Composting of anaerobic sludge: an economically feasible element of a sustainable sewage sludge management.

An investigation into the feasibility of anaerobic sludge composting, as a sustainable treatment of sewage sludge management, was carried out under actual Slovenian environmental conditions. In order to demonstrate successful composting, five pilot plant experiments were performed during the summer and winter conditions. The first three experiments were performed with pile aeration, while experiments 4 and 5 were carried out by pile turning. Anaerobic sludge to bulking agent ratios were set at 1-6.4:1. The composting was successful and thermophilic temperature being achieved in all cases. In winter conditions, the composting process was prolonged; and low ambient temperatures had a significant impact in pile turning experiments. During winter, a temperature drop of 30 °C during turning of the material doubled the necessary time for an adequate composting process. Five scenarios were considered within an economic feasibility study and in the most favourable scenario, where 60% of compost was commercialised and 40% was used as landfill cover. The payback period in this scenario was 2.9 years. The study of compost quality showed that it can be used in variety of civil engineering applications, especially as a landfill cover and for recultivation of degraded areas.

Rate, extent and concentration dependence of histamine-evoked Weibel-Palade body exocytosis determined from individual fusion events in human endothelial cells.

The rate, concentration dependence and extent of histamine-evoked Weibel-Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45+/-0.26 (s.d., n=144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0+/-4.42 s (n=9 cells) at 0.3 microM histamine and 1.57+/-0.21 s (n=15 cells) at 100 microM histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20+/-0.16 WPB s(-1) (n=9) at 0.3 microM and 3.66+/-0.45 WPB s(-1) at 100 microM histamine (n=15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 microM), for which there was a delay between calcium elevation and secretion of 1.67+/-0.24 s (n=6), and a peak fusion rate of approximately 10 WPB s(-1).

Two stage thermophilic anaerobic-aerobic mineralization-stabilization of excess activated sludge.

At the National Institute of Chemistry, Ljubljana, Slovenia, new procedure for excess activated sludge was developed and patented. The main principle of the sludge mineralization is two-step process--the first is anaerobic, and the second step is aerobic. In the first step maximal biogas production is acquired in the second step at smaller VSS the optimal VSS and COD is achieved. Anaerobic, aerobic, and combined sludge mineralization was studied. The next combinations of different successive anaerobic and aerobic mineralization have been studied: 3 + 3 (3 days of anaerobic + 3 days of aerobic digestion), 3 + 6, 5 + 5, 3 + 12, and 10 + 10 days, respectively. The best combination considering biogas production, VSS and COD reduction have been gained for the combination of 3 + 6 days, and 3 + 12 days. At 3 + 6 days digestion about 49% of VSS, and about 51% of COD reduction was gained, at 3 + 12 days about 62% of VSS, and about 57% of COD reduction was achieved. For the optimal system operation, economics, biogas production, and necessary VSS and COD reduction have to be considered.

Differential exocytosis from human endothelial cells evoked by high intracellular Ca(2+) concentration.

Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high-resolution membrane capacitance (C(m)) measurements. The total whole-cell C(m) and the amplitudes and times of discrete femtoFarad (fF)-sized C(m) steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration [Ca(2+)](i) was elevated by intracellular photolysis of calcium-DM-nitrophen to evoke secretion and monitored with the low-affinity Ca(2+) indicator furaptra. Sustained elevation of [Ca(2+)](i) to > 20 microM evoked large, slow increases in C(m) of up to 5 pF in 1-2 min. Exocytotic and endocytotic steps of amplitude 0.5-110 fF were resolved, and accounted on average for ~33 % of the total C(m) change. A prominent component of C(m) steps of 2.5-9.0 fF was seen and could be attributed to exocytosis of von-Willebrand-factor-containing Weibel-Palade bodies (WPb), based on the near-identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5-9.0 fF C(m) steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after [Ca(2+)](i) elevation, whereas total C(m) increased immediately due to the secretion of small, non-WPb granules. The results show that following a large increase of [Ca(2+)](i), corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9-110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein.

1. Vesicular secretion from single human umbilical vein endothelial cells (HUVECs) was monitored by changes in membrane capacitance (Cm). Secretion was evoked by dialysis with strongly buffered intracellular free Ca2+ concentrations ([Ca2+]i), flash photolysis of Ca2+-loaded DM-nitrophen or caged InsP3, or by thrombin. [Ca2+]i was monitored spectrofluorimetrically with furaptra. The results show that a large, slowly rising component of vesicular secretion requires prolonged exposure to high [Ca2+]i. 2. Cm increased during intracellular perfusion with [Ca2+] buffered in the range 1.0-20 microM. Changes in Cm comprised an initial slowly rising small component of 0.1-0.5 pF followed by a faster rising larger component of up to approximately 7 pF, seen when [Ca2+]i > 2 microM and which was maximal at 10-20 microM Ca2+. 3. Thrombin evoked rapid initial elevations of [Ca2+]i to a peak of 7.1 +/- 1.5 microM (mean +/- s.e. m., n = 5) that declined within approximately 20-30 s with thrombin present either to resting levels or to a maintained elevated level of 2.0 +/- 0.7 microM (mean +/- s.e.m., range 1.0-3.6 microM, n = 3). Transient [Ca2+]i rises were associated with small, slowly rising increases in Cm of 0.1-0.2 pF, that recovered to pre-application levels over 2-3 min. Maintained elevations of [Ca2+]i caused larger, faster-rising sustained increases in Cm to 1.14 +/- 0.12 pF (mean +/- s.e.m., n = 3). Separate specific enzyme-linked immunosorbent assay (ELISA) showed that 1.0 U ml-1 thrombin produced secretion of von Willebrand factor in HUVEC cultures. 4. Short-lived [Ca2+]i elevations with a peak of 3-25 microM and a duration of approximately 20 s generated by flash photolysis of caged InsP3 or DM-nitrophen produced either no net change in Cm, or small slow increases of approximately 0.1-0.6 pF at up to 5 fF s-1 that recovered to pre-flash levels over 2-3 min. 5. Maintained elevations of [Ca2+]i in the range 1-28 microM produced by flash photolysis of DM-nitrophen caused large increases in Cm, up to approximately 4 pF, corresponding to approximately 25-30 % of the initial cell Cm. The maximum rate of change of Cm was up to 50 fF s-1 at steady [Ca2+] up to 20 microM; Cm recovered towards pre-flash levels only when [Ca2+] had declined.

Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance.

We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels.

The separation of exocytosis from endocytosis in rat melanotroph membrane capacitance records.

1. Using the patch-clamp technique, we have monitored the secretory activity of single rat melanotrophs. Changes in membrane capacitance (Cm) were measured to detect small discrete femtofarad steps. These are believed to be due to interactions between single secretory organelles (granules) and plasmalemma. 2. A new approach was introduced to measure the amplitude of discrete steps in Cm. Records of Cm were converted into time derivatives, where discrete steps appeared as transients. A transient due to a 2 fF discrete step in Cm was easily distinguished from random noise, since the probability of such a transient being due to random noise was less than 0.01. To distinguish apparent steps from noise the computer-based analysis employed a threshold of 3 times the standard deviation of the noise time derivative (dCm/dt). A phase diagram was created by plotting dCm/dt versus Cm, from which the magnitude and direction of transients were determined. Transients due to 2 fF steps (equivalent to a signal-to-noise ratio of 1) were detected with a reliability of 100%, whereas steps of 1 fF were detected with a reliability of more than 60%. The amplitude of false steps detected by the program was less than 1 fF, and the frequency of false detections of 0.075 S-1 was equal for exocytotic and endocytotic events. 3. Electron microscopy was used to measure secretory organelle size and an immunogold technique was used to label the electron micrographs with an anti-adrenocorticotrophin (ACTH) antibody. Secretory organelles in cultured and non-cultured cells were of similar diameter. All sizes of secretory granules appear to contain ACTH, since secretory organelles of similar diameter stained positively with the anti-ACTH antibodies. 4. Small discrete steps in Cm, recorded with the whole-cell configuration and loosely buffered cytosolic calcium, were similar to the estimated Cm of secretory organelles from morphological data. Thus, measured discrete steps in Cm reflect interactions between single organelle size and plasma membrane. Exocytotic and endocytotic steps were found to be of similar size. 5. To separate exocytosis from endocytosis in Cm records, we assumed that the rates of exocytosis and endocytosis were related to the respective frequencies of discrete steps in Cm. A relationship between the frequency of exocytotic, but not endocytotic events, and the rate of change in Cm was observed. Thus, under our experimental conditions, an increase in Cm could be explained by an increased rate of exocytosis in rat melanotrophs.