Molecular quantitative analysis of human viruses in California stormwater.

Many human pathogenic viruses are transmitted via the oral-fecal route and water is one possible vector, representing a risk for public health. Sixty-one large-volume water samples from storm drains in California were processed by a two-step hollow fiber ultrafiltration procedure followed by molecular analysis for human enterovirus and adenovirus types. Each sample was spiked with a surrogate, the benign bacteriophage PP7. Both surrogate and human viruses were quantified by newly designed TaqMan PCR assays. Equations were developed that account for the main variables in the procedure: recovery of the ultrafiltration, efficiency of nucleic acid extraction, and effect of inhibitors on the amplification of viral targets. Adenovirus 40/41 was detected in one sample at 230 genomes per liter, and no other adenovirus or enterovirus types were found. Samples that resulted in nondetects are reported together with the corresponding sample-specific limit of detection (S(LOD)), a useful tool when estimating the public health risk associated with the contact or ingestion of water. Virus concentrations did not correlate with traditional viable indicator concentrations or any of the physicochemical parameters measured. In contrast, coliform concentrations were correlated with total suspended solids. To our knowledge, this is the first study where all factors known to influence limits of detection have been investigated and integrated into equations that are widely applicable to the quantification of viruses or other microbial targets by PCR.

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge.

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.

The effect of intermittent feeding on aerobic granule structure.

Self-immobilized biofilms, or aerobic granules without the addition of carrier material, have only been reported in one suspended growth system, the Sequencing Batch Reactor (SBR) with a very short fill time (dump fill). The SBR utilizes intermittent feeding which creates a period of high load followed by starvation (often referred to as feast-famine). In this experiment, three identical SBRs were operated with different feeding conditions to determine the role of feast-famine on granule formation. All three SBRs were operated with a total volumetric load of 2.4 kg/m3 x d. The 90 minute Fill phase was altered for each reactor, providing an increasing time of Aerated Fill. A dump fill condition was applied for one reactor, while the other two reactors were aerated for different times during Fill, resulting in a smaller COD load at the beginning of each React phase. Aerobic granules formed in all reactors, but the structural properties and content of filamentous organisms were clearly dependent on a high feast condition. Only the reactor with dump fill formed compact, stable granules. It is concluded that intermittent feeding associated with the SBR affects the selection and growth of filamentous organisms and has a critical role in granule structure and composition.

The SBR and its biofilm application potentials.

Twenty plus years of experience, innovation, and research in the field of biological wastewater treatment and biofilm applications lead to the conclusion that biofilms are in many cases more desirable in reactors than suspended activated sludge. Biofilm reactors can provide very long biomass residence times even when the hydraulic influent loading is low. This makes them particularly suitable when treatment requires slow growing organisms with poor biomass yield or when the wastewater concentration is too low to support growth of activated sludge flocs. Regardless of the settling characteristics of biological aggregates or the hydraulic influent loading the metabolic activity in the reactor can be maintained at a high level. This paper reviews the application of biofilms in sequencing batch reactor (SBR) systems to treat non-readily biodegradable substrates, volatile organic waste constituents, complex waste streams requiring co-metabolism, and particulate wastewaters. Recent research using the SBR to form aerobic granular sludge as a special application of biofilms is also discussed.

The influence of settling time on the formation of aerobic granules.

Aerobic granular sludge, without the addition of carrier material, has only been reported in one suspended growth system, the Sequencing Batch Reactor (SBR) operated with short fill and settling periods. Recent studies have demonstrated that extracellular polysaccharides increased with the formation of aerobic granules, and that the shear force may stimulate production of these polysaccharides. In the study described herein, two SBRs were operated with the same shear force (air flow rate 275 L h(-1)) and two different settling times (2 and 10 min). Only the reactor with 2 min settling formed completely granular sludge, although granules were present in both reactors. Community analysis using 16S rRNA PCR products and DGGE showed that the communities diverged quickly after reactor start-up. For samples taken at steady-state, the granular population was more stable and less diverse than the flocculent reactor. EPS extraction of samples using cation exchange resin yielded similar values for aerobic granular sludge and previously reported anaerobic granules. While differences in the protein and TOC content between the flocculent and granular reactors increased appreciably as the sludge became more granular, the protein to polysaccharide ratio was relatively constant. The experiment confirmed previous theories that short settling times in SBRs select for granular sludge. The settling time results in granular sludge having a higher EPS protein content and a less diverse but more stable population.

Biodegradation of 3-chlorophenol in a sequencing batch reactor.

The present paper shows the results obtained through a study on the biodegradation of 3-chlorophenol (3-CP) in a Sequencing Batch Reactor (SBR). To such a purpose a lab-scale SBR was fed a synthetic wastewater containing 3-CP and nutrients (nitrogen and phosphorus) diluted in tap water. The operating strategy, in terms of both the duration of either the cycle or the react phase, was changed throughout the experimental activity in order to find out the optimal one allowing to ensure constant and high removal efficiency despite the increasing 3-chlorophenol concentration in the feed. Biomass collected from a full-scale continuous flow activated sludge facility treating domestic wastewater was used as seed, after being acclimated to 3-CP by means of several batch tests. The results showed that a periodically operated activated sludge system can be successfully used for the biodegradation of chlorophenol compounds, after the needed members of the microbiological consortium are selected and enriched.

Frozen deglycerolyzed blood prevents transfusion-acquired cytomegalovirus infections in neonates.

Frozen deglycerolyzed blood (FDB) was used for routine transfusions to 63 low birth weight newborns (less than or equal to 1500 g) over a 12-month period in an effort to decrease transfusion-acquired cytomegalovirus (CMV) infections. Nine of the 63 infants also received nonfrozen blood products (platelets, liquid blood). Seventy-two percent of the donor blood units were CMV-seropositive. Urine cultures and serum titers for anti-CMV antibody (immunoglobulins G and M) were obtained prior to the initial transfusion and sequentially throughout the study. No infant (0 of 54) who received only FDB acquired CMV, whereas 3 of 9 infants (33%) who received non-frozen blood and FDB acquired CMV, as evidenced by CMV viruria and/or a 4-fold rise in immunoglobulin G anti-CMV antibody titers. These results suggest that transfusions with frozen deglycerolyzed blood decrease the risk in low birth weight infants of acquiring CMV, regardless of the CMV serologic status of the donor.

Surgical experience with carcinoma of the colon and rectum.

From September 1925 through December 1978 at Vanderbilt University Hospital, 1,279 patients with adenocarcinoma of the colon and rectum underwent operations. Reports of this cumulative series have been published previously; the last report in 1970 included 1,022 patients. The current report examines the progress made in our recognition and management of colorectal cancer. During this 54-year period, there has been a relative decrease in the incidence of carcinoma of the rectum and a relative increase in carcinomas elsewhere in large bowel. Resectability rate has steadily increased, culminating in a rate of 98.4% in the recent period (1969-1978). The operative mortality rate fell to 4.3% (1956-1960) but has shown a slight rise to 5.4% in the recent period (1969-1978). This reflects the increased number of patients in the eighth to the tenth decades of life. Five-year survival rates in 99% of 1,218 patients were computed. Absolute five-year survival for the recent period was 43.7%, compared with 17% for the initial period. Five-year survival rates for patients in the recent decade with Dukes A, B, and C lesions were 67%, 58.6%, and 33.3%, respectively. Comparison of survival data in the last two decades shows little improvement. However, in the last 20 years, 78 to 80% of patients had more advanced lesions.

Sarcoma of the gastrointestinal tract: a review of 40 cases.

In a ten-year update of the Vanderbilt University Medical Center experience, cases of malignant lymphomas (20) and soft tissue sarcomas (20) of the gastrointestinal tract were reviewed. The stomach was the most common site (63%). The three most common presenting symptoms were abdominal pain, weight loss, and hemorrhage. The average duration of symptoms prior to seeking medical consultation was 14 months. The five-year survival was 50% for soft tissue sarcomas and 60% for malignant lymphomas. It is suggested that delay is establishing a diagnosis be a consideration in recommending adjuvant therapy.

Evidence for two types of P-700 in membrane fragments from a blue-green alga.

The mathematical analysis described in the preceding paper (Biochim. Biophys. Acta (1977) 460, 65-75), in which the steady-state photooxidation of P-700 was compared with overall electron flux in Photosystem I chloroplast fragments, was applied to membrane fragments from the blue-gree alga Nostoc muscorum (Strain 7119) noted for their high activity of both Photosystem I and Photosystem II. The same analysis, which gave good agreement between the photooxidation of P-700 and the overall light-induced electron flux (measured as NADP+ reduction) in Photosystem I chloroplast fragments, revealed in the algal membrane fragments two P-700 components: one responding to high light intensity (P-700 HI), the photooxidation of which was in good agreement with the overall electron flux (measured as NADP+ reduction by reduced 2,6-dichlorophenolindophenol), and the other component responding to low light intensity (P-700 LI), the photooxidation of which was not correlated with the reduction of NADP+ by reduced 2,6-dichlorophenolindophenol.

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments.

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga.

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP+. Magnesium ions were required not only for light-induced electron transport from water to NADP+ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg2+ lost the capacity to photoreduce NADP+ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP+ by DCIPH2 was independent of Mg2+ (or Cl-) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentration of MgGl2 produced a strong inhibition of NADP+ photoreduction with DCIPH2 without significantly affecting the rate of NADP+ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP+ with different electron donors are discussed.