James D. Bryers

Local macromolecule diffusion coefficients in structurally non-uniform bacterial biofilms using fluorescence recovery after photobleaching (FRAP)

Pure culture Pseudomonas putida biofilms were cultivated under controlled conditions to a desired overall biofilm thickness, then employed within classical half-cell diffusion chambers to estimate, from transient solute concentrations, the effective diffusion coefficient for several macromolecules of increasing molecular weight and molecular complexity. Results of traditional 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 a composite transfer coefficient that may overestimate the true, local flux of solutes in the biofilm polysaccharide gel matrix. An alternative analytical technique was refined to determine the local diffusion coefficients on a micro-scale to avoid the errors created by the biofilm architectural irregularities. This technique is based upon the Fluorescence Return After Photobleaching (FRAP), which allows image analysis observation of the transport of fluorescently labeled macromolecules as they migrate into a micro-scale photobleached zone. The technique can be computerized and allows one to map the local diffusion coefficients of various solute molecules at different horizontal planes and depths in a biofilm. These mappings also indirectly indicate the distribution of water channels in the biofilm, which was corroborated independently by direct microscopic observation of the settling of fluorescently-labeled latex spheres within the biofilm. Fluorescence return after photobleaching results indicate a significant reduction in the solute transport coefficients in biofilm polymer gel vs. the same value in water, with the reduction being dependent on solute molecule size and shape.

Biofilms and the technological implications of microbial cell adhesion

Abstract Biofilms are a collection of cells entrapped within a gelatinous matrix comprising mostly insoluble extracellular polymers that the cells secrete. Although the term is applied mostly to bacterial cells and their secreted insoluble exopolymers, any biologically active layer of cells (microbial, plant, or mammalian cells) can be considered a biofilm. Any surface in contact with a biological fluid is a potential target surface for microbial cell adhesion. Once cells are attached, subsequent growth and replication of surface-attached cells, cell exopolymer production, further recruitment of planktonic cells from the fluid phase, and various biofilm detachment processes constitute what is collectively known as biofilm formation and persistence. Biofilms can play both beneficial or detrimental roles depending on whether their formation within a specific system is intentional or inadvertent. This article will review both the current and emerging technological implications of bacterial cell adhesion and biofilm formation including biomaterials preparation to prevent bacterial infections of medical implants; development of novel antibiotic therapies to control biofilm-bound bacteria; designer nanocrystalline filaments called “bionites”, fabricated from strands of bacteria, that possess unusual magnetic, optical and biocatalytic properties; specific hazardous waste detoxification by immobilized recombinant bacteria; improved recombinant plasmid retention within biofilm populations; and stable biosensors.

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, micro(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 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 (14)C 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.

Deposition of bacterial cells onto glass and biofilm surfaces

Deposition rates of Pseudomonas putida and Hyphomicrobium ZV620 onto glass and biofilm surfaces were quantified. Both species deposited to glass at a much slower rate than to biofilm. A definite bias by depositing cells for biofilms of their own species was evident in the highest attachment rates observed in this study.

Use of flow cell reactors to quantify biofilm formation kinetics

A parallel plate flow cell reactor is introduced and used to evaluate cell adhesion and biofilm formation kinetics for four different bacterial strains of the species,E. coli. The reactor allows biofilm growth under defined, well-controlled fluid dynamics while providing continuous observations and direct sampling of biofilm for biological, chemical and physical analyses as well as immunofluorescent labeling.

Activity and stability of a recombinant plasmid‐borne TCE degradative pathway in biofilm cultures

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.

Recombinant plasmid retention and expression in bacterial biofilm cultures

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 open environmental systems 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 . Thus any study regarding the fate of recombinant DNA sequences in an open system must consider processes that affect plasmid retention and expression in a biofilm culture. Biofilm cultures were cultivated in a parallel-plate flow cell reactor using E. coli DH5α which contained a recombinant plasmid with a plasmid stability factor, parB , (pTKW106) or without (pMJR1750). Using β-galactosidase as inducible reporter protein, plasmid retention and gene expression of pMJR1750 and pTKW106, in suspended versus biofilm cultures, were studied under different carbo to nitrogen ratios and plasmid induction levels. Recombinant biofilm formation under these environmental conditions was also investigated. Biofilm net accumulation rate of E. coli DH5α (pTKW106) decreases with increasing induction levels. The β-galactosidase production and ratios of β-galactosidase to total protein increase with increasing induction levels. Synthesis rates of total RNA, β-galactosidase mRNA and rRNA in biofilm cultures of E. coli DH5α (pTKW106) increase after induction by IPTG.

Cryptic growth within a binary microbial culture

SummaryThe ability of viable cells of the species Pseudomonas putida and Hyphomicrobium sp. to metabolize the particulate and soluble cellular organic constituents of both species was studied in a series of batch experiments. Both P. putida and Hyphomicrobium sp. were grown in individual batch reactors on either the 14C-labelled soluble or the particulate debris of sonicated cells of each species derived from steady-state chemostat cultures. Cell generation times (tg)observed for P. putida cultivated on soluble organic material originating from either sonicated P. putida or Hyphomicrobium sp. cells, were tg= 2.0 h and tg= 6.3 h, respectively. Corresponding tgvalues of Hyphomicrobium sp. on soluble organic material originating from sonicated P. putida and Hyphomicrobium so. were, respectively, 11.6 h and 4.3 h. While particulate debris originating from either species was solubilized by both P. putida and Hyphomicrobium sp., no increases in cell numbers were observed for either species. The data indicate that bacteria are capable of scavenging soluble material released upon cell lysis at near maximal rates; solubilization of debris also occurred but at much slower overall rates with no observable cell replication. The results reaffirm that cryptic growth and turnover of cellular biomass can be significant under situations of low substrate flux or starvation conditions.

Biopolymer particulate turnover in biological waste treatment systems: a review

Microorganisms — the major component in most biological waste treatment processes and a number of industrial fermentations — are not able to directly assimilate biopolymeric particulate material. Such organic particulates must first be solubilized into soluble polymers or monomers before they can diffuse through the capsular slime layer surrounding most bacteria, then transported across the cell membrane, to be used as either a carbon, energy or other essential nutrient source. Throughout these events, new cells are synthesized, which are themselves biopolymer particulates.The turnover of biopolymer particulates in biological treatment systems has not been examined with respect to its impact on system performance and culture physiology. The aim of this paper is to review the observations of particulate turnover in various biological treatment systems and to identify those fundamental mechanisms which govern microbial conversion of biopolymer particulates.

Effects of inducer levels on a recombinant bacterial biofilm formation and gene expression

SummaryA segregationally stable host-plasmid system, E. coli DH5α (pTKW106), was used to study the effect of induction on the accumulation rate of cells and gene expression in biofilm cultures. Isopropyl β-D-thiogalactoside (IPTG) was used to induce the expression of β-galactosidase from the plasmid. The biofilm cell net accumulation rates decreased with increasing induction levels. At 0.17 and 0.34 mM of IPTG, the biofilm cell net accumulation rates ranged between 17 and 30% when compared to the uninduced case. At 0.51 mM of IPTG, the biofilm cell density never increased. At 0.17 and 0.34 mM of IPTG, β-galactosidase contents reached maxima 36 hours after induction with both amounts representing about 7.5% of total protein. At 0.51 mM of IPTG, β-galactosidase production reached its maximum, about 16% of total protein, 48 hours after induction. The β-galactosidase mRNA synthesis rates increased with increasing inducer levels. Maximum β-galactosidase mRNA synthesis rates were reached 36 hours after induction for each IPTG concentration.