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Microbial-influenced corrosion (MIC) has been widely recognized as a significant economic and environmental problem in the oil and gas industry. MIC can be classified into two types based on the mechanisms: the extracellular electron transfer MIC (EET-MIC) and the metabolite MIC (M-MIC). The first includes electroactive bacteria that facilitate EET, while the latter includes bacteria that secrete corrosive metabolites. Sulfate-reducing bacteria (SRB) is believed to cause EET-MIC in carbon steel, a widely used metal in the oil and gas industry. In previous electroactive bacteria studies, nanowires have been shown to facilitate EET by acting as electron mediators. This study investigates the use of magnetite nanowires as electron mediators to accelerate EET-MIC of C1020 by Desulfovibrio vulgaris. The addition of 40 ppm (w/w) nanowires to carbon steel incubated with D. vulgaris, corrosive SRB species, for seven days resulted in 45% weight loss and 57% deeper pitting of carbon steel. Furthermore, electrochemical measurements of open circuit potential, linear polarization resistance and potentiodynamic polarization were found to be parallel with weight loss and pitting results. Therefore, these findings highlight the possibility of using magnetic nanowires as an electron mediator with high efficiency and selectivity to EET-MIC for future MIC studies and applications.

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Bacteria in biofilms adhere well to surfaces and are quite difficult to remove. Oral plaque is one example of a biofilm. Many researchers have studied ways to remove plaque and bacteria from surfaces. It has been found that the passage of a bubble across a surface to which bacteria has adhered can remove the bacteria from the surface. Biofilms of Streptococcus mutans were grown on glass coverslips as a simple model for oral plaque. The coverslips were mounted in a Plexiglas chamber filled with artificial saliva. A bubble stream was directed at the mounted biofilm. The velocity, gas fraction, median bubble diameter, and impingement angle were all varied to determine the effect of each parameter on removal and which parameter was the most significant. To investigate the influence of sound on removal, a Ling oscillator was attached to the chamber, and was used simultaneously with and without a bubble stream. The acoustic intensity and the frequency were varied to determine if there was any effect on biofilm removal. Biofilm removal was also video taped. The results of these experiments confirmed that biofilms are removed by a stream of bubbles. Removal of biofilm is a function of stream velocity, gas fraction, and median bubble diameter, but not of impingement angle. The results of the acoustic experiments show that sound does not affect the removal of biofilm under the conditions used in these experiments. Mathematical models relating the removal of biofilm as a function of time were also developed from the data obtained from the video recording of the experiments. Additional tests using acoustic waves to remove biofilm should be performed to determine if more intense sound can remove biofilm. The intensity of the sound used in these experiments was low and the time of exposure was only 5 sec. Additional tests that more closely simulate the conditions of the mouth during brushing, like adding a surfactant, would also provide more insight as to whether bubbles in a clinical setting would remove biofilm.

In this study, the bacterial growth dynamics of 14 pilot drinking water distribution systems were studied in order to observe water quality changes due to corrosion inhibitor addition. Empirical models were developed to quantity the effect of inhibitor type and dose on bacterial growth (biofilm and bulk water). Water and pipe coupon samples were taken and examined during the experiments. The coupons were exposed to drinking water at approximately 20°C for at least 5 weeks to allow the formation of a measurable quasi- steady-state biofilm. Bulk water samples were taken every week. In this study, two simple but practical empirical models were created. Sensitivity analysis for the bulk HPC model (for all 14 of the PDSs) showed that maintaining a chloramine residual at 2.6 mg/L instead of 1.1 mg/L would decrease bulk HPC by anywhere from 0.5 to 0.9 log, which was greater than the increase in bulk HPC from inhibitor addition at 0.31 to 0.42 log for Si and P based inhibitors respectively. This means that maintaining higher residual levels can counteract the relatively modest increases due to inhibitors. BF HPC was affected by pipe material, effluent residual and temperature in addition to a small increase due to inhibitor addition. Biofilm density was most affected by material type, with polyvinyl chloride (PVC) biofilm density consistently much lower than other materials (0.66, 0.92, and 1.22 log lower than lined cast iron (LCI), unlined cast iron (UCI), and galvanized steel (G), respectively). Temperature had a significant effect on both biofilm and bulk HPC levels but it is not practical to alter temperature for public drinking water distribution systems so temperature is not a management tool like residual. This study evaluated the effects of four different corrosion inhibitors (i.e. based on either phosphate or silica) on drinking water distribution system biofilms and bulk water HPC levels. Four different pipe materials were used in the pilot scale experiments, polyvinyl chloride (PVC), lined cast iron (LCI), unlined cast iron (UCI), and galvanized steel (G). Three kinds of phosphate based and one silica based corrosion inhibitors were added at concentrations typically applied in a drinking water distribution system for corrosion control. The data showed that there was a statistically significant increase of 0.34 log in biofilm bacterial densities (measured as HPC) with the addition of any of the phosphate based inhibitors (ortho-phosphorus, blended ortho-poly-phosphate, and zinc ortho-phosphate). A silica based inhibitor resulted in an increase of 0.36 log. The biological data also showed that there was a statistically significant increase in bulk water bacterial densities (measured as heterotrophic plates count, HPC) with the addition of any of the four inhibitors. For bulk HPC this increase was relatively small, being 15.4% (0.42 log) when using phosphate based inhibitors, and 11.0% (0.31 log) for the silica based inhibitor. Experiments with PDS influent spiked with phosphate salts, phosphate based inhibitors, and the silicate inhibitor showed that the growth response of P17 and NOx in the AOC test was increased by addition of these inorganic compounds. For this source water and the PDSs there was more than one limiting nutrient. In addition to organic compounds phosphorus was identified as a nutrient stimulating growth, and there was also an unidentified nutrient in the silica based inhibitor. However since the percentage increases due to inhibitors were no greater than 15% it is unlikely that this change would be significant for the bulk water microbial quality. In addition it was shown that increasing the chloramines residual could offset any additional growth and that the inhibitors could help compliance with the lead and copper rule. However corrosion inhibitors might result in an increase in monitoring and maintenance requirements, particularly in dead ends, reaches with long HRTs, and possibly storage facilities. In addition it is unknown what the effect of corrosion inhibitors are on the growth of coliform bacteria and opportunistic pathogens relative to ordinary heterotrophs. A method was developed to monitor precision for heterotrophic plate count (HPC) using both blind duplicates and lab replicates as part of a project looking at pilot drinking water distribution systems. Precision control charts were used to monitor for changes in assay variability with time just as they are used for chemical assays. In adapting these control charts for the HPC assay, it was determined that only plate counts ≥ 30 cfu per plate could be used for Quality Assurance (QA) purposes. In addition, four dilutions were used for all known Quality Control (QC) samples to insure counts usable for QC purposes would be obtained. As a result there was a 50% increase in the required labor for a given number of samples when blind duplicates and lab replicates were run in parallel with the samples. For bulk water HPCs the distributions of the duplicate and replicate data were found to be significantly different and separate control charts were used. A probability based analysis for setting up the warning limit (WL) and control limit (CL) was compared with the method following National Institute of Standard and Technology (NIST) guidelines.

Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks. / Doctor of Philosophy / The way in which bacteria assess and respond to their environment is of great interest to microbiologists. Bacteria transmit environmental signals via protein interactions. Some of these interactions involve the transfer of phosphate groups, and some involve a direct binding interaction between proteins. We are investigating a direct binding interaction between two proteins, RetS and GacS. These proteins control whether Pseudomonas aeruginosa, an opportunistic pathogen of humans, causes an acute infection, which is characterized by motility and invasiveness, or a chronic infection, which is characterized by a sessile biofilm lifestyle, in a human host. Through the use of structural biology techniques we have visualized the three-dimensional structure of the complex between RetS and GacS. This complex has provided insight into the role of the RetS-GacS interaction in controlling the infection state of P. aeruginosa.

The interaction between fluid dynamics and biofilm growth plays a key role in both medical and industrial applications. Biofilms, or bacteria that are embedded in a protective matrix of extracellular polymeric substances, settle on interfaces such as on implanted devices or ship hulls. These biofilms canthen cause infectious diseases or significantly increase drag. In this thesis, we investigate the influence of flow, specifically shear stress, on the development of biofilm. The first paper presents a new facility to investigate biofilm growth in laminar flow cells over extended periods of up to several weeks. Optical coherence tomography is used to obtain three-dimensional scans of the biofilm structure at regular intervals. From these time series, we derive a simple model that relates the growth of an individual microcolony to the growth of the full biofilm depending on the wall shear stress. Additionally, we show that biofilm streamers, thin, flexible filaments that extend into the bulk flow, can form on sharp biofilm structures in laminar flow, even if the substratum is a flat surface. The second contribution is a report detailing preliminary studies on biofilm experiments. We investigate the formation of biofilm in the shear layer behinda backward-facing step. The results indicate a maximum shear stress, beyond which biofilm growth is inhibited. We also provide guidelines for the design of experimental setups for the investigation of the influence of fluid dynamics on biofilm and vice-versa. / Samspelet mellan fluiddynamik och biofilmtillväxt spelar en nyckelroll i både medicinska och industriella tillämpningar. Biofilmer, eller bakterier som är inbäddade i en skyddande matris av extracellulära polymera substanser, sätter sig på ytor som på implanterade enheter eller fartygsskrov. Dessa biofilmer kan sedan orsaka infektionssjukdomar eller avsevärt öka vattenmotståndet. I den här avhandlingen undersöker vi hur flöde, speciellt skjuvspänning, påverkar utvecklingen av biofilm. I den första artikeln presenteras en ny uppställning för att undersöka biofilmstillväxt i flödesceller med laminärt flöde under längre perioder på upp till flera veckor. Optisk koherenstomografi används för att få tredimensionella skanningar av biofilmstrukturen vid regelbundna intervall. Från dessa tidsserier härleder vi en enkel modell som relaterar tillväxten av en enskild mikrokoloni till tillväxten av hela biofilmen beroende på väggskjuvspänning. Dessutom visar vi att biofilm filament som sträcker sig in i bulkflödet, kan bildas på skarpa biofilmstrukturer i laminärt flöde, även om substratum är en plan yta.  Det andra bidraget är en rapport som beskriver preliminära studier av biofilmsexperiment. Vi undersöker bildandet av biofilm i skjuvskiktet bakom ett bakåtvänt steg. Resultaten indikerar en maximal skjuvspänning, bortom vilken biofilmstillväxt hämmas. / <p>QC 240314</p>

Staphylococcus aureus is a pathogen responsible for a wide variety of life-threatening diseases such as bacteremia, endocarditis, and pneumoniae. S. aureus has been a major concern in recent years due to the rampant spread of antibiotic resistance. The ability of S. aureus to form biofilms aids in the spread of antibiotic resistance as biofilms are a known hotspot for horizontal gene transfer. Biofilms also protect cells from host immune responses and antibiotics, making these infections very difficult to treat. The matrix of S. aureus biofilms can be made of polysaccharides, protein, and DNA. In these studies, we sought to elucidate how biofilm composition correlates with source of isolation in S. aureus strains, the role of biofilm-related genes in biofilm composition, and the potential role of biofilm eDNA in horizontal gene transfer. The composition and strength of biofilms made by a variety of hospital and meat-associated strains of S. aureus was measured using crystal violet (CV) staining and DNase or proteinase K treatment. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay. We found that biofilms of hospital-associated isolates tend to have more protein and polysaccharides while those of meat isolates contain significantly more DNA. We also investigated the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes suggested by previous studies to play a role in biofilm development. Transposon insertions in agrA, atl, clfA, fnbA, purH, and sarA significantly weakened biofilms as compared to a wild-type control, whereas the acnA insertion mutant produced a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. We found that the atl and sarA insertion mutants produced biofilms with greater polysaccharide concentrations than the wild-type. Since many of the isolates produced biofilms composed of DNA, we investigated the potential role of this extracellular DNA in horizontal gene transfer. Strains with complementary antibiotic resistances and susceptibilities were paired together and co-cultured together in a biofilm and plated onto double antibiotic plates to select for possible gene transfer. Putative gene transfer was found to be largely biofilm dependent and enhanced with the addition of subinhibitory concentrations of antibiotics added to the biofilm. Potential transformation was also shown to naturally occur in many strains when naked DNA was added to a single strain biofilm and was also aided with the addition of subinhibitory antibiotics.

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