Application of multifractal wavelet analysis to spontaneous fermentation processes

"An algorithm is presented here to get more detailed information, of mixed cul-ture type, based exclusively on the biomass concentration data for fermentation processes. The analysis is performed with only the on-line measurements of the re-dox potential being available. It is a two-step procedure which includes an Artiffial Neural Network (ANN) that relates the redox potential to the biomass concen-trations in the first step. Next, a multifractal wavelet analysis is performed using the biomass estimates of the process. In this context, our results show that the redox potential is a valuable indicator of microorganism metabolic activity during the spontaneous fermentation. In this paper, the detailed design of the multifractal wavelet analysis is presented, as well as its direct experimental application at the laboratory level.

Optimization of fermentation conditions for the production of the mezcal from Agave salmiana using response surface methodology

"Response surface methodology was applied to optimize the fermentative phase for the mezcal production from Agave salmiana. A 3k factorial design was used to obtain models describing the relationship between the ethanol production, process productivity, and product yield with respect to the fermentation temperature and the initial sugar concentration. The results showed that the fermentative conditions affected the composition of higher alcohols (referred as a quality indicator) in the mezcal as well as the amount of ethanol. The highest ethanol production was attained by employing the following predicted optimum operational conditions: temperature of 28 °C and an initial sugar concentration of 105 g/l. However, the maximum productivity process was attained with 34.6 °C and 90 g/l, whereas the maximum product yield and the best quality mezcal at 28 °C and 77 g/l. Results show that the simultaneous optimization for high ethanol production and fast production rate are not compatible, since high ethanol production requires a high substrate concentration, which in turn inhibit the growth rate.

Aspectos químicos y moleculares del proceso de producción del mezcal

Tesis (Doctorado en Ciencias en Biología Molecular)"En el presente trabajo se realizó la identificación y cuantificación de compuestos volátiles presentes en mezcales comerciales producidos en la región mezcalera del país. Se investigó el efecto de las condiciones iniciales de concentración de azúcares y temperatura en el proceso de fermentación; se analizó el comportamiento del potencial redox y finalmente se llevó a cabo la identificación molecular de la microbiota presente al inicio de la fermentación. El análisis de los compuestos volátiles en los mezcales se efectuó mediante cromatografía de gases (GC) y espectrometría de masas (MS). Los destilados obtenidos a partir de Agave salmiana, producidos en la región del altiplano entre Zacatecas y San Luis Potosí, mostraron la presencia de una gran variedad de compuestos volátiles como alcoholes, ácidos orgánicos, esteres, furanos, cetonas y aldehídos. También se detectó la presencia de terpenos como limoneno, α-terpineol y α-terpineno. Únicamente los mezcales con gusano presentaron compuestos insaturados como el 6, 9-pentadecadieno-1-ol, 3-hexen-1-ol, 1-8 nonadieno y 1-dodecino. Compuestos como limoneno y el pentilbutanoato fueron identificados por primera vez, en mezcales producidos a partir de Agave salmiana. El estudio de bebidas alcohólicas como son la raicilla, sisal, tequila, mezcal, bacanora y sotol, producidos en diversas regiones del país, reveló la presencia de 105 compuestos volátiles diferentes. De los cuales once compuestos se clasificaron como mayoritarios y el resto se clasificaron como minoritarios. El análisis multivariable permitió clasificar los destilados a partir la concentración de sus compuestos. También se identificaron 17 compuestos minoritarios específicos, en los destilados de Agave, tales como el azuleno, cinnamol, butirolactona y la piridina entre otros, que podrían ser los responsables de la firma o huella de cada bebida. Finalmente se observó que el proceso de producción y la materia prima juegan un papel muy importante en el perfil sensorial de la bebida. La optimización de las variables de operación, concentración inicial de azúcares y temperatura, permitió maximizar la producción de etanol, el rendimiento y la productividad. Esto se realizó mediante la metodología de superficie de respuesta (RSM), la cual permitió optimizar simultáneamente las dos variables de operación. La máxima producción de etanol se obtuvo a 28°C y 105 g/L para el caso del rendimiento 28°C y 77g/L y por último para la productividad fue de 34.6°C y 90g/L. Se encontró que la producción de etanol está asociada al crecimiento microbiano, mientras que el crecimiento celular presentó una cinética de inhibición por sustrato. El comportamiento del potencial redox es inversamente proporcional al crecimiento microbiano y a la producción de etanol. La cuantificación por GC de los compuestos volálites mayoritarios en fermentaciones realizadas a diferentes condiciones iniciales, mostraron que la temperatura influye en la producción de alcoholes superiores y por lo tanto en la calidad de la bebida. Se emplearon técnicas microbiológicas de cultivo en placa para aislar bacterias y levaduras presentes al inicio de la fermentación. La identificación se realizó amplificando una región del DNA ribosomal, 16S rDNA en el caso de bacterias e ITS (Internal Transcriber Spacer) para las levaduras, los productos obtenidos se clonaron y secuenciaron. Los resultados mostraron que existen al menos 3 géneros distintos de levaduras, Pichia fermentans, Clavispora lusitaniae y Kluyveromyves marxianus. En el caso de las bacterias se identificó al género Lactobacillus como la población dominante. Se identificó y aisló L.kefiri, L.farraginis, L.plantarum, Weissella cibaria y W. paramesenteroides. También se identificó a Zymomonas mobilis.""In this work the identification and quantification of the volatile compounds of the commercial mezcals, produced in the mezcal region of the country is performed. Moreover, the effects of the initial substrate concentration and the temperature in the fermentation process were investigated. Finally, the molecular identification of the microbiota present at the beginning of the fermentation was carried out. The volatile compounds analysis of the mezcals was carried out using gas chromatography (GC) and mass spectrometry (MS). The distillation product of the Agave salmiana juices, produced in the altiplano region between Zacatecas and San Luis Potosi, shown a great variety of volatile compounds such as alcohols, organic acids, esters, furans, ketoses and aldehydes. In addition, the presence of terpenes (limonene, α-terpinol and α-terpinene) was detected. Only the mezcals with worms shown unsatured compounds like the 6,9-pentadecadien-1-ol, 3- hexen-1-ol, 1,8-nonodiene and 1-dodecine. We mentioned here that compounds such as limonene and pentylbutanoate were identified for first time, in mezcals produced from Agave salmiana. The study of alcoholic beverages such as raicilla, sisal, tequila, mezcal,bacanora, sotol and pulque produced in different region of the country reveled the presence of the 105 different volatile compounds. Eleven of them were classified as major compounds and the others were classified as minor compounds. The multivariate analysis allows us to establish a compositions-based classification for the distillated products. Then seventeen minor compounds, specific of the beverages, were identified providing a better fingerprinting. Finally, it is shown that the process production and the raw material play an important role in the sensorial profile of the beverage. The optimization of the operation variables such as initial sugar concentration and temperature was achieved maximizing of the ethanol concentration, yield and productivity. This was performed trough the application of the surface response methodology (RSM), optimizing at the same time the operation variables. The maximum ethanol production was obtained at 28 °C and 90 g/L. It was found that ethanol production is associated to the biomass growth, whereas the cell-growth showed a substrate inhibition behavior. The time evolution of the redox potential was inversely proportional to the microbial growth and to the ethanol production.

Inferring mixed-culture growth from total biomass data in a wavelet approach

"It is shown that the presence of mixed-culture growth in batch fermentation processes can be very accurately inferred from total biomass data by means of the wavelet analysis for singularity detection. This is accomplished by considering simple phenomenological models for the mixed growth and the more complicated case of mixed growth on a mixture of substrates. The main quantity provided by the wavelet analysis is the Holder exponent of the singularity that we determine for our illustrative examples. The numerical results point to the possibility that Holder exponents can be used to characterize the nature of the mixed-culture growth in batch fermentation processes with potential industrial applications. Moreover, the analysis of the same data affected by the common additive Gaussian noise still lead to the wavelet detection of the singularities although the Holder exponent is no longer a useful parameter.

High-gain nonlinear observer for simple genetic regulation process

"High-gain nonlinear observers occur in the nonlinear automatic control theory and are in standard usage in chemical engineering processes. We apply such a type of analysis in the context of a very simple one-gene reg-ulation circuit. In general, an observer combines an analytical di?erential-equation-based model with partial measurement of the system in order to estimate the non-measured state variables. We use one of the simplest observers, that of Gauthier et al., which is a copy of the original system plus a correction term which is easy to calculate. For the illustration of this procedure, we employ a biological model, recently adapted from Goodwin´s old book by De Jong, in which one plays with the dynamics of the concentrations of the messenger RNA coding for a given protein, the protein itself, and a single metabolite. Using the observer instead of the metabolite, it is possible to rebuild the non-measured concentrations of the mRNA and the protein.

Online monitoring of Mezcal fermentation based on redox potential measurements

"We describe an algorithm for the continuous monitoring of the biomass and ethanol concentrations as well as the growth rate in the Mezcal fermentation process. The algorithm performs its task having available only the online measurements of the redox potential. The procedure combines an artificial neural network (ANN) that relates the redox potential to the ethanol and biomass concentrations with a nonlinear observer-based algorithm that uses the ANN biomass estimations to infer the growth rate of this fermentation process. The results show that the redox potential is a valuable indicator of the metabolic activity of the microorganisms during Mezcal fermentation. In addition, the estimated growth rate can be considered as a direct evidence of the presence of mixed culture growth in the process. Usually, mixtures of microorganisms could be intuitively clear in this kind of processes; however, the total biomass data do not provide definite evidence by themselves. In this paper, the detailed design of the software sensor as well as its experimental application is presented at the laboratory level.

Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana

"Aims:  To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana. Methods and Results:  The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro‐organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus kefiri, Lactobacillus plantarum and Lactobacillus farraginis. Conclusions:  The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro‐organisms with high potential for the production of some volatile compounds in mezcal. Significance and Impact of the Study:  We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.

Riesgos potenciales de salud por consumo de agua con arsénico en Colima, México

Objetivo. Estimar los riesgos potenciales de salud debidos a la ingestión crónica de arsénico (As) en agua en Colima, México. Material y métodos. Se muestrearon aleatoriamente 36 pozos en 10 acuíferos locales. El análisis se hizo mediante ICP-OES siguiendo estándares internacionales. Se realizó una interpolación geoestadística con ArcGIS, implementando un modelo de ponderación del inverso de la distancia, para estimar la ruta de exposición de consumo en cada localidad. Se calcularon los coeficientes de peligro (HQ)y riesgo carcinogénico (R). Resultados. El HQ promedio ponderado de As para Colima es 2.41. Existen valores de HQ>1 para As que indican efectos adversos no carcinogénicos para la salud por ingestión continua y prolongada de agua; esto podría afectar a 183 832 individuos en el estado. El riesgo calculado de desarrollar cáncer debido a las concentraciones de arsénico en aguas subterráneas (R) es de 1.089E-3; estadísticamente esto podría ocasionar 446 casos de cáncer. Conclusiones. Los niveles actuales de arsénico en el agua de pozo incrementan los riesgos carcinogénicos y no carcinogénicos de salud humana en Colima

Primjena plinske kromatografije u kombinaciji s masenom spektrometrijom za karakterizaciju hlapljivih spojeva tradicionalnih meksičkih pića proizvedenih iz Agave

Ethnic Agave alcoholic beverages such as raicilla, sisal, tequila, mezcal, bacanora, sotol and pulque have been analyzed by gas chromatography and headspace solid-phase microextraction- gas chromatography-mass spectrometry (HS-SPME-GC-MS). There were 105 compounds identified, eleven were classified as major compounds and the others were classified as minor compounds. Seventeen minor compounds could be used as authenticity markers since they were beverage specific. Cluster analysis (CA) showed that Agave alcoholic beverages could be distinguished by multivariate analysis of major compounds; however, the analysis of minor compounds provided a better fingerprinting.Plinskom kromatografijom i mikroekstrakcijom na čvrstoj fazi, u kombinaciji s plinskom kromatografijom i masenom spektrometrijom (HS-SPME-GC-MS), ispitani su uzorci tradicionalnih meksičkih pića proizvedenih iz Agave (raicilla, sisal, tequila, mezcal, bacanora, sotol i pulque). Identificirano je 105 spojeva, od čega 11 glavnih sastojaka. Utvrđeno je da se za markere autentičnosti pojedinih pića može upotrijebiti 17 sporednih sastojaka. Razvrstavanjem uzoraka (engl. cluster analysis – CA) dokazano je da se tradicionalna meksička pića proizvedena iz Agave mogu razlikovati multivarijantnom analizom glavnih sastojaka, a analiza sporednih sastojaka omogućava točnije utvrđivanje porijekla pića