Effect of pH on Intracellular Accumulation of Trace Concentrations of Hg(II) in Escherichia coli under Anaerobic Conditions, as Measured Using a mer-lux Bioreporter

The effects of pH on the uptake and accumulation of Hg(II) by Escherichia coli were determined at trace, environmentally relevant, concentrations of Hg and under anaerobic conditions. Hg(II) accumulation was measured using inducible light production from E. coli HMS174 harboring a mer-lux bioreporter plasmid (pRB28). The effect of pH on the toxicity of higher concentrations of Hg(II) was measured using a constitutive lux plasmid (pRB27) in the same bacterial host. In this study, intracellular accumulation and toxicity of Hg(II) under anaerobic conditions were both significantly enhanced with decreasing pH over the pH range of 8 to 5. The pH effect on Hg(II) accumulation was most pronounced at pHs of <6, which substantially enhanced the Hg(II)-dependent light response. This enhanced response did not appear to be due to pH stress, as similar results were obtained whether cells were grown at the same pH as the assay or at a different pH. The enhanced accumulation of Hg(II) was also not related to differences in the chemical speciation of Hg(II) in the external medium resulting from the changes in pH. Experiments with Cd(II), also detectable by the mer-lux bioreporter system, showed that Cd(II) accumulation responded differently to pH changes than the net accumulation of Hg(II). Potential implications of these findings for our understanding of bacterial accumulation of Hg(II) under anaerobic conditions and for bacteria-mediated cycling of Hg(II) in aquatic ecosystems are discussed. Arguments are provided suggesting that this differential accumulation is due to changes in uptake of mercury.     

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.     

Enhancement of Pentachlorophenol Biodegradation Using Organic and Inorganic Supports

The role of soil, straw, and sawdust as supports in enhancing pentachlorophenol (PCP) mineralization by an indigenous soil consortium was examined by assessing the bioavailability of the substrate and other nutrients. PCP sorption tests were conducted in the presence of sterile supports to evaluate PCP bioavailability. Indigenous biomass, practically free of soil particles, was prepared to test the influence of sterile soil and soil components on the mineralization of increasing PCP concentrations. Organic supports such as straw and sawdust were very good sorbents for PCP, resulting in a slow, continuous desorption of substrate, high mineralization rates, and reduced toxicity to the active biomass. Soil and clay retained less PCP and desorbed it in decreasing amounts. Soil was the best amendment to enhance the mineralization of 100 mg/L PCP. Soil, soil extract, and the lowest-molecular-weight fraction of the soil extract facilitated the complete mineralization of 300 mg/L of PCP with a lag time of about 9 days, compared to 21 days for the unamended culture. Addition of soil enhanced PCP mineralization by an indigenous consortium, probably because soil particles served as an adsorbent for the contaminant to decrease its toxicity, as a support for biomass colonization, and as a source of supplementary nutrients for the biomass. This concept is of importance, particularly for the production of active and resistant biomass for the biotreatment of contaminated soils.     

Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing

The marine nitrogen (N) cycle is a complex network of biological transformations in different N pools. The linkages among these different reservoirs are often poorly understood. Traditional methods for measuring N uptake rely on bulk community properties and cannot provide taxonomic information. ¹⁵N-based stable isotope probing (SIP), however, is a technique that allows detection of uptake of individual N sources by specific microorganisms. In this study we used ¹⁵N SIP methodology to assess the use of different nitrogen substrates by Synechococcus spp. and diatoms on the west Florida shelf. Seawater was incubated in the presence of ¹⁵N-labeled ammonium, nitrate, urea, glutamic acid, and a mixture of 16 amino acids. DNA was extracted and fractionated using CsCl density gradient centrifugation. Quantitative PCR was used to quantify the amounts of Synechococcus and diatom DNA as a function of density, and ¹⁵N tracer techniques were used to measure rates of N uptake by the microbial community. The ammonium, nitrate, urea, and dissolved primary amine uptake rates were 0.077, 0.065, 0.013, and 0.055 μmol N liter⁻¹ h⁻¹, respectively. SIP data indicated that diatoms and Synechococcus spp. actively incorporated N from [¹⁵N]nitrate, [¹⁵N]ammonium, and [¹⁵N]urea. Synechococcus also incorporated nitrogen from [¹⁵N]glutamate and ¹⁵N-amino acids, but no evidence indicating uptake of labeled amino acids by diatoms was detected. These data suggest that N flow in communities containing Synechococcus spp. and diatoms has more plasticity than the new-versus-recycled production paradigm suggests and that these phytoplankters should not be viewed strictly as recycled and new producers, respectively.The marine nitrogen (N) cycle is a complex network of biological transformations in different inorganic and organic N reservoirs (58). Processes related to the N cycle can at times limit productivity in marine systems (47) and influence the rate at which carbon (C) is exported from the euphotic zone to the deep ocean and marine sediments, where it can be sequestered (21). The historical paradigm with respect to the marine N and C cycles is deeply interwoven with the concepts of new and regenerated primary production in the euphotic zone (17, 20). New and export production have traditionally been equated with large nutrient influxes, particularly influxes of nitrate, which lead to diatom productivity. When high levels of nitrate are present, diatoms often dominate and exhibit high sinking rates due to aggregation and/or packaging into fecal pellets (18, 48). By contrast, the subtropical and tropical oligotrophic surface oceans have been viewed primarily as areas where recycled productivity dominates.In recent years, however, our view of the linkages between the marine N and C cycles has become increasingly complex (58). For example, geochemical rate estimates have suggested that N fixation rates in surface waters of the tropical and subtropical oceans may be many times greater than previously thought (13, 34). The divergence between in situ observations and data obtained using the geochemical mass balance approach is attributed, among other things, to meso-scale physical forcing (38) and to diazotrophic activity of planktonic cyanobacteria (14, 57). Furthermore, it is now appreciated that the ability to use nitrate is more widely distributed among the marine bacteria than previously thought and that bacteria are capable of competing with phytoplankton for both ammonium and nitrate (29, 31). Despite these advances, direct measurements of uptake of specific forms of N by individual populations of phyto- and bacterioplankton are scarce. This is primarily due to the fact that most measurements of N uptake are made using glass fiber filters that collect autotrophs and some variable fraction of heterotrophic bacteria (3). Uptake rates thus represent bulk uptake by hundreds of different phytoplankton and bacterial species. Methods that could be used to investigate uptake of N by specific species (e.g., Synechococcus spp.) or groups of species (e.g., diatoms) would therefore greatly improve our ability to elucidate N fluxes in marine systems.DNA stable isotope probing (SIP) is a technique that is based on the observation that DNA molecules with different densities can be separated by ultracentrifugation in a concentrated solution of cesium chloride (CsCl). CsCl density gradient centrifugation has a long history in biological research and was first used to demonstrate the semiconservative nature of DNA replication (39). In their experiments, Meselson and Stahl grew Escherichia coli in medium in which all available forms of N contained the heavy, stable isotope ¹⁵N. Fully labeled with ¹⁵N, DNA has an average density of 1.722 g cm⁻³, whereas ¹⁴N-containing DNA has an average density of 1.700 g cm⁻³ (10, 11). This small, yet significant difference in density is enough to allow separation of ¹⁴N-containing DNA from ¹⁵N-containing DNA. DNA SIP has been used to study the dynamics of microbial communities (46). Radajewski et al. (46) used DNA SIP to identify the microbial species involved in the biotransformation of specific ¹³C-labeled substrates among the large pool of bacterial species that typically are present in environmental communities. These authors demonstrated that ¹³C-labeled DNA could be recovered from microbial populations after incubation. ¹³C-labeled DNA was then taxonomically characterized using routine molecular ecology methods to identify active community members, demonstrating that SIP can be a powerful technique for taxonomic identification of microbes performing specific metabolic processes under in situ conditions. A series of studies have since been performed using the ¹³C-based technique to examine microbial communities in different environments (for reviews, see references 19 and 40).More recently, ¹⁵N-based SIP techniques have been developed to facilitate identification of the free-living diazotrophs responsible for in situ N fixation in soil (10). This work demonstrated that ¹⁵N-based SIP techniques could be used to study N flow in environmental communities. In the present study, ¹⁵N-based SIP techniques (10, 11) were employed to assess the use of a suite of inorganic and organic nitrogen substrates by Synechococcus spp. and diatoms in a coastal marine system. Our goal was to investigate the traditional characterization of Synechococcus spp. as recycled producers (mainly ammonium uptake) and diatoms as new producers (nitrate uptake). To do this, seawater was incubated with a series of ¹⁵N-labeled N substrates. DNA was then extracted at the end of the incubation period, and quantitative PCR (qPCR) was used to determine the amounts of Synechococcus and diatom DNA as a function of density in fractionated gradients. Shifts in the densities of Synechococcus and diatom DNA as the result of incubation with ¹⁵N-labeled N substrates were interpreted as evidence of uptake. Our data indicate that Synechococcus spp. and diatoms both actively incorporated [¹⁵N]ammonium, [¹⁵N]nitrate, and [¹⁵N]urea. Synechococcus spp. appeared to also incorporate N from [¹⁵N]glutamate and ¹⁵N-amino acids. These data suggest that N flow in communities containing Synechococcus spp. and diatoms has more plasticity than the new-versus-regenerated production paradigm suggests and that these two types of phytoplankton should not be viewed strictly as recycled and new producers, respectively.     

Effects of Inorganic Mercury and Methylmercury on the Ionic Currents of Cultured Rat Hippocampal Neurons

1. The effects of inorganic Hg²⁺ and methylmercuric chloride on the ionic currents of cultured hippocampal neurons were studied and compared. We examined the effects of acute exposure to the two forms of mercury on the properties of voltage-activated Ca²⁺ and Na⁺ currents and N-methyl-D-aspartate (NMDA)-induced currents.2. High-voltage activated Ca²⁺ currents (L type) were inhibited by both compounds at low micromolar concentrations in an irreversible manner. Mercuric chloride was five times as potent as methylmercury in blocking L-channels.3. Both compounds caused a transient increase in the low-voltage activated (T-type) currents at low concentrations (1 M) but blocked at higher concentrations and with longer periods of time.4. Inorganic mercury blockade was partially use dependent, but that by methylmercury was not. There was no effect of exposure of either form of mercury on the I–V characteristics of Ca²⁺ currents.5. Na⁺- and NMDA-induced currents were essentially unaffected by either mercury compound, showing only a delayed nonspecific effect at a time of overall damage of the membrane.6. We conclude that both mercury compounds show a relatively selective blockade of Ca²⁺ currents, but inorganic mercury is more potent than methylmercury.     

Impacts of Labile Organic Carbon Concentration on Organic and Inorganic Nitrogen Utilization by a Stream Biofilm Bacterial Community

In aquatic ecosystems, carbon (C) availability strongly influences nitrogen (N) dynamics. One manifestation of this linkage is the importance in the dissolved organic matter (DOM) pool of dissolved organic nitrogen (DON), which can serve as both a C and an N source, yet our knowledge of how specific properties of DOM influence N dynamics are limited. To empirically examine the impact of labile DOM on the responses of bacteria to DON and dissolved inorganic nitrogen (DIN), bacterial abundance and community composition were examined in controlled laboratory microcosms subjected to various combinations of dissolved organic carbon (DOC), DON, and DIN treatments. Bacterial communities that had colonized glass beads incubated in a stream were treated with various glucose concentrations and combinations of inorganic and organic N (derived from algal exudate, bacterial protein, and humic matter). The results revealed a strong influence of C availability on bacterial utilization of DON and DIN, with preferential uptake of DON under low C concentrations. Bacterial DON uptake was affected by the concentration and by its chemical nature (labile versus recalcitrant). Labile organic N sources (algal exudate and bacterial protein) were utilized equally well as DIN as an N source, but this was not the case for the recalcitrant humic matter DON treatment. Clear differences in bacterial community composition among treatments were observed based on terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes. C, DIN, and DON treatments likely drove changes in bacterial community composition that in turn affected the rates of DON and DIN utilization under various C concentrations.     

Uptake of Cadmium by Hydroponically Grown,Mature Eucalyptus Camaldulensis Saplings and the Effect of Organic Ligands

The potential suitability of Eucalyptus camaldulensis for Cd phytoextraction was tested in a hydroponic study. Saplings were exposed to 4.5 and 89 μM Cd for one month, with and without EDTA and s,s-EDDS at 0.1, 1, and 5 mM. The saplings’ growth was not affected at the 4.5 μM Cd concentration, yet it decreased 3-fold at 89 μM, and almost all the Cd taken up was immobilized in the roots, reaching 360 and 5300 mg Cd kg^?1, respectively (approximately 75% of which was non-washable in acid). The respective Cd root-to-shoot translocation factors were 0.14 and ≈5*10^?4. At 0.1 mM concentration, EDTA and EDDS had no effect or even a positive effect on the saplings growth. This was reversed at 1 mM, and the chelants became lethal at the 5 mM concentration. At 89 μM Cd in the growth medium, 0.1 mM EDTA increased Cd translocation into the shoots by almost 10-fold, however it strongly reduced Cd content inside the roots. This hydroponic study indicates the feasibility of E. camaldulensis use for cleanup Cd-contaminated soils at environmental concentrations, both for site stabilization (phytostabilization) and gradual remediation (phytoextraction). EDTA was shown to be much more efficient in enhancing Cd translocation than s,s-EDDS.     

Effect of Inorganic and Organic Tin Compounds on ACh- and Voltage-Activated Na Currents

1. Inorganic tin and organotin compounds, occurring in aquatic ecosystems, are toxic and can cause behavioral abnormalities in living organisms. To determine the possible neuronal basis of these actions, the effects of both forms of Sn were studied on identified neurones of the mollusk, Lymnaea stagnalis L.2. SnCl₂ caused a dose-dependent decrease in the acetylcholine (Ach)-induced inward current. The effective threshold concentration, measured by a two microelectrode voltage clamp technique, was 0.1 M, and the maximal effect occurred at 5 M SnCl₂. The depression of the inward current was greater after a 10 min preapplication (20%) than after 3 min treatment (7%).3. The next series of experiments compared the actions of inorganic or organic tin compounds. In whole cell clamp experiments both (CH₃)₂SnCl₂ and (CH₃)₃SnCl, like inorganic Sn, decreased the amplitude of Ach-induced current. Increasing the duration of the preapplication time resulted in an increase in the effect, but the action was not reversible. SnCl₂ treatment caused a concentration-dependent alteration (initial potentiation followed by depression) of the amplitude of I _Na(V) over the whole voltage range and slightly shifted the I–V curves to the left. In contrast, trimethyl tin decreased the amplitude of I _Na(V) only at high concentration (100 M). The activation time course of I _Na was increased ( = 0.43 ms in control and 0.55 ms in Sn), but Sn did not alter the inactivation parameters ( = 3.43 and 3.41 ms).4. These results support earlier findings that agonist- and voltage-activated channels are direct targets of toxic metals. We conclude that tin in both inorganic and organic forms acts at neuronal membranes to modulate synaptic transmission through direct actions on agonist-activated ion channels, and suggest that these actions may be the basis of the altered behavior of animals in tin-polluted environments.     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

Effect of Organic Ligands on Accumulation of Copper in Hyperaccumulator and Nonaccumulator Commelina communis

Better understanding of copper uptake and accumulation regulation in plants is critical to the phytoremediation of copper contaminated soil. This study employed a 30-day pot experiment to assess the relationship between organic ligands and copper accumulation in plants. Hyperaccumulator and nonaccumulator varieties of Commelina communis were used, different organic ligands were applied, and the data of copper accumulation in shoots were collected. The six organic ligands included ethylenediaminetetraacetic acid and organic acids (formic acid, citric acid, malic acid, tartaric acid, and succinic acid). The results showed that organic ligands added to culture increased the copper accumulation both varieties. The results of the copper accumulation in shoots agreed with the study of the root uptake kinetics of copper influx. The addition of organic acids could increase copper accumulation in shoots because the copper influx in roots was increased. The results also indicated that the copper influx of hyperaccumulator roots was higher than that of nonaccumulator roots. This is one of the mechanisms by which a hyperaccumulator could amass large amounts of copper in its shoots. In this accumulation process, little effect on the leaf relative water content was in the hyperaccumulator and nonaccumulator of leaves and normal physiological condition of plants.     

有机肥和无机肥对稻田养分循环的影响

一、引言研究养分在农田生态系统中的循环,是农业生态学的重要内容之一。对有机肥和无机肥肥效及其对农作物产量的影响曾有过不少研究。虽然大量研究表明有机肥对水稻产量的作用与化肥一样有效,甚至更好,也有许多试验,尤其是一些长期对比试验表明有机肥对作物产量的作用不及化肥,只是使用有机肥的试验小区有很长的残效,连续使用20年家畜肥的小区停肥40年后,大麦产量仍为对照化肥小     

Production of Napiergrass as a Bioenergy Feedstock Under Organic Versus Inorganic Fertilization in the Southeast USA

Napiergrass (Pennisetum purpureum Schum.) is a high-yielding perennial biomass crop that is well adapted to the Southeast USA where poultry litter is readily available. This research was conducted to compare biomass production and nutrient utilization of napiergrass fertilized with either poultry litter or inorganic fertilizer. Each spring, approximately 100 kg ha^?1 of N, 40 kg ha^?1 P, and 90 kg ha^?1 K were applied as poultry litter or equivalent inorganic fertilizer. Biomass was harvested each winter after senescence. For the first 2 years, dry matter yield did not differ among treatments, but in the third and fourth years, yields declined in all treatments and were lowest in the unfertilized treatment. Biomass N concentration and N removal were greatest in the inorganic treatment. In general, N removal exceeded the amount applied, suggesting that higher application rates may be necessary to maintain yields. Biomass P concentration and total P uptake were greatest in the litter fertilized treatment, demonstrating that napiergrass can remove some of the excess P from applied litter. Soil cores were taken periodically to assess changes in soil properties. After 2 years of production, soil pH in the surface layer (0–15 cm) was lower in the inorganic treatment than in the other treatments. After 4 years, total soil C had increased by an average of 3,180 kg ha^?1 though fertilizer treatments did not differ. Yield declined in all treatments after 4 years and N supplementation is recommended for production in upland fields.     

The Effect of River Water Circulation on the Distribution and Functioning of Reservoir Microbial Communities as Determined by a Relative Distance Approach

The effect of river water quality, its inflow rate, and temperature on planktonic food web composition and activities were studied in the eutrophic Sau Reservoir (Catalonia, NE Spain). We analyzed 8 longitudinal transects conducted between July 1996 and April 1999 covering a wide range of variability in both seasonal and spatial circulation patterns. To compare objectively the biological longitudinal gradients under seasonally fluctuating water levels and different types of water circulation patterns, we applied a model based on the relative distance of a sampling station from the river inflow. Even under different hydrological scenarios, the model was able to characterize epilimnetic food chain successions and locations of peaks of bacteria, heterotrophic nanoflagellates, ciliates, phytoplankton, and zooplankton along the longitudinal gradient. The amplitude of microbial peaks was directly related to the proportion of nutrient and organic carbon rich river water that mixed into the reservoir epilimnion. Enhanced abundances and activities of microbes were detected in spring and summer periods, mainly during events of river water overflow when a large proportion of the river was directly mixed into the epilimnion. Thus, the relative input of river water is suggested to be a useful predictor of the amplitude of the development of the epilimnetic microbial food webs in highly loaded canyon-shaped reservoirs. These results may have important implications in the context of global change in Mediterranean regions, where expected reductions in runoff may profoundly affect river water circulation patterns in reservoirs and hence organic carbon cycling in these ecosystems.     

Effect of Substrate Concentration and Organic and Inorganic Compounds on the Occurrence and Rate of Mineralization and Cometabolism

Isopropyl N-phenylcarbamate (IPC) at 400 pg and 1 μg/ml was mineralized in samples of sewage, but only the lower concentration was mineralized in lake water samples in a 50-day period. IPC at 1 μg/ml disappeared from lake water, but it was converted to organic products. Mineralization of IPC at 400 pg/ml in lake water was enhanced by additions of inorganic nutrients or a mixture of nonchlorinated water pollutants but not by yeast extract or mixtures containing aromatic compounds or excretions of primary producers. The mineralization of 200 pg of 2,4-dichlorophenoxyacetate per ml of lake water was not affected by additions of low levels of yeast extract or compounds excreted by primary producers but was enhanced by low concentrations of mixtures of water pollutants. It is suggested that some chemicals that are found to be converted only to organic products, presumably by cometabolism, in tests using the concentrations commonly employed in laboratory evaluations may be mineralized at the lower concentrations prevailing in natural waters.     

The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

Background

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

Methods

Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.

Results

No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.

Conclusions

Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.     

Enhanced Phytoremediation of Crude Oil-Polluted Soil by Four Plant Species: Effect of Inorganic and Organic Bioaugumentation

A field experiment investigating the removal and/or uptake of Polycyclic Aromatic Hydrocarbons (PAHs) and specific metals (As, Cd, Cr) from a crude oil polluted agricultural soil was performed during the 2013 wet season using four plant species: Fimbristylis littoralis, Hevea brasilensis (Rubber plants), Cymbopogom citratus (Lemon grass), and Vigna subterranea (Bambara nuts). Soil functional diversity and soil-enzyme interactions were also investigated. The diagnostic ratios and the correlation analysis identified mixed petrogenic and pyrogenic sources as the main contributors of PAHs at the study site. A total of 16 PAHs were identified, 6 of which were carcinogenic. Up to 42.4 mg kg^?1 total PAHs was recorded prior to the experiments. At 90 d, up to 92% total PAH reduction and 96% As removal were achieved using F. littoralis, the best performing species. The organic soil amendment (poultry dung) rendered most of the studied contaminants unavailable for uptake. However, the organic amendment accounted for over 70% of the increased dehydrogenase, phosphatase, and proteolytic enzymes activities in the study. Overall, the combined use of soil amendments and phytoremediation significantly improved the microbial community activity, thus promoting the restoration of the ecosystem.     

Growth of a Dehalococcoides-Like Microorganism on Vinyl Chloride and cis-Dichloroethene as Electron Acceptors as Determined by Competitive PCR

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 ± 1.5) × 10⁸ copies of the 16S rRNA gene/μmol of Cl⁻ (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate () was determined to be 7.8 × 10⁻¹⁰ μmol of Cl⁻ (copy⁻¹ day⁻¹), indicating a maximum growth rate of 0.4 day⁻¹. These average growth yield and values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day⁻¹) and no-growth (0.09 day⁻¹) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.     

Effect of Supplemental Inorganic Zn and Mn and their Interactions on the Performance of Broiler Chicken,Mineral Bioavailability,and Immune Response

The purpose of this study was to investigate the interaction and main effects of supplemental Zn and Mn levels on growth, tissue mineral uptake, and immune response in broiler chicken. A basal diet of corn–soybean meal was supplemented with Zn at 40, 80, or 160 ppm and Mn at 60, 120, or 240 ppm in a factorial pattern to constitute nine experimental diets. Each diet was offered to nine replicates of six chicks in stainless steel battery brooders. At 35 days of age, body weight gain, feed conversion efficiency, hock joint scores, tibia weight, tibia strength, and percent ash were not influenced by Zn and Mn levels and their interactions. The concentration of Zn (207–238 ppm) and Mn (11.8–16.3 ppm) in tibia increased linearly with progressive raise of mineral inclusion in diets. Mn at 240 ppm level caused higher retention of Zn in tibia, but not vice versa. Manganese either alone or in combination with Zn (Zn160/Mn120 ppm) significantly reduced Cu retention (10.1–7.2 ppm) in tibia. Even in the hepatic tissue, Zn (93.6–98.4 ppm) and Mn (9.3–10.2 ppm) concentration increased linearly with their levels of inclusion in diets. When Zn and Mn levels were maintained at 4:3 ratio (80:60 or 160:120 ppm), the concentration of Zn (100–106 ppm) in liver was higher, while that of Mn was significantly more with low level of Zn (40 ppm) in diet. However, Mn supplementation at 120 ppm level and above significantly decreased Cu accumulation (19.5–17.1 ppm) in liver, but Mn × Zn interaction had no effect on Cu retention. The immune response measured as antibody titers to sheep RBC increased (5.9–7.9 log₂) significantly with higher Zn (80 ppm) supplementation and cell-mediated immune response to phytohemagglutinin (0.57–0.78) with Mn level at 120 ppm. In summary, Zn (40 ppm) and Mn (60 ppm) as recommended by NRC was sufficient for broiler performance and bone parameters. Mn complimented Zn retention in tibia and antagonized Cu in tibia and liver tissues. Higher levels of Zn (80 ppm) and Mn (120 ppm) than those recommended by NRC were needed for improved immune response in broilers at 35 days of age.