Scientific publications - Biofuels


Kløverpris, J., Wenzel, H., Banse, M., Milà I Canals, L., Reenberg, A.

"Conference and workshop on modelling global land use implications in the environmental assessment of biofuels"

International Journal of Life Cycle Assessment, 13 (3), pp. 178-183. (2008)

Background, Aims and Scope. On 4-5 June 2007, an international conference was held in Copenhagen. It provided an interdisciplinary forum where economists and geographers met with LCA experts to discuss the challenges of modelling the ultimate land use changes caused by an increased demand for biofuels. Main Features. The main feature of the conference was the cross-breeding of experience from the different approaches to land use modelling: The field of LCA could especially benefit from economic modelling in the identification of marginal crop production and the resulting expansion of the global agricultural area. Furthermore, the field of geography offers insights in the complexity behind new land cultivation and practical examples of where this is seen to occur on a regional scale. Results. Results presented at the conference showed that the magnitude and location of land use changes caused by biofuels demand depend on where the demand arises. For instance, mandatory blending in the EU will increase land use both within and outside of Europe, especially in South America. A key learning for the LCA society was that the response to a change in demand for a given crop is not presented by a single crop supplier or a single country, but rather by responses from a variety of suppliers of several different crops in several countries. Discussion. The intensification potential of current and future crop and biomass production was widely discussed. It was generally agreed that some parts of the third world hold large potentials for intensification, which are not realised due to a number of barriers resulting in so-called yield gaps. Conclusions. Modelling the global land use implications of biofuels requires an interdisciplinary approach optimally integrating economic, geographical, biophysical, social and possibly other aspects in the modelling. This interdisciplinary approach is necessary but also difficult due to different perspectives and mindsets in the different disciplines. Recommendations and Perspectives. The concept of a location dependent marginal land use composite should be introduced in LCA of biofuels and it should be acknowledged that the typical LCA assumption of linear substitution is not necessarily valid. Moreover, fertiliser restrictions/accessibility should be included in land use modelling and the relation between crop demand and intensification should be further explored. In addition, environmental impacts of land use intensification should be included in LCA, the powerful concept of land use curves should be further improved, and so should the modelling of diminishing returns in crop production. © 2008 Springer-Verlag.

Agbogbo, F.K., Coward-Kelly, G.

"Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis"

Biotechnology Letters, pp. 1-10. Article in Press. (2008)

Rising crude oil prices and environmental concerns have renewed interest in renewable energy. Cellulosic ethanol promises to deliver a renewable fuel from non-food feedstocks. One technical challenge producing cellulosic ethanol economically is a robust organism to utilize the different sugars present in cellulosic biomass. Unlike starch where glucose is the only sugar present, cellulosic biomass has other sugars such as xylose and arabinose, usually called C5 sugars. This review examines the most promising naturally occurring C5 fermenting organism, Pichia stipitis. In this work, the properties that make P. stipitis unique from other organisms, its physiology and fermentation results on lignocellulosic substrates have been reviewed. P. stipitis can produce 41 g ethanol/l with a potential to cleanup some of the most concentrated toxins. These results coupled with the less stringent nutritional requirements, great resistance to contamination and its thick cell walls makes P. stipitis a viable organism for scale-up. However, P. stipitis has a slower sugar consumption rate compared to Saccharomyces cerevisiae and requires microaerophilic condition for ethanol production. Finally, future studies to enhance fermentation capabilities of this yeast have been discussed. © 2008 Springer Science+Business Media B.V.

Cowan, D., Oxenbøll, K.M., Holm, H.C.

"Enzymatic bioprocessing of oils and fats"

INFORM - International News on Fats, Oils and Related Materials, 19 (4), pp. 210-212. (2008)

The industrial revolution, which started in Europe, has long since spread to much of the rest of the world and has raised living standards and brought prosperity to millions of people. However, these changes have not been without consequences, not the least of which is the coupling of increased industrialization to climate change. That this has occurred is now acknowledged by a majority of climate change researchers and by the wider community.

Andersen, N., Johansen, K.S., Michelsen, M., Stenby, E.H., Krogh, K.B.R.M., Olsson, L.

"Hydrolysis of cellulose using mono-component enzymes shows synergy during hydrolysis of phosphoric acid swollen cellulose (PASC), but competition on Avicel"

Enzyme and Microbial Technology, 42 (4), pp. 362-370. (2008)

To study the synergy between the three groups of cellulolytic enzymes, 20 mixtures of different mole percentage of Humicola insolens Cel45A (EG V) and Cel6A (CBH II), and Penicillium brasilianum Cel3A (β-glucosidase) were used to hydrolyze Avicel and phosphoric acid swollen cellulose/Avicel (PASC). In contrast to previous studies, where β-glucosidase was either not added or added in excess, we here focus on engineering binary, as well as, ternary cellulase mixtures (including a range of different mol% of Cel3A) for maximal total sugar production. Precise hydrolysis pattern based on the concentration of soluble hydrolysis products (glucose to cellohexaose measured by HPLC) was determined. The importance of proper assay selection for hydrolysis products detection was illustrated. It was found that degree of synergy (DS) for degradation of PASC were generally larger than 1 (indicating cooperativity between the enzymes), increasing as the hydrolysis proceeded. DS of binary exo-/endo-glucanase mixtures, decreased as the mol% of Cel45A increased. In contrast to hydrolysis of PASC, DS values during degradation of Avicel were less then 1, indicating inhibition of the involved enzymes. Thus, our data point to competition for the same binding sites between endo- and exo-glucanases, and preferential absorbance of exo-glucanases on crystalline substrates. © 2007 Elsevier Inc. All rights reserved.

Xu, F., Ding, H., Osborn, D., Tejirian, A., Brown, K., Albano, W., Sheehy, N., Langston, J.

"Partition of enzymes between the solvent and insoluble substrate during the hydrolysis of lignocellulose by cellulases"

Journal of Molecular Catalysis B: Enzymatic, 51 (1-2), pp. 42-48. (2008)

The interfacial and interphasic behavior of enzyme plays an important role in heterologous biocatalysis, such as the enzymatic hydrolysis of lignocellulose. The solid-solution partition of the major cellulases from a highly effective Trichoderma reesei cellulolytic system was evaluated during the enzymatic cellulolysis of a pretreated corn-stover substrate. Upon mixing with the insoluble substrate, almost all of the enzymes (including CBH-I, CBH-II, EG-I, EG-II, and BG) were adsorbed, as shown by the protein and activity assay of the solution fraction. No significant desorption was detected during as well as after the cellulolysis, indicating the enzymes' ability to function at the lignocellulose surface. The adsorption was attributed to the specific binding to and activating of cellulose during the cellulolysis, and to the non-specific binding to lignin, particularly after the cellulolysis. The presence of several representative cellulolysis enhancers, substances capable of enhancing the cellulase action on lignocellulosic substrate, led to a significant desorption of the adsorbed cellulases. The effect might be related to the enhancing effect of these substances on the cellulases. © 2007 Elsevier B.V. All rights reserved.

Erickson, B., Rath, A., Petioc, E., Carr, M.

"Media roundtable: DOE investment into small-scale biorefineries"

Industrial Biotechnology, 4 (1), pp. 24-29. (2008)



Hailing, P., Simms-Borre, P.

"Overview of lignocellulosic feedstock conversion into ethanol - Focus on sugarcane bagasse"

International Sugar Journal, 110 (1311), pp. 191-194. (2008)

Although the starch-based biofuel industry is in full swing, there is still a long way to go to make the conversion of lignocellulosic feedstock (biomass) into biofuel economical. Extensive collaboration between the major industrial participants and the subsequent integration of technologies is critical. This article overviews the general steps in processing lignocellulosic feedstock into ethanol and takes a closer look at sugarcane bagasse as a cellulosic feedstock with exceptional potential.

Petiot, E.

"On the road to cost-competitive cellulosic ethanol"

Chimica Oggi, 26 (1), pp. 20-22. (2008)

The fuel ethanol industry is making good progress towards converting lignocellulosic feedstock (biomass) to ethanol in an economical way. However, even though the cost of suitable processes and enzymes has been cut, there is still some way to go before the industry booms. Now is the time for the myriad of separate players to come together and join forces for the optimization of the entire process. Without efficient collaboration and subsequent integration of technologies between the industrial players we will not be able to fulfill the target of creating cost-competitive cellulosic ethanol.

McFarland, K.C., Ding, H., Teter, S., Vlasenko, E., Xu, F., Cherry, J.

"Development of improved cellulase mixtures in a single production organism"

ACS Symposium Series, 972, pp. 19-31. (2007)

Economical conversion of lignocellulosic biomass to sugars, ethanol or other chemical feedstocks requires an optimization of the enzymatic breakdown of plant cell walls to monosaccharides. Com Stover was converted to glucose by dilute acid pretreatment followed by enzymatic digestion with broths containing novel mixtures of proteins expressed in Trichoderma reesei. Optimization included improvement of the pretreatment, alteration of the expressed suite of enzymes, and increased productivity of the expression host, which resulted in significant reduction in the enzyme cost for utilization of lignocellulosic biomass for the production of fuels and chemicals. © 2007 American Chemical Society.

Westermann, P., Jørgensen, B., Lange, L., Ahring, B.K., Christensen, C.H.

"Maximizing renewable hydrogen production from biomass in a bio/catalytic refinery"

International Journal of Hydrogen Energy, 32 (17), pp. 4135-4141. (2007)

Biological production of hydrogen from biomass by fermentative or photofermentative microorganisms has been described in numerous research articles and reviews. The major challenge of these techniques is the low yield from fermentative production, and the large reactor volumes necessary for photofermentative production. Due to these constraints biological hydrogen production from biomass has so far not been considered a significant source in most scenarios of a future hydrogen-based economy. In this review we briefly summarize the current state of art of biomass-based hydrogen production and suggest a combination of a biorefinery for the production of multiple fuels (hydrogen, ethanol, and methane) and chemical catalytic technologies which could lead to a yield of 10-12 mol hydrogen per mol glucose derived from biological waste products. Besides the high hydrogen yield, the advantage of the suggested concept is the high versatility with respect to input of different types of biological wastes, which are abundant and cheap residues from agricultural production. Also the concept leaves the opportunity to optimize the microbiological and catalytic processes to meet specific needs for fuel flexibility. © 2007 International Association for Hydrogen Energy.

Rosgaard, L., Pedersen, S., Langston, J., Akerhielm, D., Cherry, J.R., Meyer, A.S.

"Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates"

Biotechnology Progress, 23 (6), pp. 1270-1276. (2007)

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsværd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-β-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes. © 2007 American Chemical Society and American Institute of Chemical Engineers.

Agbogbo, F.K., Wenger, K.S.

"Production of ethanol from corn stover hemicellulose hydrolyzate using Pichia stipitis"

Journal of Industrial Microbiology and Biotechnology, 34 (11), pp. 723-727. (2007)

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37-0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process. © 2007 Society for Industrial Microbiology.

Rosgaard, L., Andric, P., Dam-Johansen, K., Pedersen, S., Meyer, A.S.

"Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw"

Applied Biochemistry and Biotechnology, 143 (1), pp. 27-40. (2007)

In this study, the applicability of a "fed-batch" strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily. © Humana Press Inc. 2007.

Sørensen, H.R., Pedersen, S., Meyer, A.S.

"Characterization of solubilized arabinoxylo-oligosaccharides by MALDI-TOF MS analysis to unravel and direct enzyme catalyzed hydrolysis of insoluble wheat arabinoxylan"

Enzyme and Microbial Technology, 41 (1-2), pp. 103-110. (2007)

Hydrolysis of arabinoxylan is an important prerequisite for valorization of wheat endosperm materials, but the water insoluble arabinoxylan fraction of wheat endosperm cell walls has proven difficult to hydrolyze enzymatically. In this study, enzymatic hydrolysis of purified, water insoluble wheat arabinoxylan was examined and improved by combining detailed substrate analysis with analyses of product monosaccharides and oligosaccharides after treatments with rationally composed enzyme mixtures. Treatment of purified, water insoluble wheat arabinoxylan (substrate concentration 1.0%, w/w) for 48 h at pH 5, 50 °C with a combination of β-xylosidase from Trichoderma reesei and a hemicellulolytic fungal enzyme preparation from Humicola insolens, Ultraflo L, liberated 1.6 mg ferulic acid, 24 mg acetic acid, 51 mg arabinose, 167 mg xylose, and furthermore, solubilized 244 mg oligosaccharides per gram substrate dry matter. This yield was equivalent to solubilization of 45% by weight of the insoluble wheat arabinoxylan. Analysis of the solubilized oligomers by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed presence of different feruloylated and acetylated pentose oligomers. The data thus signified the presence of acetyl groups in wheat arabinoxylan. Addition of acetyl xylan esterase from Aspergillus aculeatus on top of the Ultraflo L and β-xylosidase addition did however not facilitate additional degradation of the insoluble wheat arabinoxylan, whereas, addition of feruloyl esterase obtained from Aspergillus niger on top of the β-xylosidase and Ultraflo L blend solubilized an additional 1.9% (w/w) dry matter from the substrate and significantly changed the MALDI-TOF MS profile of the solubilized oligomers which became significantly less feruloylated after this enzyme treatment. © 2006 Elsevier Inc. All rights reserved.

Merino, S.T., Cherry, J.

"Progress and challenges in enzyme development for biomass utilization"

Advances in Biochemical Engineering/Biotechnology, 108, pp. 95-120. (2007)

Enzymes play a critical role in the conversion of lignocellulosic waste into fuels and chemicals, but the high cost of these enzymes presents a significant barrier to commercialization. In the simplest terms, the cost is a function of the large amount of enzyme protein required to break down polymeric sugars in cellulose and hemicellulose to fermentable monomers. In the past 6 years, significant effort has been expended to reduce the cost by focusing on improving the efficiency of known enzymes, identification of new, more active enzymes, creating enzyme mixes optimized for selected pretreated substrates, and minimization of enzyme production costs. Here we describe advances in enzyme technology for use in the production of biofuels and the challenges that remain. © 2007 Springer-Verlag Berlin Heidelberg.

Dietrich, A., Nielsen, M.

"Bioethanol as renewable, economical alternative [Bioethanol als Erneuerbare, Wirtschaftliche Alternative]"

Nachrichten aus der Chemie, 55 (4), pp. 385-389. (2007)


Falholt, P.

"Novozymes' economic cellulosic ethanol strategy"

Industrial Bioprocessing, 29 (4), pp. 6-7. (2007)

At the 4th Annual World Congress on Industrial Biotechnology and Bioprocessing in Orland, Florida, the Novozymes' Per Faholt proposed a five-step strategy to achieve economically viable cellulosic ethanol. The outlined steps are as follows: 1) continued funding of research and development (R&D), specifically in the areas of biomass conversion and the development of commercial process technology; 2) establishment of flexible configuration development centers, geographically distributed to address multiple types of biomass feedstock and integrate pretreatment, hydrolysis, and fermentation processes; 3) scientific advancement to increase cost efficiency by improving underlying agricultural practices for the collection and harvesting of biomass and pretreatment methods; 4) scientific advancement in biotechnology; and 5) continued bipartisan support of a national infrastructure to support practical implementation with funding, incentives and tax credits.

Sørensen, H.R., Pedersen, S., Meyer, A.S.

"Synergistic enzyme mechanisms and effects of sequential enzyme additions on degradation of water insoluble wheat arabinoxylan"

Enzyme and Microbial Technology, 40 (4), pp. 908-918. (2007)

Generation of a fermentable hydrolysate from arabinoxylan is a first prerequisite in the utilization of wheat hemicellulose in the biofuel industry. This study examined the efficacy of four hemicellulolytic microbial enzyme preparations and one xylanase preparation in catalyzing degradation of purified water soluble and water insoluble wheat arabinoxylan-with particular emphasis on the catalytic degradation of water insoluble arabinoxylan. The effects of individual enzyme treatments were compared by assessing yields of arabinose, xylose, and xylobiose obtained under different reaction conditions of pH and temperature in response surface designs. In general, the monosaccharide yields obtained were lower on the water insoluble wheat arabinoxylan than on the water soluble. On both substrates, the Ultraflo L preparation from Humicola insolens was best in catalyzing arabinose and xylobiose release, while the Celluclast 1.5 L preparation from Trichoderma reesei was superior to all the other enzyme preparations in catalyzing xylose release. Treatments with 50:50 combinations of the enzyme preparations only resulted in a pronounced synergistic xylose release with a mixture of Ultraflo L:Celluclast 1.5 L. Examination of the progress of the substrate degradation indicated that the synergism on the insoluble arabinoxylan resembled what we have previously observed on soluble arabinoxylan, i.e. that the effect was a result of positive interaction in arabinose release and xylan depolymerization between the α-l-arabinofuranosidase (EC and endo-1,4-β-xylanase (EC activities present in Ultraflo L, and between the β-xylosidase (EC activity present in the Celluclast 1.5 L. The results of treatments with combinations of Ultraflo L and purified T. reesei β-xylosidase - with both simultaneous and sequential addition, and with and without contemporary pH adjustments to optimize individual enzyme activities - strongly corroborated this conclusion. © 2006 Elsevier Inc. All rights reserved.

Sørensen, H.R., Pedersen, S., Jørgensen, C.T., Meyer, A.S.

"Enzymatic hydrolysis of wheat arabinoxylan by a recombinant "minimal" enzyme cocktail containing β-xylosidase and novel endo-1,4-β-xylanase and α-L-arabinofuranosidase activities"

Biotechnology Progress, 23 (1), pp. 100-107. (2007)

This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel α-L-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%: 40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 α-L-arabinofuranosidase from H. insolens (Abf II), the GH51 α-L-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-β-xylanase from H. insolens (Xyl III), and a GH3 β-xylosidase from Trichoderma reesei (β-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50°C. A 10%: 40%:50% mixture of Abf II, Abf III, and β-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50°C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were ∼14, ∼18, and ∼27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei. © 2007 American Chemical Society and American Institute of Chemical Engineers.

Agbogbo, F.K., Wenger, K.S.

"Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis"

Biotechnology Letters, 28 (24), pp. 2065-2069. (2006)

Pretreatment of biomass with dilute H2SO4 results in residual acid which is neutralized with alkalis such as Ca(OH)2, NaOH and NH4OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO4 enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na 2SO4 inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH4)2SO 4 inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production. © 2006 Springer Science+Business Media B.V.

Agbogbo, F.K., Coward-Kelly, G., Torry-Smith, M., Wenger, K.S.

"Fermentation of glucose/xylose mixtures using Pichia stipitis"

Process Biochemistry, 41 (11), pp. 2333-2336. (2006)

Different synthetic glucose/xylose mixtures (60 g/L total sugar) were fermented into ethanol using P. stipitis CBS 6054. Glucose was the preferred substrate in the glucose/xylose mixtures. The high glucose fractions had higher cell biomass production rate and therefore higher substrate consumption rate and ethanol production rate compared to high xylose fractions. However, the high xylose fractions had a slightly higher ethanol yields compared to the high glucose fractions because the substrate was channeled into ethanol production rather the cell biomass. The maximum ethanol concentration was 22.7 and 24.3 g/L in 60 g/L glucose and xylose media, respectively. Using the same initial cell concentration of 2 g/L, fermentation was complete after 96 h of fermentation in 100% glucose (60 g/L), and after 120 h of fermentation in 100% xylose (60 g/L). © 2006 Elsevier Ltd. All rights reserved.

Lau, H.

"Novozymes works with China on biofuel research"

Industrial Bioprocessing, 28 (8), pp. 9-10. (2006)

Novozymes A/S and China Resources Alcohol Corporation (CRAC) will start a three-year cooperation for research on processes for producing cellulosic-based ethanol. The program will include setting up a pilot plant for cellulose-based ethanol at an existing CRAC ethanol facility at Zhaodong in Heilongjiang province. Novozymes is also developing enzymes for the process.

Olsson, L., Soerensen, H.R., Dam, B.P., Christensen, H., Krogh, K.M., Meyer, A.S.

"Separate and simultaneous enzymatic hydrolysis and fermentation of wheat hemicellulose with recombinant xylose utilizing Saccharomyces cerevisiae"

Applied Biochemistry and Biotechnology, 129 (1-3), pp. 117-129. (2006)

Fermentations with three different xylose-utilizing recombinant Saccharomyces cerevisiae strains (F12, CR4, and CB4) were performed using two different wheat hemicellulose substrates, unfermented starch free fibers, and an industrial ethanol fermentation residue, vinasse. With CR4 and F12, the maximum ethanol concentrations obtained were 4.3 and 4 g/L, respectively, but F12 converted xylose 15% faster than CR4 during the first 24 h. The comparison of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with F12 showed that the highest, maximum ethanol concentrations were obtained with SSF. In general, the volumetric ethanol productivity was initially, highest in the SHF, but the overall volumetric ethanol productivity ended up being maximal in the SSF, at 0.013 and 0.010 g/Lh, with starch free fibers and vinasse, respectively. Copyright © 2006 by Humana Press Inc. All rights of any nature whatsoever reserved.

Cherry, J.R., Gray, K.

"Introduction to session 1B"

Applied Biochemistry and Biotechnology, 129 (1-3), p. 179. (2006)


Greenwood, J., Nedwin, G., Mayer, R.

"Industry applauds Bush's call for more biofuels"

Industrial Bioprocessing, 28 (3), pp. 4-5. (2006)

In his State of the Union address, President George W. Bush talked about manufacturing motor fuel ethanol from wood chips, switch grass, and other biomass as one of the ways to end the dependence of the United States to imported oil. The administration continues to give strong support to the improvement of technology for producing fermentable sugars from lignocellulosic biomass and other technologies to bring renewable fuels closer to the market. Players involved in this area believe the US federal government must continue to fund intiatives to advance the recent technical successes in biomass conversion and assure that they are put to use in large-scale facilities.

Sørensen, H.R., Pedersen, S., Meyer, A.S.

"Optimization of reaction conditions for enzymatic viscosity reduction and hydrolysis of wheat arabinoxylan in an industrial ethanol fermentation residue"

Biotechnology Progress, 22 (2), pp. 505-513. (2006)

This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 °C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76-84%, 75-80%, and 43-47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, β-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2-6.4 and 41-49 °C for arabinose release and from pH 4.9-5.3 and 42-46 °C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields. © 2006 American Chemical Society and American Institute of Chemical Engineers.

Rosgaard, L., Pedersen, S., Cherry, J.R., Harris, P., Meyer, A.S.

"Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose"

Biotechnology Progress, 22 (2), pp. 493-498. (2006)

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a β-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 °C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reesei cellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation. © 2006 American Chemical Society and American Institute of Chemical Engineers.

Teter, S.A., Cherry, J.R.

"Improving cellulose hydrolysis with new cellulase compositions"

AIChE Annual Meeting Conference Proceedings, 2005, 7 p. (2005)

Cellulose is the most abundant organic molecule on the earth, and a renewable and seemingly inexhaustible feedstock for the production of fuels and chemicals. Bacteria and fungi have evolved complex enzymatic systems enabling their growth on plant material rich in cellulose, but these organisms typically require weeks, months, or even years to decompose a fallen log or a tilled corn stalk. For chemical or fuel production from these same materials, industry requires affordable chemical or enzymatic systems that can do the job in hours or in days. Novozymes has conducted a 4-yr project focused on improving the performance and reducing the cost of fungal cellulases for the conversion of dilute acid pretreated corn stover to fermentable sugars. A number of genes was transferred from other organisms to T. reesei, creating a super-cellulase producing fungus capable of meeting industrial needs. This is an abstract of a paper presented at the AIChE Annual Meeting and Fall Showcase (Cincinnati, OH 10/30/2005-11/4/2005).

Teter, S.A., Cherry, J.R.

"Improving cellulose hydrolysis with new cellulase compositions"

AIChE Annual Meeting, Conference Proceedings, pp. 12027-12033. (2005)

Cellulose is the most abundant organic molecule on the earth, and a renewable and seemingly inexhaustible feedstock for the production of fuels and chemicals. Evolved as a structural polymer, cellulose is remarkably recalcitrant to breakdown into its monomeric form, glucose. Bacteria and fungi have evolved complex enzymatic systems enabling their growth on plant material rich in cellulose, but these organisms typically require weeks, months, or even years to decompose a fallen log or a tilled corn stalk. For chemical or fuel production from these same materials, industry requires affordable chemical or enzymatic systems that can do the job in hours or in days. Under a research subcontract from the National Renewable Energy Lab (NREL), with funds from the U.S. Department of Energy, Novozymes has conducted a four year project focused on improving the performance and reducing the cost of fungal cellulases for the conversion of dilute acid pretreated corn stover to fermentable sugars. One aspect of this project has been the identification of novel proteins that, when added to the cellulases produced by a widely used soft rot fungus (Trichoderma reesei), increase the specific performance of the enzyme mixture. Applying state of the art tools of biotechnology to identify, clone, and express these boosting proteins, we have successfully transferred a number of genes from other organisms to T. reesei, creating a super-cellulase producing fungus capable of meeting industrial needs.

Ding, H., Xu, F., McFarland, K.C., Cherry, J.

"Enhanced enzymatic biomass conversion to fermentable sugars and fuel ethanol"

ACS National Meeting Book of Abstracts, 230, 1 p. (2005)

Expanding the use of renewable biomass as industrial feedstock is of strategic importance to reduce pollution, develop sustainable industry, and convert agriculture waste into value-added products. As a primary participant in joint government-industrial efforts, ways to significantly enhance the enzymatic conversion of pretreated corn stover into glucose and, subsequently, ethanol were studied. Using Trichoderma reesei as a starting point, the interaction between cellulolytic enzymes and lignocellulose was examined. By using a multi-facet approach, the enzymatic corn stover conversion was significantly enhanced, and reduced the cost of cellulases for biomass utilization by > 30-fold. This is an abstract of a paper presented at the 230th ACS National Meeting (Washington, DC 8/28/2005-9/1/2005).

Devantier, R., Pedersen, S., Olsson, L.

"Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain"

Applied Microbiology and Biotechnology, 68 (5), pp. 622-629. (2005)

Ethanol was produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions. The effects of glucoamylase dosage, pre-saccharification and Saccharomyces cerevisiae strain on the growth characteristics such as the ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration from 106 to 126 g/kg although the maximal specific growth rate was decreased. Ethanol production was not only growth related, as more than half of the total saccharides were consumed and more than half of the ethanol was produced during the stationary phase. Furthermore, a high stress tolerance of the applied yeast strain was found to be crucial for the outcome of the fermentation process, both with regard to residual saccharides and final ethanol concentration. The increased formation of cell mass when a well-suited strain was applied increased the final ethanol concentration, since a more complete fermentation was achieved. © Springer-Verlag 2005.

O'Connor, L., Nedwin, G.

"Frost & Sullivan technology award to Novozymes"

Industrial Bioprocessing, 27 (9), p. 10. (2005)

Novozymes has received the 2005 Technology Leadership of the Year Award from Frost & Sullivan in the field of fuel technologies for its pioneering research to reduce the enzyme cost for biomass-based fuel ethanol. The company's R&D reduced the enzyme cost for hydrolyzing cellulosic biomass from corn stover into fermentable sugars.

Devantier, R., Scheithauer, B., Villas-Bôas, S.G., Pedersen, S., Olsson, L.

"Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations"

Biotechnology and Bioengineering, 90 (6), pp. 703-714. (2005)

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites. © 2005 Wiley Periodicals, Inc.

Falhout, P., Nedwin, G.

"Novozymes looks at next steps for biomass ethanol"

Industrial Bioprocessing, 27 (5), pp. 1-2. (2005)

In 2001, Novozymes A/S and the National Renewable Energy Laboratory (NREL) started working together to reduce the cost of enzymes for producing ethanol 30-fold in laboratory trials. Novozymes cut the cost of enzymes for converting corn stover to enthanol to between $0.10 and $0.18 per gallon. The 30-fold reduction was achieved through a combination of the enzymes and improved pretreatment technology developed by NREL.

Sørensen, H.R., Pedersen, S., Viksø-Nielsen, A., Meyer, A.S.

"Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue"

Enzyme and Microbial Technology, 36 (5-6), pp. 773-784. (2005)

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei), Finizym (from Aspergillus niger), Ultraflo L (from Humicola insolens), and Viscozyme L (from Aspergillus aculeatus) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60°C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60°C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50°C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α-l-arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations. © 2005 Elsevier Inc. All rights reserved.

Bull, S., Dean, W., Nedwin, G.E.

"Award for biomass ethanol research"

Industrial Bioprocessing, 26 (8), p. 1. (2004)

NREL and two companies, Genencor International Inc., and Novozymes A/S, have been awarded for their progress in developing an economical process for the conversion of biomass into sugars that can be fermented to make ethanol or industrial chemicals. The research teams have cut the cost of enzymatic conversion of cellulosic biomass into fermentable sugars per gallon of ethanol to less than 20th of its former cost.

Nedwin, G., Kaempf, D.

"Novozymes and NREL cut ethanol enzyme costs again"

Industrial Bioprocessing, 26 (6), p. 5. (2004)

A collaboration between Novozymes and DOE's National Renewable Energy Laboratory (NREL) resulted to the development of an approach for lowering enzyme cost for converting biomass into ethanol from $5 to $0.30 per gallon. With the granting of a one-year extension of the contract, costs are expected to be lowered further to $0.05.

Traäff-Bjerre, K.L., Jeppsson, M., Hahn-Hägerdal, B., Gorwa-Grauslund, M.-F.

"Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae"

Yeast, 21 (2), pp. 141-150. (2004)

Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitol dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion. Copyright © 2003 John Wiley & Sons, Ltd.