Presentation Information
[2Biocat-04]From CO2-Derived Acetate to Biopolymers: Engineered Microbial Strains for Integrated Biocatalytic, Computational, and Scale-Up Strategies in PHBV Production
William Mawuko Siegu1,2, Lekshmi Gopakumari Satheesh Chandran1, Piotr Paneth1, Olga Marchut-Mikołajczyk2, ○Vignesh Kumaravel1 (1. International Centre for Research on Innovative Biobased Materials (ICRI-BioM), Lodz University of Technology (Poland), 2. Institute of Molecular and Industrial Biotechnology, Lodz University of Technology (Poland))
Keywords:
Engineered Strains,Biopolymers,Bioreactors,Biocatalysis,CO2 conversion
Carbon-based materials underpin modern industry and everyday life; however, their production remains largely dependent on fossil resources and energy-intensive chemical processes. Synthetic polymers, in particular, exemplify this dependence and contribute to significant environmental challenges, especially in terms of disposal.
The HELVA (https://helvaproject.eu/) project addresses these challenges by proposing a novel and potentially cost-effective method for the biosynthesis of polyhydroxyalkanoates (PHAs). This approach uses acetate, electro-synthesized from anthropogenic carbon dioxide (CO2), together with genetically modified microbial strains and environmentally friendly extraction techniques to improve PHA yields.
In the present study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was biosynthesized using Escherichia coli DH5α, with sodium acetate as the sole carbon source. Reaction parameters were optimized using an orthogonal Taguchi matrix: 30 oC, 7 g/L sodium acetate, pH 7, 0.2 medium-to-volume ratio (MVR), and 180 rpm.
Nutrient-limitation experiments revealed that nitrogen concentration exerted the strongest influence on growth, followed by oxygen availability, while phosphorus was optimal at intermediate levels. These results highlight the importance of balancing nutrient availability and oxygen transfer to maximize biomass formation and guide process development.
Experiments using acetate-containing mixtures derived from CO2 electroreduction (including ethanol, propanol, and formic acid) resulted in significantly reduced microbial growth compared to pure sodium acetate. Formic acid was identified as the primary inhibitory component responsible for reduced growth performance.
Following shake-flask optimization, scale-up to a 5 L bioreactor demonstrated stable and reproducible growth under controlled aeration, mixing, and pH conditions, confirming process robustness. FT-IR and 1H NMR analyses indicated approximately 57% similarity between the synthesized biopolymers and conventional PET, highlighting the potential of these materials as functional biobased alternatives.
Computational modeling of the PHA biosynthetic pathway included five enzymes: acetate kinase (AckA), phosphate acetyltransferase (Pta), beta-ketothiolase (PhaA), acetoacetyl-CoA transferase (PhaB), and polyhydroxyalkanoate synthase (PhaC). Density functional theory (DFT) calculations were conducted at the ωB97x-D/def2-SVP level within the QM/MM ONIOM framework, as implemented in the Gaussian software package. The results indicate that the rate-determining step involves cleavage of the C-H bond in the second acetyl-CoA molecule, concurrent protonation of Cys374, formation of a C-C bond between acetyl groups, and cleavage of the S-C bond of the acetyl enzyme.
Overall, this integrated experimental and computational approach demonstrates the feasibility of converting CO2-derived acetate into value-added biopolymers and provides a scalable framework for sustainable PHA production.
The HELVA (https://helvaproject.eu/) project addresses these challenges by proposing a novel and potentially cost-effective method for the biosynthesis of polyhydroxyalkanoates (PHAs). This approach uses acetate, electro-synthesized from anthropogenic carbon dioxide (CO2), together with genetically modified microbial strains and environmentally friendly extraction techniques to improve PHA yields.
In the present study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was biosynthesized using Escherichia coli DH5α, with sodium acetate as the sole carbon source. Reaction parameters were optimized using an orthogonal Taguchi matrix: 30 oC, 7 g/L sodium acetate, pH 7, 0.2 medium-to-volume ratio (MVR), and 180 rpm.
Nutrient-limitation experiments revealed that nitrogen concentration exerted the strongest influence on growth, followed by oxygen availability, while phosphorus was optimal at intermediate levels. These results highlight the importance of balancing nutrient availability and oxygen transfer to maximize biomass formation and guide process development.
Experiments using acetate-containing mixtures derived from CO2 electroreduction (including ethanol, propanol, and formic acid) resulted in significantly reduced microbial growth compared to pure sodium acetate. Formic acid was identified as the primary inhibitory component responsible for reduced growth performance.
Following shake-flask optimization, scale-up to a 5 L bioreactor demonstrated stable and reproducible growth under controlled aeration, mixing, and pH conditions, confirming process robustness. FT-IR and 1H NMR analyses indicated approximately 57% similarity between the synthesized biopolymers and conventional PET, highlighting the potential of these materials as functional biobased alternatives.
Computational modeling of the PHA biosynthetic pathway included five enzymes: acetate kinase (AckA), phosphate acetyltransferase (Pta), beta-ketothiolase (PhaA), acetoacetyl-CoA transferase (PhaB), and polyhydroxyalkanoate synthase (PhaC). Density functional theory (DFT) calculations were conducted at the ωB97x-D/def2-SVP level within the QM/MM ONIOM framework, as implemented in the Gaussian software package. The results indicate that the rate-determining step involves cleavage of the C-H bond in the second acetyl-CoA molecule, concurrent protonation of Cys374, formation of a C-C bond between acetyl groups, and cleavage of the S-C bond of the acetyl enzyme.
Overall, this integrated experimental and computational approach demonstrates the feasibility of converting CO2-derived acetate into value-added biopolymers and provides a scalable framework for sustainable PHA production.
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