Compact, low-threshold squeezed light source.

Strongly squeezed light finds many important applications within the fields of quantum metrology, quantum communication and quantum computation. However, due to the bulkiness and complexity of most squeezed light sources of today, they are still not a standard tool in quantum optics labs. We have taken the first steps in realizing a compact, high-performance 1550 nm squeezing source based on commercially available fiber components combined with a free-space double-resonant parametric down-conversion source. The whole setup, including single-pass second-harmonic generation in a waveguide, fits on a 30 cm×45 cm breadboard and produces 9.3 dB of squeezing at a 5 MHz sideband-frequency. The setup is currently limited by phase noise, but further optimization and development should allow for a 19" sized turn-key squeezing source capable of delivering more than 10 dB of squeezing.

Influence of temperature and predation on survival of Salmonella enterica serovar Typhimurium and expression of invA in soil and manure-amended soil.

The effects of three temperatures (5, 15, and 25 degrees C) on the survival of Salmonella enterica serovar Typhimurium in topsoil were investigated in small microcosms by three different techniques: plate counting, invA gene quantification, and invA mRNA quantification. Differences in survival were related to the effect of protozoan predation. Tetracycline-resistant Salmonella serovar Typhimurium was inoculated into soil and manure-amended soil at 1.5 x 10(8) cells g soil(-1). Population densities were determined by plate counting and by molecular methods and monitored for 42 days. Simultaneous extraction of RNA and DNA, followed by quantitative PCR, was used to investigate invA gene levels and expression. Analysis by these three techniques showed that Salmonella serovar Typhimurium survived better at 5 degrees C. Comparing DNA and CFU levels, significantly higher values were determined by DNA-based techniques. invA mRNA levels showed a fast decrease in activity, with no detectable mRNA after an incubation period of less than 4 days in any of the soil scenarios. A negative correlation was found between Salmonella serovar Typhimurium CFU levels and protozoan most probable numbers, and we propose the role of the predator-prey interaction as a factor to explain the die-off of the introduced strain by both culture- and DNA quantification-based methods. The results indicate that temperature, manure, and protozoan predation are important factors influencing the survival of Salmonella serovar Typhimurium in soil.

Field-scale variation in microbial activity and soil properties in relation to mineralization and sorption of pesticides in a sandy soil.

Pesticides applied to agricultural soils are subject to environmental concerns because leaching to groundwater reservoirs and aquatic habitats may occur. Knowledge of field variation of pesticide-related parameters is required to evaluate the vulnerability of pesticide leaching. The mineralization and sorption of the pesticides glyphosate and metribuzin and the pesticide degradation product triazinamin in a field were measured and compared with the field-scale variation of geochemical and microbiological parameters. We focused on the soil parameters clay and organic carbon (C) content and on soil respiratory and enzymatic processes and microbial biomass. These parameters were measured in soil samples taken at two depths (Ap and Bs horizon) in 51 sampling points from a 4-ha agricultural fine sandy soil field. The results indicated that the spatial variation of the soil parameters, and in particular the content of organic C, had a major influence on the variability of the microbial parameters and on sorption and pesticide mineralization in the soil. For glyphosate, with a co-metabolic pathway for degradation, the mineralization was increased in soils with high microbial activity. The spatial variability, expressed as the CV, was about five times higher in the Bs horizon than in the Ap horizon, and the local-scale variation within 100 m(2) areas were two to three times lower than the field-scale variation within the entire field of about 4 ha.

Spatial variation in 2-methyl-4-chlorophenoxyacetic acid mineralization and sorption in a sandy soil at field level.

The phenoxyacetic acid herbicide MCPA (2-methyl-4-chlorophenoxyacetic acid) is frequently detected in groundwater beneath Danish agricultural fields. We investigated spatial variation in microbial MCPA mineralization potential in a flat agricultural field of fine sandy soil (USDA classification: Humic Dystrudept) located on the Yoldia plains of Northern Jutland, Denmark. Samples for determination of MCPA mineralization and sorption were collected from the Ap and Bs horizons at 51 sampling sites located in a 200 x 220 m grid. Spatial variation in sorption was low in both horizons (distribution coefficient, 0.36-4.16 L kg(-1)). Sorption correlated strongly with soil organic carbon content in both horizons (CV, 93 and 83%, respectively) and negatively with soil pH. [Ring-(14)C]-MCPA mineralized readily in the Ap horizon, with 49 to 62% of the (14)C-MCPA being converted to (14)CO(2) during the 67-d incubation period. With the subsoil, mineralization of (14)C-MCPA varied considerably between samples (0.5-72.8%). At neither depth was there correlation between (14)C-MCPA mineralization and sorption, soil pH, organic carbon content, clay content, number of colony-forming units (CFU), pseudomonad CFU, or any of the four microbial activity parameters measured. The presence of microbial genes encoding for the TfdA enzyme was quantified using real-time polymerase chain reaction. No correlation was found between MCPA mineralization potential and the natural background number of tfdA genes present in the soil samples. The degradation kinetics suggests that the high (14)C-MCPA mineralization rate detected in soil samples was linked to growth of the MCPA-degrading soil microbial community.

An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium.

An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.

Determination of sulfonylurea degradation products in soil by liquid chromatography-ultraviolet detection followed by confirmatory liquid chromatography-tandem mass spectrometry.

A method based on liquid extraction followed by sample enrichment on reversed-phase solid-phase extraction was developed for the extraction of five degradation products of four sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, thifensulfuron-methyl and tribenuron-methyl) from soil. The compounds have been quantified by LC-UV and identified by tandem LC-MS with electrospray ionization or atmospheric pressure chemical ionization. The limits of detection for the five compounds were between 10 and 50 micrograms kg-1. The method has been applied to the extraction of soil samples after microbial degradation of sulfonylurea herbicides.

Isolation and characterisation of Rhodococcus erythropolis TA57 able to degrade the triazine amine product from hydrolysis of sulfonylurea pesticides in soils.

We isolated a bacterium capable of metabolising a methylated and methoxylated s-triazine ring as the only nitrogen source. On a weight basis, the s-triazine, commonly named triazine amine (TAM), constitutes approx. half of several sulfonylurea herbicides and is formed after hydrolysis of these herbicides. The isolate, strain TA57 was identified using multi-phasic taxonomy as a gram-positive Rhodococcus erythropolis. Strain TA57 mineralised over 50% 14C-labelled TAM within 4 days in growing cultures using all of the nitrogen for growth. The degradation capacity was found stable in cells grown on either tryptic soy broth agar plates or in minimal medium with NH4+. Among other s-triazines tested, only one other methylated, but de-methoxylated s-triazine amine supported growth. Inoculating 10(6) cells of TA57 per gram of soil (d.w.) resulted in 50% mineralisation of 14C labelled TAM (1 mg kg(-1)) within 25 days, in contrary to the indigenous population that mineralised only 6% in 50 days.

Analysis of metsulfuron-methyl in soil by liquid chromatography/tandem mass spectrometry. Application to a field dissipation study.

An analytical method is described for the extraction of metsulfuron-methyl from soil at sub-parts per billion levels (LOQ = 0.2 microgram kg(-1)). The herbicide was quantitatively determined and identified by ESI LC/MS/MS. The method has been applied to a field dissipation study in which metsulfuron-methyl was applied to spring barley at three dosage rates: 4, 8, and 16 g of active ingredient ha(-)(1). The results of 2 years are presented. The dissipation rate of metsulfuron-methyl in topsoil was very rapid, with a calculated half-life of 6.5 days. Laboratory mineralization studies with native soils in contrast to autoclaved soils indicated that microbial degradation of (14)C-labeled metsulfuron-methyl and (14)C-labeled 2-amino-4-methoxy-6-methyl-1,3,5-triazine in soil microcosms is an important factor for the complete degradation of metsulfuron-methyl in the field. However, the mineralization rate of the sulfonamide was much higher.

Mineralisation studies of 14C-labelled metsulfuron-methyl, tribenuron-methyl, chlorsulfuron and thifensulfuron-methyl in one Danish soil and groundwater sediment profile.

Bacterial mineralisation of four sulfonylurea herbicides at 20 microg kg(-1) in a sandy soil from nine different depths in a sandy soil horizon (5-780 cm) was investigated in laboratory studies. Metsulfuron-methyl, chlorsulfuron, and tribenuron-methyl were 14C-labelled in the sulfonamide ring, while thifensulfuron-methyl was labelled in the thiophene ring. The highest mineralised amount in 126 days was observed for metsulfuron-methyl (40%) followed by tribenuron-methyl (25%), and thifensulfuron-methyl (11%). Chlorsulfuron showed low mineralisation in all the soils tested (<4%). Mineralisation of the herbicides metsulfuron-methyl and tribenuron-methyl varied according to soil depth (upper profile: 5-70 cm, and lower profile: 165-780 cm) and were proven faster in soil taken from depths 5-7 and 30-35 cm, and slower in depths 45-50 and 70-75 cm. Mineralisation was absent in the lower profile (165-780 cm). As an indicator of microbial activity bacterial counts were taken at the experimental start; these counts grouped in three levels: highest in the surface layer (5-7 cm), slightly lower in the depths 30-75 cm, and lowest in the lower profile (165-780 cm). Residual concentrations of metsulfuron-methyl correlated to the accumulated amount mineralised, with high residual concentrations in soil showing low mineralisation. Also chlorsulfuron showed high residual concentrations with increasing depth in the upper profile, but the relatively high dissipation at 30-35 cm and lower one at 45-50 cm could not be related with the lack of mineralisation. This shows that hydrolysis occurs, but mineralisation of the chloro-substituted sulfonamide is restricted. Tribenuron-methyl and thifensulfuron-methyl could not be detected due to interference with other compounds.

The Hall effect in the organic conductor TTF-TCNQ: choice of geometry for accurate measurements of a highly anisotropic system.

We have measured the Hall effect on recently synthesized single crystals of the quasi-one-dimensional organic conductor TTF-TCNQ (tetrathiafulvalene-tetracyanoquinodimethane), a well known charge transfer complex that has two kinds of conductive stacks: the donor (TTF) and the acceptor (TCNQ) chains. The measurements were performed in the temperature interval 30 K < T < 300 K and for several different magnetic field and current directions through the crystal. By applying the equivalent isotropic sample approach, we have demonstrated the importance of the choice of optimal geometry for accurate Hall effect measurements. Our results show, contrary to past belief, that the Hall coefficient does not depend on the geometry of measurements and that the Hall coefficient value is approximately zero in the high temperature region (T > 150 K), implying that there is no dominance of either the TTF or the TCNQ chain. At lower temperatures our measurements clearly prove that all three phase transitions of TTF-TCNQ could be identified from Hall effect measurements.

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere.

AIMS: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. METHODS AND RESULTS: We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected for monitoring colonization of barley roots. CONCLUSIONS: We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent.

Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay.

A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.

Fate of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) in soil during water stress: effects on culturability and viability.

A sandy loam soil near field capacity moisture content (psi = -0.050 MPa) or air dried (psi = -300 MPa) was inoculated with about 3 x 10(7) CFU of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) per g and incubated in 40-g portions at 17 degrees C in closed or open Erlenmeyer flasks. In the field-moist soil, selective plating, direct viable counts, and DNA hybridization showed only minor changes in the numbers of E. cloacae and A. eutrophus cells with time (14 days), and the results obtained with the three detection methods generally agreed. In the air-dried soil, the majority of both bacteria were found as intact DNA-carrying cells that were neither culturable nor viable by the methods employed in this study. The numbers of culturable E. cloacae and A. eutrophus cells dropped to 10(5) and 10(2) CFU/g, respectively, 2 h after inoculation. Direct viable counts showed that only about 1% of the cells detected by immunofluorescence microscopy were viable, but a fraction of viable nonculturable cells of both bacteria was present. A. eutrophus did not tolerate desiccation as well as E. cloacae. Only a minor fraction of the two test organisms regained their culturability or viability after rewetting of the air-dried soil; the number of total heterotrophic culturable bacteria, however, increased more than 10-fold and reached 73% of the level found in the field-moist soil at day 14.

Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin.

A new method for the extraction of bacterial DNA from soil has been developed. Soil samples of 50 g were dispersed, and bacteria were released by use of a cation-exchange resin; subsequently, bacteria were separated from soil particles by low-speed centrifugation and lysed with lysozyme and ionic detergent, and the DNA was then purified by CsCl-ethidium bromide equilibrium density centrifugation. The extracted DNA was of high molecular weight and sufficiently pure for restriction enzyme digestion, DNA-DNA hybridization, and amplification by the polymerase chain reaction. The advantages of the new method are that the separation of bacteria from soil is considerably faster than by repeated blending, more samples can be handled, and furthermore no aerosols are formed during separation. Also, we investigated whether the CsCl-ethidium bromide equilibrium density centrifugation could be replaced by purification using Gene-Clean. However, this method produced DNAs which were insufficiently pure for several types of analysis. The new method was used to study survival of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Pseudomonas cepacia DBO1 (pRO101) in unamended soil and in soil amended with 2,4-D. We found that the degrading strain, irrespective of inoculation level, was able to grow to the same high numbers in soil amended with 2,4-D, while the strain in nonamended soil were maintained at the inoculation level. Detection based on DNA extraction and subsequent dot blot DNA-DNA hybridization was in accordance with detection by plating on selective medium.

Growth and survival of Pseudomonas cepacia DBO1 (pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid.

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10(7) CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days. The influence of 0, 0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6 x 10(4), 6 x 10(6) or 1 x 10(8) CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with Pseudomonas cepacia DBO1(pRO101), Alcaligenes eutrophus AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): effects of inoculation level and substrate concentration.

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (= pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil. Mineralization was measured as 14C-CO2 evolved during degradation of uniformly-ring-labelled 14C-2,4-D. When the strains were inoculated to a level of approximately 10(8) CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h. Mineralization of 0.05 ppm and 10 ppm 2,4-D by the two A. eutrophus strains was identical and rapid whereas mineralization by P. cepacia DBO1(pRO101) occurred more slowly. In contrast, mineralization of 500 ppm 2,4-D by the two A. eutrophus strains was very slow whereas mineralization by P. cepacia DBO1 was more rapid. Comparison of 2,4-D mineralization at different levels of inoculation with P. cepacia DBO1(pRO101) (6 x 10(4), 6 x 10(6) and 1 x 10(8) CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level. The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models. The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil. In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.

Phenotypic and genotypic characterization of phenanthrene-degrading fluorescent Pseudomonas biovars.

A total of 41 phenanthrene degraders were isolated from a former coal gasification site by using Pseudomonas-selective Gould's S1 medium. All isolates were found to belong to the fluorescent Pseudomonas group and were subjected to characterization by phenotypic methods, including classical taxonomic tests, API 20NE, and Biolog GN, and the strains were further characterized by the genotypic method repetitive extragenic palindromic PCR (REP-PCR). By using classical tests, the population was found to consist of 38 strains belonging to P. fluorescens, 2 P. putida strains, and 1 Pseudomonas sp. Bacteria in phenograms from Biolog GN and REP-PCR data were divided into groups, which were in good agreement with classical test and API 20NE results. We found a nonfluorescent group of 22 bacteria inconsistent with any Pseudomonas sp. in Bergey's Manual of Systematic Bacteriology. The group showed small differences in the genotypic test, indicating that all 22 isolates were not recent clones of the same isolate. Analyses of the nonfluorescent group indicated that it belonged to Pseudomonas, but the group could not be affiliated with P. fluorescens because of differences in DNA-DNA hybridization. Identifications using classical tests and API 20NE were found to correlate, but Biolog GN identifications after 24-h incubation resulted very often in the distantly related P. corrugata. The reproducibilities of individual tests of each phenotypic method were assessed, and low reproducibilities were mainly found to be associated with specific Biolog GN test wells. Classical tests and API 20NE proved to be the best for identification of isolates, whereas Biolog GN and REP-PCR were found to be the best tests for high resolution among these closely related isolates.

Development and utilisation of a medium to isolate phenanthrene-degrading Pseudomonas spp.

In this study, we isolated phenanthrene degraders belonging to Pseudomonas spp. by combining the selective force of two previously described media. The two compounds, sodium lauryl sarcosine and trimethoprim, from the Gould S1 medium, were added to minimal agar plates sprayed with phenanthrene. Pseudomonas spp. that could produce clearing zones were isolated in one step from the rhizosphere without first selecting for Pseudomonas spp. and subsequently screening for degraders or vice versa. Enumeration and isolation of Pseudomonas spp. attached to the rhizosphere showed clear differences between two types of soil. Rhizosphere-attached phenanthrene degraders (from Pseudomonas spp.) were isolated from a former coal gasification site, but were absent in an agricultural soil subjected to organic farming. We isolated 23 phenanthrene degraders producing clearing zones from the rhizosphere of barley roots. All of these 23 isolates (of which 16 were fluorescent in UV light) proved to be members of the Pseudomonas RNA homology group I, on the basis of results of the analytical profile index (API) test system and classic taxonomic tests.

Development and application of a most-probable-number-pcr assay to quantify flagellate populations in soil samples.

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

Pseudomonas frederiksbergensis sp. nov., isolated from soil at a coal gasification site.

Phenotypic and genotypic characterization indicated that a group of 29 closely related phenanthrene-degrading bacteria from a coal gasification site in Frederiksberg, Copenhagen, Denmark, belonged to the genus Pseudomonas. The strains were isolated at two sampling occasions 2 years apart. The isolates were phenotypically different from any known species of the genus Pseudomonas and were therefore subject to further identification. Colonies were smooth and pale yellowish and did not produce pigments fluorescent in UV light when grown on King's B agar. Cells were rod-shaped, approximately 0.5-0.8 x 1.5-3.0 microm, and grew at 4 and 30 degrees C, but not 37 degrees C. The bacteria were oxidase- and catalase-positive, accumulated poly-beta-hydroxybutyrate and denitrified, but did not utilize D-xylose. The mean G+C content was 59.6 mol%. Phenotypic data and 16S rDNA sequence data information for Pseudomonas amygdali and Pseudomonas corrugata, and 16S rDNA sequence data for Pseudomonas chlororaphis and Pseudomonas syringae showed close relationships to these strains. However, DNA-DNA hybridization data showed that the isolates belong to a new species, for which the name Pseudomonas frederiksbergensis sp. nov. is proposed. The type strain is JAJ28T (DSM 13022T).