Pieter J. Verbelen

Immobilized yeast cell systems for continuous fermentation applications

In several yeast-related industries, continuous fermentation systems offer important economical advantages in comparison with traditional systems. Fermentation rates are significantly improved, especially when continuous fermentation is combined with cell immobilization techniques to increase the yeast concentration in the fermentor. Hence the technique holds a great promise for the efficient production of fermented beverages, such as beer, wine and cider as well as bio-ethanol. However, there are some important pitfalls, and few industrial-scale continuous systems have been implemented. Here, we first review the various cell immobilization techniques and reactor setups. Then, the impact of immobilization on cell physiology and fermentation performance is discussed. In a last part, we focus on the practical use of continuous fermentation and cell immobilization systems for beer production.

Phenotypic diversity of Flo protein family-mediated adhesion in Saccharomyces cerevisiae

The Saccharomyces cerevisiae genome encodes a Flo (flocculin) adhesin family responsible for cell-cell and cell-surface adherence. In commonly used laboratory strains, these FLO genes are transcriptionally silent, because of a nonsense mutation in the transcriptional activator FLO8, concealing the potential phenotypic diversity of fungal adhesion. Here, we analyse the distinct adhesion characteristics conferred by each of the five FLO genes in the S288C strain and compare these phenotypes with a strain containing a functional copy of FLO8. Our results show that four FLO genes confer flocculation, but with divergent characteristics such as binding strength, carbohydrate recognition and floc size. Adhesion to agar surfaces, on the other hand, largely depended on two adhesins, Flo10 and Flo11. Expression of any FLO gene caused a significant increase in cell wall hydrophobicity. Nevertheless, the capacity to adhere to plastic surfaces, which is believed to depend on hydrophobic interactions, differed strongly between the adhesins. Restoring Flo8 yielded both flocculation and cell-surface adherence, such as invasive growth, a phenotype not observed when any of the single FLO genes was overexpressed. Taken together, this study reveals how S. cerevisiae carries a small reservoir of FLO genes that allows cells to display a wide variety of adhesive properties.

Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast

During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active substances, which are vital for the complex flavour of beer. In order to obtain insight into the influence of high-gravity brewing and fermentation temperature on flavour formation, we analysed flavour production and the expression level of ten genes (ADH1, BAP2, BAT1, BAT2, ILV5, ATF1, ATF2, IAH1, EHT1 and EEB1) during fermentation of a lager and an ale yeast. Higher initial wort gravity increased acetate ester production, while the influence of higher fermentation temperature on aroma compound production was rather limited. In addition, there is a good correlation between flavour production and the expression level of specific genes involved in the biosynthesis of aroma compounds. We conclude that yeasts with desired amounts of esters and higher alcohols, in accordance with specific consumer preferences, may be identified based on the expression level of flavour biosynthesis genes. Moreover, these results demonstrate that the initial wort density can determine the final concentration of important volatile aroma compounds, thereby allowing beneficial adaptation of the flavour of beer.

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.

The role of oxygen in yeast metabolism during high cell density brewery fermentations.

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.

Flocculation gene variability in industrial brewer’s yeast strains

The brewer’s yeast genome encodes a ‘Flo’ flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical ‘lager’ yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer’s yeasts.

The influence of yeast oxygenation prior to brewery fermentation on yeast metabolism and the oxidative stress response

Yeast preoxygenation can confer important advantages to brewery fermentations by means of omitting the need to oxygenate the wort. However, the impact of yeast preoxygenation on yeast metabolism has never been assessed systematically. Therefore, expression analysis was performed of genes that are of importance in oxygen-dependent pathways, oxidative stress response and general stress response during 8 h of preoxygenation. The gene expressions of both the important transcription factors Hap1 and Rox1, involved in oxygen sensing, were mainly increased in the first 3 h, while YAP1 expression, which is involved in the oxidative stress response, increased drastically only in the first 45 min. The results also show that stress-responsive genes (HSP12, SSA3, PAU5, SOD1, SOD2, CTA1 and CTT1) were induced during the process, together with the accumulation of trehalose. The accumulation of ergosterol and unsaturated fatty acids was accompanied by the expression of ERG1, ERG11 and OLE1. Genes involved in respiration (QCR9, COX15, CYC1 and CYC7) also increased during preoxygenation. Yeast viability did not decrease during the process, and the fermentation performance of the yeast reached a maximum after 5 h of preoxygenation. These results suggest that yeast cells acquire a stress response along the preoxygenation period, which makes them more resistant against the stressful conditions of the preoxygenation process and the subsequent fermentation.

Stability of high cell density brewery fermentations during serial repitching

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the decreased yeast net growth observed in these high cell density brewery fermentations can adversely affect the physiological stability throughout subsequent yeast generations. Therefore, different O2 conditions (wort aeration and yeast preoxygenation) were applied to high cell density fermentation and eight generations of fermentations were evaluated together with conventional fermentations. Freshly propagated high cell density populations adapted faster to the fermentative conditions than normal cell density populations. Preoxygenating the yeast was essential for the yeast physiological and beer flavor compound stability of high cell density fermentations during serial repitching. In contrast, the use of non-preoxygenated yeast resulted in inadequate growth which caused (1) insufficient yield of biomass to repitch all eight generations, (2) a 10% decrease in viability, (3) a moderate increase of yeast age, (4) and a dramatic increase of the unwanted flavor compounds acetaldehyde and total diacetyl during the sequence of fermentations. Therefore, to achieve sustainable high cell density fermentations throughout the economical valuable process of serial repitching, adequate yeast growth is essential.

Bioprocess Intensification of Beer Fermentation Using Immobilised Cells

Beer production with immobilised yeast has been the subject of research for approximately 30 years but has so far found limited application in the brewing industry, due to engineering problems, unrealised cost advantages, microbial contaminations and an unbalanced beer flavor (Linko et al. 1998; Branyik et al. 2005; Willaert and Nedovic 2006). The ultimate aim of this research is the production of beer of desired quality within 1–3 days. Traditional beer fermentation systems use freely suspended yeast cells to ferment wort in an unstirred batch reactor. The primary fermentation takes approximately 7 days with a subsequent secondary fermentation (maturation) of several weeks. A batch culture system employing immobilization could benefit from an increased rate of fermentation. However, it appears that in terms of increasing productivity, a continuous fermentation system with immobilization would be the best method (Verbelen et al. 2006). An important issue of the research area is whether beer can be produced by immobilised yeast in continuous culture with the same characteristic as the traditional method.