Thesis (MScEng (Process Engineering))--Stellenbosch University, 2008. / Nitriles are precursors of important amides and organic acids (e.g. acrylamide, nicotinamide, mandelic acid and acrylic acid) which are used, inter alia, as food additives, in plasticisers, detergents, make-up, medicine and as chemical intermediates in the production of various important polymers. Traditionally, chemical processes are used to convert nitriles to amides and organic acids but these processes are non-specific causing various by-products to form. Chemical processes are also environmentally unfriendly and require harsh conditions. Nitrile conversions through an enzymatic route, on the other hand, have the distinct advantages of excellent chemo-, regio- and stereo selectivities, mild process conditions and reduced downstream processing costs. The enzymatic process is mediated via an initial nitrilase catalysed conversion to amide, followed by an amidase catalysed conversion to acid. This research focused on the latter part of the enzymatic transformation of nitriles, which is the amidase catalysed biotransformation of an amide to an acid, specifically with respect to the development of a membrane immobilised amidase continuous process which has the major advantage of enzyme retention coupled with product separation. The research was conducted in three parts namely the characterisation of the free amidase, the development of the experimental bioreactor system and the quantification of the membrane immobilised amidase process.

Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.

Plastics are highly useful economically because of their resistance to diverse types of environmental and chemical agents and their ability to be molded into many types of products. Globally, plastic production is greater than 20 million metric tons per year. However, their widespread use and often their disposable nature results in significant plastic accumulation in the environment. Plastics are made of hydrocarbons, materials that are biodegradable depending on their molecular structure and size. It is hypothesized that pre-treatment of plastic materials could enhance their bioavailability, facilitating their microbial biodegradation. In this dissertation, a process was developed to treat nylon 6,6 polymers by acid hydrolysis to produce a microbial growth medium. The chemical composition of the medium was determined by low pressure liquid chromatography-spectrophotometry and electrospray ionization mass spectrometry and found that the medium was a mixture of molecules with molecular weight > 800 m/z and with similar chemical characteristics to polyamines. There was steady growth of Pseudomonas putida KT2440 in the medium with concomitant substrate biodegradation. Notably, the yeast Yarowia lipolytica grew well in the medium when supplemented with yeast extract. A similar medium derived from nylon 6,6 containing nylon-derived particles supported the growth of Beijerinckia sp. and Streptomyces sp. BAS1. Confocal laser scanning microscopy and flame ionization gas chromatography were used to identify and quantify the production of polyhydroxybutyrate, a type of “bioplastic”. The aforementioned microorganisms were cultivated in a bench-scale bioreactor that was developed as part of this dissertation. The bioreactor had a novel impeller design resulting in enhanced mixing and rotation and also a modular format allowing for diverse configurations. The bioreactor was notable for its durability and low cost. A detailed description of its design is included in the appendices. In summary, plastic materials can potentially be processed into growth media for microorganisms and can be used for production of value-added products. The media described herein can be used in bioconversion processes using a bioreactor.

Cardiovascular disease (CVD) affects more than 70 million Americans and is the number one cause of mortality in the United States. Because the regenerative capacity of adult tissues such as the heart is limited, human embryonic stem cells (hESC) have emerged as a source for potential cardiac therapies. However, despite the use of a variety of biochemical differentiation protocols, current yields of hESC-derived cardiomyocytes (CM) have been low. In the case of hESC-CM, which are inherently electromechanically active, additional forms of inducing a mature cardiac fate have not been fully explored. In order to non-invasively visualize and quantify biochemical, electrical, and mechanical stimulation on hESC-CM differentiation in future studies, a bioreactor imaging system has been developed and is described in this report. / text

<p>This master's theses was carried out on assignment from Stockholm Vatten AB as a part of a project developing new waste water treatment techniques. The goal of the theisis has been to evaluate an anaerobic membrane bioreactor for treatment of waste water from Hammarby Sjöstad. The bioreactor has not been heated and the main interest has been to study the gas production, power consumption and the reduction of organic matter and nutrients.</p><p>The system has been completed with a reverse osmosis unit and a total of four batch runs have been made with good results. The use of reverse osmosis allows nutrient in the waste water to be reintroduced into circulation as the reverse osmosis concentrate can be used as crop nutrient.</p><p>The membrane unit is of VSEP ("Vibratory Shear Enhanced Processing") type and an extensive membrane test has been conducted. This so called L-test helped determine the most suitable type of membrane for the system to allow a higher ±ux and thus lower power consumption. The L-test gave good results and a new membrane with a poresize diameter of 0,45 μm was used.</p><p>The organic load on the bioreactor has been more or less constant, around 0,7 kg COD/day, during the seven weeks of testing. The reduction over the entire system including reverse osmosis has been large, around 99 % regarding organic matter and phosporus and 93 % for nitrogen, making the system suitable for waste water treatment except for high power consumption, around 2 kWh/m3. The production of methanegas has worked although it has been quite low, with average values of 0,13 m3 CH4/kg reduced COD.</p> / <p>Examensarbetet är utfört på uppdrag av Stockholm Vatten AB som en del av det pilotprojekt som utvärderar nya tekniker för avloppsvattenrening för Hammarby Sjöstad. Målsättningen med studien har varit att utvärdera ett system bestående av en anaerob membranbioreaktor för behandling av avloppsvatten från Hammarby Sjöstad. Bioreaktorn har inte varit uppvärmd och det som har studerats är reningseffekten, biogasproduktionen samt energiåtgången.</p><p>Systemet har även innefattat en omvänd osmosanläggning och totalt har fyra försök med denna gjorts med goda resultat. Analyser har koncentrerats till att utvärdera reduktion av organiskt material över membranbioreaktorn och av närsalter och metaller över omvänd osmos anläggningen. Bakgrunden till att använda omvänd osmos är att öka återföringen av näringsämnen från avloppsvatten. Resultatet av försöken med omvänd osmos gav ett koncentrat med högt näringsinnehåll och låg halt av tungmetaller vilket ger möjligheten att sprida det på åkermark.</p><p>Membranenheten är av typen VSEP ("Vibratory Shear Enhanced Processing") och ett membrantest har även utfötts för att finna det membran som passar systemet bäst med avseende på flöde och energiförbrukning. Det så kallade L-testet var omfattande och gav en klar bild över vad som skulle vara det bästa membranet. Det membran som visade sig passa systemet bäst var ett membran med en porstorlek på 0,45 μm. Belastningen av organiskt material på reaktorn under försöksperiodens sju veckor har varit mer eller mindre konstant och låg, cirka 0,7 kg COD/dygn. Reduktionen över hela systemet inklusive omvänd osmosanläggningen med avseende på organiskt material och fosfor har varit mycket hög, omkring 99 %. Reduktionen av kväve var som högst 93 %. Gasproduktionen har fungerat och har i genomsnitt varit omkring 0,13 m3 CH4/kg reducerad COD.</p><p>Energiförbrukningen för systemet i motsvarande fullskala blev omkring 2 kwh/m3.</p>

The membrane aerated bioreactor (MABR) is a unique technological innovation where a gas permeable membrane is applied to biological processes. In an MABR, oxygen and other substrates diffuse from the opposite directions into a biofilm, and thus simultaneous chemical oxygen demand (COD) and nitrogen removal can be achieved. However, controlling biofilm thickness, stability, and attachment is challenging. The objectives of this research were to study the effect of oxygen partial pressure on process performance with respect to nitrogen removal and examine the biomass properties in MABRs at different oxygen partial pressures and COD loadings. The conditions within the bioreactors were based on a low hydrodynamic condition (average fluid velocity 22 cm/min along the membrane surface), with the intention of minimizing the impact of the hydrodynamic shear on biomass properties. Simultaneous nitrification and denitrification were achieved in the reactors, and increasing oxygen partial pressure enhanced the total nitrogen removal. The biomass at the membrane-biofilm interface was more porous at a loading of 11.3 kg COD/1000 m2/day (areal porosity about 0.9) as compared with a loading of 22.6 kg COD/1000 m2/day (areal porosity about 0.7), indicating carbon substrate was limiting near the membrane. Long-term (over 30 days) experimental results showed that at the loading of 11.3 kg COD/1000 m2/day, the oxygen partial pressures of 0.59 atm and 0.88 atm caused over 80% of the biomass to become suspended in the bulk phase while at 0.25 atm and 0.41 atm oxygen over 97% of the biomass was immobilized on the membrane. There is a critical oxygen partial pressure that can sustain the biofilm, which increases with an increasing COD loading. The nitrifying population in the reactors was examined by applying fluorescence in situ hybridization (FISH). At the loading of 22.6 kg COD/1000 m2/day, there were 12% beta-proteobacterial ammonia oxidizing bacteria (AOB) and 17%Nitrobacter in homogenized biofilm biomass at 0.59 atm oxygen while there were 7% beta-proteobacterial AOB and 4% Nitrobacter at 0.25 atm oxygen. The ratio of protein to carbohydrate in extracellular polymeric substances (EPS) of the homogenized biomass in the reactor decreased with increasing oxygen partial pressure. Surface characterization of the biomass revealed that the higher the oxygen partial pressure, the lower the biomass hydrophobicity and surface charge. The ratio of EPS protein to carbohydrate in a membrane aerated biofilm decreased when approaching the membrane-biofilm interface. The distribution of nitrifiers and dissolved oxygen profiles inside the biofilm suggested that dual substrate limitations exist, and it was concluded that the membrane aerated biofilm had an aerobic region in the inner layer and an anoxic region in the outer layer. It is proposed that the loss of EPS due to secondary substrate consumption, especially the loss of EPS proteins, at the bottom of the biofilm was responsible for biofilm detachment subjected to a critical oxygen partial pressure.

This thesis covers work carried out on algae bioreactors as a tertiary treatment process for wastewater treatment. The process was primarily assessed by the removal of Phosphorus and Nitrogen as an alternative to chemical and bacterial removal. Algal bioreactors would have the added advantage of carbon sequestration and a by-product in the energy rich algal biomass that should be exploited in the existing AD capacity. Laboratory scale bioreactors were run (4.5-30L) using the secondary treated final effluent from the local Loughborough sewage works. In a preliminary series of experiments several different bioreactor designs were tested. These included both batch feed and continuous flow feed configurations. The bioreactors were all agitated to keep the algal cells in suspension. The results demonstrated that the most effective and easy to operate was the batch feed process with the algal biomass by-product harvested by simple gravitational settling. Experiments also compared an artificial light source with natural light in outdoor experiments. Outdoor summer light produced greater growth rates but growth could not be sustained in natural UK winter light. Light intensity is proportional to productivity and algae require a minimum of around 97W/m2 to grow, an overcast winter day (the worst case scenario) was typically around 78W/m2, however this was only available for a few hours per day during Nov-Jan. The process would be better suited to areas of the world that receive year round sunlight. It was shown that phosphorus could be totally removed from wastewater by the algae in less than 24 hours depending on other operating variables. With optimisation and addition of more carbon, a HRT of 10-12 hours was predicted to achieve the EU WFD / UWWTD standard. It was further predicted that the process could be economically and sustainably more attractive than the alternatives for small to medium sized works. Biomass 3 concentrations of between 1-2g/L were found to best achieve these removals and produce the fastest average growth rates of between 125-150mg/L/d. The uptake rates of phosphorus and nitrogen were shown to be dependent on the type of algae present in the bioreactor. Nitrogen removal was shown to be less effective when using filamentous bluegreen algae whilst phosphorus removal was almost completely stopped compared to unicellular green algae that achieved a nitrogen uptake of 5.3mg/L/d and phosphorus uptake of 8mg/L/d. Soluble concentrations of Fe, Ni and Zn were also reduced by 60% in the standard 10 hours HRT. The predominant algae were shown to depend largely on these concentrations of phosphorus and nitrogen, and the strain most suited to that specific nutrient or temperature environment dominated. Nutrient uptake rates were linked to algal growth rates which correlated with the availability of Carbon as CO2. CO2 was shown to be the limiting factor for growth; becoming exhausted within 10 hours and causing the pH to rise to above 10.5. The literature showed this was a common result and the use of CO2 sparging would more than double performance making this process a good candidate for waste CO2 sequestration. Heat generated from combustion or generators with exhaust CO2 would also be ideal to maintain a year round constant temperature of between 20-25°C within the bioreactors. A number of possible uses for the algal biomass generated were examined but currently the most feasible option is wet anaerobic co-digestion. Further economic analysis was recommended on the balance between land area and complementary biomass generation for AD. It was also suggested given the interest as algae as a future fuel source, the process could also be adapted for large scale treatment and algal biomass production in areas of the world where land was available.

Master of Science / Department of Biological & Agricultural Engineering / Stacy L. Hutchinson / The shortage of clean, usable water is a global problem (Millennium Ecosystem Assessment, 2005). As much as 80% of the world’s population has been reported to be in areas of high water security risk due to a convergence of factors, such as watershed disturbance, pollution, water resource development and biotic factors (Voeroesmarty et al., 2010). Water reuse technologies are a potential solution to this problem. However, implementation of treatment technologies for improved water reuse require rapid, effective monitoring techniques capable of insuring treatment quality. Fluorescence spectroscopy has shown potential for wastewater treatment monitoring due to its sensitivity, selectivity, and capacity to be employed in-situ. Online fluorescence data and full fluorescence excitation-emission matrices coupled with parallel factor analysis (PARAFAC) were employed to evaluate the treatment performance of a membrane bioreactor (MBR) at Fort Riley, KS. Specific research goals were to evaluate the effectiveness of fluorescence for monitoring wastewater treatment and to determine the contamination detection limit of fluorescence techniques in a non-potable reuse scenario. Study results revealed a two-stage startup period, the first 60 days indicated membrane cake layer formation and the first 90 days showed signs of oxic tank maturation. Fluorescence was found to be effective at monitoring carbon concentration trends throughout the MBR system, showed preferential removal of protein-like dissolved organic matter (DOM), and an increase in biodegradation of DOM as the oxic tank matured. A ratio of the humic-like fluorescent components to the protein-like fluorescent components correlated to TOC removal (R² = .845, p < .001). Also, fluorescence was able to detect contamination in the effluent at the 0.74-1.24 mg C/L level using two wavelength pairs, indicating that effective real-time monitoring for contamination can be accomplished with minimal instrumentation and post-processing of data.

La problématique concernant la présence et les risques potentiels liés aux micropolluants dans l'environnement est devenue une préoccupation d'actualité. Aujourd'hui, les stations d'épuration ne sont pas en mesure de traiter de manière adéquate ce nouveau type de pollution. Dans le cadre de cette thèse, l'application de la technologie des bioréacteurs à membrane a été envisagée afin d'évaluer leur potentiel pour la dégradation d'un médicament anticancéreux : le cyclophosphamide (CP). Les objectifs de cette étude sont d'une part évaluer le potentiel des bioréacteurs à membrane pour la dégradation du cyclophosphamide, ainsi que pour l'élimination de sa toxicité, d'autre part rechercher l'effet du CP et de ses métabolites sur les performances globales du procédé et sur l'activité de la biomasse épuratrice ainsi que sur les propriétés physico-chimiques de la liqueur mixte et les conséquences sur le colmatage. Deux âges de boues ont été évalués, 50 jours lors de la première campagne et 70 jours lors de la deuxième. L'élimination du CP et du métabolite 4-Keto-CP durant les deux campagnes expérimentales est d'environ 80% pour les deux composés. Les processus d'adsorption et de biodégradation contribuent à l'élimination du CP de l'eau résiduaire traitée. Le cocktail de CP et ces métabolites aux conditions opératoires étudiées n'a pas d'influence significative sur l'élimination globale de la DCO et de l'azote total. Cependant, la toxicité du cocktail des composés pharmaceutiques sur la boue activée modifie les caractéristiques de la matrice biologique : Une diminution de la production de boues du BÀM R1 CP par rapport au BÀM R2 contrôle est observée. La présence du CP et ses principaux métabolites stimule les mécanismes de survie et de production des EPS avec une production légèrement plus forte des polysaccharides que des protéines. Les résultats mettent en évidence que la réponse des boues activées des BÀM au cisaillement est dépendante de la présence de ces molécules. Cette étude démontre au final l'intérêt des BÀM pour traiter ce type d'effluents, et limiter la pollution relarguée dans le milieu naturel. / In hospital or pharmaceutical discharges, but also in wastewater treatment plants and more generally in the aquatic environment, toxics pollutants have been identified. Some pharmaceuticals are not completely eliminated in the municipal wastewater treatment plants and are discharged as contaminants into receiving waters. The application of membrane bioreactor process is investigated here with the aim of evaluating the potential for removal of cyclophosphamide (CP). In this study, two membrane bioreactors (MBR) were operated: one of the MBR served as a control, whereas to the other CP and its main metabolites were continuously added. Two sludge retention times were assessed, 50 days and 70 days. Removal of CP in a MBR and its effects on the membrane performance, COD and total nitrogen (TN) removal efficiency were studied. CP and 4-Ketocyclophosphamide removals up to 80% were achieved under studied operating conditions. The sludge adsorption and biodegradation (cometabolism) play an important role in the process of CP removal. CP and its metabolites toxicity do not alter COD and total nitrogen removal efficiency of MBRs. However, it induces a modification of the biological suspended solids and in doing so a modification on the membrane fouling: a decrease in the production of sludge MBR CP compared to MBR control is observed; the presence of CP and its main metabolites stimulates mechanisms of protection and production of EPS with a slightly higher production of polysaccharides than proteins. The results underline that the response of activated sludge to shear stress is dependent on the presence of these molecules. This study demonstrates the interest of MBR to treat this type of effluent and reduce the pollution released into the environment

Shellfish waste obtained from seafood processing plants contains chitin, protein and calcium carbonate. Chitin is a versatile biopolymer with many applications. Conventionally, chitin is separated from calcium carbonate and protein by acid and alkali respectively. In this project, a biotechnological approach was applied to recover chitin from scampi (Nephrops norvegicus) waste using lactic acid bacteria (LAB) to produce lactic acid from glucose which lowers the pH of the mixture, thus preserving the waste from spoilage. The acid also dissolves the calcium carbonate and under these conditions native enzymes breakdown the protein (autolysis), thus affording a substantial amount of purification of chitin. LAB were isolated and identified from various shellfish waste fermentations. Studies on their acid-producing ability revealed a few potentially good strains, identified as Lactobacillus paracasei, Lactobacillus plantarum and Pediococcus sp. The strain of Lactobacillus paracasei was used as a starter culture in the fermentation of shellfish waste in a horizontal rotating bioreactor in order to evaluate the feasibility of the process. The design of the bioreactor was such that it enabled separation of solid and liquid end products during fermentation. Several important fermentation parameters were studied including mode of rotation, concentration of glucose, temperature, rotation rates, loading capacity, type and particle size of waste. Partial purification of the scampi waste was achieved using both batch and fed batch operation, but in the latter, improved purification was achieved at the cost of increased glucose consumption and extended fermentation times. Whilst higher temperatures increased the rates of fermentation, higher rotation rates seemed to have the reverse effect. Mincing the waste helped to increase breakdown of protein whilst larger particles tended to undergo rapid spoilage. Analysis of the chitin product enabled this method to be compared with the conventional method. The results obtained showed that this method is capable of saving large volumes of chemicals and besides producing chitin, the protein liquor by-product could also be used as an ingredient in an animal feed which is not possible by the conventional method.