The formation of bacterial biofilms on surfaces and their subsequent biofouling pose extensive safe and healthy concerns to a variety of industries. Biofilms are ubiquitous, and the biofilm state is considered the default mode of growth for the majority of the world's bacteria population. Once mature, biofilms are difficult to remove completely and have improved resistance against antibacterial agents. Given this, there has been significant interest to mitigate or at least manage biofilm formation on surfaces. One such method has been through the material design of surfaces, and to the interest of this study, through the development of antimicrobial stainless steels. Stainless steel is not an inherently antimicrobial material. Stainless steels alloyed with small amounts of either copper (Cu) or silver (Ag), both well-known natural antimicrobial agents, have been investigated since their initial development in the late 1990's onward. This class of materials have been proven to show significant antimicrobial effect over their traditional counterparts without compromising the characteristic mechanical properties of the stainless steels. However, most of the antimicrobial assessments for these materials documented within literature are conducted over a 24-hour timeframe and do not adequately account for the biofilm mode of growth. As so, this study aimed to assess how biofilms grow on this class of antimicrobial steels over a longer duration of growth and under growth conditions which more adequately modeled the biofilm mode of life.
The same strain of Escherichia coli commonly used in antimicrobial surface testing, ATCC 8739, was grown on submicron-polished coupons of a ferritic Cu-alloyed stainless steel (1.50 wt. % Cu), an austenitic Ag-alloyed stainless steel (0.042wt. % Ag), and a standard 304 series stainless steel, used as a baseline. Following ASTM-E2647-13, the E. coli/SS coupons were grown using a drip flow bioreactor under low shear conditions at either ambient temperature or 37 ± 3 degrees C with a batch phase of 6 hours and a continuous phase of 48 hours up to 96 hours. Directly after harvesting, the coupons were analyzed with an Environmental Scanning Electron Microscope (E-SEM) under low vacuum with a water vapor environment.
The effect of surface chemistry and alloy microstructure, surface roughness, rinsing the surfaces prior to inoculation and after harvesting, temperature, and growth duration on the resulting E. coli biofilms were all investigated in some capacity. Growth on the submicron finished surfaces indicated there were no significant differences between the biofilms grown on the three different steel compositions. Bacterial attachment appeared non-preferential to surface chemistry or alloy microstructure, suggesting that E. coli interacted with the surfaces effectively the same under the given growth conditions. To account for apparent randomness in bacterial attachment, it is hypothesized that the surface features of interest were on a size scale irrelevant to the size of single bacterial cells. To account for the lack of an observed biocidal effect from the Cu- and Ag-alloyed stainless steels, it is hypothesized that an organic conditioning film which developed on the surfaces from the fluid environment may have effectively inhibited the release of Cu and Ag ions from the steel surfaces. / MS / Bacteria frequently self-organize into what are commonly called bacterial biofilms, or an aggregation of bacterial cells that attach to a surface and which are embedded within a self-generated matrix of polymeric substances, such as proteins and polysaccharides. The biofilm state offers a lot of survival advantages to bacteria, and once biofilms form on a surface they are very difficult to remove. The formation of bacterial biofilms on surfaces and their subsequent biofouling pose extensive safe and healthy concerns to a variety of industries. There has been significant interest to stop or at least manage biofilm formation on surfaces. One such method has been through the design of surfaces, and to the interest of this study, through the development of antimicrobial stainless steels. Stainless steel is not an inherently antimicrobial material. Stainless steels which include small amounts of either copper or silver have been proven to show a significant antimicrobial effect over their traditional stainless steel counterparts without compromising the other desirable properties of the steels. However, most of the documented antimicrobial assessments for these materials have been conducted over a 24-hour timeframe and do not adequately account for the biofilm mode of growth.
This study aimed to assess how biofilms grow on this class of steels over a longer duration of growth and under growth conditions which more adequately modeled the biofilm mode of life. This was done by growing a single strain of E. coli bacteria onto coupons of these stainless steel materials for either a 48-hour or a 96-hour timeframe within a low-flow, continuously-fed bioreactor. The coupons were visualized with an environmental scanning electron microscope to assess the effect of the material properties on the observed biofilms grown during this study.
Overall there were little differences observed between the E. coli biofilms grown on the copper-containing stainless steel, the silver-containing stainless steel, and the standard stainless steel used within this study. Mirror finish smooth surfaces were needed in order to adequately visualize the steel coupons. The bacteria appeared to attach randomly without any preference for steel surface chemistry or other surface features. This suggested that under the given growth conditions the bacteria interacted with the smooth steel surfaces the same. To account for this randomness, it is hypothesized that the relevant surface features were significantly smaller than the size of single bacterial cells. E. coli cells are between 1 – 2 micrometers long and 0.5 – 1 micrometers in diameter. There was also no antimicrobial effect observed on the copper-containing and silver-containing stainless steels. To account for the lack of an observed antimicrobial effect, it is hypothesized that a conditioning film of carbon-based molecules formed on the surface of the steels from the liquid growth medium environment, preventing bacterial cells from being damaged by the copper and silver within the steel surfaces.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/95856 |
| Date | 01 June 2018 |
| Creators | McMullen, Amelia Marie |
| Contributors | Materials Science and Engineering, Murayama, Mitsuhiro, Corcoran, Sean G., Pruden, Amy |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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