It has previously been shown that low p0₂ stimulated the respiration of facultative bacteria suggesting that precise control of dissolved oxygen in waste treatment systems might be an effective method of increasing carbon dioxide production at the expense of cell yield. In continuous cultures of glucose limited Escherichia coli B, controlled at less than 8mmHg, we have demonstrated a 57% increase in carbon dioxide production at the expense of both cell yield and supernatant carbon. Batch cultures of Pseudomonas aeruginosa 9027 showed an even greater loss of efficiency at low oxygen tensions. Studies with mixed populations (batch culture) showed some evidence of yield reduction (in spite of inherent difficulties with floculation and natural selection). However, the reduction was considered to be too small to warrant the practical application of dissolved oxygen control in waste treatment systems. While completely anaerobic E. coli B cells appear to be uncoupled by aeration, alternating a continuous culture between anaerobic and aerobic growth did not similarily affect the cells - evidently E. coli B requires a substantial amount of time (over 4 hours) to fully adapt to anaerobic conditions. In any case, unlike E. coli B, the growth of stable anaerobic mixed populations did not become "uncoupled" when aerated - indicating that this technique would not be suitable in waste systems. Studies with an ultra-violet irradiated continuous culture of E. coli B showed considerable increases in carbon dioxide production at the expense of cell yield. In addition, the process of floculation was greatly enhanced. The practical possibilities of implementation in waste treatment systems, warrent further investigation of the effects of u.v. irradiation on bacterial growth. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Macromolecules such as proteins and polysaccharides can constitute a significant portion of dissolved organic carbon (DOC) in wastewater, but limited information is available on how these compounds are degraded in biological wastewater treatment systems. Bacteria cannot assimilate intact macromolecules but must first hydrolyze them to monomers or small oligomers. To better understand the mechanism of macromolecule degradation in wastewater treatment systems this study investigates two important questions of macromolecule metabolism. First, does hydrolysis occur in close proximity to the cell or are the hydrolytic enzymes released into bulk solution, and second, if hydrolysis is cell-associated, are hydrolyzed fragments directly assimilated into the cell or are they released back into solution? Fluorescent model substrate analogs were used to determine the location of leucine aminopeptidase and a-glucosidase activity in wastewater inoculated biofilm and suspended cultures and in trickling filter effluent. In biofilm and suspended cultures at least 93% of hydrolytic activity was cell-associated. In trickling filter effluent hydrolysis rates were at least five times higher in contact with cells and sloughed biofilm pieces than in cell-free solution. To determine whether hydrolytic fragments were directly assimilated or released into solution molecular size distributions in bulk solution were monitored using membrane ultrafiltration techniques during the degradation of the model protein, bovine serum albumin, and the model polysaccharides, dextran and dextrin, in batch and continuous suspended cultures, and in fixed-film reactor systems. Measurable amounts of macromolecule hydrolytic fragments accumulated in all reactor configurations, substrates and inocula tested. Relatively larger concentrations of hydrolytic fragments accumulated in pure culture than in wastewater culture inoculated reactors. These results support a generalized model for macromolecule degradation by bacteria that features cellbound hydrolysis of protein and polysaccharides and the subsequent release of hydrolytic fragments back into bulk solution. This hydrolysis and release is repeated until fragments are small enough ( < 1000 amu) to be assimilated by cells. A separate, but related, part of this study adapted pre-column orthophthaldialdehyde derivatization, reverse phase high performance liquid chromatographic separation and fluorometric detection to measure free and combined amino acids in unconcentrated wastewaters and to assess their treatability in wastewater treatment systems.

The purpose of this study was to extend our understanding of the ecological relationships of P. aeruginosa by investigating the differences or similarities between the strains of this organism found in sewage and those found as pathogens in human infections. This research was approached by comparing the serological types of P. aeruginosa isolated from sewage contaminated waters in Argentina (South America) to those isolated from sewage contaminated waters in Texas. They were typed with sera obtained using P. aeruginosa isolated from human infections. The data obtained revealed that bacteria isolated from sewage in Texas and from soil and water in Argentina are antigenically similar to those isolated from human infections.

Caulobacters are gram-negative bacteria that have a biphasic life cycle consisting of a swarmer and a stalked stage. As a result they have elicited interest as a simple developmental model. Less attention has focussed on their role in the environment, although they have been found in almost every aquatic environment as well as in many soils. Caulobacters are often described as oligotrophic bacteria because of their prevalence in pristine waters but have now been isolated from the relatively nutrient-rich wastewater environment. In order to learn more about this population some basic characterization was carried out and an assay system to determine their prevalence in sewage plants was designed. Most of the organisms isolated from sewage treatment facilities had similar gross morphological features, but differed in holdfast composition, total protein profile, antibiotic resistance and restriction fragment length polymorphism, thereby indicating a greater diversity than originally assumed. Most of the organisms hybridized with flagellin and surface array genes that had previously been cloned, and only one of 155 non-Caulobacter sewage isolates hybridized with the flagellin gene probe; consequently these were used in a DNA-based enumeration strategy. DNA was isolated directly from sewage and probed with the flagellin and the surface array gene probes. The signals obtained were compared to standards made up of pooled Caulobacter DNA from the sewage isolates and non-Caulobacter DNA from organisms also present in sewage. Using this assay Caulobacters could only be detected above the 1% level, which was higher than their proportion in the wastewater environment. It appears that this approach will not be useful in monitoring Caulobacters in treatment plants unless a more highly conserved or higher copy number probe is found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (M.Dip.)--Cape Technikon, 1990 / The die-off in the marine environment of micro-organisms that are present in sewage was investigated. A literature survey was carried out and studies were done in the laboratory, at an offshore outfall and at a surf-zone outfall. In the laboratory raw sewage was mixed with sea water at known dilutions and the die-off of faecal coliforms, faecal streptococci and coliphage was monitored. At the offshore sewage outfall a tracer, Rhodamine B.was released in the pump station and the distribution of the plume around the diffuser was determined by means of the Rhodamine B concentrations and faecal colifonns counts. At the factory surf-zone outlet the Rhodamine B was injected into the pipeline. Samples were taken at 50 and 100 meter distance from the outlet. Thedie-off of faecal colifonns, faecal streptococci and coliphage was monitored. The following results were obtained: Literature study: (i) Coliforms • Temperatures of 15 QC and lower are conducive to survival while nutrients, at a concentration as low as 2,5 mg/L organic carbon, stimulate growth. A rise in salinity has a negative effect on the organisms. • The T90 value in direct sunlight is <2 hours but vanes considerably in the dark (>35 days). Changes in weather• conditions, sunlight intensity, hours of sunlight, depth and turbidity of seawater and different seasons are the most important factors accounting for the variations in the T90 values. • It is, therefore, important for the initial dilution to be large enough. This will ensure that the bacterial counts conform to the water quality criteria, as the die-off rates in the marine environment are slow and not always adequate, except around mid day. • Faecal coliforms as a single parameter is not considered to be the best indicator of marine pollution. • The 1:4 ratio between faecal coliforms and faecal streptococci for human faeces and 1:0,7 ratio for animal waste are not applicable in the marine environment. The survival of faecal streptococci in seawater is better than that of faecal coliforms and the ratio therefore changes. (ii) Enterococci and pathogens • The T90 value for faecal streptococci is double that of coliforms and also varies in the dark. In direct sunlight any change in sunlight intensity, change the T90 values. • In general the numbers of faecal streptococci and pathogens occuring in sewage are smaller than those of coliforms, but their survival time is longer. This could lead to a situation where the water quality complies with the criteria according to the coliform counts, while a health risk may exist due to th survival of pathogens.

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