Stable Isotope Probing:Linking Functional Activity to Specific Members of Microbial Communities

Linking organisms or groups of organisms to specific functions within natural environments is a fundamental challenge in microbial ecology. Advances in technology for manipulating and analyzing nucleic acids have made it possible to characterize the members of microbial communities without the intervention of laboratory culturing. Results from such studies have shown that the vast majority of soil organisms have never been cultured, highlighting the risks of culture-based approaches in community analysis. The development of culture-independent techniques for following the flow of substrates through microbial communities therefore represents an important advance. These techniques, collectively known as stable isotope probing (SIP), involve introducing a stable isotope-labeled substrate into a microbial community and following the fate of the substrate by extracting diagnostic molecular species such as fatty acids and nucleic acids from the community and determining which specific molecules have incorporated the isotope. The molecules in which the isotope label appears provide identifying information about the organism that incorporated the substrate. Stable isotope probing allows direct observations of substrate assimilation in minimally disturbed communities, and thus represents an exciting new tool for linking microbial identity and function. The use of lipids or nucleic acids as the diagnostic molecule brings different strengths and weaknesses to the experimental approach, and necessitates the use of significantly different instrumentation and analytical techniques. This short review provides an overview of the lipid and nucleic acid approaches, discusses their strengths and weaknesses, gives examples of applications in various settings, and looks at prospects for the future of SIP technology.

Double neutron/proton ratio of nucleon emissions in isotopic reaction systems as a robust probe of nuclear symmetry energy

The double neutron/proton ratio of nucleon emissions taken from two reaction systems using four isotopes of the same element, namely, the neutron/proton ratio in the neutron-rich system over that in the more symmetric system, has the advantage of reducing systematically the influence of the Coulomb force and the normally poor efficiencies of detecting low energy neutrons. The double ratio thus suffers less systematic errors. Within the IBUU04 transport model the double neutron/proton ratio is shown to have about the same sensitivity to the density dependence of nuclear symmetry energy as the single neutron/proton ratio in the neutron-rich system involved. The double neutron/proton ratio is therefore more useful for further constraining the symmetry energy of neutron-rich matter.

Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. Samples for total cell counts were stored in formalin at 4°C until analysis. For sandy samples, the total cell count/CARD-FISH protocol was optimized to separate sand particles from the cells. Cells were dislodged from sediment grains and brought into solution with the supernatant by sonicating each sample onice for 2 minutes at 50W. This procedure was repeated four times and supernatants were combined. The sediment samples were brought to a final dilution of 1:2000 to 1:4000 and filtered onto 0.2µm GTTP filters (Millipore, Eschbonn, Germany).

Methodologies for an improved prediction of the isotopic content in high burnup samples. Application to Vandellos-II reactor core

Fuel cycles are designed with the aim of obtaining the highest amount of energy possible. Since higher burnup values are reached, it is necessary to improve our disposal designs, traditionally based on the conservative assumption that they contain fresh fuel. The criticality calculations involved must consider burnup by making the most of the experimental and computational capabilities developed, respectively, to measure and predict the isotopic content of the spent nuclear fuel. These high burnup scenarios encourage a review of the computational tools to find out possible weaknesses in the nuclear data libraries, in the methodologies applied and their applicability range. Experimental measurements of the spent nuclear fuel provide the perfect framework to benchmark the most well-known and established codes, both in the industry and academic research activity. For the present paper, SCALE 6.0/TRITON and MONTEBURNS 2.0 have been chosen to follow the isotopic content of four samples irradiated in the Spanish Vandellós-II pressurized water reactor up to burnup values ranging from 40 GWd/MTU to 75 GWd/MTU. By comparison with the experimental data reported for these samples, we can probe the applicability of these codes to deal with high burnup problems. We have developed new computational tools within MONTENBURNS 2.0. They make possible to handle an irradiation history that includes geometrical and positional changes of the samples within the reactor core. This paper describes the irradiation scenario against which the mentioned codes and our capabilities are to be benchmarked.

Chemical and isotopic composition of interstitial waters from the Kara Sea and Yenisey estuary

Processes of early sediment diagenesis, including chemical composition of interstitial waters, content of different sulfur compounds, and rates of sulfate reduction were investigated. Along a profile from the Yenisey River to the Kara Sea, sulfate reduction intensities were high (up to 1.5 µg S/kg/day) in the delta region and in its northern part. Intensities were comparable to those found in shallow-water sediments of the North Sea. In estuarine sediments, where different transformations were possible, lower rates of bacterial sulfate reduction were observed (0.03 to 0.06 µg S/kg/day). Annual production of reduced sulfur in sediments was 24 million tons, and only 0.5 million tons (2%) were deposited. Annual consumption of organic carbon during process of sulfate reduction in the Yenisey delta region was 17.6 million tons.

(Table T1) Trace element concentration and isotopic composition of pore waters from ODP Leg 168 sites

Pore water was collected from each of 10 sites during Ocean Drilling Program (ODP) Leg 168 on the eastern flank of the Juan de Fuca Ridge. These ten sites delineate a transect perpendicular to the present ridge axis and span a crustal age of 0.86-3.59 Ma. At nine of the ten sites the entire sediment section, which ranged from 41.3 to 613.8 m thick, was cored and attempts were made to recover at least one whole round of sediment per section of core for extraction of pore water. Several (2-5) whole-round sediment samples were taken from the uppermost and lowermost cores to constrain the chemical gradient near the sediment/water and sediment/basalt interfaces, respectively. Pore water was extracted from whole-round sediment core sections by squeezing only the most pristine sediment in a titanium squeezer designed by Manheim and Sayles (1974). Two additional water samples were collected in situ using the water-sampler temperature probe (WSTP; Barnes, 1988, doi:10.2973/odp.proc.ir.110.104.1988). Both of these samples were collected in the cased section of the open borehole from ODP Hole 1026B. Formation fluids were flowing up the cased hole into the overlying deep seawater (Fisher et al., 1997, doi:10.1029/97GL01286). Detailed descriptions of the sampling methods that were used to collect fluids are given by the Shipboard Scientific Party (Davis, Fisher, Firth, et al., 1997, doi:10.2973/odp.proc.ir.168.1997).

Synchronous fluorescence and UV–vis spectroscopic studies of interactions between the tetracycline antibiotic, aluminium ions and DNA with the aid of the Methylene Blue dye probe

Synchronous fluorescence spectroscopy (SFS) was applied for the investigation of interactions of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+). The study was facilitated by the use of the Methylene Blue (MB) dye probe, and the interpretation of the spectral data with the aid of the chemometrics method, parallel factor analysis (PARAFAC). Three-way synchronous fluorescence analysis extracted the important optimum constant wavelength differences, Δλ, and showed that for the TC–Al3+–DNA, TC–Al3+ and MB dye systems, the associated Δλ values were different (Δλ = 80, 75 and 30 nm, respectively). Subsequent PARAFAC analysis demonstrated the extraction of the equilibrium concentration profiles for the TC–Al3+, TC–Al3+–DNA and MB probe systems. This information is unobtainable by conventional means of data interpretation. The results indicated that the MB dye interacted with the TC–Al3+–DNA surface complex, presumably via a reaction intermediate, TC–Al3+–DNA–MB, leading to the displacement of the TC–Al3+ by the incoming MB dye probe.

Mapping personal trip OD from probe data

The paper analyses the expected value of OD volumes from probe with fixed error, error that is proportional to zone size and inversely proportional to zone size. To add realism to the analysis, real trip ODs in the Tokyo Metropolitan Region are synthesised. The results show that for small zone coding with average radius of 1.1km, and fixed measurement error of 100m, an accuracy of 70% can be expected. The equivalent accuracy for medium zone coding with average radius of 5km would translate into a fixed error of approximately 300m. As expected small zone coding is more sensitive than medium zone coding as the chances of the probe error envelope falling into adjacent zones are higher. For the same error radii, error proportional to zone size would deliver higher level of accuracy. As over half (54.8%) of the trip ends start or end at zone with equivalent radius of ≤ 1.2 km and only 13% of trips ends occurred at zones with equivalent radius ≥2.5km, measurement error that is proportional to zone size such as mobile phone would deliver higher level of accuracy. The synthesis of real OD with different probe error characteristics have shown that expected value of >85% is difficult to achieve for small zone coding with average radius of 1.1km. For most transport applications, OD matrix at medium zone coding is sufficient for transport management. From this study it can be drawn that GPS with error range between 2 and 5m, and at medium zone coding (average radius of 5km) would provide OD estimates greater than 90% of the expected value. However, for a typical mobile phone operating error range at medium zone coding the expected value would be lower than 85%. This paper assumes transmission of one origin and one destination positions from the probe. However, if multiple positions within the origin and destination zones are transmitted, map matching to transport network could be performed and it would greatly improve the accuracy of the probe data.

Use of Probe Vehicles to Increase Traffic Estimation Accuracy in Brisbane

Traffic congestion is an increasing problem with high costs in financial, social and personal terms. These costs include psychological and physiological stress, aggressivity and fatigue caused by lengthy delays, and increased likelihood of road crashes. Reliable and accurate traffic information is essential for the development of traffic control and management strategies. Traffic information is mostly gathered from in-road vehicle detectors such as induction loops. Traffic Message Chanel (TMC) service is popular service which wirelessly send traffic information to drivers. Traffic probes have been used in many cities to increase traffic information accuracy. A simulation to estimate the number of probe vehicles required to increase the accuracy of traffic information in Brisbane is proposed. A meso level traffic simulator has been developed to facilitate the identification of the optimal number of probe vehicles required to achieve an acceptable level of traffic reporting accuracy. Our approach to determine the optimal number of probe vehicles required to meet quality of service requirements, is to simulate runs with varying numbers of traffic probes. The simulated traffic represents Brisbane’s typical morning traffic. The road maps used in simulation are Brisbane’s TMC maps complete with speed limits and traffic lights. Experimental results show that that the optimal number of probe vehicles required for providing a useful supplement to TMC (induction loop) data lies between 0.5% and 2.5% of vehicles on the road. With less probes than 0.25%, little additional information is provided, while for more probes than 5%, there is only a negligible affect on accuracy for increasingly many probes on the road. Our findings are consistent with on-going research work on traffic probes, and show the effectiveness of using probe vehicles to supplement induction loops for accurate and timely traffic information.

Polymersomes, smaller than you think : ferrocene as a TEM probe to determine core structure

By incorporating ferrocene into the hydrophobic membrane of PEG-b-PCL polymersome nanoparticles it is possible to selectively visualize their core using Transmission Electron Microscopy (TEM). Two different sizes of ferrocene-loaded polymersomes with mean hydrodynamic diameters of approximately 40 and 90 nm were prepared. Image analysis of TEM pictures of these polymersomes found that the mean diameter of the core was 4–5 times smaller than the mean hydrodynamic diameter. The values obtained also allow the surface diameter and internal volume of the core to be calculated.

Oxidative potential of logwood and pellet burning particles assessed by a novel profluorescent nitroxide probe

This study reports the potential toxicological impact of particles produced during biomass combustion by an automatic pellet boiler and a traditional logwood stove under various combustion conditions using a novel profluorescent nitroxide probe BPEAnit. This probe is weakly fluorescent, but yields strong fluorescence emission upon radical trapping or redox activity. Samples were collected by bubbling aerosol through an impinger containing BPEAnit solution, followed by fluorescence measurement. The fluorescence of BPEAnit was measured for particles produced during various combustion phases, at the beginning of burning (cold start), stable combustion after refilling with the fuel (warm start) and poor burning conditions. For particles produced by the logwood stove under cold-start conditions significantly higher amounts of reactive species per unit of particulate mass were observed compared to emissions produced during a warm start. In addition, sampling of logwood burning emissions after passing through a thermodenuder at 250oC resulted in an 80-100% reduction of the fluorescence signal of BPEAnit probe, indicating that the majority of reactive species were semivolatile. Moreover, the amount of reactive species showed a strong correlation with the amount of particulate organic material. This indicates the importance of semivolatile organics in particle-related toxicity. Particle emissions from the pellet boiler, although of similar mass concentration, were not observed to lead to an increase in fluorescence signal during any of the combustion phases.

Regional field measurement of soil moisture content with neutron probe

The neutron logging method has been widely used for field measurement of soil moisture content. This non-destructive method permitted the measurement of in-situ soil moisture content at various depths without the need for burying any sensor. Twenty-three sites located around regional Melbourne have been selected for long term monitoring of soil moisture content using neutron probe. Soil samples collected during the installation are used for site characterisation and neutron probe calibration purposes. A linear relationship is obtained between the corrected neutron probe reading and moisture content for both the individual and combined data from seven sites. It is observed that the liner relationship, developed using combined data, can be used for all sites with an average accuracy of about 80%. Monitoring of the variation of soil moisture content with depth in six months for two sites is presented in this paper.