Person: Martínez Peinado, José
Universidad Complutense de Madrid
Faculty / Institute
Now showing 1 - 10 of 10
PublicationKinetic and Energetic Parameters of Carob Wastes Fermentation by Saccharomyces cerevisiae: Crabtree Effect, Ethanol Toxicity, and Invertase Repression(Korean Society for Microbiology and Biotechnology, 2015) Rodrigues, B.; Martínez Peinado, José; Raposo, S.; Constantino, A.; Quintas, C.; Lima Costa, M. E.Carob waste is a useful raw material for the second-generation ethanol because 50% of its dry weight is sucrose, glucose, and fructose. To optimize the process, we have studied the influence of the initial concentration of sugars on the fermentation performance of Saccharomyces cerevisiae. With initial sugar concentrations (S0) of 20 g/l, the yeasts were derepressed and the ethanol produced during the exponential phase was consumed in a diauxic phase. The rate of ethanol consumption decreased with increasing S0 and disappeared at 250 g/l when the Crabtree effect was complete and almost all the sugar consumed was transformed into ethanol with a yield factor of 0.42 g/g. Sucrose hydrolysis was delayed at high S0 because of glucose repression of invertase synthesis, which was triggered at concentrations above 40 g/l. At S0 higher than 250 g/l, even when glucose had been exhausted, sucrose was hydrolyzed very slowly, probably due to an inhibition at this low water activity. Although with lower metabolic rates and longer times of fermentation, 250 g/l is considered the optimal initial concentration because it avoids the diauxic consumption of ethanol and maintains enough invertase activity to consume all the sucrose, and also avoids the inhibitions due to lower water activities at higher S0. PublicationQuantitative analysis of morphological changes in yeast colonies growing on solid medium: the eccentricity and Fourier indices(John Wiley & sons, 2014-11) Gil de Prado, Elena; Rivas, Eva María; Siloniz Jiménez, María Isabel de; Diezma, Belén; Barreiro, Pilar; Martínez Peinado, JoséThe colony shape of four yeast species growing on agar medium was measured for 116 days by image analysis. Initially, all the colonies are circular, with regular edges. The loss of circularity can be quantitatively estimated by the eccentricity index, Ei, calculated as the ratio between their orthogonal vertical and horizontal diameters. Ei can increase from 1 (complete circularity) to a maximum of 1.17–1.30, depending on the species. One colony inhibits its neighbour only when it has reached a threshold area. Then, Ei of the inhibited colony increases proportionally to the area of the inhibitory colony. The initial distance between colonies affects those threshold values but not the proportionality, Ei /area; this inhibition affects the shape but not the total surface of the colony. The appearance of irregularities in the edges is associated, in all the species, not with age but with nutrient exhaustion. The edge irregularity can be quantified by the Fourier index, Fi , calculated by the minimum number of Fourier coefficients that are needed to describe the colony contour with 99% fitness. An ad hoc function has been developed in Matlab v. 7.0 to automate the computation of the Fourier coefficients. In young colonies, Fi has a value between 2 (circumference) and 3 (ellipse). These values are maintained in mature colonies of Debaryomyces, but can reach values up to 14 in Saccharomyces. All the species studied showed the inhibition of growth in facing colony edges, but only three species showed edge irregularities associated with substrate exhaustion. PublicationStrain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS)(Elsevier, 2010-08-15) Wrent, Petra; Rivas, Eva María; Martínez Peinado, José; Siloniz Jiménez, María Isabel deUnlike previously reported methods that need a combination of several typing techniques, we have 22 developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage 23 species. Strains belonging to other species have also been included for comparison. We have demonstrated 24 that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in 25 this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z.bailii. 26 We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns 27 with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS 28 rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of 29 Zygosaccharomyces in a separate cluster. PublicationA mesoscopic stochastic model for the specific consumption rate in substrate-limited microbial growth(PLOS, 2017-02-10) Arranz, F. J.; Martínez Peinado, JoséThe specific consumption rate of substrate, as well as the associated specific growth rate, is an essential parameter in the mathematical description of substrate-limited microbial growth. In this paper we develop a completely new kinetic model of substrate transport, based on recent knowledge on the structural biology of transport proteins, which correctly describes very accurate experimental results at near-zero substrate concentration values found in the literature, where the widespread Michaelis-Menten model fails. Additionally, our model converges asymptotically to Michaelis-Menten predictions as substrate concentration increases. Instead of the single active site enzymatic reaction of Michaelis-Menten type, the proposed model assumes a multi-site kinetics, simplified as an apparent all-or-none mechanism for the transport, which is controlled by means of the local substrate concentration in the close vicinity of the transport protein. Besides, the model also assumes that this local concentration is not equal to the mean substrate concentration experimentally determined in the culture medium. Instead, we propose that it fluctuates with a mostly exponential distribution of Weibull type. PublicationGrowth kinetics and physiological behavior of co-cultures ofSaccharomyces cerevisiae and Kluyveromyces lactis,fermenting carobsugars extracted with whey(Elsevier, 2016-10) Rodrigues, B.; Lima-Costa, M. E.; Constantino, A.; Raposo, S.; Felizardo, C.; Gonçalves, D.; Fernandes, T.; Dionísio, L.; Martínez Peinado, JoséAlcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis’ oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR–3l reactor. K. lactis was inoculated first and, with low aeration (0.13 vvm), consumed all the lactose in 48 h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80 g/l final concentration and a conversion factor of 0.4 g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal. PublicationDevelopment of species-specific primers for rapid identification of Debaryomyces hansenii(Elsevier, 2015-01-16) Wrent, Petra; Rivas, Eva María; Gil de Prado, Elena; Martínez Peinado, José; Siloniz Jiménez, María Isabel deIn this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications. PublicationAssessment of the Factors Contributing to the Growth or Spoilage of Meyerozyma guilliermondii in Organic Yogurt: Comparison of Methods for Strain Differentiation(MDPI, 2015-09) Wrent, Petra; Rivas, Eva María; Gil de Prado, Elena; Martínez Peinado, José; Siloniz Jiménez, María Isabel deIn this work we analyze the spoiling potential of Meyerozyma guilliermondii in yogurt. The analysis was based on contaminated samples sent to us by an industrial laboratory over two years. All the plain and fruit yogurt packages were heavily contaminated by yeasts, but only the last ones, containing fermentable sugars besides lactose, were spoiled by gas swelling. These strains were unable to grow and ferment lactose (as the type strain); they did grow on lactate plus galactose, fermented glucose and sucrose, and galactose (weakly), but did not compete with lactic acid bacteria for lactose. This enables them to grow in any yogurt, although only those with added jam were spoiled due to the fermentation of the fruit sugars. Fermentation, but not growth, was strongly inhibited at 8 °C. In consequence, in plain yogurt as well as in any yogurt maintained at low temperature, yeast contamination would not be detected by the consumer. The risk could be enhanced because the species has been proposed for biological control of fungal infections in organic agriculture. The combination of the IGS PCR-RFLP (amplification of the intergenic spacer region of rDNA followed by restriction fragment length polymorphism analysis) method and mitochondrial DNA-RFLP makes a good tool to trace and control the contamination by M. guilliermondii. PublicationA simple mathematical model that describes the growth of the area and the number of total and viable cells in yeast colonies(John Wiley & Sons, 2014-12) Rivas, Eva María; Gil de Prado, Elena; Wrent, Petra; Siloniz Jiménez, María Isabel de; Correa, E.C.; Conejero Meca, Francisco; Murciano Cespedosa, Antonio; Martínez Peinado, JoséWe propose a model, based on the Gompertz equation, to describe the growth of yeasts colonies on agar medium. This model presents several advantages: (i) one equation describes the colony growth, which previously needed two separate ones (linear increase of radius and of the squared radius); (ii) a similar equation can be applied to total and viable cells, colony area or colony radius, because the number of total cells in mature colonies is proportional to their area; and (iii) its parameters estimate the cell yield, the cell concentration that triggers growth limitation and the effect of this limitation on the specific growth rate. To elaborate the model, area, total and viable cells of 600 colonies of Saccharomyces cerevisiae, Debaryomyces fabryi, Zygosaccharomyces rouxii and Rhodotorula glutinis have been measured. With low inocula, viable cells showed an initial short exponential phase when colonies were not visible. This phase was shortened with higher inocula. In visible or mature colonies, cell growth displayed Gompertz-type kinetics. It was concluded that the cells growth in colonies is similar to liquid cultures only during the first hours, the rest of the time they grow, with near-zero specific growth rates, at least for 3 weeks. PublicationDevelopment of an affordable typing method for Meyerozyma guilliermondii using microsatellite markers(Elsevier, 2016-01-09) Wrent, Petra; Rivas, Eva María; Martínez Peinado, José; Siloniz Jiménez, María Isabel deDespite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only fourwere used in thiswork. Allmicrosatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction,which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair.Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool. PublicationZygosaccharomyces rouxii strains CECT 11923 and Z. rouxii CECT 10425: Two new putative hybrids?(Elsevier, 2017-01-16) Wrent, Petra; Rivas, Eva María; Martínez Peinado, José; Siloniz Jiménez, María Isabel deBased on IGS-PCR RFLP polymorphism, we previously detected two Z. rouxiistrains (CECT 11923 and CECT 10425) that clustered with hybrid strains (NCYC 1682, NCYC 3060 and NCYC 3061). Given the recently recognized important industrial role of hybrids, their detection is very useful. Based on the IGS1 rDNA region alignment of hybrid strains and the Z. rouxii CECT 11923 and CECT 10425, in this work, we developed a pair of Zygosaccharomyces hybrid-specific primers, HibZF/HibZR. Positive amplicons were only obtained in the Zygosaccharomyces spp. hybrids included in this study and the CECT 11923 and CECT 10425 strains analyzed here. In the present study, we applied molecular tools to highlight the nature of these strains; they are quite different from each other as well as from Z. rouxii type strain. Based on the presence of two heterologous copies of nuclear-encoded genes (SOD2 and HIS3), the sequences of divergent 5.8S-ITS rDNA, D1/D2 26S rDNA copies and, the amplification with species-specific primer for Z. rouxii and Z. pseudorouxii, we hypothesize that the CECT 11923 strain might be a hybrid strain. Whereas, CECT 10425, the sequence analysis of 5.8S-ITS rDNA and D1/D2 26S rDNA copies presented 99–100% sequence identity with Zygosaccharomyces sp. NBRC 10669 (LN849119.1) and Z. sapae ABT 301T. Nevertheless, we discard that it could be a Z. sapae strain based on the results obtained in this study. Namely, the amplification with hybrid-specific primer designed in this study, the number of divergent copies of HIS3 (2), the fact that it only possesses one SOD2 gene and the amplification with species-specific primer for Z. pseudorouxii, therefore it could be a new species or a hybrid strain.