The document discusses developing a screening process to identify non-Saccharomyces yeast species for use in the fermentation industry. Various wild yeast strains were isolated from different locations including the air, fruits, and water/ice samples. The isolated yeasts were identified using classical characterization methods including morphology, physiology, and biochemical tests. Preliminary tests found that habitat location may influence a strain's ability to ferment standardized wort, warranting further study. The project aims to screen strains for aroma production, tolerance, and identification using analytical and molecular methods to uncover new industry applications.

1. The fermentation industry standard is the Saccharomyces species, the main yeast species used in brewing beer (Lodolo et al., 2008). It is very well known that
Saccharomyces cerevisiae is involved in most aroma-forming processes during beer fermentation, transforming wort ingredients into alcohol and aroma compounds,
such as higher alcohols, esters and carbonyl compounds (Pires et al., 2014). However, outside of spontaneous fermentation, there is very little use of non-
Saccharomyces yeasts throughout the beer industry. Meanwhile, approximately three quarters of a million species of yeast are located in different known habitats. Out
of a total of 670,000 species, only 20 species are being used in industry (Michel et al., 2015). Towards exploiting this broad field of unknown species, aromas, and
even new aspects of taste or functionalities for greater ‘mouth-feel’, a screening process is being developed by our project, to be incorporated in the industry
screening process of the future. The screening process includes preliminary biochemical tests for ensuring that the yeasts under investigation do survive in beer wort
and, of course, will ferment it. This includes the carbohydrate fermentation of wort sugars, hop and ethanol tolerance, as well as additional various combinations.
Isolation Location and Methods: Various wild yeast species were isolated from different locations to investigate the relationship between the location and the strains
capacity to ferment on a standardized wort to see if wild yeast species had the potential to develop new sensorial profiles. Isolation methods were adapted with minor
modifications from Clemente-Jimenez et al. (2004). The first series of isolations were obtained using at wort liquid medium in an open container which was left outside
for a duration of 36 hours collecting microorganisms from the air. The second series was isolated from collected fruit samples (apple and quince) allocated from Beskid
Żywiecki, a mountain range in the Outer Eastern Carpathians in southern Poland. Isolation of yeast from fruit samples were adapted with minor modifications from
Kishori et al. (2015). The third isolation samples were collected near the Polish Polar Station in Hornsund, Svalbard, by Sławomir Sułowicz, isolated from water and ice
samples cultered on YPD medium for 7 days in 4˚ C and 18 ˚ C.
Identification by the ‚Classical' System (Kreger-van Rij et al., 1984): Evaluation of morphology and Yeast Activity by alkaline methylene blue staining was adapted
minor modifications from Sami et al. (1994). Identification of the wild yeast strains after staining was achieved by morphological and physiological characterization
according to methods described by Kurtzman et al. (1998). Images were captured using AxioCamERc5s and Zen 2011 blue edition software attached to a Zeiss Primo
Star light microscope.
Development of a screening system to effectively characterize studied yeast by
biochemical analysis (API ID 32c yeast identification), screening of secondary-
metabolites for aroma products by the analytical analysis of fermentation
products using head space gas chromatography (HS-GC-FID), hop/ethanol
resistance by cross-resistance iso-α-acid with EtOH series test, and to develop a
fast and effective way to identify yeast strains by chemotaxonomic methods
such as FAME and the use of Ramen Spectroscopy.
• Lodolo EJ et al. (2008) The yeast Saccharomyces cerevisiae- the main character in beer brewing. FEMS Yeast Res 8(7):1018-36
• Pires EJ et al. (2014) Yeast: the soul of beer's aroma-a review of flavor-active esters and higher alcohols produced by the brewing
yeast. Appl Microbiol Biotechnol 98(5):1937-49
• Michel S et al. (2015) Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora delbrueckii as model.
BMC Genet 16:13
• Clemente-Jimenez et al. (2004) The Genetics of Non-conventional Wine Yeasts: Current Knowledge and Future Challenges. Food
Microbiol 21:149–155
• Kishori et al. (2015) Isolation of yeast and ethanol production from papaya (Carica papaya) and grape (Vitis vinifera) fruits. IJSER
6(2):100-104
• Kreger-Vamn Rij et al. (1984) The yeast. A Taxonomic Study. Elsevier Science Publ., Amsterdam
• Sami et al. (1994) Evaluation of the alkaline methylene blue staining method for yeast activity determination. Jour Ferm Bioen 78(3):
212-216
• Kurtzman et al. (1998) Methods for isolation, phenotypic characterization and maintenance of yeasts. Elsevier 4:418 77-100
Image (2)*-Reniform (kidney-shaped) spores as in
Kluyveromyces marxianus. The cell wall is less
rigid than in Saccharomyces (Image 1), allowing
the forma-tion of a linear ascus.
Schizosaccharomyces spp. also form linear asci.
Image (1)*-Spores of Saccharomyces.
Heterozygous diploid cells undergo two
successive meiotic nuclear divisions to produce
four haploid spores within the original cell wall.
Lyophilized yeast in different sugar concentration 2%
4%6%.The results shows that yeast metabolism is
dependent on the amout of nutrition. The method FTIR
(infrared spectroscopy) is perfect to identified active group
of substances in mixture for lyophilized yeast studies
(Courtsey of Magdalena Biesiadecka)
Based on the evaluation of morphology and physiological characterization it was possible to
identify the wild yeast strains by the ‘classical’ system described in the methods section. Wild
yeast isolated from different locations seem to have different characteristics, however, further
testing will be needed to determine if there is correlation between the habitat and the ability
to survive on a standardized wort which would be useful for the beverage industry.
Symbol Results
+ sugar break down, with fermentation
+/- sugar break down, no fermentation
- no sugar break down, no fermentation
Image (3)*-Saturn spores, as in some Pichia spp.
and Williopsis saturn us. The ring is located
equatorially on the spherical or lens-shaped
spore, giving (with a little imagination) the
appearance of the planet Saturn.
*Isolated wild yeast strain (light microscopy, magnification x100)