Faculty of 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 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 Elucidation of microbiological-biochemical relationships in denitrification occurring during activated sludge treatment(2001) Drysdale, Gavin David; Bux, FaizalUp until now extensive work has been done to develop kinetic models and related software that can be used successfully to simulate and design nitrification denitrification (ND) and nitrification denitrification biological excess phosphorus removal (NDBEPR) systems for efficient nitrogen removal. The denitrification kinetics of these systems have primarily been determined and attributed to the ordinary heterotrophic bacteria, now also known as the OHO fraction, otherwise not involved in biological excess phosphorus removal. However, denitrification kinetics determined for ND systems have been found to vary considerably at times when applied to NDBEPR systems because of varying OHO active fraction estimates and the unexplained occurrence of anoxic phosphorus removal and anysuccess achieved to date has been some what fortuitous. Ultimately variations in process performance and kinetics are attributable to inadequate control and lack of understanding of the ecological, physiological and biochemical activities of constituent microorganisms. There is growing concern and movement towards a better understanding of the microbial community within activated sludge in order to gain optimal control of the process.Item Evaluation of biohydrogen production potential of sugarcane bagasse using activated sludge in a dark fermentation process(2016) Reddy, Karen; Bux, Faizal; Kuttun Pillai, Sheena Kumari; Gupta, Sanjay KumarAnaerobic dark fermentation is an efficient biological process to produce hydrogen from waste material. In South Africa, this technology has not been explored adequately to extract energy from biological wastes. Within the KwaZulu Natal region of South Africa, the sugar industry is a prominent venture that produces mass quantities of sugarcane bagasse amongst other waste products. This by-product can be an ideal source of substrate for biohydrogen generation. In this study, sugarcane bagasse was used as the main substrate for biohydrogen production by anaerobic fermentation using sewage sludge as the inoculum. Different pre-treatment methods were employed to maximize the release of fermentable sugars from the lignocellulosic biomass. Among the different pre-treatment methods employed, the maximum sugar yield (294.4 mg/g) was achieved with 0.25% H2SO4 for 60 minutes at 121°C. Prior to inoculation, the sewage sludge was also subjected to thermal pre-treatment to eliminate methanogens. Thermal pre-treatment of inoculum sludge for 30 min was effective in eliminating methanogens. Fluorescence in situ hybridization was used to positively identify the hydrogen producing bacteria present before and after treatment. The pre-treated substrate and inoculum was integrated into a dark fermentation process to further optimize the effect of pH, substrate to biomass, iron and magnetite nanoparticles on hydrogen production. The maximum hydrogen production (1.2 mol/mol glucose) was achieved at a pH range of 5-6, a substrate to biomass ratio of 3.5, and iron and magnetite nanoparticle concentration of 200 mg/L. Microbial analysis using quantitative polymerase chain reaction has confirmed the dominance of Clostridium spp. in the reactor. The highest hydrogenase gene activity (number of copies of hydrogenase gene expression/ng DNA) was recorded in the reactor supplemented with magnetite nanoparticles with lowest being in the raw sludge. There was a direct positive correlation between the hydrogenase gene copy number and the hydrogen yield obtained at different reactor conditions. Scanning electron microscopy was a useful to visually analyse the interaction of microorganisms with activated sludge. This study highlights the significance of anaerobic microorganisms from waste sludge being able to utilize agricultural waste material to produce biohydrogen which could be further scaled up for continuous hydrogen production. In addition, statistical tools used to predict the possible sugar (Design of experiments) and hydrogen yields (Gompertz model) produced would be helpful in saving time during full-scale operation of biohydrogen producing reactors.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 Evaluation of seasonal impacts on nitrifiers and nitrification performance of a full-scale activated sludge system(2016) Awolusi, Oluyemi Olatunji; Bux, Faizal; Sheena Kumari, S.K.Seasonal nitrification breakdown is a major problem in wastewater treatment plants which makes it difficult for the plant operators to meet discharge limits. The present study focused on understanding the seasonal impact of environmental and operational parameters on nitrifiers and nitrification, in a biological nutrient removal wastewater treatment works situated in the midlands of KwaZulu Natal. Composite sludge samples (from the aeration tank), influent and effluent water samples were collected twice a month for 237 days. A combination of fluorescent in-situ hybridization, polymerase chain reaction (PCR)-clone library, quantitative polymerase chain reaction (qPCR) were employed for characterizing and quantifying the dominant nitrifiers in the plant. In order to have more insight into the activated sludge community structure, pyrosequencing was used in profiling the amoA locus of ammonia oxidizing bacteria (AOB) community whilst Illumina sequencing was used in characterising the plant’s total bacterial community. The nonlinear effect of operating parameters and environmental conditions on nitrification was also investigated using an adaptive neuro-fuzzy inference system (ANFIS), Pearson’s correlation coefficient and quadratic models. The plant operated with higher MLSS of 6157±783 mg/L during the first phase (winter) whilst it was 4728±1282 mg/L in summer. The temperature recorded in the aeration tanks ranged from 14.2oC to 25.1oC during the period. The average ammonia removal during winter was 60.0±18% whereas it was 83±13% during summer and this was found to correlate with temperature (r = 0.7671; P = 0.0008). A significant correlation was also found between the AOB (amoA gene) copy numbers and temperature in the reactors (α= 0.05; P=0.05), with the lowest AOB abundance recorded during winter. Sanger sequencing analysis indicated that the dominant nitrifiers were Nitrosomonas spp. Nitrobacter spp. and Nitrospira spp. Pyrosequencing revealed significant differences in the AOB population which was 6 times higher during summer compared to winter. The AOB sequences related to uncultured bacterium and uncultured AOB also showed an increase of 133% and 360% respectively when the season changed from winter to summer. This study suggests that vast population of novel, ecologically significant AOB species, which remain unexploited, still inhabit the complex activated sludge communities. Based on ANFIS model, AOB increased during summer season, when temperature was 1.4-fold higher than winter (r 0.517, p 0.048), and HRT decreased by 31% as a result of rainfall (r - 0.741, p 0.002). Food: microorganism ratio (F/M) and HRT formed the optimal combination of two inputs affecting the plant’s specific nitrification (qN), and their quadratic equation showed r2-value of 0.50. This study has significantly contributed towards understanding the complex relationship between the microbial population dynamics, wastewater composition and nitrification performance in a full-scale treatment plant situated in the subtropical region. This is the first study applying ANFIS technique to describe the nitrification performance at a full-scale WWTP, subjected to dynamic operational parameters. The study also demonstrated the successful application of ANFIS for determining and ranking the impact of various operating parameters on plant’s nitrification performance, which could not be achieved by the conventional spearman correlation due to the non-linearity of the interactions during wastewater treatment. Moreover, this study also represents the first-time amoA gene targeted pyrosequencing of AOB in a full-scale activated sludge is being done.Item Development and optimization of remedial measures to control filamentous bacteria in a full-scale biological nutrient removal plant(2014) Deepnarain, Nashia; Bux, FaizalWastewater treatment plants (WWTPs) frequently experience bulking and foaming episodes, which present operational challenges by affecting sludge settling due to the excessive proliferation of filamentous bacteria. Various control strategies have been implemented over the years to minimize filamentous growth, however, filamentous bulking still remains an unresolved problem in many WWTPs worldwide. The current study focused on developing and optimizing remedial measures viz., specific and non-specific methods to reduce problematic filamentous bacteria in a full-scale WWTP. Specific methods demonstrated the influence of plant operational parameters viz. chemical oxygen demand, influent N-NH4+, food to microorganism ratio, dissolved oxygen, temperature and pH on the abundance of filamentous bacteria. A cumulative logit model was used to determine the significant relationships between the individual filamentous bacteria at present and the prevailing plant operational parameters. Using the above statistical approach, significant observations and predictions were made with respect to the individual filamentous growth under certain operational parameters. With further validation, this model could be successfully applied to other full-scale WWTPs identifying specific parameters that could contribute to filamentous bulking, thus providing a useful guide for regulating specific filamentous growth. Non-specific control methods such as chlorine, ultraviolet irradiation and ozone treatment were investigated on filamentous bacteria using a live/dead staining technique. To achieve at least 50% reduction of filamentous bacteria, a chlorine dose of 10 mg Cl2/L was required, all filaments were killed at a dose of 22 mg Cl2/L. In addition, an effective UV and ozone dose of 4418.91 μw seconds/cm2 and ±20 mg O3/L respectively, was required to kill 50% of the filamentous bacterial population. Among the three non-specific methods, ozone treatment seemed to be an effective method in controlling the filamentous population with a low negative impact to the surrounding environment. This study serves as a useful guide on the problems and control of filamentous bulking in activated sludge plants.Item Microbial community analysis of a laboratory-scale biological process for the treatment of vegetable oil effluent(2011) Degenaar, Adrian Phillip; Bux, FaizalUntreated vegetable oil effluents (VOEs) are known for creating shock-loading problems for the receiving wastewater treatment installations, resulting in poor quality final effluents being produced which do not satisfy municipal discharge standards. Onsite activated sludge treatment as an alternative has not been fully investigated. Hence, in this investigation biological treatment using the activated sludge process was chosen as the method for the treatment of VOE. The effect of VOE on measured process parameters was also determined. Novel molecular techniques such as fluorescent in situ hybridisation (FISH) and dot-blot hybridization have become powerful tools for the analysis of complex microbial communities that exist within activated sludge. The aim of this investigation was to evaluate biological treatment, optimize and apply FISH and dot-blot hybridization in order to analyze the microbial community implicated the biological treatment of VOE using probes EUBmix, ALF1b, BET42a, GAM42a and HGC69a. A laboratory-scale modified Ludzack-Ettinger (MLE) process setup and fed VOE with a COD (chemical oxygen demand) of ± 1000 mg/L. Daily monitoring of the process involved COD and TKN (total kjeldahl nitrogen) analysis of the influent and effluent as well as direct OUR (oxygen utilization rate) measurement and monitoring of the MLVSS (mixed liquor volatile suspended solids) concentration of the aerobic mixed liquor. The process exhibited overall COD and TKN removal capacities of 84% and 90% respectively. The aerobic mixed liquor had an OUR of 19 mgO/L.h and an average MLVSS concentration of 3000 mg/L. FISH results revealed that 72% of cells stained with 4‟, 6-diamidino-2-phenylindole (DAPI) within the aerobic mixed liquor bound to probe EUBmix, indicating a substantial Bacterial population within the laboratory-scale biological process. The alpha-Proteobacteria was identified as the dominant bacterial community comprising 31% of Bacterial cells, followed by the beta-Proteobacteria (17% of EUBmix), gamma-Proteobacteria (8% of EUBmix) and Actinobacteria (4% of EUBmix). Results of dot-blot hybridization were in agreement with FISH Adrian Phillip Degenaar| CHAPTER 1: General Introduction - v - results reiterating dominance of the alpha-Proteobacteria. This indicated that the class alpha-Proteobacteria could play a primary role in the biological degradation of VOE. This research will therefore aid in process design and retrofitting of biological processes treating VOE.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 Molecular analyses of pure cultures of filamentous bacteria isolated from activated sludge(2005) Naidoo, DashikaThe activated sludge process is the mostl used biological treatment process. Engineers and microbiologists are constantly seeking ways to improve process efficiency, which can be attributed to the increasing demand for fresh water supplies and proper environmental management. Since the inception of the activated sludge process, bulking and foaming have been major problems affecting its efficiency. Filamentous bacteria have been identified as the primary cause of bulking and foaming. Numerous attempts have been made to resolve this problem. Some of these attempts were effective as interim measures but failed as long term control strategies. The identification of filamentous bacteria and the study of their physiology have been hampered by the unreliability of conventional microbiological techniques. This is largely due to their morphological variations and inconsistent characteristics within different environments. To fully understand their role in promoting bulking and foaming, filamentous bacteria need to be characterized on a molecular level. The aim of this study was, therefore, to identify filamentous bacteria in pure culture with the purpose of validating these findings to the physiological traits of the pure cultures when they were isolated. Fourteen different filamentous cultures were used for this study. The cultures were identified using specific oligonucleotide probes via fluorescent in situ hybridisation and nucleotide sequencing. Prior to sequencing, an agarose gel and a denaturing gradient gel Electrophoresis profile were determined for each isolate. The various techniques were optimised specifically for the filamentous isolates. The isolates were identified as Gordonia amarae, Haliscomenobacter hydrossis, Acinetobacter sp./Type 1863, Type 021N, Thiothrix nivea, Sphaerotilus natans and Nocardioform organisms.