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

[摘要] ENGLISH ABSTRACT: Brettanomyces bruxellensis is a red wine spoilage yeast that plagues the wine industry. Itfacilitates the formation of compounds such as volatile phenols, which impart negative aromasin red wines. Its ability to survive in wine through adaptations to various stressors (e.g. highethanol 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 itssurvival mechanisms in general, as well as to detect and enumerate it accurately and toeliminate it. Nevertheless, literature on the subject is at certain times contradictory with regard tothe nutritional needs of this yeast. The survival of this yeast for extended periods of time lead usto question how it is so well adapted to this deficient environment. In other words, whatsubstrates does B. bruxellensis utilise to sustain growth or at least survival in such a nutrientdeficientmedium where stronger fermenters (e.g. Saccharomyces cerevisiae) cannot survive?This study investigates the carbon and nitrogen source assimilation of three B. bruxellensisstrains in a defined and model synthetic wine medium. In addition, the growth kinetics weredetermined (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 afocus on growth or cell maintenance (i.e. survival with no growth). The data showed that carbonconsumption followed a step wise pattern. At first, sugars were consumed, thereby leading tothe 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 thelatter. These compounds were consumed slowly and resulted in the survival of the cells for aperiod of 45 days. After this period, the consumption of ethanol allowed for the extendedfunctioning of the cell, however the assimilation of ethanol lead to an increasing degree ofimbalance (more and more NADH was produced with little to no conversion to NAD+) in the celland 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 potentialof the B. bruxellensis cell. The results displayed the partial regeneration of NAD+ during 4-EPformation, however, this metabolic pathway alone is not solely responsible.Unlike for carbon sources, the data showed that the assimilation of nitrogen compounds werestrain specific and certain strains required more nitrogen than the others. The sources of primeimportance were ammonia and arginine and were assimilated during the exponential growthphase (i.e. during sugar consumption). During the stationary phase, proline was assimilatedregardless of the presence/absence of oxygen, possibly to counteract stressors in the cell andensure survival of the population.This study contributes to an improved understanding of how B. bruxellensis survives in wine andis able to maintain cell function for extended periods of time. This leads to a betterunderstanding of the spoilage yeast B. bruxellensis and will allow for the production of wine inan integrated manner to avoid the proliferation of this microorganism.

[发布机构] Stellenbosch University