Doctoral Degrees (Food Science)

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Impact of environmental factors on the metabolic profiles of Kefir produced using different Kefir grains and subsequent enrichment of Kefir prepared with mass cultured grains
(Stellenbosch : Stellenbosch University, 2012-03) Ntsame Affane, Armelle Lyvane; Britz, T. J.; Sigge, G. O.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
ENGLISH ABSTRACT: The fermentation of milk has been known for millennia and leads to nutritious and prolonged shelf-life dairy products. In Southern Africa, traditional fermented dairy products have the same value as local staple foods and are consumed as a part of or as a whole meal. However, the retail price and the technology make many commercialised fermented dairy products unaffordable to the majority of the population. There is thus a need for a healthy nutritious low-cost easily prepared fermented dairy product. A product that could be the answer to the above need, is Kefir. The principle advantage is that the Kefir beverage is made from reusable Kefir grains, which unfortunately are not easily available and grow slowly. Kefir grains can only be obtained from pre-existing grains, which presents a problem in the marketing of the grains. A mass culturing technique was developed to produce large masses of grains but preparation of Kefir using these grains results in a product (MG Kefir) lacking in the sensory attributes of Traditional Kefir. Thus, the overall objective of this research was to determine the impact of environmental factors on the metabolic profiles of Kefir produced using different Kefir grains and this was then followed by the subsequent enrichment of Kefir prepared with mass cultured grains so as to obtain a Kefir beverage that has improved organoleptic qualities. To determine the impact of environmental factors Traditional and MG Kefir were prepared under controlled and uncontrolled conditions. Traditional Kefir was found to give the best beverage and was thus considered as the control. Under controlled conditions the optimum incubation temperature for the production of Kefir was 22ºC as over-acidification was observed at 25ºC. The metabolic profiles of both Traditional and MG Kefir showed that both contained acetaldehyde, ethanol, acetone, diacetyl and acetic acid. In addition, the metabolic profiles revealed that an inadequate ratio of diacetyl to acetaldehyde as well as the lack of ethyl acetate was responsible for the flavour defect in MG Kefir. In order to overcome this defect, citrate and ascorbate (0.015 % w.v-1) were added during Kefir fermentation to enhance the diacetyl and ethyl acetate production. This addition showed a positive impact on diacetyl but not on ethyl acetate production. Improvement of the overall flavour of Kefir was observed as the ratios of diacetyl to acetaldehyde were higher (0.21 – 0.5) in the samples with added citrate and ascorbate than in the samples without (0.12 – 0.17). The production of ethyl acetate in MG Kefir was enhanced by combining the effects of longer incubation (24 h + 18 h at 22ºC), addition of ethanol and acetic acid at 0.79% (m.v-1) and the addition of either Lactococcus lactis ssp. diacetylactis biovar diacetylactis 318 or Candida kefyr 1283. The best yields were obtained in samples containing C. kefyr 1283 and only added ethanol (9.22 mg.L-1), indicating that ethanol is an important factor in ethyl acetate production by Kefir starter strains and suggesting that the absence of ethyl acetate is an indication that the grains do not contain a yeast capable of producing sufficient ethyl acetate. During this investigation, the impact of ethyl acetate on the organoleptic quality of Kefir during storage at refrigerated and room temperatures were also studied. The results indicated that refrigerated Kefir contained up to 40 mg.L-1 of ethyl acetate and was not found defective and thus ethyl acetate was considered a positive contributor to Kefir flavour. This is of particular interest as ethyl acetate is a potent flavour compound at concentrations below 5 mg.L-1. Improvements of MG Kefir’s flavour were successful and will be of value for commercial Kefir production where the main aim is to optimise the flavour of Kefir. However, stabilising the grain microbial consortium was found to be important as it is responsible, over time, for both stable and acceptable Kefir. Acceptability of Traditional, MG and other Kefirs (Candi-Kefir and Lacto-Kefir) prepared with microbially stabilised MG was evaluated by 85 consumers. Results indicated that pH (r = 0.978; p < 0.05) was a significant driver of liking for flavour, especially for female consumers (r = 0.982; p < 0.05). In addition, three clusters, each characterised by different liking attributes were identified. Cluster I generally disliked all the products whereas slight acidic Kefir such as Candi-Kefir (7.63) and Lacto-Kefir (7.09) were preferred by Cluster III. Cluster II showed preference to Kefir with moderate acidity and high ethanol content. In that regard, Traditional Kefir obtained the best score (7.50) and MG Kefir the lowest score (4.87). The sensory study is of value as it led to the identification of the drivers of consumers liking by the different types of consumers. In the course of this project, near infrared reflectance spectroscopy was developed as a rapid method to estimate lactic and acetic acids, which are the organic acids responsible for acidity in Kefir, as well as pH and titratable acidity (TA). The results showed that the calibration models for lactic acid (RPD = 2.57 – 3.16), pH (RPD = 2.90) and TA (RPD = 2.60) were good for screening purposes (2 < RPD < 3); indicating that these models would show if the concentrations of lactic acid, the pH or the TA varied from the normal range. This study has demonstrated that the flavour of MG Kefir, prepared with enriched grains, was successfully improved and has provided some understanding on the preference liking of Kefir, an unknown fermented dairy product to South African consumers.
Impact of low-frequency high-power ultrasound on spoilage and potentially pathogenic dairy microbes
(Stellenbosch: University of Stellenbosch, 2007-12) Cameron, Michelle; Britz, T. J.; McMaster, L. D.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Food Science.
Thermal pasteurisation failures in the dairy industry have often been found to cause end-products of poor quality and short shelf-life. Therefore, alternative methods to eliminate microbial contaminants in raw milk are being studied. Ultrasonication is one such non-thermal technology that could offer the dairy industry an alternative to traditional pasteurisation. The main objective of this dissertation was to evaluate the use of high-power lowfrequency ultrasound (20 kHz, 750 W, 124 μm) applied in batch mode to eliminate a selection of spoilage and potentially pathogenic microbes, commonly associated with milk. These included Gram-positive and negative microbes, comprising of rods and cocci, an endospore-former, and a yeast (Escherichia coli, Bacillus cereus, Chryseobacterium meningosepticum, Lactobacillus acidophilus, Lactococcus lactis, Listeria monocytogenes, Micrococcus luteus, Pseudomonas fluorescens and Saccharomyces cerevisiae). Three strains of E. coli (1 x 106 cfu.ml-1) tested, viz. ATCC 11775, a wild strain from raw milk, and an O157:H7 strain from milk were sensitive to ultrasonication. Complete elimination of viable cells occurred within 10 min. Viable counts of P. fluorescens were reduced by 100% within 6 min of ultrasonication and L. monocytogenes was reduced by 99.0% within 10 min. Lactococcus lactis was reduced by 97.0% and M. luteus, B. cereus and C. meningosepticum by 88.0%, 87.0% and 85.0% respectively. Lactobacillus acidophilus showed the most resistance to ultrasound with only 78.0% of viable cells being eliminated. Under similar conditions, S. cerevisiae was reduced by 99.7%. Microbial cell morphology, size and Gram status did not necessarily influence the efficacy of ultrasonication. Sterile saline solution and UHT milk were used as the suspension media, and the reputed protective effect of milk fat was not observed under the parameters used in this study. A higher wave amplitude (100%; 124 μm) was found to be more efficient in eliminating microbes than a lower wave amplitude (50%; 62 μm). Pulsed-ultrasonication did not enhance the efficiency of ultrasonication indicating that standing waves were absent. Limited success was achieved by ultrasonication itself, and the long batch treatment time (10 min or more) was found to be unrealistic for industrial implementation. Hence the simultaneous application of ultrasound and heat (thermoultrasonication) was examined. Thermo-ultrasonication proved to be more effective than either an ultrasonic or heat treatment with all viable M. luteus cells being eliminated within 4 min (100% amplitude at 72°C). Similarly, to eliminate E. coli and Lb. acidophilus from milk, only 2 min and 4 min thermo-ultrasonication was required, respectively. Bacillus cereus endospores remained resistant and after a 10 min thermo-ultrasonic treatment only 78.04% were eliminated. During this investigation both extensive surface (SEM) and internal (TEM) cell damage caused by ultrasonication were observed in E. coli, Lb. acidophilus and S. cerevisiae. Hence ultrasonication physically/mechanically damages these microbial cells causing cell death/injury. Microbial proteins and DNA released from cells into the environment after an ultrasonic treatment was measured and an increase in released microbial proteins and DNA was found to be indicative of a decrease in the number of viable cells, providing that the initial cell concentration was high enough. It was, however, not possible to correlate the concentration of released microbial proteins and DNA with the exact number of viable cells eliminated, rendering it an ineffective quality indicator for the industry. Ultrasonication had no statistically significant influence on the protein, fat and lactose content of both raw and pasteurised milk. The somatic cell count of raw and pasteurised milk was found to decrease after ultrasonication. Unlike with heating, activity of alkaline phosphatase and lactoperoxidase were not reduced by ultrasonication. Hence neither enzyme can be used to indicate a successful ultrasonic treatment of milk. This study has demonstrated that ultrasonication offers a viable alternative to pasteurisation as it is effective in eliminating microbes, and does not alter native milk components. However, to attain a more effective killing, thermo-ultrasonication is recommended for the treatment of milk to be used for the production of different dairy products.
Integration of anaerobic biological and advanced chemical oxidation processes to facilitate biodegradation of fruit canning and winery wastewaters
(Stellenbosch : Stellenbosch University, 2005-03) Sigge, G. O.; Britz, T. J.; Fourie, P. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science .
ENGLISH ABSTRACT: Please see fulltext for abstract
Near infrared (NIR) hyperspectral imaging and X-ray computed tomography combined with statistical and multivariate data analysis to study Fusarium infection in maize
(Stellenbosch : Stellenbosch University, 2013-03) Williams, Paul James; Manley, Marena; Britz, T. J.; Geladi, Paul; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
ENGLISH ABSTRACT: Maize (Zea mays L.) is used for human and animal consumption in diverse forms, from specialised foods in developed countries, to staple food in developing countries. Unfortunately, maize is prone to infection by different Fusarium species that can produce harmful mycotoxins. Fusarium verticillioides is capable of asymptomatic infection, where infected kernels show no sign of fungal growth, but are contaminated with mycotoxins. If fungal contamination is not detected early on, mycotoxins can enter the food chain. Rapid and accurate methods are required to detect, identify and distinguish between pathogens to enable swift decisions regarding the fate of a batch or consignment of cereal. Near infrared (NIR) hyperspectral imaging and multivariate image analysis (MIA) were evaluated to investigate the fungal development in maize kernels over time. When plotting principal component (PC) 4 against PC5, with percentages sum of squares (%SS) 0.49% and 0.34%, three distinct clusters were apparent in the score plot and this was associated with degree of infection. Prominent peaks at 1900 nm and 2136 nm confirmed that the source of variation was due to changes in starch and protein. Variable importance plots (VIP) confirmed the peaks observed in the PCA loading line plots. Early detection of fungal contamination and activity (20 h after inoculation) was possible before visual symptoms of infection appeared. Using NIR hyperspectral imaging and MIA it was possible to differentiate between species of Fusarium associated with maize. It was additionally applied to examine the fungal growth kinetics on culture media. Partial least squares discriminant analysis (PLS-DA) prediction results showed that it was possible to discriminate between species, with F. verticillioides the least correctly predicted (between 16-47% pixels correctly predicted). For F. subglutinans 78-100% and for F. proliferatum 60-80% pixels were correctly predicted. Three prominent bands at 1166, 1380 and 1918 nm were considered to be responsible for the differences between the growth zones. Variations in the bands at 1166 and 1380 nm were correlated with the depletion of carbohydrates as the fungus grew while the band at 1918 nm was a possible indication of spore and new mycelial formation. By plotting the pixels from the individual growth zones as a function of time, it was possible to visualise the emergence and interaction of the growth zones as separate growth profiles. The microstructure of fungal infected maize kernels was studied over time using high resolution X-ray micro-computed tomography (μCT). The presence of voids and airspaces could be seen in two dimensional (2D) X-ray transmission images and in the three dimensional (3D) tomograms. Clear differences were detected between kernels imaged after 20 and 596 h of inoculation. This difference in voids as the fungus progressed showed the effect of fungal damage on the microstructure of the maize kernels. Imaging techniques are important for rapid, accurate and objective evaluation of products for quality and safety. NIR hyperspectral imaging offers rapid chemical evaluation of samples in 2D images while μCT offers 3D microstructural information. By combining these image techniques more value was added and this led to a comprehensive evaluation of Fusarium infection in maize.
PCR detection, denaturing gradient gel electrophoresis (DGGE) fingerprinting and identification of the microbial consortium in different types of UASB granules
(Stellenbosch: University of Stellenbosch, 2006-12) Keyser, Maricel; Witthuhn, R. C.; Britz, T. J.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Food Science.
High-rate anaerobic bioreactors are used for the treatment of various wastewaters, of which the upflow anaerobic sludge blanket (UASB) bioreactor has the widest application, especially in the food and beverage industries. In an UASB bioreactor sludge develops in a particular granular or flocculent form and the success of the anaerobic process relies on the formation of active and settable granules. These granules are formed by self-aggregation of bacteria that can be divided into different trophic groups that are responsible for the metabolic breakdown of organic substrates. The successful performance of a bioreactor is influenced by the composition of the substrate which subsequently may have an impact on the microbial consortium present in the UASB granules. In order to determine if a change in the structure of the non-methanogenic microbial community takes place, UASB brewery granules were subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium (5 g.l-1). No changes in the microbial community were observed when the granules were stressed with glucose medium as carbon source, regardless of an increase in the glucose concentration. In order to better understand the effect that different wastewaters may have on the microbial consortium present in different UASB granules, the polymerase chain reaction (PCR) based denaturing gradient gel electrophoresis (DGGE) technique and sequence analysis were used to fingerprint and identify the Bacteria and Archaea present in either, winery, brewery, distillery or peach-lye canning UASB granules. Each granule type showed distinct PCR-based DGGE fingerprints with unique bands, while other bands were found to be present in all the granules regardless of the wastewater being treated. Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes, Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas, Nitrospira, Synergistes, Rhodococcus, Rhodocyclus, Syntrophobacter and uncultured bacteria were identified, representing different acidogenic, acetogenic and homoacetogenic Bacteria.Different methanogenic bacteria such as Methanosaeta, Methanosarcina, Methanobacterium and uncultured bacteria belonging to the group Archaea were also fingerprinted and identified from different UASB granules. In both these studies a DGGE marker was constructed that may be used to assist in the identification of bacteria. The DGGE marker can also be used to monitor the presence of bacteria over a time period during anaerobic digestion. Bioaugmentation or the enrichment of granules results in tailor-made granules that may be used for the treatment of specific wastewaters. One of the most important contributions to the maintenance and enhancement of UASB granule formation is the inclusion of suitable microbes in the granule structure. Enterobacter sakazakii was isolated from raw winery wastewater and was found to produce sufficient amounts of desired fatty acids. This bacteria was, therefore, incorporated into batch cultured granular sludge. In order to identify and monitor the presence of the incorporated E. sakazakii in the tailor-made granules, 16S rRNA gene sequence primers and PCR conditions were developed. The use of molecular techniques such as PCR-based DGGE and sequence analysis proved to be successful methods to fingerprint and identify the microbial consortium present in the different UASB granules.
Studies of traditional cheese and fermented milks
(Stellenbosch : Stellenbosch University, 2001-12) Robinson, R. K. (Richard Kenneth); Britz, T. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
ENGLISH ABSTRACT: One of the curious facts about the food industry is that many of the processes in use today were being practised, in some form or other, by the Roman legions as they marched across Europe and beyond. Certainly they were familiar with the basic techniques of fermentation, and much current research into fermented foods is concerned with understanding the fundamental nature of these traditional processes, and how the individual stages in a particular fermentation can be better controlled. Recent developments in the dairy industry have tended to reflect this pattern and, over the years, my research group has done much to support the expanding markets for yoghurt and similar fermented milks. Our evaluation of the polysaccharide-producing characteristics of starter cultures, for example, encouraged yoghurt manufacturers to match physical properties to the perceived demands of consumers, and most culture suppliers followed this lead by labelling their products with precise designations as to their potential for imparting viscosity to a retail item. Similarly, my group was the first to record the unique physical properties of the concentrated yoghurt, labneh, C 230 g 1-1 total solids) that had been made for hundreds of years by draining whey from natural yoghurt hanging in a cloth or animal-skin bag. This detailed analysis of the product facilitated the application of ultra-filtration to natural yoghurt to generate a product with a quality that matched traditionallabneh and, today, factories in the Middle East, Greece and elsewhere are using modern membrane-filtration plants to satisfy a growing market demand. Our success in publicising the attractive properties of concentrated yoghurt encouraged me to devote time to yet another 'historical' concept, namely the apparent 'health benefits' derived by small communities in Eastern Europe from consuming kefir and koumiss. In the West, the flavour and texture of these latter products have never been accepted, but employing similar cultures to produce 'health-promoting' bio-yoghurts opened an entirely new avenue for research. As clinical evidence in support of the prophylactic and therapeutic properties of Lactobacillus acidophilus and a species of Bifidobacterium became available, so it became apparent that the therapeutic advantage that accompanies the regular ingestion of 'bio-yoghurts' depended on the survival of these microfloras over the stipulated shelf-lives of the retail vehicles. However, no laboratory medium was immediately available for the simultaneous enumeration of Lb. acidophilus and Bifidobacterium along with the yoghurt cultures, i.e. Streptococcus thermophilus and Lactobacillus delbrueckii sub-sp. bulgaricus. Designing such a medium became a priority for one of my students, and, even today, the procedures that he derived are being used by consumer groups that monitor the performance of the major dairy companies in England. If the improved quality of yoghurts and 'bio-yoghurts' had a major impact on consumer perceptions of fermented milks, the food sector in England gradually became aware of an even more dramatic change in consumer attitudes. Thus twenty years ago, cheese meant 'Cheddar' but, following a 'deluge' of television publicity about the attractions of 'exotic' catering, housewives began demanding mozzarella and mascarpone for lavish desserts, Feta to sprinkle over salads and Halloumi to grill or fry. In turn, exporting countries like Italy, Greece and Cyprus came under intense pressure to increase supplies of top quality products. Local manufacturers soon realised, however, that there was little information available concerning the scientific basis to the procedures employed to make some of these traditional cheeses, and my research group was selected by Funding Agencies in Greece and Cyprus to act as a focus for a series of studies of Feta and Halloumi cheese. The need to eliminate pathogens from the storage brines of Feta cheese without killing the yeasts and bacteria associated with maturation became an important consideration for exporters, and one of my students exploited a novel procedure employing furocoumarins and long-wave ultra-violet light to achieve the desired selective inactivation. At present, the economics of commercial application are somewhat dubious but, as soon as cheap, synthetic, non-toxic furocoumarins become more readily available, the system may well merit re-evaluation. We did confirm, however, that the metabolic activities of the yeasts and bacteria typically isolated from storage brines are essential for flavour development in Feta cheese, and that similar microfloras are instrumental in the development of the important charactistics of traditional Halloumi cheese. In particular, a new species of lactic acid bacterium, Lactobacillus cypricasei, was isolated from samples of the traditional ovine cheese, but whether or not the species has a unique role(s) in the maturation process remains an open question. Clearly there is still much to learn but, if the activities of my reseach group have added just a little to the scientific background essential for future studies of cheese and fermented milks, then their completion will have been worthwhile.
Thin monolithic slow-release devices for optimum in-package preservation of export table grape varieties
(Stellenbosch : Stellenbosch University, 2002-03) Opperman, Willem Jacobus; Sanderson, R. D.; Britz, T. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
ENGLISH ABSTRACT: Prototypes of a new polymer S02 gas-generating sheet for the control of Botrytis cinerea during the post-harvest storage of table grapes, were developed and manufactured for evaluation using a pilot scale production plant. Attention was paid to the appearance of the sheet, in order to make it technologically efficient as well as aesthetically acceptable to both industry and consumers. The storage quality of semi-commercial export consignments of various cultivars table grapes packed with the monolithic thin-film polymer S02 slow release sheet, was evaluated and compared to results obtained using the locally manufactured Uvasys S02 sheet. The following were investigated: the efficacy of the new polymer sheets in controlling storage decay, the stage at which S02 damage is manifested on table grapes, the level of S02 damage associated with different S02 concentrations, whether S02 damage is manifested more readily at a particular position on the bunch, and the possible effect of an increase in storage temperature, from an initial storage at -O.5°C to 10°C, on the levels of S02 bleaching. Results showed that the new polymer S02 sheet compared favourably with the existing, commercially available Uvasys S02 sheets. The exact S02 concentration required for effective decay control varied for different cultivars, as well as for the different types of grape packages. The S02 concentration incorporated within the sheet was shown to be lower for grapes packed in non-perforated bags, and slightly higher for those in perforated bags. Differences between cultivars occurred with regard to the level of control and the levels of S02 damage. Levels of S02 damage were also significantly affected by the storage period and temperature fluctuations. No significant differences in the levels of decay development and S02 damage were observed in relation to the orientation of the bunches in the carton. The extent of damage incurred to grape tissue by the absorption of S02 gas was determined by low-temperature scanning (LTSEM) and transmission electron microscopy (TEM) techniques. LTSEM and TEM micrographs of areas damaged by S02 gas revealed that exposure to S02 gas may lead to plasmolysis and the loss of cellular fluids. Although damage to the cell walls, cell wall structures and cell membranes, caused by S02 gas, was more prominent in the tissue layers nearer to the fruit surface, damage also occurred to a lesser extent in deeper tissue layers. S02 gas release-rate studies of polymer S02 sheets containing various concentrations Na2S205 revealed that levels of S02 gas emitted depended largely on the levels of Na2S205 incorporated into the sheets. Higher levels of S02 gas were released with the polymer sheets of higher concentrations Na2S205. The release curve for the commercial Uvasys S02 sheet was very different to that of the polymer sheets, with much higher levels of S02 gas emitted initially by the Uvasys S02 sheet compared to the polymer sheets, while the polymer sheets emitted low levels of S02 gas for longer periods compared to the Uvasys S02 sheet. The manufacturing process and the pilot scale production plant that was developed and constructed was successfully used to manufacture polymer S02 generating sheets that are technically sound and efficient, and aesthetically acceptable to industry. The efficacy of such sheets, regarding levels of decay control and S02 damage, was similar to that obtained with the presently available, commercially used Uvasys S02 sheet.
UASB granulation enhancement by microbial inoculum selection and process induction
(Stellenbosch: University of Stellenbosch, 2009-03) Lamprecht, Corne; Britz, T. J.; Witthuhn, R. C.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Food Science.
In the absence of anaerobic granules, anaerobically digested sewage sludge is frequently used to seed industrial upflow anaerobic sludge blanket (UASB) reactors. Because of its flocculent nature, start-up with digested sludge instead of granular sludge proceeds much slower and presents various operational problems. Any manner in which the granulation of digested sludge can be enhanced would benefit UASB reactor start-up and application in developing countries such as South Africa. The main objective of this dissertation was to improve granulation and reduce UASB reactor start-up by using pre-treated digested sludge as seed. The sludge was pre-treated based on the batch granulation-enhancement model of Britz et al. (2002). The main aim of the model was to improve extracellular polymer (ECP) production of lactate-utilising populations by applying short-term controlled organic overloading in a mechanically agitated environment. The batch granulation-enhancement (pre-treatment) process was applied to an ECP-producing digester strain, Propionibacterium jensenii S1. Non-methanogenic aggregates were formed when batch units were incubated on a roller-table instead of a linear-shake platform. Larger, more stable aggregates were obtained in the presence of apricot effluent medium. Preliminary batch granulation-enhancement studies confirmed that using the roller-table as mixing system had a positive influence on batch granulation-enhancement. The roller-table showed the most potential for handling larger volumes in comparison to a linear-shake waterbath and linear-shake platform. The addition of 450 mg.L-1 Fe2+ at the start of the study also influenced aggregate numbers positively. These studies revealed that pre-treatment results varied depending on the seed sludge source. A denaturing gradient gel electrophoresis (DGGE) method was applied for the detection of Archaea in digested sludges and UASB granules. In addition, a methanogenic marker containing methanogens important to the granulation process was constructed to aid identification. The positive influence of DMSO and “touchdown” PCR on the elimination of artifactual double bands in DGGE fingerprints were also demonstrated. Results revealed that only one of the four digested sludges tested contained Methanosaeta concilii (critical to granular nuclei formation) while it was present in all the UASB granules regardless of substrate type. Four digested sludges were obtained from stable secondary digesters. DGGE indicated the presence of M. concilii in all sludges. The Athlone 4Sb-sludge was the only sludge which exhibited measurable methanogenic activity during substrate dependent activity testing. The ST-sludge showed the highest increase in volatile suspended solids (VSS) particles ≥0.25 mm2. Laboratory-scale UASB reactor start-up was done with both sludges and start-up proceeded better in the Athlone 4Sb-reactor. Athlone 4Sb-sludge batches were pre-treated in a rolling-batch reactor in the presence of either lactate or sucrose and used to seed lab-scale UASB reactors B (sucrose seed) and C (lactate seed). Start-up efficiencies were compared to a control (Reactor A). Overall Reactor B was more efficient that the control. At the end of the study the Reactor B sludge had a higher methanogenic activity than the control reactor. It also had the highest increase in VSS ≥1.0 mm2. Pre-treatment of digested sludge in the presence of sucrose, therefore, aided granulation and reduced UASB reactor start-up time.

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