Pichia Cactophila Research Articles

Taxogenomic analysis of a novel yeast species isolated from soil, Pichia galeolata sp. nov.

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446T (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

Analysis of Prototheca and yeast species isolated from bulk tank milk collected in Tokachi District, Japan.

Bovine mastitis, a major infectious disease affecting milking cows, leads to reduced milk yield and quality, reduced animal welfare, and an increased need for culling. Although its major causative agents are bacteria, yeast species and achlorophyllous algae of the Prototheca genus are well known as causative agents of bovine refractory mastitis. Nevertheless, few studies have analyzed specific yeasts and Prototheca in this context. Herein, we present survey data of yeast species and Prototheca species isolated from bulk tank milk in the Tokachi district of Japan from April 2020 through March 2021. The species of 276 isolates were determined. Yeast species accounted for 184 isolates, of which Pichia kudriavzevii was the most prevalent species. Regarding Prototheca species, only Prototheca bovis was isolated (92 isolates). Prototheca bovis and Pichia kudriavzevii were detected throughout the year and were detected repeatedly on the same farm. Kluyveromyces marxianus was the second most frequently isolated yeast species after Pichia kudriavzevii. Candida parapsilosis, the fourth most frequently isolated yeast species, was found discontinuously. Analysis of monthly data indicated that Kluyveromyces marxianus and Candida parapsilosis were mainly found during the winter and summer months, respectively. Candida akabanensis and Pichia cactophila were the third and fifth most frequently isolated yeast species, respectively. They were detected repeatedly in bulk tank milk samples from the same farms. Results obtained from bulk tank milk underscore the prevalence of these species. These study results are expected to contribute to the elucidation of problematic yeast and Prototheca species.

Improving lipid production capacity of new natural oleaginous yeast: Pichia cactophila firstly

Biodiesel is a useful alternative source of energy and can be used as a support for oil-source diesel. Microbial lipids are considered a promising raw material for biodiesel production. In a previously completed study, Pichia cactophila was found to be the best lipid-producing yeast by lipid screening studies among 107 yeast isolates. In this study, the lipid production conditions of P. cactophila, which was chosen as a lipid producer for the first time, were partially optimized by using response surface methodology in crude glycerol. Plackett-Burman statistical experiment design was used to determine the effect of glycerol, yeast extract, peptone, urea, (NH4)2SO4, NH4Cl, NH4NO3 factors on lipid production. In order to determine the optimum levels of the most effective parameters by the result of screening (glycerol, yeast extract, urea, NH4NO3), Box-Behnken experimental design method was used. The optimum production conditions were determined as 39 g/L glycerol, 20 g/L yeast extract, 5 g/L urea, and 20 g/L NH4NO3, and the result of the experiments carried out under these conditions the lipid value (%) was determined as 91.43. Lipid content was increased 1.408-fold (40.79%) by two-step optimization with the response surface methodology and reached a very high value. The fatty acid composition obtained under optimum conditions is compatible with vegetable oils. This suggests that the microbial lipid of P. cactophila can be used as a raw material for biodiesel production and is promising for further studies.

Lipid Production from Crude Glycerol by Newly Isolated Oleaginous Yeasts: Strain Selection, Molecular Identification and Fatty Acid Analysis

Biodiesel is a renewable alternative fuel and glycerol as a main byproduct of the manufacturing process. Lipids could be produced from crude glycerol by using yeasts. The ability of 107 yeast strains to utilize glycerol was screened and 92 of these were selected. 60 strains were determined as a potential for lipid production by Sudan Black B staining. After secondary screening 25 of them showed specific growth rates (OD 600), high biomass production and lipid content. These strains were identified as Pichia cactophila, P. fermentans, P. anomala, Rhodotorula mucilaginosa, R. dairenensis, Clavispora lusitaniae, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida glabrata, C. inconspicua, C. albicans, Yarrowia lipolytica with molecular identifications based on ITS and D1/D2 26S rDNA sequences. The results showed that P. cactophila accumulated lipid up to 64.94%, the highest lipid content. C16:0, C18:0, C18:1 and C18:2 essential fatty acids for biodiesel production were detected by GC–MS in the lipids accumulated by all strains. P. cactophila and C. lusitaniae were reported for the first time as lipid-producing yeasts. The results suggest that selected 25 isolates have the ability to grow on crude glycerol and especially P. cactophila produce lipid that has potential use as a feedstock for second generation biodiesel production.

Yeast communities associated with cacti in Brazil and the description of Kluyveromyces starmeri sp. nov. based on phylogenomic analyses.

Yeast communities associated with cacti were studied in three ecosystems of Southeast, Central and North Brazil. A total of 473 yeast strains belonging to 72 species were isolated from 190 samples collected. Cactophilic yeast species were prevalent in necrotic tissues, flowers, fruits and insects of cacti collected in Southeast and North Brazil. Pichia cactophila, Candida sonorensis and species of the Sporopachydermia complex were the most prevalent cactophilic species in Southeast and Central regions. Kodamaea nitidulidarum, Candida restingae and Wickerhamiella cacticola were frequently associated with cactus flowers and fruits. The diversity of yeasts associated with the substrates studied was high. Twenty-one novel species were found. One is described here as Kluyveromyces starmeri sp. nov. based on 21 isolates obtained from necrotic tissues, flowers, fruits and associated insects of the columnar cacti Cereus saddianus, Micranthocereus dolichospermaticus and Pilosocereus arrabidae in two different ecosystems in Brazil. Phylogenetic analyses of sequences encoding the gene of the small subunit (SSU) rRNA gene, the internal transcribed spacer, the 5.8S rRNA gene and the D1/D2 domains of the large subunit (LSU) rRNA showed that the species is related to Kluyveromyces dobzhanskii, Kluyveromyces lactis and Kluyveromyces marxianus. Phylogenomic analyses based on 1264 conserved genes shared among the new species and 19 other members of the Saccharomycetaceae confirmed this phylogenetic relationship. The holotype is K. starmeri sp. nov. CBS 16103T (=UFMG-CM-Y3682T ). The Mycobank number is MB 836817.

Bacterial and Fungal Communities of Gioddu as Revealed by PCR-DGGE Analysis.

Gioddu is the sole variety of fermented milk originating in Italy. Despite the long history of consumption, Gioddu still represents an undisclosed source of microbial diversity. The present study was aimed to get an insight into the bacterial and fungal diversity of Gioddu samples collected from two artisan producers located in Sardinia. To this end 3 batches of Gioddu were collected from each producer and subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analyses. Gioddu was produced with sheep milk in accordance with the local tradition. Regarding the bacterial population, a low biodiversity emerged. In more detail, the sole species Lactobacillus delbrueckii subsp. bulgaricus was detected in all the samples, irrespective of the producer or the batch. A more ample microbial diversity was highlighted for the fungal population that included closest relatives to Pichia cactophila, Kluyveromyces marxianus and Galactomyces candidum. Based on the results, the detected bacterial and fungal species generally clustered in accordance with the producer, irrespective of the batch considered. It is noteworthy that, Gioddu revealed several microbiological similarities with kefir beverage, thus suggesting, by analogy, potential health benefits related to its consumption. More research is needed to better clarify the microbiota composition of Gioddu by using more powerful metagenomic techniques.

Pichia cactophila and Kluyveromyces lactis are Highly Efficient Microbial Cell Factories of Natural Amino Acid-Derived Aroma Compounds.

The pivotal role of non-conventional yeast (NCY) species in formation of valuable aroma compounds in various food commodities is widely acknowledged. This fact inspires endeavors aiming at exploitation of food-derived NCYs as biocatalysts in natural aromas production. In this study, we isolated, characterized and evaluated aroma-producing capacity of two NCY representatives—Pichia cactophila 7.20 and Klyuveromyces lactis 6.10 strains. The strains were isolated from food-related habitats—goat-milk regional cheese and Swiss-type ripening cheese, respectively. Aroma profiles generated by the two strains cultured in a general rich medium were analyzed through solvent extraction and GC-MS analysis of the compounds retained in the culture media. Finally, the strains were tested in bioconversion cultures with branched chain- or aromatic amino acids as the sole nitrogen source, to assess capability of the strains towards formation of amino acid-derived aromas. The results showed extraordinary capacity of both strains for production of 2-phenylethanol (at more than 3 g/L) and isoamyl alcohol (approx. 1.5 g/L). A distinctive trait of 2-phenylethyl acetate synthesis at high concentrations (0.64 g/L) was revealed for P. cactophila 7.20 strain. Highly valued disulfide dimethyl as well as methionol acetate were identified amongst the aroma compounds synthesized by the strains.

Candida inconspicua and Candida norvegensis: new insights into identification in relation to sexual reproduction and genome organization.

Candida inconspicua and Candida (Pichia) norvegensis are two emerging pathogenic species that exhibit reduced susceptibility to azole derivatives. Conventional (biochemical) approaches do not readily differentiate between the two species. The first aim of this work was to analyze the performance of biochemical, proteomic (matrix-assisted laser desorption ionization-time of flight [MALDI-TOF]), and molecular approaches in the precise identification of these species. These results then led us to sequence 3 genomic loci, i.e., the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), the D1/D2 domain of the 28S rDNA, and the elongation factor 1α (EF-1α) gene, either directly or following cloning, of 13 clinical isolates and 9 reference strains belonging to the 5 species included in the Pichia cactophila clade, namely, Pichia cactophila, Pichia insulana, C. inconspicua, C. norvegensis, and P. pseudocactophila. Finally, isolates of C. inconspicua were challenged for sexual reproduction on the appropriate medium. Our results show that EF-1α sequencing and proteic profiling by MALDI-TOF are the two most efficient approaches to distinguish between C. norvegensis and C. inconspicua. As a characteristic of the P. cactophila clade, we found multiple alleles of the rDNA regions in certain strains belonging to the tested species, making ITS or D1/D2 sequencing not appropriate for identification. Whatever the method of identification, including MALDI-TOF and EF-1α sequencing, none could differentiate C. inconspicua from P. cactophila. The results of phylogenetic analysis and the generation of asci from pure cultures of all C. inconspicua strains both support the identification of P. cactophila as the teleomorph of C. inconspicua.

Pichia insulana sp. nov., a novel cactophilic yeast from the Caribbean

A novel species of ascomycetous yeast, Pichia insulana sp. nov., is described from necrotic tissue of columnar cacti on Caribbean islands. P. insulana is closely related to and phenotypically very similar to Pichia cactophila and Pichia pseudocactophila. There are few distinctions between these taxa besides spore type, host preference and locality. Sporogenous strains of P. insulana that produce asci with four hat-shaped spores have been found only on Curaçao, whereas there was no evidence of sporogenous P. cactophila from that island. In addition, sequences of the D1/D2 fragment of the large-subunit rDNA from 12 Curaçao strains showed consistent differences from the sequences of the type strains of P. cactophila and P. pseudocactophila. The type strain of P. insulana is TSU00-106.5(T) (=CBS 11169(T) =UCD-FST 09-160(T)).

Preliminary notes on yeasts associated with necrotic cactus stems from different localities in Brazil

The yeast species found in necrotic stems of three columnar cacti (Pilosocereus machrisii, Pilosocereus vilaboensis, and Praecereus euchlorus) at eight localities in Brazil were described and a similarity analysis using Sorensen distances was used to compare the composition of yeast species at these localities. Of 56 necrotic cactus stems sampled, 32 produced yeast colonies. Ten species of yeast or yeast-like microorganisms were identified from 53 isolates, with Pichia cactophila, Candida sonorensis, Geotrichum sp., and Sporopachydermia cereana being the most common. The remaining species occurred in low proportions in the cacti surveyed. The similarity analysis provided a dendogram (UPGMA) that clustered the yeast communities from different cactus species and indicated that host cactus species was unimportant in this clustering.

Analysis of population structure of cactophilic yeast from the genus Pichia: P. cactophila and P. norvegensis.

DNAs from 40 strains of Pichia cactophila and Pichia norvegensis, yeasts characteristic of cactus necroses, were compared using randomly amplified polymorphic DNA (RAPD) banding patterns and killer/sensitive phenotypes. Both species belong to the same species complex within the genus. The levels of between-strain RAPD variation were high in both species (higher in the automictic P. cactophila than in the heterothallic P. norvegensis), although there is little variation in physiological abilities within either species. Although each species was a separate lineage, RAPD analysis confirms that the species are related. Within each species, RAPD variation was related to the geographic origin of the strains. Pichia cactophila strains from southern Florida were more related to those from Antigua than to those from northern Florida. These results correlated well with the differences among killer/sensitive phenotypes of strains. Principal component analysis indicated that the phenotypes of each species differ. Here too, strains from southern Florida were more similar to those from Antigua than to those from northern Florida. Previous work had identified differences in the cactophilic yeast communities from southern and northern Florida, and these results indicate that the differences are detectable at the population levels as well.

Yeast communities associated with different plant resources in sandy coastal plains of southeastern Brazil

The distribution of yeasts was studied in different plant substrata of beach and restinga ecosystems in southeastern Brazil. Candida sonorensis, Clavispora opuntiae, Pichia barkeri, Pichia cactophila and Geotrichum sp. were generally prevalent in yeast communities associated with necrotic tissues of the cacti Cereus pernambucensis, Selenicereus rizzini, Opuntia vulgaris and Opuntia sp. Cactus yeast communities were unique and no cactophilic yeast species were isolated from plant substrata other than cacti in these ecosystems. Flowers of the cactus Cereus pernambucensis had a highly specific yeast community composed almost exclusively of Pichia sp. C, a Candida domercqii -like species and Candida sp. C. Flowers of Ipomoea litoralis and Ipomoea pes-caprae , and extrafloral nectaries of Senna australis and Senna bicapsularis had communities composed predominantly of basidiomycetous yeast anamorphs. Insect vectors, the chemistry of each plant resource, and environmental conditions of these microhabitats are among the most important factors determining the species composition of these communities.

Genotype-specific habitat selection for oviposition sites in the cactophilic species Drosophila buzzatii

Isofemale lines of the cactophilic species, Drosophila buzzatii, exhibit genetic variation for their oviposition response to cactus yeast species in the laboratory. In general, interactions between yeast species preclude the use of pairwise preferences as predictors of preferences in three-way choice experiments. Two isofemale lines with relatively high laboratory preference for ovipositing on the yeast Pichia cactophila (as opposed to Cryptococcus cereanus) and two isofemale lines with relatively low preference for P. cactophila were used in a series of field release experiments to determine if laboratory preferences were also realized under field conditions. The influence of yeast species on both settling behaviour (long-distance response) and oviposition preference (short-distance response) were tested. The four lines were identical in their settling behaviour, preferring P. cactophila. The analysis of the oviposition preference tests showed significant line effects which correlated with the laboratory results. Thus a genetic component for oviposition preference under laboratory and field conditions was demonstrated and this strengthens the evidence for genotype-specific habitat selection in D. buzzatii. One low line, however, did not differ significantly from the two high lines under field conditions. A laboratory retest of this low line showed that the laboratory preference had not changed. The reason for the difference in the two situations is unknown but undoubtedly is attributable to uncontrolled variables under the field situation. Settling behaviour and oviposition response, in general, appear to be proximately linked to differences in the volatiles produced by the different yeast species.

Yeast communities of the cactus Pilosocereus arrabidae and associated insects in the Sandy coastal plains of southeastern Brazil.

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactus Pilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent species Pichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp., Myxozyma mucilagina and Sporopachydermia sp. A, representing about 80% of the total isolates. Pichia sp. A and a Candida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genus Kloeckera, basidiomicetous anamorphs of the genus Cryptococcus, the black yeast Aureobasidium pullulans, Pichia sp. A, a Candida domercqii-like species, and some cactophilic yeasts, especially Clavispora opuntiae. The feeding site of Sigelgaita sp. larvae had Clavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.

Yeast communities of the cactus Pilosocereus arrabidae as resources for larval and adult stages of Drosophila serido.

The feeding behavior of Drosophila serido on the yeast communities of necrotic stem tissue of Pilosocereus arrabidae were studied in a sand dune ecosystem of Rio de Janeiro, Brazil. The prevalence of cactophilic yeasts including Pichia barkeri, Candida sonorensis and Geotrichum sp. in the crops and external surfaces of D. serido reflected its association with the cactus habitat. The effective number of yeasts vectored on the surface of flies was higher than that in the crops. Also overlap between the yeasts from stems and from crops was partial suggesting selective feeding by the flies in the substrates visited. The females had a higher effective number of yeast species and a lower similarity than males with the yeast community of P. arrabidae. This was probably related to the search for oviposition sites by females. The presence of Pichia thermotolerans-like and Pichia amethionina var pachycereana in the flies, but not in P. arrabidae stems, indicated that D. serido was not limited to this cactus species. The larvae and adults lived in different patches with the adults feeding in patches with higher yeast species richness. The larvae had a narrower feeding niche and higher overlap with P. arrabidae, and preferred P. barkeri and Pichia cactophila as food. Adult flies fed on patches with the most frequent yeasts except for P. cactophila. Pichia caribaea was found in higher frequency in the adult crops than in the stems. Our data suggested that there was food selection and diet partitioning between adult and larval stages of D. serido.

Clavispora opuntiae and other yeasts associated with the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae of Rio de Janeiro, Brazil.

Clavispora opuntiae was the prevalent yeast associated with the feeding sites of Sigelgaita sp. larvae in the cactus Pilosocereus arrabidae. Also associated with this habitat were Candida sonorensis, Pichia cactophila, Pichia barkeri, Candida sp. A, Geotrichum sp., Geotrichum sericeum and the yeast like organisms Prototheca zopfii and Acremonium sp. Atypical yeast biotypes that may represent new species of Pichia, Sporopachydermia and Candida were isolated. Mating types of Clavispora opuntiae were at a ratio 70 h- to 3 h- and reduced levels of sporulation suggested low pressure for sexual reproduction in this habitat. Sigelgaita sp. probably was not an important vector for Clavispora opuntiae because it was not isolated from an adult or eggs of this moth.

Adaptations of Drosophila and Yeasts: their Interactions with the Volatile 2-propanol in the Cactus-Micro organism-Drosophila Model System

The interactions of yeasts growing in decaying cactus tissue with and without 2-propanol were studied with respect to the costs and benefits provided to three cactophilic Drosophila species (D. mojavensis, D. arizonensis and D. buzzatii). Two common cactus yeasts, Candida sonorensis and Cryptococcus cereanus, which can tolerate and metabolize 2-propanol, provide benefits to the three Drosophila species in the presence of the alcohol, as compared with another common cactus yeast, Pichia cactophila, which has less tolerance and cannot metabolize 2-propanol. Because 2-propanol is commonly found in decaying cactus tissue and C. sonorensis and Cr. cereanus are also frequently recovered from the rotting tissue being utilized by the Drosophila species, the interactions described here are viewed as a possible adaptation in which the yeast provides benefits to one of its vectors by metabolism of 2-propanol in the habitat.

Pichia pseudocactophila, a new species of yeast occurring in necrotic tissue of columnar cacti in the North American Sonoran Desert

A description is given of Pichia pseudocactophila, a new species of yeast which is closely related to Pichia cactophila. Pichia pseudocactophila is heterothallic and produces four-spored asci, whereas P. cactophila is homothallic and its asci contain a maximum of two spores. Their morphological and physiological properties and G + C contents of their nuclear deoxyribonucleic acids (DNAs) are similar, but the DNA of P. pseudocactophila shows only 35% homology with the DNA of P. cactophila. In addition, strains of P. pseudocactophila have electrophoretic enzyme patterns for glucose-6-phosphate dehydrogenase, hexokinase-1, hexokinase-2, and phosphoglucomutase that allow them to be distinguished from P. cactophila strains. Lastly, P. pseudocactophila has been found only in rotting tissue of columnar cacti (Pachycereus pringlei and close relatives) in Mexico and southern Arizona, whereas P. cactophila is a host-plant generalist and is distributed worldwide in all cactus species so far investigated.

Heterogeneity of the yeast flora in the breeding sites of cactophilic Drosophila

Yeasts were isolated from 278 rots in cladodes and basal stems of Opuntia stricta sampled over 15 months at one locality. The most common yeast species among 370 nonidentical isolates representing 20 species were Candida sonorensis (27% of isolates) and Pichia cactophila (24%). These species generally were present in all months and throughout the area sampled. Other relatively common species (3–9% of alt isolates) had much more restricted distributions in both time and space. The mean number of yeast species per rot was 1.27 ± 0.082, and no yeasts were isolated from 20% of the rots sampled. Significant differences among months or seasons in average number of yeast species per rot were due primarily to lower numbers in the summer and winter months, while differences among rot types resulted mainly from a higher number in the longer persisting basal stem rots. Both hierarchical diversity analysis and analysis of the distributions of the 10 most common yeast species showed temporal effects (month or season) to be the major component contributing to heterogeneity, while microgeographic effects and rot type also were significant. The seasonal and microgeographic heterogeneity are interpreted in terms of longer persisting rots in part of the locality providing a reservoir for yeast species through the winter and are discussed in relation to the maintenance of genetic variation in cactophilic Drosophila that utilize the rots as feeding and breeding sites.

Larval selectivity for yeast species by Drosophila mojavensis in natural substrates

The yeast flora found in the major substrates of Drosophila mojavensis and in larval guts was studied both qualitatively and quantitatively. Quantitative studies show that, in four of the five substrates tested, the larvae did not contain a random sample of the yeasts present in the substrates. One widely distributed cactus yeast, Pichia cactophila, was typically in greater frequency in the larvae than in the substrates. Another cactus yeast, Candida sonorensis, typically exhibited the opposite relationship. Laboratory tests support larval preference behavior rather than differential digestion as being primarily responsible. Larvae are capable of distinguishing between patches of different yeast species and spend more time in patches of preferred yeasts. D. mojavensis appear to be ecological (host plants) generalists and physiological (yeasts) specialists in contrast to the other cactophilic Drosophila. Selective feeding by D. mojavensis larvae in natural substrates may represent optimal foraging behavior.