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Horvat P.,University of Zagreb | Koller M.,University of Graz | Braunegg G.,ARENA Association for Resource Efficient and Sustainable Technologies
World Journal of Microbiology and Biotechnology | Year: 2015

A review of the use of elementary flux modes (EFMs) and their applications in metabolic engineering covered with yield space analysis (YSA) is presented. EFMs are an invaluable tool in mathematical modeling of biochemical processes. They are described from their inception in 1994, followed by various improvements of their computation in later years. YSA constitutes another precious tool for metabolic network modeling, and is presented in details along with EFMs in this article. The application of these techniques is discussed for several case studies of metabolic network modeling provided in respective original articles. The article is concluded by some case studies in which the application of EFMs and YSA turned out to be most useful, such as the analysis of intracellular polyhydroxyalkanoate (PHA) formation and consumption in Cupriavidus necator, including the constraint-based description of the steady-state flux cone of the strain’s metabolic network, the profound analysis of a continuous five-stage bioreactor cascade for PHA production by C. necator using EFMs and, finally, the study of metabolic fluxes in the metabolic network of C. necator cultivated on glycerol. © 2015, Springer Science+Business Media Dordrecht. Source

Koller M.,University of Graz | Chiellini E.,University of Pisa | Braunegg G.,ARENA Association for Resource Efficient and Sustainable Technologies
Chemical and Biochemical Engineering Quarterly | Year: 2015

The halobacterium Haloferax mediterranei was used to study the production of two types of biopolymers: The biopolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was accumulated as intracellular granules, whereas an extracelluar polysaccharide was excreted in parallel to biopolyester synthesis. After production, microbial re-use and degradation of these polymers under different conditions were investigated to assess the requirements for handling the product-rich fermentation broth prior to the downstream processing for product recovery. Degradation kinetics of the polymers and the impact of different storage conditions on molar mass of PHBV were studied. It turned out that the biotechnological fermentation process can be run without any sterility precautions. No major product losses were observed without pasteurization of fermentation broth after the stop of fermentation. In addition, neither PHBV nor EPS are re-utilized by the cells for biomass formation even if the culture is maintained under conditions of carbon starvation for an extended time. Source

Koller M.,University of Graz | Marsalek L.,University of Natural Resources and Life Sciences, Vienna | de Sousa Dias M.M.,French Institute of Health and Medical Research | Braunegg G.,ARENA Association for Resource Efficient and Sustainable Technologies
New Biotechnology | Year: 2016

Sustainable production of microbial polyhydroxyalkanoate (PHA) biopolyesters on a larger scale has to consider the ". four magic e": economic, ethical, environmental, and engineering aspects. Moreover, sustainability of PHA production can be quantified by modern tools of Life Cycle Assessment.Economic issues are to a large extent affected by the applied production mode, downstream processing, and, most of all, by the selection of carbon-rich raw materials as feedstocks for PHA production by safe and naturally occurring wild type microorganisms. In order to comply with ethics, such raw materials should be used which do not interfere with human nutrition and animal feed supply chains, and shall be convertible towards accessible carbon feedstocks by simple methods of upstream processing. Examples were identified in carbon-rich waste materials from various industrial braches closely connected to food production. Therefore, the article shines a light on hetero-, mixo-, and autotrophic PHA production based on various industrial residues from different branches. Emphasis is devoted to the integration of PHA-production based on selected raw materials into the holistic patterns of sustainability; this encompasses the choice of new, powerful microbial production strains, non-hazardous, environmentally benign methods for PHA recovery, and reutilization of waste streams from the PHA production process itself. © 2016 Elsevier B.V. Source

Koller M.,University of Graz | Dias M.M.S.,Institute Of La Vision | Rodriguez-Contreras A.,Polytechnic University of Catalonia | Kunaver M.,Slovenian National Institute of Chemistry | And 3 more authors.
Materials | Year: 2015

Liquefied wood (LW) prepared in a microwave process was applied as a novel; inexpensive precursor feedstock for incorporation of (R)-3-hydroxyvalerate (3HV) into polyhydroxyalkanoate (PHA) biopolyesters in order to improve the biopolyester's material quality; Cupriavidus necator was applied as microbial production strain. For proof of concept, pre-experiments were carried out on a shake flask scale using different mixtures of glucose and LW as carbon source. The results indicate that LW definitely acts as a 3HV precursor, but, at the same time, displays toxic effects on C. necator at concentrations exceeding 10 g/L. Based on these findings, PHA biosynthesis under controlled conditions was performed using a fed-batch feeding regime on a bioreactor scale. As major outcome, a poly(3HB-co-0.8%-3HV) copolyester was obtained displaying a desired high molar mass of Mw = 5.39 * 105 g/mol at low molar-mass dispersity (DM of 1.53), a degree of crystallinity (Xc) of 62.1%, and melting temperature Tm (176.3 °C) slightly lower than values reported for poly([R]-3-hydroxybutyrate) (PHB) homopolyester produced by C. necator; thus, the produced biopolyester is expected to be more suitable for polymer processing purposes. © 2015 by the authors. Source

Koller M.,University of Graz | Salerno A.,University of Graz | Muhr A.,University of Graz | Reiterer A.,University of Graz | Braunegg G.,ARENA Association for Resource Efficient and Sustainable Technologies
Materiali in Tehnologije | Year: 2013

Polyhydroxyalkanoates (PHAs) attract considerable attention as sustainable "green plastics" with a real potential to replace their petrol-based competitors in some applications in the not-too-distant future. To reach this goal PHAs must be able to compete with the established petrol-based plastics in both technical and economic terms. The current PHA production is based on prized substrates of high nutritional value such as sucrose, starch or vegetable oils. An alternative, carbon-rich industrial waste can be used as a suitable feedstock. This would contribute to making PHAs economically competitive and would avoid the conflict with human nutrition or animal feeding. Consequently, the decision about the location of the PHA-production facilities depends on the preferable in-house availability of such waste streams. The issue of competitive, large-scale PHA production in Europe was the topic of the ANIMPOL and the WHEYPOL projects. Both intended to develop novel processes for the transformation of abundant, locally available, renewable wastes. In the ANIMPOL case, waste lipids from slaughterhouses are converted to glycerol and a mixture of saturated and unsaturated fatty acid esters (FAEs), better known as biodiesel. The production of saturated FAEs is 50 000 t per year in Europe, decreasing the biodiesel performance as an engine fuel. However, within the project it was demonstrated that they can be efficiently metabolized to PHAs. In the WHEYPOL project attention was focused on 1.4 × 108 t per year of whey from dairies. This waste is of limited use and causes environmental concern. However, lactose, the main carbohydrate found in whey, can be used as a substrate in the WHEYPOL bioprocesses. These strategies demonstrate the feasibility of making "green plastics" competitive by integrating their manufacturing directly into the existing production lines, where the convertible waste streams accrue. Source

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