Ultrastructure of a bio-electrolytic methanogenic/methanotrophic granular biofilm for the complete degradation of tetrachloroethylene in contaminated groundwater.

The electrolytical methanogenic/methanotrophic coupling (eMaMoC) process was tested in a laboratory-scale single-stage reactor for the treatment of tetrachloroethene (PCE)-contaminated waters. A water electrolysis cell was placed directly in the effluent recirculation loop for the supply of both O2 and H2 to the system: H2 serving as the electron donor for both carbonate reduction into CH4 and reductive dechlorination. The concurrent presence of O2 and CH4 could be used by the methanotrophs for co-metabolically oxidising the chlorinated intermediates left over by the anaerobic transformation of PCE. At a PCE inlet of 33-52 microM and a hydraulic residence time (HRT) of 5.6 days, PCE reductive dechlorination to dichloroethene (DCE) was over 95% with a maximum DCE mineralisation of 83%. Fluorescence in situ hybridisation with 16S rRNA probes related to type I and type II methanotrophic bacteria were utilised to localise the methanotrophic communities in the anaerobic/aerobic granules. It evidenced that with operational time, along with increasing oxygenation rate, methanotrophic communities were specifically colonising onto the outermost layer of the anaerobic/aerobic granule.

Comparison of the microbial population dynamics and phylogenetic characterization of a CANOXIS reactor and a UASB reactor degrading trichloroethene.

AIMS: To understand the microbial ecology underlying trichloethene (TCE) degradation in a coupled anaerobic/aerobic single stage (CANOXIS) reactor oxygenated with hydrogen peroxide (H2O2) and in an upflow anaerobic sludge bed (UASB) reactor. METHODS AND RESULTS: The molecular study of the microbial population dynamics and a phylogenetic characterization were conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). In both reactors, TCE had a toxic effect on two uncultured bacterial populations whereas oxygen favoured the growth of aerobic species belonging to Rhizobiaceae and Dechloromonas. No methanotrophic bacteria were detected when targeting 16S rRNA gene with universal primers. Alternatively, pmo gene encoding the particulate methane monooxygenase of Methylomonas sp. LW21 could be detected in the coupled reactor when H2O2 was supplied at 0.7 g O2 l day(-1). CONCLUSIONS: Methylomonas sp. LW21 that could be responsible for the aerobic degradation of the TCE by-products is not among the predominant bacterial populations in the coupled reactor. It seems to have been outcompeted by heterotrophic bacteria (Rhizobiaceae and Dechloromonas sp.) for oxygen. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained show the limitations of the coupled reactor examined in this study. Further investigations should focus on the operating conditions of this reactor in order to favour the growth of the methanotrophs.

Strategies for augmenting the pentachlorophenol degradation potential of UASB anaerobic granules.

Anaerobic degradation of pentachlorophenol (PCP) is an example of a process that may benefit from enrichment or bioaugmentation. In one approach, enrichment acceleration was attempted by applying an on-line control-based selective stress strategy to a native anaerobic upflow sludge bed (UASB) system; this strategy linked PCP loading rate to methane production. As a result, the reactor biomass potential for PCP complete dechlorination reached a rate of 4 mg g(-1) volatile suspended solid (VSS) day(-1) within a period of 120 days. In another approach, a pure culture, Desulfitobacterium frappieri PCP-1, a strictly anaerobic Gram-positive bacterium, was used to augment the granular biomass of the UASB reactor. This also resulted in a specific degradation rate of 4 mg PCPg(-1) VSS day(-1); however, this potential was attained within 56 days. Fluorescent in situ hybridization (FISH) showed that the PCP-1 strain was able to rapidly attach to the granule and densely colonize the outer biofilm layer.

A social relations variance partitioning of dyadic behavior.

The authors investigate the relative importance of actor and interaction partner as determinants of dyadic behavior. Using the social relations model (D. A. Kenny, 1994a; D. A. Kenny & L. La Voie, 1984), the authors estimate the variance attributable to each determinant plus the reciprocity of behavioral responses from 7 studies. The authors find evidence for moderate behavioral consistency in a person's behavior across interaction partners, little or no evidence that people consistently engender the same behavioral response from others, and preliminary evidence of unique responding to particular partners. They also consider several methodological issues concerning behavioral measurement as well as the implications of the results for the study of accuracy.

Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Desulfitobacterium frappieri strain PCP-1.

Anaerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil from a wood-treating industrial site was studied in soil slurry microcosms inoculated with a PCP-degrading methanogenic consortium. When the microcosms containing 10%-40% (w/v) soil were inoculated with the consortium, more than 90% of the PCP was removed in less than 30 days at 29 degrees C. Less-chlorinated phenols, mainly 3-chlorophenol were slowly degraded and accumulated in the cultures. Addition of glucose and sodium formate to the microcosms was not necessary, suggesting that the organic compounds in the soil can sustain the dechlorinating activity. Inoculation of Desulfitobacterium frappieri strain PCP-1 along with a 3-chlorophenol-degrading consortium in the microcosms also resulted in the rapid dechlorination of PCP and the slow degradation of 3-chlorophenol. Competitive polymerase chain reaction experiments showed that PCP-1 was present at the same level throughout the 21-day biotreatment. D. frappieri, strain PCP-1, inoculated into the soil microcosms, was able to remove PCP from soil containing up to 200 mg PCP/kg soil. However, reinoculation of the strain was necessary to achieve more than 95% PCP removal with a concentration of 300 mg and 500 mg PCP/kg soil. These results demonstrate that D. frappieri strain PCP-1 can be used effectively to dechlorinate PCP to 3-chlorophenol in contaminated soils.

Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction.

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 microgram soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time.

Meta-accuracy among acquainted individuals: a social relations analysis of interpersonal perception and metaperception.

Meta-accuracy, knowing how others view the self, was examined using the Social Relations Model. Fifteen groups of 4-6 acquainted individuals gave self-ratings, perceptions of other group members, and estimated others' perceptions of self (metaperceptions) on the Big Five and Interesting. Individuals also rated liking and metaperceptions of liking. Trait perceptions were consensual, and self-other agreement emerged for most traits. Affect judgments were entirely relational; individuals differentiated among targets. Trait metaperceptions were dominated by perceiver variance. Individuals differed in the impression they believed others generally held about them. Affect metaperceptions, however, were relational in nature. Correlations between perceptions and metaperceptions assessed 2 types of meta-accuracy. Generalised meta-accuracy was obtained for some trait ratings. Affect judgments revealed significant dyadic meta-accuracy.

Constructing success and failure: age differences in perceptions and explanations of success and failure.

Age differences in attributions for self-reported successes and failures in both important and daily situations were examined. Sixty-one young adults (M age = 19.2), twenty-one middle-aged adults (M age = 45), and fifteen older adults (M age = 71.4) gave attributions and affects for self-chosen situations, which were classified as social or nonsocial. The attributions and affects were coded according to Blank's scheme with attributions dichotomized into internal and external, and stable and unstable. Middle-age and older adults were more likely than young adults to attribute failure to external causes and to describe more social than nonsocial situations. Consistent age differences in attributional stability were not found nor were there age differences in attributional internality for success outcomes.

Vascular endothelial cell proliferation in culture and the influence of flow.

The influence of laminar shear stress on cell proliferation was investigated for subconfluent bovine aortic endothelial cell monolayers seeded on either glass or Thermanox. The effect of both steady and pulsatile shear stress was studied. For bovine aortic endothelial cells on Thermanox exposed to steady flow, shear stress levels greater than 15 dyne/cm2 resulted in a dose-related reduction in the rate of cell proliferation. At 90 dyne/cm2, the rate of proliferation was virtually totally arrested for 48 h, but then resumed. Pulsatile shear stress produced an exaggeration of the effect observed in response to steady shear stress. Bovine aortic endothelial cells seeded on glass, exhibited a similar but more sensitive response, with a significant reduction in growth rate observed after 24 h at shear stress levels greater than 5 dyne/cm2 and a near cessation of proliferation at 13 dyne/cm2.

The study of rheological effects on vascular endothelial cells in culture.

A number of cell culture studies have been reported on the influence of shear stress on vascular endothelial cells. Although through such studies much has been learned about the effect of an endothelial cell's hydrodynamic environment on its structure and function, the reports indicate significant differences in methodology. Using cell shape as an indicator of differences that might result from differing methodologies, an investigation of the influence of selected variables has been carried out. The results presented indicate that not only are such variables as the level of shear stress and the duration of exposure important, but also substrate, media composition, characteristics of the cell itself, and the nature of the flow, e.g. whether it is steady state or pulsatile.

The application of a homogeneous half-space model in the analysis of endothelial cell micropipette measurements.

Experimental studies have shown that endothelial cells which have been exposed to shear stress maintain a flattened and elongated shape after detachment. Their mechanical properties, which are studied using the micropipette experiments, are influenced by the level as well as the duration of the shear stress. In the present paper, we analyze these mechanical properties with the aid of two mathematical models suggested by the micropipette technique and by the geometry peculiar to these cells in their detached post-exposure state. The two models differ in their treatment of the contact zone between the cell and the micropipette. The main results are expressions for an effective Young's modulus for the cells, which are used in conjunction with the micropipette data to determine an effective Young's modulus for bovine endothelial cells, and to discuss the dependence of this modulus upon exposure to shear stress.

Epicardial coronary blood flow including the presence of stenoses and aorto-coronary bypasses--II: Experimental comparison and parametric investigations.

This article is the second in a series which presents a computer model of the left coronary arteries. The first article discussed the geometry, the governing equations, and the numerical method employed. This paper details an acute canine experiment used to validate the approach as well as the systematic investigation of several important parameters governing the left coronary circulation. These parameters include peripheral resistance, wall properties, and altered geometric properties through various stenosis/bypass configurations. With appropriate selection of parameters, the model reproduces an in vivo waveform very closely. The model also predicts many clinical phenomena, such as the "critical" value of stenosis, the dramatic reduction in flow through a stenosis when bypassed, and the restorative effect of the bypass upon flow to the distal bed. The model also is used to show that the autonomic state of the animal profoundly affects the influence of various factors, e.g., the critical value of a stenosis is much higher under resting conditions than under hyperemic conditions.

Micropipette aspiration of cultured bovine aortic endothelial cells exposed to shear stress.

The mechanical properties of cultured bovine aortic endothelial cells exposed to a fluid-imposed shear stress were studied using the micropipette technique. The cells, which were attached to a Thermanox plastic substrate, were exposed to a specific steady shear stress of either 10, 30, or 85 dynes/cm2 and for a duration ranging from 0.5 to 24 hours. Morphological changes in shape and orientation were observed, and following each experiment, the mechanical properties were measured using the micropipette aspiration technique applied to cells detached from the substrate. Fluorescent microscopy was carried out to observe cytoskeletal F-actin filaments stained with rhodamine phalloidin. During exposure to shear, the en face shape of the endothelial cells on the substrate became more elongated and their long axis became oriented to the direction of flow. There was also an alteration in the F-actin filaments. These changes were dependent on both the level of shear stress and the duration of exposure. After detachment, the cells exposed to shear maintained their deformed shape. This is in contrast to cells in a static, no-flow environment which became spherical in shape upon detachment. Cells exposed to shear stress demonstrated a mechanical stiffness significantly greater than that of control cells, which was dependent on both the level of shear stress and the duration of exposure. Furthermore, it appears that the influence of shear stress on endothelial cell mechanical stiffness may be related to alterations in cytoskeletal structure.

An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells.

The mechanical properties of endothelial cells were measured using the micropipette technique. The cells employed were collected from bovine aortic endothelium and cultured in our laboratory. Endothelial cells from confluent monolayers under no-flow conditions were detached from their substrate by trypsin or by a mechanical method and suspended in modified Dulbecco medium (MDM). In the micropipette technique, a part of the cell is aspirated into the tip of the micropipette under a microscope, and the deformation measured from a photograph. In this study, the data obtained were analyzed using a model where the cytoskeletal elements, which are considered to be the primary stress bearing components, are assumed to reside in a submembranous, cortical layer. Detached cells were found to have almost homogeneous mechanical properties based on measurements from different regions of the surface of a single cell. However, a hysteresis loop was observed in the relation between pressure and cell deformation during the loading and unloading processes. The calculated elastic shear moduli obtained for the trypsin-detached cells were as much as 10-20 times larger than those of a red blood cell. Mechanically-detached cells had moduli approximately twice that of the trypsin detached cells. Passage time, i.e., cell culture age, had no influence on the mechanical properties of the trypsin-detached cells, but did have an effect on the mechanically-detached cells, with both the younger and older cells being somewhat stiffer.

Correlation of endothelial cell shape and wall shear stress in a stenosed dog aorta.

The pattern of endothelial geometry at various locations along stenosed dog aortas was examined. This was done to test the hypotheses that the shape of an endothelial cell is related to the local wall shear stress associated with the flowing blood and that alterations in hemodynamics, produced by vascular geometrical changes, influence endothelial cell geometry. Aortic stenosis with a reduction of 71% of the cross-sectional area was produced. The animals were sacrificed 12 weeks later, and the endothelial cell geometry and orientation were studied using the vascular casting technique and a computerized analysis to determine cell area and shape index. The regions of the stenosis examined were those known to experience different hemodynamic conditions. The value of the shape index was found to fall rapidly in the convergent region of the stenosis and to increase suddenly in the divergent region, eventually returning to the prestenotic value at a more distal site. Using a model of a stenosis made from a vascular cast, laser Doppler anemometry was applied to measure velocity profiles and to estimate the local wall shear stress in a stenosed aorta. It is shown that the shape index distribution along these stenosed vessels may be correlated with the level of wall shear stress, with more elongated cells occurring in regions of higher shear stress.

The elongation and orientation of cultured endothelial cells in response to shear stress.

Vascular endothelial cells appear to be aligned with the flow in the immediate vicinity of the arterial wall and have a shape which is more ellipsoidal in regions of high shear and more polygonal in regions of low shear stress. In order to study quantitatively the nature of this response, bovine aortic endothelial cells grown on Thermanox plastic coverslips were exposed to shear stress levels of 10, 30, and 85 dynes/cm2 for periods up to 24 hr using a parallel plate flow chamber. A computer-based analysis system was used to quantify the degree of cell elongation with respect to the change in cell angle of orientation and with time. The results show that (i) endothelial cells orient with the flow direction under the influence of shear stress, (ii) the time required for cell alignment with flow direction is somewhat longer than that required for cell elongation, (iii) there is a strong correlation between the degree of alignment and endothelial cell shape, and (iv) endothelial cells become more elongated when exposed to higher shear stresses.

Does an unfavorable metabolic environment for red cells develop within the cat spleen when abnormal cells become trapped?

Intrasplenic pH, blood gas tensions, and glucose concentration were deduced from measurements of blood drained from cat spleen during contraction with the inflow occluded. During this procedure the hematocrit of the outflow rises gradually from 35% to 40% (arterial) to 75% to 80%, the last fraction representing a pure sample of blood from the splenic pulp. In normoxic animals no evidence was found of an unfavorable metabolic environment for red cells within the spleen on account of low pH, low O2 tension, and substrate deprivation, as is generally believed. However, red cell flow through the red pulp can be impeded rheologically after sequestration of 10(9) heat-treated (HT) autologous red cells, and we have tested the hypothesis that under these conditions the availability of O2 and glucose might be reduced and a decline in pH might occur. One hour after injection of the HT cells into the splenic artery, splenic contraction was induced with the arterial inflow occluded; the blood expelled from the splenic vein was collected anaerobically as successive 1 ml fractions. Values of pH, O2 tension, and glucose concentration in the final samples expelled were not significantly different from those in corresponding samples from control spleens. Thus, even when stasis of 50% of intrasplenic red cells occurs, caused by the sequestration of 10(9) abnormal cells, no hostile metabolic environment develops within the red pulp. Presumably the residual plasma flow through the pulp is sufficient to maintain a normal metabolic environment.

Intramyocardial pressure and distribution of coronary blood flow during systole and diastole in the horse.

Transmural myocardial blood flow was measured with microspheres in systole and in diastole, along with intramyocardial pressure, in seven anaesthetised horses. Intramyocardial pressures were measured with a miniature manometer implanted in the tip of a 16-gauge needle. Peak systolic intramyocardial pressure decreased from subendocardium to subepicardium and never exceeded intraventricular pressure. Systolic blood flow decreased from epicardium to endocardium where it did not differ from zero. Diastolic blood flow increased from epicardium to subendocardium, but then decreased in the most endocardial layer to a level not different from the immediate subepicardial layer. The horse was a useful model for studying these parameters because the ventricular walls are so thick and the heart rate is so slow that injections may be made during various phases of the cardiac cycle. These results of transmural myocardial blood flow and intramyocardial pressure measured in the same animal are identical with those of others, except for the reduction in subendocardial blood flow compared with the layers just epicardial to that.