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Evaluation of Molecular Techniques in Characterization of Deep Terrestrial Biosphere  [PDF]
Malin Bomberg, Mari Nyyss?nen, Aura Nousiainen, Jenni Hultman, Lars Paulin, Petri Auvinen, Merja It?vaara
Open Journal of Ecology (OJE) , 2014, DOI: 10.4236/oje.2014.48040

A suite of molecular methods targeting 16S rRNA genes (i.e., DGGE, clone and high-throughput [HTP] amplicon library sequencing) was used to profile the microbial communities in deep Fennoscandian crystalline bedrock fracture fluids. Variation among bacterial 16S rRNA genes was examined with two commonly used primer pairs: P1/P2 and U968f/U1401r. DGGE using U968f/ U1401r mostly detected β-, γ-proteobacteria and Firmicutes, while P1/P2 primers additionally detected other proteobacterial clades and candidate divisions. However, in combination with clone libraries the U968f/U1401r primers detected a higher bacterial diversity than DGGE alone. HTP amplicon sequencing with P1/P2 revealed an abundance of the DGGE bacterial groups as well as many other bacterial taxa likely representing minor components of these communities. Archaeal diversity was investigated via DGGE or HTP amplicon sequencingusing primers A344F/ 519RP. The majority of archaea detected with HTP amplicon sequencing belonged to uncultured Thermoplasmatales and Pendant 33/DHVE3, 4, 6 groups. DGGE of the same samples detected mostly SAGMEG and Methanosarcinales archaea, but almost none of those were revealed by HTP amplicon sequencing. Overall, our results show that the inferred diversity and composition of microbial communities in deep fracture fluids is highly dependent on analytical technique and that the method should be carefully selected with this in mind.

Application of hybridization control probe to increase accuracy on ligation detection or minisequencing diagnostic microarrays
Jarmo Ritari, Lars Paulin, Jenni Hultman, Petri Auvinen
BMC Research Notes , 2009, DOI: 10.1186/1756-0500-2-249
Abstract: Here we demonstrate the application of a per-spot hybridization control oligonucleotide probe and a novel way of computing normalization for tag array data. The method takes into account the absolute value of the detection probe signal and the variability in the control probe signal to significantly alleviate problems caused by artefacts and noise on low quality microarrays.Diagnostic microarray platforms require experimental and computational tools to enable efficient correction of array artefacts. The techniques presented here improve the signal to noise ratio and help in determining true positives with better statistical significance and in allowing the use of arrays with poor quality that would otherwise be discarded.Nucleic acid detection by ligation and single-nucleotide extension minisequencing techniques take advantage of the catalytic selectivity of DNA ligase and polymerase enzymes, respectively, to recognize a unique position in a target DNA strand. In ligation assays, two specific ssDNA oligonucleotide detection probes are designed to hybridize adjacently on target DNA strand so that the 3' end of the label-carrying probe recognizes a discriminating position and is ligated to the phosphorylated 5' end of the other probe in the presence of a matching target molecule (figure 1A) [1,2]. Ligation detection can also be implemented as a single probe which is circularized upon ligation [3]. In minisequencing, the target is recognized through the addition of a specific labeled dideoxynucleotide to the 3' end of the oligonucleotide detection primer annealed immediately upstream of a discriminating position in the target (figure 1B) [4,5]. Both methods allow tagging of the probes for detection on a microarray platform containing complementary tag sequences providing uniform thermodynamic hybridization properties for all probes. The relatively high throughput and superior accuracy over traditional microarray and PCR based methods have motivated the application of l
Universal ligation-detection-reaction microarray applied for compost microbes
Jenni Hultman, Jarmo Ritari, Martin Romantschuk, Lars Paulin, Petri Auvinen
BMC Microbiology , 2008, DOI: 10.1186/1471-2180-8-237
Abstract: Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS) area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array.This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.Composting is one of the principal methods to treat separately collected biodegradable waste. In composting, organic material is aerobically decomposed into humus-like material by bacteria, fungi and, to a lesser extent, other larger organisms [1]. To understand the composting process and the ecological processes of composting, the microbes present in the process need to be tracked. The compost microbiota have been characterised with a variety of molecular and cultivation based methods in both laboratory and full-scale processes (e.g. [2-6]. In order to follow the industrial composting process and to confirm the hygienisation of the compost, the microbiology needs to be understood and followed. Several approaches has been used to resolve compost microbiology, such as single-stranded conformational polymorphism (SSCP) [7], automated rRNA intergenic spacer analysis (ARISA) [5] denaturing gradient gel electrophoresis (DGGE) [3,4] and cloning and sequencing (Hultman et al. unpublished, Partanen et al. unpublished). Common to the technologies mentioned above is that they are rather time consuming and not suitable for routine determination of compost
Bacterial diversity at different stages of the composting process
Pasi Partanen, Jenni Hultman, Lars Paulin, Petri Auvinen, Martin Romantschuk
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-94
Abstract: Over 1500 almost full-length 16S rRNA gene sequences were analysed and of these, over 500 were present only as singletons. Most of the sequences observed in either one or both of the composting processes studied here were similar to the bacterial species reported earlier in composts, including bacteria from the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Deinococcus-Thermus. In addition, a number of previously undetected bacterial phylotypes were observed. Statistical calculations estimated a total bacterial diversity of over 2000 different phylotypes in the studied composts.Interestingly, locally enriched or evolved bacterial variants of familiar compost species were observed in both composts. A detailed comparison of the bacterial diversity revealed a large difference in composts at the species and strain level from the different composting plants. However, at the genus level, the difference was much smaller and illustrated a delay of the composting process in the full-scale, sub-optimally performing plants.Composting is an aerobic process, during which organic waste is biologically degraded by micro-organisms to humus-like material. The end product should not contain pathogens or viable seeds, and it should be stable and suitable for use as a soil amendment [1]. Many factors such as oxygen content, moisture, composition of the feed, pH, and temperature, affect the composting process and ultimately the end product. Furthermore, these parameters are strongly connected.The source of separated biowaste, as collected and treated in the Nordic countries and other cold climate areas, primarily consists of food waste which in itself has a low pH and contains high quantities of carbohydrates that form organic acids upon degradation. The low initial pH limits microbial activity and delays the increase in temperature [2,3].In recent years, composting has attracted much attention as a viable and environmentally sensible alternative for treatment of or
Electronic Structure and Chemical Bonding of Amorphous Chromium Carbide Thin Films
Martin Magnuson,Matilda Andersson,Jun Lu,Lars Hultman,Ulf Jansson
Physics , 2012, DOI: 10.1088/0953-8984/24/22/225004
Abstract: The microstructure, electronic structure, and chemical bonding of chromium carbide thin films with different carbon contents have been investigated with high-resolution transmission electron microscopy, electron energy loss spectroscopy and soft x-ray absorption-emission spectroscopies. Most of the films can be described as amorphous nanocomposites with non-crystalline CrCx in an amorphous carbon matrix. At high carbon contents, graphene-like structures are formed in the amorphous carbon matrix. At 47 at% carbon content, randomly oriented nanocrystallites are formed creating a complex microstructure of three components. The soft x-ray absorption-emission study shows additional peak structures exhibiting non-octahedral coordination and bonding.
Structure and bonding in amorphous iron carbide thin films
Andrej Furlan,Ulf Jansson,Jun Lu,Lars Hultman,Martin Magnuson
Physics , 2015, DOI: 10.1088/0953-8984/27/4/045002
Abstract: We investigate the amorphous structure, chemical bonding, and electrical properties of magnetron sputtered Fe1-xCx (0.21
Crystallization characteristics and chemical bonding properties of nickel carbide thin film nanocomposites
Andrej Furlan,Jun Lu,Lars Hultman,Ulf Jansson,Martin Magnuson
Physics , 2014, DOI: 10.1088/0953-8984/26/41/415501
Abstract: The crystal structure and chemical bonding of magnetron-sputtering deposited nickel carbide Ni$_{1-x}$C$_{x}$ (0.05$\leq$x$\leq$0.62) thin films have been investigated by high-resolution X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and soft X-ray absorption spectroscopy. By using X-ray as well as electron diffraction, we found carbon-containing hcp-Ni (hcp-NiC$_{y}$ phase), instead of the expected rhombohedral-Ni$_{3}$C. At low carbon content (4.9 at\%) the thin film consists of hcp-NiC$_{y}$ nanocrystallites mixed with a smaller amount of fcc-NiC$_{x}$. The average grain size is about 10-20 nm. With the increase of carbon content to 16.3 at\%, the film contains single-phase hcp-NiC$_{y}$ nanocrystallites with expanded lattice parameters. With further increase of carbon content to 38 at\%, and 62 at\%, the films transform to X-ray amorphous materials with hcp-NiC$_{y}$ and fcc-NiC$_{x }$ nanodomain structures in an amorphous carbon-rich matrix. Raman spectra of carbon indicate dominant $sp^{2}$ hybridization, consistent with photoelectron spectra that show a decreasing amount of C-Ni phase with increasing carbon content. The Ni $3d$ - C $2p$ hybridization in the hexagonal structure gives rise to the salient double-peak structure in Ni $2p$ soft X-ray absorption spectra at 16.3 at\% that changes with carbon content. We also show that the resistivity is not only governed by the amount of carbon, but increases by more than a factor of two when the samples transform from crystalline to amorphous.
Electronic structure and chemical bonding of nc-TiC/a-C nanocomposites
Martin Magnuson,Erik Lewin,Lars Hultman,Ulf Jansson
Physics , 2011, DOI: 10.1103/PhysRevB.80.235108
Abstract: The electronic structure of nanocrystalline (nc-) TiC/amorphous C nanocomposites has been investigated by soft x-ray absorption and emission spectroscopy. The measured spectra at the Ti 2p and C 1s thresholds of the nanocomposites are compared to those of Ti metal and amorphous C. The corresponding intensities of the electronic states for the valence and conduction bands in the nanocomposites are shown to strongly depend on the TiC carbide grain size. An increased charge-transfer between the Ti 3d-eg states and the C 2p states has been identified as the grain size decreases, causing an increased ionicity of the TiC nanocrystallites. It is suggested that the charge-transfer occurs at the interface between the nanocrystalline TiC and the amorphous C matrix and represents an interface bonding which may be essential for the understanding of the properties of nc-TiC/amorphous C and similar nanocomposites.
The electronic-structure origin of the anisotropic thermopower of nanolaminated Ti3SiC2 determined by polarized x-ray spectroscopy and Seebeck measurements
Martin Magnuson,Maurizio Mattesini,Ngo Van Nong,Per Eklund,Lars Hultman
Physics , 2012, DOI: 10.1103/PhysRevB.85.195134
Abstract: Nanolaminated materials exhibit characteristic magnetic, mechanical, and thermoelectric properties, with large contemporary scientific and technological interest. Here, we report on the anisotropic Seebeck coefficient in nanolaminated Ti3SiC2 single-crystal thin films and trace the origin to anisotropies in element-specific electronic states. In bulk polycrystalline form, Ti3SiC2 has a virtually zero Seebeck coefficient over a wide temperature range. In contrast, we find that the in-plane (basal ab) Seebeck coefficient of Ti3SiC2, measured on single-crystal films has a substantial and positive value of 4-6 muV/K. Employing a combination of polarized angle-dependent x-ray spectroscopy and density functional theory we directly show electronic structure anisotropy in inherently nanolaminated Ti3SiC2 single-crystal thin films as a model system. The density of Ti 3d and C 2p states at the Fermi level in the basal ab-plane is about 40 % higher than along the c-axis. The Seebeck coefficient is related to electron and hole-like bands close to the Fermi level but in contrast to ground state density functional theory modeling, the electronic structure is also influenced by phonons that need to be taken into account. Positive contribution to the Seebeck coefficient of the element-specific electronic occupations in the basal plane is compensated by 73 % enhanced Si 3d electronic states across the laminate plane that give rise to a negative Seebeck coefficient in that direction. Strong phonon vibration modes with three to four times higher frequency along the c-axis than along the basal ab-plane also influence the electronic population and the measured spectra by the asymmetric average displacements of the Si atoms. These results constitute experimental evidence explaining why the average Seebeck coefficient of Ti3SiC2 in polycrystals is negligible over a wide temperature range.
The electronic structure of GaN and Ga investigated by soft x-ray spectroscopy and first-principles methods
Martin Magnuson,Maurizio Mattesini,Carina H?glund,Jens Birch,Lars Hultman
Physics , 2011, DOI: 10.1103/PhysRevB.81.085125
Abstract: The electronic structure and chemical bonding of wurtzite-GaN investigated by N 1s soft x-ray absorption spectroscopy and N K, Ga M1, and Ga M2,3 emission spectroscopy is compared to that of pure Ga. The measurements are interpreted by calculated spectra using first-principles density-functional theory (DFT) including dipole transition matrix elements and additional on-site Coulomb interaction (WC-GGA+U). The Ga 4p - N 2p and Ga 4s - N 2p hybridization and chemical bond regions are identified at the top of the valence band between -1.0 and -2.0 and further down between -5.5 and -6.5 eV, respectively. In addition, N 2s - N 2p - Ga 4s and N 2s - N 2p - Ga 3d hybridization regions occur at the bottom of the valence band between -13 and -15 eV, and between -17.0 and -18.0 eV, respectively. A band-like satellite feature is also found around -10 eV in the Ga M1 and Ga M2,3 emission from GaN, but is absent in pure Ga and the calculated ground state spectra. The difference between the identified spectroscopic features of GaN and Ga are discussed in relation to the various hybridization regions calculated within band-structure methods.
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