Austrian Centre of Industrial Biotechnology (Austria)

Organic fraction of municipal solid waste (OFMSW) is a valuable feedstock within a circular economy. At well-defined process conditions, enzymatic hydrolysis followed by microbial conversion of hydrolysis products can lead to valuable carboxylic acids. The present study assesses enzymatic hydrolysis of OFMSW at 15% dry matter and thermophilic conditions (55°C). Torque measurements from a four-vessel parallel bioreactor system were made to monitor changes in viscosity of the OFMSW slurry. Additionally, the influence of minimum pH setpoints at pH 5.0 and pH 6.0 on enzymatic hydrolysis as well as on the formation of organic acids was evaluated. Adding enzymes, which are tailored to the substrate, led to a rapid drop in torque at the beginning of the incubation. This reduction in viscosity by liquefaction of the OFMSW slurry can improve the processability of the slurry as well as reduce the energy consumption during stirring. It was possible to correlate the release of glucose as well as the changes in torque to the enzyme addition. A minimum pH setpoint of 6.0 appears to favour organic acid formation from indigenous microorganisms. This resulted in an 26.6% increase of lactic acid to 27.9 ± 0.49 g L -1 and a metabolisation into butyric acid of up to 15.4 ± 1.8 g L -1 . The presented results provide valuable information about the processing of OFMSW for future valorisation within biorefinery processes. • Enzyme addition led to rapid reduction in torque and viscosity. • Liquefaction and reduced disturbances during stirring were achieved. • Substantial concentrations of lactic acid and butyric acid were obtained.

Natural spider silk fibers are recognized for their outstanding mechanical properties. The production of underlying recombinant spider silk proteins in scalable quantities unlocks their potential for technical and biomedical applications. The recombinant spider silk technology further enables the introduction of new functions via genetic encoding. In the present study, the non-canonical amino acid L-azidohomoalanine is incorporated into the engineered Araneus diadematus fibroin 4, eADF4(C16), at positions explicitly defined by methionine codons in an N-terminal peptide tag. The azido-functionalized eADF4(C16) is processed into particles, films, or electrospun into nanofibers. As functional entities, high molecular weight molecules, such as antibodies, are modified with bio-orthogonal groups suitable for strain-promoted cycloaddition to the exposed azido-groups on the spider silk supports. The antibody-activated spider silk nonwoven meshes exemplarily show their applicability as bio-capturing membranes.

The performance of enzyme electrodes depends critically on the interactions between the enzyme and the electrode surface. In this study, L-lactate oxidase with an N-terminal His-tag (His-LOx) was immobilized on planar gold electrode modified with self-assembled monolayers (SAMs) engineered to provide distinct surface characteristics: hydrophilic, hydrophobic, or Ni²⁺-chelating for His-tag affinity binding. Electrochemical measurements revealed that the hydrophilic 1-thioglycerol (TG) SAM supported the highest initial activity (10.5 µA/cm²), whereas the hydrophobic 1-octanethiolled to a substantially lowered activity. Ni²⁺-complexed nitrilotriacetic acid (Ni-NTA) SAM markedly enhanced the operational stability of His-LOx, retaining over 65% of activity after 20 h compared to less than 20% for TG. A mixed SAM combining TG and Ni-NTA enabled the simultaneous achievement of high activity and improved stability, demonstrating a synergistic effect of hydrophilic and affinity-based interactions. These results provide mechanistic insight into how SAM surface properties and enzyme-surface interactions govern both activity and stability of LOx, offering practical design principles for the development of robust and sensitive LOx electrodes for biosensing applications.

Water-soluble polymers have exceptional properties and are therefore used in many applications. Many of these products end up down the drain; therefore biodegradability, especially in wastewater treatment plants, is essential to prevent accumulation in the environment. This study examined recombinant hydrolases PahZ1KT-1 and PahZ2KT-1 from Sphingomonas sp. KT-1 and PahZ1KP-2 from Pedobacter sp. KP-2 for their role in biodegrading water-soluble poly(aspartic acid) (tPAA). Analysis by a turbidity assay and Gel Permeation Chromatography (GPC) revealed highest activity of PahZ1KT-1 and PahZ1KP-2 at pH 8 and 40°C on tPAA, whereas PahZ2KT-1 showed no activity on the polymer but yet on its oligopeptides with highest values at pH 7 and 55°C. GPC analysis revealed that PahZ1KT-1 and PahZ1KP-2 hydrolyzed tPAA (MN>17,000Da) into oligopeptides (>500Da), while PahZ2KT-1 further degraded them to α-di(l-aspartic acid) and l-aspartic acid. Combined, these enzymes synergistically decomposed tPAA completely into l-aspartic acid within 24h. Supplementation of hydrolases into standardized biodegradation test improved biodegradation (54%) of tPAA after 28d. Supplementing PahZ1KT-1 or PahZ1KP-2 alone achieved 52% and 54% tPAA biodegradation, respectively. Preincubating tPAA with hydrolases boosted degradation to 71% in 28d. These findings highlight enzymes' crucial role in breaking down macromolecules into lower molecular weight species for effective tPAA biodegradation.

Butanol is currently produced on an industrial scale, primarily from fossil-based raw materials. An alternative method involves gas fermentation. To improve the efficiency of microbial processes, one promising approach is electrofermentation, which involves the application of an electric current to stimulate microbial growth or modulate metabolic pathways. This study examined the production of biobutanol from gas fermentation supported by electrofermentation, as assessed through a Life Cycle Assessment (LCA). The LCA was conducted for a biobutanol production technology developed within the framework of the BesTECH project, funded in the ERA-NET Bioenergy programme. Two environmental impact assessment methods were applied: ReCiPe 2016 and IPCC 2021 GWP 100 (with CO2 absorption considered). The results of the LCA indicated that the most significant environmental impact is associated with greenhouse gas emissions from fossil fuel combustion used to generate electricity (based on Austria’s energy mix). An additional environmental burden is related to the production of the fermentation medium. Sensitivity analysis revealed that the environmental performance of the process is strongly influenced by the source of electricity used in biobutanol production.

On a cellular level, proteinogenic amino acids (AAs) are the building blocks of proteins. On a global scale, AAs serve as important nutrients for humans and animals. Beyond their nutritional value, AAs are of relevance in medicine, and, due to their chemical properties, they are indispensable for applications in many different realms, such as pharmaceutics, cosmetics, animal feed, food, and the beverage industry. Here, we report the first archaeal cell factory for leucine production from CO2 that has been generated by rational design, random mutagenesis and pathway engineering. The cell factory has been bioprocess-technologically examined and successfully scaled-up with regard to productivity, product quality, and operational stability. In a 2-day fed-batch campaign, we produced 181 g of leucine from CO2 at 150 L pilot-plant scale at a mean volumetric leucine productivity of 65 mg L-1 h-1. A thorough techno-economic analysis indicates that the roll-out of leucine production from CO2 is nearly economically feasible on a global scale.

ABSTRACTThe development of virus‐like particle (VLP) production processes is often constrained by the extensive number of analytical methods required for their quantification and characterization, as well as the significant labor demands associated with these techniques. Asymmetrical flow field‐flow fractionation (AF4) coupled with in‐line detectors, such as ultraviolet (UV) and multi‐angle light scattering (MALS), presents a promising label‐free and rapid approach to simultaneously assess the quantity and quality of VLP samples. While AF4‐MALS has been widely applied for bionanoparticle characterization and quantification in final products and process development, the influence of host cell‐derived impurities on the outcome of the analysis remains underexplored.This study investigates the impact of host cell‐derived impurities, particularly host cell DNA and chromatin, on AF4‐MALS‐DLS analysis of both unpurified and purified VLP samples, using HIV‐1 gag VLPs produced in CHO cells as a model system. Our results demonstrate that DNA, chromatin, and VLPs can co‐elute due to their overlapping size distribution, which, if overlooked, may lead to imprecise determination of VLP concentrations in early process samples and inaccurate yield calculations at later stages. Nevertheless, for total particle quantification, AF4‐MALS was shown to be a suitable surrogate for nanoparticle tracking analysis, as the 90° light scattering peak area exhibited a strong linear correlation with total particle concentration. This substitution enables faster sample processing and reduces sample volume requirements.Additionally, our findings highlight the importance of particle concentration and method parameter selection, particularly the detector flow rate, when characterizing samples based on hydrodynamic radius (Rhyd). Underestimation of Rhyd due to high detector flow rates was proposed as the possible explanation for the higher‐than‐expected shape factors obtained for VLPs. These results emphasize the need for further optimization of AF4 methods to improve the separation of VLPs from host cell impurities and to ensure reliable characterization of bionanoparticles in complex mixtures.

Besides the simplicity of genetic modification and convenient low-effort cultivation, Pichia pastoris (today mostly Komagataella phaffii species are used for biotechnology) comes with another great advantage: simple targeting of recombinant proteins to different compartments of the host cell. Indeed, the most prominent pathway for recombinant proteins is the secretory pathway, whereby the protein is guided through the endoplasmic reticulum (ER) and Golgi apparatus, resulting in secretion at the cell surface. Secreted proteins can be separated from the cells, forgoing expensive cell disruption and purification processes. However, there are further localization sites for recombinant proteins (e.g., mitochondria, nucleus, peroxisomes, vacuole, cytoplasmic membrane, cell wall, etc.), technologically relevant.