Browsing by Subject "Biofilm"
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Item Adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment(2013-12) Huse, Holly Kristen; Whiteley, MarvinChronic microbial infections result from persistent host colonization that is not cleared via the immune response or therapeutics. Within the host, microbes can undergo adaptive evolution, whereby beneficial traits promoting persistence arise due to selection; these traits can therefore affect disease outcomes and treatment strategies. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic, fatal respiratory infections in individuals with the heritable disease cystic fibrosis (CF). The goal of this dissertation is to identify adaptations that allow P. aeruginosa to persist in the host during chronic CF lung infection. To achieve this goal, P. aeruginosa was chronologically sampled from 3 CF patients, ranging from the first infecting bacterium (the ancestor) to ~40,000 generations post-infection. By comparing gene expression profiles of ancestral and evolved isolates sampled from multiple patients, I identified 24 parallel gene expression changes that occurred over time within each lineage, suggesting that these traits are beneficial to the bacterium. Because most of these traits had unknown physiological roles, I sought to characterize their biological significance. I used a gain-of-function genetic screen and discovered that a subset of these genes enhance biofilm formation, a sessile mode of growth proposed to be important during chronic CF lung infection. I showed that enhanced biofilm formation is due to increased production of the exopolysaccharide Psl, which is traditionally viewed as less critical for maintaining chronic infections than other virulence factors. Lastly, I demonstrated that a majority (~72%) of chronic P. aeruginosa isolates produce more Psl than their corresponding ancestor, suggesting that this exopolysaccharide is important during chronic infection and an adaptive trait.Item Comparison of wire and plastic belts on microbial attachment and biofilm formation(Texas Tech University, 2006-08) Pitchiah, Shankaralingam; Brashears, Mindy M.; Alvarado, Christine Z.; Jeter, Randall M.Food processing equipment is subject to attachment of bacteria, which can lead to product contamination, spoilage, and surface destruction. Prevention of bacterial contamination during processing of poultry products is a major concern for food processors. During processing of poultry products, bacteria from the carcass can attach to wet surfaces which can lead to biofilm formation providing a source for cross contamination for subsequent carcasses. The purpose of this study was to determine susceptibility to bacterial attachment and biofilm formation with and without poultry products of different conveyor belts including polyurethane with mono polyester fabric, acetal (3.2 % open mesh), polypropylene- mesh top (24% open mesh), polypropylene (48% open mesh), stainless steel – single loop (80% open mesh) and stainless steel – balance weave (70% open mesh). To perform the sanitizer efficacy against attached bacteria on different conveyor belts test chips were prepared. Test chips were made in duplicate and inoculated with raw chicken. One test chip was removed to check aerobic plate count and coliform count while other test chip was exposed to quaternary ammonia (200 ppm). Except polyurethane with mono polyester fabric others belts showed significant reduction (P < 0.05) after exposed to sanitizer. Surfaces were inoculated with Salmonella / Listeria monocytogenes cocktail (without any poultry product) in BPW to achieve final inoculum level of 5 log CFU/ml. Test surfaces were analyzed by sponge and swab method at 0, 1, 2, 4, 6, 8, 12, 24 and 48 hr. Initial attachment (0 hr) of Salmonella / Listeria monocytogenes did not show any significant (P < 0.05) change in all belt types; however, polyurethane with mono polyester fabric belts showed a significant attachment over time. Attachment of bacteria with poultry products was determined with test chips. Poultry products were inoculated with Salmonella or Listeria monocytogenes culture for one hour and rinsed with PBS. Test chips were immersed in this solution and evaluated for 1, 6, 12, 24 and 48 hr, through vortexing with glass beads. At 1 and 48 hr, attachment of Salmonella and Listeria monocytogenes was lowest in the stainless steel single loop, and stainless steel –balance weave. Attachment of Listeria monocytogenes to form a biolfim was determined. Initial biofilm formation was very low on the stainless steel belts but by day 4 all belts had all belts showed significant biofilm formation. An increased understanding of bacterial attachment and biofilm formation with respect different design of conveyor belts will assist in the development of interventions to counter act these processes and thereby, enhance plant sanitation and pathogen control.Item Control of Listeria monocytogenes on contact and non-contact surfaces by electrostatic spraying of quaternary ammonia(2008-12) Dow, Andrea E.; Alvarado, Christine Z.; Brashears, Mindy M.; Takhar, Pawan S.The attachment of bacteria on food processing equipment and in the environment can cause potential cross- contamination, which can lead to food spoilage, possible food safety concerns, and surface destruction. Food contact surfaces used for food handling, storage or processing as well as environmental surfaces such as drains, walls, and floors are areas where microbial contamination commonly occurs. Even with proper cleaning and sanitation regimes or practices in place, bacteria can remain attached to the surfaces and this attachment can lead to biofilm formation. The purpose of this research was to determine if electrostatic spraying of quaternary ammonium compounds would provide a more efficient means for sanitizing food contact and environmental surfaces to prevent bacterial attachment and biofilm formation compared to conventional application. The surfaces subjected to 200 ppm of ala-quaternary ammonium treatments were ceramic tile, FRP (plastic wall board), polypropylene conveyor belt- mesh top (24% open mesh) and stainless steel conveyor – single loop (80% open mesh). To determine the efficacy of the electrostatic sprayer in comparison to pressure spraying for sanitizing, the surfaces were inoculated with a Listeria monocytogenes cocktail with a final concentration of 106 cfu/ ml. The test surfaces were analyzed with a swab method prior to inoculation, after 1 hr of attachment and after sanitizing treatment. Overall there were significant (P< 0.05) reductions in the amount of LM that remained on the surfaces after being treated with both the electrostatic spray and the air- pressure spray. There were no significant differences in the bacterial loads between the two treatments (P< 0.05).To determine electrostatic spray could prevent biofilm formation, surfaces were treated with quaternary ammonium electrostatic spray or a pressure sprayer. The surfaces that were used were ceramic tile, FRP, stainless steel coupons (306 food grade), and polyethylene (plastic cutting board). After the surfaces were subjected to the treatment, they were allowed to air dry for 30 minutes. The surfaces were then placed in wells containing a Listeria monocytogenes solution with a concentration of 106 cfu/ mL. The test surfaces were analyzed after 24 hrs by crystal method analysis and scanning electron microscopy (SEM). Results from the crystal method analysis indicated that electrostatic spray significantly (P< 0.05) reduced the biofilm formation on all the surfaces. Overall, electrostatic spray of quaternary ammonium provided a more efficient means of biofilm prevention on multiple food contact and non- contact surfaces.Item Coupling algae biofilm cultivation with wastewater treatment for sustainable biomass production and nutrient recycling(2015-05) Campbell, Daniel Martin; Berberoglu, Halil; Murphy, ThomasThe main objective of this study was to model the transport phenomena through which algal biofilms are able to recover nutrients contained within secondarily treated wastewater and to gauge the potential of hydrothermal liquefaction process water as a viable nutrient source. The cultivation of algae in the form of biofilms has shown promise due to this technique's greater energy return on investment during cultivation. These biofilms are known to be nutrient transport limited, thus an understanding of the phenomena associated with nutrient transport into algal biofilms is necessary for the optimized design and effective implementation of fixed cultivation techniques. This thesis reports a numerical study that investigates the transport of dissolved nutrients from wastewater into biofilms and an experimental study of the suitability of hydrothermal liquefaction process water as a nutrient source for algal cultivation. For the numerical study, the biofilm is modeled as a flat surface whose composition and growth characteristics were obtained from values in the literature. The fluid over the biofilm is modeled as a one-dimensional flow over a flat surface. The transport of dissolved nutrients was assumed to be diffusion dominated into the biofilm and was described through Fick's law. A novel derivation of the concentration boundary layer was demonstrated for use in a fully developed, thin fluid layer. The areal algal productivity and the nutrient removal rate by the biofilm were determined using the generated model and were validated against the literature data. The results showed that biofilm productivity and nutrient removal rates are highly sensitive to the presence of boundary layers, with a nearly order of magnitude decrease in productivity within the first meter of the modeled reactor. Finally, a parametric study on the effects of fluid velocity and depth on nutrient removal rates was performed. The study indicated the nutrient uptake rates increased by 24% with a 50% reduction in fluid depth and increased by 118% with a ten-fold increase in fluid velocity In the second part of this thesis, nitrate and phosphate concentrations in the process water of hydrothermal liquefaction of municipal biosolids were determined by ion chromatography to gauge its potential as a nutrient medium for algal cultivation. A parametric study on the effect of process conditions with the intent of determining the best nutrient source for algal cultivation indicated that the aqueous nutrient content was largely unaffected by heating rate, but was highly sensitive to the type of biosolid.Item Development of a novel algae biofilm photobioreactor for biofuel production(2012-08) Ozkan, Altan; Berberoglu, Halil; Kinney, Kerry; Katz, Lynn; Kirisits, Mary J.; Lawler, Desmond; Brand, Jerry; Cetiner, SelimAlgae are photosynthetic microorganisms that convert carbon dioxide and sunlight into biomass that can be used for biofuel production. Although they are usually cultivated in suspension, these microorganisms are capable of forming productive biofilms over substrata given the right conditions. This dissertation focuses on algal biofilms and their application in biofuel feedstock production. In particular it reports the construction and performance of an algae biofilm photobioreactor, the physico-chemical surface properties of different algal species and adhesion substrata, and cell-surface interactions based on experimental results and theoretical models. A novel algae biofilm photobioreactor was constructed and operated (i) to demonstrate the proof of concept, (ii) to analyze the performance of the system, and (iii) to determine the key advantages and short comings for further research. The results indicated that significant reductions in water and energy requirements were possible with the biofilm photobioreactor. Although the system achieved net energy ratio of about 6, the overall productivity was low as Botryococcus branunii is notoriously slow growing algae. Thus, further studies were focused on identification of algal species capable of biofilm growth with larger biomass and lipid productivities. Adhesion of cells to substrata precedes the formation of all biofilms. A comprehensive study has been conducted to determine the interactions of a planktonic and a benthic algal species with hydrophilic and hydrophobic substrata. The physico-chemical surface properties of the algal cells and substrata were determined and using these data, cell-substrata interactions were modeled with the thermodynamic, Derjaguin, Landau Verwey, Overbeek (DLVO) and Extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) approaches and critical parameters for algal adhesion were identified. Finally, the adhesion rate and strength of algal species were quantified with parallel plate flow chamber experiments. The results indicated that both cell and substrata surface hydrophobicity played a critical role for the adhesion rate and strength of the cells and XDLVO approach was the most accurate model. Finally, based on these findings the physico-chemical surface properties of ten algal species and six substrata were quantified and a screening was done to determine algae species substratum couples favoring adhesion and biofilm formation.Item Effect of Nutrition on In Vitro Biofilm Formation of Gastrointestinal Associated Microbes(2013-04-28) Hokazono, AsukaGastrointestinal (GI) health is an important contributor to one?s overall well-being. In the past decade, the focus of this aspect of health has been on the organisms that inhabit the intestines: gut microbes. A concept central to understanding bacterial behavior for health or disease promotion is biological film (biofilm) formation. The predominant form of growth for bacteria is biofilm formation, an as yet poorly described phenomenon for gut microbes. In order to better understand bacterial behavior in response to nutrients that pass through the GI system, a high throughput system to assess biofilm formation was developed. Gastrointestinal-associated microbes, Escherichia coli and Enterococcus faecalis, were assayed for biofilm formation in 96-well plates after 24 hours of incubation. Nutrients, inter-and intrakingdom signaling molecules such as monosaccharides, calcium, insulin, endocannabinoids, and AI-1, AI-2 like signaling compounds, respectively, were added to cultures in order to test their effects on biofilm formation. Biofilm was quantified spectrophotometrically by the measurement of optical density of each well measured at 580nm following crystal violet staining of adherent biofilm. Values were expressed as means ? standard error of the mean (SEM); differences between means were assessed using t-testing and ANOVA using GraphPad Prism, with mean differences considered significant at P < 0.05. Results showed that biofilm formation by E. coli was enhanced by glucose, galactose, lactose, AI-1 like signaling compound and insulin at 50 and 100?U/ml, while AI-2 like compound and calcium inhibited biofilm formation. Biofilm formation by E. faecalis was also enhanced by AI-1 like compound and insulin at 50?U/ml in RPMI medium and inhibited in cultures grown in BHI medium or with added calcium. We conclude that gastrointestinal-associated microbes are influenced by nutrients as well as various factors, including the culture medium, signaling compounds, as well as host-signaling compounds such as insulin and calcium. This study provides a platform required for future studies involving nutrient effect on biofilm formation.Item The effects of polymicrobial metabolism on pathogenesis and survival in Aggregatibacter actinomycetemcomitans(2011-08) Ramsey, Matthew M.; Whiteley, Marvin; Marcotte, Edward; Meyer, Richard; Trent, Stephen M.; Walker, JamesIn this dissertation I describe a model system to characterize the response of an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans to the metabolic byproducts of a representative member of the oral flora, Streptococcus gordonii. A. actinomycetemcomitans is a causative agent of periodontal infections in humans. To cause infection, A. actinomycetemcomitans must overcome numerous challenges, including the host immune system and toxic metabolite production from other microbes. The most numerically dominant flora in the oral cavity are oral streptococci, which are well known for their ability to produce copious amounts of lactic acid and H₂O₂. By studying the response to H₂O₂ and lactic acid in pure and co-cultures, I have demonstrated that A. actinomycetemcomitans responds to these metabolites by several novel mechanisms that both enhance its survival in the presence of the host immune system and in the presence of the model oral streptococci S. gordonii. These studies have demonstrated that metabolites produced by normal flora can impact the survival of a single species in vivo as much as previously known virulence factors have done. In addition, I present a new method for measuring metabolite production in an attached cell population. This method is a novel application of scanning electrochemical microscopy (SECM) and I used this technique to study H₂O₂ production in the three dimensional space surrounding a multispecies biofilm in real time. In a related study I present the use of SECM to discover a novel redox chemistry phenomenon in the opportunistic pathogen Pseudomonas aeruginosa.Item Radiant and thermal energy transport in planktonic and benthic algae systems for sustainable biofuel production(2011-05) Murphy, Thomas Eugene; Berberoglu, Halil; Howell, John R.Biofuel production from microalgal biomass offers a clean and sustainable liquid fuel alternative to fossil fuels. In addition, algae cultivation is advantageous over traditional biofuel feedstocks as (i) it does not compete with food production, (ii) it potentially has a much greater areal productivity, (iii) it does not require arable land, and (iv) it can use marginal sources of water not suitable for irrigation or drinking. However, current algae cultivation technologies suffer from (i) low solar energy conversion effiencies, (ii) large thermal fluctuations which negatively affect the productivity, and (iii) large evaporative losses which make the process highly water intensive. This thesis reports a numerical study that address these key issues of planktonic as well as benthic algal photobioreactor technologies. First, radiant energy transfer in planktonic algal photobioreactors containing cells with different levels of pigmentation was studied. Chlamydomonas reinhardtii and its truncated chlorophyll antenna transformant tla1 were used as model organisms. Based on these simulations guidelines are derived for scaling the size and microorganism concentration of photobioreactors cultivating cells with different levels of pigmentation to achieve maximum photosynthetic productivity. To achieve this, the local irradiance obtained from the solution of the radiative transport equation (RTE) was coupled with the specific photosynthetic rates of the microorganisms to predict both the local and total photosynthetic rates in a photobioreactor. For irradiances less than 50 W/m2, the use of genetically modified strains with reduced pigmentation was shown to have negligible effect on increasing photobioreactor productivity. However, at irradiances up to 1000 W/m2, improvements of up to 30% were possible with cells having 63% less pigment concentration. It was determined that the ability of tla1 to transmit light deeper into the photobioreactor was the primary mechanism by which a photobioreactor using the modified strain can achieve greater productivity. Furthermore, it was determined photobioreactors using each strain have dead zones in which the local photosynthetic rate is negligible due to nearly complete light attenuation. These dead zones occur at local optical thicknesses greater than 169 and 275 in photobioreactors using the wild strain and the genetically modified strain, respectively. In addition, a thermal model of an algae biofilm photobioreactor was developed to assess the thermal fluctuations and evaporative loss rate of these novel photobioreactors under varying outdoor conditions. The model took into account air temperature, irradiance, relative humidity, and wind speed as inputs and computed the temperature and evaporative loss rate as a function of time and location in the photobioreactor. The model was run for a week-long period in each season using weather data from Memphis, TN. The range of the daily algae temperature variation was observed to be 13.2C, 12.4C, 12.8C, and 9.4C in the spring, summer, winter, and fall, respectively. Furthermore, without active cooling, the characteristic evaporative water loss from the system is approximately 6.3 L/m2-day, 7.0 L/m2-day, 4.9 L/m2-day, and 1.5 L/m2-day in the spring, summer, fall, and winter, respectively.Item Role of protein acetylation, formation and dispersal of biofilms, and their impact on insects(2012-07-16) Ma, QunBacterial biofilms form on liquid/air and liquid/solid surfaces and consist of cells combined with an extracellular matrix such as exopolysaccharides, extracellular DNA, and glycoproteins. Bacteria have up to a 1000-fold increase of antibiotic resistance in biofilms compared to planktonic cells. Furthermore, biofilm cells show better tolerance to adverse environmental conditions such as nutrition limitations, temperature changes, pH changes, and non-optimal osmotic conditions. In Escherichia coli, the outer membrane protein OmpA increased biofilm formation on polystyrene, polypropylene, and polyvinyl chloride surfaces while it decreased biofilm formation on glass surfaces. This surface-dependent phenotype was because OmpA inhibits cellulose production by inducing the CpxRA two-component signal transduction pathway, and cellulose inhibits biofilm formation on plastic due to its hydrophilic nature. We discovered, and then engineered, BdcA (formerly YjgI), for biofilm dispersal. We found that in E. coli, BdcA increases motility and extracellular DNA production while it decreases exopolysaccharide production, cell length, and aggregation. We reasoned that the 3, 5-cyclic diguanylic acid (c-di-GMP) levels increase upon deleting bdcA, and showed that BdcA binds c-di-GMP in vitro. In addition, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete biofilm dispersal. We isolated Proteus mirabilis from the blowfly Lucilia sericata, which swarmed significantly. By motility screening and complementation with putative interkingdom signal molecules that have been shown to attract flies, we found lactic acid, phenol, NaOH, KOH, putrescine, and ammonia restore the swarming motility of seven different swarming deficient mutants. These mutants and putative signal molecules will be further tested for fly attraction and oviposition. Acetylation of lysine residues is conserved in all three kingdoms although its role in bacteria is not clear. We demonstrated that acetylation enables E. coli to withstand environmental stresses. Specifically, the bacteria became more resistant to heat and oxidative stress. Furthermore, we showed that the increase in oxidative stress resistance is due to the induction of catalase gene katG. Hence we demonstrate for the first time a specific physiological role for acetylation in prokaryotes.Item Role of TapY1 and PlsB in Biofilm formation in aeromonas(2009-08) Khan, Mohammed R.; Jeter, Randall M.; Francisco, Michael J. D. S.; Zak, JohnType IV pili in Aeromonas are involved in biofilm formation and colonization of biotic and aboitic surfaces. The tapY1 gene in Aeromonas encodes a type IV pili biogenesis protein. The ability of a tapY1 mutant of A. salmonicida strain 92 to be transformed is greatly reduced when compared to the wild type. In a microtiter plate biofilm assay, the tapY1 mutant showed an adhesion-deficient phenotype with an efficiency of only 37% of the wild-type A. salmonicida strain 92. In Aeromonas species, type IV pili play an important role in natural transformation and competence development. Type IV pili bind DNA in the competent cells and promote cell aggregation. Following growth for 8 hr, the highest concentration of extracellular DNA was found in the supernatant of the tapY1 mutant culture but was in association with the pelleted cells in the wild-type bacterial culture. The tapY1 mutant consistently showed higher concentrations of DNA in the supernatant than the wild type at different incubation times. Microscopic examination of the adhesion phenotype and biofilm formation revealed that the wild-type strain is able to make a stable attachment on the surface of the microtiter plate well and form a structured biofilm, whereas the tapY1 mutant showed no stable attachment on the abiotic surface. Since a previous study in our laboratory reported that the tapY1 mutant is greatly reduced in its ability to be transformed when compared to the wild-type, these results suggest that type IV pili in A. salmonicida strain 92 play a dual role in natural transformation and biofilm formation. This study also identified two other genes, by random transposon mutagenesis that play role in biofilm formation in A. hydrophila ATCC 7965. One gene encodes glycerol-3-phosphate acyltransferase (PlsB), which catalyzes the first step in phospholipid synthesis and is known to play a role in persister cell generation in Escherichia coli. The plsB mutant showed a partial biofilm-deficient phenotype, with an adhesion efficiency of 58% of the wild-type A. hydrophila ATCC 7965 strain. The other identified gene encodes a conserved hypothetical protein in A. hydrophila ATCC 7965. The conserved hypothetical protein mutant also showed an adhesion-deficient phenotype in the biofilm assay. This conserved protein of A. hydrophila ATCC 7965 shares the conserved domain of a pili assembly protein superfamily of Escherichia coli and Gram-negative pili assembly chaperone. Therefore, it can be assumed that this conserved hypothetical protein might have a role in pili assembly and biofilm formation in Aeromonas.Item The ability of polymicrobial biofilms to promote wound infection chronicity(2011-05) Dalton, Trevor; Rumbaugh, Kendra P.; Dowd, Scot E.; Hamood, Abdul N.Chronic wound infections have a profound effect on the morbidity and mortality of a large patient population and cost billions of dollars in direct medical costs annually in the United States. Chronic wound infections are typically polymicrobial and biofilm-associated, however little is known about how they affect the host immune system and impair the healing process. This project was designed to compare single versus polymicrobial biofilm infections in a murine chronic wound model. We hypothesized that multispecies bacterial biofilms, or communities of bacteria that reside in a polysaccharide shell, contribute to the severity and antibacterial resistance of wounds. To test this hypothesis we developed a chronic-wound mouse model to determine the ability of multispecies bacterial biofilms to increase wound chronicity in biofilm-associated infections. Multispecies biofilms consisting of both gram negative and gram positive strains, as well as aerobes and anaerobes, were grown in vitro and then transplanted onto the wounds of mice. These in vitro-to-in vivo multispecies biofilm transplants generated polymicrobial wound infections, which remained heterogeneous with all four bacterial species throughout the experiment. In support of our hypothesis, we observed that wounded mice given multispecies biofilm infections displayed impaired wound healing over non-infected mice, or mice infected with a single species of bacteria. In addition, the bacteria in the polymicrobial wound biofilms displayed increased antimicrobial tolerance in comparison to those in single species biofilms. These data suggest that synergistic interactions between different bacterial species in wounds may contribute to healing delays and/or antibiotic tolerance.Item The effect of various amino acids as nitrogen source on biofilm formation of Aeromans spp.(2012-12) Price, Rachael; Jeter, Randall M.; Zak, John; Rumbaugh, Kendra P.; Hamood, Abdul N.For generations, scientists have studied microorganisms in their planktonic form to discover basic information about microorganisms and to perform more detailed experiments, including testing the effectiveness of antimicrobial agents. The term biofilm was defined and the concept of the biofilm was formulated very recently. Therefore, relatively little is known about biofilm growth in otherwise well-characterized microbial species. This would be a minor issue if microbial growth within a biofilm was similar to planktonic growth, but this is not the case. The difference in microbial growth within a biofilm compared to that of planktonic bacteria is immense. The perspective on uses for and treatments of biofilms are changing as we learn more about microorganisms within biofilms. For this reason, it is important to further explore the bacterial biofilm. A correlation between biofilm growth and nutrient environment has previously been observed but needs clarification. In this thesis project, I have isolated an Aeromonas environmental strain from the playa lake in Maxey Park in Lubbock, TX and analyzed the effect of varied nutrients, specifically nitrogen source, on biofilm formation of the environmental Aeromonas strain, comparing its biofilm formation capabilities to those of a reference Aeromonas hydrophila ATCC 7965 strain with the crystal violet assay. I have also assessed the accuracy of the crystal violet assay in quantifying cell concentrations by observing crystal violet absorbance of planktonic Aeromonas cells. Both the environmental Aeromonas isolate and the reference ATCC 7965 Aeromonas strain formed thicker biofilms in the presence of rich nutrient environments as compared to the more dilute nutrient environments. Our data suggests that Aeromonas ATCC 7965 is a more effective biofilm-former than the environmental isolate. It also reveals extreme variance in biofilm formation of both Aeromonas strains observed in the presence of different amino acids as nitrogen source. Finally, our assessment of the accuracy of the crystal violet assay and the calibration curve demonstrated the limitations of a common technique used in preliminary biofilm research.Item The tolerance of a Rhodococcus drinking water isolate and Zoogloea ramigera to silver nanoparticles in biofilm and planktonic cultures(2011-08) Gao, Qiao Huan; Kirisits, Mary Jo; Katz, Lynn E.Spurred by a host of beneficial uses, the global use of nanoparticles is rapidly growing. Silver nanoparticles (Ag NPs) are used widely in consumer products, medicine, and the semiconductor industry. As nanoparticles become more commonly used, the transport of nanoparticles into the environment might negatively affect microorganisms in natural and engineered systems. The effects of Ag NPs on microorganisms have primarily been studied in planktonic or free-swimming cultures, but little work has been done to look at biofilm susceptibility to Ag NPs. This thesis describes bacterial tolerance, or the ability of an organism to survive exposure to an insult, to Ag NPs. The tolerance of planktonic and biofilm cells of the common wastewater treatment bacterium Zoogloea ramigera and a Rhodococcus strain isolated from drinking water was tested. These bacteria were exposed to different concentrations of Ag NPs, ranging from 0 to 25 mg/L, for a period of 5 hours. Results showed decreased tolerance with increasing Ag NP concentrations for both bacterial species. Z. ramigera biofilm cells are slightly more tolerant to Ag NPs than are planktonic cells. On the other hand, Rhodococcus planktonic and biofilm cells exhibit similar tolerance. However, in both cases, biofilm cells do not exhibit a striking protective effect against Ag NPs as compared to planktonic cells. This study shows that even short-term insults with Ag NPs can affect bacteria in engineered systems. A preliminary study of the shedding of free silver ions as a possible mechanism of Ag NP toxicity demonstrated that free silver ions were toxic to Escherichia coli in a 0.14M chloride environment. The data suggest that free silver ions can be pulled into solution from Ag NPs in chloride environments via ligand-promoted dissolution. Further work is needed to examine the antibacterial mechanism of Ag NPs against planktonic and biofilm cells to better understand how the release of nanoparticles into the environment can affect microorganisms in natural and engineered water systems.