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Based on IGS-PCR RFLP polymorphism, we previously detected two Z. rouxii strains (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.

Despite 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 four were used in this work. All microsatellite 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.

Unlike 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.

Copper is one of the most abundant toxic heavy metals in municipal wastewaters and, in consequence, in sewage sludge and compost. The ability of a strain of the yeast Pichia guilliermondii, which was isolated from sewage sludge, to eliminate copper has been evaluated, using both viable and nonviable biomass. It has been found that raising concentrations of copper affected both morphology and physiological parameters of the viable yeast, and it is thought that a process of bioaccumulation may be involved in its copper uptake. The growth rate of nonadapted cells decreased with increasing concentrations of copper, mainly due to a decrease in the biomass yield. The cells could be adapted by training with increasing copper concentrations up to 317.7 mg/l. This adaptation was an all-or-nothing process: once cells had adapted, the biomass yield, metabolic flux and consequent growth rate were constant and independent of the external copper concentration. Also, it was determined that up to 20 mg of copper per gram of viable adapted biomass could accumulate from the medium (i.e., double the amount when using nonadapted viable biomass). Finally, adsorption data on nonviable cells were found to be well modeled by the Langmuir isotherm ( q max =9.09 mg/g of biomass).