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

Abstract

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.

Keywords: Lactobacillus bulgaricus; Streptococcus thermophilus; bioprocess engineering; lactic acid bacteria; metabolomics; microbial consortia.

Figure 1

(left) Image of the membrane unit. The inlet and outlet of the channel were connected to vessels or tubes to allow the circulation of cells. Two polycarbonate elements were used to clamp a semi-permeable membrane that was aligned and fixed. (right) Mass balance of a compartment with inflow (feed), outflow (harvest), and diffusion flows in the membrane unit. The mixing pump allowed the circulation of the cultivation broth within the compartment.

Figure 2

(A) Diagram of a vessel bioreactor system. The vessels were connected to the membrane unit, and circulation of medium in each compartment was achieved by mixing pumps. (B) Diagram of a tube bioreactor system. The inlets and outlets of the membrane unit were connected by tubes, and circulation of medium in each compartment was achieved by mixing pumps. Additionally, attached tubes for feeds and harvests allowed sampling and continuous cultivation by using feed pumps for each compartment. (C) Technical parameters and results of co-cultivations in respective two-compartment systems (2cs) with Lactobacillus delbrueckii subs. bulgaricus in synthetic medium (SM) containing casein and lactose and Streptococcus thermophilus in SM containing lactose. Strains were cultivated in co-culture in the 2cs, enabling exchange of metabolites, and strain-specific growth rates were determined from biomass measurements (Figures S4 and S5 in Supplementary Materials). V, volume.

Figure 3

Amino acid profiles in the compartment containing Streptococcus thermophilus during co-cultivation with Lactobacillus delbrueckii subs. bulgaricus in the tube bioreactor system. (rhomb) Extracellular amino acid concentrations (µM) in the compartment containing S. thermophilus during the continuous mode. (bars) Extracellular peptide-bound amino acid concentrations (µM) in the compartment containing S. thermophilus during the continuous mode. S. thermophilus was cultivated in co-culture with L. bulgaricus in the tube bioreactor system containing synthetic medium (SM) with casein and glucose in the L. bulgaricus compartment and SM with glucose in the S. thermophilus compartment. * Profile data for these peptide-bound amino acids not measured.

Figure 4

Metabolic productivity of Lactobacillus delbrueckii subs. bulgaricus (rhomb) and Streptococcus thermophilus (triangle) cultivated in the tube bioreactor system as a co-culture. Positive values indicate the release or production of amino acids; negative values indicate the uptake of amino acids. Strains were cultivated in a tube bioreactor system containing synthetic medium (SM) with casein and glucose in the L. bulgaricus compartment and SM with glucose in the S. thermophilus compartment. Amino acids were sorted in rows according to the mol-fraction in casein, except tyrosine, proline, tryptophan, and methionine.

Figure 5

Biomass-specific activity of Streptococcus thermophilus. Amino acid production or consumption rates of S. thermophilus bridging amino acid productivity in mono-culture and co-culture. (Filled) S. thermophilus grown in co-culture with Lactobacillus delbrueckii subs. bulgaricus. Strains were cultivated in a tube bioreactor system containing synthetic medium (SM) with casein and glucose in the L. bulgaricus compartment and SM with glucose in the S. thermophilus compartment. (Non-filled) S. thermophilus grown in a crimp-top serum bottle containing SM with amino acids and lactose (modified from [62]). Amino acids were sorted in rows according to mol-fraction in casein, except tyrosine, proline, tryptophan, and methionine.

Figure 6

Alanine production and consumption of Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus cultivated in the tube bioreactor system. (A) Illustration of alanine pools in the tube bioreactor system. r₁ and r₃ are the production and consumption rates of non-labelled alanine; r₂ and r₄ are the production and consumption rates of 13-C alanine; r_diff is the diffusion rate of alanine in the membrane unit according to concentration differences; and D is the dilution rate in compartment 1 or compartment 2. (B) Compartment 1 was filled with L. bulgaricus and synthetic medium (SM) with casein and 13-C glucose. Compartment 2 was filled with S. thermophilus and SM with 13-C glucose. Concentrations of non-labelled (triangle) and 13-C alanine (circle) were measured via LC-MS. Strain-specific rates were calculated by balancing each compartment. Positive rates: production; negative rates: consumption.

Figure 7

Illustration of terms to estimate the Damkoehler number (Da) during the continuous mode. Trans-membrane transport provided amino acids; Streptococcus thermophilus or Lactobacillus delbrueckii subs. bulgaricus consumed amino acids; and the continuous mode provoked amino acid washout. The initial concentration for some amino acids was above zero at the start of the continuous mode.

Figure 8

Damkoehler numbers (Da_I) of individual amino acids. (A) Da_total = Da_dilution + Da_consumption in the compartment containing Streptococcus thermophilus. (B) Da_dilution + Da_consumption separated in the compartment containing S. thermophilus. Strains were cultivated in the tube bioreactor system, and Da was calculated for each hour of continuous cultivation. Da numbers were only calculated if amino acid uptake was present within 1 h. The red line indicates Da = 1.