Improvement of Tryptophan Analysis by Liquid Chromatography-Single Quadrupole Mass Spectrometry Through the Evaluation of Multiple Parameters.

Tryptophan is a key component in many biological processes and an essential amino acid in food and feed materials. Analysis of the tryptophan content in proteins or protein-containing matrices has always been a challenge. We show here that the preparation of samples prior to tryptophan analysis can be significantly simplified, and the time consumption reduced, by using ascorbic acid as antioxidant to eliminate the problem of tryptophan degradation during alkaline hydrolysis. Combined with separation by HPLC and detection by Single Quadrupole Mass Spectrometry, this allows the analytical run time to be reduced to 10 min. The alkaline hydrolysate obtained in the method presented here may be combined with the oxidized hydrolysate obtained when sulfur-containing amino acids are to be measured, thus essentially providing two analyses for the time of one.

Breeding for dual-purpose wheat varieties using marker-trait associations for biomass yield and quality traits.

KEY MESSAGE: This study demonstrates that an active breeding nursery with rotation can be used to identify marker-trait associations for biomass yield and quality parameters that are important for biorefinery purposes. Wheat straw is a valuable feedstock for bioethanol production, but due to the recalcitrant nature of lignocellulose, its efficient use in biorefineries is limited by its low digestibility and difficult conversion of structural carbohydrates into free sugars. A genome-wide association study (GWAS) was conducted to search for significant SNP markers that could be used in a breeding programme to improve the value of wheat straw in a biorefinery setting. As part of a 3-year breeding programme (2013-2016), 190 winter wheat lines were phenotyped for traits that affect the yield and quality of the harvested biomass. These traits included straw yield, plant height, lodging at three growth stages and Septoria tritici blotch (STB) susceptibility. Release of glucose, xylose and arabinose was determined after hydrothermal pretreatment and enzymatic hydrolysis of the straw. The lines were genotyped using 15 K SNP markers and 5552 SNP markers could be used after filtering. Heritability for all traits ranged from 0.02 to 0.74. GWASs were conducted using CMLM, SUPER and FarmCPU algorithms, to analyse which algorithm could detect the highest number of marker-trait associations (MTAs). Comparable tendencies were obtained from CMLM and FarmCPU, but FarmCPU produced the most significant results. MTAs were obtained for lodging, harvest index, plant height, STB, glucose, xylose and arabinose at a significance level of p < 9.01 × 10-6. MTAs in chromosome 6A were observed for glucose, xylose and arabinose, and could be of importance for increasing sugar release for bioethanol production.

Test of Efficacy of Cellulases for Biomass Degradation.

Testing of cellulases on real biomass samples is required to do a true assessment of their efficacy for biomass degradation. Cellulase enzymes belong to a number of different glycosyl hydrolase families, all with different activity, specificity and modes of action. The concerted and synergistic action of these different cellulases determines the efficacy for plant cell wall deconstruction and cellulose hydrolysis. However, the plant cell wall of lignocellulosic materials is a very complex matrix and the efficacy of a cellulase preparation to degrade lignocellulosic materials is influenced by many factors. In this chapter, two protocols for testing efficacy of cellulases on pretreated biomass samples are described. The first protocol describes a small-scale setup employing low solids concentration that easily enables the testing of a larger number of samples. The second protocol describes a method for testing the efficacy of cellulases at conditions more closely resembling industrial conditions, i.e., high solids concentrations. Both protocols can be used to test the cellulases under a variety of substrate types, substrate concentrations, enzyme loadings and process conditions. The protocols can also be used to evaluate different feedstocks.

Lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier rather than by inducing nonproductive adsorption of enzymes.

BACKGROUND: Lignin is known to hinder efficient enzymatic conversion of lignocellulose in biorefining processes. In particular, nonproductive adsorption of cellulases onto lignin is considered a key mechanism to explain how lignin retards enzymatic cellulose conversion in extended reactions. RESULTS: Lignin-rich residues (LRRs) were prepared via extensive enzymatic cellulose degradation of corn stover (Zea mays subsp. mays L.), Miscanthus × giganteus stalks (MS) and wheat straw (Triticum aestivum L.) (WS) samples that each had been hydrothermally pretreated at three severity factors (log R0) of 3.65, 3.83 and 3.97. The LRRs had different residual carbohydrate levels-the highest in MS; the lowest in WS. The residual carbohydrate was not traceable at the surface of the LRRs particles by ATR-FTIR analysis. The chemical properties of the lignin in the LRRs varied across the three types of biomass, but monolignols composition was not affected by the severity factor. When pure cellulose was added to a mixture of LRRs and a commercial cellulolytic enzyme preparation, the rate and extent of glucose release were unaffected by the presence of LRRs regardless of biomass type and severity factor, despite adsorption of the enzymes to the LRRs. Since the surface of the LRRs particles were covered by lignin, the data suggest that the retardation of enzymatic cellulose degradation during extended reaction on lignocellulosic substrates is due to physical blockage of the access of enzymes to the cellulose caused by the gradual accumulation of lignin at the surface of the biomass particles rather than by nonproductive enzyme adsorption. CONCLUSIONS: The study suggests that lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier blocking the access of enzymes to cellulose rather than by inducing retardation through nonproductive adsorption of enzymes.

High-throughput analysis of amino acids in plant materials by single quadrupole mass spectrometry.

BACKGROUND: The amino acid profile of plants is an important parameter in assessments of their growth potential, resource-use efficiency and/or quality as food and feed. Screening studies may involve large number of samples but the classical amino acid analysis is limited by the fact that it is very time consuming with typical chromatographic run times of 70 min or more. RESULTS: We have here developed a high-throughput method for analysis of amino acid profiles in plant materials. The method combines classical protein hydrolysis and derivatization with fast separation by UHPLC and detection by a single quadrupole (QDa) mass spectrometer. The chromatographic run time is reduced to 10 min and the precision, accuracy and sensitivity of the method are in line with other recent methods utilizing advanced and more expensive mass spectrometers. The sensitivity of the method is at least a factor 10 better than that of methods relying on detection by fluorescence or UV. It is possible to downscale sample size to 20 mg without compromising reproducibility, which makes the method ideal for analysis of very small sample amounts. CONCLUSION: The developed method allows high-throughput analysis of amino acid profiles in plant materials. The analysis is robust and accurate as well as compatible with both free amino acids and protein hydrolysates. The QDa detector offers high sensitivity and accuracy, while at the same time being relatively simple to operate and cheap to purchase, thus significantly reducing the overall analytical costs compared to methods based on more advanced mass spectrometers.

Surface properties correlate to the digestibility of hydrothermally pretreated lignocellulosic Poaceae biomass feedstocks.

BACKGROUND: Understanding factors that govern lignocellulosic biomass recalcitrance is a prerequisite for designing efficient 2nd generation biorefining processes. However, the reasons and mechanisms responsible for quantitative differences in enzymatic digestibility of various biomass feedstocks in response to hydrothermal pretreatment at different severities are still not sufficiently understood. RESULTS: Potentially important lignocellulosic feedstocks for biorefining, corn stover (Zea mays subsp. mays L.), stalks of Miscanthus × giganteus, and wheat straw (Triticum aestivum L.) were systematically hydrothermally pretreated; each at three different severities of 3.65, 3.83, and 3.97, respectively, and the enzymatic digestibility was assessed. Pretreated samples of Miscanthus × giganteus stalks were the least digestible among the biomass feedstocks producing ~24 to 66.6% lower glucose yields than the other feedstocks depending on pretreatment severity and enzyme dosage. Bulk biomass composition analyses, 2D nuclear magnetic resonance, and comprehensive microarray polymer profiling were not able to explain the observed differences in recalcitrance among the pretreated feedstocks. However, methods characterizing physical and chemical features of the biomass surfaces, specifically contact angle measurements (wettability) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (surface biopolymer composition) produced data correlating pretreatment severity and enzymatic digestibility, and they also revealed differences that correlated to enzymatic glucose yield responses among the three different biomass types. CONCLUSION: The study revealed that to a large extent, factors related to physico-chemical surface properties, namely surface wettability as assessed by contact angle measurements and surface content of hemicellulose, lignin, and wax as assessed by ATR-FTIR rather than bulk biomass chemical composition correlated to the recalcitrance of the tested biomass types. The data provide new insight into how hydrothermal pretreatment severity affects surface properties of key Poaceae lignocellulosic biomass and may help design new approaches to overcome biomass recalcitrance.

An Aspergillus nidulans GH26 endo-ß-mannanase with a novel degradation pattern on highly substituted galactomannans.

The activity and substrate degradation pattern of a novel Aspergillus nidulans GH26 endo-ß-mannanase (AnMan26A) was investigated using two galactomannan substrates with varying amounts of galactopyranosyl residues. The AnMan26A was characterized in parallel with the GH26 endomannanase from Podospora anserina (PaMan26A) and three GH5 endomannanases from A. nidulans and Trichoderma reesei (AnMan5A, AnMan5C and TrMan5A). The initial rates and the maximal degree of enzymatically catalyzed conversion of locust bean gum and guar gum galactomannans were determined. The hydrolysis product profile at maximal degree of conversion was determined using DNA sequencer-Assisted Saccharide analysis in High throughput (DASH). This is the first reported use of this method for analyzing galactomannooligosaccharides. AnMan26A and PaMan26A were found to have a novel substrate degradation pattern on the two galactomannan substrates. On the highly substituted guar gum AnMan26A and PaMan26A reached 35-40% as their maximal degree of conversion whereas the three tested GH5 endomannanases only reached 8-10% as their maximal degree of conversion. α-Galactosyl-mannose was identified as the dominant degradation product resulting from AnMan26A and PaMan26A action on guar gum, strongly indicating that these two enzymes can accommodate galactopyranosyl residues in the -1 and in the +1 subsite. The degradation of α-6(4)-6(3)-di-galactosyl-mannopentaose by AnMan26A revealed accommodation of galactopyranosyl residues in the -2, -1 and +1 subsite of the enzyme. Accommodation of galactopyranosyl residues in subsites -2 and +1 has not been observed for other characterized endomannanases to date. Docking analysis of galactomannooligosaccharides in available crystal structures and homology models supported the conclusions drawn from the experimental results. This newly discovered diversity of substrate degradation patterns demonstrates an expanded functionality of fungal endomannanases, than hitherto reported.

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer.

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.

Cellobiohydrolase and endoglucanase respond differently to surfactants during the hydrolysis of cellulose.

BACKGROUND: Non-ionic surfactants such as polyethylene glycol (PEG) can increase the glucose yield obtained from enzymatic saccharification of lignocellulosic substrates. Various explanations behind this effect include the ability of PEG to increase the stability of the cellulases, decrease non-productive cellulase adsorption to the substrate, and increase the desorption of enzymes from the substrate. Here, using lignin-free model substrates, we propose that PEG also alters the solvent properties, for example, water, leading the cellulases to increase hydrolysis yields. RESULTS: The effect of PEG differs for the individual cellulases. During hydrolysis of Avicel and PASC with a processive monocomponent exo-cellulase cellobiohydrolase (CBH) I, the presence of PEG leads to an increase in the final glucose concentration, while PEG caused no change in glucose production with a non-processive endoglucanase (EG). Also, no effect of PEG was seen on the activity of ß-glucosidases. While PEG has a small effect on the thermostability of both cellulases, only the activity of CBH I increases with PEG. Using commercial enzyme mixtures, the hydrolysis yields increased with the addition of PEG. In parallel, we observed that the relaxation time of the hydrolysis liquid phase, as measured by LF-NMR, directly correlated with the final glucose yield. PEG was able to boost the glucose production even in highly concentrated solutions of up to 150 g/L of glucose. CONCLUSIONS: The hydrolysis boosting effect of PEG appears to be specific for CBH I. The mechanism could be due to an increase in the apparent activity of the enzyme on the substrate surface. The addition of PEG increases the relaxation time of the liquid-phase water, which from the data presented points towards a mechanism related to PEG-water interactions rather than PEG-protein or PEG-substrate interactions.

Influence of high gravity process conditions on the environmental impact of ethanol production from wheat straw.

Biofuel production processes at high gravity are currently under development. Most of these processes however use sugars or first generation feedstocks as substrate. This paper presents the results of a life cycle assessment (LCA) of the production of bio-ethanol at high gravity conditions from a second generation feedstock, namely, wheat straw. The LCA used lab results of a set of 36 process configurations in which dry matter content, enzyme preparation and loading, and process strategy were varied. The LCA results show that higher dry matter content leads to a higher environmental impact of the ethanol production, but this can be compensated by reducing the impact of enzyme production and use, and by polyethylene glycol addition at high dry matter content. The results also show that the renewable and non-renewable energy use resulting from the different process configurations ultimately determine their environmental impact.

Structure and enzymatic accessibility of leaf and stem from wheat straw before and after hydrothermal pretreatment.

BACKGROUND: Biomass recalcitrance is affected by a number of chemical, physical and biological factors. In this study we looked into the differences in recalcitrance between two major anatomical fractions of wheat straw biomass, leaf and stem. A set of twenty-one wheat cultivars was fractionated and illustrated the substantial variation in leaf-to-stem ratio between cultivars. The two fractions were compared in terms of chemical composition, enzymatic convertibility, cellulose crystallinity and glucan accessibility. The use of water as a probe for assessing glucan accessibility was explored using low field nuclear magnetic resonance and infrared spectroscopy in combination with hydrogen-deuterium exchange. RESULTS: Leaves were clearly more degradable by lignocellulolytic enzymes than stems, and it was demonstrated that xylose removal was more linked to glucose yield for stems than for leaves. Comparing the locations of water in leaf and stem by low field NMR and FT-IR revealed that the glucan hydroxyl groups in leaves were more accessible to water than glucan hydroxyl groups in stems. No difference in crystallinity between leaf and stem was observed using wide angle x-ray diffraction. Hydrothermal pretreatment increased the accessibility towards water in stems but not in leaves. The results in this study indicate a correlation between the accessibility of glucan to water and to enzymes. CONCLUSIONS: Enzymatic degradability of wheat straw anatomical fractions can be indicated by the accessibility of the hydroxyl groups to water. This suggests that water may be used to assess glucan accessibility in biomass samples.

Cellulase inhibition by high concentrations of monosaccharides.

Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other monosaccharides such as mannose and galactose (stereoisomers of glucose) decrease glucose yields as well. NMR relaxometry measurements showed direct correlations between the initial T2 of the liquid phase in which hydrolysis takes place and the total glucose production during cellulose hydrolysis, indicating that low free water availability contributes to cellulase inhibition. Of the hydrolytic enzymes involved, those acting on the cellulose substrate, that is, exo- and endoglucanases, were the most inhibited. The ß-glucosidases were shown to be less sensitive to high monosaccharide concentrations except glucose. Protein adsorption studies showed that this inhibition effect was most likely due to catalytic, and not binding, inhibition of the cellulases.

Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation.

BACKGROUND: Microbial bioconversion of photosynthetic biomass is a promising approach to the generation of biofuels and other bioproducts. However, rapid, high-yield, and simple processes are essential for successful applications. Here, biomass from the rapidly growing photosynthetic marine cyanobacterium Synechococcus sp. PCC 7002 was fermented using yeast into bioethanol. RESULTS: The cyanobacterium accumulated a total carbohydrate content of about 60% of cell dry weight when cultivated under nitrate limitation. The cyanobacterial cells were harvested by centrifugation and subjected to enzymatic hydrolysis using lysozyme and two alpha-glucanases. This enzymatic hydrolysate was fermented into ethanol by Saccharomyces cerevisiae without further treatment. All enzyme treatments and fermentations were carried out in the residual growth medium of the cyanobacteria with the only modification being that pH was adjusted to the optimal value. The highest ethanol yield and concentration obtained was 0.27 g ethanol per g cell dry weight and 30 g ethanol L(-1), respectively. About 90% of the glucose in the biomass was converted to ethanol. The cyanobacterial hydrolysate was rapidly fermented (up to 20 g ethanol L(-1) day(-1)) even in the absence of any other nutrient additions to the fermentation medium. CONCLUSIONS: Cyanobacterial biomass was hydrolyzed using a simple enzymatic treatment and fermented into ethanol more rapidly and to higher concentrations than previously reported for similar approaches using cyanobacteria or microalgae. Importantly, as well as fermentable carbohydrates, the cyanobacterial hydrolysate contained additional nutrients that promoted fermentation. This hydrolysate is therefore a promising substitute for the relatively expensive nutrient additives (such as yeast extract) commonly used for Saccharomyces fermentations.

The role of endoglucanase and endoxylanase in liquefaction of hydrothermally pretreated wheat straw.

The role of endocellulases and endoxylanase during liquefaction and saccharification of hydrothermally pretreated wheat straw was studied. The use of a flow-loop setup with in-line magnetic resonance imaging enabled frequent measurements of viscosity at 55°C during saccharification at 6% total solids content. Viscosity data were complemented with off-line measurements of fiber lengths and release of soluble sugars. A clear correlation between fiber attrition and a decrease in viscosity was found. Fiber lengths and viscosity dropped quickly within the first hour and then stagnated, while sugar yields increased substantially thereafter, illustrating that liquefaction and saccharification are separate mechanisms. Both endoglucanase and endoxylanase were shown to have a significant effect on viscosity during liquefaction while the addition of endoxylanase also increased sugar yield.

Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit.

Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K D was performed using a novel distillation setup allowing stripping of ethanol at 50-65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55-65 °C) and exposure to gas-liquid and liquid-heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.

Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?

Production of ethanol from lignocellulosic materials has a promising market potential, but the process is still only at pilot/demonstration scale due to the technical and economical difficulties of the process. Operating the process at very high solids concentrations (above 20% dry matter-DM) has proven essential for economic feasibility at industrial scale. Historically, simultaneous saccharification and fermentation (SSF) was found to give better ethanol yields compared to separate hydrolysis and fermentation (SHF), but data in literature are typically based on operating the process at low dry matter conditions. In this work the impact of selected enzyme preparation and processing strategy (SHF, presaccharification and simultaneous saccharification and fermentation-PSSF, and SSF) on final ethanol yield and overall performance was investigated with pretreated wheat straw up to 30% DM. The experiments revealed that an SSF strategy was indeed better than SHF when applying an older generation enzyme cocktail (Celluclast-Novozym 188). In case of the newer product Cellic CTec 2, SHF resulted in 20% higher final ethanol yield compared to SSF. It was possible to close the mass balance around cellulose to around 94%, revealing that the most relevant products could be accounted for. One observation was the presence of oxidized sugar (gluconic acid) upon enzymatic hydrolysis with the latest enzyme preparation. Experiments showed gluconic acid formation by recently discovered enzymatic class of lytic polysaccharides monoxygenases (LPMO's) to be depending on the processing strategy. The lowest concentration was achieved in SSF, which could be correlated with less available oxygen due to simultaneous oxygen consumption by the yeast. Quantity of glycerol and cell mass was also depending on the selected processing strategy.

The challenging measurement of protein in complex biomass-derived samples.

Measurement of the protein content in samples from production of lignocellulosic bioethanol is an important tool when studying the adsorption of cellulases. Several methods have been used for this, and after reviewing the literature, we concluded that one of the most promising assays for simple and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay-alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant interference from these compounds when using acid hydrolysis, which was not the case when using the alkaline hydrolysis. We evaluated the interference from glucose, cellulose, xylose, xylan, lignin and ethanol on protein determination of BSA, Accellerase(®) 1500 and Cellic(®) CTec2. The experiments demonstrated that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin.

Adsorption of ß-glucosidases in two commercial preparations onto pretreated biomass and lignin.

BACKGROUND: Enzyme recycling is a method to reduce the production costs for advanced bioethanol by lowering the overall use of enzymes. Commercial cellulase preparations consist of many different enzymes that are important for efficient and complete cellulose (and hemicellulose) hydrolysis. This abundance of different activities complicates enzyme recycling since the individual enzymes behave differently in the process. Previously, the general perception was that ß-glucosidases could easily be recycled via the liquid phase, as they have mostly been observed not to adsorb to pretreated biomass or only adsorb to a minor extent. RESULTS: The results from this study with Cellic® CTec2 revealed that the vast majority of the ß-glucosidase activity was lost from the liquid phase and was adsorbed to the residual biomass during hydrolysis and fermentation. Adsorption studies with ß-glucosidases in two commercial preparations (Novozym 188 and Cellic® CTec2) to substrates mimicking the components in pretreated wheat straw revealed that the Aspergillus niger ß-glucosidase in Novozym 188 did not adsorb significantly to any of the components in pretreated wheat straw, whereas the ß-glucosidase in Cellic® CTec2 adsorbed strongly to lignin.The extent of adsorption of ß-glucosidase from Cellic® CTec2 was affected by both type of biomass and pretreatment method. With approximately 65% of the ß-glucosidases from Cellic® CTec2 adsorbed onto lignin from pretreated wheat straw, the activity of the ß-glucosidases in the slurry decreased by only 15%. This demonstrated that some enzyme remained active despite being bound. It was possible to reduce the adsorption of Cellic® CTec2 ß-glucosidase to lignin from pretreated wheat straw by addition of bovine serum albumin or poly(ethylene glycol). CONCLUSIONS: Contrary to the ß-glucosidases in Novozym 188, the ß-glucosidases in Cellic® CTec2 adsorb significantly to lignin. The lignin adsorption observed for Cellic® CTec2 is usually not a problem during hydrolysis and fermentation since most of the catalytic activity is retained. However, adsorption of ß-glucosidases to lignin may prove to be a problem when trying to recycle enzymes in the production of advanced bioethanol.

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling.

Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes.

Lignocellulolytic enzymes are among the most costly part in production of bioethanol. Therefore, recycling of enzymes is interesting as a concept for reduction of process costs. However, stability of the enzymes during the process is critical. In this work, focus has been on investigating the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5 % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation.