Öljyllä saastuneen maaperän molekulaarinen biomonitorointi ritsoremediaation aikana

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEXSuomessa on tilastoitu tuhansia öljyllä saastuneita maa-alueita, kuten hylättyjä huoltoasemia. Näitä voitaisiin puhdistaa kasvien juuristossa elävien mikrobien avulla eli ritsoremediaatiolla. Tässä työssä tutkittiin typpeä sitovan palkokasvin, vuohenherneen juuriston bakteerien osallistumista monoaromaattisten hiilivetyjen, kuten bentseenin, tolueenin ja ksyleenin, hajottamiseen öljyllä saastuneessa maaperässä. Biologisten tekijöiden tuntemus (mm. bakteerit ja niiden hajotusgeenit) on oleellista kemiallisen (esim. myrkkypitoisuus) ja fysikaalisen (esim. maan rakenne) tiedon lisäksi, jotta ritsoremediaatiosta voidaan kehittää hallittu ja tehokas biopuhdistusmenetelmä. Siksi myös luotettavien biologisten seurantamenetelmien kehittämisen tarve on ilmeinen. Tämän väitöskirjatyön tarkoituksena oli 1) arvioida vuohenherneen ja sen ritsobin eli symbioottisen typensitojabakteerin soveltuvuus öljyllä saastuneen maaperän puhdistamiseen, 2) kehittää molekyylitason menetelmiä biomonitorointia varten ja 3) soveltaa näitä menetelmiä tutkittaessa mikrobiologisia ilmiöitä ritsoremediaation aikana. Ritsobin korkea aromaattisten aineiden sietokyky ja vuohenherneen elinkykyisyys öljymaissa osoittivat tämän palkokasvi-bakteeri -yhdistelmän soveltuvan hyvin öljyllä saastuneen maaperän ritsoremediaatioon. Molekyylibiologisia menetelmiä suunniteltiin ja/tai kehitettiin bakteerien ja niiden hajotusgeenien seuraamiseksi maassa. Viljeltävissä olevia, heterogeenisia juuristobakteereita ryhmiteltiin erään vähemmän käytetyn, mutta tässä työssä tehokkaaksi osoitetun, genomisen sormenjälkimenetelmän, taksonomisen ribotyyppauksen ja sekvenoinnin avulla. Yhteisötasolla, ja siten myös ei-viljeltävissä olevien bakteerien havainnoimiseksi, käytettiin denaturoivaa gradienttigeelielektroforeesia. Geenispesifinen PCR-menetelmä suunniteltiin TOL-plasmidien tunnistamiseksi. Tehoste-PCR kehitettiin TOL-plasmidien havaitsemiseksi herkästi suoraan maasta. Sekä TOL-plasmideja että laajemminkin erilaisia aromaatteja hajottavia entsyymejä koodaavia plasmideja profiloitiin DNA:n pilkonta-analyysillä. Vuohenherneen juuristovaikutukset öljyllä saastuneessa maapeässä olivat seuraavat: 1) Vuohenherne lisäsi juuristobakteerien määrää, mutta erityisesti yhdessä TOL-plasmidin sisältävän Pseudomonas-bakteerin kanssa m-toluaattia hajottavien bakteerien lukumäärää. Tämä osoitti hajotuspotentiaalin olevan suurempaa kasvillisissa maissa kuin kasvittomissa maissa. 2) Vuohenherneen juuristossa oli kasvitonta maata enemmän eri bakteerilajeja, mutta ei kuitenkaan merkittävästi enemmän m-toluaattia hajottavia bakteerilajeja. Myös yhteisötasolla suurin bakteerilajien monimuotoisuus havaittiin kasvillisissa maissa. Monipuoliset yhteisöt saattavatkin parhaiten taata tehokkaan ritsoremediaation tarjoamalla lukuisia geneettisiä koneistoja hajotusprosessien käyttöön. 3) Kokeen lopussa TOL-plasmideja ei kyetty enää havaitsemaan suoralla DNA-analyysillä maasta, jossa oli sekä vuohenherne että Pseudomonas. TOL-plasmidien havaintoraja oli siis ylittynyt osoittaen hajotusplasmidien määrän ja siten katabolisen aktiivisuuden vähentyneen. Tämä viittasi ritosremediaation onnistumiseen. 4) Eräältä P. oryzihabitans -kannalta löytyi kokeissa käyttämämme P. putidan TOL-plasmidi. Lisäksi eräiltä P. migulae - ja P. oryzihabitans -kannoilta löydettiin TOL-plasmidi, jota niillä ei tiedetty olevan. Nämä tapahtumat osoittivat mahdollista hajotusplasmidin siirtymistä bakteerikannasta toiseen ja siten puhdistumisen tehostumista vuohenherneen juuristossa. Vuohenherneen käyttö ritsoremediaatiossa on maailmanlaajuisestikin ainutlaatuista. Ympäristönsuojelu luonnon omia voimavaroja käyttäen, ja siten kestävän kehityksen periaatteen mukaisesti toimien, olisi sekä taloudellisesti että viihtyisän ja puhtaan ympäristön säilymisen kannalta hyödyllistä yhteiskunnalle. Tässä väitöskirjatyössä uutta tietoa saatiin vuohenherneen juuristovaikutuksista monoaromaattisilla hiilivedyillä saastuneissa maissa. Hajotusplasmidien liikkuminen bakteerikannasta tai -lajista toiseen osoittautui ilmiöksi, jota kannattaa seurata jatkossa tarkemmin, jotta puhdistumisen aikaisista dynaamisista muutoksista ja geeniekologisista lainalaisuuksista saataisiin enemmän tietoa. Kehitettyjä molekulaarisia biomonitorointimenetelmiä voidaan soveltaa moneen tarkoitukseen ympäristöbioteknologian alalla, kuten maan luontaisen biohajotuspotentiaalin kartoittamiseen, bioremediaation tehostamisen seurantaan ja puhdistusprosessin onnistumisen arviointiin

High-latitude artificial aurora using the EISCAT high-gain HF facility

The EISCAT high-frequency (HF) transmitter facility at Ramfjord, Norway, has been used to accelerate F-region electrons sufficiently to excite the oxygen atoms and nitrogen molecules, resulting in optical emissions at 630, 557.7 and 427.8 nm. During O-mode transmissions at 5.423 MHz, using 630 MW effective radiated power, in the hours after sunset on 12 November 2001 several new observations were made, including: (1) The first high-latitude observation of an HF induced optical emission at 427.8 nm and (2) Optical rings being formed at HF on followed by their collapse into a central blob. Both discoveries remain unexplained with current theories

Efficient Certified Resolution Proof Checking

We present a novel propositional proof tracing format that eliminates complex processing, thus enabling efficient (formal) proof checking. The benefits of this format are demonstrated by implementing a proof checker in C, which outperforms a state-of-the-art checker by two orders of magnitude. We then formalize the theory underlying propositional proof checking in Coq, and extract a correct-by-construction proof checker for our format from the formalization. An empirical evaluation using 280 unsatisfiable instances from the 2015 and 2016 SAT competitions shows that this certified checker usually performs comparably to a state-of-the-art non-certified proof checker. Using this format, we formally verify the recent 200 TB proof of the Boolean Pythagorean Triples conjecture

On QBF Proofs and Preprocessing

QBFs (quantified boolean formulas), which are a superset of propositional formulas, provide a canonical representation for PSPACE problems. To overcome the inherent complexity of QBF, significant effort has been invested in developing QBF solvers as well as the underlying proof systems. At the same time, formula preprocessing is crucial for the application of QBF solvers. This paper focuses on a missing link in currently-available technology: How to obtain a certificate (e.g. proof) for a formula that had been preprocessed before it was given to a solver? The paper targets a suite of commonly-used preprocessing techniques and shows how to reconstruct certificates for them. On the negative side, the paper discusses certain limitations of the currently-used proof systems in the light of preprocessing. The presented techniques were implemented and evaluated in the state-of-the-art QBF preprocessor bloqqer.Comment: LPAR 201

Incrementally Computing Minimal Unsatisfiable Cores of QBFs via a Clause Group Solver API

We consider the incremental computation of minimal unsatisfiable cores (MUCs) of QBFs. To this end, we equipped our incremental QBF solver DepQBF with a novel API to allow for incremental solving based on clause groups. A clause group is a set of clauses which is incrementally added to or removed from a previously solved QBF. Our implementation of the novel API is related to incremental SAT solving based on selector variables and assumptions. However, the API entirely hides selector variables and assumptions from the user, which facilitates the integration of DepQBF in other tools. We present implementation details and, for the first time, report on experiments related to the computation of MUCs of QBFs using DepQBF's novel clause group API.Comment: (fixed typo), camera-ready version, 6-page tool paper, to appear in proceedings of SAT 2015, LNCS, Springe

Infants born to women with substance use: Exploring early neurobehavior with the Dubowitz neurological examination

BackgroundThere is a special concern regarding substance using pregnant women due to the possible adverse effects on the infant. While the immediate effects of prenatal substance exposure are well known, the long-term data on the infants' neurodevelopment is inconclusive.AimsThe purpose of this study was to assess early neurobehavior of infants of mothers with substance use using the Dubowitz examination and to follow their neuromotor development until one year of age.Study design and subjectsNinety-five pregnant women with a recent history of substance use were recruited and followed up at the maternity outpatient clinic. Follow-up data was collected from hospital records and maternal interviews. The Dubowitz neurological examination was performed to the 54 clinically healthy term infants. The results were converted into optimality scores and compared to normative values from clinically healthy term infants derived from a separate normative population. The infant's neuromotor development was followed up to one year of age.ResultsOnly 7% of the infants born to women with recent or current substance use reached optimal scores (n = 30) of these infants was found normal.ConclusionsA high percentage of infants of mothers who were referred prenatally to hospital due to substance use showed suboptimal neurological findings during their first days of life.</div

Yield and leakage currents of large area lattice matched InP/InGaAs heterostructures

Demonstrating and harnessing electroluminescent cooling at technologically viable cooling powers requires the ability to routinely fabricate large area high quality light-emitting diodes (LEDs). Detailed information on the performance and yield of relevant large area devices is not available, however. Here, we report extensive information on the yield and related large area scaling of InP/InGaAs LEDs and discuss the origin of the failure mechanisms based on lock-in thermographic imaging. The studied LEDs were fabricated as mesa structures of various sizes on epistructures grown at five different facilities specialized in the growth of III-V compound semiconductors. While the smaller mesas generally showed relatively good electrical characteristics and low leakage current densities, some of them also exhibited unusually large leakage current densities. The provided information is critical for the development and design of the optical cooling technologies relying on large area devices.Peer reviewe

High-k GaAs metal insulator semiconductor capacitors passivated by ex-situ plasma-enhanced atomic layer deposited AlN for Fermi-level unpinning

This paper examines the utilization of plasma-enhanced atomic layer deposition grown AlN in the fabrication of a high-kinsulator layer on GaAs. It is shown that high-kGaAsMIS capacitors with an unpinned Fermi level can be fabricated utilizing a thin ex-situ deposited AlNpassivation layer. The illumination and temperature induced changes in the inversion side capacitance, and the maximum band bending of 1.2 eV indicates that the MIS capacitor reaches inversion. Removal of surface oxide is not required in contrast to many common ex-situ approaches.Peer reviewe

Syzeuctus irrisorius (Hymenoptera: Ichneumonidae: Banchinae): New genus and species record for the fauna of Iran

مطالعه­ فونستیک زنبورهای زیرخانواده­ Banchinae (Hymenoptera: Ichneumonidae) در جنوب استان کرمان، در سال­های 1393 و 1394 با استفاده از 8 تله­ مالیز در شهرستان­های جیرفت و فاریاب انجام شد. سه گونه از این زیرخانواده به نام­های Exetastes adperssorius (Thunberg, 1824)، Lissonota pleuralis Brischke, 1880 و Syzeuctus irrisorius (Rossi, 1794) جمع­آوری و شناسائی گردیدند. گونه­ L. pleuralis برای استان کرمان و جنس و گونه­ S. irrisorius، برای فون ایران گزارش جدید می­باشند