Dissertations / Theses on the topic 'Biofilm monitoring'

Hospital Acquired Infections and equipment contamination are some of the biggest issues faced by the healthcare industry worldwide. These infections generally range from mild to life threatening human infections which lead to increased costs and prolonged hospitalization time. The primary factor which caused these issues were biofilm forming bacteria which are able to withstand medications and defend themselves from various cleaning procedures. Another aspect which make these bacteria troublesome is that they are able to hide inside the biofilm, thus evading a lot of diagnostic tests. The methods used to detect these biofilms are unfortunately toxic to cells and cannot be used in vivo. Though there is enough data on the formation of biofilm on abiotic surfaces, the data present on the biophysical properties, structural organizations within the biofilm or their viscoelastic properties is very limited. In this master’s degree project, a dynamic monitoring platform is made for 2 different strains of the Salmonella Enteritidis bacteria where their structural and biophysical properties was investigated. Each strain lacks either curli or cellulose which are major components responsible for proper biofilm formation so performing these experiments on them gave us important information on how their properties get affected over time. Bacterial growth monitoring for all the strains were performed by measuring the absorbance every hour over a period of 5h and it was observed that all the strains had a very similar growth pattern until the end of the 4th hour after which they showed very mild differences. The next set of experiments involved using an eQCM to monitor the formation of biofilm on the surface of a quartz chip over 48h. There were differences observed in the biofilm formation pattern and mass deposition which provided clues as to how the biofilm formation and their viscoelastic properties are affected due to the mutations. This led to finding further clues regarding which aspect of biofilm formation is targeted by a specific mutant.

Orientador : Profª. Ph.D Regina Tiemy Kishi
Coorientador : Prof. D.Sc. Stephan Fuchs
Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Recursos Hídricos e Ambiental. Defesa: Curitiba, 15/03/2016
Inclui referências : f.75-78
Resumo: Os metais traço são utilizados na indústria e na agricultura e podem estar presentes em efluentes de mineração e esgoto. Assim, esses elementos atingem o ambiente e podem ser prejudiciais aos organismos, meio ambiente e às pessoas. Um monitoramento representativo é essencial para a gestão dos recursos hídricos e consequente prevenção à poluição. Monitoramentos convencionais da água algumas vezes podem não revelar a real condição do ambiente. Isto acontece devido às condições de lançamento e chegada dos poluentes ao sistema, à tecnologia disponível para quantificar a concentração e devido às características da própria substância monitorada. No caso dos metais traço, estes têm uma atração maior por outras partículas (sólidos suspensos, solo, sedimento, carbono orgânico dissolvido), as quais sedimentam, não permanecendo na coluna d'água. Outro fator a ser considerado é que as fontes de metais traço são geralmente intermitentes e as coletas são não contínuas. Além disto, os limites de detecção são altos e não detectam tais elementos na água. Desta forma, o biofilme é uma técnica alternativa de monitoramento, pois analisa o nível de contaminação em um intervalo de tempo. Para este estudo foram construídos dois amostradores. Amostras de biofilme, água e sedimento foram coletadas ao longo de oito meses para dois pontos de monitoramento, um na bacia do rio Barigüi e outro na bacia do rio Miringuava. Parâmetros de qualidade da água, granulometria e conteúdo de metais traço foram estimados. Os resultados mostram que o biofilme identificou os metais traço em praticamente todas as campanhas, enquanto que para as amostras de água isto não aconteceu. As amostras de sedimento representaram a poluição, porém não foi possível determinar o tempo da contaminação pelo método de coleta utilizado. O biofilme representou as diferenças no uso e ocupação do solo, representando poluição consistente com cada bacia hidrográfica. Palavras-chave: biofilme, metais traço, monitoramento, rio Miringuava, rio Barigüi.
Abstract: Trace metals are used in industries and agriculture and can be present in mining and sewer effluents. In such context, these elements can enter the environment and be very harmful to organisms, environment and people. A representative monitoring is essential for water resources management and, consequently, pollution prevention. Conventional water monitoring do not always show real environment condition. That happens because of effluent release conditions, pollution arrival system conditions, available technology to identify element concentrations and monitoring substance characteristics. Specifically for trace elements, they tend to adhere to other particles (suspended matter, soil, sediment, DOC) and deposit in riverbed. Other factor is that trace metal sources are usually from intermittent discharges and collections are not continuous. Besides that, usual techniques have high quantification limit and do not identify these elements in water. Thus, biofilm is an alternative monitoring technique for trace metals evaluation since it analyses contamination level in a time space. For this study, two biofilm samplers were constructed. Biofilm, water and sediment samples were collected for an eight month period in two monitoring sites, Barigüi and Miringuava watershed. Water quality parameters, granulometry, and trace metals content were estimated. Results showed that biofilm identified metals in almost every campaign, while water samples did not. On the other hand, sediment samples represented pollution but it was not possible to determine the contamination time by the used collection method. Biofilm also represented differences in soil use and occupation, representing consistent pollution potential for each basin. Keywords: biofilm, trace metals, monitoring, Miringuava River, Barigüi River.

It is considered that partial nitrification combined with anammox, named deammonification, is more environmental friendly compared with conventional nitrification/denitrification due to decrease energy requirement, low emission of CO2 and N2O. Dissolved oxygen (DO) is a significant parameter influencing the nitrogen removal rate and activity of different microorganisms. A proper level of DO concentration is needed to allow ammonium oxidizing bacteria (AOB) to produce a sufficient amount of NO2--N for anammox reaction. Too high NO2--N levels should be avoided as they cause inhibition effects on anammox bacteria or increase growth of nitrite oxidizing bacteria (NOB). In this study, investigations have been carried out, both in laboratory and pilot scales to evaluate the influence of different aeration strategies (characterized by dissolved oxygen concentration - DO and the ratio between non-aerated and aerated phase duration – R) on the deammonification process applied in the moving bed biofilm reactor (MBBR). Three series of batch tests were conducted in laboratory scale with different DO concentrations (2, 3, 4 mg/l) and R values (0 - continuous aeration; 1/3, 1, 3 – intermittent aeration), the same initial ammonium concentration, volume of the reject water and temperature. It was found that the impact of DO on deammonification was dependent on the R value. At R=0 and R=1/3, an increase of DO caused a significant increase in nitrogen removal rate, whereas for R=1 and R=3 similar rates of the process were observed irrespectively of the DO. The highest nitrogen removal rate of 3.33 gN/m2·d was obtained at R=1/3 and DO=4 mg/l. Significantly lower nitrogen removal rates (1.17 - 1.58 gN/m2·d) were observed at R=1 and R=3 for each examined DO. It was a consequence of reduced aerated phase duration times and lower amounts of residual nitrite in non - aerated phases as compared to R=1/3. Pilot scale experiments were carried out in a MBBR with a working volume of 200 L. The pilot plant has been operated for 1.5 years to remove nitrogen from reject water after dewatering of digested sludge. The activity of different groups of microorganisms in the biofilm was measured by specific anammox activity (SAA), oxygen uptake rate (OUR) and nitrate utilization rate (NUR) tests. The whole operation was divided into seven periods according to different nitrogen loads and different aeration strategies. The highest nitrogen removal rate and efficiency was obtained when DO was 3.5 mg/l and R equaled to 1/3. Activity tests showed that anammox bacteria and AOB play the dominating roles in the biofilm. The average and maximum values of specific anammox activity (SAA) were 3.01 gN/m2·d and 4.3 gN/m2·d, respectively. An average value of 4.0 gO2/m2·d and the maximum value of 5.1 gO2/m2·d was obtained in the oxygen uptake rate for AOB activity tests. Study results showed that application of an appropriate selected aeration strategy reduced energy consumption without any negative impacts on the process. Introduction of anaerobic phases and high nitrogen load enhanced the activity of anammox bacteria and NOB activity was limited.
Partiell nitrifikation i kombination med anammoxprocess, som kallas för deammonifikationprocess, anses vara mer miljövänlig jämfört med konventionell nitrifikation/denitrifikation pga minskat energibehov samt låga utsläpp av CO2 och N2O. Löst syre (DO) är en viktig parameter som påverkar hastigheten för kväverening och aktiviteten hos olika mikroorganismer. DO koncentrationer bör vara på en viss nivå för att ammoniumoxiderande bakterier (AOB) skall producera en tillräcklig mängd NO2-N för anammoxreaktionen, men inte heller för hög då hög NO2-N nivå ger en anammoxhämmande effekt eller ökad tillväxt av nitritoxiderande bakterier (NOB). I denna studie har undersökningar utförts både i laboratorie- och pilotskala för att utvärdera inverkan av olika luftningsstrategier, (som kännetecknas av koncentrationen av löst syre - DO och förhållandet (R) mellan tider för icke luftade och luftade faser), på deammonifikationprocessen i en MBBR (Moving Bed Biofilm Reactor). Tre serier av satsvisa försök utfördes i laboratorieskala med olika syre koncentrationer (2, 3, 4 mg/l) och R värden (0 - kontinuerlig luftning; 1/3, 1, 3 - intermittent luftning), men med samma initiala ammonium-koncentration, volym av den rörliga bädden och temperatur. Man fann att effekten av löst syre (DO) på deammonifikationen var beroende på R-värde. Vid R = 0 och R = 1/3, gav en ökning av löst syre (DO) en signifikant ökning i kvävereningshastigheten, medan för R = 1 och R = 3 observerades samma hastighet i processen oberoende av löst syrehalt (DO). Den högsta hastigheten för kväveavskiljning 3,33 gN/m2.d (avskiljningsgraden var lika med 69,5%) erhölls vid R=1/3 och DO=4 mg/l. Betydligt lägre värden (från 1,17 till 1,58 gN/m2.d) observerades vid R=1 och R=3 för varje undersökt halt av löst syre (DO). Det var en följd av minskad varaktighet av luftad fas och mindre mängd av kvarvarande nitrit i icke luftade faser jämfört med R= 1/3. Pilotskaleförsök utfördes i en MBBR med en arbetsvolym på 200 L. Pilotanläggningen har drivits i 1,5 år med att avlägsna kväve från rejektvatten från avvattning av rötslam. Aktiviteten hos olika grupper av mikroorganismer i biofilmen mättes genom tester av specifik anammoxaktivitet (SAA), syreupptagningshastighet (OUR) och nitratutnyttjandegrad (NUR). Driften var uppdelat i 7 perioder med olika kvävebelastning och luftningsstrategier. Den högsta hastigheten och grad av kväveavskiljning erhölls då DO var 3,5 mg/l och R uppgick till 1/3. Aktivitetstester visade att anammoxbakterier och AOB spelade dominerande roller i biofilmen. De genomsnittliga och maximala värden för specifika anammoxaktiviteten (SAA) var 3,01 gO2/m2.d och 4,3 gO2/m2.d, respektive. 4,0 gO2/m2.d som medelvärde och högsta värde på 5,1 gO2/m2.d erhölls för syreupptagningen för AOB aktivitetstester.  Studien visade att användning av en lämpligt vald luftningsstrategi minskar energiförbrukningen utan några negativa effekter på processen. Införande av anaeroba faser och hög kvävetillförseln ökar aktiviteten för anammoxbakterier och NOB-aktiviteten begränsades.
QC 20120628

Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.

El treball presentat en aquesta tesi doctoral te com objectiu principal la contribució en el camp de la microbiologia per entendre el biofilms i el possible control de desenvolupament mitjançant l’ús de mètodes i enfoc multidisciplinari. Els biofilms estan definits com comunitats de microorganismes que creixen envoltats en una matriu exopolisacárida i s’adhereixen a una superfície inert o teixit viu. La formació dels biofilms bacterians tenen un gran interès en microbiologia clínica degut al desenvolupament d’infeccions que son causades pel contacte directe o per colonització de dispositius mèdics implantats i pròtesis. Actualment es consideren causa de més del 60 % de les infeccions bacterianes. El problema dels biofilms bacterians a nivell clínic es que mostren millor resistència a antibiòtics arribant inclús a ser de 500 a 5000 cops més resistents a agents antimicrobians comparant amb la mateixa bactèria planctònica (bactèria en suspensió). Hi ha hagut moltes temptatives d’adaptar mètodes a laboratoris clínics on es reprodueixen les condicions pel desenvolupament de biofilms, però encara no s’ha arribat a obtenir òptims protocols estàndard per a aquest propòsit de monitoritzar la formació i toxicitat a temps real. Ha crescut l’interès en disseny, desenvolupament i utilització de dispositius de microfluídica que poden emular els fenòmens biològics que ocorren amb diferents geometries, dinàmica de fluids i restriccions de transport de biomassa en microambients fisiològics. La recerca descrita en aquesta tesis s’ha dut a terme amb diferents mètodes “label-free” basats en la variació acústica y/o propietats elèctriques per a la monitorització de biofilms. El treball presentat en la monografia descriu un dispositiu “custom-made” per a la utilització d’Espectroscòpia de impedància electroquímica com a eina útil per a l’obtenció d’informació d’adherència i formació de biofilms. El fet d’afegir nanopartícules com a segon biosensor permet la correlació de biofilm amb la seva toxicitat a temps real per a la detecció del punt òptim de tractament de biofilms. Finalment el disseny d’aquesta tecnologia s’utilitza per l’assaig de la resposta de biofilms a antibiòtics com a model in vitro d’infeccions causades per biofilms.
El trabajo presentado en esta tesis doctoral tiene como principal objetivo la contribución en el campo de la microbiología para entender los biofilms y el posible control de desarrollo mediante el uso de métodos y enfoque multidisciplinar. Los biofilms están definidos como comunidades de microorganismos que crecen embebidos en una matriz exopolisacárida y se adhieren a una superficie inerte o tejido vivo. La formación de los biofilms bacterianos tiene un gran interés en microbiología clínica debido al desarrollo de infecciones que son causadas por contacto directo o por colonización de dispositivos médicos implantados y prótesis. Actualmente se consideran la causa de más del 60 % de las infecciones bacterianas. El problema de los biofilms bacterianos a nivel clínico es que muestran mejor resistencia a antibióticos llegando incluso a ser de 500 a 5000 veces más resistentes a agentes antimicrobianos comparado a la misma bacteria planctónica (bacteria en suspensión). Ha habido muchas tentativas de adaptar métodos a laboratorios clínicos donde se reproducen las condiciones para el desarrollo de biofilms, pero aún no se ha llegado a obtener óptimos protocolos estándar para este propósito de monitorizar la formación y toxicidad en tiempo real. Ha crecido el interés en diseño, desarrollo y utilización de dispositivos de microfluídica que puedan emular los fenómenos biológicos que ocurren con diferentes geometrías, dinámica de fluidos y restricciones de transporte de biomasa en microambientes fisiológicos. La investigación descrita en esta tesis se lleva a cabo con diferentes métodos “label-free” basados en variación acústica y/o propiedades eléctricas para la monitorización de biofilms. El trabajo presentado en esta monografía describe un dispositivo “custom-made” para la utilización de Espectroscopia de impedancia electroquímica como herramienta útil para obtener información de adherencia y formación de biofilms. El hecho de añadir nanopartículas como segundo biosensor permite la correlación de biofilm con su toxicidad en tiempo real para la detección del punto óptimo del tratamiento de biofilms. Finalmente el diseño de esta tecnología es usada para el ensayo de la respuesta de biofilms a antibióticos como modelo in vitro de infecciones causadas por biofilms.
The work presented in this thesis has the main aim to contribute in the field of clinical microbiology to understand the biofilms and the possible of development through the use of methods with multidisciplinary approach. Biofilms are defined as communities of microorganisms that grow embedded in a matrix of exopolysaccharides and adhering to an inert surface or living tissue. The formation of bacterial biofilms has an interest in clinical microbiology with the development of infections that usually arise from either direct contact or the colonization of implanted medical devices and prostheses. Currently they are considered the cause of over 60% of bacterial infections. The problem of bacterial biofilms at clinical level is showing great resistance to antibiotics, so that the biofilm bacteria are 500 to 5000 times more resistant to antimicrobial agents that the same bacteria grown in planktonic cultures (bacteria in suspension). There have been attempts to adapt methods to clinical laboratories where they reproduce the conditions of biofilms, but have not yet adopted an optimal standard protocol for this purpose to follow-up the formation and toxicity in real-time. There has been a growing interest in design, development and utilization of microfluidic devices that can emulate biological phenomena that occur in different geometries, fluid dynamics and mass transport restrictions in physiological microenvironments. The research described in this thesis deals with different label-free methods based on variation of acoustic and electric properties for biofilm monitoring. The work presented in this monograph describe a custom-made device for using electrochemical impedance spectroscopy (EIS) as useful tool to obtain information of adherence and formation of biofilms. The addition of nanoparticles as toxicity biomarker allows the correlation of biofilm formation with its toxicity in real-time for detention of the optimal point for biofilm treatment. Finally the design of this technology is used for testing the biofilm response to antibiotic as in vitro model of biofilm-related infection.

Freshwater is essential for socioeconomic development, health of the population and equilibrium of the ecosystem. However, pollution generated by human activities often degrade the quality of this vital resource. In order to address this problem with effective management strategies, continuous assessment of freshwater and wastewater status is necessary. Microbial electrochemical systems (MESs) are attracting increasing attention as sensing tools, due to their low-cost, sustainability, portability, generation of continuous on-line signal and low involvement of operators. In this dissertation, the implementation of MESs as sensors in two different environmental contexts is discussed: wastewater treatment monitoring and agriculture-related pollutants detection. Different prototypes were designed according to their specific application and their detection capabilities were investigated. Regarding the monitoring of wastewater treatment process, floating Microbial Fuel Cells (MFCs) were tested as sensors for organic matter, a parameter that is currently measured with the labour-intensive and time-consuming BOD5 test. The first prototype consisted of a floating frame holding two carbon cloth electrodes separated by a polypropylene felt and a clay layer. The biosensor was tested in the lab using real wastewater as both electrolyte and inoculum, and a calibration curve was obtained. However, when the device was tested in the plant of Carimate (CO), a strong influence of light irradiation on the signal was noticed. During a month of operations, a correspondence between the peaks of voltage and of the peaks of organic load was present, but the signal was delayed of about two days. To further understand the influence of different environmental factors on the current signal, a new device was conceived and compared with the previous configuration. This time the experimentation was carried out at the plant of Bresso Niguarda (MI). The new setup consisted in a cylindrical terracotta separator sealed at one end and held perpendicularly to the wastewater surface by a plastic floater. The carbon cloth anode was placed outside the cylinder, while a cathode of the same material was located inside. Light irradiation, temperature, sCOD and nitrates were continuously monitored for about 20 days. An automatic sampler was built to obtain wastewater samples every two hour every day. The correlation between the physical-chemical parameters and the cell voltages was quite poor, especially for the floating type, which was often subjected to the inversion of the electrodes potential. This was probably due to oxygen diffusion in the first layer of wastewater. The microbial analysis of the biofilms confirmed this aspect, as aerobic strains (e.g. Nocardicaceae) were found on the anodes, and electrogenic bacteria usually present on anodes were found on the cathodes (i.e. Geobacteriaceae). To monitor agriculture-related pollutants (i.e. herbicides), amperometric biosensors based on the inhibition of cyanobacterial photocurrent were studied. Many herbicide compounds are harmful to humans and the environment, and they are currently measured with classic analytic techniques (e.g. HPLC, GC-MS, etc.), which are expensive and time consuming. To build the first prototype, Anabaena variabilis cells were entrapped on a carbon felt electrodes using an alginate hydrogel, and p-benzoquinone was used as electron shuttle to sustain the electron transfer. With this device it was possible to obtain concentration-current calibration curves for two commonly used herbicides (i.e. diuron and atrazine), and the obtained linear range was suitable for environmental analysis. However, to build a long lasting device, a mediatorless configuration is preferable, as the redox mediator is cytotoxic and can undergo photodegradation. For this reason, a new biosensor was created: a paper-based electrode coated with carbon nanotube paint and a titanium nanolayer was used as substrate for the formation of a Synechocystis wt. biofilm. Whit this configuration, only a presence/absence inhibition signal could be obtained for atrazine and diuron, while the herbicide paraquat temporarily enhanced the electron transfer due to its redox mediator capabilities. Nevertheless, this biosensor was able to maintain its sensitivity even after it was kept in the fridge for 22 days, proving its potential as long-lasting device which can be easily stored or shipped after preparation. These results demonstrated that microbial electrochemical sensors are a promising technology for sensing applications, with a great potential for the creation of a smart, diffuse grid of low-cost sensors for the continuous monitoring of water quality. However, further improvements are needed in order to reduce the response time, improve the sensitivity and discern between the influence of different environmental factors on the signal.

The water distribution system represents the final barrier to the degradation of treated water prior to consumption. Maintaining the integrity of these systems therefore is essential for public health. Current strategies for monitoring bacteriological water quality within distribution systems are dependent upon culture-based methods, which are affected by many factors, including conventional disinfection strategies (chlorine) that have been shown to impact the culturability of bacteria on media. Improved methods to assess the bacteriological loads and responses to treatment are needed. This thesis investigated the development and application of alternate means of analysis of water quality analytes, enabling a toolbox approach for bacteriological water quality monitoring. Tools used in this study include biomass concentration using DNA quantification, bacterial community characterisation using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), targeted population assessment using quantitative PCR (QPCR) and in situ phylogenetic abundance using fluorescence in situ hybridisation (FISH). Conventional methods such as heterotrophic plate counts were also included. Results show that chlorine does not eradicate bacteria by the proportions generally reported using culture-reliant methods. Within both drinking and recycled water distribution systems, bacteria belonging to Enterobacteriaceae and species of Pseudomonas were detected in water containing 2 ppm free chlorine concentrations. The persistence of such bacteria in conditions prevalent within distribution systems necessitates the revision of techniques of analysis as well as improved treatment and disinfection management strategies. Furthermore, current strategies for water quality monitoring are reliant on the assessment of planktonic bacterial populations, and do not consider attached (biofilm) populations. Molecular methods of analysis showed up to 5-log more bacteria are present in biofilms compared to planktonic sources. Thus, improved methods for water quality maintenance in distribution systems need to consider the contributions of biofilms. This thesis also studied the effects of a nitric oxide (NO) donor, as NO has been shown to encourage detachment of bacterial cells from the biofilm which then enter the planktonic phase of the life cycle. The molecular toolbox analysis approach showed that effects are variable on the diverse bacterial populations present in distribution systems, and some bacteria show heightened resistance compared to other populations. Ongoing work targeting biofilms in distribution systems is required to improve conventional practices of water quality assurance in distribution systems.

Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to continuously monitor cell attachment and growth ofStreptococcus lIIutans (+1-1 % (w/v) sucrose) and Candida albicans (+1-10 % (v/v) horse serum) as biofilms within an overnight period of approximately 20 h. S. 11I11tans biofilms generated using a 'continuous flow' method had a greater mass and were more dissipative (more viscoelastic) than those established using an 'attach and flow' strategy with or without 1% sucrose. Biofilms of C. albieans grown with 10 % horse serum had a greater mass and were more dissipative than those grown without the addition of horse serum. Cell numbers (as colony forming units, c.f.u.) in QCM-D derived biofilms after a 2-h attachment phase and during a -20-h growth period could be related to frequency (f) changes and these increased after growth of S. lIIutans and C. albicans. The energy losses displayed by the increases in the dissipative factor (D) indicated an increase in 'softness' of the attached cells. The ratio of D/f was used to provide information of the way in which viscoelasticity changed per unit mass. For S. 1II11tans flow conditions over the cells on the surface appeared to be important in creating biofilms of a greater complexity and stability and the QCM-D enabled properties of cells during attachment and binding, proliferation and removal to be monitored continuously. The percentage surface coverage on the QCM-D crystals by biofilms was estimated using the surface analysis features of the atomic force microscope and image analysis software. Mean percentage coverage also increased after the growth of these organisms. Mean percentage coverage was also increased after the overnight growth of C. albicans in the presence of horse serum. QCM-D was also used to create in situ a Zetag® 17 nm diameter silica nanoparticulate surface and a silvernanoparticulate surface (AgNPs). The influence of the Zetag 1silica nanoparticulate surface was assessed in terms of the responses in f and D on the growth of S. 11I11tans and C. albicans as biofilms. Biofilms of S. 1II11tans had a reduced mass and viscoelasticity when grown on this modified surface and also showed increased variability in terms of their complexity and stability as shown by the D/f ratio. Biofilms of C. albicans showed a reduced level of viscoelasticity as indicated by a reduced D factor and reduced complexity indicated by the D/fratio. Biofilms of C. albicans grown upon an AgNPs surface showed reduced mass and viscoelasticity in comparison with the growth on an unmodified surface and growth upon the various stages of surface creation i.e. an MUA Self Assembled Monolayer and a hydrazide surface. AFM images and analyses supported the presence of cells on these surfaces, as well as the reduction in cell number due to inhibition by nanopartic1es.

Little is known about the distribution of bacterial indicators and pathogens in biofilms on different surface types in natural aquatic environments. This study was conducted to examine the distribution of pathogens and indicator bacteria in biofilms in an agricultural watershed. The study particularly focused on whether biofilms can act as sinks for pathogenic organisms and could be monitored to protect public health. To do so, we monitored the presence of faecal contamination indicators (heterotrophic plate counts, faecal coliforms, enterococci, and E. coli) and particular pathogens (E. coli 0157, Campylobacter sp. and Salmonella sp.) in water, sediment, and in biofilms on river and slate rock, wood, sandpaper, and Lexan™ in Elk Creek (British Columbia, Canada) from December 2005 to April 2007. Faecal indicator concentrations and pathogen presence were evaluated using standard culturing and isolation methods. The results showed that both faecal indicators and pathogens were present at the headwaters and that the use of water column grab samples underestimated faecal indicator numbers. Also, water column grab samples during the dry season were not representative of pathogens present in the creek. This indicates that biofilms might be the main reservoir of Salmonella sp. and pathogenic E. coli O157 in the summer when rainfall (which results in flow changes and sloughing) is limited. Campylobacter sp. was not retrieved in the dry season. Campylobacter on sediment, slate rock and wood showed high correlations with nitrates and enterococci, which could be used as faecal contamination surrogates. Numbers of indicator organisms and pathogens in one-month biofilms were compared to those in long-term biofilms (colonized 12 and 24 weeks) and short-term biofilms (colonized one to three weeks). The comparison showed that surface type, colonization period and water quality all affected the concentration of indicator organisms and pathogens present in biofilms. Finally, results showed high levels of phenotypic antibiotic resistance of E. coli and pathogenic E. coli O157 isolated from the watershed (even at the headwaters), particularly to tetracycline, ampicillin and streptomycin. This study highlights the potential biofilms could play in prediction of water quality changes, the improvement of sampling methods, and the study of aquatic environments.

A research report submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Engineering. August, 2017
The foremost aim of potable water treatment is to produce water that does not pose a health risk when consumed and/or otherwise used. Nevertheless, research has established that the quality of treated water deteriorates during distribution. The nature and extent of this deterioration varies from system to system and from time to time. The aim of this research study was to monitor the parameters that are known to significantly affect biostability and biofilm formation potential in drinking water distribution systems. Biweekly water samples were collected from thirteen sites, across a section of Johannesburg Water’s network, between September 2015 and August 2016. All samples were assayed for a suite of fifteen water quality parameters using standard methods. Heightened temperature, dearth of chlorine residuals, availability of biodegradable dissolved organic carbon (BDOC), and advanced water age all engendered the loss of biostability (instability). Biostability controlling parameters varied seasonally and spatially. Samples collected during spring and summer, in general, were most likely to be characterized by instability than samples collected during winter and autumn. Samples collected from sites RW80, RW81, RW82, RW83, RW104 and RW253 were more prone to instability compared to samples from other sites. From the results, it is clear that chlorine residuals ought to be kept above 0.2 mg/l, and, BDOC below 0.3mg/l to prevent the loss of heterotrophic stability in distributed water. BDOC concentrations can be decreased by, flushing the pipes, cleaning reservoirs regularly and by further treating feed water before distributing. Booster disinfection can be relied upon to ensure that chlorine residuals are maintained throughout the network. Apart from potential health risks, biological instability and biofilm growth can result in non-compliance with regulations.
MT2018

Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.

A research report submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in Partial Fulfilment of the requirements for the degree of Masters of Science in Engineering, 2017
Access to good quality drinking water is essential for the maintenance of public health. To guarantee a steady supply of good quality water, water treatments plants are designed to provide potable water that meets national and, where necessary, local water quality standards. While the protection of natural water resources against pollution, and proper treatment of water at treatment plants are both crucial to the provision of safe drinking water, the reality is that the quality of treated water can degrade during distribution. Microbial proliferation within distribution systems can cause problems such as unpleasant tastes and odours as well as the proliferation of pathogenic microorganisms. For most utilities, it is biofilm that grows on pipe surfaces that act as permanent inocula continuously inoculating the bulk water as it flows through the distribution system. Distribution system biofilm growth and the resulting impact on water quality can be minimized by various treatment processes, designed to remove biodegradable organic matter (BOM) from the water. The removal of BOM is of great importance to water utilities because it eliminates bacterial regrowth and the many associated water quality issues. Hence, the spatial and temporal mapping of biodegradable organic carbon (BDOC) offers water utilities an effective strategy in managing the BOM in the distribution system. This research is aimed at evaluating the applicability of BOM measurement protocols to monitoring biostability and biofilm formation potential within a drinking water distribution system (DWDS). This study specifically investigated the efficacy of a simplified version of the high-density BDOC test as a protocol for monitoring BDOC in finished water. The high-density BDOC protocol was found to be a more streamlined approach in contrast to the assimilable organic carbon (AOC), and provides a suitable monitoring mechanism for lowering biofilm formation potential in DWDSs.
CK2018

碩士
國立交通大學
應用化學系碩博士班
100
Most bacteria in natural habitats live as biofilms, in which bacterial cells are embedded in matrices of extracellular polymeric substances adherent to a surface. Recent studies have revealed that the biofilm mode of living increases the survival rate of bacteria by e.g., acquiring extremely high antibiotic resistance. Although unique structures and functions of biofilms have attracted much attention in many disciplines of science, it remains challenging to study biofilms in vivo and in situ as well as with high chemical specificity. In the present study, we focus on the biofilms of the bacterial species of practical interest known as Rhodococcus sp. SD-74 and monitor the process of its biofilm development using Raman microspectroscopy and imaging. Because Raman spectroscopy is nondestructive and label-free, and is less hampered by the presence of water, Raman microspectroscopy and imaging are an ideal tool to study biofilms in situ. We successfully obtained detailed distribution and composition changes within R. sp. SD-74 biofilm during the development process. We found that the concentration of carotenoids in the biofilm matrix drastically increases as biofilm development proceeds. To interpret this phenomenon of carotenoid accumulation, we hypothesize that carotenoids act as an antioxidant in R. sp. SD-74 biofilms and counter possible oxidative stress. Raman imaging experiments performed under different light-illumination conditions support this hypothesis.

The aim of this study was to design and optimize new PCR primers for detection of potential cyanobacterial producers of cytotoxic lipopeptides puwainaphycins and minutissamides in environmental samples. Samples from two distinct localities were tested, as suggested based on preliminary data. The first set of samples consisted of cyanobacterial soil biofilms from sheep pastures affected by Alveld illness in Norway. The other one contained samples of planktic cyanobacaterial blooms from Protected Landscape Area Třeboň and its vicinity. Three different approaches were used for evaluation of the presence of cyanobacterial lipopeptide producers: microscopy, PCR with the designed primeres, and liquid chromatography-mass spectrometry analysis. Results of this study confirmed the specificity of the newly designed PCR primers. The presence of producers of puwainaphycins/minutissamides was proven at both tested localities.

碩士
國立臺灣科技大學
化學工程系
105
Bacteriorhodopsin (BR) is a retinal protein residing in Halobacterium salinarum purple membrane (PM). When illuminated, BR pumps a proton gradient across PM, leading to photocurrent generation. To improve the selectivity of antibody-PM and aptamer-PM composite sensor chips for Streptococcus mutans detection, which were previously developed by using the principles that PM photocurrent correlates linearly with illumination intensity and that bacteria scatter light, this study used glycine to block the residual active moiety of the homobifunctional crosslinker fabricated on PM-coated chips for recognition-element conjugation. For the detection of 106 CFU/mL bacteria, without prior glycine blocking, the antibody-PM chips exhibited not only a 53 % photocurrent reduction on S. mutans detection, but also 21% and 24% reductions for E. coli and L. acidophilus, respectively. On the other hand, with prior glycine blocking, the photocurrent reductions of the chips decreased to 8% and 7% on E. coli and L. acidophilus detections, respectively, indicating selectivity improvement of the antibody-PM chips. The effect of glycine blocking was less pronounced with the aptamer-PM chips because the photocurrent reductions decreased from 14% and 19% to only 12% and 15% for E. coli and L. acidophilus, respectively. Furthermore, a double-decked microfluidics device was designed and constructed to real-time monitor the formation of S. mutans biofilm on the substrate placed in the upper deck with a PM-coated photoelectric chip mounted in the lower deck. A diluted growth medium and an electrolyte buffer were injected into the upper and lower decks, respectively. The comparison of ITO glass modified with self-assembled monolayers with different terminal groups suggested that the hydrophobic and positively charged surface most easily caused S. mutans to form a mature 3D-strucutred biofilm. Moreover, the anti-biofilm peptides coated on the amine-terminated substrate effectively inhibited biofilm formation, with the inhibition efficiency increasing with their coating amounts. Therefore, the results confirmed the feasibility of real-time monitoring biofilm formation with the developed double-decked microfluidics.

Hygienistic food monitoring can be performed at various supply chain levels; the thesis “Food chains Hygiene: monitoring of retail products and characterization of isolated microbial species (virulence factors, ability to form biofilm, antibiotic resistance)” focused attention on the final phase of this distribution chain. Different types of meat and vegetables samples were investigated on microbiological basis. Samples were collected in Naples city (Italy). According to the the CE Regulation 2073/2005, the following microbiological protocols were assessed: total bacterial count, Escherichia coli, Salmonella spp. . The microbiological monitoring was additionally performed to evaluate: Sulphite-reducing Clostridia, Clostridium difficile, Listeria spp and L. monocytogenes, Staphylococcus spp. and S. aureus, Campylobacter spp., E. coli O157: H7, Clostridium perfringens, Enterococcus spp., Aeromonas spp .. In addition, virulence factors studies were realized. The statistical study involved the verification of positivities registered and the frequency distributions analysis. These evaluations provided useful input to the risk assessment associated with production chains.

碩士
國立臺灣大學
農業化學研究所
90
Pseudomonas aeruginosa is an opportunistic pathogen that can cause serious infections in immunocompromised hosts. When forming biofilms, P. aeruginosa will increase the resistence to antimicrobial agents. In this study, we have constructed the quorum-sensing systems, lasI and rhlI, recomplementary plasmids to examine the contribution of these two genes to the biofilm development. After 72 hours of cultivation, the cell densities of the PAO1(wild type), PDO100(ΔrhlI) and JP2(ΔlasIΔrhlI) biofilm reached 5.0±0.3x109 CFU/cm2, 6.4±3.9x108 CFU/cm2 and 1.6±1.1x108 CFU/cm2 respectively. The rhlI recomplementary to PDO100 biofilm reached 1.5±0.6x109 CFU/cm2 while lasI recomplementary to JP2 was 1.9±0.6x108 CFU/cm2. The polysaccharide and protein ratios of PAO1(wild type), PDO100(ΔrhlI) and JP2(ΔlasIΔrhlI) biofilm reached 0.8±0.1, 0.7±0.1, and 0.4±0.2 respectively. The rhlI recomplementary to PDO100 biofilm reached 0.8±0.2 while lasI recomplementary to JP2 was 0.7±0.2. Combined the above data with in conjuction of cryoembedding and cryosectioning couple fluorescent staining results, the lasI gene seems to play an important role in polysaccharide production while rhlI gene associated with the cell density in biofilms. To further elucidate the quorum-sensing gene expression within biofilms, we utilized green fluorescent protein to be the reporter gene to directly visulize the gene expression. The result indicated that the quorum-sensing gene expressed instinctly around biofilm.

La présence de matière biologique (biofilms) dans les sites de stockage géologique profond, d'éléments toxiques ou encore de l'eau potable des aquifères est maintenant clairement démontrée. Cette biomasse est à l'origine de processus physiques et chimiques qui modifient considérablement la durabilité et la pérennité des sites concernés. Ces processus, principalement de type oxydo-réductif, sont encore mal compris. Ceci est principalement dû aux méthodes d'investigation, principalement macroscopiques, loin de l'échelle micrométrique caractéristique des bactéries. Seules des études, basées sur des méthodes d'investigation locale, peuvent apporter les informations requises. Ainsi, nous avons développé un dispositif expérimental basé sur l'utilisation combinée de la microscopie optique (en transmission), la microscopie à force atomique (AFM) et la microscopie AFM en mode électrique et électrochimique (EC_AFM) afin d'obtenir des informations simultanées sur la topographie de l'échantillon et sur les processus électrochimiques à l'échelle des bactéries. La première étape sensible consistait à utiliser l'AFM sur des échantillons biologiques en milieu liquide: nous présentons ici les résultats de l'imagerie AFM en milieu liquide de plusieurs types de bactéries dans leurs conditions physiologiques naturelles (conditions in vivo). Aucun protocole d'immobilisation, ni chimique ni mécanique, n'a été nécessaire; et pour la première fois, les mouvements de reptation de cyanobactéries Nostoc ont été étudiés par l'AFM. Les études AFM ont permis d'acquérir des données topographiques mais aussi mécaniques : nous avons pu ainsi mesurer le module d'Young, la pression de turgescence de différentes souches bactériennes (Anabaenopsis circularis, Rhodococcus wratislaviensis). Cette étude complète, a révélé que l'imagerie AFM est donc possible sur des espèces vivantes en mouvement. Ces résultats ouvrent une grande fenêtre sur de nouvelles études d'intérêts tels que la formation de biofilms et les propriétés dynamiques de bactéries dans des conditions physiologiques réelles. La deuxième étape délicate était de combiner l'AFM aux mesures optiques et électriques. Nous avons développé un nouveau dispositif expérimental permettant (i) le suivi de l'évolution de la croissance bactérienne par la mesure des propriétés optiques comme la densité optique DO (pour le développement bactérien en volume - milieu planctonique) , ou l'analyse de l'image du substrat par comptage du nombre de bactéries sur la surface de l'échantillon (biofilm), et (ii) les mesures électriques et électrochimiques. L'ensemble de ces résultats sera prochainement appliqué au développement de nouveaux outils de surveillance d'une biodépollution de terrain contaminé par les hydrocarbures, par le suivi in situ et en temps réel de l'activité de bactéries dépolluantes (ECOTECH_BIOPHY ANR).