Endophytes as source of polypeptide and polyketide antibiotics fengycin and bacillaene
Endophytic bacteria, which populate plant tissues, can influence physiological processes in plants. They also have antagonistic effects against many phytopathogens. Utilization of these bacteria as a biological agent for protection of plants is an environmentally friendly alternative to chemical methods. Moreover, since plants belong to the eukaryotic organisms, antibiotics produced by endophytes may have reduced cell toxicity. Therefore, the antimicrobial compounds isolated from endophytes have a big potential for the pharmaceutical industry and agriculture. Potato plants were chosen as source of endophytes due to their ability to protect themselves from a broad spectrum of phytopathogens. Several bacterial strains having antifungal activities were isolated from potato tubers. One strain, which exhibited the strongest fungicide potential, was characterized and identified as Bacillus subtilis subsp. subtilis. It was deposited under the number DSM 21393 at the public collection of the DSMZ GmbH (Germany). The substances exhibiting antifungal activities were purified and after structure elucidation identified as the lipopeptide antibiotic fengycin. Due to its very strong fungicide as well as bactericide and anti-cancer properties this antibiotic has a good potential for applications in the pharmaceutical industry and agriculture. Furthermore, it exhibits strong surface active properties, which makes it of great interest as biosurfactant. Despite the great potential of lipopeptides, the industrial use of these antibiotics is still limited due to their high production costs, which result primarily from low strain productivity and difficulties in bioprocess design as well as downstream processing. Therefore, a further aim of this project was the development of a cost-effective production process for fengycin. Different complex substrates such as malt-, potato extracts and molasses as well as their mixtures were used as culture media. The maximum fengycin yield of 325mg/L was obtained in the optimized medium containing malt and potato extract. As the preparation of potato extract for a larger cultivation scale is not practicable, a side-product of starch production - potato fruit juice (PFJ) was used as an alternative. A fengycin yield up to 550mg/L could be achieved by applying this medium. The utilization of the medium containing malt extract and PFJ led not only to an enhanced fengycin yield but also reduced the production costs of fengycins. Moreover, during the purification experiments it was seen, that another antibiotic family called bacillaene was co-synthesized at high concentrations (up to 400mgL−1). This antibiotic having a broad spectrum of antibacterial activities belongs to the polyketide antibiotics. Other cultivation parameters such as temperature and inoculum quantity were shown to have influence on the cultivation process and fengycin yield. Moreover, it was shown, that the aeration strategy during cultivation was a crucial parameter influencing the production of both antibiotics fengycin and bacillaene. Thereby, a high aeration rate was favorable for bacillaene synthesis whereas the limited aeration rate induced and even could enhance fengycin synthesis rate. Moreover, process selectivity toward fengycin production could be increased from 37% up to 82% by decreasing the aeration rate. A key bottleneck for the application of lipopeptides on an industrial scale is the process development under extreme foam formation. In this project an in situ product removal (ISPR) strategy based on a foam collection was developed. The specialty of this system was the combination of foam fractionation with an adsorption-desorption process on polystyrene resins, which is more efficient than other commonly used ISPR techniques. Application of this ISPR technique not only allowed for a stable and efficient cultivation of B.subtilis DSM 21393 under intensive foaming conditions without the need to add anti-foam agents but also led to an increase in fengycin yield up to 1g/L. Using the developed ISPR strategy bacillaene yield could be also enhanced up to 550mg/L. Due to their low concentration, downstream processing of lipopeptides is relatively expensive. A novel cost-effective method based on a removal and separation of biosurfactants from the culture broth by compressing and harvesting of the liquid surface layer using a new device called Flounder was investigated. The Flounder can be efficiently applied for the separation and concentration of fengycin. The fengycin concentration in the collected fractions was found to be four times higher than in the culture centrifugate. A separation of fengycin from the less surface-active bacillaene could be achieved due to a stronger surface activity of fengycin. The process design for fengycin production from strain isolation via integration of an ISPR technique and production in 60L scale up to downstream of the target product was shown in this project. Thereby, the crucial production parameters for fengycin were determined. The proposed bioprocess development strategy could be easily adapted for the production of other lipopetides/biosurfactants.
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