京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21829号 / 農博第2342号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5201(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

: Filamentous hemagglutinin (FHA) is adhesive protein molecule that is secreted by Gram- negative bacterium Bordetella pertusis, the causative agent of whooping cough (pertussis). The C-terminal segment of FHA plays a crucial role in host-pathogen interaction, however, the structural features are still unknown. Here, we identified the C-terminal residue of FHA and processed form of FHA (FHA*) as alanine residues in position 2304 and 2228, respectively. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy demonstrated that the C-terminal segment of FHA(FHA 1995-2228) is characterized by alpha-helical contribution without any compact protein fold. Moreover, suppression of transcription of small regulatory RNA pairing to the 5'-end of fhaB transcript resulted in two- fold increase of FHA production. These data suggested that the C-terminal segment of FHA appear to be an unstructured protein and FHA secretion is negatively regulated by small regulatory RNA. (In Czech) Keywords: Bordetella pertussis, filamentous hemagglutinin, small RNA

Filamentösa svampar används inom många biotekniska områden. Svamparnas biomassa kan användas till föda eller djurfoder, medan biprodukter som svampar producerar kan användas inom områden som industri samt medicin. Kultiveringen av Neurospora intermedia och Aspergillius oryzae genomförs på smör, raps och frityrolja i detta arbete för att få en större förståelse för hur svamparna kan växa på olika lipider samt skillnaden som kan uppstå mellan olika arter av filamentösa svampar. Under arbetes studeras biomassakoncentration, proteinhalt samt pH. Även problem som att svamparna kan lagra lipider i cellerna och att det kan påverka resultaten genom en ökning av biomassakoncentrationen diskuteras.  Kultiveringarna genomfördes i Erlenmeyerflaskor med 20 g/L lipid samt saltblandning för att bestå med nödvändiga näringsämnen. För A.oryzae genomförs även kultivering i en airliftbioreaktor för att förstå hur svampen beter sig i större skala. Högst koncentrationen torrbiomassa som erhålls ifrån Erlenmeyerflaskorna är 18,49±1,90 g/L för A.oryzae samt17,31±1,14 g/L för N.intermedia med en proteinhalt för torr biomassa upp till ca 14%.Utbytetmellan torr biomassa och lipid som tillsatts i kultiveringen hamnade på 94,00%±0,06%(rapsolja) för A.oryzae samt 81,69%±0,007% (frityrolja) för N.intermedia. De höga utbytena kan förklaras med att svamparna kan lagra lipider i cellerna vilket även ger de höga koncentrationen av torr biomassa. För att förstå morfologin, lipidhalt i biomassan samt pH beteende behövs mer arbete utföras med ändring av parametrar som pH justeringar, temperatur, luftflöde samt analys av biomassan för att utreda hur mycket lipider som lagras. / Filamentous fungi are used in many different biotechnological fields. The fungal biomass can be used for food or feed, while by-products that the fungi produce can be used in different industries or medical applications. The cultivations of Neurospora intermedia and Aspergillius oryzae implemented on butter, rapeseed and frying oil that is used in this work is for the larger understanding of how the fungi can grow on different lipids and the difference between species of fungi. Biomass concentration, protein content and pH are the main focus during this work, but problems like lipids storage in the biomass that can affect the results by increase of the total biomass concentration is discussed. The cultivations were implemented in Erlenmeyerflasks with 20 g/L lipid with added saltsolution with necessary nutrients. For A.oryzae cultivation in an airlift bioreactor was carried out for the understanding of larger scale cultivation. Highest concentration of dry biomass obtained from Erlenmeyerflasks are 18,49±1,90 g/L for A.oryzae and 17,31±1,14 g/L for N.intermedia with protein content for dry biomass up to 14%.The yield for dry biomass perlipid added during the cultivation ended at 94,00%±0,06% (rapeseed oil) for A.oryzae and 381,69%±0,007% (frying oil) for N.intermedia. During cultivation the cells can store lipids and that explains the high yields and biomass concentrations. For the understanding of morphology, the amount of lipids in the biomass and pH behaviour needs more work that includes changes of parameters like pH adjustment, temperature, airflow and analysis of the biomass for lipid content.

No description available.

La phyllotaxie, arrangement d’éléments botaniques autour de l’axe primaire de la plante, suit unpatron spatio-temporel robuste. Elle est établie au niveau du Méristème Apical Caulinaire (MAC),qui est la niche de cellules souches post-embryonnaires des parties aériennes de la plante.L’accumulation locale de la phytohormone auxine déclenche la formation des organes latéraux auniveau du MAC. En même temps, la déplétion d’auxine dans les cellules environnantes crée unchamp inhibiteur, où toute nouvelle organogenèse est impossible. La croissance permet auxanciens organes de constamment s’éloigner du MAC, ce qui libère l’espace nécessaire auxnouvelles organogenèses. C’est un exemple frappant de processus biologique auto-organisé etitératif. Des mécanismes moléculaires et génétiques régulant la phyllotaxie ont été identifiés,majoritairement dans le contexte d’une phyllotaxie commune, qu’est la spirale de Fibonacci. Iln’est pas actuellement démontré que ces mécanismes expliquent aussi d’autres types dephyllotaxies. Nous avons identifié DRB27, un mutant d’Arabidopsis thaliana présentant une fortetendance à générer des pseudo-verticilles. Cela rappelle la phyllotaxie observée chez presquetoutes les fleurs et quelques tiges d’espèces non parentes. La quantification de la phyllotaxie deDRB27 ainsi que des expériences d’imagerie in vivo, ont révélé que ces groupes d’organes ne sontpas des verticilles, mais correspondent à des éruptions d’organogenèses se produisant en croissantà la périphérie du MAC quand, à l’inverse, de larges domaines restent inactifs. Ces observationsvont à l’encontre des règles classiques de positionnement d’organes lors de la mise en place de laphyllotaxie. De façon surprenante, nous avons identifié deux mutations candidates affectant lesrégulateurs abaxiaux FILAMENTOUS FLOWER et MIR166A, potentiellement toutes deuximpliquées dans le phénotype phyllotactique de DRB27. Alors que ces gènes sont exprimés dansles organes en développement, cela suggère des retours d’information sur la phyllotaxie, de façonnon autonome. Nous avons identifié des séries d’anomalies dans le MAC de DRB27, incluant despatrons de signalisation d’auxine anormaux, la perturbation des frontières entre les organes et leméristème, la modification des domaines WUS-CLV3 et de la géométrie du MAC, ainsi qu’unemodification de la dureté de la surface du méristème. Toutes ces données suggèrent que l’identitéet le développement des organes latéraux agissent sur l’homéostasie du MAC et l’établissement dupatron phyllotactique. / Phyllotaxis, the arrangement of botanical elements around plant axis,conforms to a robust spacial-temporal pattern. It is primarily established atthe shoot apical meristem (SAM), the post-embryonic aerial stem-cellniche. Local accumulation of the phytohormone auxin locally triggersorgan formation at the SAM, while depletion of auxin in the surroundingcells creates an inhibitory field, where no new organ can be initiated.Growth constantly moves older organs away from the SAM, clearingspace for new organogenesis. This is a striking example of an iterative and self-organized process driven by inhibitory fields. Molecular and geneticmechanisms regulating phyllotaxis are now being identified, but mostly inthe context of the most common Fibonacci spiral. Whether or not thesame mechanisms explain other types of phyllotaxis remains to beexplored. We identified DRB27, an Arabidopsis thaliana mutant with astrong tendency to generate clusters of organs. This is reminiscent of thewhorled phyllotaxis, observed in almost all angiosperm flowers and insome shoots of unrelated species. Quantification of DRB27 phyllotaxisand live imaging revealed that clusters are not whorls but correspond toburst of organs initiating in crescent domains at the periphery of the SAM.Conversely, large crescent domains remain devoid of organ initiation.Organogenesis in these clusters violates classical rules of organ spacing inphyllotactic systems. Surprisingly, we identified two candidate mutationsaffecting the two abaxial regulators FILAMENTOUS FLOWER andMIR166A, which likely combines to produce DRB27 peculiar phyllotaxis.Since these genes are expressed in developing organs, it suggests non-cellautonomous feedbacks on phyllotaxis. We identify a series of anomaliesin DRB27 SAM, including abnormal patterns of auxin signalling,perturbation of organ boundary formation, modification of CLV3/WUSdomains and SAM geometry and increase in cell wall stiffness. Takentogether our data questions how lateral organ identity and developmentfeedbacks on SAM homeostasis and phyllotaxis patterning.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: It is known that many hyphomycetous fungi are dispersed by wind, water and insects. However, very little is known about how these fungi may differ from each other regarding their ability to be disseminated by different environmental vectors. Consequently, to obtain an indication of the primary means of spore dispersal employed by representatives of the genera Acremonium, Aspergillus and Penicillium, isolated from soil and indoor environments, we monitored spore liberation of cultures representing these genera in an airflow cell. The experimental data obtained, of plate counts conducted of the air at the outlet of the airflow cell, were subjected to an appropriate analysis of variance (ANOVA), using SAS statistical software. Intraspecific differences occurred regarding aerial spore release. Under humid conditions, however, Penicillium species were more successful in releasing their spores than Aspergillus and the Acremonium strain. Under desiccated conditions the Aspergillus took longer to release their spores than representatives of Acremonium and Penicillium. The taxa that were investigated did not differ from each other regarding the release of spores in physiological salt solution (PSS). Although not proven, indications are that water may act as an important dispersion agent for these fungi, because washing of cultures with PSS resulted in all cases in an immediate massive release of colony forming units. Subsequently, using standard plate count techniques, conidial adhesion of the fungi mentioned above to synthetic membranes, leaf cuttings and insect exoskeletons differing in hydrophobicity and electrostatic charge were investigated. We found that the different genera showed different adhesion profiles for the series of test surfaces, indicating differences in physico-chemical characteristics of the fungal spore surfaces. In general, the Penicillium strains showed a greater ability to adhere to the test surfaces, than the aspergilli, while the representative of Acremonium showed the least adherence. No significant difference in the percentage spore adhesion was found between hydrophobic and hydrophilic materials. Furthermore, evidence was uncovered supporting the contention that, under dry conditions, electrostatic surface charges play a role in the adherence of fungal spores to surfaces, because adherence was positively correlated (Correlation coefficient = 0.70898, p = 0.001) to positive electrostatic charges on the lamellar surfaces. In the next part of the study, standard plate count methods were used to determine the relative adhesion of the above mentioned hyphomycetous fungi, as well as a polyphyletic group of yeasts, to the test surfaces submerged in 10 mM sodium phosphate buffer (pH 7.0). As was found with the experiments with the dry surfaces, both intraspecific and intergenus differences were uncovered. Overall, the fungi adhered better to hydrophilic surfaces than to hydrophobic surfaces. This indicated that the fungal surfaces were covered with relatively hydrophilic compounds such as carbohydrates. Subsequently, it was demonstrated that all the fungi adhered to plasma membrane glycoprotein coated polystyrene and the presence of fungal carbohydrates on the surfaces of the fungal propagules was confirmed using epi-fluorescence microscopy. Differences in the strategy of the fungal genera to release their airborne spores, as well as differences in their adhesion profiles for the series of test materials, may be indicative of a unique environmental niche for each genus. In future, this phenomenon should be investigated further. / AFRIKAANSE OPSOMMING: Hifomisete fungi is daarvoor bekend om te versprei deur middel van wind, water, en insek vektore. Maar nietemin, daar is bykans geen kennis m.b.t. hoe hierdie fungi van mekaar verskil t.o.v. hul vermoë om versprei te word deur omgewings vektore nie. Gevolglik was spoorvrystelling van kulture, verteenwoordigend van die genera Acremonium, Aspergillus en Penicillium gemoniteer om ‘n aanduiding te kry van primêre wyse van spoorverspreiding waardeur verteenwoordigers van die onderskeie genera ingespan word. Eksperimentele data ingewin, vanaf plaat tellings wat uitgevoer was op lug afkomstig vanuit die uitlaat-klep van die lugvloei kapsule, was onderwerp aan ‘n toepaslike analise van afwyking (ANOVA), deur gebruik te maak van ‘n SAS statistiese pakket. Intraspesie verskille is waargeneem t.o.v. lug spoorvrystelling. Desnieteenstaande was Penicillium meer suksesvol onder vogtige kondisies t.o.v. spoorvrystelling in vergelyking met Aspergillus en die Acremonium stam. Onder droë kondisies het verteenwoordigers van Aspergillus langer geneem om hul spore vry te stel as verteenwoordigers van onderskeidelik, Penicillium en Acremonium. Geen verskille was waargeneem m.b.t. spoorvrystelling in fisiologiese soutoplossing (FSO) tussen die verskillende filogenetiese stamme nie. Alhoewel dit nie bewys is nie, wil dit voorkom asof water as belangrike verspreidingsagent van die betrokke fungi dien, aangesien die spoel van kulture met FSO tot ‘n oombliklike enorme vrystelling van kolonie-vormende eenhede gelei het. Gevolglik, deur gebruik te maak van standaard plaattellings tegnieke, was spoor aanhegting van bogenoemde fungi aan sintetiese membrane, blaar snitte en insek eksoskelette wat verskil in terme van hidrofobisiteit en elektriese lading, ondersoek. Daar was gevind dat die aanhegtingsprofiele m.b.t. hierdie reeks toetsoppervlaktes van die verskillende genera verskil, wat op sigself ‘n aanduiding was van verskille in fisieschemiese eienskappe van die swamspoor oppervlaktes. Penicillium stamme het ‘n hoër aanhegtings vermoë aan die toetsoppervlaktes getoon as die aspergilli, terwyl die verteenwoordiger van Acremonium die laagste aanhegting getoon het. Geen betekenisvolle verskille i.t.v. persentasie spoor aanhegting was gevind tussen hidrofobiese en hidrofiliese oppervlakte nie. Daarbenewens was die argument dat spoorvrystelling onder droë kondisies beïnvloed word deur elektrostatiese oppervlak ladings, bevestig deur ons bevindinge, want aanhegting het positief gekoreleer (Korrelasie koëffisient = 0.70898, p = 0.001) met positiewe ladings op die oppervlaktes. ‘n Standaard plaattellingstegniek was aangewend in die volgende fasset van die studie om die relatiewe aanhegting van bogenoemde hifomisete fungi, sowel as ‘n polifilitiese groep giste aan die toetsoppervlaktes, gedompel in 10 mM natrium fosfaat buffer (pH 7.0) vas te stel. Intraspesie en intragenus verskille was weereens waargeneem, net soos in die geval van die eksperimente met die droë oppervlakte. In die algemeen het die swamme baie beter geheg aan hidrofiliese oppervlaktes in vergelyking met hidrofobiese oppervlakte. Dit was ‘n aanduiding dat die swamspoor oppervlaktes bedek was met relatiewe hidrofiliese verbindings bv. koolhidrate. Verder was daar bewys dat alle swamme ingesluit in hierdie studie die vermoë het om plasmamembraan glikoproteïn bedekte polistireen te bind, en gevolglik was die teenwoordigheid van van koolhidrate op die swamspore bevestig m.b.v epi-fluoresensie mikroskopie. Verskille in die strategie van swamme om spore in die lug vry te stel, sowel as verskille in die aanhegtingsprofiele vir ‘n reeks toetsmateriale, mag net ‘n aanduiding wees van ‘n unieke omgewings nis vir elke genus wat in hierdie studie ondersoek is. Hierdie verskynsel moet dus in die nabye toekoms nagevors word.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2006. / Filamentous fungi have been employed for production of heterologous proteins such as enzymes, antibiotics and vaccines due to their good secretion capacities and effective posttranslational modifications of these proteins. With an improvent in recombinant DNA technologies it has become possible to express many useful proteins in species such as the Aspergilli. However the submerged cultivation of filamentous fungi is complicated by the difficulties in mixing and oxygen and nutrient transfer in the highly viscous culture fluids that result. The purpose of the project was to investigate the potential of simultaneous control of morphology and production of enzymes in the dimorphic fungus, Mucor circinelloides, in order to overcome problems associated with the submerged cultivation of filamentous fungi. Dimorphic M. circinelloides, a zygomycete in the order Mucorales, occurs in a filamentous form or a yeast-like morphology in response to environmental conditions. Recently, advances were made in transformation of Mucor, and it has become possible to transform M. circinelloides to express heterologous proteins. The first example of a strong, regulated promoter from M. circinelloides being used for recombinant protein production was the expression of the glucose oxidase gene (from Aspergillus niger) under the control of the glyceradehyde-3-phosphate dehydrogenase (gpd1) promoter. Glucose oxidase (GOX) is an enzyme used to prevent oxidation of foods to extend shelf-life, to produce low-kilojoule beverages and to measure glucose levels in medical diagnostic applications. The scope of this project was to establish the conditions for yeast and filamentous growth of M. circinelloides in order to allow control of morphology, and to evaluate enzyme production under these conditions. Enzyme production of the GOX producing mutant strain, that was recently constructed, was compared to that of a wild type M.circinelloides strain. M. circinelloides was cultured in two-stage batch fermentations, firstly a yeast stage and then a filamentous stage. The yeast morphology was induced by anaerobic conditions while the filamentous morphology was achieved by exposure to air. The enzyme, biomass and metabolite production of the glucose-oxidase producing mutant strain and the wild type were monitored during the two-stage fermentations. GOX from the mutant and native amylase activity levels from the wild type were compared to each other and to other production systems for these enzymes. The morphology could be maintained in a yeast form under N2 with addition of ergosterol and Tween 80. The GOX activity levels in the culture fluid were comparable to some of the unoptimized GOX production systems in literature, but much lower than the optimized, recombinant GOX production systems that employ certain yeasts, or Aspergilli or Penicillium. The intracellular GOX levels were almost 6-fold higher than the extracellular levels which was unexpected as GOX is usually well-secreted. The morphological control improved the morphology for the initial yeast-stage of the fermentation but did not improve the morphology during the filamentous, enzyme-producing stage and it decreased the biomass yield and enzyme production by 50%. The constraint of Mucor to its yeast-like form did not improve the broth homogeneity or enzyme production and increased the time required for enzyme production. In this study M. circinelloides did not perform that well against other species already used to produce these enzymes. However, M. circinelloides could be used to produce enzymes from zygomycetes that systems such as A. niger do not produce well.

<p>Sedan december 2003 har Kalmar läns slakteris (KLS) nya reningsverk varit i drift. Entreprenör för det nya reningsverket samt driftansvariga under det två första åren är Läckeby Water Group. Verket är av SBR-typ (Sekventiell Biologisk Rening) med biologisk kväverening och kemisk fällning av fosfor med hjälp av järnklorid. Från slakteriet leds avloppsvattnet genom en 2 km lång ledning ner till reningsanläggningen. Verket hade under 2004 problem med höga halter fosfor i utgående vatten, flertalet mekaniska haverier samt två perioder av slamsvällning. Slamsvällningen orsakades av filamentösa (trådformiga) bakterier, första gången av Thiothrix spp. och andra gången av Typ 021N. Syftet med examensarbetet var att finna orsaken till den senare slamsvällningen samt att söka förebyggande åtgärder mot Typ 021N. Examensarbetet utfördes genom litteraturstudier, laboratorieförsök, fullskaleförsök, genomgång av driftsdata samt mikroskopering av aktivt slam vid verket.</p><p>Utifrån litteraturstudier konstaterades att filamentösa bakterier kan gynnas under perioder av låga syrehalter samt av låg näringstillförsel eftersom dessa bakterier har en högre tillväxthastighet vid låga substratkoncentrationer än flockbildande bakterier. Specifikt för Typ 021N är att dessa har möjlighet att utnyttja reducerat svavel som energikälla samt gynnas vid tillgång på korta lättnedbrytbara kolföreningar. Laboratorieförsök visade inte entydigt att låga fosfor eller syrehalter gynnade de filamentösa bakterierna. Inverkan av FeCl3, Ecofloc, PAXXL60, NaOCl och H2O2 studerades under korttids laboratorieförsök och effekten utvärderades i mikroskop. I några fall hämmades filamenten men aldrig utan att även påverka övriga mikroorganismer negativt. PAX-XL60 hämmade filamentförkomsten mest och påverkade andra organismer förhållandevis lite. Tillsats av PAX i filamenthämmande och flockbildande syfte utfördes därefter i fullskala. Effekten av tidigare tillsatser av NaOCl och H2O2 i filamenthämmande syfte studerades och visade sig ha givit varierande resultat. NaOCl visade sig effektivt bekämpa filamentösa bakterier i processen då inblandning skedde under rätt förutsättningar.</p><p>Processdata för våren 2004 jämfördes med data från en period under hösten, vilken följdes av en slamsvällning. Perioderna visade stora skillnader m.a.p. syrehalt, temperatur, dosering av järnklorid och organisk belastning. En on-line mätning i inkommande vatten visade på mycket höga halter av svavelväte. Svavelväte bildas under anaeroba förhållanden t.ex. i stillastående avloppsvatten. Orsaker till slamsvällningen i september-oktober 2004 tros vara höga halter av svavelväte, perioder med låga syrehalter, höga vattentemperaturer samt tillgång på lättnedbrytbart organiskt material. Svavelvätet kan förslagsvis elimineras genom en tidsstyrd dosering av CaNO3 i inkommande ledning. Noggrann övervakning av syre samt tillgång på syre måste garanteras i processen. Det inkommande vattnets mikroflora kan förändras genom installation av en aerob selektor för att gynna de flockformande bakterierna. För att sänka fosforhalterna i utgående vatten samt att inte riskera fosforbrist i processen har en tillfällig efterfällning med extra tillsats av FeCl3 och polymer installerats.</p> / <p>In December 2003 the new plant treating slaughterhouse wastewater from KLS was taken into operation. Läckeby Water Group was entrepreneur and responsible for the maintenance during the following two years. The treatment plant is of SBR-type and has biological nitrate removal and chemical precipitation of phosphate with iron chloride. The wastewater from the slaughterhouse passes a 2 km long pipeline before entering the treatment plant. During 2004, the plant had problems with high levels of phosphorous in the effluent, several mechanical problems and two occasions of sludge bulking caused by filamentous bacteria. The first incident was caused by Thiothrix spp. and the second by Type 021N. The aim with the thesis was to find causes for the latest period of sludge bulking as well as investigate preparatory actions against Type 021N. The thesis included literature studies, laboratory and full-scale tests, evaluation of prior process data and continuous microscopic analysis of the activated sludge at the plant.</p><p>The literature study showed that filamentous bacteria are favoured by low oxygen and low nutrient concentrations due to their possibly higher growth rate during low substrate concentrations. Type 021N, specifically, can use reduced sulphides as energy source and benefits from an excess of low molecular substrates. Laboratory experiments did not verify that the filamentous bacteria were favoured by low oxygen concentration or low phosphate levels. The effect of FeCl3, Ecofloc, PAX-XL60, NaOCl and H2O2 added to a bulking sludge was evaluated by microscopic analysis. No chemical was found to suppress the filamentous bacteria without also affecting the floc-forming bacteria negatively. PAX-XL60 showed the largest negative effects on filamentous bacteria and only a minor impact on other microorganisms. Full-scale tests with PAX were thereafter performed in order to suppress filamentous bacteria as well as flocculate particulate solids. The effect of earlier additions of NaOCl and H2O2 into the process gave varied results. NaOCl was efficient against filamentous bacteria when addition was made during correct circumstances.</p><p>Process data from two separate periods during 2004 was compared. One period was followed by good effluent values and another period by a sludge bulking period. Large differences between the two periods were seen in oxygen conditions, temperature, FeCl3 dosage and organic load. Measurements on influent wastewater showed high levels of hydrogen sulphide, which can be produced during anaerobe conditions i.e. in stagnant sewage pipes. Likely causes for the sludge bulking in September-October 2004 were high levels of hydrogen sulphide in the influent, periods of insufficient oxygen concentrations, high water temperatures and access to easy degradable substrate. The hydrogen sulphide can be eliminated through time-controlled dosage of CaNO3 in influent pipeline. Sufficient oxygen levels must be guaranteed in the process. The microbiological fauna in influent can be changed by installation of an aerobe selector to benefit floc-forming bacteria. To lower the phosphorous levels in effluent water and not risk phosphorous deficiency in the process a post-precipitation have been installed. The post-precipitation include extra dosage of FeCl3 and polymer and a drum screen to minimize suspended solids.</p>

A number of advanced pre-treatment techniques and methods have been evaluated for the sole purpose of improving digestibility of waste activated sludge. The pre-treatment of waste activated sludge (WAS) offers the benefit of releasing solubilized substrates, making them readily available to be utilized in the anaerobic digestion process. Other potential benefits include: reducing shock loading to the digester, improving overall digestibility and potentially providing filament / foaming control. Chlorine dioxide, a well-known disinfectant and oxidizing agent has been utilized in many drinking water processes around the world. Its use in wastewater treatment processes however is limited; especially in Canada where legislation has prevented its use for final effluent disinfection. As an oxidizing agent, chlorine dioxide induces cell rupture resulting in the release of soluble material, which when fed into the digester, may serve as readily available substrate for active microorganisms. This mode of action creates the potential for chlorine dioxide to be used as a sludge pre-treatment agent to improve digester performance and in alleviating pre-existing filamentous sludge bulking. This study was conducted using waste activated sludge obtained from the City of Winnipeg’s South End Water Pollution Control Centre (SEWPCC), with the following objectives: 1. Determine the efficacy of chlorine dioxide in alleviating pre-existing filamentous sludge bulking; 2. Determine chlorine dioxide ability to increase WAS solubilization; and 3. Define impact of chlorine dioxide on anaerobic digester performance. WAS pre-treatment using chlorine dioxide was found to be effective in alleviating filamentous bulking. This is significant as filamentous bulking in the activated sludge may lead several problems downstream. Following pre-treatment, sludge bulking was determined to be alleviated as observed by photomicrographic evidence and as measured by a 57% decrease in the stirred sludge volume index (sSVI). Particulate COD solubilization increased by 60%, 76%, and 74% over the untreated sludge for WAS pre-treated with 25, 50, and 100 mg ClO2/L (v/v), respectively. The pre-treatment of sludge using chlorine dioxide did not have any negative impact on digester performance although it also did not lead to improved performance. The volatile solids destruction and COD removal remained unchanged for both untreated and pre-treated sludge. Chlorine dioxide pre-treatment did not affect anaerobic digestion even at the lowest SRT evaluated; it is possible to decrease the digester SRT to as low as 6 days while maintaining the solids destruction and COD removal capability. Biogas production did not improve with increasing chlorine dioxide dosage during pre-treatment but also was not hindered by the pre-treatment agent. Chlorine dioxide was shown to alleviate filamentous bulking and improve solubility and has the potential to improve digester performance without negative impacts to the digester. However, the full benefit of the pre-treatment method may only be realized for complex “difficult to disintegrate” sludge types. / October 2016

ABSTRACT 1: Abrasive treatment and subsequent electroporation of the basidiomycete Pleurotus ostreatus in a hypertonic buffer was investigated as a potential method of transforming of intact hyphæ. Mycelia, which are not capable of being transformed via electroporation due to interference by the cell wall, were abraded in an attempt to mechanically degrade the cell wall prior to electroporation. An electroporation field strength of 12,500 V/cm for 500 μs to 1.25 ms was found to be optimal based upon mortality effects. A 32 μm carborundum abrasive was initially evaluated but was quickly found to be inappropriately large so a corundum abrasive with an average size of 300 nm was focused on. Vortexing as well as low and medium power ultrasonic agitation with the corundum abrasive were investigated for their potential to cause mechanical degradation of the cell walls. Vortexing and low power sonication were found to be ineffective at causing adequate degradation while medium power sonication was found to be both ineffective and super lethal. While the possibility of mechanical abrasion facilitating transformation via electroporation remains, it is unlikely that conventional methods of agitation will be effective. ABSTRACT 2: The dose response of the basidiomycetes Pleurotus ostreatus, Agrocybe aegerita and Cordyceps millitaris to the antibiotic hygromycin B was tested on two common and one in-house solid growth media. The three species were grown on Potato dextrose agar, malt extract agar and an agar containing maltose, yeast-extract, peptone and glucose with concentrations of hygromycin B from zero to 100 μg/ml for 11 to 14 days. Micrometer measurements were used to determine the growth rate of each species on each media. Significant differences in hygromycin B tolerance for each species between the three media types were evident (p-value < .0001 by ANOVA for all). Neither the media type nor the growth rate on hygromycin B free plates were useful predictors of effective hygromycin B doses so optimization should be performed on every strain and media type used for selection.