Growth and survival of Saccharomyces cerevisiae in soil

Bester, Reinhard (Stellenbosch : University of Stellenbosch, 2011-10)

Thesis

ENGLISH ABSTRACT: Saccharomyces cerevisiae is commonly associated with the wine industry. However, this yeast was also isolated from soils not associated with vines. Despite the fact that S. cerevisiae is not perceived as an autochthonous soil yeast, its interaction with other soil microbiota suggests the contrary. Aside from a few in vitro studies, the fate of S. cerevisiae in soil is largely unknown. This may partly be ascribed to the lack of reliable methods to enumerate fermentative yeasts in soil. Consequently, we evaluated an enumeration procedure for fermentative yeasts in soil, whereby yeast malt extract (YM) agar plates containing selective agents, were incubated in anaerobic jars before the colonies were enumerated. This procedure proved to be selective for fermentative yeasts, such as industrial strains of S. cerevisiae. We then commenced studying the growth and survival of S. cerevisiae in soil differing in moisture content and nutrient levels, using S. cerevisiae strain S92 and the genetically modified strain S. cerevisiae ML01, as well as two autochthonous soil yeasts, Cryptococcus laurentii and Cryptococcus podzolicus. The yeast strains were each inoculated into three series of microcosms containing sterile soil with a moisture content of ca. 30% (v/w), a moisture content of ca. 15% (v/w), or a moisture content of ca. 30% supplemented with nutrients used in agriculture. Growth of each strain was monitored for a period of 48 days and all the yeasts were found to grow or survive under these conditions, up until the end of the incubation period. Generally, the cryptococci reached larger population sizes in the soil than the Saccharomyces strains, which may be due to their ability to utilize a wider range of carbon sources and to survive in semi-arid soils. Aside from cell numbers observed in nutrient supplemented soil, in which S. cerevisiae ML01 reached higher numbers than S92, there was no significant difference between the growth and survival of the Saccharomyces strains. In all the microcosms, metabolic rates, as determined by measuring CO2 emissions from soil, reached a maximum within the first day and then declined over the remainder of the trial, possibly due to depletion of nutrients. Differences in CO2 emissions from the different series of microcosms were attributed to different metabolic rates and energy expenditure needed to maintain yeast populations under different conditions. Each of the above-mentioned yeasts was subsequently inoculated in a microcosm prepared from non-sterile soil and monitored using selective enumeration procedures. The Saccharomyces strains were enumerated using the above-mentioned soil dilution plates incubated in anaerobic jars. The presence of natural soil biota caused a decrease in viable yeast numbers for all strains and this was ascribed to competition with and predation by other soil borne organisms. Further evidence for competition and/or amensalism impacting on Saccharomyces populations in soil was obtained when monitoring co-cultures of Saccharomyces with C. laurentii 1f and C. podzolicus 3f in soil microcosms, revealed a significant reduction in Saccharomyces numbers during a 28 day incubation period. However, when the two Saccharomyces strains were cultured in soil microcosms inoculated with a protistan predator, populations of both strains increased and remained at these high levels for the duration of the trial. These findings point to a possible symbiosis between Saccharomyces and the protista whereby the predators ensure continuous nutrient cycling within the soil microcosms. In the final part of the study, epifluorescence microscopy revealed that, similar to known soil cryptococci, the two Saccharomyces strains were able to form biofilms in oligotrophic conditions. The results of this study showed that in the presence of natural soil microbes, no differences exist between the growth and survival of S. cerevisiae S92 and S. cerevisiae ML01. Also, the findings point to a natural niche for this species somewhere in the soil habitat.

AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae word algemeen met die wynindustrie geassosieer. Hierdie gis is egter ook uit grond geïsoleer wat nie met wingerd geassosieer word nie. Ten spyte van die feit dat S. cerevisiae nie as ‘n outogtoniese grondgis beskou word nie, dui sy interaksie met ander grondmikrobiota op die teendeel. Behalwe vir ‘n paar in vitro studies, is die lot van S. cerevisiae in grond grootliks onbekend. Dit mag gedeeltelik aan die gebrek aan betroubare metodes om fermenterende giste in grond te tel, toegeskryf word. Ons het gevolglik ‘n tellingsmetode vir fermenterende giste in grond geëvalueer waarin gis-mout ekstrak (GM) agar plate, bevattende selektiewe agente, in anaërobiese flesse geïnkubeer is voordat die kolonies getel is. Hierdie metode was selektief vir fermenterende giste, soos die industriële stamme van S. cerevisiae. Hierna is die groei en oorlewing van S. cerevisiae bestudeer in gronde met verskillende vog- en nutriëntvlakke deur gebruik te maak van S. cerevisiae stam S92 en die geneties gemodifiseerde stam S. cerevisiae ML01, asook twee outogtoniese grondgiste, Cryptococcus laurentii en Cryptococcus podzolicus. Die gisstamme is elk geïnokuleer in drie reekse van mikrokosmosse bestaande uit steriele grond met ‘n vogvlak van ca. 30% (v/w), ‘n vogvlak van ca. 15% (v/w), of ‘n vogvlak van ca. 30% aangevul met landbounutriënte. Die groei van elke stam is waargeneem vir ‘n tydperk van 48 dae en al die giste het onder hierdie omstandighede tot aan die einde van die inkubasietydperk gegroei of oorleef. Oor die algemeen het die cryptococci groter populasies in die grond gevorm as die Saccharomyces stamme, wat toegereken kan word aan hul vermoë om ‘n wyer reeks koolstofbronne te benut en om in droë gronde te oorleef. Behalwe dat S. cerevisiae ML01 ‘n hoër aantal selle in nutriënt aangevulde grond behaal het as S92, was daar geen beduidende verskil tussen die groei en oorlewing van die Saccharomyces stamme nie. In al hierdie mikrokosmosse het die metaboliese tempo, soos bepaal deur CO2 vrystellings vanuit grond te meet, ‘n maksimum bereik binne die eerste dag en dan het dit afgeneem oor die res van die toetsperiode, waarskynlik as gevolg van die uitputting van die nutriënte. Verskille in die CO2 vrystellings wat vir die verskillende reekse van mikrokosmosse aangeteken is, is te wyte aan die verskillende metaboliese tempo’s en energiegebruik benodig om gispopulasies onder verskillende omstandighede in stand te hou. Elk van bogenoemde giste is vervolgens geïnokuleer in ‘n mikrokosmos wat voorberei is van nie-steriele grond, en waargeneem deur selektiewe enumerasie prosedures toe te pas. Die Saccharomyces stamme is getel deur gebruik te maak van bogenoemde grondverdunningsplate wat in anaërobiese flesse geïnkubeer is. Die teenwoordigheid van natuurlike grondbiota het in alle stamme ‘n afname in lewensvatbare gisgetalle veroorsaak en is toegeskryf aan die kompetisie met en predasie deur ander grondorganismes. Verdere bewys van die impak van kompetisie en/of amensalisme op Saccharomyces populasies in die grond, is die beduidende afname in Saccharomyces getalle tydens ‘n 28 dag inkubasie tydperk, waartydens ko-kulture van Saccharomyces stamme met C. laurentii 1f en C. podzolicus 3f in grond mikrokosmosse ondersoek is. Toe die twee Saccharomyces stamme egter in grond mikrokosmosse opgekweek is wat met ‘n protistiese predator geïnokuleer is, het populasies van albei stamme gegroei en om hierdie hoë vlakke gebly tot aan die einde van die toets. Hierdie bevindings dui ‘n moontlike simbiose tussen Saccharomyces en die protista aan waardeur die predatore deurlopende nutriëntsiklering binne die grondmikrokosmos verseker. In die laaste deel van die studie toon epifluoressensie mikroskopie aan dat, net soos bekende grond cryptococci, die twee Saccharomyces stamme in staat is om biofilms in oligotrofiese omstandighede te vorm. Die resultaat van die studie toon aan dat in die teenwoordigheid van natuurlike grondmikrobe daar geen verskil tussen die groei en oorlewing van S. cerevisiae S92 en S. cerevisiae ML01 is nie. Die bevindings dui ook aan dat daar ‘n natuurlike nis vir hierdie spesie iewers in die grondhabitat is.

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