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“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.

SUBMITTER: Boruta T

PROVIDER: S-EPMC8511322 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCulturing cells as cell spheres results in a tissue-like environment that drives unique cell phenotypes, making it useful for generating cell populations intended for therapeutic use. Unfortunately, common methods that utilize static suspension culture have limited scalability, making commercialization of such cell therapies challenging. Our team is developing an allogeneic cell therapy for the treatment of lumbar disc degeneration comprised of discogenic cells, which are progenitor cells expanded from human nucleus pulposus cells that are grown in a sphere configuration.MethodsWe evaluate sphere production in Erlenmeyer, horizontal axis wheel, stirred tank bioreactor, and rocking bag format. We then explore the use of ramped agitation profiles and computational fluid dynamics to overcome obstacles related to cell settling and the undesired impact of mechanical forces on cell characteristics. Finally, we grow discogenic cells in stirred tank reactors (STRs) and test outcomes in vitro (potency via aggrecan production and identity) and in vivo (rabbit model of disc degeneration).ResultsComputation fluid dynamics were used to model hydrodynamic conditions in STR systems and develop statistically significant correlations to cell attributes including potency (measured by aggrecan production), cell doublings, cell settling, and sphere size. Subsequent model-based optimization and testing resulted in growth of cells with comparable attributes to the original static process, as measured using both in vitro and in vivo models. Maximum shear rate (1/s) was maintained between scales to demonstrate feasibility in a 50 L STR (200-fold scale-up).ConclusionsTransition of discogenic cell production from static culture to a stirred-tank bioreactor enables cell sphere production in a scalable format. This work shows significant progress towards establishing a large-scale bioprocess methodology for this novel cell therapy that can be used for other, similar cell therapies.

Project description:BackgroundIn Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption.ResultsAn automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P.?pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved.ConclusionThe RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.

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“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

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