS. The effect of acetic acid, formic acid, levulinic acid furfural, and 5-HMF on fermentability was assayed in model fermentations Ethanol yield and volumetric productivity decreased with increasing concentrations of acetic acid, formic acid, and levulinic acid. Furfural and 5-HMF decreased the volumetric productivity but did not influence the final yield of ethanol. The decrease in volumetric productivity was more pronounced when 5-HMF was added to the fermentation, and this compound was depleted at a lower rate than furfural. The inhibition observed in hydrolysates produced in higher CS could not be fully explained by the effect of the by-products furfural, 5-HMF, acetic acid, formic acid: and levulinic acid. (C) 1998 Elsevier Science Inc

Comparison of SO2 and H2SO4 impregnation of softwood prior to steam pretreatment on ethanol production

The pretreatment of softwood with sulfuric acid impregnation in the production of ethanol, based on enzymatic hydrolysis, has been investigated. The parameters investigated were: H2SO4 concentration (0.5-4.4% w/w liquid), temperature (180-240 degrees C), and residence time (1-20 minutes). The combined severity (log Ro-pH) was used to combine the parameters into a single reaction ordinate. The highest yields of fermentable sugars, i.e., glucose and mannose, were obtained at a combined severity of 3. At this severity, however, the fermentability declined and the ethanol yield decreased. In a comparison with previous results, SO2 impregnation was found to be preferable, since it resulted in approximately the same sugar yields, but better fermentability

Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics

A bench-scale unit for the development of an enzymatic process for the bioconversion of lignocellulosics to ethanol has been used to study the recycling of waste-water streams to minimize fresh-water requirements and reduce effluent streams. Willow, after impregnation with sulphur dioxide, was steam-pretreated, enzymatically hydrolysed, and the sugars produced were fermented using S. cerevisiae. The fermentation broth was distilled and the stillage was fractionated by evaporation into six separate condensate fractions and a residue. The overall yield of ethanol from willow was 65% of the theoretical yield based on total fermentable sugars. The inhibitory effect of the evaporation condensates was assessed by fermentation using S. cerevisiae. The non-volatile residue of the stillage was found to be inhibitory to fermentation. The ethanol yield decreased from 0.37 g/g in a pure sugar reference to 0.31 g/g in the residue and the average ethanol fermentation rate decreased fi om 6.3 g/(l h) to 2.7 g/(l h), respectively. The evaporation condensates, containing the volatile components, showed no negative effects on fermentation. The intermediate evaporation condensate fractions, fractions 4 and 5, had the lowest chemical oxygen demand (GOD), 1560 and 1120 mg/l, compared with 33 300 mg/l for the stillage. Therefore, these fractions can be released directly into the effluent without further treatment. Copyright (C) 1997 Elsevier Science Ltd