DNA Helix

Cofund on Biotechnologies

Innovation for Europe – life science meets market application

6th Joint Call

Projects recommended for funding

In 2014, ERA-IB-2 and EuroTransBio (ETB) launched the 6th transnational joint call for multilateral research projects using Industrial Biotechnology (IB).

Funding was available for innovative industry-relevant Industrial Biotechnology projects on the following topics: 

  1. Conversion of industrial by-products and biomass into value-added products;
  2. Novel systems for new or more sustainable processes; e.g. the use of enzymes, micro-organisms and cell-free biosynthesis systems;
  3. New compounds from existing but previously under-studied biological systems, through a greater understanding of their metabolic pathways;
  4. Modeling and optimization of biological processes;
  5. Process development, intensification and/or integration in existing industrial processes; e.g. upstream or downstream design, scale-up of biotechnological processes, etc. 

The submitted projects needed to identify the product (produced by biotechnological processes) and market to be addressed. For example: 1. Bio-based materials; 2. Platform chemicals, such as bio-monomers, oligomers and polymers; 3. Pharmaceuticals, functional food/feed ingredients.

With this joint call, ERA-IB and ETB wished to foster the integration of the different steps throughout the entire value chain. This has resulted in the following 13 projects funded out of the 28 full proposals submitted:

  • ASTINPROD: Enhancing production of the anti-tumor compound astin by a novel fungal endophyte of Aster tataricus
  • BioProMo: Biotechnological production of monoterpenoids
  • Convert-Si: Conversion of phytogenic silica reach food industry by-products into value-added products
  • CrossCat: Symbiosis of bio - and chemo-catalysts for the sustainable conversion of hemicelluloses
  • FAPA: Fumaric acid for polymer applications
  • FunBioPlas: Novel synthetic biocomposites for biomedical devices
  • FunChi: Fungal chitosans from fermentation mycelia for plant biostimulants
  • No-P: Enzymatic sugar coupling
  • SurfGlyco: Enhanced fermentative and biocatalytic conversion strategies of renewables to tailor-made glycolipid biosurfactants
  • TTRaFFIC: Toxicity and transport for fungal production of industrial compounds
  • YeastTempTation: Tailoring thermo-tolerant yeasts for more sustainable, eco-efficient and competitive industrial fermentations
  • Z-Fuels: A novel bacterial system with integrated micro-bubble distillation for the production of acetaldehyde
  • 2GEnzymes: Enzymes for 2G sugars

The projects started in 2016 and are expected to end in 2019.

Granted Projects

Enhancing production of the antitumor compound astin by a novel fungal endophyte of Aster tataricus


Jutta Ludwig-Müller Technische Universität Dresden Germany
Didier Allaer Diagenode Belgium (Wallonia)
Arnaud Delecroix Lipofabrik Belgium SPRL Belgium (Wallonia)
Phillipe Jacques Université de Liège Belgium (Wallonia)
Willem van Berkel Wageningen University Netherlands
Karl-Heinz van Pee Technische Universität Dresden Germany
Luc Willems Université de Liège Belgium (Wallonia)



Extracts of dried roots of the plant Aster tataricus are successfully used in traditional Chinese medicine. Astins, secondary metabolites isolated from these root extracts, show promising antitumor activities. Astins are cyclic pentapeptides and ca 20 different astins have been now isolated from aster roots. For antitumor activity, cyclization and a rarely observed dichlorinated proline residue are necessary. Since astins have been isolated from dried aster roots, they were assumed to being produced by the plant itself. However, we showed that the producer of astins is not the plant but a novel, not yet described fungus that we named Pelliciarosea asterica. We detected at least three astins in fermentation broths of the new fungus, among them astin C, which has been one of the active compounds in antitumor studies. We have sequenced the genome of the astin-producing fungus and we could detect nonribosomal peptide synthetase gene clusters likely to code for the synthesis of the cyclic pentapeptide core and additional genes for the formation of nonproteinogenic amino acids required for astin biosynthesis. The discovery that astins can be produced biotechnologically by fermentation of the new fungus now offers the possibility for large-scale production in microorganisms. This will save valuable resources and will be less
time-consuming. In addition, microbial production systems also enable the engineering of product formation via feeding of specific precursors such as amino acids or other halides than chloride. Moreover, the fungus and heterologous expression hosts can be optimized in their astin production rates by genetic engineering. Still, astin production needs to be further improved to make these compounds available in large scale for extensive bioactivity and pharmacological studies. Finally, we will assess the bioactivities of a variety of astin derivatives using, among others, different tumor cell lines as models.


Biotechnological Production of Monoterpenoids


Jens Schrader DECHEMA-Forschunginstitut Germany
Harro Bouwmeester Wageningen University Netherlands
Manuel Rodriguez-Concepcion Centre Dd Recerca En Agrigenomica (CRAG) CSIC-IRTA-UAB-UB Spain
Johannes Panten Symrise AG Germany



Monoterpenoids are challenging products for IB processes due to their toxicity. Despite all efforts monoterpenoid yields of respective research projects, usually focused on increasing production instead of adjusting it to host cell capacities, have remained below expectations. The BioProMo consortium will create an IB complement to fossil-resources-based chemical processes for industrial monoterpenoid production. A sustainable and competitive platform technology based on the solvent resistant microbe mseudomonas putida will be established by combining biotechnological methods such as functional genomics, metabolic engineering, synthetic biology and bioprocess engineering.

Key aspects addressed by BioProMo are:

  • increasing resilience of a monoterpenoid tolerant P. putida strain at the level of efflux pumps
  • the mitigation of monoterpenoid toxicity by producing them as glycosides
  • the usage of metabolic switches, shunt pathways and enzyme coupling to create a resilient and efficient production host

The microbial platform aimed at will create two novel production routes: a) a whole-cell biocatalysis to selectively oxyfunctionalize a monoterpene hydrocarbon, a cheap by-product of the food processing industry (short –term goal) and b) a self-regulated de novo production circuit starting from the renewable raw material glycerol, a by-product of biodiesel production (mid -term goal). The use of renewable industrial by-products and waste streams as raw material for monoterpenoid production will create novel value-added chains for the European industry. We unite the complementary know-how and expertise of European research groups from three different countries to accomplish the goal of establishing a microbial production platform for monoterpenoids. The industrial partner of BioProMo will not only advice the research project from its market-oriented  viewpoint but also actively participate in every work package and intends to transfer the envisaged IB process into application.


Conversion of phytogenic silica reach food industry by-products into value-added products


Florin Oancea National Research and Development Institute for Chemistry and Petrochemistry Romania
Petruta Cornea University of Agronomic Sciences and Veterinary Medicines Romania
Karol Leluk Wroclaw University of Technology Poland
Sergy Shaposhnikov NorGenoTech AS Norway
Ionut Moraru Laboratoarele Medica Sri Romania
Krzysztof Garman MKF-Ergis Sp. z.o.o.z Poland



Convert-Si project aims to develop optimized processes for a total conversion of industrial plants by-products into value-added products. Main innovative contribution is related to the introduction of a (pre)treatment step based on new microbial products: non-catalytic small proteins from cerato-platanin family (CP), which weaken (ligno)cellulose structure, metabolites from silicon solubilizing microorganisms (SiM) and super-active ligno-cellulolytic enzymes.

Convert-all project integrated approach is intended to close a biomimetic industrial symbiosis. The by-products of one conversion process represent raw material for other conversion steps.

Convert-Si proposed cascade process is applied to two value-added chains: cereal husks and/or bran, fruit/grape pomace. From these the following ingredients are recovered: anti-oxidant polyphenols and/or feruloylated oligosaccharides, useful for new cosmetics and dietary supplement products; essential oil terpenes and (brassino)steroids, trapped and/or bounded  by/to lignocellulose network, with plant growth stimulating and/or insects repellent activities; biosilica (BSi); fermentable carbohydrates, recalcitrant lignin.

BSi, fermentable carbohydrates and recalcitrant lignin are converted into mesoporous silica nanoparticles, value added fermented food / feed (probiotics) supplements and, respectively, substrate for plant biostimulants formulation. Mesoporous silica nanoparticles are used for the “ smart”, controlled released formulation of the recovered polyphenols, essential oils, and (brassino)steroids. Recalcitrant lignin, together with the biomass of CP producing microorganisms and, respectively, SiM will be used for the production of a complex plant biostimulant.  Safety and efficacy of biosilica nano-particles and “smart” formulated phytoextracts will be tested by state of the art high through-output cell assay.


Symbiosis of bio- & chemo-catalysts for the sustainable conversion of hemicelluloses


Thomas Bley Technische Universität Dresden Germany
Rüdiger Lange Technische Universität Dresden Germany
Tapio Salmi Abo Akademi University Finland
Patrick Sagmeister EXPUTEC GmbH Austria



In our day-to-day life, most of us need items and energy which still strongly depend on fossil resources. But the increasing scarcity and prices, as well as environmental aspects of these resources require a shift towards renewable raw materials such as plant biomass. During the last decades, as biorefinery technology started to grow out of its infant stage, the research community recognized that of the three main biomass components, hemicelluloses are still relatively under-represented. Therefore, hemicelluloses - the second most abundant component - recently started to receive nearly equal attention as cellulose and lignin.

A prominent example of hemicellulose-based chemical products is xylitol (E 967) which is in strong demand for applications in food, pharmaceuticals, cosmetics and dental care due to its insulin independence, high sweetening power and anti-cariogenic effect. However, the conventional production of xylitol via Raney-nickel catalysts is ineffective and expensive, compared to the potential of a future process based on industrial biotechnology.

Therefore, we propose to develop a hybrid process to convert lignocellulosic biomass into platform chemicals (e.g. sugars and polyols) for food, pharma and cosmetic industries. Key will be the combination of chemical catalysts, enzymes and extremophiles (whole cells) in a novel one-pot process. This innovative approach connects the advantages of chemical catalysis and white biotechnology and minimizes disadvantageous factors, such as high energy consumption and long residence time. Thus, both economic efficiency and environmental sustainability of a future bio-economy will be increased significantly. The conversion of hemicelluloses into sugar monomers and alcohols will be investigated using xylan as example. The intermediate product xylose is an established platform chemical and the final product xylitol has high added-value for existing markets for chemicals, pharmaceuticals, paper and food industry.


Fumaric Acid for Polymer Applicants


Ulf Prüße Thünen-Institute of Agricultural Technology Germany
Miguel Ladero University Complutense Madrid Spain
Antonia Rojas Biopolis S.L. Spain
Arno Cordes ASA Spezialenzyme GmbH Germany
Victor Cost UBE Corporation Europe, SA Spain
Emre Yöntem Ekodeng Turkey



The project objective is the development of an efficient fermentative process for the production of fumaric acid from the renewable feedstocks raw glycerol, orange peels and apple pomace.

Fumaric acid producing fungi are able to convert numerous carbohydrates as well as glycerol, but studies regarding the mentioned substrates are rather scarce. Hence, a screening of microorganisms able to convert the different substrates most efficiently will be carried out. Two different process variants shall be considered: separate hydrolysis and fermentation (SHF) as well as simultaneous saccharification and fermentation (SSF). In case of SHF, the substrates need to be pretreated and hydrolyzed first. These processes will be optimized for the different substrates and new efficient enzymes shall be developed for this purpose. In case of SSF, hydrolysis will be achieved by the hydrolytic enzymes known to be segregated by Rhizopus spp.

The fermentation process itself needs to be optimized for the different substrate/microorganism combinations. More specifically, various parameters, such as the pH value and neutralization strategy, oxygen demand, media composition and fungal morphology need to be optimized to reach a high yield coupled with a high productivity. Additionally, efficient downstream processes will be investigated in combination with the fermentation process to maximize the efficiency of the microbial fumaric acid production.

The whole research work will additionally be accompanied by complete Life Cycle Assessment (LCA) studies. LCA will be used to evaluate the process sustainability with realization of the whole production process. In order to evaluate the cost considerations, Life Cycle Cost (LCC) assessment will be used by evaluating the material and energy flows with respect to their unit prices. Project outcomes will aim to reach sustainable product design by multi-objective optimization and
cost-benefit analysis tool.


Novel synthetic biocomposites for biomedical devices


Raul Machado University of Minho Portugal
José Carlos Rodriguez-Cabllo Universidad de Valladolid Spain
Stephanie Lesage Oxford Biomaterials Ltd United Kingdom



Innovative biomedical products rely on newly developed materials tailored for specific biomedical applications. The FunBioPlas project aims to functionalize bioplastics specifically developed for the fabrication of vascular grafts, temporary skin substitutes and catheters. This will be achievable through the biotechnological production of functionalized recombinant protein-based polymers (rPBPs) and their use as fillers in the formulation of biopolymer matrices and further integration into industrial plastic processing routes. The challenge of FunBioPlas is to drive rPBPs closer to the marketplace, which will be achievable by: i) scaling-up the use of agro-industrial wastes to reduce costs of rPBPs biotechnological production, ii) optimizing the processing conditions and iii) formulating novel materials based on rPBPs.

rPBPs are an emerging class of biomaterials with unique chemical, physical and biological characteristics, produced with the precise control of the polymer chain composition and length. Synthetic protein biotechnology approaches allow us to tailor-make the molecular structure of rPBPs and incorporate biologically active functionalities. We will therefore create medical devices with enhanced biological performance, including better infection control and cell function, as well as improved mechanical properties through the integration of rPBPs in the fabrication processes. The present proposal involves the cooperation of academic institutions from Portugal (University of Minho, UMinho, project coordination) and Spain (Universidad de Valladolid, UvA), as well as an SME from UK (Oxford Biomaterials Ltd, OBM) to provide expertise across the whole value chain of the project: from the designing and bioproduction of rPBPs, materials characterization and processing, to the proof-of-concept and market validation.

The FunBioPlas project thus represent a unique opportunity to develop highly innovative cross-disciplinary research.


Fungal Chitosans from Fermentation Mycelia for Plant Biostimulants


Bruno M. Moerschbacher WWU Münster University Germany
Arthur F.J. Ram Leiden University Netherlands
Johannes de Bie WeissBioTech Germany
Peter J. Punt TNO Netherlands
Antonio Molina Universidad Politecnica de Madrid Spain
Marise Borja Plant Response Biotech Spain



The FunChi consortium of two SME and four academic partners from Germany, Netherlands, and Spain will overcome two main problems in industrial scale fungal fermentation as required for the transition from an oil-based to a bio-based economy, namely high viscosity at high cell densities hindering stirring and oxygen transfer, and large amounts of mycelial wastes. Both problems will be addressed by targeting the cell wall biosynthetic machinery of a high performance albino production strain of Aspergillus niger. Viscosity will be reduced by aiming at shorter, more branched hyphae leading to micro-pelleted growth. The chitin content of the fungal cell wall will be increased and its incorporation into the complex cell wall will be modified so that it can be more easily extracted with better yields and higher quality in terms of polymer size and purity. Thus, the mycelial waste fraction will be converted into a high added-value product.

Chitosan is one of the most promising functional biopolymers, but commercial chitosans are mostly derived from chitin extracted from shrimp shell wastes. This animal origin involves two potential problems, i.e. limited supply leading to rather high costs, and the possibility of allergen or viral contamination. Fungal cell walls are a known alternative source of chitin, but its covalent incorporation into the fungal cell wall has so far hindered the development of a commercially viable process for the large scale extraction of high quality chitin from fungal mycelia. By biotechnologically modifying the cell wall of a fungus which is used at large industrial scale, such that it contains more chitin which is more easily extractable, we will overcome both of these hurdles for market entry and penetration. Proof-of-principle will be reached by the knowledge-based development of a plant biostimulant based on fungal chitosan that will allow significant reductions of chemical inputs in agriculture, benefiting both the environment and the consumer.


Enzymatic sugar coupling


Ulf Hanefeld Technische Universiteit Delft Netherlands
Dirk Tischler TU Bergakademie Freiberg Germany
Rob Schoevaart ChiralVision B.V. Netherlands
Andrzej Jarzębski Silesian University of Technology Poland
Arkadiusz Chruściel MEXEO Poland
Isabel Bento European Molecular Biology Laboratory (EMBL), Hamburg outstation Germany



In today’s world our societies are not sustainable. To ensure that all humans can live on our current level of health and wealth without destroying the perspective of future generations to do this as well, we need to convert our societies into fully sustainable civilisations that do not dependent on limited resources. The way we propose to achieve this is to utilise enzymes. Enzymes allow for very benign processes under mild conditions with very high selectivity, drastically reducing energy consumption and waste. These enzymes we propose to immobilise to ensure maximum stability and recycling thereof. At the same time the immobilisation will allow a different reactor concept. With microfluidics we will ensure that already the reaction conditions developed in the laboratory can be directly scaled out to industrial production. Thus enzymes and their immobilisation ensure sustainable processes that can be implemented straightforwardly, as will be proven by the industrial partners in this project.

The process we will develop is the coupling of sugars, enabling technology to utilise natural resources. A major problem in this area is that current technology does not allow high yields unless very unsustainable conditions are employed. We propose a combination of modified enzymes that makes it possible to obtain essentially complete yields without waste. To achieve this we will use enzymes that rely on phosphorus activated compounds. Since phosphorus is a limited element on this planet we will modify the enzymes in such a manner that they will utilise less phosphorus. Additionally we will design the process in such a manner that the small amount of phosphorus still necessary can be fully recycled so that the overall phosphorus consumption is zero.

Overall a straightforward, environmentally benign coupling of sustainable sugars under mild conditions will be developed and proven in industrial settings.


Enhanced fermentative and biocatalytic conversion strategies of renewables to tailor-made glycolipid biosurfactants


Steffen Rupp Fraunhofer Institute for Interfacial Engineering and Biotechnology Germany
Christoph Syldatk Karlsruhe Institute for Technology Germany
Dirk Verdoes The Netherlands Organization of Applied Scientific Research Netherlands
Douglas Cossar CRODA Europe Ltd United Kingdom
Maria Cuellar Delft University of Technology Netherlands
Bruno Ferreira Biotrend SA Portugal
Matti Heikkilae MetGen Oy Finland



Surfactants are surface-active molecules which we encounter daily for instance in cleaning agents or personal care products. The major amount of surfactants is still produced petro-chemically. Surfactants from chemical synthesis based on renewables are on the rise but the required oils originate from tropical plants. Thus there is a special interest in new tailor-made, biodegradable surfactants from renewable substrate flexible processes with native European sustainable resources. Natural biosurfactants, especially glycolipids, can be produced using microbial or enzymatic processes which results in a wide range of molecules with varying sugar groups and hydrophobic lipid moieties. Their unique molecular structure often leads to beneficial effects like antimicrobial or skin repair activity which create an added value for the desired applications. Only few microbial glycolipids are already manufactured on an industrial scale due to a low microbial productivity of desired glycolipid derivatives and a cost-intensive downstream processing. One aim of SurfGlyco is to enhance the potential of the underexploited microbial glycolipids MEL and CL which show promising yields and an enormous molecular variability. By adjusted feeding strategies and fermentations connected to an effective downstream processing we want to produce tailor-made biosurfactants with optimized performance in the area of personal care products, cleaning agents and natural plant protection. As second aim SurfGlyco will generate novel glycolipids of varying sugar and lipid components by using highly selective enzymatic reactions under mild reaction conditions. Currently, enzymatic synthesis still suffers from low space-time yields and a narrow range of products. SurfGlyco wants to overcome these problems by using stable enzymes with altered substrate specificities and the use of deep eutectic solvents as non-toxic reaction media which recently have been shown to enable high glycolipid yields.


Toxicity and Transport for Fungal production of Industrial Compounds


Gertien Smits Swammerdam Institute for Life Sciences University of Amsterdam Netherlands
Nick Wierckx RWTH Aachen University Germany
Nuno Mira Instituto Superior Técnico Portugal
Matthias Steiger Austrian Centre of Industrial Biotechnology Austria
Mustafa Turker Pak Gida Uretim Pazarlama A.S. Turkey
Guido Meurer BRAIN AG Germany
Peter Punt TNO Netherlands



Organic acids are important emerging industrial building blocks. Yeasts and fungi are often natural producers of a range of organic acids, and are particularly efficient hosts because of their high tolerance to weak acids as well as to low pH, a highly desirable trait for industrial application due to reduced downstream processing costs. In concert we aim to develop two major improvements to the production of itaconic acid (IA) by fungal hosts. 1) We will optimize the production pathway, taking into account and making use of the multiple compartments in the cell. Because of these multiple compartments, we need to understand how metabolic properties of the compartments suit the production process. 2) We will address transport between compartments and eventually out of the cell. At the same time we need to improve the host organism, by reducing sensitivity to the compound and by reducing the deleterious effects that the production of IA in the cell can exert within the specific compartments in which production takes place.

To do this, we take an integrative approach of genome-wide phenotypic screens to understand and reduce toxicity, metabolic modeling and engineering to understand the effects of and improve pathway topology, and detailed kinetics and novel screening for transporters to improve pathway productivity by reducing rate limiting transport steps. Such an integrated approach, focused on the simultaneous optimization of host and pathway for the production of one specific compound, has not been previously addressed. We expect to improve the production rates of IA to levels suitable for commercialization of the fungal production process for novel bulk-scale applications that will open up new opportunities in the framework of a bio-based economy.


Tailoring thermotolerant yeasts for more sustainable, eco-efficient and competitive industrial fermentations


Jose Manuel Guillamon National Research Council (CSIC), Institute of Agrochemistry and Food Technology (IATA) Spain
Walter M. van Gulik Delft University of Technology (TU Delft) Netherlands
José María Heras Lallemand Bio, S.L. Spain
Maria Manuel Dantas UNICER Portugal
José Antonio Teixeira University of Minho Portugal



Production of useful products through microbial fermentation is a core activity of industrial biotechnology, and yeasts are one of the most widely used microorganisms in industrial biotechnology. This industry spends huge amounts of energy to cool or heat these processes in order to fine-tune temperature as closely as possible to the optimum growth temperature of the fermentative strains. Notwithstanding, this optimum temperature quite often does not very well match the cost-effectivity or quality of the end product. Thus, new processing concepts demand greater efficiency and expose microbes to extreme conditions. Adaptation and tolerance of yeast strains to temperatures beyond the optimum range is crucial for economic and eco-efficient production processes for new and traditional fermentations. Our proposal attempts to address a systems biology approach to identify the key pathways, enzymes and genes related to a particular phenotype adapted to grow at different temperatures. From an industrial point, such knowledge is important to come up with better metabolic engineering strategies that consider the impact of novel genes and pathways on cellular economics to develop cost-effective bio-based processes. In line with this, we will design rational genetic improvement strategies to obtain more robust and adapted yeast strains to grow and ferment at low and high temperatures, with benefits for different fermentation processes. This will be put into practice on industrial scale by our industrial partners Lallemand and UNICER. Therefore, the main general aim of this project is the generation of improved thermotolerant yeast strains and their application to industrial fermentation processes (wine, beer, cider and bioethanol).


A novel bacterial system with integrated micro-bubble distillation for the production of acetaldehyde


Graeme Hitchen Perlemax Limited United Kingdom
Uldis Kalnenieks University of Latvia Latvia
Per Bruheim NTNU Norwegian University of Science and Technology Norway
Katja Bettenbrock Max-Planck-Institute for Dynamics of Complex Technical Systems Germany
Germany University of Stuttgart Germany



Butanol is a desirable biofuel as its characteristics most closely match gasoline and allows using current gasoline fuel infrastruture and vehicles. Its production however is limited by the energy requirements needed for its synthesis from ethanol and the poor yields.  A known intermediary in butanol production is acetaldehyde, the production of which involves high cost.  Microorganisms exist which can produce acetaldehyde directly. The bacterium wymomonas mobilis is the most prominent example, which bears a great potential for bioprocess improvement by metabolic engineering. However, the process yields and productivity are restricted by the metabolic inhibition caused by the produced acetaldehyde. The concept of the present project is to design, construct and operate a bacterial process, based on genetically engineered Z. mobilis with an integrated microbubble distillation system to convert complex sugary feedstocks and crude glycerol to acetaldehyde.  Effective removal of acetaldehyde during the metabolic process will alleviate the inhibition and give higher yields.  From the higher production quantities of acetaldehyde a more competitive and efficient route to butanol production can be obtained compared to current practise.


Enzymes for 2G Sugars


Tino Elter Fraunhofer Center for Chemical-Biotechnological Processes CBP Germany
Juan Lema University of Santiago de Compostela Spain
Matti Heikkilä MetGen Oy Finland
Christian Wilhelm University Leipzig Germany



The ambition of this project is to design a new integrated process for the production of second generation sugars (2GS) using ligninolytic enzymes (LE) and novel cellulases (CA). Basis for the generation of 2GS is the organosolv process of Fraunhofer CBP which allows the fractionation of lignocellulosic material into cellulose, hemicellulose and lignin. This is carried out under relatively severe conditions and requires the use of energy, chemicals and specialized equipment. The resulting cellulose is hydrolyzed enzymatically to glucose. The innovative idea lies in the combined application of CA and LE in both steps of the 2GS production. The application of LE before and during wood fractionation will lead to a reduced consumption of energy and chemicals by using less severe conditions in the pretreatment and to lower lignin contents in the resulting cellulose fraction. In the hydrolysis step CA and LE will be applied simultaneously to the resulting fibers. This will lower inhibitory effects of lignin on CA and will likely lead to higher yields of fermentable sugars. Also lower necessary CA concentrations and a faster reaction are expected. As LE have already been used to detoxify 2GS, it is also expected to improve the fermentability of the gained sugars. Another aspect is the use of novel CA from Penicillium verruculosum. This cellulase is favored for saccharification of pulp because of the higher content of ß-glucosidase and the tolerance towards CA inhibitors compared to commercial enzyme preparations. It is intended to integrate CA production into the overall process from lignocellulosics to 2GS by using cellulose from the wood fractionation as substrate. We further aim to improve our production strain genetically to optimize the production of cellulolytic enzymes by P. verruculosum. Finally, the modified organosolv process as well as the optimized CA fermentation process is demonstrated in pilot scale to obtain scale-up data for an industrial relevant scale.