Theses and dissertations (Applied Sciences)
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Item Physiological and biochemical evaluation of pure cultures of problematic filamentous bacteria isolated from activated sludge(2004) Ramothokang, Tshireletso R.; Bux, FaizalSince the development of the activated sludge process, bulking and foaming have been a major problem affecting treatment efficiency. Filamentous bacteria have long been known to be the primary cause of bulking and foaming problems in activated sludge wastewater treatment systems. Attempts to cure filamentous bulking and foaming have thus far not shown great success in effective long-term control measures due to a lack of understanding of these organisms. Chemical methods such as chlorination and the use of hydrogen peroxide are still used to cure bulking but are only effective as interim measures. This could be due to the main factors stimulating filamentous bacterial growth not being changed by these methods for curing bulking and also, a lack of in-depth understanding of filamentous bacteria by scientists. It is therefore important to gain a proper understanding of these bacteria on the basis of their physiological, biochemical and growth characteristics. For all this to be successfully attained, filamentous bacteria need to be studied in pure culture so as to facilitate a better understanding of bulking and foaming and the control thereof during wastewater treatment. The aim of this study was therefore, to isolate and cultivate problematic filamentous bacteria and determine the physiological, biochemical and morphological traits of these organisms in pure culture, with the purpose of being able to integrate these findings to in situ analysis. Using four different isolation techniques, a total of 14 isolates from 7 different wastewater systems were obtained and evaluated for a range of physical, chemical, redox and substrate conditions. Results of the study indicate that filamentous survival and proliferation in BNR systems is largely due to varied phosphate uptake capacities and widespread ability to denitrify both nitrate and nitrite. Lipid hydrolysis is also a major component of filamentous bacterial metabolism with hydrolysis of other large compounds, as revealed by Biolog, such as starch, dextrin, proteins/peptides, Tween 40, Tween 80 and nucleosides indicating an affinity for larger slowly biodegradable substrates. They also strive on a variety of amino acids and sugars. The results obtained in this study revealed that filamentous bacteria are more diverse and complex in their biochemistry and physiology hence the difficulty in achieving long- term optimal control of filamentous bulking in activated sludge. It was concluded that filamentous bacteria have the ability to survive during times of starvation where growth factors are limiting and, this may be attributed to their ability to store storage compounds such as PHB, glycogen and polyP. The filaments' ability to store storage compounds and denitrify, suggests that they may in fact play significant roles in denitrification and EBPR. It is also concluded from this study that the filamentous bacteria under study are sensitive to aromatic compounds and that they have an affinity for slowly biodegradable polymers such as lipids, nucleosides, proteins/ peptides, dextrin and starch. Also concluded is that, the use of Biolog for biochemical profiling! fingerprinting of filamentous bacteria is useful, however, due to the possibility that some organisms may in fact, not grow and! or may give negative results on some and! or all substrates, other strategies. for biochemical profiling be established and used in this regard. Identification and evaluation of filamentous bacteria based on morphological traits is limiting and requires development and optimization of in situ techniques, such as DNAIRNA based probes and micro autoradiography. Bulking and BNR are elaborate and still not fully understood. The filaments' ability to take up phosphates and denitrify means that an advanced understanding of the roles they play in BNR systems and AA- bulking (Anoxic- Aerobic) is still required. Physiological and biochemical fingerprinting of pure cultures of filamentous bacteria is an important basis to understanding these organisms, and establishing potential bulking and foaming criteria for in situ evaluation and verification. It is from a study such as this that the main goal of curing bulking and gaining an enhanced understanding of BNR may be achieved.Item Investigations on aerobic thermophilic treatment of pulp mill effluent(2004) Reddy, Prenaven; Singh, Suren; Pillay, Visvanathan Lingamurti; Buckley, ChrisThermophilic treatment of wastewaters increases in importance as industries shift from end-of-pipe treatment towards integrated process water treatment. The need for treatment of process water becomes evident, as the levels of pollutants in industrial water circuits need to be controlled whereas the intake of fresh water generally diminishes. In the paper and pulp industry, high process water temperatures prevail and thus wastewater treatment needs to take place under thermophilic conditions. This thesis describes research in which the aerobic treatment of paper and pulp mill effluent was investigated under thermophilic conditions. The objectives of this study were, firstly, to identify a suitable inoculum for thermophilic degradation of pulp mill effluent and this was achieved by screening from various sources i.e., activated sludge, heating water, soil and compost. The second objective was to determine the feasibility of aerobic thermophilic degradation of pulp mill effluent using temperatures of 40°C, 50°C and 60°C. Batch, fed-batch and continuous experiments would enable the feasibility of degradation of pulp mill effluent. Prior to fed-batch systems, batch systems were optimised by determining the following parameters: 1) Effect of temperature on degradation of pulp mill effluent, 2) Effect of biomass concentration on degradation of pulp mill effluent, 3) Effect of aeration on degradation of pulp mill effluent, and 4) the effect of nutrient supplementation. I I I I I I I I I I I I I I I I I I I I I Once batch systems were optimised, the fed-batch systems came into play, and this system demonstrated much potential for thermophilic degradation of pulp mill effluent, which finally led to developing a continuous system, were degradation was optimal.Item Optimisation of food to microorganism ratios during activated sludge respirometric batch assays(2003) Ismail, Arshad Abdool Hak; Bux, FaizalThe measured kinetics of a bacterial culture degrading a single organic compound as a sole carbon source in a batch reactor depends on the history of the culture, the identifiability of the parameters, and the manner in which the experiment to measure them is run. The initial substrate to biomass ratio (So/Xo) used in the experiment is particularly important because it influences both parameter identifiability and the expression of the culture history.Item Determination of the relationship between epiphytes and selected filamentous bacteria in activated sludge(2016) Conco, Thobela; Bux, Faizal; Sheena Kumari, S.K.; Stenström, Thor-AxelActivated 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.Item Determination of the heterotrophic and autotrophic active biomass during activated sludge respirometric batch assays using molecular techniques(2008) Ismail, ArshadActivated sludge models now in use worldwide for the design and operation of treatment systems use hypothetical concentrations of active organisms. In order to validate and calibrate model outputs, concentrations and activities of organisms responsible for nitrification and denitrification need to be reflected by actual measurements. This research has been initiated by the observation of an increasing gap of suitable techniques that exist in the direct measurement and separation of active biomass components, responsible for COD removal and denitrification.Item Characterisation of the microbial communities present in an anaerobic baffled reactor utilising molecular techniques(2005) Lalbahadur, Tharnija; Bux, FaizalThe provision of safe and sanitary water is a constitutional right and above all, a necessity of life. As a result of the rapid urbanisation and the past policies of apartheid, a large population of South Africa dwell in informal settlements, where there is very little hope of development, as the government does not possess the resources that are necessary for a full-scale sanitation programme. Therefore, on-site treatments have been considered to provide sanitation in these dense peri-urban areas. The anaerobic baffled reactor (ABR) is one such sanitation system. This reactor utilises the phenomenon of anaerobic digestion to degrade substrates. One of the major disadvantages of any anaerobic treatment processes is the extreme sensitivity of the bacterial communities, thus inducing slow recovery rates following toxic shocks. Therefore, an understanding of these microbial consortia is essential to effectively control, operate and optimise the anaerobic reactor. Fluorescence in situ hybridization, 4’,6-diamidino-2-phenylindole (DAPI) staining and DNA sequencing techniques were applied to determine the microbial consortium, as well as their reactions to daily operating conditions. With an understanding of these populations and their responses to perturbations within the system, it is possible to construct an anaerobic system that is successful in its treatment of domestic wastewater. In situ hybridizations were conducted for three operating periods, each characterised by specific flow rates. Results showed Eubacterial population dominance over the Archaeal population throughout both of the operating periods investigated. However, these cells cumulatively consisted of 50% of the total biomass fraction, as determined by DAPI staining. Group-probes utilised revealed a high concentration of fermentative acidogenic bacteria, which lead to a decrease in the pH values. It was noted that the ABR did not separate the acidogenic and methanogenic phases, as expected. Therefore, the decrease in pH further inhibited the proliferation of Archaeal acetoclastic methanogens, which were not present in the second operating period. DNA sequencing results revealed the occurrence of the hydrogenotrophic Methanobacterium and Methanococcus genera and confirmed the presence of Methanosarcina. Sequencing of the bacterial DNA confirmed the presence of the low G+ C Gram Positives (Streptococcus), the high G+C Gram Positives (Propionibacterium) and the sulfate reducing bacteria (Desulfovibrio vulgaris). However, justifications were highly subjective due to a lack of supportive analytical data, such as acetate, volatile fatty acids and methane concentrations. Despite this, findings served to add valuable information, providing details on the specific microbial groups associated with ABR treatment processes.Item Molecular characterization of filamentous bacteria isolated from full-scale activated sludge processes(2007) Marrengane, ZinhleActivated sludge flocs are responsible for flocculation, settling and dewaterability. It is important to maintain the growth off loc-forming bacteria for efficient sludge settleability and compaction for good quality effluent. Filamentous bacteria on the other hand are believed to provide rigid support network or backbone upon which floc-forming bacteria adhere to form stable activated sludge flocs (Wilderer et al., 2002; Ramothokang et al., 2003). Filamentous bacteria can also be detrimental to the process when they outgrow floc-forming bacteria. Morphologically filamentous bacteria are at an advantage as they have higher outward growth velocity and can extend freely to bulk liquid substrate. Proliferation of filamentous bacteria causes foaming and bulking (Martins et al., 2004). Although chemical alleviation measures to circumvent bulking are present, they are symptomatic (Chang et al., 2004). Eikelboom (1975) developed the first identification keys for the classification of filamentous bacteria that is primarily based on morphological characteristics and microscopic examination. Although very useful, this type of identification has its limitations. For instance some filamentous bacteria can change morphology in response to changes in the environment and although some of them can be morphologically similar they may vary considerably in their physiology and taxonomy (Martins et al., 2004). A vast number of filamentous bacteria are still very poorly understood which could be due to the problems of cultivation due to their slow growing nature and maintenance of cultures (Rossetti et al., 2006). This limitation necessitates a molecular approach to resolve the taxonomy of filamentous bacteria as it is a culture-independent technique which is highly accurate. This project was undertaken to verify the identity of pure cultures of filamentous bacteria isolated previously through the application of molecular techniques. The 16S rDNA are conserved regions in bacterial cells and they can be extracted and specific nucleic acid fragments amplified. Denaturation gradient gel electrophoresis enabled the separation of fragments of identical length but different size and served as an indication of purity (Muyzer et al., 1993).