Doctoral Dissertations

Date of Award

8-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

David C. White

Committee Members

Walter Farkas, Thomas Montie, Dwayne Savage

Abstract

Bacterial biofilm formation on inanimate substrata in freshwater, marine, and physiological environments often precedes microbially influenced corrosion and other biofouling activities. Colonization of and attachment to surfaces in these environments is mediated by environmental factors as fluid dynamics, bulk-phase biotic and abiotic constituents, and the physicochemisty of the substrata. A considerable effort has been applied to resolving questions of biofilm effects on hydraulic regimes and substrata properties. However, fundamental research into the biomass constituents, community structure, and activity of bacterial biofilms as a function of bulk-phase and substrata variables has been more limited. In part, this knowledge gap has resulted from a lack of test systems and analytical techniques for examining bacterial colonization and biofilm formation under conditions which approximate those of in situ environments. This dissertation explores the effects of bulk-phase environments and surfaces on bacterial biofilms under dynamic-flow conditions. Radial-flow, cell adhesion measurement modules (CAMM) were used to demonstrate that changes in cellular concentration, composition, and metabolic activity of a monoculture of Alteromonas atlantica biofilms on 316 stainless steel (SS) were a function of the applied shear force. At shear forces in the range of between 1 and 10 dynes cm-2, acridine orange direct counts (AODC) on substrata ranged from 0.15 to 57 X 106 cells cm-2. Biomass protein and total fatty acids, carbohydrate:protein ratios, and unit area metabolic activity as measured by in situ 14C-acetate incorporation into cellular lipids were positively correlated with the applied shear force. On a per cell basis, however, metabolic activity decreased with shear, indicating a possible shift in metabolism. A series of laminar-flow adhesion cells were designed, validated, and constructed using qualitative and quantitative measures of fluid dynamics and bacterial biofilm development. The flow cell design incorporated provisions for on-line, non-destructive measurements of open circuit potential (OCP), bioluminescence, and pO2 for monitoring bacterial colonization and succession as influenced by a systematic change in bulk phase conditions. The cells consisted of two high density polyethylene blocks, with a 1 mm deep flow channel milled in the top block. A glass viewing window enabled direct observation of a removable, flush-mounted 25 X 50 mm coupon, which was recessed into the bottom block. The overall dimensions of the cells were 32 mm high X 65 mm wide X 178 mm long. Flow laminarity was validated at 1.3 cm s-1 (20 mL min-1) . Colonization by monocultures of Pseudomonas fluorescens was reproducible, with 72 h AODC and viable plate counts values ranging from between 5.1 to 10.4 X 107 and 1.4 to 2.2 X 107 cells cm-2, respectively, for replicated flow cells (n=20). On-line OOP measurements were stable over 80 h test periods; onset of P. fluorescence visible biofilm formation coincided with a significant perturbation in OCP. Bulk-phase bacterial composition and successional order after incubation of 4 organisms from continuous culture were found to significantly affect the community structure, biomass constituents, and metabolic activity of biofilms on 316 SS. Laminar-flow adhesion cells were employed in colonization studies which demonstrated that biofilms exposed to sequential seeding of bulk—phase bacteria in the order of P. fluorescens, Hafnia alvei, Desulfovibrio gigas, and B. subtilis had the highest AODC, protein, and unit area metabolic activity. The freshwater, adhesive aerobe, P. fluorescens, was the dominant member of all biofilm communities, with 5-day biofilms containing on the order of 108 cells cm2. Viable B. subtilis were not recovered from SS surfaces; recovery of D. gigas was poor. Significant perturbations in OCP response coincided with visible biofilm formation; however, no significant differences in the shape or magnitude of the response curves between the various communities tested were observed. No significant effects on biofilm biomass parameters of AODC, viable counts, protein, carbohydrate:protein ratios, or per cell metabolic activity were attributed to metallurgical inhomogeneities in 316/E308 welded SS coupons in an oligotrophic, laminar-flow environment. The community structure of 5-day biofilms exposed to various successional orders of bulk-phase cultures of P. fluorescens, H. alvei, D. gigas, and B. subtilis was not significantly affected by surface topology or the presence of weldments. Comparison of OCP plots of 1 μm polished/etched coupons and 600 grit polished coupons did not reveal significant differences. Transmission electron micrographs of embedded and thin-sectioned 5-day biofilms taken from various surfaces revealed the presence of extensive intracellular inclusions reminiscent of poly beta-hydroxyalkanoate and polyphosphate granules seen in microbial mats. Bacterial bioluminescence by a P. fluorescens (lux) construct was utilized as an endpoint for adhesion in a fluid shear gradient. The CAMM were used to differentially colonize the bioluminescent bacteria on glass and 316 SS surfaces. When induced by sodium salicylate, bulk phase and adherent cells were assayed quantitatively with an ammeter-photomultiplier-fiber optic system. The detection limit was 2 X 105 cells cm-2 on glass coupons. Light production was found to positively correlate with biofilm lipid synthesis on a per unit area basis. In the range of 105 to 107 cells cm-2, light was found to correlate directly (r2= 0.826) with AODC values, providing on-line detection of both biofilm and bulk-phase biomass and metabolic activity when induced.

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