Bärbel Hahn-Hägerdal

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

Fermentation of lignocellulosic hydrolysates for ethanol production.

Ethanol production from lignocellulosic hydrolysates in an economically feasible process requires microorganisms that produce ethanol with a high yield from all sugars present (hexoses as well as pentoses) and have a high ethanol productivity in lignocellulosic hydrolysates, i.e., can withstand potential inhibitors. Different fermentation organisms among bacteria, yeasts, and fungi (natural as well as recombinant) are reviewed with emphasis on their performance in lignocellulosic hydrolysates. Depending on the type of lignocellulosic hydrolysate, the composition of inhibitors will differ and their influence on the microorganisms and the fermentation performance will consequently vary. The inhibition may be partly overcome by the removal of inhibitors, i.e., detoxification. Microbial constraints on parameters such as pH, temperature, and nutrient supplementation are discussed in relation to their implication on the process economy. Not only are the properties of the microorganism of importance in the process, but also the choice of fermentation strategies such as batch culture, continuous culture with cell recycling and in situ ethanol removal. For the realization of the ethanol production from lignocellulosic materials, the fermentation step has to be integrated with the rest of the process. These aspects are also discussed.

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

ABSTRACT For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiaehas been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenousXKS1 gene, encoding xylulokinase (XK), under control of thePGK1 promoter were integrated into the chromosomalHIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h−1 g (dry weight) of cells−1 (0.24 to 0.30 g h−1 g [dry weight] of cells−1) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h−1. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h−1 g (dry weight) of cells−1 when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h−1 g (dry weight) of cells−1 when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.

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.

New improvements for lignocellulosic ethanol

The use of lignocellulosic biomass for the production of biofuels will be unavoidable if liquid fossil fuels are to be replaced by renewable and sustainable alternatives. Ethanol accounts for the majority of biofuel use worldwide, and the prospect of its biological production from abundant lignocellulosic feedstocks is attractive. The recalcitrance of these raw materials still renders proposed processes complex and costly, but there are grounds for optimism. The application of new, engineered enzyme systems for cellulose hydrolysis, the construction of inhibitor-tolerant pentose-fermenting industrial yeast strains, combined with optimized process integration promise significant improvements. The opportunity to test these advances in pilot plants paves the way for large-scale units. This review summarizes recent progress in this field, including the validation at pilot scale, and the economic and environmental impacts of this production pathway.

Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization

For an economically feasible production of ethanol from plant biomass by microbial cells, the fermentation of xylose is important. As xylose uptake might be a limiting step for xylose fermentation by recombinant xylose-utilizing Saccharomyces cerevisiae cells a study of xylose uptake was performed. After deletion of all of the 18 hexose-transporter genes, the ability of the cells to take up and to grow on xylose was lost. Reintroduction of individual hexose-transporter genes in this strain revealed that at intermediate xylose concentrations the yeast high- and intermediate-affinity transporters Hxt4, Hxt5, Hxt7 and Gal2 are important xylose-transporting proteins. Several heterologous monosaccharide transporters from bacteria and plant cells did not confer sufficient uptake activity to restore growth on xylose. Overexpression of the xylose-transporting proteins in a xylose-utilizing PUA yeast strain did not result in faster growth on xylose under aerobic conditions nor did it enhance the xylose fermentation rate under anaerobic conditions. The results of this study suggest that xylose uptake does not determine the xylose flux under the conditions and in the yeast strains investigated.

Metabolic engineering for pentose utilization in Saccharomyces cerevisiae

The introduction of pentose utilization pathways in bakers yeast Saccharomyces cerevisiae is summarized together with metabolic engineering strategies to improve ethanolic pentose fermentation. Bacterial and fungal xylose and arabinose pathways have been expressed in S. cerevisiae but do not generally convey significant ethanolic fermentation traits to this yeast. A large number of rational metabolic engineering strategies directed among others toward sugar transport, initial pentose conversion, the pentose phosphate pathway, and the cellular redox metabolism have been exploited. The directed metabolic engineering approach has often been combined with random approaches including adaptation, mutagenesis, and hybridization. The knowledge gained about pentose fermentation in S. cerevisiae is primarily limited to genetically and physiologically well-characterized laboratory strains. The translation of this knowledge to strains performing in an industrial context is discussed.

Ethanol production from enzymatic hydrolysates of sugarcane bagasse using recombinant xylose-utilising Saccharomyces cerevisiae

Sugarcane bagasse was pre-treated by steam explosion at 205 and 215degreesC and hydrolysed with cellulolytic enzymes. The hydrolysates were subjected to enzymatic detoxification by treatment with the phenoloxidase laccase and to chemical detoxification by overliming. Approximately 80% of the phenolic compounds were specifically removed by the laccase treatment. Overliming partially removed the phenolic compounds, but also other fermentation inhibitors such as acetic acid, furfural and 5-hydroxy-methyl-furfural. The hydrolysates were fermented with the recombinant xylose-utilising Saccharomyces cerevisiae laboratory strain TMB 3001, a CEN.PK derivative with over-expressed xylulokinase activity and expressing the xylose reductase and xylitol dehydrogenase of Pichia stipitis, and the S. cerevisiae strain ATCC 9658 1, isolated from a spent sulphite liquor fermentation plant. The fermentative performance of the lab strain in undetoxified hydrolysate was better than the performance of the industrial strain. An almost two-fold increase of the specific productivity of the strain TMB 3001 in the detoxified hydrolysates compared to the undetoxified hydrolysates was observed. The ethanol yield in the fermentation of the hydrolysate detoxified by overliming was 0.18 g/g dry bagasse, whereas it reached only 0.13 g/g dry bagasse in the undetoxified hydrolysate. Partial xylose utilisation with low xylitol formation was observed. (Less)