Production of lipids by the oleaginous yeast Cutaneotrichosporon oleaginosus using the organic fraction of municipal solid waste as the feedstock
Author
Nielsen, Line
Term
4. term
Education
Publication year
2022
Submitted on
2022-06-10
Pages
71
Abstract
Biodiesel made from lipids is a promising alternative to fossil fuels, but progress is limited by low lipid yields and high production costs. This thesis explores whether the organic fraction of municipal solid waste (OFMSW) can serve as a low-cost feedstock to grow oil-producing yeast for biodiesel. We compared two process setups: Separate Hydrolysis and Fermentation (SHF), where enzymes first break the waste down into sugars before fermentation, and Simultaneous Saccharification and Fermentation (SSF), where breakdown and fermentation occur at the same time. Untreated waste was enzymatically hydrolyzed for 24 hours to release sugars, and the non-detoxified hydrolysate (i.e., without removing potential inhibitors) was used directly as medium in small-scale shake-flask fermentations. Using the oleaginous yeast Cutaneotrichosporon oleaginosus, we achieved maximum total lipids of 34.53 g per liter of waste medium with SHF after 4 days and 35.30 g per liter with SSF after 7 days. These represent increases of 10.20 g/L and 9.67 g/L, respectively, over a 50% (w/v) OFMSW medium baseline, corresponding to yields of 61.41 g of lipids per kg of dry OFMSW for SHF and 57.80 g per kg for SSF. The results suggest both setups give similar lipid yields, with SHF reaching its maximum faster. In addition, a new vector system, pFLEXI-hyg-yfp, was developed to support future genetic engineering of C. oleaginosus. The plasmid was introduced by Agrobacterium tumefaciens-mediated transformation (ATMT), mutants were verified by colony PCR (a rapid DNA screen of colonies), and an optimized ATMT protocol for C. oleaginosus was established. Finally, the impact of random genomic integration on expression was assessed by tracking YFP fluorescence over time in multiple C. oleaginosus mutants. Overall, this work demonstrates that household organic waste can be converted into yeast oils for biodiesel and provides genetic tools to further improve the production organism.
Biodiesel lavet af lipider er et lovende alternativ til fossile brændsler, men udviklingen bremses af lave lipidudbytter og høje produktionsomkostninger. Denne afhandling undersøger, om den organiske fraktion af kommunalt husholdningsaffald (OFMSW) kan bruges som billig råvare til at dyrke gær, der producerer olie til biodiesel. Vi sammenlignede to gæroprocesser: Separate Hydrolysis and Fermentation (SHF), hvor affaldet først enzymatisk nedbrydes til sukker og derefter fermenteres, og Simultaneous Saccharification and Fermentation (SSF), hvor nedbrydning og gæring sker samtidig. Affaldet blev enzymatisk hydrolyseret i 24 timer for at frigive sukker, og den ikke-detoxificerede hydrolysat (dvs. uden fjernelse af mulige hæmmere) blev brugt direkte som medie i små skalerede rystekolbe-fermenteringer. Med den olieakkumulerende gær Cutaneotrichosporon oleaginosus opnåede vi maksimale totale lipidmængder på 34,53 g pr. liter affaldsmedie med SHF efter 4 dage og 35,30 g pr. liter med SSF efter 7 dage. Det svarer til stigninger på henholdsvis 10,20 g/L og 9,67 g/L i forhold til udgangspunktet med 50 % (w/v) OFMSW, og til udbytter på 61,41 g lipider pr. kg tørt OFMSW for SHF og 57,80 g pr. kg for SSF. Resultaterne indikerer, at de to opsætninger giver sammenlignelige lipidudbytter, men at SHF når sit maksimum hurtigere. Derudover blev et nyt vektorsystem, pFLEXI-hyg-yfp, udviklet for at styrke fremtidige muligheder for genetisk modificering af C. oleaginosus. Vektoren blev indført i gæren ved Agrobacterium tumefaciens-medieret transformation (ATMT), de opnåede mutanter blev bekræftet med colony PCR (en hurtig DNA-test af kolonier), og en optimeret ATMT-protokol for C. oleaginosus blev etableret. Endelig blev effekten af tilfældig integration på genekspression undersøgt ved at måle YFP-fluorescens over tid i flere forskellige C. oleaginosus-mutanter. Samlet viser arbejdet, at organisk husholdningsaffald kan omdannes til gær-olier til biodiesel, samtidig med at der opbygges genetiske værktøjer til at forbedre produktionsorganismen.
[This apstract has been rewritten with the help of AI based on the project's original abstract]