New federal regulations with regards to ammonia in wastewater effluent discharge will require over 1000 existing wastewater treatment facilities to be upgraded. Although biological treatment is the most common and economical means of wastewater ammonia removal, nitrification rates can be completely impeded at cold temperatures. Moving bed biofilm reactors (MBBR) have shown promise as an upgrade nitrifying unit at pilot-scale and full-scale applications with respect to low temperature nitrification. MBBR technologies offfer the advantages of less space requirement, utilizing the whole tank volume, no sludge recycling, and no backwashing, over other attached growth systems. Two laboratory MBBRs were used in this study to investigate MBBR nitrification rates at 20deg.C, after long-term exposure to 1deg.C, and at the kinetic threshold temperature of 5deg.C. Furthermore, the biologically produced solids from the MBBR system 20deg.C and after long-term exposure to 1deg.C, and the Arrhenius temperature correction models used to predict nitrification rates after long-term exposure to 1deg.C. The nitrification rates at 1deg.C over a four month exposure period as compared to the rate at 20deg.C were 18.7 + 5.5% and 15.7 + 4.7% for the two reactors. The nitrification rate at 5deg.C was 66.2 + 3.9% and 64.4 + 3.7% compared to the rate measured at 20deg.C for reactors 1 and 2, respectively, and as such was identified as the kinetic temperature threshold. The quantity of solids detached from the nitrifying MBBR biocarriers was low and did not vary significantly at 20deg.C and after long-term exposure to 1deg.C. Lastly, a temperature correction model based on exposure time to cold temperatures, developed by Delatolla et al. (2009) showed a strong correlation to the calculated ammonia removal rates relative to 20deg.C following a gradual acclimatization period to cold temperatures. Biofilm morphology along with biomass viability at various depths in the biofilm were investigated using variable pressure electron scanning microscope imaging (VPSEM) and confocal laser scanning microscope (CLSM) imaging in combination with viability live/dead staining. The biofilm thickness along with the number of viable cells showed significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant changes. Hence, this study observed higher cell activities at warm temperatures and a slightly greater quantity of biomass with lower activities at cold temperatures in nitrifying MBBR biofilms. Using DNA sequencing analysis, 'Nitrosomonas' and 'Nitrosospira' (ammonia oxidizers)as well as 'Ntrospira' (nitrite oxidizer) were identified in which no population shift was observed during 20deg.C and after long-term exposure to 1deg.C. Furthermore, a number of non-nitrifiers were identified int he biofilm during warm and cold temperatures presenting the possibility that their presence may have provided some form of protection to the nitrifiers during long-term temperature exposure.

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch. VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner. V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq. In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530. Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.

Natürliche Biofilme von zwei urankontaminierten Standorten, dem ehemaligen Uranbergwerk in Königstein (Sachsen) und dem Gebiet der ehemaligen Aufstandsfläche der Gessenhalde (Thüringen), wurden in dieser Arbeit näher untersucht. An beiden Standorten konnte in den Minenwässern die hochmobile, gelöste Uranspezies Uranylsulfat (UO2SO4) als dominierend nachgewiesen werden. Aufgrund der Instabilität vieler kommerzieller Fluoreszenzfarbstoffe bei niedrigen pH-Werten war eine gezielte Anfärbung der Mikroorganismen in den sauren Biofilmen nicht möglich, ohne den pH-Wert der Biofilmproben anzuheben, was die Probenchemie maßgeblich verändert. In Kooperation mit der Firma DYOMICS (Jena, Deutschland) wurden neue, kommerziell nicht erhältliche, säurestabile Farbstoffe erstmals hinsichtlich ihrer Eignung zur Anfärbung von Mikroorganismen in sauren Biofilmen ohne Veränderung des pH-Wertes sowie der sonstigen Probenchemie getestet. Die neuen Farbstoffe DY-601XL, V07-04118, V07-04146 und DY-613 zeigten eine Eignung für solche Färbungen, da sie eine intensive Anfärbung der Mikroorganismen bei niedrigen pH-Werten unter pH 3 – 4 herbeiführen und außerhalb des Emissionsbereiches von Uran fluoreszieren. In dieser Arbeit wurde die Fähigkeit von Euglena Mutabilis-Zellen zur Bioakkumulation des Urans im pH-Wertbereich 3 – 6 in den Hintergrundmedien Natriumperchlorat (9 g/l) oder Natriumsulfat (3,48 g/l) an lebenden Zellen untersucht. Unabhängig vom Medium konnte bei sauren pH-Werten um pH 3 – 4 über 90 % des vorgelegten Urans aus den Probelösungen abgetrennt werden. Die Speziation des an den Euglena-Zellen akkumulierten Urans, wurde mittels laserinduzierter Fluoreszenzspektroskopie (LIFS) untersucht. Es zeigte sich, dass unabhängig vom Hintergrundmedium, Lebenszustand und pH-Wert eine vergleichbare neue Uranspezies an den Zellen gebildet wird. Durch den Vergleich der Daten aus den LIFS-Messungen mit Referenzwerten, konnte die gebildete Uranspezies auf eine Anbindung durch (organo)phosphatische und/oder carboxylische funktionelle Gruppen eingegrenzt werden. Mit Hilfe der zeitaufgelösten FT-IR-Spektroskopie konnte die carboxylische Anbindung des Urans an toten Zellen nachgewiesen werden. Ein Ausschluss der (organo)phosphatischen Komplexierung konnte jedoch mit dieser Methode nicht geführt werden. Untersuchungen zur Lokalisation des Urans an bzw. in den Zellen, mittels der gekoppelten CLSM/LIFS-Technik zeigten erstmals ein Indiz für die intrazelluläre Akkumulation von Uran in den lebenden Zellen. Ergänzende TEM/EDX-Messungen bestätigten die intrazelluläre Aufnahme und belegen eine Akkumulation in runden bis ovalen Zellorganellen, bei denen es sich vermutlich um Vakuolen oder Vakuolen-ähnliche Vesikel handelt. An den toten Zellen konnte mit diesen Methoden kein Uran detektiert werden. Dies lässt auf eine passive, homogen verteilte Biosorption des Urans an die verfügbaren Bindungsplätze an der Zelloberfläche der toten Biomasse schließen.

The application of membrane bioreactors (MBRs) to wastewater treatment is increasing due to their ability to operate at high biomass concentrations and to deliver effluents of high quality. The major challenges associated with the application of MBRs is fouling which can shorten the useful life of the membrane, increase in the amount of energy consumed, and the cost for membrane cleaning. The main reasons for fouling are the deposition of solids as a cake layer, pore plugging by colloidal particles, adsorption of soluble compounds and biofouling. Fouling is a particular problem for activated sludge membrane bioreactors (AS-MBRs) since this process deals with liquors having a high concentration of total solids as well as dissolved compounds such as extracellular polymeric substances (EPS). The combination of a moving bed biofilm reactor and a membrane reactor (MBBR-MR) has significant potential. It may be considered as a compact wastewater treatment process which can compensate for the drawbacks of AS-MBRs. Readily biodegradable COD is removed in the MBBR while particulate matter is separated by the membrane. To further reduce the membrane fouling the effects of adding an intermediate coagulation stage was investigated critically on membrane fouling. The present study includes an overall assessment of the performance of a combined MBBR-MR system, based on the chemical oxygen demand (COD) removal efficiency and membrane fouling mechanism. The required test runs were conducted using pilot-scale MBBR and ultra filtration membrane. The pilot MBBR had a working volume of 1.8 m3 with a 60% carrier fill fraction. The MBBR was operated with loading rate of 78 ± 21 g/m2/d (HRT of 4 h). The ultra-filtration was spiral wound and composed of polyethersulfone (PES) with a pore size of 0.03 microns. The MBBR feed was obtained from a final treated wastewater effluent in a food processing plant located in SW Ontario. In this research, ferric chloride was also employed as a coagulant and influences of different coagulant doses and permeate fluxes on membrane fouling were studied. Based on the experimental results, it was found that the combination of MBBR with membrane filtration can produce a constant high quality permeate that is appropriate for water reuse purposes. The composition analysis of permeate showed that the stream is free of suspended solids and the average COD turns to 75 ± 25 mg/l. In addition, the MBBR had a SCOD removal of 76% ± 7% which is considered as a reasonable efficiency for a single reactor. Operating the membrane without adding coagulant caused rapid fouling in a short time period and the Trans Membrane Pressure (TMP) reached the maximum allowable pressure of 10 psi. However, addition of coagulant was found to decrease the fouling of the membrane as well as increasing the filtration time. The extent of the pre-coagulation effect on membrane fouling was found to strongly depend on the dosage of the coagulant and the MBBR effluent characteristics. A coagulant dose of 400 mg/l with a permeate flux of 7.6 LMH performed the best at reducing membrane fouling. Colloidal fouling was found to be a significant fouling mechanism at low coagulant dose (e.g. 200 mg/l), while cake formation appeared to be mainly responsible for fouling at higher coagulant doses. Permeate flux was found to have a significant effect on the fouling of the membrane. The presence of colloidal matters at low fluxes and TSS at higher fluxes were responsible for fouling of the membrane by blocking the pores and formation of the cake layer on the membrane surface, respectively. Then later addition of Dissolved Air Flotation (DAF) inside the factory had a noticeable effect on wastewater characteristics and consequently on fouling of the membrane. A 22% and 31% improvement in TCOD and TSS in the wastewater was observed leading to reduction in the fouling.

Es calcula que prop d'un 95% de la biomassa bacteriana que es pot trobar en els sistemes de distribució d'aigua potable es troba localitzada adherida a les superfícies internes de les canonades en forma de biopel·lícules, i que només un 5% es troba lliure en la fase aquosa. El creixement dels microorganismes formant biofilms sobre les superfícies altera la composició dels materials. En el cas de les superfícies metàl·liques, les reaccions fisicoquímiques es tradueixen en canvis indesitjables que es veuen agreujades per la biocorrosió o la corrosió influenciada per microorganismes. El coure és un element traça essencial, però ingerit de forma massiva és tòxic. S'han descrit casos d'intoxicacions a causa de problemes de corrosió i cuprosolvència on els microorganismes juguen un paper primordial. Alhora, alguns bacteris formadors del biofilms tenen la capacitat d'acumular el metall, incrementant el risc per la salut pública. Aquest treball posa de manifest com Agrobacterium radiobacter EPS-916 és capaç de captar coure provinent de solucions aquoses de CuSO4.5H2O, a diferents concentracions, així com el seu exopolisacàrid. Mitjançant microscòpia electrònica de transmissió s'ha comprovat que la captació del coure per part del microorganisme és intracel·lular, observant-se cúmuls densos en el citoplasma dels bacteris. A més s'ha aïllat Pseudomonas putida-M16 de les canonades de coure d'ús domèstic i ha demostrat una resistència elevada a la sal de coure, essent també capaç d'acumular-lo. A més les dues soques han estat capaces de captar el metall quan aquest es presenta en forma de superfície cúprica en cupons de coure i superficies de canonades. Per concloure l'estudi les imatges obtingues mitjançant microscòpia de rastreig han demostrat la presència de biopel·lícules tant en les superfícies cúpriques estudiades, així com en la superfície interior d'una secció de canonada de coure després d'un període aproximat de dos anys en contacte amb aigua. / In their natural environments, bacteria don't exist as isolated cells but grow and survive in organized communities commonly referred to as biofilms. Biofilms are differentiaded masses of microbes that form on surfaces and are surrounded by an extracellular matrix (EPS). Generally, biofouling can be defined as the undesired accumulation of deposits of a biological nature on surface. The former was defined as the process resulting from the accumulation of microbial biofilms consisting of: microorganisms, polymeric materials secreted by theses organisms, particulate matter of different origin, and, mainly of water. The accumulation of biofilms on copper plumbing pipes is well established. Biofilms have been implicated in the corrosion of copper plumbing, Known as cuprosolvency, which results in increases in soluble copper concentrations in drinking water supplies.This has significant implications for public health as the consumption of copper in water at levels exceeding 2mg/mL can cause adverse gastrointestinal symptoms in humans. This study investigated the capacity of Agrobacterium radiobacter EPS-916 to uptake copper from aqueous solutions of copper sulfate, and its skill to form biofilms in a copper plumbing. On the other hand our work want to show how biofilm bacteria isolated from domestic copper plumbing contributed to cuprosolvency and by scanning electron microscopy we can see the biofilm formation in copper surfaces.

Catheter-associated urinary tract infection (CAUTI) is one of the most common nosocomial infections and is caused by a range of different uropathogens, particularly by uropathogenic Escherichia coli (UPEC). Amongst the different virulence factors, biofilm formation and bacterial aggregation, often mediated by cell surface structures such as fimbriae, are common traits among uropathogens that cause CAUTI. In this study, a collection of UPEC isolates were screened for virulence genes and phenotypes associated with urinary tract infections such as biofilm formation and mannose-sensitive haemagglutination. Two strains, E. coli MS2027 (which formed a strong biofilm) and E. coli M184 (which aggregated strongly) were analysed in detail to determine the molecular mechanisms associated with these phenotypes. Transposon mutagenesis of E. coli MS2027 identified type 3 fimbriae as the factor responsible for its strong biofilm growth. Further screening revealed the presence of type 3 fimbriae in uropathogenic Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae and other E. coli. Phylogenetic analysis of the type 3 fimbrial (mrkABCD) genes from these strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of the sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. We demonstrated that type 3 fimbriae are functionally expressed by different Gram negative nosocomial pathogens and present evidence to suggest that they contribute significantly to catheter colonisation. The type 3 fimbrial genes from E. coli MS2027 were found to be located on a conjugative plasmid. Sequencing and annotation revealed that this 42,644 bp plasmid, named pMAS2027, contains 58 putative genes. Bioinformatic analysis identified pMAS2027 as an incompatibility X (IncX1) plasmid. Plasmid pMAS2027 contained genes encoding two important virulence factors, type 3 fimbriae and a type IV secretion (T4S) system. The biofilm ability was solely based on the expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027. Differential tagging with fluorescent reporter genes demonstrated conjugative transfer of pMAS2027 between cells during biofilm growth. Finaly, transposon mutagenesis of E. coli M184 revealed a number of putative genes potentially responsible for bacterial aggregation. Of these, genes involved in the synthesis of the enterobacterial common antigen (ECA) were shown to be associated with an aggregation phenotype.

Catheter-associated urinary tract infection (CAUTI) is one of the most common nosocomial infections and is caused by a range of different uropathogens, particularly by uropathogenic Escherichia coli (UPEC). Amongst the different virulence factors, biofilm formation and bacterial aggregation, often mediated by cell surface structures such as fimbriae, are common traits among uropathogens that cause CAUTI. In this study, a collection of UPEC isolates were screened for virulence genes and phenotypes associated with urinary tract infections such as biofilm formation and mannose-sensitive haemagglutination. Two strains, E. coli MS2027 (which formed a strong biofilm) and E. coli M184 (which aggregated strongly) were analysed in detail to determine the molecular mechanisms associated with these phenotypes. Transposon mutagenesis of E. coli MS2027 identified type 3 fimbriae as the factor responsible for its strong biofilm growth. Further screening revealed the presence of type 3 fimbriae in uropathogenic Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae and other E. coli. Phylogenetic analysis of the type 3 fimbrial (mrkABCD) genes from these strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of the sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. We demonstrated that type 3 fimbriae are functionally expressed by different Gram negative nosocomial pathogens and present evidence to suggest that they contribute significantly to catheter colonisation. The type 3 fimbrial genes from E. coli MS2027 were found to be located on a conjugative plasmid. Sequencing and annotation revealed that this 42,644 bp plasmid, named pMAS2027, contains 58 putative genes. Bioinformatic analysis identified pMAS2027 as an incompatibility X (IncX1) plasmid. Plasmid pMAS2027 contained genes encoding two important virulence factors, type 3 fimbriae and a type IV secretion (T4S) system. The biofilm ability was solely based on the expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027. Differential tagging with fluorescent reporter genes demonstrated conjugative transfer of pMAS2027 between cells during biofilm growth. Finaly, transposon mutagenesis of E. coli M184 revealed a number of putative genes potentially responsible for bacterial aggregation. Of these, genes involved in the synthesis of the enterobacterial common antigen (ECA) were shown to be associated with an aggregation phenotype.

Catheter-associated urinary tract infection (CAUTI) is one of the most common nosocomial infections and is caused by a range of different uropathogens, particularly by uropathogenic Escherichia coli (UPEC). Amongst the different virulence factors, biofilm formation and bacterial aggregation, often mediated by cell surface structures such as fimbriae, are common traits among uropathogens that cause CAUTI. In this study, a collection of UPEC isolates were screened for virulence genes and phenotypes associated with urinary tract infections such as biofilm formation and mannose-sensitive haemagglutination. Two strains, E. coli MS2027 (which formed a strong biofilm) and E. coli M184 (which aggregated strongly) were analysed in detail to determine the molecular mechanisms associated with these phenotypes. Transposon mutagenesis of E. coli MS2027 identified type 3 fimbriae as the factor responsible for its strong biofilm growth. Further screening revealed the presence of type 3 fimbriae in uropathogenic Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae and other E. coli. Phylogenetic analysis of the type 3 fimbrial (mrkABCD) genes from these strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of the sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. We demonstrated that type 3 fimbriae are functionally expressed by different Gram negative nosocomial pathogens and present evidence to suggest that they contribute significantly to catheter colonisation. The type 3 fimbrial genes from E. coli MS2027 were found to be located on a conjugative plasmid. Sequencing and annotation revealed that this 42,644 bp plasmid, named pMAS2027, contains 58 putative genes. Bioinformatic analysis identified pMAS2027 as an incompatibility X (IncX1) plasmid. Plasmid pMAS2027 contained genes encoding two important virulence factors, type 3 fimbriae and a type IV secretion (T4S) system. The biofilm ability was solely based on the expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027. Differential tagging with fluorescent reporter genes demonstrated conjugative transfer of pMAS2027 between cells during biofilm growth. Finaly, transposon mutagenesis of E. coli M184 revealed a number of putative genes potentially responsible for bacterial aggregation. Of these, genes involved in the synthesis of the enterobacterial common antigen (ECA) were shown to be associated with an aggregation phenotype.

Konstanz, Univ., Diss., 2007.

Zugl.: Braunschweig, Techn. Univ., Diss., 2006