“Microbial Wars” in a Stirred Tank Bioreactor: Investigating the Co-Cultures of Streptomyces rimosus and Aspergillus terreus, Filamentous Microorganisms Equipped With a Rich Arsenal of Secondary Metabolites

Abstract

Microbial co-cultivation is an approach frequently used for the induction of secondary metabolic pathways and the discovery of novel molecules. The studies of this kind are typically focused on the chemical and ecological aspects of inter-species interactions rather than on the bioprocess characterization. In the present work, the co-cultivation of two textbook producers of secondary metabolites, namely Aspergillus terreus (a filamentous fungus used for the manufacturing of lovastatin, a cholesterol-lowering drug) and Streptomyces rimosus (an actinobacterial producer of an antibiotic oxytetracycline) in a 5.5-L stirred tank bioreactor was investigated in the context of metabolic production, utilization of carbon substrates and dissolved oxygen levels. The cultivation runs differed in terms of the applied co-culture initiation strategy and the composition of growth medium. All the experiments were performed in three bioreactors running in parallel (corresponding to a co-culture and two respective monoculture controls). The analysis based upon mass spectrometry and liquid chromatography revealed a broad spectrum of more than 40 secondary metabolites, including the molecules identified as the oxidized derivatives of rimocidin and milbemycin that were observed solely under the conditions of co-cultivation. S. rimosus showed a tendency to dominate over A. terreus, except for the runs where S. rimosus was inoculated into the already developed bioreactor cultures of A. terreus. Despite being dominated, the less aggressive strain still had an observable influence on the production of secondary metabolites and the utilization of substrates in co-culture. The monitoring of dissolved oxygen levels was evaluated as a fast approach of identifying the dominant microorganism during the co-cultivation process.

Quantitative Morphological Analysis of Filamentous Microorganisms in Cocultures and Monocultures: Aspergillus terreus and Streptomyces rimosus Warfare in Bioreactors

“…The experiments presented in this paper were described in terms of microbial morphology. Furthermore, the production of secondary metabolites and bioprocess kinetics was also tested in detail by Boruta et al [ 30 ], which allows for a wider discussion of the relationship between morphology and metabolism of the cocultivated filamentous microorganisms. In the experiments from ATSR2 to ATSR6, where the cocultures were initiated by the parallel introduction of precultures to the bioreactors (preculture versus preculture, Figure 1 , Figure 2 , Figure 3 and Figure 4 ), S. rimosus gained the morphological advantage over A. terreus , however its domination was not as strong as in the ATSR9 experiment.…”

“…This was demonstrated by a significant decrease in the A. terreus pellets’ projected area in cocultures (e.g., 24 h of ATSR2 A. terreus pellets’ projected area: coculture A = 1.3 × 10 6 µm 2 , monoculture A = 1.3 × 10 7 µm 2 , p < 0.001) while the sizes of the S. rimosus pellets remained the same in the mono- and co-cultures (e. g. 168 h of ATSR2 S. rimosus pellets’ projected area: coculture 1.5 × 10 5 µm 2 , monoculture A = 1.7 × 10 5 , p > 0.05). The total ion chromatograms presented by Boruta et al [ 30 ] showed that metabolic profiles of the broth composition of the cocultures and corresponding S. rimosus monocultures in ATSR2-ATSR6 were very similar. Particular attention should be paid to two S. rimosus metabolites, oxytetracycline and rimocidin, which show antibacterial and antifungal activity, respectively.…”

“…In the ATSR9 experiment, in which the coculture was initiated by the parallel introduction of A. terreus ans S. rimosus spores, the actinomycete was the dominant microorganism both morphologically ( Figure 6 ) and metabolically [ 30 ]. In this experiment, S. rimosus morphological domination was complete since it caused vanishing of the A. terreus pellets.…”

“…The first stage of the study included 5 experiments (from ATSR2 to ATSR6) performed with the use of the following coculture initiation strategy: 24 h shaking flask precultures of A. terreus and S. rimosus were introduced to the bioreactor in the same moment. The precultures’ preparation was previously described by Boruta et al [ 30 ]. Due to fact that the initial number of spores in the precultures should be equal to 10 9 per liter, spores were counted in a Thoma cell counting chamber and their number corrected, if required.…”

“…The method was based on artificial intelligence and could be used for maintaining cultivated microorganisms at the targeted levels. The issue of the metabolism of filamentous microorganisms in the stirred tank bioreactor cocultures was for the first time reported by Boruta et al [ 30 ]. They studied the production of secondary metabolites and bioprocess kinetics in A. terreus and S. rimosus bioreactor cocultures and respective parallel monocultures of these microorganisms.…”