Safe-and-Sustainable-by-Design (SSbD) is a promising approach to develop new chemicals. However, well-elaborated tools to guide selection of SSbD alternatives that fullfill the desired and essential function in a given application are missing. In this proposal, we will focus on Persistent, Mobile and Toxic chemicals, as they pose a risk to the watercycle and human and ecological health. This TOSS project will develop integrated tools to select and produce SSbD alternatives for PMTs, will gain experience with putting SSbD in practice including active contributions of different stakeholders, and will formulate lessons for industry, academic research and policy.

Sugar and oxygen gradients frequently occur in industrial-scale bioreactors and commonly have a detrimental effect on yield, productivity, and product quality. Reducing or even preventing the occurrence of these gradients is therefore of eminent importance but attempts to realize this by improving the bioreactors have so far only been partially successful. We are proposing a completely new approach to solve this challenge by changing the microorganisms, not the bioreactor.

Integral Engineering of Acetic Acid Tolerance in Yeast Acronym: INTACT Carbon efficiency and food security dictate that a substantial replacement of current petrochemical production by industrial biotechnology should be based on crude plant biomass hydrolysates as feedstocks rather than on refined, food-grade carbohydrates. The presence of acetylated polymers in these crude hydrolysates implies that acetic acid tolerance of industrial microorganisms is and will remain a key issue in the implementation of sustainable, non-food feedstocks in industrial biotechnology. The yeast Saccharomyces cerevisiae, one of the most important microorganisms in industrial production and in metabolic engineering, already has an innate degree of tolerance to weak organic acids and low pH. However, better understanding and improvement of the tolerance to acetic acid is essential for development, diversification and intensification of yeast-based bioprocesses in industrial biotechnology. Finding solutions to this problem is urgent, since the first full-scale factories for yeast-based production processes from lignocellulosic feedstocks (the first products will be biofuels) are anticipated within the next 5 years. Our highly complementary consortium will integrate classical genetic mapping, comparative genomics, genome-wide expression analysis, evolutionary engineering and global transcription machinery engineering with targeted genetic modification, with the aim to understand and rationally improve acetic tolerance in S. cerevisiae. Key deliverables from the project will include: - Target genes for functional analysis and metabolic engineering through the integration of classical genetic mapping and comparative genomics - Selection procedures to improve acetic acid tolerance - Metabolic engineering strategies to rationally improve acetic acid tolerance - S. cerevisiae strains with improved acetic acid tolerance

Agroindustrial production processes are associated with the generation of enormous quantities of organic residues, often in a water diluted form. Effective valorization of these organic residues is of critical importance for the sustainability of agroindustrial production processes. Here we propose an innovative route for processing agroindustrial residues for the production of novel biofuels. The hybrid processing route proposed is based on two steps: In the first step a microbial ecology based biotechnological process is used to concentrate the water dissolved organic substrates in the form of intracellular storage polymers: polyhydroxyalkanoates (PHAs). This process has demonstrated to be capable of production of biomass that consists for 85-90% of PHAs, does not require aseptic conditions, and can be based on different types of ?waste? organic substrates. In the second step of the process, the PHAs are used for the chemical production of hydroxyalkanoate methyl esters (HAMEs). This esterification process has shown to yield HMEs with more than 95% purities, which can be used directly as fuel additives and have combustion heat values comparable to propanol and butanol. A major advantage of the HME production process is that it does not require highly purified PHA-based raw material, excluding the need for energy intensive PHA purification steps. For both steps in the HME production process a proof of principle has been established, but many questions remain to be answered to enable implementation of the process proposed. In this project Delft University of Technology will work on the influence of combinations of different substrates on the metabolic fluxes involved in PHA production and PHA-composition in a mixed microbial community. The relation between community stability and process stability will be evaluated. The objective of the work conducted at Tsinghua University is to optimize the HAME production process in terms of yields, solvent recovery, and upscaling. Both groups will work on the optimized process design and the interaction between e.g. PHA composition and fuel value.