Submitted in fulfillment for the Degree of Masters of Applied Sciences (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Activated sludge (AS) flocs are paramount in biological treatment of wastewater, are comprised of microbial consortia with organic and inorganic material bound together by extra polymeric substances (EPS). The filamentous bacteria play a vital role in the floc formation process by providing the necessary structural support. Presence of epiphytic attachment on selected filamentous bacteria is a commonly occurring phenomenon in activated sludge samples. Different theories have been proposed to describe this phenomenon; however, not much research has been carried out to explore the profundity of the attachment. In this study, an attempt has been made to elucidate the intrinsic nature of the epiphytic attachment between the bacterial rods and filamentous bacteria based on microscopic (morphological and structural) analysis. Characterization of these epiphytes were performed using fluorescence in situ hybridization (FISH) at group level using Alpha, Beta and Gamma Proteo-bacterial probes. Morphological characteristics of filament hosts and the bacterial rods at the interface region was assessed using scanning electron microscopy (SEM). The SEM micrographs indicated that the attachment was facilitated by more than the EPS layer. Further ultrastructural examination using transmission electron microscopy (TEM) indicated a possible cell-to-cell interaction between epiphytes and the selected filaments. Fibrillar structures resembling amyloid-like proteins were observed within the filament cell targeted by the epiphytes. An interaction was apparent between the amyloid like proteins and the epiphytes as exhibited by the direction of fibrillar structures pointing towards the approaching epiphytes. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on the epiphytic bacteria protruding towards the targeted filamentous cell. The sheath of host filaments however, remained intact and unpenetrated, during colonization. Amyloid-like fibrils at interface may potentially play the role of attachment sites for the attaching epiphytes, as attachment facilitating appendages were not visualized. / M

The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.

In recent years there has been pressure exerted on the feed industry in Southern Africa to produce enough animal feed to meet the region’s nutritional requirements. The increase in the cost of animal feed eventually affects the affordability and availability of high quality food to low income communities. However, the overall national production of protein feed can easily be surpassed by the amount of protein that could be extracted from sludge. For example, the amount of protein wasted through sludge in one province alone (Gauteng, South Africa) amounts to 106,763 metric tonnes/yr, and slightly lower than the national protein requirement of approximately 145,000 tonnes/yr. Waste Activated Sludge (WAS) from wastewater treatment plants treating domestic wastewater is shown to contain protein in a ratio of 2:1 against fishmeal. However, some of this protein content could be lost during processing. In this study, the protein content in sludge and fishmeal was evaluated in laboratory analyses conducted as a preliminary step towards designing a protein supplement substitute. A pilot test was conducted with 5 batches (10 chicken per batch), with fishmeal to sludge substitutions of 0%, 25%, 50%, 75%, and 100%. Metal content in the sludge was lowered by a rudimentary leaching process and its impact on the protein content was also evaluated. The initial mass gain rate, mortality rate, initial and operational costs analyses showed that protein from Waste Activated Sludge (WAS) could successfully replace the commercial feed supplements with a significant cost saving without adversely affecting the health of the animals. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Chemical Engineering / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Diversity of micro-organism communities in activated sludge have been analyzed by culture -dependent methods, which exclude the majority of endogenous microbes due to the selective nature of the media. Molecular and biochemical techniques have been evaluated, but they are time - consuming, complex and the results are difficult to interpret. Methods such as community level carbon source utilization patterns (i.e. Biolog) are easy to use and detect different patterns, which could be related to diversity and function, in this and other studies. Our aim was not to try and detect each and every metabolic reaction of all the individuals in the community, but the collective pattern for a specific community. Since, 1) a high species diversity should lead to a higher relative number of substrates utilized, because there are more possibilities and 2) upon dilution, some organisms will be lost (causing a decrease in species diversity) from the community, depending on their abundance and the relative contribution (perhaps only one metabolic reaction in the system), reducing the number of possibilities. The extent of the reduction of the possibilities upon dilution, should theoretically reflect something about the community structure. The key, therefore, lies in the interpretation of the results. The Biolog system unlike traditional culture - dependent methods, which are generally selective for the component of the community that has to be cultured, can reflect the activities of a broad range of bacteria. In this study the Biolog system was not considered as a culture - dependent method, but rather as a collection of metabolic tests (database) used for the purpose of generating a recognizable pattern for a specific community. Our hypothesis was that microbial community level carbon source utilization could be used to determine diversity and evenness in activated sludge systems. In our study we used activated sludge systems representative of an environment with a high species diversity and uneven distribution of species, indicated that upon dilution some of the substrates where no longer utilized due to the loss of some of the species. / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology: Biotechnology, Durban Institute of Technology, 2004. / M

HT biomass sonicated at ES inputs < 56KJ/gTS decreased floc size by 41% and enhanced its metabolic activity by 50-250% compared to control. ES inputs >118 KJ/gTS caused HT biomass solubilization and irreversible loss of its metabolic activity and reflocculation ability. During continuous activated sludge processing (ASP) of real primary effluent the observed yield (Yobs) decreased by 20% compared to control ASP at SF (stress factor) of 1 (biomass exchanged without USPT). At SF of 0.5, 1 and 1.5 (biomass exchanged with USPT) the Yobs further decreased by 33, 25 and 44% respectively as compared to control. This indicated that combining biomass anoxic exposure with USPT enhanced sludge reduction by increasing microbial maintenance metabolism likely in combination with microbial flora shift in the ASP depending on SF.

The objective of this investigation was to evaluate Holmberg's method for simultaneous dissolved oxygen control and parameter estimation in a completely mixed diffused air activated sludge reactor.

An investigation into the removal of phosphorus in the activated sludge process showed that: (1) Luxury uptake of phosphorus may be induced by the presence of an anoxic zone (2) If a primary and secondary anoxic reactor are present in the system, the actual retention time of the primary anoxic reactor will have the dominant effect on the degree of phosphorus removal. (3) In a system where only a primary anoxic reactor is present, there is an optimum actual anoxic retention time for obtaining maximum phosphorus removal. This optimum value appears to coincide with the condition where there is no nett release of phosphorus in the anoxic zone. Decreasing or increasing the actual anoxic retention time either side of the optimum value has a detrimental effect on phosphorus removal. (4) Increasing the influent ammonia-nitrogen and hence the reactor nitrate concentration, enhances phosphorus removal. (5) An increase in aeration reactor pH to between pH 7-8 enhances phosphorus removal. (6) The mechanism of removal does not appear to be precipitation. Although an increase in pH through the anoxic reactor was observed due to denitrification, phosphorus was released into solution. (7) The degree of phosphorus removal is independent of the influent phosphorus concentration. (8) Anoxic stripping of phosphorus is not a pre-requisite for luxury uptake of phosphorus to occur. (9) The concentration of phosphorus removed is a function of the COD utilized. The ratio of phosphorus removed to the COD utilized is constant at a particular sludge age, so that as the concentration of COD utilized increases, the concentration of phosphorus removed increases. (10) The concentration of phosphorus removed is a function of the sludge age. For a fixed biodegradable influent COD, the longer the sludge age, the less phosphorus removed.

In this investigation, two separate methods for determining oxygen transfer rates were applied to the oxidation ditches of an activated sludge plant. Steady state oxygen uptake rate testing and an oxygen mass balance technique were used to propose an in-process procedure for monitoring aeration efficiency using available resources. Although some overall averages offered promise, the testing results revealed that the mass balance analysis yielded results that do not accurately represent the oxygen transfer capabilities within the individual reactors due to shared variables that control the oxygen transfer rate. The steady state method provided more favorable results. Overall averages of daily oxygen transfer rates determined using the steady state method displayed a ratio of oxygen transfer rate between the reactors that corresponds to the expected ratio of 5/6 derived from the linear feet of aerator rotor present in each reactor.