AKADEMIN FÖR HÄLSA OCH ARBETSLIV Avdelningen för arbets- och folkhälsovetenskap Growth and biodegradation by Sporidiobolales yeasts in vanillin-supplemented medium Natalia González Gaarslev 2017 Examensarbete, G2E, 15 hp Biologi Examensarbete i Biologi Handledare: Sandra A. I. Wright Examinator: Mikael Lönn Summary Studies of biodegradation in lignins by basidiomycetes yeasts show the conversion of lignin in various degradation products among which vanillin, a valuable substance, suggested to be a strong inhibitor of both fermentation and growth of yeasts, stands. Sporidiobolales yeasts used in these experiments were aimed to be identified by their highly conserved ITS region as well as studied in vanillin- supplemented medium through, vanillin-supplemented plates, TLC and Neubauer’s chamber to find out which, among the several isolates tested, were the most resistant ones, understand how they take up vanillin and how their growth is affected by the presence of the phenolic compound. Two strains were identified as Rhodotorula babjevae. One of them, L4, together with LS22, Rhodosporidium kratochvilovae, could withstand and biodegrade high concentrations of vanillin, producing biodegradation products with Rf values similar to the ones know for vanillic acid and vanillyl alcohol. Better growth in medium supplemented with small doses of vanillin was found, as well as disparity among the same species and their metabolic features, therefore, herbicides resistance was suggested as a reason for strains divergence. Further morphological-species comparison could also describe if there exist a relation between them. Resumen Estudios sobre la biodegradación de ligninas por levaduras basidiomicetes muestran la conversión de lignina en distintos productos de degradación, entre los cuales se encuentra la vainillina, un fuerte inhibidor de la fermentación y el crecimiento de levaduras. Las levaduras Sporidiobolales utilizadas en estos experimentos han intentado ser identificadas a través de la región ETI, muy conservada, además de estudiadas en medios suplementados con vainillina mediante placas suplementadas con vainillina, CCF y cámara de Neubauer para averiguar cuáles son las cepas más resistentes, entender cómo metabolizan la vainillina y cómo su crecimiento se ve afectado por la presencia de dicho compuesto. Dos cepas fueron identificadas como Rhodotorula babjevae. Una de ellas, L4, junto con con la cepa LS22, Rhodosporidium kratochvilovae, pudieron soportar y biodegradar elevadas concentraciones de vainillina, originando productos de biodegradación con valores de Rf similares a los del ácido vanílico y alcohol vanílico previamente conocidos. Se encontró un crecimiento mejor en medios suplementados con pequeñas dosis de vainillina además de una disparidad entre mismas especies y sus características metabólicas, así, herbicidas han sido sugeridos como una posible causa en dicha divergencia. Una futura comparación morfología-especie podrá describir si existe relación entre ambos. Key words/Palabras clave: Sporobolomyces/Sporobolomyces, Vanillin/Vainillina, Internal Transcribed Spacer (ITS)/Espaciador Transcribible Interno (ETI), Thin layer chromatography (TLC)/Cromatografia en capa fina (CCF), Neubauer’s chamber/Cámara de Neubauer. Contents Summary ........................................................................................................................... 2 1. Introduction .................................................................................................................. 2 1.1. Background ............................................................................................................ 2 1.1.1. Lignin, an abundant organic polymer in Earth ............................................... 2 1.1.2. Vanillin as a biodegradation product from lignin ........................................... 2 1.1.3. Biodegradation of vanillin by microorganisms .............................................. 3 1.1.4. Sporidiobolales yeasts .................................................................................... 3 1.1.5. Ecological uses of Sporidiobolales yeasts ...................................................... 4 1.1.6. Methods of identification of fungi .................................................................. 4 1.2. Aims, objectives .................................................................................................... 4 2. Materials and methods .................................................................................................. 5 2.1. Culture media ........................................................................................................ 5 2.1.1. Yeast extract-peptone-dextrose (YEPD) ........................................................ 5 2.1.2. Lilly-Barnet, LiBa (Lilly VG, 1951) .............................................................. 5 2.1.3. LiBa-Vanillin (VA) stock solution 0.1 M ...................................................... 5 2.2. Yeast collection ..................................................................................................... 5 2.3. Identification experiments ..................................................................................... 7 2.3.1. Genomic DNA extraction ............................................................................... 8 2.3.2. Polymerase chain reaction (PCR), amplification of ITS regions ................. 10 2.3.3. PCR product classification ........................................................................... 11 2.3.4. Purification of the PCR product gel bands ................................................... 11 2.3.5. Verification of an adequate amount of DNA in the samples ........................ 12 2.3.6. Preparation of sequencing samples .............................................................. 12 2.3.7. Sequencing ................................................................................................... 12 2.3.8. Identification ................................................................................................. 13 2.4. Vanillin resistance experiments ........................................................................... 13 2.4.1. Selection test of the most resistant isolates .................................................. 13 2.4.2. Vanillin resistance and degradation in liquid cultures ................................. 14 2.5. Yeast growth in vanillin supplemented medium ................................................. 15 2.5.1. Cell count in Neubauer’s chamber ............................................................... 16 3. Results ........................................................................................................................ 16 3.1. Identification experiments ................................................................................... 16 3.1.1. DNA extraction ............................................................................................ 16 3.1.2. PCR product ................................................................................................. 16 3.1.3. Gel elution and recovery of purified ITS fragments .................................... 18 3.1.4. Verification of required minimum concentration of template DNA ............ 18 3.1.5. Identification results ..................................................................................... 18 3.2. Vanillin experiment: selection of most vanillin resistant isolates ....................... 19 3.3. Vanillin biodegradation in liquid cultures, TLC results ...................................... 20 3.4. Vanillin resistance: Neubauer’s chamber results................................................. 22 4. Conclusions and discussion ........................................................................................ 23 5. References .................................................................................................................. 24 1 1. Introduction 1.1. Background 1.1.1. Lignin, an abundant organic polymer in Earth Lignin is a high molecular weight three-dimensional macromolecule (Hedges & Mann, 1979), a complex heteropolymer (Campbell & Sederoff, 1996) synthesized in vascular plants through the dehydrogenative polymerization of three cinnamyl alcohols: 4- hydroxycinnamyl alcohol, coniferyl alcohol and synapyl alcohol (Boudet, et al., 1995) . Lignin is the main structural component in plants secondary wall (Guo, et al., 2001) being deposited in wall cells at the last stages of their differentiation (Boudet et al., 1995). Besides mechanical support, lignin plays a significant role in both water transport and defence in vascular plants (Campbell & Sederoff, 1996). Vascular plants, mainly constituted of lignin, cellulose and hemicellulose (Demain, et al., 2005) when dead, remain in the environment as lignocellulosic biomass, considered a natural renewable resource due to its abundance (Demain et al., 2005). Lignin, therefore, is one of the most abundant polymers in nature (Fengel & Wegener, 1984) and due to its non-water solubility and optical inactivity, the study of its degradation is very complicated (Fengel & Wegener, 1984). Lignin is known to be very resistant to microbial degradation (Higuchi, 1990). Studies of biodegradation in decayed wood lignins by whiterot basidiomycetes groups (Hata, 1966; Kirk & Chang, 1974) show the conversion of lignin in several degradation products among which vanillin, considered a valuable substance, stands (Henderson, 1961; Higuchi, 1981; Zimmermann, 1990).