Submerged vinegar fermentation in small scale culture systems

• Submerse Essigfermentation in Kleinkultursystemen

Schlepütz, Tino; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2013)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2013

Abstract

In industry, vinegar is widely produced by submerged fermentation. Although vinegar production is established on the large scale, small scale culture methods, in which bioprocesses are typically developed or optimized nowadays, are missing. To reinvestigate the established fermentation process, the aim of this thesis was the development of suitable small scale culture methods and systems for submerged vinegar fermentation. Since obligatory aerobic acetic acid bacteria in vinegar production suffer even from short oxygen depletion during traditional precultivation steps, the reproducibility of results in the main culture is insufficient. A reproducible small scale cultivation method for obligatory aerobic acetic acid bacteria at industrially relevant high ethanol and acetic acid concentrations was established by ensuring constant oxygen transfer in the whole inoculation procedure. An acetic acid bacteria preculture was drained off from a laboratory-scale bioreactor into an aerated mobile bubble column and transferred to an already shaking RAMOS shake flask device. Whereas the respiration curves of the traditionally processed acetic acid bacteria cultures were low and greatly diverged, those of the preculture transferred first into the bubble column and then into the already shaking flasks were high and coincided with one another. Shutting off aeration in the mobile bubble column led to a rapid decrease in bacterial activity. In conclusion, traditional precultivation steps are not suitable for obligatory aerobic acetic acid bacteria in vinegar fermentation. Maintaining constant oxygen transfer is necessary to guarantee the reproducibility of main culture experiments with such bacteria. In industry, vinegar is produced in repeated batch or repeated fed-batch processes. As yet, there was no small scale culture system for repeated batch bioprocesses. Therefore, a new shaken culture system for parallel repeated batch vinegar fermentation was explored. A new operation mode - the flushing repeated batch - was developed. Parallel repeated batch vinegar fermentation could be established in shaken overflow vessels in an automated operation with only one pump per reactor. This flushing repeated batch was theoretically investigated and empirically tested. The ethanol concentration was monitored during repeated batch fermentation by semiconductor gas sensors. The switch from one ethanol substrate lot to different ethanol substrate lots resulted in prolonged lag phases and durations of the first batches. In the subsequent batches the length of the fermentations decreased considerably. This decrease in the respective lag phases indicates an adaptation of the acetic acid bacteria mixed culture to the specific ethanol substrate lot. Consequently, flushing repeated batch fermentations on small scale are valuable for screening fermentation conditions and, thereby, improving bioprocesses in terms of robustness, stability and productivity. To improve the shaken repeated batch system, the system was expanded with electrochemical oxygen sensors. The measurement of the oxygen partial pressure in the headspace of the shaken reactors allowed the compensation of the oxygen partial pressure dependency of the semiconductor ethanol gas sensors. Furthermore, the oxygen transfer rate in the repeated batch fermentation system could be determined during cultivation. The oxygen transfer rate was with 28 mmol/L/h in the same range as in batch cultivations in the RAMOS device. To calibrate the oxygen sensors online during cultivation, a method applying gas flow change was theoretically considered and practically implemented. After the vinegar fermentation was accomplished in milliliter shake flask scale in batch as well as repeated batch mode, the vinegar fermentation was scaled-down to microliter scale in microtiter plates of this thesis. In order to minimize evaporation losses of ethanol and acetic acid in a 48-well microtiter plate during vinegar fermentation a new custom-made lid was developed. A diffusion model was used to calculate the dimensions of a hole in the lid to guarantee a suitable oxygen supply and level of ventilation. A reference fermentation was conducted in a 9 L-bioreactor to enable the calculation of the proper cultivation conditions in the microtiter plate. The minimum dissolved oxygen tensions in the microtiter plate were between 7.5% and 23% of air saturation and in the same range as in the 9 L-bioreactor. Evaporation losses of ethanol and acetic acid were less than 5% after 47 h and considerably reduced compared to those of microtiter plate fermentations with a conventional gas-permeable seal. Furthermore, cultivation times in the microtiter plate were with about 40 h as long as in the 9 L-bioreactor. In conclusion, microtiter plate cultivations with the new custom-made lid provide a platform for high-throughput studies on vinegar fermentation. Results are comparable to those in the 9 L-bioreactor.