Eva Palmqvist

Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition

Abstract During hydrolysis of lignocellulosic materials a wide range of compounds which are inhibitory to microorganisms are formed or released. Based on their origin the inhibitors are usually divided in three major groups: weak acids, furan derivatives, and phenolic compounds. These compounds limit efficient utilisation of the hydrolysates for ethanol production by fermentation. If the inhibitors are identified and the mechanisms of inhibition elucidated, fermentation can be improved by developing specific detoxification methods, choosing an adapted microorganism, or optimising the fermentation strategy. The present review discusses the generation of inhibitors during degradation of lignocellulosic materials, and the effect of these on fermentation yield and productivity. Inhibiting mechanisms of individual compounds present in the hydrolysates and their interaction effects are reviewed.

Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification

The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during hydrolysis. This review describes the effect of various detoxification methods on the fermentability and chemical composition of the hydrolysates. Inhibition of fermentation can be relieved upon treatment with the ligninolytic enzyme laccase, pre-fermentation by the filamentous fungus Trichoderma reesei, removal of non-volatile compounds, extraction with ether or ethyl acetate, and treatment with alkali or sulfite. Various fermentation strategies can also be used to improve yield and productivity in lignocellulosic hydrolysates. Batch, fed-batch, and continuous fermentation are discussed in relation to inhibition of fermentation in lignocellulosic hydrolysates.

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 (Bakers 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

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Abstract Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO2. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process.

The effect of water-soluble inhibitors from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation

In the process of producing ethanol from lignocellulosic materials, compounds inhibitory to enzymatic hydrolysis and fermentation are generated during the pretreatment of the wood. In an industrial process, these compounds will accumulate due to the recirculation of process streams. The inhibitory effects of the accumulation of volatile and nonvolatile compounds released during stream pretreatment on enzymatic hydrolysis and fermentation were studied. The volatile compounds did not affect either the enzymatic hydrolysis or the fermentation significantly even at high concentrations. In contrast, the nonvolatile compounds severely affected both the hydrolysis and the fermentation: the effect was more pronounced in the latter case. For the effective use of a lignocellulosic material as a substrate for ethanol production, the nonvolatile compounds must thus be removed. (Less)

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°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.

Simultaneous detoxification and enzyme production of hemicellulose hydrolysates obtained after steam pretreatment

In the process of making ethanol from lignocellulosic materials, compounds inhibitory to microorganisms are generated during steam pretreatment of the wood. Water-soluble inhibitors and pentoses are liberated and washed from the cellulose structure which is further enzymatically hydrolyzed. To make the process economically feasible, the pentoses have to be fermented to ethanol. A major drawback with the pentose-fermenting organisms that have been suggested for this purpose is that they do not tolerate an inhibitory environment and therefore, the pentose stream has to be detoxified prior to fermentation. An alternative use of the hemicellulose hydrolysate obtained after steam-pretreatment of willow is to use it for enzyme production by the cellulolytic fungus Trichoderma reesei. The sugars in the pentose fraction are almost completely utilized, and simultaneously the hemicellulose hydrolysate is detoxified and can be recirculated in the process to minimize the need for freshwater.

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. (Less)

Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C-30

The growth and enzyme production by Trichoderma reesei Rut C-30 using different lignocellulosic materials as carbon source were investigated. Cellulose, sugar beet pulp and alkaline extracted sugar beet pulp (resulting in partial removal of hemicellulose, lignin and pectin) or mixtures thereof were used as carbon sources. It was found that endoglucanase and endoxylanse activities were produced throughout the cultivations, whereas α-arabinosidase was induced late during the cultivation. The highest amounts of endoglucanse, could be measured when T. reesei Rut C-30 was grown on cellulose or cellulose containing mixtures. Endoxylanase was produced on all substrates, but the presence of cellulose was favourable for the production. Polygalacturonase activity could be measured at high varying levels throughout the cultivations, except during growth on cellulose. The varying levels might originate from the production of different isoenzymes of polygalacturonase.

POPULATION EXTINCTIONS IN CORRELATED ENVIRONMENTS

For a spatial population assemblage, extinction risk should be greatly affected by features of local population dynamics and interpatch migration patterns. In a variable environment, the magnitude of environmental correlation between local population patches may have great impact on local dynamics and thereby global extinction risk. We examined the effect of correlated environmental variation on global extinction risk in a coupled lattice model consisting of local populations governed by density dependent population growth and density independent interpatch migration. We let each local population experience a stochastic environment expressed as a variation in maximum birth rate and let this environmental variation be correlated among local populations. We simulated global population growth under different magnitudes of environmental variability, correlation of environmental variability, emigration rate and migration survival, in order to evaluate the magnitude of their effect on local population dynamics and global extinction risk. The risk of global extinction increases with increasing magnitude of environmental correlation and environmental variability. The major determinant of global extinction risk is the balance between local population variability and the synchrony in local population fluctuations. A low rate of successful interpatch migration connects the local populations to each other, exposing them to less extinction risk than when they are isolated. High levels of interpatch migration are often negative for population persistence. The reason for this is that increased migration survival causes an increased risk of population crashes, due to overcompensatory population growth. This effect is amplified by a high emigration rate. Thus, local dynamics are affected by temporal and spatial variability in birth rates as well as interpatch migration levels. An assemblage of local populations in a variable environment will suffer least risk of global extinction when environmental correlation is low and interpatch migration is moderate.