Nutritional requirements and survival of the red wine spoilage yeast Brettanomyces bruxellensis

Abstract

Brettanomyces bruxellensis is a red wine spoilage yeast that plagues the wine industry. It facilitates the formation of compounds such as volatile phenols, which impart negative aromas in red wines. Its ability to survive in wine through adaptations to various stressors (e.g. high ethanol and sulphur dioxide concentrations, low pH) have made it the subject of several studies. These studies aim to understand its biology (and in particular its nutrient requirements) and its survival mechanisms in general, as well as to detect and enumerate it accurately and to eliminate it. Nevertheless, literature on the subject is at certain times contradictory with regard to the nutritional needs of this yeast. The survival of this yeast for extended periods of time lead us to question how it is so well adapted to this deficient environment. In other words, what substrates does B. bruxellensis utilise to sustain growth or at least survival in such a nutrientdeficient medium where stronger fermenters (e.g. Saccharomyces cerevisiae) cannot survive? This study investigates the carbon and nitrogen source assimilation of three B. bruxellensis strains in a defined and model synthetic wine medium. In addition, the growth kinetics were determined (as well as the consumption pattern of the various carbon and nitrogen sources). This was performed in conditions similar to bottle ageing (anaerobic) and barrel maturation (semi-anaerobic). Furthermore, the purpose of assimilating these sources is explored with a focus on growth or cell maintenance (i.e. survival with no growth). The data showed that carbon consumption followed a step wise pattern. At first, sugars were consumed, thereby leading to the production of ethanol and biomass concurrently. Upon complete consumption of the sugars, malic acid was consumed together with ethanol, but only when oxygen was present for the latter. These compounds were consumed slowly and resulted in the survival of the cells for a period of 45 days. After this period, the consumption of ethanol allowed for the extended functioning of the cell, however the assimilation of ethanol lead to an increasing degree of imbalance (more and more NADH was produced with little to no conversion to NAD+) in the cell and eventually lead to a slow but steady decline of the population. The formation of 4- Ethylphenol (4-EP) was investigated in order to ascertain its ability to correct the redox potential of the B. bruxellensis cell. The results displayed the partial regeneration of NAD+ during 4-EP formation, however, this metabolic pathway alone is not solely responsible. Unlike for carbon sources, the data showed that the assimilation of nitrogen compounds were strain specific and certain strains required more nitrogen than the others. The sources of prime importance were ammonia and arginine and were assimilated during the exponential growth phase (i.e. during sugar consumption). During the stationary phase, proline was assimilated regardless of the presence/absence of oxygen, possibly to counteract stressors in the cell and ensure survival of the population. This study contributes to an improved understanding of how B. bruxellensis survives in wine and is able to maintain cell function for extended periods of time. This leads to a better understanding of the spoilage yeast B. bruxellensis and will allow for the production of wine in an integrated manner to avoid the proliferation of this microorganism.National Research Foundatio