Scholarly Work - Center for Biofilm Engineering
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Item Activity and stability of a recombinant plasmid-borne tce degradative pathway in biofilm cultures(1998-08) Sharp, Robert R.; Bryers, James D.; Jones, Wallace E.The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term. Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic.Item Mobilization of a broad host range plasmid from pseudomonas putida to an established biofilm of bacillus azotoformans part ii: modeling(1998-02) Beaudoin, D. L.; Bryers, James D.; Cunningham, Alfred B.; Peretti, Steven W.A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited.Item Mobilization of a broad host range plasmid from pseudomonas putida to an established biofilm of bacillus azotoformans part i: experiments(1998-02) Beaudoin, D. L.; Bryers, James D.; Cunningham, Alfred B.; Peretti, Steven W.A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization.Item Activity and stability of a recombinant plasmid-borne tce degradative pathway in suspended cultures(1998-02) Sharp, Robert R.; Bryers, James D.; Jones, Warren G.; Shields, Malcom S.The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h−1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min · mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 287–296, 1998.Item Retention and expression of recombinant plasmids in suspended and biofilm-bound bacteria degrading trichloroethene (tce)(1997) Bryers, James D.; Sharp, Robert R.Exposure of plasmid recombinant microorganisms to an open environment, either inadvertently or intentionally, requires research into those fundamental processes that govern plasmid retention, transfer and expression. In the open environment, a majority of the microbial activity occurs associated with an interface, within thin biological layers consisting of cells and their insoluble extracellular polymer, layers known as biofilms. Current toxic wastewater or wastegas treatment reactors exploit bacterial biofilm systems for certain system operating advantages. Using recombinant bacteria within a biofilm reactor to degrade xenobiotic wastes requires finding a suitable host to harbor and express the desired plasmid phenotype. Suitable host characteristics include: the ability to produce copious amounts of biofilm, resistance to waste-related injury and toxicity, and the ability to retain and express the desired plasmid during long term operation. This paper reports on a laboratory evaluation of factors governing plasmid retention and the expression of trichloroethene (TCE) degradative capacity in both suspended and biofilm cultures.Item A dynamic model for receptor-mediated specific adhesion of bacteria under uniform shear flow(1997-09) Wang, Grace Tein-Ya; Bryers, James D.A dynamic mathematical model is proposed to describe bacterial cell adhesion in viscous shear flow that is mediated by specific receptor: ligand binding. Bacterial cells are assumed here to be ideal spheres covered uniformly with spring‐like receptors. The model describes the specific binding between receptor molecules on the cell and ligands associated with the target surface. Accounting for the processes of attachment, detachment, and growth of attached bacteria, a set of non‐linear ordinary differential equations (ODEs) is derived that governs the net accumulation of cell numbers per surface area (S) of ligand‐coated surface, cell numbers (X) and growth‐limiting substrate concentration (S) per volume of solution passing over the target substratum. The non‐linear ODEs are numerically solved by using the fourth‐order Runge‐Kutta algorithm. Results of numerical simulations reveal that various stages of bacterial attachment are pertinent at different stages of biofilm development. The recommendation of a proper time duration for experimental adhesion research is possible, based on model solutions. A sensitivity analysis is carried out and the results indicate that bacterial deposition is dominated by the attachment and detachment rate constants rather than by ligand density or shear rate. Specific adhesion of a defined mixed culture is also simulated with an expanded version of the basic model.Item Toluene degradation kinetics for planktonic and biofilm-grown cells of pseudomonas putida 54g(1997-03) Mirpuri, Rajesh G.; Jones, Warren L.; Bryers, James D.Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of 14C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, μmax = 10.08 ± 1.2/day; half-saturation constant, KS = 3.98 ± 1.28 mg/L; substrate inhibition constant, KI = 42.78 ± 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate 14C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng53: 535–546, 1997.Item Local mass transfer coefficients in bacterial biofilms using fluorescence recovery after photobleaching (frap)(1996) Bryers, James D.; Drummond, F.Pure culture Pseudomonas putida biofilms were cultivated under controlled conditions to desired overall biofilm thicknesses. Said biofilms were placed within half-cell diffusion chambers to estimate, from transient solute concentrations in each chamber, the effective diffusion coefficient for severed macromolecules of increasing molecular weight and molecular complexity. Results of the half-cell studies were found to be erroneous due to the existence of microscopic water channels or crevasses that perforate the polysaccharidic gel matrix of the biofilm, sometimes completely to the supporting substratum. Thus, half-cell devices measure an average transfer coefficient that overestimates the true, local flux of solutes in the biofilm alginate gel matrix. An alternative analytical technique was refined to determine the local diffusion coefficients on a micro-scale in order to avoid the errors created by the water channels. This technique is based upon the Fluorescence Return After Photobleaching (FRAP) which allows image analysis observation of the transport of fluorescently labeled molecules as they migrate into a micro-scale photobleached zone. The technique allowed us to map the local diffusion coefficients of various solute molecules at different horizontal planes and depths in a biofilm. These maps also indirectly indicate the distribution of water channels in the biofilm. FRAP results illustrate a significant reduction in macromolecule transport coefficients in biofilm polymer gel versus the same value in water, with the reduction being dependent on solute molecule size and shape.Item Comparison of retention and expression of recombinant plasmids between suspended and biofilm-bound bacteria degrading tce(1996) Bryers, James D.; Sharp, Robert R.Any exposure of plasmid recombinant microorganisms to an open system environment, either inadvertently or intentionally, mandates research into those fundamental organism:plasmid processes that influence plasmid retention, transfer, and expression. In the open environment, a majority of the microbial activity occurs associated with an interface, within thin biological layers consisting of the cells and their insoluble extracellular polymer; layers known as biofilms. In addition, current toxic wastewater or wastegas treatment reactors exploit bacteria biofilms for certain system operating advantages. Thus any study regarding the fate of recombinant DNA sequences in either an open environment or closed reactor system must consider processes that impact plasmid retention and expression in a biofilm culture. Using recombinant bacteria within a biofilm reactor to degrade a recalcitrant waste requires finding a suitable host to harbor and express the desired degrading plasmid phenotype. Suitable host characteristics include: ability to produce copious amounts of biofilm, resistance to waste-related injury and toxicity, and ability to retain and express the desired plasmid during long term operation. This paper reports on a laboratory evaluation of factors governing plasmid retention and the expression of TCE degradative capacity in both suspended and biofilm cultures.Item Xenobiotic biodegradation test using attached bacteria in synthetic seawater(1995-08) Osswald, P.; Courtes, R.; Bauda, P.; Block, James C.; Bryers, James D.; Sunde, E.The aerobic biodegradability of aniline, used as reference chemical, has been performed in synthetic seawater with attached biomass in a continuously fed reactor (biofilm chemostat reactor, BCR). Marine bacteria inocula came from local marine fish aquarium filters to limit the geographic and seasonal variations in quality. A pretreatment of these inocula combining 5-μm filtration and centrifugation was used to concentrate bacteria and remove organic carbon contamination of the test. The performances of the BCR were tested in comparison with simple shake flask tests. Among the different variables tested, the ratio S0X0 (initial concentration of xenobiotic to initial density of the inoculum), the presence of dissolved oxygen, and the hydraulic residence time appear to be the key parameters controlling the length of the biodegradation process. On the other hand, the addition of a co-substrate (easily biodegradable compound) does not provide advantages. Thus, marine biofilm chemostat reactors with a high density of attached bacteria (around 107 cells cm−2) and fed with synthetic seawater plus nitrogen provide good tools for screening biodegradability of chemicals in the marine environment.