Application of dielectric spectroscopy to unravel the physiological state of microorganisms: current state, prospects and limits.

Microbial physiology is an essential characteristic to be considered in the research and industrial use of microorganisms. Conventionally, the study of microbial physiology has been limited to carrying out qualitative and quantitative analysis of the role of individual components in global cell behaviour at a specific time and under certain growth conditions. In this framework, groups of observable cell physiological variables that remain over time define the physiological states. Recently, with advances in omics techniques, it has been possible to demonstrate that microbial physiology is a dynamic process and that, even with low variations in environmental culture conditions, physiological changes in the cell are provoked. However, the changes cannot be detected at a macroscopic level, and it is not possible to observe these changes in real time. As an alternative to solve this inconvenience, dielectric spectroscopy has been used as a complementary technique to monitor on-line cell physiology variations to avoid long waiting times during measurements. In this review, we discuss the state-of-the-art application of dielectric spectroscopy to unravel the physiological state of microorganisms, its current state, prospects and limitations during fermentation processes. Key points • Summary of the state of the art of several issues of dielectric spectroscopy. • Discussion of correlation among dielectric properties and cell physiological states. • View of the potential use of dielectric spectroscopy in monitoring bioprocesses.

Dielectric property measurements as a method to determine the physiological state of Kluyveromyces marxianus and Saccharomyces cerevisiae stressed with furan aldehydes.

Cell physiology parameters are essential aspects of biological processes; however, they are difficult to determine on-line. Dielectric spectroscopy allows the on-line estimation of viable cells and can provide important information about cell physiology during culture. In this study, we investigated the dielectric property variations in Kluyveromyces marxianus SLP1 and Saccharomyces cerevisiae ERD yeasts stressed by 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde during aerobic growth. The dielectric properties of cell permittivity, specific membrane capacitance (Cm), and intracellular conductivity (σIn) were considerably affected by furan aldehydes in the same way that the cell population, viability, cell size, substrate consumption, organic acid production, and respiratory parameters were. The yeasts stressed with furan aldehydes exhibited three physiological states (φ): adaptation, replicating, and nonreplicating states. During the adaptation state, there were small and stable signs of permittivity, Cm, and σIn; additionally, no cell growth was observed. During the replicating state, cell growth was restored, and the cell viability increased; in addition, the permittivity and σIn increased rapidly and reached their maximum values, while the Cm decreased. In the nonreplicating state, the permittivity and σIn were stable, and Cm decreased to its minimum value. Our results demonstrated that knowing dielectric properties allowed us to obtain information about the physiological state of the cells under control and stressed conditions. Since the permittivity, Cm, and σIn are directly associated with the physiological state of the yeast, these results should contribute to a better understanding of the stress response of yeasts and open the possibility to on-line monitor and control the physiological state of the cell in the near future.

Fermentative capabilities of native yeast strains grown on juices from different Agave species used for tequila and mezcal production.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48-52 g L-1) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1-2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. Graphical abstract.

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation.

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.