Isolation and Identification of oleaginous yeasts Margarida dos Reis Pereira Pataco1 1Institute for Bioengineering and Biosciences, Instituto Superior Técnico, University of Lisbon Abstract: An alternative for a sustainable biodiesel production is the accumulation of single-cell oils by oleaginous yeasts. Xylose is the second most abundant sugar in plant biomass after glucose, but its metabolization by yeasts is usually not efficient, limiting the utilization of lignocellulosic materials for the production of single-cell oils. The main objective of this work is to find efficient oleaginous yeasts with the ability to metabolize xylose. Yeast isolation from several samples was performed and the yeast isolates obtained were taxonomically identified based on the sequencing of D1/D2 and ITS regions from ribosomal DNA, yielding 78 strains from 32 species. The 67 non-pigmented strains isolated and 58 additional strains from the IST Yeast Culture Collection were screened for lipid production using a qualitative method based on Rhodamine B staining. This allowed the selection of 37 potential oleaginous strains grown in glucose and 21 grown in xylose. The species Candida boidinii and Pichia membranifaciens were considered potential oleaginous xylose metabolizing yeasts. A genome mining was performed to identify putative sugar transporters in their genomes, allowing the identification of 33 sugar transporters for Candida boidinii and 10 for Pichia membranifaciens, to which a phylogenetic analysis was done. The phylogenetic trees obtained for Candida boidinii and Pichia membranifaciens sugar transporters showed, respectively, twelve and eight transporters close to known xylose transporters. Future studies will involve the qualitative and quantitative characterization of the lipids produced by these species, as well as the optimization of growth conditions, higher lipid productions and accumulations. Key words: Oleaginous yeast; Intracellular lipids; Bio-oils; Xylose utilization; Lignocellulose utilization; Biodiesel. Introduction enhance the productivity of yeasts in fatty acid production, which is not an easy task, since the Fossil fuel energy is responsible for a substantial part production and composition of lipids by oleaginous of today's demand of industry and economy1. yeasts is subject to change, depending on the However, their exploration and use are not cultivation conditions and stress under growth7. Also, sustainable and are responsible for carbon dioxide one of the main reasons for the high cost of biodiesel emissions to the atmosphere, increasing the production, is the price of the raw material used, since greenhouse effect and contributing to global it makes 70-85% of the total value of this fuel warming1. production8. The solution is to use a cheap fermentation medium, therefore, organic wastes and A small number of microorganisms accumulate lipids agro-food-forest residues are an interesting raw above 20% of their cell mass as a reserve storage material for the synthesis of lipids by oleaginous 2 material . These microorganisms include some yeasts yeasts9. Consequently, when biomass is used for the and fungi and a few algae, and are called oleaginous generation of fuels, the waste is converted into a species, being known for their oil accumulation that resource, making this an efficient process10. may reach values as high as 70% of their cell biomass2. Bio-oils are composed by triglycerides and Xylose is the second most abundant sugar in plant are very similar to vegetable oils, which increases the biomass after glucose, but most of the oleaginous interest in its use for the synthesis of fatty acid methyl yeasts use glucose preferentially to other sugars, due esters into biodiesel3. to carbon catabolite repression, increasing cultivation times when a mixture of xylose and glucose is present, Notwithstanding other microorganisms accumulating consequently reducing lipid productivity11,12. lipids, yeasts show several advantages, not only the Additionally, xylose uptake into cells have been fact that they can accumulate triglycerides, but also highlighted as a significant limitation in the efficient because they include a broader metabolism of feed utilization of lignocellulosic sugars by yeasts and sources and have tolerance to higher ranges of pH, efforts have been made to find specific efficient xylose 4 inhibitors and ionic strength . Additionally, oleaginous transporters13. Therefore, yeasts that successfully co- yeasts are usually non-pathogenic, most are suitable utilize glucose and xylose, would allow an effective for larger fermentations and are easy to manipulate and sustainable process for lipid production from 5,4 genetically . renewable cheap materials11. However, at the moment, biodiesel production is not Nowadays, there are more than 40 known oleaginous competitive with fossil fuels, being necessary to yeast species already identified and distributed among reduce the cost of its production6. It is necessary to 1 several clades and with different inter- and intra- dilutions were plated on agar media (isolation medium specific lipid accumulation potential14,15. Moreover, the with glucose and isolation medium with xylose, with search for new oleaginous species and strains with 20g/L agar) and incubated at 30ºC. After this step, potential for higher lipid accumulation is crucial and is single colonies of the different strains from the in constant actualization15. Therefore, this work has isolation step were selected based on their the purpose of finding effective oleaginous yeast morphology and streaked into new media. strains that also have the ability of assimilating xylose. Soil samples: For the isolation of yeasts from soil Materials and Methods samples, in the primary enrichment, one table spoon of soil (~1g of soil, that usually contains 103 yeast Part of the yeast isolation and identification work here cells) was inoculated in 50 mL of enrichment medium described was done in collaboration with my colleague (with glucose and xylose as carbon sources) and Érica Vieira from the MSc degree in Biotechnology. incubated at 30ºC, 150 rpm, for 48 h. In the case of samples of olive tree soil 1 and garden soil, a half Sampling: Samples were collected from various tablespoon was used for medium inoculation. After, natural sites, in order to proceed with the isolation of the scale-up enrichment consisting of in inoculating 1 yeasts. Samples were collected from soil near two mL from Primary Enrichment in 49 mL of enrichment different olive trees (1-from Póvoa-Cadaval and 2- medium followed by incubation at 30ºC, 150 rpm, for from Ferreira do Alentejo) as well as from soil near an 48 h. This was followed by Differential Enrichment, oak tree, from a vegetable garden, both from Póvoa- also inoculating 1 mL from Scale-up Enrichment into Cadaval, and from surface seawater collected in 49 mL of isolation medium with glucose (glucose as Berlengas and Arrábida. Olives (from the olive tree 2), carbon source) and isolation medium with xylose and olives curing water were also used, as well as (xylose as carbon source) media and incubation at physalis, juniper berries, plums and walnut green 30ºC, 150 rpm for 48 h. All these media were done husk. Finally, sediments from a permafrost lake with ddH2O and Chloramphenicol was added at a located in the Artic provided by Dr. João Canário from concentration of 100 μg/mL after autoclaving. Next, Centro de Química Estrutural of IST and soils the isolation consisted in the steps done for marine contaminated with fuel provided by Dr. Ricardo Santos samples. from Laboratório de análises of IST were also used to isolate yeasts. Fruit samples: To isolate the strains from olives, plums, physalis and juniper berries, demineralized sterile water was added to a tube containing the Marine samples: The Differential Isolation: sample and vortexed. After, 1 mL or 5 mL of the water isolation was based on the method described Zaky et from plums’ sample and 10 mL from physalis and al. (2016)16. This method starts with three enrichment juniper berries (no growth was observed with only cycles. In the primary enrichment, 500 mL, 1 L, 2 L, 1mL) were inoculated in 50 mL of enrichment medium and 3 L of surface seawater were filtered, using a and incubated at 30ºC, 150 rpm, for 48 h. The metallic filtration system (SS Filter Holder 100mL, subsequent steps were made as described for the 47mm; by Mili-Q). The filter was introduced into 100 isolation from marine and soil samples. mL of enrichment medium, which contains glucose and xylose as carbon sources (30 g/L Glucose; 30 g/L Olives-curing water sample: To isolate the strains Xylose ; 3 g/L Malt extract ; 3 g/L yeast extract ; 5 g/L from olives-curing water, the procedure was similar to Peptone; 1 g/L (NH4)2SO4 ; 0.25 g/L KH2PO4 ; pH 5)), the one described for soil samples, but in the first step, and incubated at 30ºC, 150 rpm. In the scale-up 500 μL of this water were inoculated in 50 mL of enrichment, 20 mL from Primary Enrichment were enrichment medium and incubated at 30ºC, 150 rpm, added to 180 mL of enrichment medium and for 48 h. incubated at 30ºC, 150 rpm, for 48 h. In the differential enrichment, 10 mL from Scale-up Enrichment were Direct isolation: Additionally, the strains obtained added to 90 mL of isolation medium with glucose, a from cabbage, nuts and sediments or from a medium with glucose as the carbon source (60 g/L permafrost lake located in the Artic were isolated in a Glucose; 3 g/L yeast extract; 5 g/L Peptone; 1 g/L direct way: demineralized sterile water was added to (NH4)2SO4; 0.25 g/L KH2PO4; pH 5) and isolation a tube containing the cabbage or nuts sample and medium with xylose, a medium with xylose as the vortexed for 2 minutes. The sample of sediments from carbon source (60 g/L Xylose; 3 g/L yeast extract; 5 a permafrost lake from Artic was already suspended g/L Peptone; 1 g/L (NH4)2SO4; 0.25 g/L KH2PO4; pH in the lake water.