RESUMEN
The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.
RESUMEN
Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%-90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.
RESUMEN
Bacillus spp. endospores are important dormant cell forms and are distributed widely in environmental samples. While these endospores can have important industrial value (e.g. use in animal feed as probiotics), they can also be pathogenic for humans and animals, emphasizing the need for effective endospore detection. Standard spore detection by colony forming units (CFU) is time-consuming, elaborate and prone to error. Manual spore detection by spore count in cell counting chambers via phase-contrast microscopy is less time-consuming. However, it requires a trained person to conduct. Thus, the development of a facilitated spore detection tool is necessary. This work presents two alternative quantification methods: first, a colorimetric assay for detecting the biomarker dipicolinic acid (DPA) adapted to modern needs and applied for Bacillus spp. and second, a model-based automated spore detection algorithm for spore count in phase-contrast microscopic pictures. This automated spore count tool advances manual spore detection in cell counting chambers, and does not require human overview after sample preparation. In conclusion, this developed model detected various Bacillus spp. endospores with a correctness of 85-89%, and allows an automation and time-saving of Bacillus endospore detection. In the laboratory routine, endospore detection and counting was achieved within 5-10 min, compared to up to 48 h with conventional methods. The DPA-assay on the other hand enabled very accurate spore detection by simple colorimetric measurement and can thus be applied as a reference method.