Submitted in partial fulfillment of academic requirements for the degree of Masters of Technology: Department of Chemical Engineering, Durban University of Technology, 2009. / The primary objective of this project was to establish a screening protocol which could be used to access high strength/toxic effluent for toxicity and degradability prior to being disposed in wastewater treatment works. The serum bottle method (materials and method section) is simple, makes use of small glass vials (125 mℓ-volume were used in this research) which do not require any stirring nor feeding device or other engineered tool: a serum bottle is sealed immediately after all components are poured inside and thereafter conducted in a batch mode and occasionally shaken to ensure adequate homogenisation of the components. The only variables which are regularly measured are the volume of biogas produced and gas composition. The two assays, originally developed by Owen et al. (1979) to address the toxicity and the biodegradability have been combined in a single test called AAT, Anaerobic Activity Test, which enables one to assess simultaneously the inhibitory effect on the methanogenic biomass and the biodegradability of the test material as well as the ability of the biomass to adapt to the test material and therefore to overcome the initial inhibition. The screening protocol is illustrated in Annexure A. The protocol consists of a sequence of assays which employ the serum bottle methodology. A first step of the procedure is aimed at rapidly estimating whether the effluent is potentially toxic to the methanogenic biomass and in what concentration. The second step is a more extensive screening, aimed at precisely characterising the toxicity of the effluent, the extent of biodegradation that can be achieved, as well as at establishing whether a potential for adaptation of the biomass exists upon exposure. If the sample passes the screening stage, the same serum bottle method will be used to conduct a series of batch co-digestion experiments aimed at evaluating a convenient volumetric ratio between the test material and the readily biodegradable substrate. Finally, a laboratory-scale codigestion trial could simulate the full-scale process, thus enabling the selection of appropriate operating conditions for the start-up of the full-scale implementation. This the protocol has been used to assess the amenability to be anaerobically (co)digested of four industrial effluents, i.e. size and distillery effluents which are classified as high strength and scour and synthetic dye effluents classified as toxic. From the biodegradability and toxicity assays the following conclusions were drawn. The size and distillery effluent were found to be ii degradable at 32 g COD/ℓ and 16 g COD /ℓ concentrations respectively. Concentrations higher than these stipulated above were found inhibitory. Scour effluent was found to be recalcitrant at all concentration tested and synthetic dye was 100 % degradable at 0.12 g COD/ℓ and lower and highly inhibitory at concentration higher than 1.1 g COD/ℓ. Co-digestion experiment using serum bottle AAT method were undertaken between effluents i.e. size + distillery, size + scour, distillery + synthetic dye in an attempt to verify whether the digestion performance benefits from simultaneous presence of the two substrates. The volumetric ratios between the effluents were 1:1, 1:2, 2:1. The presence of two mixtures in the case of size and distillery had better methane production compared to individual substrate i.e. size or distillery separate. The mixture with volumetric flow rate ratio of 2:1 (size: distillery) was preferable in terms of process performance as it had highest COD removal compared to the other mixtures /ratios and individual substrates. The mixture of size and scour (2:1) had highest degradation percentage compared to other ratios but not high enough to qualify as degradable (less than 50 %). The mixture of distillery and synthetic dye had the same pattern with ratio of 2:1 giving the best COD conversion. The pattern than can be drawn from the degradability of mixtures is: the degradability of mixtures increase with the increasing amount of the most biodegradable compound/effluent in the mixture. Serum bottle results provided the detailed information regarding the safe operating parameters which should be used during the starting point for the larger scale investigation i.e. lab-scale investigations. The lab scale investigations were conducted primarily to validate screening and monitor how the digestion progresses and also to provide data for future project i.e. pilot plant investigation. Other effluents i.e. scour and synthetic dye and their co-digestion mixture were excluded from the lab-scale investigations since they were found to be non- biodegradable i.e. their COD conversion was less the 50 % in the screening protocol. Due to time constrains and other technical difficulties in the laboratory, the co-digestion of size and distillery mixture trials we not conducted on the laboratory scale. Laboratory-scale digestion trials showed that the best organic loading rate for distillery effluent in terms of reactor performance and stability was 1.0g COD/ℓ with efficiency of about 45 %, and for size was 2.0g COD/ℓ with an efficiency of 40 %. The efficiencies obtained in both effluents trials could be greatly improved by acclimation; however these results showed that the digestion of these effluents on the bigger scale is possible.

The effect of untreated roughages on digestibility and rumen fill of the gut was reviewed as physical mechanism influencing the regulation of roughage intake. The review of literature also focused on identifying factors that affect the way in which urea-ammoniation alters the roughage intake, digestibility and performance of ruminant animals. Trials were carried out with fistulated cows to address to what extent concentrate proportion and urea-ammoniation affected microbial colonization and degradation of roughage diets in the rumen. One interest of this study was to develop a model that would help to predict the benefit associated with urea-treatment of roughages. The first trial (Chapter 3) investigated the effect of urea-ammoniation of roughage and concentrate proportion of the diet on degradation of roughages, and the benefit associated with the treatment of roughages. Four rumen-fistulated Jersey cows were fed on a basal diet composed of either urea treated (3 kg of urea per 100 kg of straw) or untreated Eragrostis curvula hay. These basal diets were supplemented with concentrate composed of maize meal (78%) and cotton seed cake (22%). The concentrate contributed 0, 25, 50 and 75% of the total ration and hay the rest. The experiment consisted of 6 periods. Each period lasted 19 days, comprising 12 days of adaptation to the experimental diet followed by 6 days degradability measurements and 1-day rumen fluid collection. During each period the 4 Jersey cows were randomly allocated to 4 of the 8 dietary treatments, ensuring that each diet was fed to 3 animals during the entire experimental period. The experimental roughages used in this trial were wheat (Triticum sativum) straw, barley (Hordeum Vulgare) straw, coastcross (k11) (Cynodon hybrid) hay, veld hay (natural grass), oat (Avena sativa) straw, oat (Avena sativa) hay, maize (Zea mays) stover, kikuyu (Pennisetum clandestinum) grass, weeping love grass (Eragrostsis curvula) and Italian rye (Lolium multiflorum) grass. Each roughage (sample) was subdivided into two equal portions, one of which was then treated with urea. The urea solution was prepared by dissolving 30 g of urea in 0.4 liter of water. The solution was fully distributed over I kg of roughage. Treated roughages were sealed tightly and stored at room temperature for 5 weeks in plastic bags. Immediately after opening, the different roughages, including the untreated ones, were sun dried, chopped fine by hand and ground through a 2-mm screen in a laboratory mill. About 3 g of each sample was weighed into labeled nylon bags. The bags were tied to a stainless steel disc with 10 evenly spaced small holes drilled through the periphery of the disc serving as anchor points. The bags were incubated (in duplicate per time interval) in the rumen for 120, 96, 72, 48, 24, 12, 6 and 3 h, sequentially. The treated samples were incubated in animals fed treated hay, while untreated samples were incubated in animals given untreated hay. Immediately after removal from the rumen, the bags, including the 0 hour ones, which had not been incubated but soaked in warm water for I hour, were washed in 6 cycles (each lasting 4 minutes) in a semi-automatic washing machine. The washed bags were then dried in a forced draught oven at 60 degrees C for 48 hours, cooled in a desicator and weighed. The pH of the rumen fluid ranged between 6.5 and 6.8 for all diets. Rumen ammonia concentration was higher (P<0.002) when the basal diet consisted of urea treated hay. Increasing the concentrate proportion in the diet had the desired effect of increasing rumen ammonia concentration without severely affecting pH. Urea-ammoniation increased (P<O.OOO1) the slowly degradable fraction (B), potential degradability (PD), effective degradability (ED) of dry matter and neutral detergent fiber (NDF), decreased (P>0.05) lag time (LT) but had no effect on the rate of degradation (c) of dry matter. Concentrate proportions affected (P<0.05) the slowly degradable fraction, potential degradability, lag time and effective degradability but had no effect (P>0.05) on the rate of degradation of dry matter (DM). Maximum and minimum values of the slowly degradable fraction, potential degradability and effective degradability of DM and NDF were obtained at the 25 and 75% concentrate levels, respectively. Within urea-ammoniation, roughage type affected (P<O.OO1) the B-fraction, PO and EO of OM and NDF degradation. Rate of degradation of DM of untreated roughages varied from 0.022 h(-1) in wheat straw to 0.087 h(-1) in rye grass, while for urea treated roughages it varied from 0.022 h(-1) in oat straw to 0.082 h(-1) in rye grass. Rye grass degraded almost three to four times faster than urea treated oat or untreated wheat straw. Urea-ammoniation was less effective in increasing DM and cell wall degradation rates (c) of rye grass compared to wheat straw. The results showed that low quality roughages such as wheat straw benefited relatively the most from urea-ammoniation. The effect of urea-ammoniation and dietary manipulation on microbial colonization (Chapter 4) of fiber particles in the rumen of animals was also investigated in two experiments. In Experiment 1, the cows were fed on rations comprising either urea-ammoniated or untreated Eragrostis curvula hay supplemented with concentrate at hay to concentrate ratio of 100:0, 75:25, 50:50, 25:75, resulting in eight different rumen environments. The experiment consisted of two periods. Each period lasted 12 days of adaptation to the experimental diet followed by one-day incubation of urea-ammoniated and untreated barley straw. Experiment 2 consisted of two urea-ammoniated (7.5 kg of urea per 100 kg of hay) hay levels (20 and 40% of the total ration) and concentrate levels (60 and 80%). Fistulated Jersey cows were adapted for 12 days after assigning to the dietary treatment. Feed was given at the rate of 9.0 kg day(-1) per animal portioned into equal meals of 4.50 kg each and offered at 08:00 and 16:00 every day. About 3 g of urea-ammoniated or untreated barley (Hordeum vulgare) straw, ground through a 2-mm screen, was weighed into a labelled nylon bag and incubated for 3, 6 or 12 h in the rumen of the fistulated cows. Microbes adhering to incubated fiber particles were examined under the Environmental Scanning Electron Microscopy (ESEM) and analysed on the image analyser. Depending on morphology , the microbes were divided into three groups: bacilli (rod), cocci (round) and others (spiral, fimbrea and cluster ; not specifically defined or undefined microbes). Urea-ammoniation of dietary roughage decreased (P<O.OO1) bacilli counts and total bacteria count but had no effect on count of the undefined group of microbes on fiber particles in the rumen of cows (Experiment 1). Concentrate proportions had no effect (P>0.05) on bacilli, cocci and total bacterial count on fiber particles. However, the results from electron micrograph observations revealed that the total bacterial count tended to decrease as the concentrate level increased in the diet of cows. Bacilli, cocci, undefined group of microbes and total count of microbes increased (P<0.05) as length of incubation increased. In Experiment 2, incubated feed, concentrate proportion and time of incubation had no effect (P>O.05) on bacilli , others (undefined group of microbes) and total count of fiber-adhering microbes in the rumen of cows. However, increasing concentrate in the diet of cows tended to decrease (P<O.07) the count of fiber-adhering cocci. The total count of microbes on fiber particles was higher in animals fed 80% concentrate as compared to 60% concentrate. The benefit derived from urea treatment in terms of B-fraction, effective degradability and potential degradability of DM and fiber of roughages increased with increasing the NDF content. Therefore, the important conclusions drawn from the results of the present study is that urea-ammoniation of roughages should be done strategically and that high quality roughages may give little return per unit of cost of ammoniation. This means that the benefit associated with urea-ammoniation would be justified for poor quality roughages only. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Chapter 2: Avian frugivores have been somewhat poorly studied with regards to the effects that different fruit sugar types and concentrations have on their digestive efficiencies. Digestion of sugars in equicaloric and equimolar artificial fruit of different sugar types at varying concentrations and molarities were therefore investigated in two relatively large South African frugivores, the Knysna (Tauraco corythaix) and the Purple-crested (Gallirex porphyreolophus) Turaco. Artificial fruits of 6.6, 12.4 and 22% sucrose and glucose respectively and artificial fruits of 0.42, 0.83 and 1.66 mol L-1 sucrose and glucose respectively were used to determine daily food and energy intake, digestive efficiencies and digestive transit times. Digestive transit times of both Turaco species were slower with an increase in sugar concentration and molarity, irrespective of sugar type. Food intake of both Turaco species increased with decreasing concentration and molarity, irrespective of sugar type suggesting compensatory mechanisms for energy requirements. Apparent assimilation efficiencies of both Turaco species ranged from 61.4-90.0% and 60.2-92.4% for equicaloric and equimolar artificial fruit diets respectively. Consequently these two frugivores appear to be tolerant of sugar type and would be expected to select fruits based on energy yields. Future studies of the composition of indigenous forest fruit sugars may give insight into food preferences of the Turaco species and their role as potential seed dispersers of fruiting tree species. Chapter 3: The effects that different fruit sugar types and concentrations have on food preferences of avian frugivores have been relatively poorly studied. Although it has been recently advocated that preference is based on equicaloric energy it is also important to note whether preferences change as energy content changes. Therefore, sugar preferences of equicaloric and equimolar artificial fruit of different sugar types at varying concentrations and molarities were investigated in two relatively large South African frugivores, Knysna (Tauraco corythaix) and Purple-crested (Gallirex porphyreolophus) Turacos. Artificial fruits of 6.6, 12.4 and 22% sucrose and glucose respectively and artificial fruits of 0.42, 0.83 and 1.66 mol L-1 sucrose and glucose respectively were used to determine sugar preferences. Knysna Turacos preferred the sucrose to the glucose equicaloric artificial fruit diet at low concentrations whereas Purple-crested Turacos showed no preference for either diet. Both Turacos species preferred the sucrose equimolar artificial fruit diet to the glucose at low concentrations. At high concentrations neither species showed a preference for either equicaloric or equimolar artificial fruit diets. This suggests that energy requirements influence food preferences more than sugar type and that birds will select fruit that is higher in energy irrespective of sugar type. This complements an earlier study on digestion of sugars at varying concentrations of differing equicaloric and equimolar artificial fruit sugar types. It again emphasizes the need for future studies looking at the composition of indigenous forest fruit sugars in order to obtain insight into the role of these avian frugivores as potential seed dispersers of fruiting tree species. Chapter 4: Avian frugivores may select fruit based on their seed loads and pulp to seed ratios and this may have important implications for their role as seed dispersal agents. Consequently the effect of different seed packaging was investigated in two relatively large South African frugivores, Knysna (Tauraco corythaix) and Purple-crested (Gallirex porphyreolophus) Turacos. Small-seeded artificial fruits containing on average five black plastic beads (2.24 mm diameter) and large-seeded artificial fruits containing one average one single black plastic bead (3.85 mm diameter) were used to investigate transit rates and food preference. Total seed volume in the two fruits was on average approximately equal (29.42 mm3 and 29.88 mm3 for small- and large-seeded fruits, respectively). Bead transit rates ranged from 38-45 min for Knysna Turacos and 36-50 min for Purple-crested Turacos with faster transit rates on the small-seeded diet. Pulp transit rates ranged from 25-39 min for Purple-crested Turacos and 34-40 min for Knysna Turacos. Purple-crested Turacos had significantly shorter pulp transit rates on the large-seeded than the small-seeded diet whereas Knysna Turacos had no significant difference between diets. Knysna Turacos preferred the small-seeded to the large-seeded artificial fruits whereas Purple-crested Turacos showed no preference for either diet. Further studies on avian dispersers will contribute to a greater understanding of their evolutionary relationship with fruiting plants. In particular, future studies establishing suitable seed sizes and volumes for use in determining the effect of seed packaging on larger birds are required as well as studies looking at seed packaging of indigenous forest fruit and the effects of seed packaging on digestion and food preference in indigenous avian frugivores may give insight into the role of avian frugivores as fruit consumers and seed dispersers. Chapter 5: Fleshy-fruited plants and frugivores share a mutualistic relationship with plants offering the frugivores a reward of fleshy fruit pulp in order to disperse their seeds. In return frugivores receive a food source and consequently a source of energy. However all fruits are not equal in terms of available energy and consequently the digestion efficiency of six indigenous tree species was investigated in an avian frugivore, the Knysna Turaco (Tauraco corythaix). It was predicted that the Turacos would process these indigenous fruits efficiently and have fast transit rates and high intake rates irrespective of species. Fruits were fed to the Turacos in separate trials to determine daily food and energy intake, digestive efficiencies and digestive transit times. Digestive transit times of Knysna Turacos (c. 12-28 min) fall within the range of published examples for other frugivorous birds. Apparent assimilation efficiencies of the Turacos ranged from c. 15-84% and were generally lower than those observed in other avian frugivores. Future studies looking at the nutritional composition of indigenous forest fruit may provide insight into the Knysna Turacos digestive efficiency and consequently their food preferences and role as potential seed dispersers of fruiting tree species. Chapter 6: Seed dispersal plays an important role in the persistence, regeneration and maintenance of plant communities. It is therefore not surprising that much attention has been paid to the germination potential of seeds ingested by frugivorous animals. Consequently the aim of this study was to determine what effect ingestion of seeds by Knysna Turacos (Tauraco corythaix) has on the germination rate and germination percentage of indigenous South African tree species. Fruits from twelve tree species were fed to the Turacos in separate trials and seed retention times were determined as it has been suggested that a longer seed retention time may increase germination rates. At the end of each trial, seeds were extracted from excreta of individual birds and planted in trays containing potting soil. Germination was recorded daily until 14 days post of no germination. Knysna Turacos seed retention times were in the range of those obtained by other studies with Ficus sur seeds having the fastest retention times (12.4 ± 0.8 min) and Celtis africana the slowest (34.6 ± 5.6 min). Mean percentage germination of ingested seeds was not significantly different to pulp removed and whole fruit seeds indicating that ingestion by Knysna Turacos did not enhance percentage germination through either seed coat abrasion or pulp removal. Seed ingestion by Knysna Turacos did not influence the rate at which seeds germinated in 75 % of the tree species while it did for ingested F. lutea, F. natalensis and F. sur seeds which germinated significantly sooner than whole fruit seeds. Future studies relating the composition of indigenous forest fruits to food preferences of Knysna Turacos may give insight into their role as potential seed dispersers of indigenous fruiting tree species. Chapter 7: Frugivorous animals play a major role in dispersing tropical, and to a lesser extent, temperate tree species. In order to attract potential seed dispersers, plants generally offer a reward of fleshy fruit pulp. Criteria for fruit choice by avian frugivores are influenced by a number of non-nutritive (e.g. fruit size and colour) factors; and nutritional composition of the fruit. There is a paucity of nutritional composition and other fruit trait data of indigenous South African fruit. This information is necessary in order to determine which frugivores are likely to ingest which fruits and consequently act as potential seed dispersal agents. This information would provide us with an understanding of the inter-relationships between indigenous fruit and frugivores in South Africa. Consequently nutritional composition was investigated in various indigenous fruit species that avian frugivores feed on. Fruits were collected from 38 indigenous tree species found in KwaZulu-Natal afromontane and coastal forests. Pulp was freeze-dried to constant mass and then analyzed for sugar, lipid and protein content; and for water content determination. Fruit width in this study ranged from 4 mm (Searsia rehmanniana and Trema orientalis) to 40 mm (Annona senegalensis, Ficus sur and Xylotheca kraussiana). Of the fruits examined in this study 29% were black and 43% red when ripe. Most (84%) fruit species analysed for sugar content were hexose dominant with 50% being fructose and 34% being glucose dominant. Only 16% of the fruit species analysed were sucrose dominant. Fruits in this study were generally observed to be high (mean: 68.1 ± 3.3%; n = 30) in water content; and low in protein and lipid content respectively (mean: 8.2 ± 0.5%; 9.3 ± 2.2%; n = 30) indicating that these fruits species could be considered as nutrient-dilute. Future studies need to determine the nutritional composition of the remaining indigenous South Africa fruit in order to develop a comprehensive database as well as examining non-nutritive factors. Together this information would play a key role in understanding fruit preferences and consequently seed dispersal and would therefore be useful in developing management plans for forest conservation. Chapter 8: Many seasonal thermoregulation studies have been conducted on Holarctic birds that live in predictable, highly seasonal climates with severe winters. However, relatively few studies have been conducted on their southern hemisphere Afrotropical counterparts that encounter less predictable climates with milder winters. These latter birds are expected to conserve energy in winter by down-regulating their metabolic rates. Therefore in this study, metabolic rate was measured during summer and winter in Knysna Turaco, Tauraco corythaix (Musophagiformes, Musophagidae) (c. 310 g), a non-passerine, in order to test whether there is energy conservation in winter. No overall significant differences in resting metabolic rates over a range of ambient temperatures were observed between winter and summer. However, whole-organism basal metabolic rates were 18.5 % higher (p = 0.005) in winter than in summer (210.83 ± 15.97 vs. 186.70 ± 10.52 O2 h-1). Knysna Turacos had broad thermoneutral zones ranging from 20-28 oC in winter and 10-30 oC in summer. These results suggest that Knysna Turacos show seasonal thermoregulatory responses that represent cold defense rather than energy conservation, which is contrary to what was expected. Keywords: sugar digestion, sugar preference, seed packaging, basal metabolic rate (BMR), resting metabolic rate (RMR), phenotypic flexibility, indigenous fruit, germination percentage, germination rate, fruit sugars, protein, lipids, fruit size, fruit colour, Knysna Turaco, Purple-crested Turaco. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Anaerobic digestion is a process that simultaneously treats waste and produces renewable energy in the form of biogas. Applications include swine and cattle waste management, which is still dominated by aerobic digestion, a less environmental alternative. The low adoption rates of anaerobic digestion is partly caused by the lack of modelling basis for the technology. This is due to the complexity of the process, as it involves dozens of interrelated biochemical reactions driven by hundreds of species of micro-organisms, immersed in a three-phase, non-Newtonian fluid. As a consequence, no practical computer models exist, and therefore, unlike most other engineering fields, the design process for anaerobic digesters still relies heavily on traditional methods such as trial and error. The current state-of-the-art model is Anaerobic Digestion Model No. 1 (ADM1), published by the International Water Association in 2001. ADM1 is a bulk model, therefore it does not account for the effects of concentration gradients, stagnation regions, and particle settling. To address this, this thesis works toward the creation of the first three-dimensional spatially resolved anaerobic digestion model, called Anaerobic Digestion Model with Multi-Dimensional Architecture (ADM-MDA), by developing a framework. The framework, called Coupled Reaction-Advection Flow Transient Solver (CRAFTS), is a general reaction solver for single-phase, incompressible fluid flows. It is a novel partial differential and algebraic equation (PDAE) solver that also employs a novel programmable logic controller (PLC) emulator, allowing users to define their own control logic. All aspects of the framework are verified for proper function, but still need validation against experimental results. The biochemistry from ADM1 is input into CRAFTS, resulting in a manifestation of ADM-MDA; however the numerical stiffness of ADM1 is found to conflict with the second order accuracy of CRAFTS, and the resulting model can only operate under restricted conditions. Preliminary results show spatial effects predicted by the CRAFTS model, and non-observable in the bulk model, impact the digester in a non-trivial manner and lead to measurable differences in their respective outputs. A detailed discussion of suggested work to arrive at a practical spatially resolved anaerobic digestion model is also provided.

A static in vitro digestion method was developed as a strategy to understand the effects of modelled digestion on the solubility of nutrients and selected bioactive compounds of autoclaved and untreated wheat bran. Brans from common soft, hard, and durum wheats were evaluated. Results indicated that the in vitro digestion protocol was suitable and effective. Effects of gastrointestinal simulation were considerably larger than gastric digestion alone. Digestibility of the brans ranged from 28.1 to 47.9%. Digestibility of bran minerals, starch and protein was substantial compared to lesser, but still significant, effects on fibre. Fibre solubility was significantly enhanced due to autoclaving. Total phenolic content, free radical scavenging and metal chelation activity were all substantially increased in soluble digests. Yields of these factors indicated that digestion of wheat bran releases ample levels of antioxidants that would be available for absorption in the small intestine to promote beneficial health effects.

The effect of engineered nanoparticles (ENPs) mixture consisting of silver oxide, (Agg0[Silver Oxide Nanopartical], 20 nm), titanium dioxide, (TiO2[Titanium dioxide], 30-40 nm) and zinc oxide, (ZnO, 20 nm) compared with their bulk metal salts was evaluated against unspiked activated sludge (control) using 3 parallel pilot-scale treatment plants. The total concentration of the ionic species of Ag+ Ti[Silver + Titanium] and Zn(2+) in the effluent of the ENP spiked activated sludge (AS) was below limits of detection and> 99% of the spiked ENP were found in the waste activated sludge (WAS), whereas 39 – 58 % of Ag0[Silver Oxide Nanopartical], 51 – 63 % and 58 – 74 % of ZnO ion concentrations were recovered in the anaerobic digestate (AD) cake suggesting higher affinity of ENPs to WAS than to anaerobic digestate. ENPs induced a 2-fold increase of the microbial community specific oxygen uptake rate (SOUR) compared with the control and > 98 % of ammonia and 80 % of COD were removed from the AS suggesting that the heterotrophic biomass retained their ability to nitrify and degrade organic matter at the spiked ENP concentration. The floc size and cultivable microbial abundance was reduced in the ENP spiked AS with no apparent disruption of the overall AS process efficiency. However, scanning electron microscopic analysis clearly showed damage to specific microbial cells. The lipid fingerprint and 16S rRNA gene-based pyrosequencing evidenced the dominance of Proteobacteria, Firmicutes, and Bacteriodetes with a clear temporal shift in microbial community structure. The prominent nano-tolerant bacterial species identified were Acidovorax, Rhodoferax, and Comamonas whereas Methanocorpusculum and Methanosarcina were recovered in AS and were the dominant Archaea in the AD with 99 and 98 % similarities to the closest culturable relative. Their presence in the AS suggests tolerance to ENPs and oxygen-dependent respiration. V. fisheri activity was not sensitive to the ionic concentrations of the ENP or metal salt mixture in the digestate samples and illustrates the need to develop bioassay using indigenous wastewater microorganisms to detect the potential effect of ENP. Overall, unlike other xenobiotic compounds, ENPs can hasten the natural selection of microbial species in activated sludge and anaerobic digestion processes.

Many of the current methods of assessing anaerobic biodegradability of solid samples require sample modification prior to testing. Steps like sample drying, grinding, re-drying and re-grinding to 2mm or less make the test results difficult to apply to field conditions and could lead to oxygen exposure, possibly distorting the results. Finally, because of a small sample size of about 10-50g w/w, the test result may not be representative of the bulk material. A new tool dubbed ‘tube’ has been developed, made of 10 cm diameter PVC pipe measuring 43.5 cm long with 3600 ml capacity with caps at both ends. For easy sample introduction, one endcap is fixed while the other is screw capped. A distinctive feature is the wide neck opening of about 10 cm where solid samples can be introduced as is, without further sample modification. Research has proven the tube applicable across various types of solid organic waste and conditions provided that a suitable organic loading rate is determined. The tube is best operated using 5-7 days pre-digested digested sewage sludge as seed, with minimal mixing and without the addition of nutrients or alkali solution. The test result can be obtained within 4-6 days to 20 days, signifying a 50-75% and 95% substrate degradation, respectively. Irreproducibility seen in some experiments may not only be a function of the seed and the substrate. The organic loading rate (OLR) at which the test is conducted is also influential especially if test is conducted closer to its maximum OLR tolerance where anaerobic process is more erratic. The performance of a continuous reactor digesting on a similar substrate can be estimated using this new tool. Food waste is established by the tubes to have an ultimate methane potential (B0) of 0.45L CH4/g VS. The same substrate when digested in a continuous reactor will produce about (B) 0.32 L CH4/g VS. The first order rate constant for both systems; batch and continuous are identical at 0.12 to 0.28 d-1. First order kinetics is efficient at modelling the anaerobic degradation when the process is healthy but may be less reliable under an unstable process. This research recommends the use of kinetics in combination with the experimental data (e.g. HRT, OLR, yield) when planning and designing an industrial plant to avoid overdesign and unnecessary building, maintenance and operating costs.

Content of free- (FPC) and bound- phenolics (BPC) significantly (p<0.05) increased during mixing, fermenting and baking. Bread crust and crumb contained the highest FPC and BPC, respectively. Antioxidant activities (AOA) followed the trends of their respective phenolic contents. HPLC analysis demonstrated that different phenolic acids showed various responses to the bread-making process. Total anthocyanin content (TAC) was significantly (p<0.05) reduced through mixing and baking, but fermentation elevated the levels. Anthocyanin extract of purple wheat exerted higher AOA than those of common wheat. Digested purple wheat extracts after in-vitro digestion demonstrated significantly (p<0.05) higher AOA than common wheat. During in-vitro testing, extracts exhibited concentration-dependent effects, while the use of different cell lines exhibited varying levels of cellular antioxidant and pro-oxidant properties. Purple wheat demonstrated higher cytoprotectivity and cellular AOA than those of common wheat. Our findings suggest that purple wheat has the potential to act as functional food in bakery products.

Anaerobic digestion is a process that simultaneously treats waste and produces renewable energy in the form of biogas. Applications include swine and cattle waste management, which is still dominated by aerobic digestion, a less environmental alternative. The low adoption rates of anaerobic digestion is partly caused by the lack of modelling basis for the technology. This is due to the complexity of the process, as it involves dozens of interrelated biochemical reactions driven by hundreds of species of micro-organisms, immersed in a three-phase, non-Newtonian fluid. As a consequence, no practical computer models exist, and therefore, unlike most other engineering fields, the design process for anaerobic digesters still relies heavily on traditional methods such as trial and error. The current state-of-the-art model is Anaerobic Digestion Model No. 1 (ADM1), published by the International Water Association in 2001. ADM1 is a bulk model, therefore it does not account for the effects of concentration gradients, stagnation regions, and particle settling. To address this, this thesis works toward the creation of the first three-dimensional spatially resolved anaerobic digestion model, called Anaerobic Digestion Model with Multi-Dimensional Architecture (ADM-MDA), by developing a framework. The framework, called Coupled Reaction-Advection Flow Transient Solver (CRAFTS), is a general reaction solver for single-phase, incompressible fluid flows. It is a novel partial differential and algebraic equation (PDAE) solver that also employs a novel programmable logic controller (PLC) emulator, allowing users to define their own control logic. All aspects of the framework are verified for proper function, but still need validation against experimental results. The biochemistry from ADM1 is input into CRAFTS, resulting in a manifestation of ADM-MDA; however the numerical stiffness of ADM1 is found to conflict with the second order accuracy of CRAFTS, and the resulting model can only operate under restricted conditions. Preliminary results show spatial effects predicted by the CRAFTS model, and non-observable in the bulk model, impact the digester in a non-trivial manner and lead to measurable differences in their respective outputs. A detailed discussion of suggested work to arrive at a practical spatially resolved anaerobic digestion model is also provided.

A static in vitro digestion method was developed as a strategy to understand the effects of modelled digestion on the solubility of nutrients and selected bioactive compounds of autoclaved and untreated wheat bran. Brans from common soft, hard, and durum wheats were evaluated. Results indicated that the in vitro digestion protocol was suitable and effective. Effects of gastrointestinal simulation were considerably larger than gastric digestion alone. Digestibility of the brans ranged from 28.1 to 47.9%. Digestibility of bran minerals, starch and protein was substantial compared to lesser, but still significant, effects on fibre. Fibre solubility was significantly enhanced due to autoclaving. Total phenolic content, free radical scavenging and metal chelation activity were all substantially increased in soluble digests. Yields of these factors indicated that digestion of wheat bran releases ample levels of antioxidants that would be available for absorption in the small intestine to promote beneficial health effects.