Developing a metabolic model-based fed-batch feeding strategy for Pichia pastoris fermentation through fine-tuning of the methanol utilization pathway.

Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed-batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine-tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Muts ) growth during the induction phase for three fed-batch strategies, conducted at constant specific growth rates (µ-stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed-batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl2 ) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (Rmeoh/Δx ), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of Rmeoh/Δx compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L-1 to 928 mg L-1 ), while production yield improved by 85% (24.8 mg gDCW -1 to 46 mg gDCW -1 ).

Investigating the Evolution and Development of Biological Systems from the Perspective of Thermo-Kinetics and Systems Theory.

Life itself is grander than the sum of its constituent molecules. Any living organism may be regarded as a part of a dissipative process that connects irreversible energy consumption with growth, reproduction, and evolution. Under energy-fuelled, far-from-equilibrium conditions, chemical systems capable of exponential growth can manifest a specific form of stability- dynamic kinetic stability (DKS) - indicating the persistence of self-reproducible entities. This kinetic behavior is associated with thermodynamic conditions far from equilibrium leading to an evolutionary view of the origin of life in which increasing entities have to be associated with the dissipation of free energy. This review aims to reformulate Darwinian theory in physicochemical terms so that it can handle both animate and inanimate systems, thus helping to overcome this theoretical divide. The expanded formulation is based on the principle of dynamic kinetic stability and evidence from the emerging field of systems chemistry. Although the classic Darwinian theory is useful for understanding the origins and evolution of species, it is not meant to primarily build an explicit framework for predicting potential evolution routes. Throughout the last century, the inherently systemic and dynamic nature of the biological systems has been brought to the attention of researchers. During the last decades, "systems" approaches to biology and genome evolution are gaining ever greater significance providing the possibility of a deeper interpretation of the basic concepts of life. Further progress of this approach depends on crossing disciplinary boundaries and complex simulations of biological systems. Evolutionary systems biology (ESB) through the integration of methods from evolutionary biology and systems biology aims to the understanding of the fundamental principles of life as well as the prediction of biological systems evolution.

Performance assessment of gas-phase toluene removal in one- and two-liquid phase biotrickling filters using artificial neural networks.

The main aim of this work is to study gas-phase toluene removal in one- and two-liquid phase biotrickling filters (O/TLP-BTF) and model the BTF performance using artificial neural networks (ANNs). The TLP-BTF was operated for 60 d in the presence of silicone oil at empty bed residence times (EBRTs) of 120, 60, and 45 s, respectively, and toluene concentrations in the range of 0.9-3.1 g m-3. A t-test analysis indicated that increasing the silicone oil volume ratio from 5 to 10% v/v, did not significantly improve the TLP-BTF performance (p-value = 0.65 > 0.05). The results from ANN modeling showed that toluene removal was more negatively affected by the inlet concentration (casual index, CI = -5.63) due to the kinetic limitation. The CI values for inlet concentration (+4.01) and liquid trickling rate (-2.45) indicated that the diffusion-limited regime controlled the removal process in the OLP-BTF.