Biotehnoloogia magistritööd - Master's theses

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Browsing Biotehnoloogia magistritööd - Master's theses by Subject "Acetogens"

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    Insights into gas fermentation optimisation for enhanced acetate production
    (Tartu Ülikool, 2024) Mishchuk, Anatolii; Acuna Lopez, Pedro, juhendaja; Quataert, Koen, juhendaja; Valgepea, Kaspar, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Conversion of CO2 utilising gas fermenting acetogens is a feasible and environmentally beneficial solution to the global problem of greenhouse gas emissions. Homoacetogens are an especially intriguing type of microorganisms since they can yield acetate as their primary metabolic product via the Wood–Ljungdahl pathway. A product inhibition mechanism is observed when acetate accumulates in high concentrations, adversely impacting bacterial growth and acetate production. The tolerance of these microorganisms towards varying concentrations of acetate and under identical conditions has not previously been investigated. However, it has been defined as crucial for selecting the most robust acetogen for its use at industrial scale. The designed experimental setup for acetate tolerance studies, including positive and negative controls, was intended to answer this scientific question. Following the pre-screening test, it was observed that the carbon source affected the microbial ability to tolerate acetate; bacteria performed substantially better when a more energy-rich carbon source, glucose, was supplemented. The latter finding was accounted for when designing the main screening setup for acetate tolerance in four well-known homoacetogenic strains, where Moorella thermoacetica and Thermoanaerobacter kivui performed the best. Then, T. kivui was utilised to perform pH-controlled fermentations in pressurised gas bioreactors. Furthermore, the outcomes of this master's thesis support the hypothesis that gram-positive anaerobes undergo variation in their Gram staining due to oxygen exposure. This thesis provides valuable information for the future selection and screening of homoacetogenic strains and the effect of pH-controlled cultivation in a pressurised bioreactor.
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    Optimization of plasmid curing from genetically engineered Clostridium autoethanogenum
    (Tartu Ülikool, 2024) Udemezue, Victoria Chinonyerem; Valgepea, Kaspar, juhendaja; Shaikh, Kurshedaktar Majibullah, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    The accumulation of greenhouse gases (GHGs) released by harmful human activities involving the combustion of fossil-fuels is a driver of climate change that threatens biosustainability on Earth. Microbial gas fermentation provides an attractive option to capture CO2 while also enabling biomanufacturing of chemicals, fuels, and proteins. Acetogens are the preferred biocatalysts for gas fermentation as they can use CO2 as their sole carbon source (with H2 as energy source). However, genetic engineering of acetogens to better understand their metabolism and develop cell factories is challenged by slow growth, very low transformation efficiencies, and inefficient plasmid curing. In this thesis, we developed a CRISPR/Cas9-based curing plasmid (C-plasmid) tool for optimized plasmid curing from the model-acetogen Clostridium autoethanogenum. Firstly, the C-plasmid was constructed to express Cas9 and a gRNA targeting the ColE1 origin of replication in both the C-plasmid and an editing plasmid (E-plasmid). Next, the C-plasmid and the non-template gRNA plasmid (N-plasmid) were electroporated into C. autoethanogenum harboring an E-plasmid used for gene deletion and culture were plated on agar. Plate counting and PCR screening showed no presence of plasmids in colonies transformed with either C-plasmid or N-plasmid. This implies that cells were cured of plasmids by the act of electroporation and transformation of a C-plasmid might not be needed. In any case, this thesis seems to have identified a significantly more efficient plasmid curing method for C. autoethanogenum. Further tests are needed to confirm these observations and its applicability to other genetically-engineered C. autoethanogenum strains. The methodology has potential to contribute towards improving genetic engineering workflows for acetogens.