The pH-dependent lactose metabolism of Lactobacillus delbrueckii subsp. bulgaricus: An integrative view through a mechanistic computational model.

The fermentation process of milk to yoghurt using Lactobacillus delbrueckii subsp. bulgaricus in co-culture with Streptococcus thermophilus is hallmarked by the breakdown of lactose to organic acids such as lactate. This leads to a substantial decrease in pH - both in the medium, as well as cytosolic. The latter impairs metabolic activities due to the pH-dependence of enzymes, which compromises microbial growth. To quantitatively elucidate the impact of the acidification on metabolism of L. bulgaricus in an integrated way, we have developed a proton-dependent computational model of lactose metabolism and casein degradation based on experimental data. The model accounts for the influence of pH on enzyme activities as well as cellular growth and proliferation of the bacterial population. We used a machine learning approach to quantify the cell volume throughout fermentation. Simulation results show a decrease in metabolic flux with acidification of the cytosol. Additionally, the validated model predicts a similar metabolic behaviour within a wide range of non-limiting substrate concentrations. This computational model provides a deeper understanding of the intricate relationships between metabolic activity and acidification and paves the way for further optimization of yoghurt production under industrial settings.

A Two-Compartment Fermentation System to Quantify Strain-Specific Interactions in Microbial Co-Cultures.

To fulfil the growing interest in investigating microbial interactions in co-cultures, a novel two-compartment bioreactor system was developed, characterised, and implemented. The system allowed for the exchange of amino acids and peptides via a polyethersulfone membrane that retained biomass. Further system characterisation revealed a Bodenstein number of 18, which hints at backmixing. Together with other physical settings, the existence of unwanted inner-compartment substrate gradients could be ruled out. Furthermore, the study of Damkoehler numbers indicated that a proper metabolite supply between compartments was enabled. Implementing the two-compartment system (2cs) for growing Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus, which are microorganisms commonly used in yogurt starter cultures, revealed only a small variance between the one-compartment and two-compartment approaches. The 2cs enabled the quantification of the strain-specific production and consumption rates of amino acids in an interacting S. thermophilus-L. bulgaricus co-culture. Therefore, comparisons between mono- and co-culture performance could be achieved. Both species produce and release amino acids. Only alanine was produced de novo from glucose through potential transaminase activity by L. bulgaricus and consumed by S. thermophilus. Arginine availability in peptides was limited to S. thermophilus' growth, indicating active biosynthesis and dependency on the proteolytic activity of L. bulgaricus. The application of the 2cs not only opens the door for the quantification of exchange fluxes between microbes but also enables continuous production modes, for example, for targeted evolution studies.

Differential Amino Acid Uptake and Depletion in Mono-Cultures and Co-Cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in a Novel Semi-Synthetic Medium.

The mechanistic understanding of the physiology and interactions of microorganisms in starter cultures is critical for the targeted improvement of fermented milk products, such as yogurt, which is produced by Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus. However, the use of complex growth media or milk is a major challenge for quantifying metabolite production, consumption, and exchange in co-cultures. This study developed a synthetic medium that enables the establishment of defined culturing conditions and the application of flow cytometry for measuring species-specific biomass values. Time courses of amino acid concentrations in mono-cultures and co-cultures of L. bulgaricus ATCC BAA-365 with the proteinase-deficient S. thermophilus LMG 18311 and with a proteinase-positive S. thermophilus strain were determined. The analysis revealed that amino acid release rates in co-culture were not equivalent to the sum of amino acid release rates in mono-cultures. Data-driven and pH-dependent amino acid release models were developed and applied for comparison. Histidine displayed higher concentrations in co-cultures, whereas isoleucine and arginine were depleted. Amino acid measurements in co-cultures also confirmed that some amino acids, such as lysine, are produced and then consumed, thus being suitable candidates to investigate the inter-species interactions in the co-culture and contribute to the required knowledge for targeted shaping of yogurt qualities.

Organ-on-a-disc: A platform technology for the centrifugal generation and culture of microphysiological 3D cell constructs amenable for automation and parallelization.

Organ-on-a-chip (OoC) systems have evolved to a promising alternative to animal testing and traditional cell assays in drug development and enable personalization for precision medicine. So far, most OoCs do not fully exploit the potential of microfluidic systems regarding parallelization and automation. To date, many OoCs still consist of individual units, integrating only one single tissue per chip, and rely on manual, error-prone handling. However, with limited parallelization and automation, OoCs remain a low-throughput technology, preventing their widespread application in industry. To advance the concept of microphysiological systems and to overcome the limitations of current OoCs, we developed the Organ-on-a-disc (Organ-Disc) technology. Driven only by rotation, Organ-Discs enable the parallelized generation and culture of multiple 3D cell constructs per disc. We fabricated polydimethylsiloxane-free Organ-Discs using thermoplastic materials and scalable fabrication techniques. Utilizing precisely controllable centrifugal forces, cells were loaded simultaneously into 20 tissue chambers, where they formed uniform cell pellets. Subsequently, the cells compacted into dense 3D cell constructs and were cultured under vasculature-like perfusion through pump- and tubing-free, centrifugal pumping, solely requiring a low-speed rotation (<1 g) of the Organ-Disc. Here, we provide a proof-of-concept of the Organ-Disc technology, showing the parallelized generation of tissue-like cell constructs and demonstrating the controlled centrifugal perfusion. Furthermore, Organ-Discs enable versatile tissue engineering, generating cell constructs with a customizable shape and a layered multi-cell type structure. Overall, the Organ-Disc provides a user-friendly platform technology for the parallelization and automation of microphysiological systems, bringing this technology one-step closer to high-throughput applications in industry.