VLB Research: Production of acetic acid bacteria as starter cultures

 KOMBUCHA & CO.

Production of acetic acid bacteria as starter cultures: influence of yeast extract on biomass

Maximilian Schmacht, Dr.-Ing. Martin Senz, VLB Berlin, Research Institute for Biotechnology and Water, Department Bioprocess Engineering and Applied Microbiology

Fermented low or non-alcoholic beverages such as Kombucha or water kefir enjoy increasing popularity against the background of an increasing awareness of a healthy and conscious lifestyle. The traditional production methods applying undefined microbial consortia, however, are contrasting the need for reproducible and scalable industrial production processes. The VLB Research Institute for Biotechnology and Water (FIBW) serves its customers with appropriate starter cultures.

Traditionally fermented beverages, such as Kombucha, water kefir or kvass are getting increasing attention by costumers interested in natural beverages that potentially aid in fostering a healthy lifestyle. The traditional production methods, however, rely on the usage of non-defined microbial communities. Thus, a reproducible production of products showing always the same characteristics is hard to achieve. A solution to this issue is the usage of defined starter cultures resulting in a more controllable fermentation process. The Research Institute for Biotechnology and Water has served its customers with starter cultures for a wide variety of beverages for decades and is also currently active in expanding the range of offerings. In the case of Kombucha, the socalled SCOBY (symbiotic culture of bacteria and yeast) is deployed for the fermentation of sugared tea to an acetic refreshing drink. It mainly consists of acetic acid bacteria (e.g. Acetobacter, Komagataeibacter) and yeast (e.g. Brettanomyces, Zygosaccharomyces). In order to achieve high biomasses, the fermentation media need to be adapted to the requirements of the respective strains. In general, the usage of complex media is usually the most efficient way for high biomass production as they provide a broad range of nutrients. However, the selection of specific components, such as yeast extract, can have a high impact on the final

Product

Tab. 1: Different yeast derivatives used for fermentation studies with K. hansenii Ko-0201

Product Feature

Reference yeast extract Commonly used in microbiology laboratories

X‐SEED® KAT(Ohly GmbH, Germany) Yeast extract rich in free amino acids

X‐SEED® Nucleo Advanced (Ohly GmbH, Germany) Yeast extract rich in free ribonucleotides

X‐SEED® Nucleo Max (Ohly GmbH, Germany) Yeast extract with highest content of free ribonucleotides

result. Herein, a study on the influence of different yeast derivatives on the growth of Komagataeibacter hansenii was conducted using the miniaturized fermentation platform BioLector® Pro (m2plabs GmbH). This state-of-the-art fermentation unit was funded by the Federal Ministry for Economic Affairs and Energy (INNO-KOM module IZ: IZ150029) and allows high-throughput screening of different experimental conditions in batch as well as fed-batch mode in microliter scale.

Batch screening experiments without pH control

As a first step, fermentation media containing 50 g/L glucose and 10 g/L of different yeast derivatives (see Table 1) were used for biomass growth of Komagataeibacter hansenii Ko-0201 (VLB strain collection). The fermentations in the different media proceeded equally in the first stage. Within the phase of exponential growth, however, XSEED® KAT allowed a significantly improved growth compared to all other used yeast derivatives by achieving 25.37 AU, which corresponded to 5.52∙109 cells/mL (Figure 1). The usage of X-SEED Peptone resulted approximately in the same final biomass, yet taking additional 10 h. Concluding, it can be assumed that K. hansenii

Ko-0201 requires free amino acids for growth rather than ribonucleotides. The provision of these amino acids in the form of peptides necessitates their enzymatic cleavage resulting in a delayed growth compared to direct provision of free amino acids.

Batch screening experiments at controlled pH

Following up these results, experiments were conducted investigating the influence of pH on the further potential to improve the biomass yield. Therefore, fermentations applying media including X-SEED KAT as best performing yeast extract and pH control to 5.5 as well as 4.5 were performed using the microfluidic BioLector microtiter plates. The highest final biomass was achieved using X-SEED KAT at pH 4.5 with 34.55 AU corresponding to 7.20 ∙ 109 cells/mL (n=2), which was slightly higher compared to pH 5.5 (28.24 AU; 6.46 ∙ 109 cells/mL (n=3)) (Figure 2). Correspondingly, the production and reconsumption of gluconic acid was a bit faster at pH 4.5. However, this trend was not statistically significant (student’s t-test > 0.05). Anyway, the control of the pH value allowed for a higher biomass than seen without pH control suggesting that the enzymes and the overall cell metabolism is enhanced at slightly acidic conditions in contrast to lower pH values < 3.0. By means of online biomass measurement, the harvesting time can directly be determined and compared between different experimental conditions leading to an economically viable starter culture production.

Summary

The choice of the right cultivation medium providing all essential nutrients for effective biomass

Figures: VLB Berlin

Fig. 1: Biomass progress of K. hansenii Ko-0201 in media containing different yeast derivatives. Experiments were performed in biological duplicates (displayed are mean values ± mean deviation)

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production is crucial for industrially feasible starter culture manufacturing. Five different yeast derivatives were tested for their influence on the growth of Komagataeibacter hansenii Ko-0201 and it was revealed that the provision of free amino acids by X-SEED KAT was the most advantageous way of achieving high biomasses. This effect was even more pronounced at controlled pH levels. The screening approach in the miniaturized fermentation platform BioLector Pro provided essential data for all the experimental conditions in a short amount of time.

Photos: ew

Contact: Dr.-Ing. Martin Senz Head of Research Institute for Biotechnology and Water m.senz@vlb-berlin.org

Contact: Maximilian Schmacht Research Institute for Biotechnology and Water m.schmacht@vlb-berlin.org Fig. 2: Cultivation of K. hansenii Ko-0201 in medium containing X-SEED® KAT yeast extract controlled to pH 4.5 and pH 5.5, respectively. Due to sampling, the number of biological replicates decreases over time. However, values represent a minimum of three biological replicates for pH 5.5 and at least duplicates for pH 4.5

Acknowledgement

The authors would like to express their sincere gratitude for the good cooperation with Anna Korona (m2p-labs GmbH) and Dr. Abhishek Somani (Ohly GmbH).

The state-of-the-art fermentation unit BioLector® Pro was funded by the Federal Ministry for Economic Affairs and Energy, INNO-KOM module IZ: IZ150029