In the post-genomic era, increasing efforts have been made to describe the relationship between the genome and the phenotype in cells and organisms. It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype. Therefore, researchers have started to study the metabolome (or the metabolic complement of functional genomics). Within this context, mass spectrometry (MS) has increasingly occupied a central position in the methodologies developed for determination of the metabolic state. This review is mainly focused on the status of MS in the metabolome field, trying to direct the reader to the main approaches for analysis of metabolites, reviewing basic methodologies in sample preparation, and the most recent MS techniques introduced. Apart from the description of the different methods, this review will try to state a general comparison between the several different techniques that involve MS and metabolite analysis, and will highlight their limitations and preferred applicability.

Recent technical advances in mass spectrometry (MS) have propelled this technology to the forefront of methods employed in metabolome analysis. Here, we compare two distinct analytical approaches based on MS for their potential in revealing specific metabolic footprints of yeast single-deletion mutants. Filtered fermentation broth samples were analyzed by GC-MS and direct infusion ESI-MS. The potential of both methods in producing specific and, therefore, discriminant metabolite profiles was evaluated using samples from several yeast deletion mutants grown in batch-culture conditions with glucose as the carbon source. The mutants evaluated were cat8, gln3, ino2, opi1, and nil1, all with deletion of genes involved in nutrient sensing and regulation. From the analysis, we found that both methods can be used to classify mutants, but the classification depends on which metabolites are measured. Thus, the GC-MS method is good for classification of mutants with altered nitrogen regulation as it primarily measures amino acids, whereas this method cannot classify mutants involved in regulation of phospholipids metabolism as well as the direct infusion MS (DI-MS) method. From the analysis, we find that it is possible to discriminate the mutants in both the exponential and stationary growth phase, but the data from the exponential growth phase provide more physiological relevant information. Based on the data, we identified metabolites that are primarily involved in discrimination of the different mutants, and hereby providing a link between high-throughput metabolome analysis, strain classification, and physiology.

Alcohol fermentation productivity can be strongly improved using a flocculation-based yeast recycle. However, the efficiency of the biomass retention system depends strongly on the yeast particle size. Accordingly, the monitoring and control of yeast floc diameter are of primary importance. The on-line measurement of mean floc diameter has been achieved using on-line image analysis, based on the evaluation of image texture. The texture analysis method consisted in the building of a co-occurrence matrix from which the so-called "Energy parameter" was extracted. While image texture is usually used for classification purposes, it has been used here as a quantitative descriptor: a correlation has been found between this statistical image feature and off-line manual floc-size determinations. In the floc-size range investigated (X 0.5-4.3 mm), the evaluated mean diameter was in good agreement with the actual particle size, with a determination coefficient equal to 0.980. In contrast with manual measurements, slow and tedious, this method gave the value of the mean particle diameter in real-time, without sampling. This novel tool has been used to investigate the behavior of yeast aggregates as a function of fermentation conditions. While biomass concentration was kept constant, step increases of the feed rate led to a decrease of the mean floc diameter. Image analysis showed that the particle-size reduction could occur within a few minutes after modification of the medium dilution rate, demonstrating the disruptive effect of the CO(2) efflux. The kinetic of aggregate formation was dependent on the gas-phase composition. Instead of recycling fermentation gas, sparging the fermentor with nitrogen, to reduce dissolved CO(2) concentration, increased the rate of floc-size growth.

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