Degradation of lignin and other 14C-labelled compounds in compost and soil with an emphasis on white-rot fungi

Puu, ruohokasvit ja näistä tehdyt tuotteet kuten mekaanisesta massasta valmistettu sanomalehtipaperi sisältävät ligniiniä, joka hajoaa yleensä hyvin hitaasti luonnossa. Valkolahosienet hajottavat ligniiniä tehokkaimmin, ja koska niiden tuottamat entsyymit hajottavat myös muita vaikeasti hajoavia yhdisteitä, voidaan valkolahosienten avulla mahdollisesti puhdistaa saastuneita maita. Tässä työssä haluttiin selvittää, säilyttävätkö valkolahosienet (Abortiporus biennis, Bjerkandera adusta, Dichomitus squalens, Phanerochaete chrysosporium, Phanerochaete sordida, Phlebia radiata, Pleurotus ostreatus, Trametes hirsuta ja Trametes versicolor) aktiivisuutensa ja kasvavatko ne maassa. Aktiivisuutta mitattiin seuraamalla sienten synteettisen ligniinin (14C-DHP) hajotuskykyä. T. versicolor (silkkivyökääpä) osoittautui tehokkaimmaksi ligniinin hajottajaksi ja sen pentakloorifenolin (PCP) hajotuskykyä tutkittiin erillisessä kokeessa. Entiset tai pitkään käytössä olleet saha-alueet ovat yhä saastuneet puun käsittelyaineista peräisin olevilla kloorifenoleilla. Biohajoavien muovien kehitystyö sekä kompostoinnin yleistyminen jätteiden käsittelymenetelmänä ovat luoneet tarpeen materiaalien biohajoavuuden määrittämiseen. Euroopan standardisoimisjärjestön (CEN) kontrolloidussa kompostitestissä biohajoavuus määritetään materiaalin hajoamisen aikana muodostuvan hiilidioksidin perusteella. Hiilidioksidin tuotto mitataan sekä näytettä sisältävästä kompostista että kompostista ilman näytettä, ja tällöin oletetaan, että kompostin orgaaninen aines molemmissa komposteissa (tausta) tuottaa yhtä paljon hiilidioksidia. Testin puutteeksi saattaa osoittautua kompostissa tai maassa esiintyvä "priming effect". Tällä tarkoitetaan materiaalin lisäämisen jälkeen esiintyvää epänormaalin suurita tai pientä hiilidioksidin muodostusta, minkä seurauksena testin tulosksena saatava biohajoavuus on virheellinen. Ligniinin hajotessa muodostuu enemmän humusta kuin hiilidioksidia, koska ligniini on humuksen tärkein lähtöaine. Näin ollen ligniiniä sisältävät paperituotteet saattavat testin mukaan vaikuttaa biologisesti hajoamattomilta. Valkolahosienet hajottivat 4-23% ligniinistä hiilidioksidiksi ja T. versicolor 29% PCP:sta. Kompostissa ligniini hajosi hiilidioksidiksi 58°C:ssa huomattavasti vähemmän (8%) kuin lämpötiloissa 35°C ja 50°C (23-24%). Kompostin todennäköisesti tärkeimpien ligniinin hajottajien, termofiilisten sienten, tyypillinen optimilämpötila on 45°C, eivätkä ne ole enää aktiivisia 58°C:ssa. Sekä maassa että kompostissa ligniini sitoutui kuitenkin suurimmaksi osaksi humukseen. Valkolahosienet hajottivat sekä humukseen sitoutunutta ligniiniä että PCP:ia, mutta kompostin sekapopulaatio ei tähän pystynyt, ja ligniiniä sitoutui humukseen yhä enemmän kompostoinnin aikana. T. versicolor hajotti PCP:ia tehokkaasti, eikä se tuottanut myrkyllisiä kloorianisoleja, joita jotkut valkolahosienet saattavat muodostaa kloorifenoleista. Priming effect ilmiötä tutkittiin eri ikäisissä ja kypsyydeltään erilaisissa komposteissa. Kompostit erosvat toisistaan myös hajoamattoman jätteen määrän ja mikrobipopulaation suhteen. Negatiivinen priming effect havaittiin kaikissa epästabiileissa komposteissa (ikä enintään 6 kk), ja sen lisäksi yhdessä näistä komposteista positiivinen priming effect kokeen lopussa. Stabiileissa komposteissa (ikä vähintään 6 kk) ilmiötä ei sen sijaan havaittu. Epästabiileissa komposteissa biohajoavuudelle saadut tulokset eivät siis ole luotettavia. Työn tulosten perusteella valkolahosienet, ja erityisesti T. versicolor, ovat lupaavia saastuneen maan puhdistukseen, joskin sienirihmaston mahdollisuudet säilyä aktiivisena maan alkuperäisen mikrobipopulaation kanssa täytyy vielä selvittää. Kompostin sekapopulaatio, joka ei sisällä valkolahosieniä, hajotti ligniiniä yllättävän tehokkaasti termofiilisille sienille sopivissa lämpötiloissa, vaikka ligniini sitoutuikin pääasiallisesti humukseen. Kompostin kypsyys osoittautui tärkeäksi tekijäksi kontrolloidun kompostitestin onnistumisen kannalta. Priming effect ilmiön välttämiseksi on varmistettava, että testissä käytetty komposti on riittävän kypsä. Kompostien mikrobipopulaation koostumusta kompostoinnin eri vaiheissa tulisi tarkemmin selvittää, koska stabiilien ja epästabiilien kompostien ero aiheutui todennäköisesti populaatioiden rakenteessa vallitsevista eroista. Näin myös priming effect ilmiön syyt voitaisiin selittää paremmin.

Lignocellulose degradation and humus modification by the fungus Paecilomyces inflatus

Composting is the biological conversion of solid organic waste into usable end products such as fertilizers, substrates for mushroom production and biogas. Although composts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and vegetable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident microbial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic polymers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation. In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces inflatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocellulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes. Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. inflatus mineralised 6-10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly mineralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic compounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromatics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degradative features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment, with a special emphasis on the role of ubiquitous microfungi.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Pystynävertäjän (Tomicus piniperda) vaikutus männyn (Pinus sylvestris) fotosynteesiin ja VOC-emissioihin

Pystynävertäjä on Suomessa yksi merkittävimpiä mäntyjen tuhohyönteisiä. Se syö männynversoja ontoiksi, minkä seurauksena nämä tippuvat maahan. Tästä aiheutuu puulle kasvutappioita ja metsänomistajalle tulonmenetyksiä. Pystynävertäjän ekologiaa on tutkittu paljon, mutta ei tiedetä, miten se vaikuttaa versojen fotosynteesiin ja haihtuvien orgaanisten yhdisteiden eli VOC-yhdisteiden emissioihin ennen versojen putoamista. Monissa muissa tutkimuksissa on havaittu herbivoreilla olevan vaikutusta isäntäkasviensa fotosynteesiin. Lisäksi herbivorian seurauksena isäntäkasvin VOC-emissiot ovat voineet muuttua laadultaan tai määrältään. Pystynävertäjän vaikutuksia versoihin tutkittiin kokeellisesti SMEAR II -asemalla Pirkanmaalla. Pystynävertäjän vaurioittamien ja kontrolliversojen fotosynteesiä mittattiin kahden männyn latvuksessa heinä–syyskuussa 2010. Mittaukset tehtiin kannettavalla fotosynteesimittauslaitteistolla. Samalla otettiin VOC-näytteitä neulasten emissioista adsorbenttiputkiin. Lisäksi toisesta koepuusta suljettiin pystynävertäjäja kontrolliverso raamikyvettiin, johon mahtui oksan kärkeä n. 20 cm. Raamikyvetin läpi kierrätetystä ilmasta kerättiin VOC-näytteitä SPME-kuituun ja siihen sitoutuneet yhdisteet analysoitiin enantiomeereittain. Pystynävertäjä- ja kontrolliversojen välillä ei ollut selvää eroa hiilen assimilaatiossa, transpiraatiossa, ilmarakokonduktanssissa, vedenkäytön tehokkuudessa tai fotokemiallisessa saannossa. Vain pahimmin vaurioituneissa versoissa vaurio vaikutti fotosynteesiin huomattavasti. Esimerkiksi fotokemiallinen saanto laski selvästi vasta, kun verso oli juuri katkeamaisillaan pystynävertäjän sisäänmenoreiän kohdalta. Neulaskyvettimittausten perusteella vaikuttaa siltä, että pystynävertäjä toisella koepuulla lisää monoterpeeniemissioita ja toisella vähentää niitä. Raamikyvettimittauksessa pystynävertäjä lisäsi (+)-enantiomeerin osuutta ?- ja ?-pineeni- sekä limoneeniemissioista, mutta emissioiden kokonaismäärä ei juuri eronnut kontrolliversosta. Pystynävertäjän vaikutukset versojen fotosynteesiin jäivät vähäisiksi, vaikka pystynävertäjä olikin tehnyt versoihin yhdestä neljään reikää ja niiden kohdalta poistanut suuren osan verson poikkileikkauspinta-alasta. Pystynävertäjän aiheuttamat kasvutappiot vaikuttavatkin olevan seurausta lähes yksinomaan versojen putoamisesta. Pystynävertäjä ei juuri vaikuttanut versosta haihtuvien yhdisteiden kokonaismäärään, mutta se muutti niiden keskinäisiä runsaussuhteita ja erityisesti enantiomeerien runsaussuhteita. Pystynävertäjien vaikutukset VOC-emissioihin näyttävät riippuvan suuresti kemotyypistä. Laajempien johtopäätösten tekeminen pystynävertäjän vaikutuksista männyn VOC-emissioihin vaatisi tutkimuksen toistamista suuremmalla koepuumäärällä.

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.