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.

Breve Descripción de la Biología Sintética y la Importancia de su Relación con otras Disciplinas / Brief description of Synthetic Biology and the importance of its relationship with other disciplines

Resumen La biología sintética (SynBio) es una disciplina de reciente aparición que sirve para diseñar o re-diseñar sistemas biológicos y otorgarles cualidades mejoradas o nuevas cualidades. En la SynBio el diseño de nuevos sistemas biológicos requiere de herramientas moleculares muy precisas, tales como: a) la bioinformática, b) la secuenciación NGS (Next Generation Sequencing), el ensamble y/o síntesis de ADN c) y la edición de genomas a través de CRISPR-Cas9. En la SynBio encontramos además otras disciplinas con un perfil más hacia el ámbito social, las cuales tocan aspectos éticos, legales, filosóficos y económicos, considerándose así una multidisciplina. La SynBio está propiciando el desarrollo de nuevas tecnologías (emergentes) partiendo de una óptica ingenieril. En la SynBio, al ADN se le entiende de forma práctica y abstracta como una serie de partes que se pueden ensamblar en cierto orden para obtener los productos deseados una vez que se conoce la funcionalidad de cada parte. La SynBio ha dado pie a una nueva concepción de la economía a nivel mundial y por consecuencia se ha tomado muy seriamente el termino Bioeconomía como una nueva disciplina que transformará a las sociedades.

Abstract Synthetic biology (SynBio) it is considered a very recent discipline. View as a tool serves to design or re-design biological systems, giving them improved qualities or new qualities. In the SynBio, the design of new biological systems requires very precise molecular tools, such as: a) bioinformatics, b) sequencing NGS (Next Generation Sequencing), assembly and synthesis of DNA c) and CRISPR- Cas9 genome editing. Within the SynBio there are other social profile disciplines which concerned to ethical, legal, philosophical, and economic, and for that reason it is considered a multidiscipline. The SynBio is promoting the development of new (emerging) technologies based on an engineering perspective. In SynBio, DNA is understood in a practical and abstract way as a series of parts that can be assembled in a certain order to obtain the desired products once the functionality of each part is known. The SynBio has given rise to a new conception of the economy worldwide and consequently the term Bioeconomy is already taken very seriously as a new discipline that will transform societies.

Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches.

The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected.

Yeast communities associated with artisanal mezcal fermentations from Agave salmiana.

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts.

AIMS: The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. METHODS AND RESULTS: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. CONCLUSIONS: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.