Browsing by Author "Sharp, Robert R."

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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.
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.
Bacterial transport issues related to subsurface biobarriers
(1999) Sharp, Robert R.; Gerlach, Robin; Cunningham, Alfred B.
Biofilm barriers to contain and degrade dissolved trichloroethylene
(2004-04) Komlos, John; Cunningham, Alfred B.; Camper, Anne K.; Sharp, Robert R.
Biologically produced subsurface barriers (i.e., biofilm barriers) are a viable technology for controlling contaminant migration from hazardous waste sites. Biofilm barriers are created through the injection of bacteria and selective growth medium into a series of wells downstream of a contaminant plume. Adequate substrate addition enables the bacteria to grow and form thick biofilms capable of uniform plugging of the subsurface. This technology has been successful in significantly reducing porous media permeability in bench-scale and field-scale applications. The research presented herein expands on current biofilm. barrier technology by examining the feasibility of using a biofilm barrier to not only control contaminant migration through permeability reduction, but also facilitate contaminant biodegradation. ne experimental scenario involved the creation of a dual-species biofilm matrix: one organism to reduce porous media permeability through thick biofilm formation and another organism to degrade a contaminant, in this case trichloroethylene (TCE). Porous medium column experiments demonstrated that a dual-species biofilm barrier can be created and that growth medium concentration was a very important variable in controlling simultaneous TCE degradation and permeability reduction.
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.
Effect of substrate concentration on dual-species biofilm population densities of Klebsiella oxytoca and Burkholderia cepacia in porous media
(2006) Komlos, John; Cunningham, Alfred B.; Camper, Anne K.; Sharp, Robert R.
The long-term operation of bioremediation technologies relies on the success of the contaminant-degrading microorganism(s) to compete for available resources with microorganisms already present in an aquifer or those that may contaminate a bioreactor. Though research has been performed studying the interaction of multiple species in batch and chemostat reactors, little work has been done looking at multi-species interactions in environments that more closely resemble field-scale applications. The research presented herein examined the interaction of Burkholderia cepacia PR1-pTOM31c, an aerobic trichloroethylene (TCE)-degrading bacterium, with Klebsiella oxytoca, a facultative bacterium, in a flow-through porous media (PM) reactor. Growth characteristics and population distributions in PM were compared to previously reported values from batch and chemostat reactors. The faster growing organism in batch experiments (K. oxytoca) did not always have the greater population density in dual-species PM experiments. The biofilm population distribution was influenced by substrate concentration, with B. cepacia having a greater dual-species population density than K. oxytoca at a low (30 mg/L dissolved organic carbon [DOC]) substrate concentration and K. oxytoca having a greater population density at a high (700 mg/L DOC) substrate concentration. This change in species population distribution with change in substrate concentration, which was not observed in batch reactors, was also observed in chemostat reactors. Therefore, manipulation of substrate concentration enabled the control of species dominance to the advantage of the TCE degrading population in this dual-species PM system and may provide a mechanism to enhance bioremediation scenarios involving TCE or other contaminants of concern. © 2005 Wiley Periodicals, Inc.
Effects of starvation on bacterial transport through porous media
(2007-06) Cunningham, Alfred B.; Sharp, Robert R.; Caccavo, Frank; Gerlach, Robin
A major problem preventing widespread implementation of microbial injection strategies for bioremediation and/or microbially enhanced oil recovery is the tendency of bacteria to strongly adhere to surfaces in the immediate vicinity of the injection point. Long term (weeks to months) nutrient starvation of bacteria prior to injection can decrease attachment and enhance transport through porous media. This paper summarizes results of starvation-enhanced transport experiments in sand columns of 30 cm, 3 m, and 16 m in length. The 16 m column experiments compared transport, breakthrough and distribution of adhered cells for starved and vegetative cultures of Klebsiella oxytoca, a copious biofilm producer. Results from these experiments were subsequently used to design and construct a field-scale biofilm barrier using starved Pseudomonas fluorescens. The 30 cm and 3 m sand columns experiments investigated starvation-enhanced transport of Shewanella algae BrY, a dissimilatory metal-reducing bacterium. In both cases the vegetative cells adsorbed onto the sand in higher numbers than the starved cells, especially near the entrance of the column. These results, taken together with studies cited in the literature, indicate that starved cells penetrate farther (i.e. higher breakthrough concentration) and adsorb more uniformly along the flow path than vegetative cells.
Evaluation of drinking water biostability using biofilm methods
(2001-05) Sharp, Robert R.; Camper, Anne K.; Crippen, J. J.; Schneider, O. D.; Leggiero, S.
A biofilm based, annular reactor method was developed and used to measure the biological regrowth potential of effluent water from various pilot treatment processes at the New York City Croton Lake Pilot Plant. A series of studies were carried out over the year-long study to collect bacterial growth and organic carbon biodegradation data for waters from six treatment options, including the raw source water. Quantitative and qualitative evaluations were made to determine the effects of filter media type, direct filtration, preozonation, and primary chlorination on the relative biostability of the produced waters compared to that of the original source water and water currently being distributed to consumers. In addition, results were compared to those obtained using traditional biodegradable organic material measuring methods such as assimilable organic carbon and biodegradable organic carbon. Quantitative biostability factors were developed that take into account both biological growth potential and biodegradability of the tested waters. Results from these studies were used to compare various piloted treatment processes and to assess pilot plant operation, design parameters, and seasonal source water quality.
Interaction of Klebsiella oxytoca and Burkholderia cepacia in dual-species batch cultures and biofilms as a function of growth rate and substrate concentration
(2005-01) Komlos, John; Cunningham, Alfred B.; Camper, Anne K.; Sharp, Robert R.
Dual-species microbial interactions have been extensively reported for batch and continuous culture environments. However, little research has been performed on dual-species interaction in a biofilm. This research examined the effects of growth rate and substrate concentration on dual-species population densities in batch and biofilm reactors. In addition, the feasibility of using batch reactor kinetics to describe dual-species biofilm interactions was explored. The scope of the research was directed toward creating a dual-species biofilm for the biodegradation of trichloroethylene, but the findings are a significant contribution to the study of dual-species interactions in general. The two bacterial species used were Burkholderia cepacia PR1-pTOM(31c), an aerobic organism capable of constitutively mineralizing trichloroethylene (TCE), and Klebsiella oxytoca, a highly mucoid, facultative anaerobic organism. The substrate concentrations used were different dilutions of a nutrient-rich medium resulting in dissolved organic carbon (DOC) concentrations on the order of 30, 70, and 700 mg/L. Presented herein are single- and dual-species population densities and growth rates for these two organisms grown in batch and continuous-flow biofilm reactors. In batch reactors, planktonic growth rates predicted dual-species planktonic species dominance, with the faster-growing organism (K. oxytoca) outcompeting the slower-growing organism (B. cepacia). In a dual-species biofilm, however, dual-species planktonic growth rates did not predict which organism would have the higher dual-species biofilm population density. The relative fraction of each organism in a dual-species biofilm did correlate with substrate concentration, with B. cepacia having a greater proportional density in the dual-species culture with K. oxytoca at low (30 and 70 mg/L DOC) substrate concentrations and K. oxytoca having a greater dual-species population density at a high (700 mg/L DOC) substrate concentration. Results from this research demonstrate the effectiveness of using substrate concentration to control population density in this dual-species biofilm.
Predictive model for toluene degradation and microbial phenotypic profiles in flat plate vapor phase bioreactor
(1997-06) Mirpuri, Rajesh G.; Sharp, Robert R.; Lewandowski, Zbigniew; Cunningham, Alfred B.
A predictive model has been developed to describe degradation of toluene in a flat-plate vapor phase bioreactor (VPBR). The VPBR model incorporates kinetic, stoichiometric, injury, and irreversible loss coefficients from suspended culture studies for toluene degradation by P. putida 54G and measured values of Henry's law constant and boundary layer thickness at the gas-liquid and liquid-biofilm interface. The model is used to estimate the performance of the reactor with respect to toluene degradation and to predict profiles of toluene concentration and bacterial physiological state within the biofilm. These results have been compared with experimentally determined values from a flat plate VPBR under electron acceptor and electron donor limiting conditions. The model accurately predicts toluene concentrations in the vapor phase and toluene degradation rate by adjusting only three parameters: biomass density and rates of death and endogenous decay. Qualitatively, the model also predicts gradients in the physiological state cells in the biofilm. This model provides a rational design for predicting an upper limit of toluene degradation capability in a VPBR and is currently being tested to assess applications for predicting performance of bench and pilot-scale column reactors.
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.
Scale-up implications of respirometrically determined microbial kinetics parameters
(1994) Sturman, Paul J.; Sharp, Robert R.; DeBar, J. B.; Stewart, Philip S.; Cunningham, Alfred B.; Wolfram, James H.
Subsurface biofilm barriers for the containment and remediation of contaminated groundwater
(2003-07) Cunningham, Alfred B.; Sharp, Robert R.; Hiebert, Dwight Randall; James, Garth A.
An engineered microbial biofilm barrier capable of reducing aquifer hydraulic conductivity while simultaneously biodegrading nitrate has been developed and tested at a field-relevant scale. The 22-month demonstration project was conducted at the MSE Technology Applications Inc. test facility in Butte, Montana, which consisted of a 130 ft wide, 180 ft long, 21 ft deep, polyvinylchloride (PVC)-lined test cell, with an initial hydraulic conductivity of 4.2 x 10-2 cm/s. A flow field was established across the test cell by injecting water up-gradient while simultaneously pumping from an effluent well located approximately 82 ft down gradient. A 30 ft wide biofilm barrier was developed along the centerline of the test cell by injecting a starved bacterial inoculum of Pseudomonas fluorescens strain CPC211a, followed by injection of a growth nutrient mixture composed of molasses, nitrate, and other additives. A 99% reduction of average hydraulic conductivity across the barrier was accomplished after three months of weekly or bi-weekly injections at intervals ranging from three to ten months. After the barrier was in place, a sustained concentration of 100 mg/l nitrate nitrogen, along with a 100 mg/l concentration of conservative (chloride) tracer, was added to the test cell influent over a six-month period. At the test cell effluent the concentration of chloride increased to about 80 mg/l while the effluent nitrate concentration varied between 0.0 and 6.4mg/l.
Visualization and characterization of dynamic patterns of flow, growth, and activity of biofilms growing in porous media
(2005) Sharp, Robert R.; Stoodley, Paul; Adgie, M.; Gerlach, Robin; Cunningham, Alfred B.
Using a mesoscale porous media flat plate reactor we utilized a naturally bioluminescent biofilm (V. fischeri) and dye studies to obtain valuable information on the interactions between biofilms and reactive flow in porous media. The growth and development of the V. fischeri biofilm in a porous media geometry was studied using digital time lapse images of the bioluminescent signal given off by the developing biofilm. The effect of biofilm development on porous media hydrodynamics was examined using dye tracer studies and image analysis. The natural bioluminescence of the V. fischeri allowed real-time, in-situ study of biofilm development in porous media, without destruction of the biofilm. Dye studies and image analysis enabled the study of effects of biofilm accumulation on porous media hydraulics, with comparisons to plug flow and completely mixed systems with varying degrees of biofilm accumulation. The real-time nature of the study permitted us to visualize dynamic flow channel formation within the biofilm/porous media system. In addition, the sensitivity of the V. fischeri biofilm to dissolved oxygen allowed us to capture real-time images of reactive transport within the system. This work is the first meso-scale visualization of the interactions between biofilm and flow in porous media.