Immobilized yeast in continuous fermentation of beer: Dissertation

Continuous primary and secondary fermentation of beer with immobilized yeast was examined in laboratory scale and pilot scale packed bed reactors of cylindrical or tapered geometry. The purpose was to determine the basic prerequisites for rapid production of high quality beer in a continuous process. Secondary fermentation was the first objective of this work. The pilot scale secondary fermentation process consisted of three steps: Firstly, removal of yeast from green beer by centrifugation, secondly, decarboxylation of Ç-acetolactate by heat treatment and finally reduction of diacetyl in immobilized yeast bioreactors (50 dm3). DEAE-cellulose was used as the carrier material. The quality of the beer was as good as that of conventional products. The residence time was only 2.5 hours, compared with several weeks in conventional fermentation. The sensitivity of the secondary fermentation system to potential brewery contaminants was examined in laboratory scale (1.6 dm3). Lactobacillus brevis, Pediococcus damnosus and Enterobacterium agglomerans were incapable of contaminating the bioreactors at inoculation levels of 104 - 106 cells cm-3. Wild yeasts, for example Saccharomyces cerevisiae (ex. diastaticus) were, however, able to attach and grow in the reactors, but even they were incapable of producing significant amounts of off-flavours in beer. A study of the main fermentation was the second objective of this work. It was established that flavour formation could be controlled by choice of a suitable carrier material, moderate aeration and a two-stage process set-up. Porous glass was chosen for the carrier material in pilot scale (25 dm3), resulting in a stable fermentation and a well balanced formation of acetate esters. Furthermore, less clogging problems were encountered with porous glass than with DEAE- cellulose. The residence time in most experiments was 40 hours to reach a final (apparent) attenuation of 80 %, but even higher flow rates could be maintained. A genetically modified brewer's yeast with an alfa-ald (budA) gene from Klebsiella terrigena was used in the immobilized yeast system. The integrant strain is a producer of the enzyme alfa-acetolactate decarboxylase. Using this strain the formation of diacetyl was significantly reduced and the secondary fermentation could be considerably shortened or even omitted. No alterations in yeast metabolism were observed, except for a very slight decrease in yeast viability. Simultaneously with the fermentation experiments, electrochemical measurements of viable biomass were performed both for suspended and for immobilized yeast samples. A capacitance probe was used for this purpose. The probe was well suited for monitoring of both suspended and immobilized yeast. Conventional methods did not give satisfactory results for immobilized yeast, probably because of the presence of non-viable matter (in gravimetric methods) or because of incomplete desorption of yeast cells (in cell counting methods).