We are coming to understand that the ecological importance of diatoms is not limited to primary productivity, as many diatoms produce extracellular polymeric substances (EPS), which are vital components in algal and bacterial “biofilms.” While great effort has been made to chemically identify the types of molecules and polymers used to create and modify diatom EPS there is still much about the process we do not know. Rather than studying this process chemically, we have elected to search for the genes involved in EPS production and secretion. We assembled transcriptomes from three EPS-producing diatoms (Cyclophora tenuis, Lucanicum concatenatum, Thalassionema frauenfeldii) and two diatoms which do not (Astrosyne radiata, Thalassionema sp. ‘BlueH20’). In an attempt to limit the differences to EPS-related transcripts, the taxa were selected in a phylogenetic framework (which is also discussed in this dissertation), where EPS-producing taxa were closely-related to taxa which did not produce EPS (A. radiata, C. tenuis, L. concatenatum as one set, T. frauenfeldii and T. sp. ‘BlueH20’ as the other). The resulting pool of transcripts sorted for contigs which appeared in the EPS-producing taxa but not their closely-related non EPS-producing counterparts, and those contigs were then compared to two annotated diatom genomes and sorted by function, looking specifically for genes related to secretion, polysaccharide assembly or modification and carbohydrate metabolism. In the Thalassionema clade, 41 contigs with the aforementioned annotations were found, while 22 such contigs were found in the Cyclophora/Lucanicum/Astrosyne clade. These putative EPS-related markers are identified in this dissertation for further study on their function and evolution across diatoms. / text

Dans l’environnement marin, les surfaces artificielles sont rapidement colonisées par des bactéries qui s’organisent en communautés appelées biofilms, s’entourant d’une matrice de substances polymériques extracellulaires (EPS). La formation d’un biofilm est une étape critique du processus nommé biofouling, c’est-à-dire l’accumulation de micro- et de macro-organismes sur une surface immergée, pouvant conduire à des conséquences néfastes dans le secteur marin. Dans cette étude, il s’agit d’identifier des souches bactériennes isolées de supports immergés en Mer Méditerranée et de les caractériser phénotypiquement par diverses approches. Leur capacité à former un biofilm in vitro a été évaluée dans différentes conditions avec une attention particulière portée sur leurs capacités à produire une matrice polymérique abondante riche en polysaccharides; l’objectif étant d’isoler des exopolysaccharides originaux à activité antifouling. Treize souches ont ainsi fait l’objet d’analyses phylogénétiques et d’une caractérisation phénotypique. Sept genres et douze espèces différentes ont été identifiés au sein desquelles deux isolats peuvent être affiliés à une nouvelle espèce, nommée Persicivirga mediterranea. Ce genre n’a jamais été décrit en Mer Méditerranée jusqu’à présent. L’extraction des EPS de chaque souche cultivée en biofilm a permis de déterminer leur composition générale en glucides, protéines, acides nucléiques et lipides. Une souche, Pseudoalteromonas ulvae TC14, se distingue par sa capacité à produire des exopolysaccharides en quantité importante. Il s’agit essentiellement de polymères du glucose dont les analyses chromatographiques et spectroscopiques ont révélé la diversité de taille (Mw ~ 1–4000 kDa), de charge (neutre ou anionique) et de fonction associée (lactate ou acétate). Les fractions d’EPS enrichies en polysaccharides inhibent la formation de biofilm par d’autres souches marines. Ces derniers sont également synthétisés par les bactéries en culture planctonique mais en proportions très différentes. / In marine environment, artificial surfaces are promptly colonized by biofilms, which are communities of bacteria surrounded by matrix of extracellular polymeric substances (EPS). Formation of biofilm is a critical step of biofouling development, which corresponds to the accumulation of micro and macro-organisms on immersed surfaces and which can have important negative ramifications in particular in the marine sector. In this study, bacteria isolated from the Mediterranean Sea have been identified and characterized using different phenotypical tools. Their capacity to form a biofilm in vitro has been studied in different conditions, with a particular focus on their ability to produce abundant carbohydrate-rich EPS, the overall objective of the study being the isolation of original antifouling-active exopolysaccharides. Thirteen strains have been phylogenetically and phenotypically characterized. Seven genera and twelve species were identified among which two isolates were affiliated to a new species, named Persicivirga mediterranea. This genus has never been described in the Mediterranean Sea. Extraction of EPS of each strain, grown in biofilm conditions, allowed the determination of their general composition in carbohydrates, proteins, nucleic acids and lipids. One strain, Pseudoalteromonas ulvae TC14, was able to produce large quantities of exopolysaccharides, comprising in majority polymers of glucose whose chromatographic and spectroscopic analyzes revealed a diversity in size (Mw ~ 1-4000 kDa), charge (neutral or anionic) and associated function (acetate or lactate). These polysaccharides inhibited biofilm formed by other marine strains isolated from the Mediterranean Sea. They can also be synthesized by planktonic TC14, but in very different proportions.

Microbial biofilms are significant in a variety of settings including the human microbiome, infectious disease, industrial processes, and environmental remediation. Due to the ubiquitous nature of biofilms, there is a great interest in understanding cellular activities within the biofilm matrix. Biofilm cells are able to better withstand environmental stress, experience increased horizontal gene transfer, and live longer. The purpose of this research is to grow Pseudomonas sp. strain ADP as a biofilm and examine the chemical and physical characteristics the microbe undertakes in a sticky extracellular matrix. ADP is the organism of choice because of its ability to metabolize atrazine. Cells are grown in a drip biofilm reactor and flow cells under varying time lapse to gain insight to biofilm formation. Some cells are grown with atrazine as the sole nitrogen source, while others are grown in a nutrient-rich medium to compare cells response under nutrient-limited conditions with atrazine particles in the matrix. As a positive control, Escherichia coli are grown in a similar manner. Raman spectroscopy was the main analytical technique used to evaluate the chemical and molecular characteristics of this system. Scanning electron microscopy is used to examine cellular distribution, and several assays are performed for molecular composition analysis. Raman analysis in the fingerprint region revealed distinct differences between free cells and cells in biofilm. Soluble extracellular polymeric substances (EPS) were found to be more prevalent than tightly bound EPS and lightly bound EPS in the biofilm matrix. Comparison of relative peak intensity ratios suggests that it is possible to track atrazine degradation by means of intermediates using Raman spectroscopy. SEM micrographs revealed EPS role as an immobilizing agent when in contact with compounds, such as atrazine. Further research is needed to determine if atrazine can bind to EPS fractions outside the presence of cells and whether its affinity to EPS is mostly attributed to physical conditions, due to the architecture of biofilm, or chemical, based on functional groups presents. The results obtained from this research will contribute to the development of a less invasive microscale approach to address the acquisition and induction of biotransformation activity occurring in xenobiotic degrading systems. The extracellular interactions observed can be used to further characterize biofilm-mediated bioremediation. Results have contributed to the Raman spectra library for microorganisms and organic compounds. .

Water and wastewater treatment solutions incorporating biofilm systems are becoming increasingly popular due to more stringent regulations pertaining to drinking water and wastewater effluent discharge in Canada and in other parts of the world. As a major component of biofilm, extracellular polymeric substances (EPS) have been considered as an important factor affecting the physical and chemical properties of biofilm. Further, the selected method of EPS extraction and the methods of detecting the composition of the EPS have shown to affect the results of EPS measurements. In this research, protocols for EPS extraction and EPS composition analysis were investigated and optimized for nitrifying moving bed biofilm reactor (MBBR) biofilm. In addition, the confocal Raman microscopy (CRM) spectra of EPS in nitrifying MBBR biofilm and the protein, polysaccharide and extracellular DNA (eDNA) percent concentrations of the EPS were investigated at various operating temperatures. Further, the CRM spectra and the protein, polysaccharide and eDNA percent concentration of EPS in nitrifying MBBR biofilm along with the biofilm morphology and thickness and the viability of the embedded cells were investigated at various hydraulic retention times (HRTs). The EPS was characterized at various temperatures and HRTs in order to investigate potential correlation between the EPS components of the nitrifying biofilm and the ammonia removal kinetics. The biofilm morphology and thickness along with the bacterial viability of the biofilm were also investigated at various HRTs. Biofilm morphology images and thickness measurements were acquired using a variable pressure scanning electron microscope (VPSEM). The percentages of viable embedded cells in the biofilm were quantified using live/dead staining in combination with confocal laser microscopy (CLSM) imaging. The research demonstrates that an increase in protein content and subsequently a decrease in polysaccharides and eDNA contents in the EPS of nitrifying MBBR biofilm were observed at the lowest operational HRT and the highest temperature in this work. In particular, the EPS protein to polysaccharide (PN/PS) ratio of nitrifying MBBR systems was shown to significantly decrease below a value of 3 when the system was underloaded (observed at the highest operational temperature in this study) or hydraulically overloaded (observed at the lowest HRT in this study). As such, data obtained at lower operational temperatures, with the system no longer underloaded, and at longer HRTs, with the system no longer hydraulically overloaded, all demonstrate an EPS PN/PS ratio of approximately 3. Correlations were observed between the chemically measured EPS PN/PS ratios and the measured Raman spectra intensity ratios; supporting the concept of higher PN/PS ratios of EPS in more optimal nitrifying MBBR operations. Further, the ammonia removal kinetics and EPS response at HRT values of 0.75 and 1.0 h indicate that nitrifying MBBR systems may be optimized to operate at HRTs as low as 0.75 to 1.0 hour as opposed to conventional HRTs of 2.0 to 6.0 h.

Research performed at the Division of Environmental Microbiology has over the last years resulted in the isolation of possible bacterial key-organisms with efficient nutrient removal properties (Comamonas denitrificans, Brachymonas denitrificans, Aeromonas hydrophila). Effective use of these organisms for enhanced nutrient removal in wastewater treatment applications requires the strains to be retained, to proliferate and to maintain biological activity within theprocess. This can be achieved by immobilization of the organisms using an appropriate system.Two putative immobilization systems, agar entrapment and biofilm formation, wereassessed. Surface attached biofilm growth provided better results with respect to cell retention,proliferation and microbial activity than immobilization in agar beads. Thus, biofilm physiology was further characterized using simplified systems of single, dual or multi strain bacterial consortia containing the key-organisms as well as other wastewater treatment isolates. Mechanisms for initial adherence, biofilm formation over time, dynamics and characteristics of extracellular polymeric substances (EPS) and exopolysaccharides, nutrient removal activity as well as the effect of bacterial interactions were investigated. The results showed that all theassessed bacterial strains could form single strain biofilm providing that a suitable nutrientsupply was given. Production of EPS was found to be critical for biofilm development and both EPS and polysaccharide residue composition varied with bacterial strain, culture conditions and biofilm age. Denitrification and phosphorus removal activity of the keyorganisms was maintained in biofilm growth. Co-culturing of two or more strains resulted in both synergistic and antagonistic effects on biofilm formation as well as the microbial activitywithin the biofilm. Bacterial interactions also induced the synthesis of new polysaccharideswhich were not produced in pure strain biofilms.The complexity of single and mixed strain biofilm development and the implications of interactions on biofilm performance were underlined in this study. The data presented can be useful for modeling of biofilm systems, serve as a tool for selection of bacterial strain combinations to use for bioaugmentation/bioremediation or provide a base for further experiment design. / QC 20100622

Aerobic (AeMBRs) and Anaerobic Membrane Bioreactors (AnMBRs) are an essential part of the advanced wastewater treatment options, which offer advantages in terms of higher effluent discharge and smaller footprints over the traditional wastewater treatment. This study evaluates the performance of (AnMBRs) by analyzing the cumulative effect of eleven physico-chemical parameters from the data obtained from the studies conducted from year 2000 onwards. Effect of various parameters such as Solid Retention Time (SRT), Hydraulic Retention Time (HRT), Mixed Liquor Suspended Solids (MLSS), influent Chemical Oxygen Demand (COD), Organic Loading Rate (OLR), influent COD, and temperature on the COD removal, methane production and membrane fouling were evaluated. Spearman’s correlation analysis was performed to investigate the impact of environmental and operational parameters on membrane fouling, COD reduction, EPS/SMP and methane production and explain the results. It should be noted that the literature used has all needed variables; incomplete data sets were removed for the regression analysis, in this case, the fouling rate may be estimated. Of these variables, the fouling rate was significantly correlated only with flux (r = 0.291, p =

No description available.

A contributing factor in recurrent Listeria monocytogenes (L. monocytogenes) food contamination is that this bacterium produces biofilms on surfaces to persist in food-processing environments. Quorum sensing (QS) is a cell-to-cell communication system utilized by bacteria within biofilms to collaborate and adapt to environmental stresses. However, the details of how the QS-dependent network contributes to biofilm development of L. monocytogenes have yet to be well understood. By comparing the transfer rates of planktonic and biofilm (sessile) L. monocytogenes from stainless steel blades to bologna slices, we found that sessile bacteria had reduced transferability onto a single slice but caused the increase in the number of contaminated slices. This suggests that physiological adaptions derived during biofilm development affect bacterial dissemination. Given the contribution of proteins and environmental temperatures to the extracellular polymeric substances (EPS) synthesis and biofilm integrity, we evaluated the exoproteomes of biofilms formed at 25 and 37°C using 2D-gel electrophoresis and LC-MS/MS. We found exoproteases Lmo0186, Cwh, and Spl exclusively in biofilms formed at 25°C and their greater expression in the gene level at 25°C. By using the zymography and crystal-violet-staining assay with a protease inhibitor, we observed a greater proteolytic activity at lower temperatures and showed that the attenuated proteolytic activity of proteases is positively correlated with increased biofilmorming ability at 25°C. Considering the transcriptional role of QS systems during biofilm development, we investigated how the accessory gene regulator (Agr)-based and metabolite S-Adenosylmethionine (SAM)-involved QS systems modulate nutrient availability and EPS synthesis. The results revealed that the SAM signal interacts with the Agr QS at the transcriptional level during biofilm development, whereas SAM and Agr QS regulate distinct EPS synthesis pathways. Additionally, this interaction is dependent on bacterial life modes (planktonic and sessile). Overall, we conclude that L. monocytogenes manipulates the synthesis of EPS with the coregulation of metabolism and QS for biofilm formation and the production of exoproteases for biofilm dispersion. These precise regulations on EPS enable L. monocytogenes to prolong its survival and promote its dissemination in environments.

Extracellular polymeric substances (EPS) in activated sludge are known to account for the flocculent nature of activated sludge. Extensive studies over the last few decades have attempted to extract and characterize activated sludge EPS, but a lack of agreement between studies has also been quite common. The molecular makeup of EPS has, however, remained nearly unexplored, leaving their identity, function, and fate over various stages in the activated sludge system mainly unknown. In spite of their critical involvement in bioflocculation and long history of related research, our understanding of EPS is still greatly limited and better elucidation of their composition and structure is needed. The hypothesis of this research was that activated sludge floc contains different fractions of EPS that are distinguishable by their association with certain cations and that each fraction behaves differently when subjected to shear, aerobic digestion, anaerobic digestion and other processes. In order to examine this floc hypothesis, the research mainly consisted of three sections: 1) development of EPS extraction methods that target cations of interest (divalent cations, especially calcium and magnesium, iron, and aluminum) from activated sludge; 2) molecular investigations on activated sludge EPS using metaproteomic analyses, comprising sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein identification by liquid chromatography tandem mass spectrometry (LC/MS/MS), and hemaagglutination (HA)/HA inhibition assays; and 3) investigating the fate of EPS in sludge digestion using SDS-PAGE. Evaluation of prior research and data from preliminary studies led to the development of the three extraction methods that were used to target specific cations from activated sludge and to release their associated EPS into solution. These methods are the cation exchange resin (CER) procedure for extracting Ca²⁺+Mg²⁺, sulfide extraction for removing Fe, and base treatment (pH 10.5) for dissolving Al. The cation selectivity in the three extraction methods, the composition of EPS (protein/polysaccharide), amino acid composition, and a series of sequential extraction data established initial research evidence that activated sludge EPS that are associated with different cations are not the same. SDS-PAGE was successfully applied to study extracellular proteins from several sources of both full- and bench-scale activated sludges. The three extraction methods led to different SDS-PAGE profiles, providing direct evidence that proteins released by the three methods were indeed different sludge proteins. Another important outcome from this stage of research was finding the similarity and differences of extracellular proteins between different sources of activated sludge. SDS-PAGE data showed that many of CER-extracted proteins were well conserved in all the sludges investigated, indicating that a significant fraction of Ca²⁺ and Mg²⁺-bound proteins are universal in activated sludge. On the other hand, protein profiles resulting from sulfide and base extraction were more diverse for different sludges, indicating that Al and Fe and their associated proteins are quite dynamic in activated sludge systems. Protein bands at high densities were analyzed for identifications by LC/MS/MS and several bacterial proteins and polypeptides originating from influent sewage were identified in this study. This was also thought to be the first account of protein identification work for full-scale activated sludge. The analysis of SDS-PAGE post sludge digestion revealed that CER-extracted proteins remained intact in anaerobic digestion while they were degraded in aerobic digestion. While the fate of sulfide-and base-extracted proteins in aerobic digestion was not as clearly resolved, their changes in anaerobic digestion were well determined in this research. Sulfide-extracted protein bands were reduced by anaerobic digestion, indicating that Fe-bound EPS were degraded under anaerobic conditions. While parts of base-extracted proteins disappeared after anaerobic digestion, others became more extractable along with the extraction of new proteins, indicating that the fate of base-extractable proteins, including Al-bound proteins, is more complex in anaerobic digestion than CER-extracted and sulfide-extracted proteins. These results show that Ca²⁺+Mg²⁺, Fe³⁺, and Al³⁺ play unique roles in floc formation and that each cation-associated EPS fraction imparts unique digestion characteristics to activated sludge. Finally, since a considerably different cation content is quite common for different wastewaters, it is postulated that this variability is one important factor that leads to different characteristics of activated sludge and sludge digestibility across facilities. The incorporation of the impact of cations and EPS on floc properties into an activated sludge model might be challenging but will assure a better engineering application of the activated sludge process. / Ph. D.

Bioflocculation happens naturally and microorganisms aggregate into flocs during wastewater treatment. It is critical to understand the mechanisms of bioflocculation and its impact on the following solid/liquid separation process since seperation by settling is one of the key aspects that determine the efficiency and the overall economy of activated sludge systems. Bioflocculation occurs via extracellular polymeric substances (EPS) and cations by creating a matrix to hold various floc components together so the cations become an important part of the floc structure. The main objective of this study is to investigate the effects of monovalent cations specifically potassium and sodium (K and Na) on the bioflocculation, settleability and dewaterability of activated sludge. The particular aim is to grow the mixed culture microorganisms in the presence of specific cation so that the effect of cation on the stimulation of EPS production can be seen. In order to achieve this aim, semi-continuous reactors were separately operated at concentrations of 5, 10, and 20 meq/L of each cation with mixed culture bacteria and fed with synthetic feed medium representing influent to the activated sludge systems. Also, a control reactor at low cation dose was operated for each reactor set. The effective volume of the reactors was 2 L with 8 days of sludge residence time (SRT) and pH was kept at 7.7&plusmn / 0.3. The activated sludge reactors were operated until the reactors reached steady state and then related analyses were conducted. It was found that addition of potassium and sodium ions at increasing concentrations resulted in increase in total polymer concentration. However, potassium ions promoted the synthesis of both polysaccharide and protein type polymers whereas sodium ions tended to stimulate production of protein type polymers and had an affinity to bind more protein within the floc structure. Sodium sludges had lower hydrophobicity and higher surface charges, so sodium ions led to deterioration in flocculation of sludges. Addition of both these ions decreased the dewaterability, sodium ions had more detrimental effect on dewaterability of sludges compared to potassium ions. The examination of data related to settleability showed that potassium ions led to no drastic deterioration in settling characteristics of the activated sludge but the addition of sodium ions deteriorated the settleability. In addition, it was seen that while the addition of potassium ions to the feed led to a decrease in viscosity, increase in sodium concentration correlated with an increase in viscosity. Finally, the comparison of chemical oxygen demand (COD) removal efficiency of these cations showed that sodium is more efficient in COD removal.