Browsing by Subject "Mikrobiologi"

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  • Jäppinen, Sanni (Helsingfors universitet, 2013)
    Filamentous cyanobacteria taxa Nostocales and Stigonematales cells can differentiate into heterocysts nitrogen fixing cells when nitrogen is limiting the growth and into resting cells akinetes when nutrients decrease or the growth conditions become unfavorable for growth. Akinetes overwinter in the water sediments during the unfavorable growth time. When the growing condition improves akinetes germinate and can start a new cyanobacterial bloom. Akinete differentiation remains unclear. It is known from the literature that only a few akinete specific genes exist. First described akinete specific gene was avak. The morphological changes of akinete differentiation are known but the changes at molecular genetics level in regulation and differentiation remains unclear. The aim of this study was to design a method for akinete differentiation-related genes, avak, hepA and hap, for an Anabaena 1TU33s10 strain and to monitor the gene expression changes in a seven-week growth experiment. Primers for the differentiation related genes were designed based on the known whole genome sequence of the Anabaena 1TU3310 strain. In this study it was managed to design a quantitative reverse transcriptase polymerase chain reaction, qRT-PCR method, based on the genes involved in the akinete differentiation process. It was observed that gene expression changed when akinetes began to differentiate into the filaments. In the growth experiment II avaK-gene expression was increased 2-fold between the 14. and 30. days, and hap-gene showed 1.5 fold growth between 14. and 30. days. The number of akinetes was also increasing at the same time. In the growth experiment I heap-gene showed 1-8 fold growth between the days 21. and 27. –30. days when the number of heterocysts were also increasing. The number of akinetes was relatively low compared to number of vegetative cells which also explains the small expression fold-differences in the cultures during the experiment time when compared to expression fold-differences described in the literature. Designed method can thus also detect minor changes in gene expression. The designed and built qRT-PCR method can be used in the future for monitoring gene expression changes also for new akinete specific genes, and the method can be further optimized for screening natural water samples.
  • Partanen, Asta (Helsingfors universitet, 2013)
    Cyanobacteria are aquatic and terrestrial organisms that have photosynthetic cababilities. Cyanobacteria are classified to five orders according to their morphology. These are Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales. Anabaena sp. 90 is a filamentous cyanobacterium in the order of Nostocales. A sample of this cyanobacterium was isolated from the lake Vesijärvi in 1986. Anabaena sp. 90 produces a variety of bioactive peptides such as microcystins, anabaenopeptins and anabaenopeptilides which are the most commonly studied of the peptides produced by the strain. Majority of the bioactive peptides are produced by non-ribosomal peptide synthetases (NRPS). The way these compounds affect cyanobacteria is currently unclear. Anabaenopeptilide deficient mutant (apd-) variety has been developed from the Anabaena sp 90 wild type (WT). The mutant is unable to produce anabaenopeptilides. The aim of my Master´s thesis was to study the changes in proteomes of WT and apd- strains by conducting a six day cultivation experiment. The purpose was to evaluate the possible differences in the amount of proteins that the different cultivation stocks were able to produce. The secondary purpose was to examine the transcription of anabaenopeptilide synthetase genes. Proteomic study was performed using a procedure called two dimensional differential gel electrophoresis (2D DIGE). The differences in the proteomes´ statistics were examined by using the Student´s t test. Transcription of anabaenopeptilide synthetase genes were studied using a reverse transcriptase polymerase chain reaction (RT-PCR). The data collected from these studies concluded that there were differences in the proteomes of WT and apd- strains. In eighteen cases there were significant differences in the protein amounts between the strains. The single protein counts were often higher in the apd- than in the WT strain. Anabaenopeptilide synthetase genes were transcribed in both strains which suggests that the mutation in the apd- strain did not hinder the ability of the strain to produce anabaenopeptilide transcripts. The identification of significant proteins would yield more detailed knowledge of the metabolical functions of WT and apd- strains.
  • Jokela, Roosa (Helsingin yliopisto, 2019)
    The purpose of this thesis was to study the antibiotic perturbations in bacterial populations. Perturbations are common in ecosystems and they can change the composition and functionality of a community substantially. The response of a community is governed by ecological and evolutionary factors: perturbations change the competitive ability of species in the community, but rapid evolution can further affect species fate. This thesis focuses more on the ecological effects. Understanding the community response to a disturbance would be interesting both from a general point of view and from the more practical approach of understanding natural communities under perturbations caused by antibiotics but also, for example, by climate change or chemicals. Thus far, most studies have been performed in one- or two-species systems, not taking into account the effects communities have on the fate of a single species. To study antibiotic perturbations, a multi-species bacterial community was exposed to a streptomycin pulse of three different levels concentrations. Changes in community composition were studied in the end of the pulse (ecological resistance) and after a recovery period (resilience) from the antibiotic perturbation comparing to the pre-perturbed communities. Further, the presence of species flow was manipulated to examine if it could enhance community resistance and resilience. Based on the analysis, even low antibiotic concentrations can have a long-lasting effect on community composition, but the magnitude of the effect is dependent on the concentration. Community diversity was recovered better than the composition, especially after the weaker perturbations. Species flow aids in community recovery but does not affect resistance. The results were relatively reproducible between replicate communities, and species traits steered the species fate in, pointing to deterministic ecological processes driving the community response. However, repeatability decreased in communities perturbed with the highest antibiotic concentration, which could point to evolution.
  • Christodoulou, Maria (Helsingin yliopisto, 2019)
    The rapid emergence of drug resistant pathogens prevents effective treatment of diseases and threatens the lives of millions of people. Similarly, resistance to chemotherapeutic agents has been observed in several types of cancer. Therefore, screening for novel antimicrobial and antileukemic substances is urgently needed. Screening microorganisms for bioactive molecules has resulted in the discovery of several substances that are currently used for disease treatment. Cyanobacteria represent an ancient group of oxygenic, photosynthetic prokaryotes that produce a variety of functionally diverse and structurally complex natural compounds, some of which have already been used as source of inspiration in drug development process. In this study, I evaluated the antimicrobial and antileukemic potential of filamentous cyanobacteria against Gram-positive, Gram-negative and fungal potential pathogens and acute myeloid leukemia (AML) MOLM-13 cell line. Extracts showing antibiotic and/or antileukemic activity were subjected to reversed-phase HPLC and individual fractions were re-evaluated for their ability to kill the abovementioned pathogens and/or induce cell death in MOLM-13 cell line. Metabolites present in the active HPLC fractions were analysed by UPLC/ESI/Q-TOF and elemental compositions were obtained. Identification of metabolites was accomplished by searching online databases for compounds with identical elemental composition. Chemical structures were further confirmed by comparing mass spectrometry data with publicly available data. New bioactive metabolites, new variants of known metabolites and a number of yet unidentified metabolites exhibiting antimicrobial and/or antileukemic activity are reported herein. In detail, novel metabolites belonging to aromatic polyketides and their new variants were present in the active fractions of Nostoc sp. CENA69. The cyanobacterium Aliinostoc sp. CENA513 produced the recently discovered metabolite nocuolin A, a compound with antimicrobial and antiproliferative properties, lipids and unidentified lipidic compounds that showed only bactericidal activity against B. cereus. Interestingly, none of the abovementioned strains had any effect on the growth of Gram-negative pathogens. Planktothrix agardhii UHCC 0018 and Anabaena sp. UHCC 0187 strains showed only antileukemic activity. The majority of the bioactive fractions deriving from these two strains contained either lipids or pigments and their derivatives. The remaining active HPLC fractions of these two strains contained a great number of unidentified compounds. Further studies are required to identify the unknown compounds and purify the novel metabolites and antibacterial lipids. The results presented herein clearly show that Cyanobacteria are an emerging source of bioactive metabolites that can be used in drug development process or act as a source of inspiration for the production of novel synthetic drugs.
  • Chen, Yongchen (Helsingfors universitet, 2013)
    Soil contamination with oily products poses great healthy and environmental risks to the polluted sites. The remediation difficulty mainly comes from the complexity of hydrocarbons. Different kinds of remediation technologies have been applied for hydrocarbon removal from soil. New technologies especially in situ bioremediation technologies are emerging constantly. Soil assessment is a key step in the remediation processes since it provides information about the contamination level and potential risks. In the present study, hydrocarbon contaminated soil samples were collected from two sites (one site was contaminated by weathered oily sludge waste with some vegetated plots; the other was contaminated with fuel oil with short-chain hydrocarbons). The samples were analyzed for physicochemical properties and hydrocarbon degraders were enumerated. Four degrading strains were isolated from the samples and their 16S rRNA genes were sequenced. The samples and isolates were investigated to check the existence of three catabolic genes involved in petroleum degradation. The objective was to reveal the intrinsic bioremediation potential of contaminated soils by investigating the key remediation “players” i.e. the degrader microorganisms and catabolic genes. The coexistence of abundant degraders and diverse catabolic genes give the soil a good potential for bioremediation. In addition, the relationships between degrader counts, genes detection and soil contamination levels can reveal how the contaminants affect the indigenous microbial community. The differences between vegetated and nonvegetated plots can also suggest if vegetation with legumes has good potential for hydrocarbon bioremediation. According to the results, both sites were moderately contaminated with different hydrocarbon composition. In the landfarming site, the TPH depletion in vegetated fields was higher than the unvegetated bulk soil areas. However, the degrading microorganism counts had no significant differences between vegetated and nonvegetated plots. The hydrocarbon contamination level had no correlation with the degrader counts. In subsurface soils where aeration was quite limited, degrading microorganisms were much lower than those in surface soils. Catabolic genes were detected from the isolated strains but rarely from the contaminated soil samples. The contaminants co-extracted with soil DNA may inhibit the PCR-based gene detection. With more primer sets or primers targeting broader genetic diversity ranges, more detection results can be expected.
  • Valo, Karoliina (Helsingfors universitet, 2014)
    Studies have revealed that members of Archaea exist in various environments such as human gut and the sporocarps of some fungi in addition to the extreme environments in which they were first discovered. It is yet unknown how some archaea end up in these environments and what is their role there. To study the archaea efficiently in different environments, enrichment or pure cultures are required. In this work liquid media were developed for cultivation and enrichment of archaea from the edible sporocarps of Suillus bovinus, Craterellus tubaeformis, Agaricus bisporus and Boletus pinophilus. The source of carbon in each medium was determined to be yeast extract, crushed and sterile filtered sporocarp of either Craterellus tubaeformis or Agaricus bisporus, or casaminoacids. The highest amount of prokaryotes grew on the casaminoacid medium but it was also a very good medium for the bacteria in sporocarps. The media were anaerobic to prevent the fastest aerobic bacteria from growing. Antibiotics kanamycin and ampicillin were added to reduce bacterial growth but with further inoculation resistant bacteria outgrew the archaea. Adding lysozyme with the antibiotics reduced bacterial growth also with later cultivations and the archaea-bacteria ratio turned favourable. The amount of both archaea and bacteria was determined with the quantitative PCR of the 16S rRNA gene. The most archaea was enriched from the sporocarps of Suillus bovinus. On the contrary, prokaryotes that grew from the sporocarp of Craterellus tubaeformis were mostly bacteria. The Sporocarps of both Boletus pinophilus and Agaricus bisporus didn t harbour many archaea or bacteria that could have been detected or enriched in this study. It was also noted that there was great variation between individual sporocarps of the same species in the amount of prokaryotes they originally harboured. Archaea were successfully enriched from the sporocarps of Suillus bovinus for the duration of three consecutive cultivations on the casaminoacid medium with the addition of kanamycin, ampicillin and lysozyme.
  • Puhakainen, Kai (Helsingfors universitet, 2016)
    Bakteereita ja eukaryootteja esiintyy erittäin runsaasti jätevedessä, jossa ne voivat muodostaa fyysisiä vuorovaikutuksia toistensa kanssa. Fyysisiä vuorovaikutuksia bakteerien ja eukaryoottien välille voi muodostua symbioosien tai saalis-saalistaja suhteen välityksellä. Bakteerit ja eukaryootit osallistuvat myös jäteveden puhdistusprosesseihin, joten näillä molemmilla on iso merkitys puhdistusprosessissa. EpicPCR-menetelmän avulla voidaan tutkia isoja mikrobipopulaatioita sekvensoimalla genomin alueita suuresta määrästä yksittäisiä soluja. Aiemmin menetelmää on käytetty selvittämään sulfaatin pelkistämiseen osallistuvien bakteerien fylogeneettista diversiteettiä järven sedimenteissä. Tämän työn tarkoituksena oli tutkia bakteerien ja eukaryoottien fyysisiä vuorovaikutuksia jätevedessä käyttäen apuna epicPCR-menetelmää. Tutkimuksessa yhdistettiin tiiviissä fyysisessä yhteydessä olleen bakteerin 16S rRNA- ja eukaryootin 18S rRNA -geeni. Samalla selvitettiin miten epicPCR-menetelmä soveltuu käytettäväksi kahden fylogeneettisen 16S rRNA- ja 18S rRNA -geenin yhdistämiseen ja miten jätevesi soveltuu näytteeksi. Tulosten perusteella pohdittiin millaisia voisivat olla bakteerien ja eukaryoottien fyysiset vuorovaikutukset ja mitä ne merkitsevät kummallekin osapuolelle. Fyysisiä yhteisvaikutuksia tutkittiin käyttämällä yksinkertaiseen teknologiaan perustuvaa epicPCR-menetelmää ja sekvensointi suoritettiin Illumina MiSeq -menetelmällä. Sekvenssien käsittely ja analyysi tehtiin käyttäen Unixin Bash -komentorivikieltä ja R-ohjelmointikieltä CSC:n Taito-laskentaklusterilla käyttäen apuna Qiime-työkalupakettia. Tulosten perusteella bakteerien ja eukaryoottien välillä havaittiin fyysisiä yhteyksiä, jotka jakaantuivat kahdella eri tavalla. Jätevedestä löytyi eukaryoottiryhmiä, jotka muodostivat yhteyden useamman bakteeriryhmän kanssa ja eukaryoottiryhmiä, jotka muodostivat yhteyden vain tietyn bakteeriryhmän kanssa. Saalistukseen pystyvät eukaryoottiryhmät muodostivat yhteyksiä useamman bakteeriryhmän kanssa kuten myös määrällisesti enemmän kuin fotosynteettiset eukaryoottiryhmät.
  • Pehkonen, Kati (Helsingfors universitet, 2013)
    Fungi play a crucial role in the ecosystem by recycling nutrients and forming mycorrhizal roots with plants. Many of the decomposer and mycorrhizal fungi are Bacidiomycetes. In the sexual reproduction stage, Bacidiomycetes produce fruiting bodies which enable them to produce and disseminate spores allowing fungi to spread to new growing sites. Fruiting bodies have been discovered to contain bacteria which may have a role in differentiation and maintenance of the fruiting body. They might also protect fruiting bodies against animals and diseases, and influence the nutritional value of the fruiting body. There is little knowledge about the amount of bacteria in the fruiting bodies. All previous research has been carried out entirely by cultivation-based methods and it shows that different fungal species contain very different amounts of bacteria. Some fruiting bodies have been shown not to contain easily cultivatable bacteria. The occurrence of archaea in fruiting bodies has not been previously studied and investigation into their function in fungi has only recently begun. In the present work significant amounts of bacterial and archaeal 16S rRNA -gene copies were discovered in the fruiting bodies of three ectomycorrhizal and three decomposer fungi species. This is the first time fruiting bodies have been shown to contain archaea. The occurrence of bacteria and archaea and the abundance of their 16S rRNA -genes in the fruiting bodies were determined using PCR ja quantitative PCR methods. Suillus bovinus and Boletus pinophilus fruiting bodies contained significantly more archaeal than bacterial gene copies. Cantharellus cibarius and Lycoperdon perlatum contained more bacterial than archaeal 16S rRNA -gene copies. In two decomposer fungi fruiting bodies, Agaricus arvensis and Piptoporus betulinus, the abundance of bacterial and archaeal gene copy numbers were equal. Suillus bovinus fruiting bodies had the largest copy number of archaeal 16S rRNA -genes from all species investigated. According to the results obtained in this work, the occurrence of bacteria and archaea might be common in fruiting bodies. The presence of bacteria and archaea in significant amounts in fruiting bodies may indicate their necessity for the development and sustainability of the fruiting body and hence to the whole life cycle of fungi.
  • Humisto, Anu (Helsingin yliopisto, 2014)
    Syanobakteerit tuottavat bioaktiivisia sekundääri- eli erikoismetabolian tuotteita. Syanobakteerien bioaktiivisia yhdisteitä on rakenteiltaan lukuisia erilaisia ja ne voidaan jakaa pääryhmiin peptidit, polyketidit, alkaloidit ja lipopolysakkaridit. Yhdisteistä on löydetty muun muassa antibakteerisia, antifungaalisia ja antiviraalisia ominaisuuksia. Lisäksi näihin bioaktiivisiin aineisiin kuuluu terveysriskin aiheuttavia toksisia yhdisteitä. Planktiset syanobakteerit ovat saaneet runsaasti huomiota niiden muodostamien massaesiintymien vuoksi, mutta myös benttiset eli sedimentissä tai erilaisilla pinnoilla elävät syanobakteerit voivat aiheuttaa terveysriskin. Benttisillä syanobakteereilla on myös potentiaalia uusien bioaktiivisten yhdisteiden löytymisessä biotekniikan tai lääketeollisuuden tarkoituksiin. Tutkimuksessa seulottiin Itämeren ja järven benttisten syanobakteerien tuottamia bioaktiivisia yhdisteitä. Syanobakteerikannat tunnistettiin 16S rRNA geenin avulla ja niiden bioaktiivisuutta tarkasteltiin maljadiffuusiomenetelmän avulla, PCR:n avulla etsittiin tunnettuja biosynteesigeenejä. Bioaktiivisia yhdisteitä pyrittiin tunnistamaan LC/MS-menetelmällä. Tunnistetut syanobakteerit muodostivat monipuolisen benttisille syanobakteereille tyypillisen lajikirjon, jossa bioaktiivisia ominaisuuksia havaittiin 24 % tutkituista kannoista. Bioaktiivisia ominaisuuksia löydettiin niin meri- kuin järviympäristöistä eristetyiltä kannoilta. Useita bioaktiivisuuksia esiintyi muun muassa Nostoc- ja Anabaena-syanobakteereilla. Benttiset syanobakteerit tuottivat antifungaalisia yhdisteitä, kuten hassallidiinia ja skytofysiiniä sekä sytotoksista anabaenolysiiniä. Lisäksi löydettiin useita syanobaktiinien biosynteesigeenejä. Benttisiltä syanobakteereilta ei havaittu mikrokystiinin tai anatoksiini-a:n biosynteesigeenejä. Antimikrobisia ominaisuuksia löydettiin kahdesta syanobakteerikannasta, joiden tuottamia bioaktiivisia yhdisteitä ei vielä tunnistettu. Benttiset syanobakteerit tuottavat siis useita tunnettuja bioaktiivisia yhdisteitä ja ovat myös potentiaalinen uusien bioaktiivisten yhdisteiden lähde.
  • Jouhten, Hanne (Helsingfors universitet, 2015)
    Clostridium difficile infection (CDI) is a microbiota-related disease. Typically, antibiotic-induced perturbation of gut microbiota precedes the infection, while a healthy gut microbiota provides protection, i.e. colonization resistance, against it. Furthermore, in the case of recurrent CDI that is not resolved by antibiotics, restoring the gut microbiota with a fecal microbiota transplantation (FMT) is among the few treatment options that really work. Although FMT is effective in the treatment of CDI, the factors behind treatment success remain unclear. Both, the key species and the functions that are necessary to restore the healthy microbiota and eradicate C. difficile, are a matter of speculation. This study was based on the hypothesis that the adherence of some commensal bacteria to the gut epithelial cells could play a role in eradicating C. difficile by competing for epithelial binding with it. Furthermore, the isolation of those bacteria from the donor feces would enable more detailed mechanistic studies and development of a bacterial product for the treatment of CDI in the future. As a pre-selection step, bacterial adhesion to Caco-2 cells was utilized to isolate and cultivate epithelium-adherent bacteria from the donor feces. Microbiota composition of fecal sample, and the adhered and cultured sub-populations thereof, was determined by partial 16S rDNA amplicon sequencing using MiSeq method. The pre-selection approach was successful, since the obtained populations were different, both after the adhesion and cultivation, as compared to the original fecal sample. In addition, most obtained pure isolates adhered well to enterocytes. The ability of fecal bacteria to compete with C. difficile for binding to gut epithelial cells in vitro was also studied. Isolated bacteria from Caco-2-adhered populations were applied in competition and exclusion assays with C. difficile as purified or multi-species cultures, and reduction in C. difficile binding was observed due to the certain bacteria or bacterial populations. These assays still need developing and the results must be confirmed with more repetitions. However, the results are promising and a useful ground for future work in developing bacteriotherapeutic formulations for the treatment of CDI.
  • Virta, Susanna (Helsingin yliopisto, 2015)
    Compared to many other countries, Salmonella prevalence in finnish poultry, bovines and pigs are at a low level. This low level has been obtained because of the national salmonella control programme. However, Salmonella still occur in feeds and at broiler farms in Finland and EU. Bioactive glass (Bioglass) could be a potential feed additive for preventing Salmonella in poultry since many new feed additives have got EU ac-ceptance. Bioactive glasses have been found bactericidic when used in dentistry and artificial tissue technology as implant material. The aim of this thesis was to study if and how bioglass effects Salmonella enterica bacteria in chicken feed and gut contents. Further, the purpose was to determine if bioglass can reduce the amount of Salmonella in the sample matrixes using the spread plate method. Six strains of Salmonella enter-ica from Hambi culture collection (HAMBI 1111, 1112, 1306, 1513, 2317 ja 2331) was added to the matrixes. Commercial XLD medium was used to determine the amount of Sal-monella in each time point of the test. 0 - 10 % of bioglass was tested for bactericidic effects. The ability of bioglass to inhibit the growth of Salmonella was attempted to demonstrate with transmission electron microscopy and using gram staining and light microscopy. The results indicate that the bactericidic effects of bioglass work in the sample matrix-es nearly as well as in liquids. The added amount of Salmonella was significantly re-duced with the higher concentrations of bioglass. Lower levels of bioglass did not show bactericidic effects or the inhibition of the growth of Salmonella was minor. The bacte-ricidic effect is thought to be based on the rise of pH and the adherence to bacterial cell wall. TEM imagines illustrated the attachment of the bioglass particles to degrad-ing cells of Salmonella Typhimurium (HAMBI 1112). Bioglass could be a potential feed additive for the prevention of Salmonella in broilers.
  • Hartikainen, Anna (Helsingin yliopisto, 2019)
    Nykyinen jälkiteollinen ihmiskunta tuottaa energiansa pääasiassa fossiilisilla polttoaineilla. Pariisin ilmastosopimuksen osapuolet sitoutuivat pitämään ilmaston lämpenemisen alle kahdessa celsiusasteessa verrattuna esiteolliseen aikaan ja muuttamaan energiantuotantotapojaan. Yhtenä ratkaisuna nähdään uusiutuvat energianlähteet, joita kuuluvat muun muassa biopolttoaineet. Bioetanoli on maailmanlaajuisesti käytetyin liikenteen biopolttoaine, jonka tuotanto on huomattavasti kasvanut 1980-luvulta lähtien. Tällä hetkellä bioetanolia tuotetaan etupäässä ruoantuotannon kanssa kilpailevista sokeri- ja tärkkelyspitoisista viljelykasveista, mutta niin kutsuttujen toisen sukupolven biopolttoaineiden tuotanto olisi ihmiskunnan ja maapallon kannalta kestävämmällä pohjalla. Toisen sukupolven bioetanolia valmistetaan kasvi- ja puupohjaisesta jätemateriaalista, kuten maa- ja metsätalouden sekä elintarviketeollisuuden jätteistä. Aikaisemmissa tutkimuksissa hyväksi lignoselluloosan ja puuperäisten jätteiden hajottajaksi ja monipuoliseksi entsyymien tuottajaksi tunnistettu, valkolahottajiin kuuluva kääpämäinen kantasieni rusorypykkä Phlebia radiata kykenee samanaikaiseen kiinteän kasvualustan hajotukseen ja etanolifermentaatioon. Tämän vuoksi rusorypykän etanolin tuottoa ja siihen liittyviä perusaineenvaihduntareittejä haluttiin tutkia tarkemmin. Sienen rihmastoa kasvatettiin jätelignoselluloosamateriaaleilla hapettomiksi muuttuvissa fermentaatio-olosuhteissa. Etanolin tuoton ja kasvualustan pilkkoutumisen lisäksi tutkittiin käänteistranskription kvantitatiivisella PCR-reaktiolla lignoselluloosan hajotukseen ja etanolifermentaatioon liittyviä entsyymejä koodaavia geenejä ja näiden geenien ilmentymistä eri kasvuaikapisteissä. Tulosten perusteella havaittiin rusorypykän tuottavan noin 13,5 massa-% etanolia kasvualustan hiilihydraattien sokeripitoisuuden perusteella lasketusta teoreettisesta maksimiarvosta. Lignoselluloosan hajotukseen ja etanolin fermentointiin oleellisesti liittyvien entsyymien geenit ilmentyivät kasvatusten aikana jätelignoselluloosalla tilastollisesti merkitsevin eroin eri aikapisteissä, mikä osoitti hajotustoiminnan, rihmaston kasvun ja etanolin tuoton olevan muuttuvia ja toisiinsa vaikuttavia prosesseja. Sieni pystyi hyödyntämään kiinteää kasvualustaansa irrottaen siitä käyttöönsä sokereita. Rihmaston kasvusta ja elävästä sienibiomassasta viitteitä antavaa ergosterolia havaittiin koko kuukauden pituisen kasvatuksen ajan. Lisäksi kaasukehässä havaittiin odotettuja fermentaatiomuutoksia, kuten hiilidioksidin kertymistä, ja happikaasu oli neljässä viikossa lähes kulutettu loppuun. Tulosten perusteella rusorypykkä osoittautui tehokkaaksi jätelignoselluloosan pilkkojaksi ja kestäväksi etanolin tuottajaksi. RNA-eristys kiinteältä kasvualustalta sekä aineenvaihduntageenien ilmentymisen analysointi RT-qPCR-menetelmällä mahdollistivat rusorypykän transkriptomin jatkotutkimuksen geenien säätelyn selvittämiseksi fermentoivissa ja lähes hapettomissa olosuhteissa.
  • Raitosalo, Amanda (Helsingin yliopisto, 2020)
    Heavy traffic and transportation of crude oil makes the Baltic Sea vulnerable to accidental oil spills. Currently mechanical removal is the recommended response method in the Baltic Sea region. However, in bad weather conditions, mechanical oil removal can be challenging, and it may be justifiable to consider alternative response methods in order to protect valuable natural sites. Chemical dispersants are mixtures of surfactants and solvents and are used to treat spilled oil in marine environments. Dispersants break oil slicks into small droplets, which are more readily available for microbial biodegradation. Large-scale studies on the effects of dispersants on microbial communities and biodegradation rates have not been conducted in the Baltic Sea. During this 40-day microcosm study, native microbial communities from the Gulf of Finland were exposed to crude oil, dispersant or their combination. Additional nutrients were not supplied during the study. The experiment was carried out at 10 °C, and samples were collected at days 1, 2, 5, 14 and 40. The amount and expression of genes related to microbial abundance, oil biodegradation and nutrient cycling was assessed by quantitative PCR. Extracellular enzymatic activities of microbes were studied utilizing high throughput robotics. The results indicated that microbial response was primarily elevated in samples containing dispersant. Dispersant was degraded almost completely during the study period. Biodegradation of C21-40 hydrocarbons in crude oil was not enhanced with the addition of dispersant. On genetic level, the abundance and expression of alkane monooxygenase gene (alkB) was elevated in samples with dispersant, whereas the abundance and expression of polycyclic aromatic ring-hydroxylating dioxygenase gene (PAH-RHD) was enhanced also in samples containing crude oil.
  • Manninen, Anna (Helsingin yliopisto, 2018)
    In Finland, the final disposal of spent nuclear fuel is due to begin in the 2020s. The spent nuclear fuel will be stored in the deep bedrock of Olkiluoto, Eurajoki. Biological sulphate reduction is one of the most significant mechanisms possibly disrupting the integrity of the final disposal concept. Microorganisms living in groundwater may produce sulphide, which induces corrosion of the metallic waste capsules designed for spent nuclear fuel. In addition, metallic waste present in the low-level and intermediate-level nuclear waste repository may be subject to corrosion. The rate of biological sulphate reduction in groundwater samples from Olkiluoto and in waters from experimental set-ups was studied with 35SO42- -radiolabel method. Furthermore, effects of supplemental nutrients and incubation time on detected sulphate reduction rates were studied. First, the method was optimised for the oligotrophic, deep groundwaters with low cell numbers. Abundance of marker genes was determined with qPCR, including dissimilatory sulphite reductase gene dsrB (beta subunit) and 16S rRNA gene of bacteria and archaea. Several improvements were made to the sulphate reduction rate measurement method during this thesis work. The method was demonstrated to be applicable for environmental samples. However, statistical matters, such as setting the minimum detection limit, need to be considered separately for each water type studied. Due to low cell numbers of the studied waters, variation between parallel samples was considerable throughout the work. Biological sulphate reduction was detected at various sites in the repository area. Sulphate reduction rates in the studied waters varied from approximately 0 to 1 nmol cm-3 d-1, whereas sulphate reduction rate of reference strain Desulfovibrio desulfuricans was up to 24 nmol cm-3 d-1. Supplemental nutrients and incubation time had variable effects on sulphate reduction rates. Microbial communities were demonstrated to be well-adapted to changing environmental conditions, as they were able to utilise introduced nutrients. Sulphate reduction rates detected in this study were modest. However, as the time scale for nuclear waste disposal is extremely long, estimating microbial metabolism over such period is challenging.
  • Pussila, Susanna (Helsingin yliopisto, 2014)
    The development of new antibiotics is very challenging work. However, it has become less attractive area of research for the pharmaceutical industry due to the rapid development of antibiotic resistance among bacteria. As a result, nowadays there are less new antimicrobial medicines appearing on the market. Increase in antibiotic resistance challenges the effective treatment of infectious diseases. Therefore, it is important to know about the existence and prevalence of antibiotic resistant bacteria and resistance genes. Waste water has been found to contain substantial amounts of antibiotics or their derivatives. Furthermore the bacterial density of the waste water is high. These factors may cause the selection pressure that assists the evolution, preservation and spread of antibiotic resistance in bacterial population. Wastewater treatment plants are believed to act as a reservoir of antibiotic resistant bacteria and antibiotic resistance genes. In this study, from samples taken from Viikinmäki wastewater treatment plant it was determined the quantity of resistance genes making bacteria resistance to a wide range of beta-lactam antibiotics. The samples were taken at the different stages of wastewater treatment process during three sampling days in June, September and December 2010. The samples were taken from the incoming wastewater and, from the final effluent and from the dried sludge. The DNA from wastewaters and sludge was isolated and the gene copy numbers of blaCTX-M-32 and blaOXA-58 antibiotic resistance genes were measured by quantitative PCR method. Antibiotic resistance gene copy numbers were normalized with 16S ribosomal RNA gene copy numbers. The antibiotic resistance genes investigated in this work make bacteria resistant to broad-spectrum penicillins, different groups of cephalosporins, monobactams, and carbapenems. It was found that blaOXA-58 and blaCTX-M-32 gene copies do exist in both incoming wastewater and dried sludge. From the incoming wastewater we found on average 3x10-3 blaOXA-58 gene copies/16S rRNA gene and 7x10-5 blaCTX-M-32 gene copies/16S rRNA gene. From dried sludge we found on average 3x10-3- blaOXA-58 gene copies/16S rRNA gene and 2x10-2 blaCTX-M-32 gene copies/16S rRNA. Both antibiotic resistance genes were also found in the final effluents. The amounts of antibiotic resistance genes /16S rRNA genes were found to decrease during of the wastewater treatment. In the final effluent the amount of blaOXA-58 genes was found to be two orders of magnitude less than in the incoming wastewater and for, blaCTX-M-32 gene copies it was found the decrease by one order of magnitude. Although the amount of antibiotic resistance genes decreased during of the wastewater treatment, the amount left in the final effluent is still notable and when it ends up in the water or soil it might have an effect to the antibiotic resistance in the nature.
  • Heinilä, Lassi Matti Petteri (Helsingin yliopisto, 2017)
    Cyanobacteria, also referred as blue-green algae, are abundant everywhere on earth inhabi-ting terrestrial and marine environments and living in symbiosis with several other organisms. Cyanobacteria were the first oxygenic photosynthetic organisms on earth and many species are also capable of fixing atmospheric nitrogen which makes them important primary produ-cers in many ecosystems. Cyanobacteria are nevertheless best known for producing toxic compounds. Cyanobacteria produce a variety of bioactive compounds including toxic ones. Some of these compounds have a potential as drugs. Most of these compounds are produ-ced by nonribosomal peptide synthetases and polyketide synthases. Nostoc sp. CENA543 is a cyanobacteria isolated from Brazilian wetland Pantanal. The strain was found to produce hepatptoxic nodularin, bioactive anabaenopeptins and a pre-viously unkown peptide. In this work the chemical structure and the biosynthetic gene cluster of the unkown peptide were characterized. Amount of produced nodularin was measured and biosynthetic genes of nodularin and anabaenopeptins were examined. Also the optimal growth conditions for the strain were studied. Essential methods applied in this work were liquid cromatography and mass spectromet-ry. To examine the biosynthetic genes DNA extraction methods and bioinformatic tools such as AntiSMASH, BLAST, Artemis and BioEdit were applied. The growth conditions were in-vestigated on different growth media. Bioactivity of the compounds were examined on disc diffusion assays and with the Kawabata method for enzyme activity. The results of this study show that Nostoc sp. CENA543 produces toxic amounts of no-dularin. The novel peptide group was named pseudospumigins. Six variants of pseudospu-migins were observed, the main variant being pseudospumigin A. Amino acid sequence of pseudospumigin A is Hpla-D-Hty-L-Ile-Argininal with mass of 612,4 Da. Gene clusters of nodularin and anabaenopeptin match corresponding genes of Nodularia spuimigena CCY9414. The biosynthetic genes of pseudospumigin have high resemblance to spumigin genecluster of Nodularia spuimigena CCY9414. Change in adenylation domain substrate specificity and deletion of three additional genes would axplain the difference between pseu-dospumigins and spumigins.
  • Suutarinen, Maiju (Helsingin yliopisto, 2019)
    Imbalance of intestinal microbiota is called dysbiosis. Signs of dysbiosis are altered abundance of different bacterial species and reduced diversity together with altered interactions between bacterial species and microbiota and the host. Dysbiosis of intestinal microbiota is connected to many intestinal diseases and today many studies are focused to find so called “next generation” probiotics to be used for the alleviation of dysbiosis instead of traditional antibiotic treatments. The study was made in the Human Microbiome Research Program, Faculty of Medicine, University of Helsinki. Aim of the study was to isolate spore-forming bacterial species for the treatment of intestinal inflammation and infections with bacterial therapy. For this purpose, feces from a healthy adult who had acted as a donor for fecal microbiota transplantation was used to isolate spore-forming commensal bacteria. The isolated bacteria were identified and their ability to adhere into intestinal epithelium and strengthen it was investigated. Also anti-inflammatory potential of these isolated bacterial strains was investigated. For isolating bacteria three different heat treatments and ethanol and methanol treatments were used as a pre-treatment step. Pre-treated samples were cultivated on YCFA-media and isolates were picked from plates at different growth points for further cultivation. Selected isolates were purified, their DNA was isolated and they were identified by partial 16S rRNA -gene sequencing. From these identified isolates four isolates were chosen for further investigation and their full length 16S rRNA -gene was sequenced. These isolates were studied also by using API and aerotolerance tests. Potential anti-inflammatory and adhesion properties of the isolates were investigated by attenuation, adhesion and TER-experiments. In the isolation, the effect of different pre-treatments on the recovery of isolates was clear and based on sequencing isolates that were spore-forming anaerobic bacteria were selected for further investigation. Three of the isolates were Clostridium butyricum and one Blautia wexlerae species. Anti- and pro-inflammatory properties of these isolates were very different depending on isolate and one of them was potentially anti-inflammatory. Isolates also adhered differentially and two of them possibly strengthened gut epithelial barrier so they are promising for further research and in the future investigation with these isolates continues. Experience and results with different cultivation methods can be used to for further development of cultivation for anaerobic intestinal bacteria.
  • Zhen, Zeng (Helsingfors universitet, 2014)
    Rhizobia are agriculturally important bacteria that possess the ability to fix nitrogen for their host legumes, an attribute ascribed to the presence of symbiosis-related genes usually clustered on plasmids called symbiotic plasmids (pSyms). Many pSyms have been proven self-transmissible, capable of transferring themselves to other bacteria through conjugation, thereby propagating their symbiotic features. Rhizobium galegae symbiovar (sv.) officinalis has a pSym, on which typical conjugation genes have been revealed. A Type IV secretion system (T4SS) functioning as a conjugation system has also been computationally predicted on a chromid, another replicon in R. galegae sv. officinalis. In addition, the transfer of the pSym of R. galegae sv. officinalis to a non-nodulating mutant strain of R. galegae sv. orientalis has been previously observed under laboratory conditions. Therefore, this thesis was aimed at investigating the self-transmissibility of the pSym of R. galegae sv. officinalis and the necessity of the T4SS’ presence for the pSym transfer. Two derivatives of the R. galegae sv. officinalis were generated with one strain cured of its pSym by using Tn5-mob-sacB transposon and the other strain excised the T4SS from the chromid by Cre-lox site specific recombination system. Conjugation were then performed between these two derivatives as well as between the wild-type strain and the plasmid-cured derivative, followed by the host plant nodulation tests. The tests showed no formation of a single nodule in either pair, which was unexpectedly inconsistent with the previous experimental observation. No solid explanations could be proposed at this stage. It might be due to the low transfer frequency resulted from complex associations with subtle environmental signal molecules or recipient cell recognition that presumably disabled the transmissibility of the pSym.
  • Rautapää, Henriikka (Helsingin yliopisto, 2018)
    Cryptosporidium parvum is a coccidian parasite. It causes an intestinal infection, named cryptosporidiosis. C. parvum infects mainly young calves and can be transmitted zoonotically to humans. C. parvum causes watery diarrhea, which is usually self-limiting. Cryptosporidiosis can be life-threatening to small children, immunocompromised patients and weak calves. C. parvum spreads mainly by the fecal-oral route by oocysts. It multiplies intracellularly, usually in the enterocytes of the small intestine. The life cycle is spent in a single host. Oocysts are extremely resilient and can cause large epidemics when household water or food gets exposed. Aim of this study was to research subtypes of protozoan C. parvum that occurred in calves in Finland 2012-2017, and research their divergence geographically and temporally. Additionally subtypes of 10 C. parvum -isolates from humans were studied and results were compared to subtypes found in calves. Fassisi BoDia, a rapid test designed to identify the four most common pathogens that infect young calves (rotavirus, coronavirus, C. parvum and E. coli K99/F5-type) was tested. The rapid test also detects possible mixed infections. Test is immunocromatographic and the diagnostic method to find C. parvum’s specific protein is based on monoclonal antibodies. Sample collection for determination of subtypes consists of 100 fecal samples from calves and 10 human fecal samples. Fassisi BoDia rapid test was tested with 67 fecal samples from calves. Subtyping was made by multiplying the protein gp60 coding gene with nested-PCR and by analyzing sequences of the gene. Research was made in Evira research unit of veterinary bacteriology and pathology in Kuopio. Subtype IIaA15G2R1 was the most common in calves. Distribution of this subtype had increased in Finland after subtype results from earlier years. Subtype IIaA15G2R1 was mainly found in western Finland, especially around Pohjanmaa. Subtype IIaA18G1R1 was the only one found in human samples, the same subtype was not found from calf samples in this study. Sensitivity of the Fassisi BoDia rapid test for C. parvum was 91,7 % and specificity was 83,9 % reliability.
  • Klingenberg, Daniela (Helsingfors universitet, 2011)
    The bacterial genus Stenotrophomonas comprises 12 species. They are widely found throughout the environment and particularly S. maltophilia, S. rhizophila and S. pavanii are closely associated with plants. Strains of the most common Stenotrophomonas species, S. maltophilia, promote plant growth and health, degrade natural and man-made pollutants and produce biomolecules of biotechnological and economical value. Many S. maltophilia –strains are also multidrug resistant and can act as opportunistic human pathogens. During an INCO-project (1998-2002) rhizobia were collected from root nodules of the tropical leguminous tree Calliandra calothyrsus Meisn. from several countries in Central America, Africa and New Caledonia. The strains were identified by the N2-group (Helsinki university) and some strains turned out to be members of the genus Stenotrophomonas. Several Stenotrophomonas strains induced white tumor- or nodule-like structures on Calliandra?s roots in plant experiments. The strains could, besides from root nodules, also be isolated from surface sterilized roots and stems. The purpose of my work was to investigate if the Stenotrophomonas strains i) belong to a new Stenotrophomonas species, ii) have the same origin, iii) if there are other differences than colony morphology between phase variations of the same strain, iv) have plant growth-promoting (PGP) activity or other advantageous effects on plants, and v) like rhizobia have ability to induce root nodule formation. The genetic diversity and clustering of the Stenotrophomonas strains were analyzed with AFLP fingerprinting to get indications about their geographical origin. Differences in enzymatic properties and ability to use different carbon and energy sources were tested between the two phases of each strain with commercial API tests for bacterial identification. The ability to infect root hairs and induce root nodule formation was investigated both using plant tests with the host plant Calliandra and PCR amplification of nodA and nodC genes for nodulation. The PGP activity of the strains was tested in vitro mainly with plate methods. The impact on growth, nitrogen content and nodulation in vivo was investigated through greenhouse experiments with the legumes Phaseolus vulgaris and Galega orientalis. Both the genetic and phenotypic diversity among the Stenotrophomonas strains was small, which proposes that they have the same origin. The strains brought about changes on the root hairs of Calliandra and they also increased the amount of root hairs. However, no root nodules were detected. The strains produced IAA, protease and lipase in vitro. They also showed plant a growth-promoting effect on G. orientalis, both alone and together with R. galegae HAMBI 540, and also activated nodulation among efficient rhizobia on P. vulgaris in greenhouse. It requires further research to get a better picture about the mechanisms behind the positive effects. The results in this thesis, however, confirm earlier studies concerning Stenotrophomonas positive impact on plants.