This thesis presents a systematic investigation ofyeasts associated with wine grapes cultivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc, Semilion) grape varieties were examined throughout grape cultivation. The yeast-like fungus, Aureobasidium pullulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodotorula and Sporobolomyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hanseniaspora and Metschnikowia species for the 2001-2002 seasons, but not for the drought affected, 2002-2003 seasons. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast populations and species diversity than intact healthy berries. PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 103 CFU/g were not detected. In some cases, PCR-DGGE revealed the presence ofyeasts (Candida galli, C. zemplinina) not isolated by culture. Fermentative wine species (Kluyveromyces, Torulaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures. Significant morphological and genetic variability were detected among A. pullulans and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings ofAureobasidium, Hormonema and Kabatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Bacillus thuringiensis, several wine yeasts, and some spoilage and mycotoxigenic fungi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice. A species-specific probe was developed for the identification of the wine spoilage yeast, Zygosaccharomyces bailii in a microtitre plate hybridization assay. The probe detected 102 cells/ml in wine, reliably differentiating Z. bailii from other Zygosaccharomyces and other wine-related yeasts.

Hammarby Sjöstadsverk is a pilot plant operated by IVL Swedish Environmental Research Institute and Royal Institute of Technology (KTH) which treats municipal wastewater. Different processes are used of which one is a Sequential Batch Reactor (SBR) with activated sludge process. The activated sludge process is an effective biological municipal wastewater treatment process. Foaming and floating sludge is a significant problem for municipal wastewater treatment plants globally, that appears from time to time and a full understanding of the process causing this phenomenon is still not available. The problem can have multiple causes such as imbalance of microorganisms, nutrients, toxicity, pH, DO, temperature, denitrification, nitrification etc. The scope of this thesis project is to find out which microorganism’s cause foaming and bulking and how to control it. For this investigation, the Hammarby Sjöstadsverk pilot wastewater treatment plant in Stockholm was utilized. The laboratory facilities at KTH were used during thesis work. Laboratory methods were used to cultivate and isolate filamentous organisms from foam, sludge and inlet wastewater samples to investigate by microscopy and see which microorganisms exist in the activated sludge process were performed. General cultivation media for bacteria and fungi were used for culturing microorganisms. Isolation of some filamentous forms was based on colony morphology on an agar plate and microscopy. Microscopic observation of foam and isolates were performed, comparing cell morphology with literature studies. The sludge showed very few types of protozoa. The isolated and cultivated samples were fungi, yeast-like fungi and bacteria. The organisms are most likely the reason for foaming and bulking. Other reasons were also investigated by comparing retrieved data from IVL with literature studies; the process levels such as pH, DO, temperature and nutrients proved to be low or varying. The reason to why fungi and yeast-like microorganisms habitate in the basin depend on a low pH level average of 6.7. A method for effective settling is to withstand a cutting-pump to the container. This was tested during this project and resulted in increased levels of floating sludge during a period of time. Hence this is not a recommended treatment solution. Suggested treatment methods for further studies are to introduce protozoa to treat foaming and bulking, or by raising the pH and DO. This has not been covered within this project. / Hammarby Sjöstadsverk är en testanläggning som drivs av IVL Svenska Miljöinstitutet och KTH som består av ett avloppsreningsverk i pilotskala. Verket behandlar kommunalt avloppsvatten i en Sekvenserad Batch Reaktor (SBR) med aktiv slam processen. Den aktiva slamprocessen är en effektiv biologisk reningsmetod av avloppsvatten. Skumning och flytslam är ett stort problem för avloppsreningsverk globalt sett. Orsaken till dessa problem kan vara många, som t.ex. obalans av mikroorganismer, näringsämnen, toxicitet, pH, löst syre, temperatur, denitrifikation, nitrifikation osv. Målet med projektet är att ta reda på vilka organismer orsakar skumning och flytslam hos Hammarby Sjöstadsverk i Stockholm. Vad som orsakar dessa samt vilka kontrollmetoder som fungerar mot skumning och flytslam. Under projektet användes laboratoriet på KTH campus för att kultivera och isolera filamentösa organismer för att undersöka dessa under mikroskop och även se vilka mikroorganismer faktiskt existerar i skummet och flytslammet. Ympning av bakterier och fungi användes med hjälp av media. Isolering av vissa filamentösa organismer baserades på dess morfologi på agar plattor. Mikroskopiska observationer av skummet och isoleringar utvärderades och jämfördes med relevant litteratur om cellmorfologi. Resultaten av isolaten var fungi, jäst-liknande fungi och bakterier. Organismerna är mest sannolikt anledningen till skumning och flytslam. Andra anledningar så som datajämförelser med litteraturstudier visade att processens parametrar så som pH, löst syre, temperatur och näringsämnen visades vara låga eller varierande. Anledningen till att exempelvis fungi och jäst-liknande organismer trivdes berodde på låga pH nivåer som i snitt var 6.7. En föreslagen metod för att gynna sedimentering är att inte använda en skärpump till bassängen. Detta testades under projektets gång och resulterade under en period i ökade nivåer av flytslam. Därmed är denna metod inte att föredra. Ett förslag till en biologisk metod introduceras med att tillföra protozoer för att behandla skumning och flytslam, eller att höja pH och löst syre i bassängen. Detta har dock inte undersökts inom ramen för detta projekt.