Functional genes and gene array analysis as tools for monitoring hydrocarbon biodegradation

Bioremediation, which is the exploitation of the intrinsic ability of environmental microbes to degrade and remove harmful compounds from nature, is considered to be an environmentally sustainable and cost-effective means for environmental clean-up. However, a comprehensive understanding of the biodegradation potential of microbial communities and their response to decontamination measures is required for the effective management of bioremediation processes. In this thesis, the potential to use hydrocarbon-degradative genes as indicators of aerobic hydrocarbon biodegradation was investigated. Small-scale functional gene macro- and microarrays targeting aliphatic, monoaromatic and low molecular weight polyaromatic hydrocarbon biodegradation were developed in order to simultaneously monitor the biodegradation of mixtures of hydrocarbons. The validity of the array analysis in monitoring hydrocarbon biodegradation was evaluated in microcosm studies and field-scale bioremediation processes by comparing the hybridization signal intensities to hydrocarbon mineralization, real-time polymerase chain reaction (PCR), dot blot hybridization and both chemical and microbiological monitoring data. The results obtained by real-time PCR, dot blot hybridization and gene array analysis were in good agreement with hydrocarbon biodegradation in laboratory-scale microcosms. Mineralization of several hydrocarbons could be monitored simultaneously using gene array analysis. In the field-scale bioremediation processes, the detection and enumeration of hydrocarbon-degradative genes provided important additional information for process optimization and design. In creosote-contaminated groundwater, gene array analysis demonstrated that the aerobic biodegradation potential that was present at the site, but restrained under the oxygen-limited conditions, could be successfully stimulated with aeration and nutrient infiltration. During ex situ bioremediation of diesel oil- and lubrication oil-contaminated soil, the functional gene array analysis revealed inefficient hydrocarbon biodegradation, caused by poor aeration during composting. The functional gene array specifically detected upper and lower biodegradation pathways required for complete mineralization of hydrocarbons. Bacteria representing 1 % of the microbial community could be detected without prior PCR amplification. Molecular biological monitoring methods based on functional genes provide powerful tools for the development of more efficient remediation processes. The parallel detection of several functional genes using functional gene array analysis is an especially promising tool for monitoring the biodegradation of mixtures of hydrocarbons.Biopuhdistuksessa pilaantunutta ympäristöä kunnostetaan haitallisia yhdisteitä hajottavia, ympäristössä luonnollisesti esiintyviä mikrobeja hyväksi käyttäen. Biopuhdistusta pidetään ympäristön kannalta kestävänä sekä taloudellisesti kannattavana kunnostusmenetelmänä. Biologisten puhdistusprosessien hallitseminen vaatii kuitenkin hyvää ymmärrystä mikrobiyhteisöjen hajotuspotentiaalista sekä haitta-aineita hajottavissa mikrobeissa kunnostuksen seurauksena tapahtuvista muutoksista. Tässä väitöskirjatutkimuksessa tutkittiin hiilivetyjen hajotuksesta vastaavien geenien soveltuvuutta hiilivetyjen biohajoamisen seurantaan. Jotta useiden hiilivetyjen biohajoamista olisi mahdollista seurata samanaikaisesti, tutkimuksessa kehitettiin suoraketjuisten sekä monoaromaattisten ja polyaromaattisten hiilivetyjen hajotusgeeneistä koostuvat pienimuotoiset geeniarrayt. Menetelmien toimivuutta hiilivetyjen biohajoamisen seurannassa arvioitiin sekä laboratoriomittakaavan biohajoamiskokeissa että kenttämittakaavan biopuhdistusprosesseissa. Laboratoriomittakaavan biohajoamiskokeissa molekyylibiologisilla seurantamenetelmillä mitatut muutokset hajotusgeenien määrissä olivat yhdenmukaisia hiilivetyjen biohajoamisen kanssa. Geeniarray-analyysi mahdollisti useiden hajotusgeenien ja siten useiden erilaisten hiilivetyjen biohajoamisen tutkimisen samanaikaisesti. Hajotusgeenien tutkiminen molekyylibiologisten menetelmien avulla lisäsi biologisten kunnostusprosessien optimoinnissa ja suunnittelussa tarvittavaa tietoa myös kenttämittakaavan biopuhdistusprosesseissa. Esimerkiksi kreosootilla pilaantuneessa pohjavedessä geeniarray-analyysi osoitti, että kohteessa esiintyvää, mutta hapettomien olosuhteiden rajoittamaa, hiilivetyjen hajotuspotentiaalia oli mahdollista aktivoida hapettamalla sekä lisäämällä ravinteita. Tutkimuksen tulokset osoittavat, että hajotusgeenit ja niihin perustuvat molekyylibiologiset seurantamenetelmät muodostavat tehokkaita seurantatyökaluja, joiden avulla on mahdollista kehittää entistä tehokkaampia ja hallittavampia kunnostusprosesseja. Useiden hajotusgeenien samanaikainen tunnistaminen geeniarray-analyysiin perustuen on erityisen lupaava menetelmä hiilivetyseosten biohajoamisen seurantaan

Response of Deep Subsurface Microbial Community to Different Carbon Sources and Electron Acceptors during similar to 2 months Incubation in Microcosms

Acetate plays a key role as electron donor and acceptor and serves as carbon source in oligotrophic deep subsurface environments. It can be produced from inorganic carbon by acetogenic microbes or through breakdown of more complex organic matter. Acetate is an important molecule for sulfate reducers that are substantially present in several deep bedrock environments. Aceticlastic methanogens use acetate as an electron donor and/or a carbon source. The goal of this study was to shed light on carbon cycling and competition in microbial communities in fracture fluids of Finnish crystalline bedrock groundwater system. Fracture fluid was anaerobically collected from a fracture zone at 967 m depth of the Outokumpu Deep Drill Hole and amended with acetate, acetate + sulfate, sulfate only or left unamended as a control and incubated up to 68 days. The headspace atmosphere of microcosms consisted of 80% hydrogen and 20% CO2. We studied the changes in the microbial communities with community fingerprinting technique as well as high-throughput 16S rRNA gene amplicon sequencing. The amended microcosms hosted more diverse bacterial communities compared to the intrinsic fracture zone community and the control treatment without amendments. The majority of the bacterial populations enriched with acetate belonged to clostridial hydrogenotrophic thiosulfate reducers and Alphaproteobacteria affiliating with groups earlier found from subsurface and groundwater environments. We detected a slight increase in the number of sulfate reducers after the 68 days of incubation. The microbial community changed significantly during the experiment, but increase in specifically acetate-cycling microbial groups was not observed.Peer reviewe

Rapid Reactivation of Deep Subsurface Microbes in the Presence of C-1 Compounds

Microorganisms in the deep biosphere are believed to conduct little metabolic activity due to low nutrient availability in these environments. However, destructive penetration to long-isolated bedrock environments during construction of underground waste repositories can lead to increased nutrient availability and potentially affect the long-term stability of the repository systems, Here, we studied how microorganisms present in fracture fluid from a depth of 500 m in Outokumpu, Finland, respond to simple carbon compounds (C-1 compounds) in the presence or absence of sulphate as an electron acceptor. C-1 compounds such as methane and methanol are important intermediates in the deep subsurface carbon cycle, and electron acceptors such as sulphate are critical components of oxidation processes. Fracture fluid samples were incubated in vitro with either methane or methanol in the presence or absence of sulphate as an electron acceptor. Metabolic response was measured by staining the microbial cells with fluorescent dyes that indicate metabolic activity and transcriptional response with RT-qPCR. Our results show that deep subsurface microbes exist in dormant states but rapidly reactivate their transcription and respiration systems in the presence of C-1 substrates, particularly methane. Microbial activity was further enhanced by the addition of sulphate as an electron acceptor. Sulphate- and nitrate-reducing microbes were particularly responsive to the addition of C-1 compounds and sulphate. These taxa are common in deep biosphere environments and may be affected by conditions disturbed by bedrock intrusion, as from drilling and excavation for long-term storage of hazardous waste.Peer reviewe

Active Microbial Communities Inhabit Sulphate-Methane Interphase in Deep Bedrock Fracture Fluids in Olkiluoto, Finland

Active microbial communities of deep crystalline bedrock fracture water were investigated from seven different boreholes in Olkiluoto (Western Finland) using bacterial and archaeal 16S rRNA, dsrB, and mcrA gene transcript targeted 454 pyrosequencing. Over a depth range of 296–798 m below ground surface the microbial communities changed according to depth, salinity gradient, and sulphate and methane concentrations. The highest bacterial diversity was observed in the sulphate-methane mixing zone (SMMZ) at 250–350 m depth, whereas archaeal diversity was highest in the lowest boundaries of the SMMZ. Sulphide-oxidizing ε-proteobacteria (Sulfurimonas sp.) dominated in the SMMZ and γ-proteobacteria (Pseudomonas spp.) below the SMMZ. The active archaeal communities consisted mostly of ANME-2D and Thermoplasmatales groups, although Methermicoccaceae, Methanobacteriaceae, and Thermoplasmatales (SAGMEG, TMG) were more common at 415–559 m depth. Typical indicator microorganisms for sulphate-methane transition zones in marine sediments, such as ANME-1 archaea, α-, β- and δ-proteobacteria, JS1, Actinomycetes, Planctomycetes, Chloroflexi, and MBGB Crenarchaeota were detected at specific depths. DsrB genes were most numerous and most actively transcribed in the SMMZ while the mcrA gene concentration was highest in the deep methane rich groundwater. Our results demonstrate that active and highly diverse but sparse and stratified microbial communities inhabit the Fennoscandian deep bedrock ecosystems

Oil degradation potential of microbial communities in water and sediment of Baltic Sea coastal area

Two long-term potentially oil exposed Baltic Sea coastal sites near old oil refineries and harbours were compared to nearby less exposed sites in terms of bacterial, archaeal and fungal microbiomes and oil degradation potential. The bacterial, archaeal and fungal diversities were similar in oil exposed and less exposed sampling sites based on bacterial and archaeal 16S rRNA gene and fungal 5.8S rRNA gene amplicon sequencing from both DNA and RNA fractions. The number of genes participating in alkane degradation (alkB) or PAH-ring hydroxylation (PAH–RHDα) were detected by qPCR in all water and sediment samples. These numbers correlated with the number of bacterial 16S rRNA gene copies in sediment samples but not with the concentration of petroleum hydrocarbons or PAHs. This indicates that both the clean and the more polluted sites at the Baltic Sea coastal areas have a potential for petroleum hydrocarbon degradation. The active community (based on RNA) of the coastal Baltic Sea water differed largely from the total community (based on DNA). The most noticeable difference was seen in the bacterial community in the water samples were the active community was dominated by Cyanobacteria and Proteobacteria whereas in total bacterial community Actinobacteria was the most abundant phylum. The abundance, richness and diversity of Fungi present in water and sediment samples was in general lower than that of Bacteria and Archaea. Furthermore, the sampling location influenced the fungal community composition, whereas the bacterial and archaeal communities were not influenced. This may indicate that the fungal species that are adapted to the Baltic Sea environments are few and that Fungi are potentially more vulnerable to or affected by the Baltic Sea conditions than Bacteria and Archaea

Katse Ryhmässä - Ryhmän merkitys kouluviihtyvyyteen nuoren näkökulmasta

Tässä opinnäytetyössä tarkastellaan kahden peruskoululuokan oppilaiden käsityksiä kouluviihtyvyydestä omassa luokassaan ja tutkimuksen tulosten avulla on tarkoitus löytää keinoja oppilaiden kouluviihtyvyyden parantamiseen ja yhteisöllisyyden lisäämiseen. Aihetta tutkittiin laaditun kyselylomakkeen avulla. Tutkimusaineisto kerättiin keväällä 2012 kahdelta 9.-luokan oppilailta. Toinen luokista on musiikkipainotteinen ja luokan jäsenet ovat olleet tiiviinä ryhmänä kauemmin kuin toinen tutkimuskohteena ollut luokka, tutkimuksessa vertaillaan näiden kahden eri ryhmän vastauksia ryhmädynamiikan ja sosiaalisten suhteiden merkittävyydestä heidän kouluviihtyvyydessään. Tuloksista nousee esille sosiaalisten suhteiden merkittävyys ja yhteisöllisyyden tarve. Myös koulukiusaaminen ja siihen puuttumisen tärkeys nousee esille tutkimustuloksista. Opinnäytetyön tulokset tukevat vanhoja tutkimuksia sosiaalisten suhteiden merkittävyydestä nuoren kouluviihtyvyyteen