Effects of Bio-Ash Amendments on the Metabolism of Ectomycorrhizal Fungi : A Method Development and Metabolomic Study

Forest ecosystems have played a fundamental role in the development of our society. Since the beginning of the civilization, forest have provided us with wood as a product for construction, tools, furniture and domestic heating. The well-being of the forest is therefore fundamental to our existence. Today, our growing societies have increased energy needs; the resulting depletion of fossil reserves and the effects of their use has again shown how the forest is among the most important alternatives for sustainability of our ecosystem. In order to responsibly make this resource a key part of our energy and material supply, we need to understand how forestry practices influence the different processes taking place in the forest ecosystems. The use of raw material from forest as energy source produces huge amounts of ash. The ash contains the base cations that have once been translocated from soil to the upper parts of the trees. Ash recycling has therefore been suggested as a measure to counteract soil acidification due to extensive harvest. Since spreading of ash can have great effects on the forest, it is important to understand which these effects are and how big they might be. This thesis focuses on the effects that such an ash recycling may have on the metabolism of ectomycorrhizal fungi; that is, fungi that are able to colonize root of trees, and contribute to the acquisition of nutrients and water from soil. The work presented here utilized an in vitro metabolomic approach on eight species of ectomycorrhizal fungi normally found in boreal forests. A targeted metabolomic study addressed the effects of ash amendments on growth, external pH and the exudation of low molecular mass organic acids, amino acids and hydroxamate siderophores. This was complemented by an untargeted metabolomic study to address the effects of ash amendment on the general metabolism of the fungal species. Analyses were performed with well-established chemical methods, and some that had to be developed specifically for this thesis work. A method for the analysis of amino acids without derivatization and yet compatible with mass spectrometry had to be developed and validated. The result was a robust method that works well with external calibration, shows good long-term stability, relatively low detections limits and high sample throughput. A screening protocol for the determination of siderophores from mass spectrometry data was also established. The metabolomic studies showed that bio-ash amendment increased the exudation of low molecular mass organic compounds from all the studied species. This means that the species tended to exude more of the same compounds compared to the controls without ash. In some cases, the bio-ash also triggered the exudation of new compounds. There was some exceptions, though; bio-ash amendment had negative effects on the exudation of certain metabolites, but these negative effects were of lower magnitude compared to the positive effects. Both metabolomics studies showed a differentiation between the ascomycetes and the basidiomycetes species. The targeted metabolomic study, indicated a trade-off in the utilization of carbon for accumulation of biomass or for the exudation of low molecular mass organic compounds, in which the ascomycetes accumulated more carbon as biomass compared to the basidiomycetes. According to the untargeted metabolomic study, the ascomycetes species presented the greatest number of metabolites that were influenced significantly by ash treatment, either as increase or decrease. Adding extracted ash to the culturing medium at the beginning of the experiment increased the pH, but this was counteracted by species metabolism as exudation of organic acids correlated with a drop in external pH. Ash treatment triggered the total exudation of low molecular mass organic acids in five of the eight studied species and especially in Cortinarius glaucopus. Ash treatment also triggered the exudation of amino acids from Tomentellopsis submollis and ferricrocin from Hymenoscyphus ericae. Of note was that no metabolite significantly influenced by ash was found to be common to all species, indicating that the ash amendments mainly affected the secondary metabolism under the culturing conditions used. Additionally, the ascomycetes Hymenoscyphus ericae exuded the greatest number of metabolites affected by ash that were exuded only by a single species. Conversely, Piloderma olivaceum exuded the largest number of unique metabolites not influenced by ash.Skogsekosystem har genom alla tider spelat en central roll i utvecklingen av vårt samhälle. Trä för konstruktioner, möbler och hushållsuppvärmning har varit tjänster från skogar sedan civilisationens början. Skogens välbefinnande kan därför anses grundläggande för vår existens. Idag har vårt samhälle utvecklats och behovet av skogsråvara ökar kontinuerligt. Detta i kombination  med minskande fossila reserver och den inverkan som användningen av dessa reserver har haft på vår miljö har återigen gjort skogen till ett av de viktigaste alternativen för att åstadkomma en hållbar utveckling. Detta innebär att vi måste öka vår kunskap om hur olika former av skogsbruk påverkar miljön i skogsekosystemen.  Användningen av skogsråvara som energikälla ger upphov till stora mängder aska. Askan innehåller höga halter av de baskatjoner som en gång har transporteras från jorden till de övre delarna av träden. Återföring av aska har därför föreslagits som en skogsbrukspraxis för att motverka den försurning som en omfattande avverkning kan ge upphov till. Eftersom återföring av aska kan ha stora effekter på skogen är det viktigt att förstå vilka dessa effekter är och hur omfattande de kan vara. Denna avhandling fokuserar på de effekter som askåterföring till skogsmark kan ha på metabolismen hos ektomykorrhizasvampar, det vill säga svampar som kan kolonisera rötter och bidra till trädens upptag av näringsämnen och vatten från jorden. Arbetet uppnåddes med ett in vitro metabolomiskt tillvägagångsätt på åtta arter av ektomykorrhizasvampar som förekommer normalt i boreala skogar. En riktad metabolomisk studie genomfördes för att studera effekterna av olika doser av aska på tillväxt, yttre pH och utsöndring av lågmolekylära organiska syror samt aminosyror och hydroxamatsideroforer. Denna studie kompletterades med en mer generell metabolomikstudie där effekterna av aska på svamparnas totala metabolism studerades. I ett arbete som detta är det viktigt med tillförlitliga metoder. En stor del av detta arbete har varit att utveckla nya kemiska analysmetoder. En metod för analys av aminosyror utan derivatisering och samtidigt kompatibel med masspektrometrisk detektion behövde utvecklas och valideras. Detta resulterade i en robust metod som fungerade väl med extern kalibrering och uppvisade bra detektionsnivåer samt förhållandevis hög genomströmning. Vidare så utvecklades även en metod för att kunna upptäcka förekomst av låga halter av sideroforer i komplexa prover med stöd av masspektrometri. De metabolomiska studierna visade att exponering for bioaska ledde till ökad utsöndring av lågmolekylära organiska föreningar hos alla studerade arter. Huvudsakligen tenderade svamparna till att utsöndra mer av samma föreningar som registrerades för kontrollerna utan tillförsel av aska. I vissa fall ledde exponeringen även till utsöndring av nya föreningar. Det fanns också undantag där askan snarare hade negativ inverkan på utsöndringen av vissa metaboliter. Generellt var dessa negativa effekter av lägre magnitud än de positiva effekterna. Båda metabolomikstudierna visade en differentiering mellan basidiomyceter och ascomyceter. Den riktade metabolomiska studien antydde en kompromiss mellan användningen av kol för ackumulering av biomassa respektive utsöndring av organiska föreningar med låg molekylvikt, där ascomyceterna tycktes ackumulera mer kol som biomassa jämfört med de basidiomyceter som ingick i studien. Den mer generella metabolomikstudien, visade också att ascomyceterna var de arter som utsöndrade flest antal metaboliter som påverkades av bioaska, antingen i positiv eller i negativ riktning. Tillsats av extraherad bioaska till odlingsmediet vid experimentets början ledde till ökat pH, men detta motverkades med tiden av metabolismen genom utsöndringen av organiska syror. Askbehandlingen resulterade i ökad utsöndring av organiska syror med låg molekylvikt för fem av de åtta studerade arterna och speciellt för Cortinarius glaucopus. Behandlingen resulterade också i ökad utsöndring av aminosyror från Tomentellopsis submollis och av ferricrocin från Hymenoscyphus ericae. Ingen av de metaboliter som påverkades signifikant av bioaska visade sig vara gemensam för alla studerade arter. Detta indikerar att under de odlingsbetingelser som användes så var det huvudsakligen den sekundära metabolismen som påverkades.  Vid tidpunkten för disputationen var följande delarbeten opublicerade: delarbete 2 (inskickat), delarbete 4 (manuskript).At the time of the doctoral defence the following papers were unpublished: paper 2 (submitted), paper 4 (manuscript).</p

ENHANCED NATURAL BIODEGRADATION OF DIESEL FUEL CONTAMINANTS IN SOIL BY ADDITION OF WHEY AND NUTRIENTS

The contamination of soils by petroleum hydrocarbons, such as diesel fuel, has since many years been a serious environmental problem. Treatment of contaminated areas is a concern for governments and environmental authorities in several countries and efforts have been done with the purpose to eliminate this problem. Different methods have been tested and today the most common technique involves the excavation and transportation of contaminated soil to special treatment facilities. In earlier studies we have demonstrated the effect of adding organic amendments, such as fermented whey, on the biodegradation of n-alkanes in diesel contaminated soil. Non-fermented sweet whey also proved significantly to enhance the biodegradation of an aromatic substance (phenanthrene) in contaminated soil. The current paper presents the results of an in-situ field test at a former gas station in the north of Sweden. In parallel to the field study, biodegradation profiles were monitored under controlled laboratory conditions by taking soil samples from the contaminated site and spike them with diesel fuel. The experiments were carried out by adding whey and mineral nutrients (NPK) to the test area and to the laboratory samples, and monitor the degradation of hydrocarbons by gas chromatographic analysis of extracted soil samples. Significant effects on the degradation rates were achieved in the laboratory tests. For the in-situ test, however, no such positive effects could be registered.Bioremediatio

Supercapacitors with graphene coated paper electrodes

Paper based supercapacitors are prepared by stacking a paper between two graphene electrodes and soaking these in an aqueous electrolyte. We demonstrate that supercapacitors can easily be manufactured by using proven paper technologies. Several different electrode materials were compared and two types of contacting material, silver and graphite foil were tested. The influence of the paper used as separator was also investigated. The supercapacitors with a graphene-gold nanoparticle composite as electrodes showed a specific capacitance of up to 100 F/g and an energy density of 1.27 Wh/kg. The energy density can further be increased by using other electrolytes. The silver contacts showed a pseudo capacitance, which the graphite contacts did not. The papers tested had a minor effect on the capacitance, but they have an influence on the weight and the volume of the supercapacitor