Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments

Synergistic degradation of lignocellulose by fungi and bacteria in boreal forest soil

Thesis (M.S.) University of Alaska Fairbanks, 2015Boreal forests contain an estimated 28% of the world's soil carbon, and currently act as a significant global carbon sink. Plant-derived lignocellulose is a major component of soil carbon, and its decomposition is dependent on soil bacteria and fungi. In order to predict the fate of this soil carbon and its potential feedbacks to climate change, the identities, activity, and interactions of soil microbial decomposer communities must be better understood. This study used stable isotope probing (SIP) with ¹³C-labeled lignocellulose and two of its constituents, cellulose and vanillin, to identify microbes responsible for the processing of lignocellulose-derived carbon and examine the specific roles that they perform. Results indicate that multiple taxa are involved in lignocellulose processing, and that certain taxa target specific portions of the lignocellulose macromolecule; specifically, fungi dominate the degradation of lignocellulose and cellulose macromolecules, while bacteria scavenge aromatic lignocellulose monomers. Major fungal taxa involved in lignocellulose degradation include Ceratobasidium, Geomyces, and Sebacina, among others. Bacterial taxa processing lignocellulose and cellulose included Cellvibrio and Mesorhizobium in high abundance relative to other taxa, although Burkholderia were the primary vanillin consumers. These results elucidate some of the major players in lignocellulose decomposition and their specific roles in boreal forest soil. This information provides knowledge of small-scale microbial processes that dictate ecosystem-level carbon cycling, and can assist in predictions of the fate of boreal forest carbon stocks

Targeted metagenomics of active microbial populations with stable-isotope probing

The ability to explore microbial diversity and function has been enhanced by novel experimental and computational tools. The incorporation of stable isotopes into microbial biomass enables the recovery of labeled nucleic acids from active microorganisms, despite their initial abundance and culturability. Combining stable-isotope probing (SIP) with metagenomics provides access to genomes from microorganisms involved in metabolic processes of interest. Studies using metagenomic analysis on DNA obtained from DNA-SIP incubations can be ideal for the recovery of novel enzymes for biotechnology applications, including biodegradation, biotransformation, and biosynthesis. This chapter introduces metagenomic and DNA-SIP methodologies, highlights biotechnology-focused studies that combine these approaches, and provides perspectives on future uses of these methods as analysis tools for applied and environmental microbiology

Associated bacteria affect sexual reproduction by altering gene expression and metabolic processes in a biofilm inhabiting diatom

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally coexisting bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations

Time-lapse monitoring of an electrokinetic soil remediation process through frequency-domain electrical measurements

The electrokinetic (EK) method is an emerging technique for soil remediation, even though a monitoring system of the contaminant removal through geophysical methods has not been developed yet. In this paper, frequency-domain time-lapse measurements are used on heavy-metal contaminated sediments for monitoring an EK remediation process in a small-scale measuring cell. Our goal is to monitor the development of the electrokinetic process within the sediment and to evaluate the total time needed for the treatment. In fact, frequency-domain electrical monitoring provides complex resistivity spectra at different time steps that can be correlated to changes in the physical properties of the sediments. We perform laboratory spectral induced polarization (SIP) measurements on different samples before, during and after the EK treatment, using different electrolyte solutions (acids and tap water), commonly employed in EK remediation. Direct-current measurements (resistivity and chargeability) were also acquired on one sample for testing the reliability of the system by a comparison with a widespread commercial instrumentation for field measurements. Results indicate that resistivity is a diagnostic parameter as long as it is linked to changes in water saturation, pH and ionic concentration and not to the percentage of metal extraction. The resistivity exhibited well-defined signatures as a function of time that changes depending on the conditioning agent and the grain size distribution. These peculiarities were used to understand the physical processes occurring within the cell and consequently to assess the effectiveness of the electrokinetic treatment. Conversely, the polarization effect was negligible using acids as conditioning agents at the electrolyte chambers. Therefore, the SIP method is not effective under these conditions, being the polarization effect significant only when tap water was used at both ends of the measuring cell. In this case, we were able to correlate changes in water saturation with the time-shift observed on relaxation time distributions (RTDs) after inversion of SIP data and to observe, using normalized chargeability, that polarization is stronger at high pH values. On these basis, resistivity is suitable to monitor the development of the remediation, to optimise the energy levels required for treatment and to assess the end time of the EK process (time when metal mobilization ends). In fact, the end time of treatment can be associated with the time at which resistivity becomes stable. This time is highly dependent on the particular working conditions and sediment grain size as demonstrated by our experiments

Microphytobenthic extracellular polymeric substances (EPS) in intertidal sediments fuel both generalist and specialist EPS-degrading bacteria

Microphytobenthic biofilms contain high concentrations of carbohydrate-rich extracellular polymeric substances (EPS) that are important in sediment carbon cycling. Field measurements at two locations in the Colne Estuary, U.K., showed that a significant curvilinear relationship explained 50% of the variability in chlorophyll a and EPS content. Estimates of EPS production, based on field data and published rates of production by diatoms, revealed that EPS turnover of 52% to 369% over the tidal cycle was required to account for field standing stocks. We investigated EPS degradation in sediment slurries using purified 13C-EPS produced by the diatom Nitzschia tubicola. Although EPS constituted only 5% of the sediment dissolved organic carbon (DOC) pool, 100% of the added EPS was utilized within 30 h, before decreases in other sediment-carbohydrate fractions and DOC concentrations. A general 13C enrichment of phospholipid fatty acids (PLFAs), representative of Gram-positive and Gram-negative bacteria, occurred within 6 h, with the PLFAs a15:0, i15:0, and 18:1?7c being highly enriched. The diatom PLFA 20:5?3 had relatively low but significant 13C enrichment. Stable isotope probing of 16S ribosomal ribonucleic acid (RNA-SIP) at 30 h revealed 13C-enriched sequences from the diatom genus Navicula; further evidence that diatoms assimilated the EPS, or EPS-breakdown products, from other diatom taxa. RNA-SIP also demonstrated a diverse range of highly 13C-enriched bacterial taxa, including a distinct subset (Alphaproteobacteria and Gammaproteobacteria) found only in the heavily labeled microbial assemblages. Thus, cycling of diatom EPS is rapid, and involves a wide range of microbial taxa, including some apparent specialists

Phase Stability and Segregation in Alloy 22 Base Metal and Weldments

The current design of the waste disposal containers relies heavily on encasement in a multi-layered container, featuring a corrosion barrier of Alloy 22, a Ni-Cr-Mo-W based alloy with excellent corrosion resistance over a wide range of conditions. The fundamental concern from the perspective of the Yucca Mountain Project, however, is the inherent uncertainty in the (very) long-term stability of the base metal and welds. Should the properties of the selected materials change over the long service life of the waste packages, it is conceivable that the desired performance characteristics (such as corrosion reistance) will become compromised, leading to premature failure of the system. To address this, we will study the phase stability and solute segregation characteristics of Alloy 22 base metal and welds. A better understanding of the underlying microstructural evolution tendencies, and their connections with corrosion behavior will (in turn) produce a higher confidence in the extrapolated behavior of the container materials over time periods that are not feasibly tested in a laboratory. Additionally, the knowledge gained here may potentially lead to cost savings through development of safe and realistic design constraints and model assumptions throughout the entire disposal system

Closing the loop of SIEM analysis to Secure Critical Infrastructures

Critical Infrastructure Protection is one of the main challenges of last years. Security Information and Event Management (SIEM) systems are widely used for coping with this challenge. However, they currently present several limitations that have to be overcome. In this paper we propose an enhanced SIEM system in which we have introduced novel components to i) enable multiple layer data analysis; ii) resolve conflicts among security policies, and discover unauthorized data paths in such a way to be able to reconfigure network devices. Furthermore, the system is enriched by a Resilient Event Storage that ensures integrity and unforgeability of events stored.Comment: EDCC-2014, BIG4CIP-2014, Security Information and Event Management, Decision Support System, Hydroelectric Da