A bioluminescence assay using Nitrosomonas europaea for rapid and sensitive detection of nitrification inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O2 uptake and NO2- production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

Phylogenetic differences between particle-associated and planktonic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Northwestern Mediterranean Sea

The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to Nitrosomonas eutropha, while the sequences obtained from the planktonic samples were related to a novel marine Nitrosospira group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as Nitrosomonas clones, while 96% of these clones were identified as clones that were related to the marine Nitrosospira species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the Nitrosomonas group. This is the first indication that Nitrosomonas species and Nitrosospira species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments.     

Changes in the microbial community in Japan Trench sediment from a depth of 6292 m during cultivation without decompression

A sample of deep-sea sediment was obtained from the Japan Trench at a depth of 6292 m using a pressure-retaining sediment sampler. Microorganisms in the sediment sample were cultivated in marine broth 2216 at ambient pressure (65 MPa) without decompression, and at atmospheric pressure (0.1 MPa) as a control experiment. 16S ribosomal RNA genes (rDNA) were amplified by PCR from DNA extracted from the original sediment sample and the mixed cultures, and the nucleotide sequences were determined. The results of phylogenetic analysis based on 16S rDNA sequences indicated that microbial diversity in the original sediment samples showed a wide distribution of types in the domain Bacteria. Furthermore, in the mixed cultures incubated at 65 MPa without decompression, bacterial strains belonging to the Shewanella barophiles branch and the genus Moritella existed together at the beginning of cultivation, and Moritella strains became dominant towards the end of the cultivation period. Finally, in the mixed cultures incubated at atmospheric pressure, strains belonging to the genus Pseudomonas were dominant at all times. Analysis of fatty acids extracted from the cultures supported the phylogenetic results.     

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.     

Prevalence of Clostridium botulinum in Finnish trout farms: pulsed-field gel electrophoresis typing reveals extensive genetic diversity among type E isolates

The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg-1, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.     

Altered gene expression and genetic damage in North American fish populations

Populations of marine, estuarine, and freshwater fish from highly urban and industrialized sites in North America often exhibit elevated prevalences of neoplastic, preneoplastic, and nonneoplastic hepatic lesions, and sometimes epidermal neoplasms compared to conspecifics from more pristine reference locales. Positive statistical associations with environmental concentrations of PAHs and other xenobiotics and experimental laboratory studies suggest a chemical etiology to these epizootics. Studies have investigated the expression of carcinogenically relevant genes, the extent of overall DNA damage, somatic cell mutations, germ line polymorphisms, and overall levels of genetic diversity in fish from these populations and other polluted sites. In general, elevated levels of cytochrome P4501A expression have been found in fish from contaminated locales; however, inhibition of gene induction has been seen in hepatic lesions and in normal tissue in fish from the most contaminated sites, perhaps due to genetic adaptation or physiological acclimation. Levels of bulky hepatic DNA adducts, as detected by 32P-postlabeling, are almost always elevated in fish from populations that are exposed to highly contaminated environments. However, levels of DNA adducts were not always predictive of the vulnerability to neoplasia of populations and species from polluted sites. Elevated levels of oxygen radical-induced DNA damage have been observed in hepatic tumors, preneoplastic lesions, and normal livers in a single species of flatfish from contaminated sites; however, the prevalences of these alterations in other species and at other polluted sites has yet to be evaluated. Frequent alterations in the K-ras oncogene have been reported in hepatic neoplasms in several species from highly contaminated sites and also in embryos that were experimentally exposed to oil-contaminated sediments. Studies also suggest that heritable germ line polymorphisms, altered allelic frequencies, and reductions in overall genetic diversity may have occurred in some highly impacted populations; however, the origin and functional significance of altered allelic frequencies have largely yet to be evaluated. In summary, feral fish appear particularly sensitive to DNA alterations from xenobiotics, perhaps due to their unusually high levels of exposure, relatively inefficient DNA repair, and the high frequency of polyploidy in some taxa and provide excellent models to explore the relationships between xenobiotic exposure and altered gene structure and expression.     

In situ morphology of nitrifying-like bacteria in aquaculture systems

The in situ microbiota from several aquaculture facilities with active nitrification was examined by transmission electron microscopy of thin sections for the presence of bacteria that contained intracytoplasmic membranes characteristic of the nitrifying bacteria. Colonies of bacteria with the cellular morphology of a species of Nitrosomonas were found to be present in both the culture water and in the biological filter slime of a freshwater chinook salmon (Oncorhynchus tshawytscha) culture system. bacteria in the water possessed the normal nitrosomonas type of ultrastructure, whereas similar bacteria in the slime had an aberrant morphology due to multiple invaginations of the cell wall and cyto-membranes and a significantly greater number of ribosomes. These nitrosomonas-like bacteria lysed during enrichment in commonly used media. Bacteria with the morphology of species of Nitrosomonas and Nitrosococcus were also observed in colonies in the surface slimes of marine culture systems for striped bass (Morone saxatilis) and quahaug (Mercenaria mercenaria).     

The anaerobic oxidation of ammonium

From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist in nature. 'Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium oxidation was about 2 nmol NH4+ min-1 (mg protein)-1 using nitrogen dioxide as electron acceptor. This reaction, which may involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH4+ min-1 (mg protein)-1 was determined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h-1 (doubling time 11 days). 15N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this new process. A novel type of hydroxylamine oxidoreductase containing an unusual P468 cytochrome has been purified from the Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for future application in the removal of ammonium from wastewater with high ammonium concentrations.     

16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.     

Fate and biological effects of polymeric MDI (4,4'-diphenylmethane diisocyanate and homologs) in small artificial ponds

The effects from a simulated accidental pollution event in a pond with polymeric MDI (4,4'-diphenylmethane diisocyanate and homologs) on different trophic levels of the aquatic ecosystem were investigated in small artificial ponds. Three 4.5-m3 volume ponds, interconnected with closable locks, were provided with natural lake sediment and ground water. Caged fish (rainbow trout, Oncorhynchus mykiss) were added to each pond, and the interconnecting locks were kept open to establish nearly identical physicochemical and biological conditions. At this stage, the ponds were isolated from one another and MDI was added at a dosage of 1 g/liter on top of the sediment of treated part of the first pond, 10 g/liter to the second pond, and 0 g/liter to the third pond (untreated control). Neither the applied monomer MDI nor its potential reaction product MDA (4,4'-diphenylmethanediamine) was detected in water or accumulated by fish. The MDI polymerized to inert polyurea on the sediment of the test ponds. This polymerization formed carbon dioxide, released as bubbles which floated to the water surface. Some carbon dioxide was solubilized in water and reduced the water pH of about 9 by 2.0 units as an average in the high-dosed pond and 0.7 in the low-dosed pond. This reduction caused some other minor changes in the physicochemical characteristics of the pond water. Neither application rate caused any direct effect on the pelagic community (phytoplankton, zooplankton, fish, macrophytes) of the test ponds. Some minor indirect effects caused by the production of carbon dioxide were observed in phyto- and zooplankton community structures. Also, an increase of macrophyte growth was noted. Organisms living in the untreated part of the sediment (macrobenthos) were affected as a result of physical obstructions in this habitat. These populations, however, regained densities equivalent to the control after some weeks, except for Bivalvia which have too long of a generation time for the test period of this study.     

Accudosetm aerosol--an effective house fly (Diptera; Muscidae: Musca domestica L.) adulticide control system for cage type poultry houses

An automatic piped aerosol system (Accudose TM) using 0.7% synergized pyrethrin insecticide, was tested at a typical narrow cage poultry farm for the control of adult house flies, Musca domestica L. A similar narrow cage poultry farm was used as a control with all house fly control measures left up to the cooperating poultryman. AccudoseTM was compared with other house fly control methods at other similar farms which included three types of man-portable ultra volume (ULV) generators and an integrated (biological-chemical) program. Results of the five month test demonstrated that the AccudoseTM system suppressed house fly populations better than other control methods.     

稀土矿区低碳氨氮废水短程硝化SBR过程研究

采用SBR工艺处理稀土矿区低碳氨氮废水, 对活性污泥进行驯化培养并考察了曝气量、曝气时间及碳氮比对短程硝化系统的影响。试验结果表明:温度为(28±1) ℃、曝气量为65 L/h, pH值为8的条件下, 经过69 d的驯化培养后, 系统对氨氮的去除率达92%, 亚硝态氮积累率稳定在90%以上, 对短程硝化过程启动前后样品进行高通量测序, 结果表明:污泥中微生物种类减少, 多样性降低, 亚硝化单胞菌属成为优势种群, 占比达11.5%。提高曝气量至120 L/h并在此条件下运行7 d后, 亚硝态氮积累率下降至82%;维持C/N在3.5~7.6之间, 系统中NH₄+-N的去除率均能稳定在95%左右, NO₂--N积累率也均可达93%以上;过度曝气会破坏短程硝化系统, 过度曝气至第8天, 亚硝态氮的积累率降至48.89%。      

Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.     

Evaluation of ADCP Apparent Bed Load Velocity in a Large Sand-Bed River: Moving versus Stationary Boat Conditions

Detailed mapping of bathymetry and apparent bed load velocity using a boat-mounted acoustic Doppler current profiler (ADCP) was carried out along a 388-m section of the lower Missouri River near Columbia, Missouri. Sampling transects (moving boat) were completed at 5- and 20-m spacing along the study section. Stationary (fixed-boat) measurements were made by maintaining constant boat position over a target point where the position of the boat did not deviate more than 3?m in any direction. For each transect and stationary measurement, apparent bed load velocity (vb) was estimated using ADCP bottom tracking data and high precision real-time kinematic (RTK) global positioning system (GPS). The principal objectives of this research are to (1)?determine whether boat motion introduces a bias in apparent bed load velocity measurements; and (2)?evaluate the reliability of ADCP bed velocity measurements for a range of sediment transport environments. Results indicate that both high transport (b>0.6??m/s) and moving-boat conditions (for both high and low transport environments) increase the relative variability in estimates of mean bed velocity. Despite this, the spatially dense single-transect measurements were capable of producing detailed bed velocity maps that correspond closely with the expected pattern of sediment transport over large dunes.     

Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.     

Heme packing motifs revealed by the crystal structure of the tetra-heme cytochrome c554 from Nitrosomonas europaea

Cytochrome c554 (cyt c554), a tetra-heme cytochrome from Nitrosomonas europaea, is an essential component in the biological nitrification pathway. In N. europaea, ammonia is converted to hydroxylamine, which is then oxidized to nitrite by hydroxylamine oxidoreductase (HAO). Cyt c554 functions in the latter process by accepting pairs of electrons from HAO and transferring them to a cytochrome acceptor. The crystal structure of cyt c554 at 2.6 A resolution shows a predominantly alpha-helical protein with four covalently attached hemes. The four hemes are arranged in two pairs such that the planes of the porphyrin rings are almost parallel and overlapping at the edge; corresponding heme arrangements are observed in other multi-heme proteins. Striking structural similarities are evident between the tetra-heme core of cyt c554 and hemes 3-6 of HAO, which suggests an evolutionary relationship between these redox partners.     

Organic halogen removal from chlorinated humic ground water and lake water by nitrifying fluidized-bed biomass characterised by electron microscopy and molecular methods

The dechlorinating and genotoxicity-removing activities of nitrifying fluidized-bed reactor biomass towards chlorinated organic compounds in water were shown at level below 1 ppm. The removal rates of adsorbable organic halogens were 200 micrograms Cl (g VS day)-1 for chlorinated humic ground water and 50 micrograms Cl (g VS day)-1 for chlorinated lake water when studied in batch mode. In a sequenced batch mode the removal rates [micrograms Cl (g VS day)-1] were 2000 from chlorohumus, 1400-1800 from chlorophenols in chlorinated ground water, and 430-720 from chlorohumus in chlorinated lake water. Genotoxicity was removed to a large extent (60%-80%) from the chlorinated waters upon incubation with nitrifying reactor biomass. 2,6-Di-, 2,4,6-tri and 2,3,4,6-tetrachlorophenols competed with chlorinated water organohalogens for dechlorination. The dechlorination of chlorophenols and chlorohumus required no ammonia and was not prevented by inhibitors of ammonia oxidation, nitrapyrin, parathion, sodium diethyldithiocarbamate, or allylthiourea. Electron microscopical inspection of the biomass showed the dominance of clusters of bacteria resembling known nitrifying species, Nitrosomonas, Nitrobacter, and Nitrosospira. This was supported by polymerase chain reaction amplification of the biomass DNA with four different primers, revealing the presence of 16S rDNA sequences assignable to the same species. The most intensive band obtained with the Nitroso4E primer was shown to be closely related to Nitrosomonas europaea by restriction analysis.     

Hydraulic and Sedimentological Characterizations of a Reach on the Anaktuvuk River, Alaska

Rivers located on the North Slope of Alaska’s Brooks Range are typically not well characterized with respect to basic hydraulic and sedimentological data. In order to obtain basic hydrosedimentological information on the Anaktuvuk River, a pristine stream located in the Colville River basin, we conducted an extensive field campaign from late May to early June 2009. The study reach was located at N69°27.785′, W151°09.858′, latitude and longitude, respectively. The Anaktuvuk River flows north from the Brooks Range to the Colville River, drains an area of 7,058?km2, and encompasses 2,063 m of vertical relief. During the field campaign, the field crew measured discharge and water-surface slope, collected water samples, and characterized bed sediment. As a result of fieldwork and laboratory work, we present an initial rating curve for the Anaktuvuk River, as well as the calculated roughness coefficient and suspended sediment concentrations. In addition, we compare the observed bankfull discharges with the bankfull discharge predicted by a recently published model.