Evaluation of the metabolic diversity of microbial communities in four different filter layers of a constructed wetland with vertical flow by Biolog analysis.

The community-level substrate utilization test based on direct incubation of environmental samples in Biolog EcoPlates is a suitable and sensitive tool to characterize microbial communities. The aim of this study was to investigate the influence of plant roots and soil structure on the metabolic diversity of microorganisms in a constructed wetland with vertical flow. Sediment samples were taken from different filter depths representing specific filter layers. The color development representing the substrate utilization was measured with the samples over a period of 10 days. The average well color development (AWCD) for all carbon sources was calculated as an indicator of total activity and in order to compensate the influence of the inoculum's density on the color development in the plates. After transformation by dividing by the AWCD, the optical density data were analysed by principal component analysis (PCA). An analysis of the kinetic profile of the AWCD was carried out to increase the analytical power of the method. The corrected data have been successfully fit to the logistic growth equation. Three kinetic model parameters, the asymptote (K), the exponential rate of color change (p) and the time to the midpoint of the exponential portion of the curve (s), were used for statistical analysis of the physiological profile of the microbial community in the different filter layers of the constructed wetland. We found out that in the upper two horizons, which were rooted most densely, mainly easily degradable materials like specific carbohydrates were utilized, while in the lower layers, where only single roots occur, more biochemically inert compounds, e.g. 2-hydroxy benzoic acid, were utilized. Furthermore it could be shown that microorganisms in the surface layer benefited from the plant litter because they can utilize decay products of these. In the lower filter layers specialists took advantage because they had to cope with the biochemically inert materials and the lower nutrient supply.

Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

Fixation procedures for flow cytometric analysis of environmental bacteria.

Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.

The root surface as the definitive detail for microbial transformation processes in constructed wetlands--a biofilm characteristic.

It was the goal of the investigations to characterise the biofilm on the plant roots because of the demonstrable major role of these associated bacteria. The essential criteria for the research were to look at the structure of the microbial colonisation (pattern, density) and to determine properties of the rhizoplane biofilm such as thickness and structure. The root material from a hydroponic system, planted with Glyceria maxima and used for nitrogen removal, has been used for the investigations. Several properties of the bacteria became visible due to the application of specific dyes. The evaluation of the samples was performed by scanning confocal laser microscopy (CLSM). It was shown that the microbial colonisation of the root surface of Glyceria maxima was on an unexpected high level and seems to be related mainly to the permeability and therefore to the age of the plant roots. The thickness of the rhizoplane biofilm is remarkably thin; no inactive layers could be observed in contrast to biofilm growing on technical carrier material. Caused by the untypically two-sided supply with nutrients the whole biofilm is in interaction with the surroundings. This indicates the importance of the plant roots for the microbial transformation processes in wetlands and underlines the especialness of the root as carrier for microorganisms.

Detection of phages carrying the Shiga toxin 1 and 2 genes in waste water and river water samples.

AIMS: To evaluate the occurrence and abundance of phages that carry the stx(1) and stx(2) gene in water samples of different quality. METHODS AND RESULTS: Phages growing on the Shiga toxin-negative Escherichia coli O157:H7 (ATCC 43,888) strain were enumerated by a plaque assay in concentrated raw and treated waste water samples and river water samples. Plaques were investigated for the presence of stx(1) and stx(2) genes by a multiplex/nested PCR procedure. An overall number of 805 plaques were tested for the presence of stx-carrying phages. Stx genes could be demonstrated in 2% (stx(1)) and 16% (stx(2)) of the plaques. Stx-phages were eliminated with approximately the same efficiency in comparison with somatic coliphages during the waste water treatment process. CONCLUSIONS: Due to the low numbers of phages carrying the stx genes 1 and 2 in treated waste water and river water, the dilution and inactivation of host bacteria and the unsuitable conditions for the transduction of host organisms in aquatic environments, it is difficult to derive from the data the direct evidence for a public health problem. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show the quantitative occurrence of stx-carrying phages in waste and river water and confirm the frequent circulation of these viruses in the aquatic environment.

Root stimulated nitrogen removal: only a local effect or important for water treatment?

Plants in constructed wetlands serve as carriers for attached microbial growth. They mainly transfer oxygen and release exsudates to the root zone. In consequence of this an area around the roots, called the rhizosphere exists, in which bacteria are stimulated by root growth. The goals were to ascertain whether stimulating the microbial cenosis only has a local effect on the rhizoplane, and to establish the importance of this stimulation for the water purification process in the root zone. Observations were carried out in a laboratory batch reactor filled with sand and planted with reeds (Phragmites australis). A small section was separated with gauze to avoid root growth outside this zone. The reactor was incubated with an artificial waste water containing a high concentration of ammonium. Samples were taken at intervals of 10 mm away from the gauze. The chemical and physical conditions and enzyme activities in soil sections at different distances from the roots affecting the efficiency of nitrogen removal were characterized. An influence was detectable by several parameters up to a specific root distance. Indirect parameters such as the total bacterial number and the DNA amount seem to be affected up to a distance of 50 mm from the root whereas the oxygen amount and DOC are unaffected at a distance exceeding 20-30 mm. This is an initial indication that improved nitrogen removal is also possible in the wider root surroundings. In view of the average root-to-root distance of 35 mm, the root-influenced area could therefore be expanded to the whole rooted zone in a constructed wetland. The influence on bacteria by roots is not just a local effect but may also play an important role in the whole purification process.

[The EU Bathing Water Directive. Risk assessment and standards]. / Die Novelle der EU-Badegewässerrichtlinie. Aspekte der Risikobewertung bei der Grenzwertsetzung.

In an attempt to reduce the risk of infection in natural bathing waters the European Union is in the process of improving the Bathing Water Directive 76/160/EWG, which regulates the safety of such waters. The proposal contains several positive innovations which will improve the protection of the bathers: (1) health-related indicators, (2) harmonized detection methods, (3) requirements for active bathing water management, and (4) stricter standards for coastal waters. One of the most salient features of the current draft is the introduction of bacterial standards that are more stringent for coastal than for fresh waters. This decision on different standards seems unjustified: it was taken solely on the grounds that in two epidemiological studies-one carried out in coastal, the other in fresh waters-the maximum excess rate of gastroenteritis among bathers in coastal waters was higher than among bathers in fresh waters. However, it was not taken into account that the concentrations of bacterial indicators at which the gastroenteritis rate began to increase was nearly identical in both studies. The ratio between the standard concentrations of E. coli and intestinal enterococci in the draft was set at 2.5. This value does not correspond to the ratio found in German surface waters with low pollution levels, with ratios ranging from 2.7 to 4.0, and to the even higher ratios found in raw and treated sewage effluents. As a consequence in a majority of cases the non-compliance of bathing waters in Germany would be caused exclusively by a violation of E. coli standards. In assessing risks of infection it must also be taken into account that the adequacy of E. coli and intestinal enterococci for signaling the presence of viruses in water is far from optimal. The decay of viruses in water-estimated by the decay of bacteriophages-was found to be substantially slower than the die-off of indicator bacteria.

Detection of enteric viruses and bacterial indicators in German environmental waters.

A German mining lake and the supplying surface waters, which are located downstream of a sewage plant, were examined regarding their microbiological and virological quality. Between October 2002 and September 2003, specific PCR methods were used to determine the occurrence of enteric viruses in 123 water specimens drawn at different sites downstream of the waste water treatment plant and in 9 samples from the sewage plant influent. Detection rates in sewage plant effluents and surface water samples depended on sampling sites and were: 29-76% for enterovirus (EntV), 24-42% (astrovirus, AstV), 15-53% (norovirus, NV), 3-24% (rotavirus, RoV), 5-20% (hepatitis A virus, HAV) and 20% (adenovirus, AdV). AstV genome load of selected samples was between 3.7 x 10(3) to 1.2 x 10(8) genome equivalents per liter (gen.equ./l), depending on sampling location; NV average genome load ranged from 1.8 x 10(4) to 9.7 x 10(5) gen.equ./l. Cell culture methods showed that three out of 18 PCR positive samples contained infectious EntV. Even though microbiological parameters such as Escherichia coli, enterococci and coliphages indicated acceptable microbiological water quality, the virological data of this study suggest the possibility that surface waters may be a source for enteric viral infections.

Effect of chronic intermittent immobilization stress on Fos-like immunoreactivity in rat brain and adrenal medulla.

The present study examined the influence of short- and long-term chronic intermittent immobilization stress throughout the brain and on the adrenal medulla of intact rats using Fos-like immunoreactivity (Fos-LI) as a marker of cellular activation. The effect of adreno-medullectomy on the central nervous system (CNS) response to chronic immobilization stress was also examined. It was found that control unoperated, unstressed rats had no Fos-LI cells in the brain or in the adrenal medulla. In intact rats, neither short term (1 week) nor long term (4 weeks) chronic intermittent immobilization stress produced significant increases in Fos-LI in the CNS compared with control animals. However, marked increase in the number of Fos-LI cells was observed in the adrenal medulla of animals stressed for 4 weeks compared with control, unstressed animals or those stressed for 1 or 2 weeks. In adreno-medullectomised rats, 4 weeks, but not 1 week, chronic immobilization stress produced significant increases in numbers of Fos-LI neurons in the paraventricular hypothalamic and supraoptic nuclei and the medial amygdala compared with intact animals stressed for a similar period of time. It is concluded that long term stress produces chronic Fos-LI in the adrenal medulla and that adreno-medullectomy increases the Fos response of the PVN, supraoptic nucleus and medial amygdala to long term stress.

Stability of enhanced biological phosphorus removal and composition of polyphosphate granules.

The influence of varying Ca- and Mg-concentration of the influent wastewater on the enhanced biological phosphorus removal was investigated in an anaerobic-aerobic bench-scale plant. The artificial enhancement of the Mg-concentration in the influent from 15 to 24 mg l(-1) and 31 mg l(-1), respectively, caused a raise of the mean P-removal efficiency from 85 to 97%. The P-elimination was very stable in time. A chemical precipitation of magnesium ammonium phosphate could be excluded. The elemental composition of polyphosphate granules was investigated by electron microscopy and energy dispersive X-ray spectroscopy. The elements Ca, Mg and K were the principal metal components of polyphosphate granules. Concerning the metal composition, different types of granules could be distinguished. The quantitative ratios of Ca, Mg and K varied in dependence on the influent concentration of these metals. A relation between the Mg/Ca-ratio of the granules and the efficiency of enhanced biological phosphorus removal can be supposed.

Alcohol induces formation of morphine precursors in the striatum of rats.

Dopamine-derived alkaloids, the tetrahydroisoquinolines (TIQs), are suspected to play a role in the pathogenesis of alcoholism. The present study describes the alcohol induced formation of the S-enantiomer of Tetrahydropapaveroline and Norcoclaurine in the rat brain. These compounds are of special interest since both were found as being intermediates in the biosynthesis of morphine in the opium poppy. The concentration of both TIQs were determined in different brain regions of Wistar rats after 6 and 18 months alcohol consumption ad libitum. Gas chromatography/mass spectrometry was used for the detection and quantification of the enantiomers. Tetrahydropapaveroline and norcoclaurine were detected only in the striatum of those rats which had consumed alcohol for 18 month, neither in other brain regions nor in any brain regions of the controls or the short term (6 month) alcohol treated rats. These findings and the fact that those tetrahydroisoquinolines were only detected in the striatum of the alcohol fed rats and were present only as the S-enantiomers suggest that an alcohol induced biosynthetic pathway exists in the mammalian brain.

Effects of voluntary ethanol ingestion on the POMC gene expression in the rat pituitary and on the plasma beta-endorphin content.

Studies investigating the influence of chronic ethanol treatment on the beta-endorphin content and the proopiomelanocortin (POMC) gene expression in the rat pituitary revealed contradictory results. Because of this we decided to start a more complex study to investigate the effects of isolation stress, chronic ethanol treatment and voluntary ethanol consumption on the POMC mRNA level in the rat pituitary. The immediately prepared total RNA from rat pituitaries was used in hybridization experiments (Northern- and Dot-blots). The results suggest a correlation between the POMC gene expression and the different fashions of 'living conditions' tested. So the POMC gene expression in long-term alcohol-treated animals was decreased supporting the theory of beta-endorphin deficiency in alcoholism. More interestingly, data obtained from the group of voluntary ethanol consumption suggest an inverse correlation between total ethanol ingestion and POMC gene expression. This indicates the importance of the method of ethanol administration. Consistent with a decreased POMC gene expression in the pituitary during chronic ethanol treatment are previous studies showing a decrease in the plasma beta-endorphin content in such situations. Surprisingly, in the present study the plasma beta-endorphin levels measured by radioimmunoassay were only slightly decreased in chronically ethanol-treated rats. This may be due to dysregulatory effects of ethanol on post-translational processing, degradation and/or release of beta-endorphin.

Does a relationship exist between the quality of stress and the motivation to ingest alcohol?

Previous experimental findings on the relationship between emotional stress and motivation to ingest alcohol are contradictory. To obtain information about this relationship we tested the effects on alcohol consumption in rats subjected to two types of chronic unavoidable stressors, intermittent immobilization and social isolation, which differ in their influence on the functional state of the endogenous opioid system. To characterize the nature and magnitude of the stress induced by these stressors, we measured their effects on functional parameters which have a close relationship to the regulatory influence of endogenous opioid peptides (endogenous opioid dependence, pain sensitivity, blood pressure). Our investigations have shown that chronic intermittent immobilization, which induced development of endogenous opioid dependence, presumably due to activation of endogenous opioid systems, did not produce increased alcohol consumption. On the contrary, chronic social isolation, which did not induce development of endogenous opioid dependence, was followed by a significant increase in alcohol consumption. It is concluded that not all types of stress produce increased alcohol consumption, but that the effect on the endogenous opioid system may be a decisive factor in determining whether a stressor produces increased alcohol consumption.

Endogenous opioid dependence--a basic pathophysiological phenomenon of stress-induced and genetically fixed disturbances in adaptation--influence of substance P.

Disturbances in adaptive processes can be induced by chronic exposition to stress or can result from a genetical predisposition. Experimental data of chronically stressed Wistar rats and of spontaneously hypertensive rats (SHR) demonstrate a relation between a decreased level of substance P (SP) in adrenals, the existence of a dependence on endogenous opioid peptides and an increased regulatory level of blood pressure. The endogenous level of SP was determined by using a RIA. The dependence (physical) on endogenous opioid peptides was detected by using the method of "gut dependence". SP injection i.p. once a day for 4 d antagonized the dependence on endogenous opioid peptides and normalized the increased level of blood pressure in both animal models. Investigations on SHR had shown that the adaptive effect of SP on blood pressure and endogenous opioid dependence is bound to the premise of an acute stimulated endogenous opioid system at the moment of SP-application. Experimental findings suggest that different systems of opioid peptides take part in the etiopathogenesis of genetically predisposed hypertension of SHR and in stress-induced increase of blood pressure level of Wistar rats. The effect of SP on blood pressure and endogenous opioid dependence will be discussed as a result of the modulatory influence on the cholinergic-opioid-peptidergic interaction.

Stress-induced dependence on endogenous opioid peptides--a fundamental process in the pathophysiology of a disturbed adaptation--its influence by substance P.

Using previous findings of stress-induced disturbances in regulation of peripheral and central function within the general adaptation process and hints from the literature to a multiple participation of opioid peptides in adaptive processes, this paper represents experimental data which gives hints to a functional relation in the etiopathophysiology between the development of stress-induced dependence on endogenous opioid peptides and stress-induced disturbances in blood pressure regulation and to the adaptive effect of substance P within the peptidergic interaction with endogenous opioid peptides and its importance for the physiology or pathophysiology of adaptive processes.

Possible role of substance P on regulation of pituitary-adrenal axis.

The effects of chronic immobilization stress on the physiological responses of male rats were studied. The results indicate that the SP-like immunoreactivity (SPLIR) is diminished in the adrenals and pituitary after chronic stress. In vitro noradrenaline (NA) release from adrenals was increased. The i.p. administration of SP during the stress procedure normalized the increased NA release in vitro indicating that the catecholamine secretion may be influenced by SP. On the other hand, in demedullated animals the SPLIR in the pituitary was partly reduced and the blood pressure was increased. In such animals chronic stress resulted in an increase of SPLIR in the pituitary in comparison with nonstressed, demedullated animals, but was without effect on the blood pressure. It is concluded that exposure to SP and the resulting decrease of noradrenaline release may have a significant influence on the pituitary-adrenal responsiveness to stress.

Stress-induced changes of the noradrenergic transmitter release in adrenals and the influence of substance P.

The paper presents results about the role of Substance P (SP) in stressful situations, especially on the spontaneous and nicotine-evoked noradrenaline (3H-NA) outflow in adrenal gland slices. Chronic stress increases the spontaneous as well as the stimulated 3H-NA outflow. SP applied i.p. during stress exposure inhibits the stress-induced elevation of the spontaneous and stimulated 3H-NA outflow. Application of SP direct to the superfusion solution influences the stimulated 3H-NA outflow but it does not change the spontaneous 3H-NA outflow in adrenal gland slices of stressed rats. The data support the hypothesis that SP modulates the transmitter release in adrenals during stressful situations.

Influence of adrenal demedullation on stress induced alterations within the general adaptation behaviour of August-rats.

Based on new theoretical aspects of functional participation of adrenal glands in adaptation process, resulting from supplement of Selye's stress concept by integration of peptidergic systems, stress related adaptation behaviour of sham operated and demedullated August-rats, characterized by a high stress-sensitivity, were investigated. To characterize the stress related behaviour the following parameters were investigated: Number of rats surviving the exposure, alterations of body mass, occurrence of ulcers and hyperaemia in stomach, blood pressure behaviour, organ mass of adrenals, pituitaries and hypothalami in total, SPLIR in adrenals, pituitaries, hypothalami in total as well as in their special nuclei areas of N. ventromedialis and N. lateralis. The following conclusions can be drawn from experimental results: The important role of adrenal medulla in regulation of adaptive processes is given by the interaction between of its catecholamine system and its regulatory system of peptides (substance P, Opioids). Whereas demedullation itself makes visible alterations which are connected with the loss of adrenal-catecholamine system, additional stressor exposition makes visible alterations connected with the loss of adrenal-regulatory system. Investigations show differences in the sensitivity of reaction against the influence of stressors between catecholamine system (more sensitive) and regulatory system of adrenal medulla of August-rats. Demedullation has a different influence on alterations induced by mild manipulation stress (activation of catecholamine system) and by immobilization (activation of regulatory system). The influence of manipulation stress is inhibited and the influence of immobilization is increased by demedullation. Results of this investigations refer to the existence of feedback relations between the adrenal medulla and pituitary as well as between adrenal medulla and hypothalamus. Furthermore there are indications, that the adrenal medulla takes part in the regulation of extraadrenal sympathetic activity. The experimental data give furthermore a hint for the existence of a high stress sensitivity of August-rats primary caused by the CNS.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal influence of substance P on blood pressure and stress sensitivity of spontaneously hypertensive rats.

Substance P, administered i.p. to female spontaneously hypertensive rats in the last 6 d of gestation, diminishes the age dependent increase of blood pressure especially in male offspring and the additionally stress related blood pressure increase in both sexes.