Browsing by master's degree program "Magisterprogrammet i mikrobiologi och mikrobiell bioteknik"

The objective of this thesis was to isolate and characterize new bacteriophages (phages) against clinical Klebsiella pneumoniae strains for phage therapy. K. pneumoniae is causing an emerging threat to global health due to its broad antibiotic resistance profile and hypervirulent strains. New treatment options are urgently needed to defeat the crisis. Phage therapy could provide one option to treat multiresistant K. pneumoniae infections. In this thesis, five new phages were isolated and characterized from Finnish wastewater and Georgian river water against two clinical K. pneumoniae strains. The three phages from Georgian river water, fMtkKpn01, fMtkKpn03, and fMtkKpn04, resembled Drulisviruses based on phylogenetic analysis. The two phages from Finnish wastewater, fJoKpn03 and fJoKpn05 were phylogenetically distinct. fJoKpn03 couldn’t be classified. fJoKpn05 resembled Weberviruses. Based on sequence analysis, none of the phage genomes included any harmful genes that would prevent their use in phage therapy. All phages demonstrated a 6-hour total inhibition to host bacterial growth. Their host range was determined to be narrow, only infecting their respective host strains from the 80 bacterial strains tested. All the phages tolerated high pH well. fJoKpn03 was the only one tolerating very low pH. All phages showed a synergistic effect on the inhibition of bacterial growth when applied together with piperacillin. In conclusion, all five phages proved potential for phage therapy. They demonstrated inhibitory action against K. pneumoniae strains with capsule types against which there previously were no phages in our collection. Due to their narrow host range, they could be suited for personalized phage therapy or used in combination therapy with antibiotics to increase efficacy and duration of action. fJoKpn03 could provide an opportunity for oral administration due to its broad pH stability profile.

The increase of antibiotic resistance is one of the major healthcare threats globally. One potential way to battle against antibiotic resistant bacterial infections is to treat them with the natural opponents of bacteria, bacteriophages, known as phage therapy. The aim of this thesis was to identify new bacteriophages against clinically notable bacterial species such as Escherichia coli, Burkholderia cepacia, Enterococcus faecalis and Enterococcus faecium. Bacteriophages were screened from various origins such as hospital sewage samples, soil samples and manure samples, collected in between 2019 and 2022. The isolated bacteriophages were then initially characterized to evaluate their potential use in phage therapy. In this thesis, two phages (fHo-Eco16, fHo-Eco17) against clinical E. coli isolate and one phage (fHo-Efa06) against clinical E. faecalis isolate were found from the recently collected Finnish hospital sewage sample pool. Both E. coli phages were classified as Felixounaviruses belonging to family of Ounavirinae and class of Caudoviricetes. Enterococcus phage fHo-Efa06 was characterized as Saphexavirus belonging to class of Caudoviricetes. Preliminary genome annotation did not reveal any characteristics of lysogenic lifecycle, or antibiotic resistance or bacterial toxin genes, which would prevent the use of phages in phage therapy. Both E. coli phages (fHo-Eco16, fHo-Eco17) showed narrow host range infecting only the primary host bacterial isolate but none of 29 other tested clinical E. coli isolates. Phage fHo-Efa06 showed relatively broad host range properties infecting nine tested E. faecalis isolates out of 20 tested E. faecalis isolates but no infection capabilities against six tested clinical E. faecium isolates. In conclusion, freshly collected hospital sewage seemed to be optimal environment to find bacteriophages against clinical bacterial isolates. Furthermore, phages fHo-Eco16, fHo-Eco17 and fHo-Efa06 did not display any strictly unsuitable properties which could prevent their use in phage therapy. In turn, to obtain the definitive certainty on the usability of the phages in therapeutic use, in-depth host range screening together with detailed functional and structural annotation for the phage genomes of fHo-Efa06, fHo-Eco16 and fHo-Eco17 should be completed.

The aim of this master’s thesis was to examine university teachers’ professional vision and misconceptions from the perspective of the role of students’ prior knowledge in learning. We also examined how participants’ professional vision and concepts changed during the pedagogical course. University students can also have a lot of misconceptions which differ from scientific view. Those misconceptions can make learning harder and even hinder it. Teachers should recognise these misconceptions and they should be able to support students’ conceptual change in their teaching. Participants (N=73) were life science university teachers. They were selected to this study because they participated in two university pedagogical courses with the same content. Participants’ professional vision, conceptions and beliefs were investigated with a video annotation and two questionnaires. Study is quasi-experimental research with pretest-posttest design. Video annotation and one of the questionnaires were tested before and after the pedagogical course. The delayed questionnaire was collected six months after the course. Purpose of the delayed questionnaire was to know if the teachers have been using the things they learn in a course in their own teaching. At the analysis phase participants were divided in to three groups according to their previous teaching experience and pedagogical courses (novices, experienced teachers, and most experienced teachers). Then we were able to compare these three groups and examine if the teaching experience had any effect on the answers. This study utilized a mixed methods approach and analysis was made with both quantitative and qualitative methods. The results show that pedagogical course changed teachers’ concepts considering learning and teaching. All participants’ groups got better scores in professional vision after the pedagogical course despite their previous teaching experience or pedagogical courses. Novices got lowest scores in the pretest which was expected because they didn’t have any previous experience. Their answers changed significantly in all research aspects. Experienced and most experienced teachers also got better scores in posttest especially in professional vision. Developed professional vision was related to more constructivist beliefs of learning. These findings support previous studies that even short pedagogical course can change teachers’ beliefs and concepts about teaching and learning.

Lignocellulosic biomass is an abundant and sustainable resource to produce valuable products through biorefinery processes. However, the challenge of lignin degradation remains complex. My thesis aimed to investigate the potential of Pleurotus ostreatus, a white rot fungus, for use in biorefinery applications. This study investigated the capability of P. ostreatus to grow and consume technical lignin (organosolv) with great potential for utilization in lignocellulose biorefineries. To further enhance its bioconversion potential, the study also explored the metabolic engineering of P. ostreatus to produce protocatechuate. Additionally, the P. ostreatus PC9 monokaryon strain was found to be suitable for genetic engineering using the CRISPR Cas9 system; however, the inability to disrupt the KU80 gene may have contributed to the lower efficiency of gene targeting observed in this study. The lignin degradation potential of P. ostreatus secretomes was investigated by measuring enzyme activities and using model compounds. The use of P. ostreatus secretomes has shown potential for improving lignin degradation in co-culture with an engineered Aspergillus niger strain which accumulates the protocatechuate, and the addition of nucleophilic thiols, such as L-cysteine, showed promise in promoting lignin bioconversion.

The use of plastics has increased globally and more and more of it ends up in the environment. Microbes can be used to produce polyhydroxyalkanoates (PHA), a biodegradable plastic substrate. Instead of nonrenewable fossil raw materials, such as renewable sewage sludge can be used as a carbon source for polyhydroxyalkanoates. The aim of this work was to investigate the microbial diversity and metabolism of the first hydrolysis step of the three-step polyhydroxyalkanoate (PHA) production process. In the first step, organic polymers, such as carbohydrates, lipids and proteins are hydrolyzed into monomers, which are then converted into short-chain volatile fatty acids (VFA). The volatile fatty acids are used in the third step as a substrate in microbial PHA production. In the second step, the polyhydroxyalkanoates accumulating microbes are enriched, and used in the third step for PHA synthesis. In this work, different types of sludges were used as a carbon source. Sludges were sludge for biogas production, sludge after biogas production and sludge after nitrogen removal in stripping. The concentration of volatile fatty acids, cellulose and lignin was determined in the bioreactors. Volatile fatty acids were determined by gas chromatography. Cellulose and lignin were determined after hydrolysis by filtering and drying the samples. The nucleotide sequence of the 16S rRNA gene was determined from pure materials. Shotgun metagenome sequencing was performed on bioreactor DNA samples to sequence the entire microbial genomes. Hydrolysis bioreactors were maintained for 12 days. Microbes did not degrade cellulose and lignin well. The best volatile fatty acids yields were obtained from the sludge for biogas production (172 mg/g organic matter ± 6.25). Proteobacteria and Firmicutes were the major phyla in the bioreactors, and microbial genera differed greatly between bioreactors. Microbial genes coding for carbohydrate and protein metabolism were predominant in the bioreactors.

Perusopetuksen opetussuunnitelman perusteet (POPS) painottaa biologian opetuksen kohdalla tutkivaa oppimista ja tutustumista biologialle ominaisiin tutkimusmenetelmiin. POPS:n biologian sisältöalueisiin kuuluu myös mikrobiologiaa. Tässä tutkimuksessa selvitettiin sisällönanalyysin keinoin miten yläkoulun biologian Koodi- ja Elo-oppikirjasarjojen tehtävissä ilmeni mikrobiologia ja tutkimuksellisuus. Tutkimuskysymykset olivat: 1. Kuinka paljon mikrobiologiaan liittyviä tehtäviä on ja mihin aiheeseen ne liittyvät? 2. Kuinka paljon mikrobiologian tutkimuksellisia tehtäviä on ja millaista tutkimuksellisuutta ne edustavat? 3. Onko mikrobiologian tehtävien määrällä ja laadulla eroavaisuutta kirjasarjojen välillä? Tulosten perusteella pohdittiin vastasivatko oppikirjat POPS:n sisältöjä ja tavoitteita mikrobiologiaan liittyvien tehtävien osalta. Molemmissa kirjasarjoissa oli mikrobiologiaan liittyviä tehtäviä lähes yhtä paljon. Koodi-kirjasarjassa tehtäviä oli selvästi eniten Elämä-kirjassa ja Elo-kirjasarjassa tehtäviä oli eniten Ihminen-kirjassa. Koodi-kirjasarjassa erilaisia mikrobiryhmiä oli käsitelty hieman kattavammin ja tasaisemmin kuin Elo-kirjasarjassa. Elo-kirjasarjasta myös puuttuivat kokonaan yhteen POPS:n sisältöalueeseen, elämän kehitykseen ja evoluutioon, liittyvät mikrobiologiset tehtävät, joita Koodi-kirjasarjassa oli useita. Sen sijaan vain Elo-kirjasarjassa oli POPS:ssa mainittua biotekniikka-sisältöä mikrobiologiaan liittyen. Tutkimuksellisia tehtäviä oli molemmissa kirjasarjoissa vähän alle puolet mikrobiologian tehtävistä. Elo-sarjassa niitä oli hieman enemmän, tutkimuksellisuus oli monipuolisempaa ja myös kokonaisia tutkimuksia oli useita, toisin kuin Koodi-kirjasarjassa. Molemmissa kirjasarjoissa oli useita mikroskopointitehtäviä. Tuloksista voitiin päätellä, että molemmat kirjasarjat vastaavat POPS:n tutkimuksellisuustavoitteeseen, vaikkakin Elo-kirjasarja hieman paremmin. Mikrobiologian aihesisältöjen osalta kirjoissa oli painotuseroja ja joitain puutteita verrattuna POPS:n sisältöihin. Tutkimuksen tulokset voivat olla biologian opettajan apuna oppikirjan valinnassa ja sen POPS:n vastaavuuden tarkistamisessa. Jatkotutkimuksena oppikirjojen tekstin analysointi antaisi kokonaiskuvan oppikirjojen mikrobiologian sisällöstä. Oppikirjojen mikrobiologian sisältöjen näkymistä opetuksessa voisi myös tutkia.

Biologia on tieteenalana jatkuvassa muutoksessa. Geenitutkimusten kehittyessä saadaan uutta tietoa eliöiden perimästä ja sukulaissuhteista, jolloin niitä voidaan myös luokitella tarkemmin. Opinnäytetyössä tarkastellaan biologian tieteenalalla tapahtuvien muutoksien vaikutuksia biologiaan oppiaineena peruskoulussa ja lukiossa. Tutkimuksessa selvitän, miten biologian opettajat suhteutuvat päivitettyyn eliöiden luokitteluun ja se otettu osaksi opetusta. Lisäksi tavoitteenani on selvittää mitkä eliöryhmät opettajat kokevat merkittäväksi opettaa kullakin opetusasteella, ja ovatko jotkin eliöryhmät opettajien mielestä merkityksettömiä yleissivistävän koulutuksen kannalta. Selvitän myös mitä aineistoja biologian aineenopettajat käyttävät merkittävimpänä lähteenään opetuksessa. Tutkimus toteutettiin puolistrukturoituna kyselylomakkeena, jossa oli sekä avoimia kysymyksiä, että monivalintakysymyksiä. Tutkimuksen taustatiedot osio on tehty kirjallisuuskatsauksena luokittelun muutokseen liittyviin artikkeleihin sekä peruskoulun ja lukion opetussuunnitelmaan pohjautuen. Tutkimuksen tuloksia analysoitiin sekä kvantitatiivisesti että kvalitatiivisesti. Osa opettajista ei ole ollut tietoinen eliökunnan luokittelun päivityksestä, mikä on vaikuttanut sen käyttöönottamiseen opetuksessa. Päivitetty luokittelu on koettu haastavimmaksi yläkoulun opettajien keskuudessa, eikä sitä ole otettu yhtä laajasti käyttöön kuin lukioissa. Opettajilla on melko yhtenäinen käsitys siitä, mitä eliöryhmiä tulisi opettaa kullakin opetusasteella. Vastausten perusteella opettajat käyttävät tiedonlähteenä pääasiassa opettajille tuotettuja materiaaleja. Tämän vuoksi olisi tärkeää, että keskeisistä uudistuksista tiedotetaan riittävästi ja niistä tehtäisiin helposti saatavilla olevia lisämateriaaleja.

Filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a crucial production organism for enzymes used in industrial applications, such as in feed, food, textile, and biofuel production, due to its ability to secrete high amounts of homologous and heterologous enzymes. Therefore, development of genetic tools to improve the properties of industrial T. reesei strains for even better production yields is essential. In this study, a polyethylene glycol mediated CRISPR-Cas9 transformation method for industrial T. reesei production strains was aimed to be optimised by testing an alternative Cas9 enzyme and varying the stoichiometry and total amount of Cas9 enzyme and single guide RNA in the ribonucleoprotein complex. Correct integration of the gene constructions in the obtained transformants was determined by colony PCR and Southern blot analysis. In addition, two selected background activity encoding genes, endoglucanase 6 and α-glucuronidase 1, were individually deleted from T. reesei xylanase production strain utilising the improved CRISPR-Cas9 transformation protocol. The effect of background activity deletions on the strain growth and protein production were analysed from culture supernatants by pH measurement, Bradford protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and enzyme activity assays. An improved CRISPR-Cas9 transformation protocol for T. reesei was successfully established basing on high number of transformants and improved DNA integration fidelity. No negative effects were observed in the growth or protein production properties of the background activity deletion strains compared to the xylanase production strain. Thus, further cleansing of T. reesei secretome can be continued to refine the industrial production strains.

The increasing use of antimicrobials causes a heavy pollution load on the environment and can enhance antimicrobial resistance of pathogenic bacteria, thus having a negative impact on human and animal health. Antibiotic concentrations in the environment are constantly monitored, however traditional chemical analyses fail to provide data on the bioavailability of antimicrobials. Whole-cell bacterial bioreporters have been developed to detect a wide variety of environmental pollutants including antimicrobials. These living, genetically engineered organisms can also be used for the measurement of the bioavailable fraction in a sample and thus bioreporters could give insights on the role of antimicrobial pollution in the dissemination of antimicrobial resistance. The aim of this study was to design and construct an improved bioluminescent bioreporter for detection of macrolide antibiotics. The mrx gene and the mph(A)R repressor gene were coupled with the mph(A) promotor of the macrolide resistance operon mph(A) and the reporter genes of the luciferase operon luxCDABE. The main objective was to determine whether Mrx, the hydrophobic and putative transmembrane transport protein of the macrolide resistance operon mph(A), would improve the sensitivity and reduce the induction time. Another aim was to optimize the use of three existing bioreporters with other macrolides than erythromycin, which was used earlier in testing their performance. The mrx-mph(A)R fragment was cloned into the pmph(A)luxCDABE vector, and the bioreporter plasmid was introduced to Escherichia coli strain DH10B. After verification of the construct pmph(A)luxCDABE-mrx-mph(A)R, the usability of the new whole-cell biosensor was compared against the three existing macrolide bioreporters with three different macrolide and lincosamide antibiotics, erythromycin, tylosin and clindamycin. The cloning of the new bioluminescent bioreporter for macrolides was performed successfully. However, the addition of erythromycin, tylosin or clindamycin to a suspension of E. coli DH10B(pmph(A)luxCDABE-mrx-mph(A)R) did not stimulate the expression of the lux genes. High concentrations of all three antibiotics triggered a light response with the existing bioreporters although the response was slow. The results indicated that further studies on the mrx gene and its encoded Mrx protein are still needed. The response of the mph(A) operon to other macrolide and lincosamide antibiotics than erythromycin was a positive and encouraging finding, since this enables detection of other synthetic macrolides than erythromycin as well as lincosamides with the existing bioreporters after optimization.

Methanococcus maripaludis is a hydrogenotrophic methanogen which has become a model organism for archaea and for the study of methanogenesis. New genetic and genomic tools hold promise to convert M. maripaludis into a host organism for synthetic biology and metabolic engineering. However, an extensive characterization of suitable promoters in this organism for their use in metabolic engineering has been lacking. In this study, the strength of 25 promoter sequences was quantified by use of a β-glucuronidase reporter gene assay, establishing it as an adequate method for this purpose. PglnA seemed to evade the control exerted by the native transcriptional regulator NrpR and thus became the strongest promoter of all tested. Pmtr, Pmcr, , Pmcr_JJ and Ppor_JJ could also be regarded as strong promoters in this organism. On the other hand, inclusion of a putative transcription factor downstream of the eha operon increased the strength of Peha and Peha_JJ, suggesting its role as the activator of the operon. Overall, these results provide valuable information for the implementation of genetic, metabolic and promoter engineering in M. maripaludis. Genetic manipulation of M. maripaludis was done via a recently developed CRISPR/Cas toolbox, serving as an example of its efficiency.