Schiedam, Netherlands
Schiedam, Netherlands

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Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: KBBE.2011.3.4-02 | Award Amount: 13.25M | Year: 2012

BioConSepT aims to demonstrate the technically feasibility of White Biotech processes for the conversion of 2nd generation biomass into platform chemicals, which are 30% cheaper and 30% more sustainable than existing chemical routes or 1st generation processes. BioConSepT uses lignocellulose and non-edible oils & fats as cheap, abundantly available feedstocks, which cannot be used as food. The main achievements expected for BioConSepT are: (1) to develop the robust enzymes and micro-organisms suited for the more dirty 2nd generation feedstocks; (2) to reduce equipment costs and the number of process steps by the integration of bioconversion and highly selective separation technologies; (3) to facilitate easy integration in existing production chains by deploying combinations of bio- and chemical conversions and by proving the suitability of the produced platform chemicals for bio-based polymers, resins, plasticizers, solvents and surfactants and (4) by realisation of the 1st demonstration of integrated production chains from 2nd generation feedstocks to platform chemicals at industrially relevant scale. BioConSepT will bring novel technologies from lab to pilot scale by high level applied research. The consortium consists of 15 SMEs (suppliers of equipment, bioconversions, separation technologies and services), 10 large industrial parties (producers, end-users, engineering and consultancy companies) and 5 leading RTOs from 11 different countries. The large industrial parties and SMEs expect new products, processes, services and customers with a potential value of hundreds of M. BioConSepT will reduce the total processing costs and thus improve the competitiveness of the European agro/food and chemical industries. The use of renewable biomass will lead to a significant reduction of Green House Gas emissions and a more secure supply of feedstocks, energy and water as well as reduction of waste generation.


Koopman F.,Bio based Sustainable Industrial Chemistry B Basic | Koopman F.,Technical University of Delft | Koopman F.,Kluyver Center for Genomics of Industrial Fermentation | Wierckx N.,TNO | And 7 more authors.
Bioresource Technology | Year: 2010

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based platform chemical that may serve as a 'green' substitute for terephthalate in polyesters. Recently, a novel HMF/furfural oxidoreductase from Cupriavidus basilensis HMF14 was identified that converts 5-(hydroxymethyl)furfural (HMF) into FDCA. The hmfH gene encoding this oxidoreductase was introduced into Pseudomonas putida S12 and the resulting wholecell biocatalyst was employed to produce FDCA from HMF. In fed-batch experiments using glycerol as the carbon source, 30.1 g l-1 of FDCA was produced from HMF at a yield of 97%. FDCA was recovered from the culture broth as a 99.4% pure dry powder, at 76% recovery using acid precipitation and subsequent tetrahydrofuran extraction. © 2010 Elsevier Ltd. All rights reserved.


Meijnen J.-P.,Technical University of Delft | Meijnen J.-P.,Kluyver Center for Genomics of Industrial Fermentation | De Winde J.H.,Technical University of Delft | De Winde J.H.,Kluyver Center for Genomics of Industrial Fermentation | And 3 more authors.
International Sugar Journal | Year: 2011

The rising price of oil and impending deficit of fossil resources stimulate the development of alternative processes for the production of chemicals. The production of chemicals from lignocellulosic biomass is a promising alternative. Lignocellulosic biomass consists of a mixture of sugars that can be converted into valuable products or chemicals by means of bioconversion. It is essential that, in order to establish an economically sound process, the feedstock is utilized as close to completion as possible. However, due to its heterogeneous nature, lignocellulosic feedstock is often metabolized incompletely. This situation is also encountered during the production of aromatic compounds by engineered strains of the solvent-tolerant micro-organism Pseudomonas putida S12. This bacterial strain is not able to use all sugars from biomass, most notably the pentose fraction. Therefore, strategies were explored to engineer D-xylose metabolic pathways in P. putida S12, to enable the consumption of the most abundant pentose sugar present in lignocellulosic biomass, thereby lowering production costs of commodity chemicals.


Meijnen J.-P.,Technical University of Delft | Meijnen J.-P.,Kluyver Center for Genomics of Industrial Fermentation | Meijnen J.-P.,Institute of Botany and Microbiology | Meijnen J.-P.,Vlaams Institute for Biotechnology | And 5 more authors.
Journal of Biological Chemistry | Year: 2012

Previously, an efficient D-xylose utilizing Pseudomonas putida S12 strain was obtained by introducing the D-xylose isomerase pathway from Escherichia coli, followed by evolutionary selection. In the present study, systemic changes associated with the evolved phenotype were identified by transcriptomics, enzyme activity analysis, and inverse engineering. A key element in improving the initially poor D-xylose utilization was the redistribution of 6-phospho-D-gluconate (6-PG) between the Entner-Doudoroff pathway and the oxidative pentose phosphate (PP) pathway. This redistribution increased the availability of 6-PG for oxidative decarboxylation to D-ribose-5-phosphate, which is essential for the utilization of D-xylose via the nonoxidative PP pathway. The metabolic redistribution of 6-PG was procured by modified HexR regulation, which in addition appeared to control periplasmic sugar oxidation. Because the absence of periplasmic D-xylonate formation was previously demonstrated to be essential for achieving a high biomass yield on D-xylose, the aberrant HexR control appeared to underlie both the improved growth rate and biomass yield of the evolved D-xylose utilizing P. putida strain. The increased oxidative PP pathway activity furthermore resulted in an elevated NADH/NAD + ratio that caused the metabolic flux to be redirected from the TCA cycle to the glyoxylate shunt, which was also activated transcriptionally. Clearly, these findings may serve as an important case in point to engineer and improve the utilization of non-natural carbon sources in a wide range of industrial microorganisms. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Meijnen J.-P.,Technical University of Delft | Meijnen J.-P.,Kluyver Center for Genomics of Industrial Fermentation | Meijnen J.-P.,Catholic University of Leuven | Verhoef S.,Technical University of Delft | And 7 more authors.
Applied Microbiology and Biotechnology | Year: 2011

The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol. © 2011 The Author(s).


Bandounas L.,B Basic | Bandounas L.,Technical University of Delft | Bandounas L.,Kluyver Center for Genomics of Industrial Fermentation | Wierckx N.J.P.,B Basic | And 9 more authors.
BMC Biotechnology | Year: 2011

Background: To expand on the range of products which can be obtained from lignocellulosic biomass, the lignin component should be utilized as feedstock for value-added chemicals such as substituted aromatics, instead of being incinerated for heat and energy. Enzymes could provide an effective means for lignin depolymerization into products of interest. In this study, soil bacteria were isolated by enrichment on Kraft lignin and evaluated for their ligninolytic potential as a source of novel enzymes for waste lignin valorization.Results: Based on 16S rRNA gene sequencing and phenotypic characterization, the organisms were identified as Pandoraea norimbergensis LD001, Pseudomonas sp LD002 and Bacillus sp LD003. The ligninolytic capability of each of these isolates was assessed by growth on high-molecular weight and low-molecular weight lignin fractions, utilization of lignin-associated aromatic monomers and degradation of ligninolytic indicator dyes. Pandoraea norimbergensis LD001 and Pseudomonas sp. LD002 exhibited best growth on lignin fractions, but limited dye-decolourizing capacity. Bacillus sp. LD003, however, showed least efficient growth on lignin fractions but extensive dye-decolourizing capacity, with a particular preference for the recalcitrant phenothiazine dye class (Azure B, Methylene Blue and Toluidene Blue O).Conclusions: Bacillus sp. LD003 was selected as a promising source of novel types of ligninolytic enzymes. Our observations suggested that lignin mineralization and depolymerization are separate events which place additional challenges on the screening of ligninolytic microorganisms for specific ligninolytic enzymes. © 2011 Bandounas et al; licensee BioMed Central Ltd.


Bandounas L.,B Basic | Bandounas L.,Technical University of Delft | Bandounas L.,Kluyver Center for Genomics of Industrial Fermentation | Bandounas L.,TNO | And 9 more authors.
New Biotechnology | Year: 2013

In this study we have investigated the molecular background of the previously reported dye decolourization potential of Bacillus sp. LD003. Strain LD003 was previously isolated on Kraft lignin and was able to decolourize various lignin model dyes. Specifically Azure B (AB) was decolourized efficiently. Proteins possibly involved in AB decolourization were partially purified, fractionated by gel electrophoresis and identified via mass spectrometry. Five candidate enzymes were selected and expressed in Escherichia coli. Of these, only a quinone dehydrogenase was shown to decolourize AB. Thus, this quinone dehydrogenase was identified as an AB decolourizing enzyme of Bacillus sp. LD003. © 2012 Elsevier B.V.


Foti M.,B Basic | Foti M.,TNO | Foti M.,MicroDish BV | Medici R.,B Basic | And 5 more authors.
Journal of Biotechnology | Year: 2013

Pseudomonas putida S12 was engineered for the production of monoethanolamine (MEA) from glucose via the decarboxylation of the central metabolite l-serine, which is catalyzed by the enzyme l-serine decarboxylase (SDC).The host was first evaluated for its tolerance towards MEA as well as its endogenous ability to degrade this alkanolamine. Growth inhibition was observed at MEA concentrations above 100. mM, but growth was never completely arrested even at 750. mM of MEA. P. putida S12 was able to catabolize MEA in the absence of ammonia, but deletion of the eutBC genes that encode ethanolamine ammonia-lyase (EAL) enzyme sufficed to eliminate this capacity.For the biological production of MEA, the sdc genes from Arabidopsis thaliana (full-length and a truncated version) and Volvox carteri were expressed in P. putida S12. From 20mM of glucose, negligible amounts of MEA were produced by P. putida S12 δeutBC expressing the sdc genes from A. thaliana and V. carteri. However, 0.07mmol of MEA was obtained per g of cell dry weight of P. putida S12 δeutBC expressing the truncated variant of the A. thaliana SDC. When the medium was supplemented with l-serine (30mM), MEA production increased to 1.25mmolMEAg-1 CDW, demonstrating that l-serine availability was limiting MEA production. © 2013 Published by Elsevier B.V.


Wierckx N.,BIRD Engineering BV | Wierckx N.,RWTH Aachen | Koopman F.,Technical University of Delft | Ruijssenaars H.J.,BIRD Engineering BV | De Winde J.H.,Technical University of Delft
Applied Microbiology and Biotechnology | Year: 2011

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid. © 2011 The Author(s).

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