Permeability of a growing biofilm in a porous media fluid flow analyzed by magnetic resonance displacement‐relaxation correlations

Biofilm growth in porous media is difficult to study non‐invasively due to the opaqueness and heterogeneity of the systems. Magnetic resonance is utilized to non‐invasively study water dynamics within porous media. Displacement‐relaxation correlation experiments were performed on fluid flow during biofilm growth in a model porous media of mono‐dispersed polystyrene beads. The spin–spin T₂ magnetic relaxation distinguishes between the biofilm phase and bulk fluid phase due to water–biopolymer interactions present in the biofilm, and the flow dynamics are measured using PGSE NMR experiments. By correlating these two measurements, the effects of biofilm growth on the fluid dynamics can be separated into a detailed analysis of both the biofilm phase and the fluid phase simultaneously within the same experiment. Within the displacement resolution of these experiments, no convective flow was measured through the biomass. An increased amount of longitudinal hydrodynamic dispersion indicates increased hydrodynamic mixing due to fluid channeling caused by biofilm growth. The effect of different biofilm growth conditions was measured by varying the strength of the bacterial growth medium. Biotechnol. Bioeng. 2013; 110: 1366–1375. © 2012 Wiley Periodicals, Inc.     

Incorporating toxin hypothesis into a mathematical model of persister formation and dynamics

Biofilms are well known for their extreme tolerance to antibiotics. Recent experimental evidence has indicated the existence of a small fraction of specialized persister cells may be responsible for this tolerance. Although persister cells seem to exist in planktonic bacterial populations, within a biofilm the additional protection offered by the polymeric matrix allows persister cells to evade elimination and serve as a source for re-population. Whether persister cells develop through interactions with toxin/antitoxin modules or are senescent bacteria is an open question. In this investigation we contrast results of the analysis of a mathematical model of the toxin/antitoxin hypothesis for bacteria in a chemostat with results incorporating the senescence hypothesis. We find that the persister fraction of the population as a function of washout rate provides a viable distinction between the two hypotheses. We also give simulation results that indicate that a strategy of alternating dose/withdrawal disinfection can be effective in clearing the entire persister and susceptible populations of bacteria. This strategy was considered previously in analysis of a generic model of persister formation. We find that extending the model of persister formation to include the toxin/antitoxin interactions in a chemostat does not alter the qualitative results that success of the dosing strategy depends on the withdrawal time. While this treatment is restricted to planktonic bacterial populations, it serves as a framework for including persister cells in a spatially dependent biofilm model.     

The use of biocides to control sulphate‐reducing bacteria in biofilms on mild steel surfaces

Three different types of biocides, viz. formaldehyde (FM), glutaraldehyde (GA) and isothiozolone (ITZ) were used to control planktonic and sessile populations of two marine isolates of sulphate‐reducing bacteria (SRB). The influence of these biocides on the initial attachment of cells to mild steel surfaces, on subsequent biofilm formation and on the activity of hydrogenase enzymes within developed biofilms was evaluated. In the presence of biocides the rate and degree of colonization of mild steel by SRB depended on incubation time, bacterial isolate and the type of biocide used. Although SRB differed in their susceptibility to biocides, for all isolates the biofilm population was more resistant to the treatment than the planktonic population. GA showed highest efficiency in controlling planktonic and sessile SRB compared with the other two biocides. The activity of the enzyme hydrogenase measured in SRB biofilms varied between isolates and with the biocide treatment. No correlation was found between the number of sessile cells and hydrogenase activity.     

Pore‐network modeling of biofilm evolution in porous media

The influence of bacterial biomass on hydraulic properties of porous media (bioclogging) has been explored as a viable means for optimizing subsurface bioremediation and microbial enhanced oil recovery. In this study, we present a pore network simulator for modeling biofilm evolution in porous media including hydrodynamics and nutrient transport based on coupling of advection transport with Fickian diffusion and a reaction term to account for nutrient consumption. Biofilm has non‐zero permeability permitting liquid flow and transport through the biofilm itself. To handle simultaneous mass transfer in both liquid and biofilm in a pore element, a dual‐diffusion mass transfer model is introduced. The influence of nutrient limitation on predicted results is explored. Nutrient concentration in the network is affected by diffusion coefficient for nutrient transfer across biofilm (compared to water/water diffusion coefficient) under advection dominated transport, represented by mass transport Péclet number >1. The model correctly predicts a dependence of rate of biomass accumulation on inlet concentration. Poor network connectivity shows a significantly large reduction of permeability, for a small biomass pore volume. Biotechnol. Bioeng. 2011;108: 2413–2423. © 2011 Wiley Periodicals, Inc.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

The effect of biofilm permeability on bio-clogging of porous media

A 3D Biofilm model, appropriate for complex porous media support structures, is successfully modified such that non‐zero permeability of biofilms structures is enabled. A systematic study is then conducted into the influence of biofilm permeability on overall biomass growth rate. This reveals a significant influence at large biofilm concentrations; even when the permeability of the biomass is 1.25% of that of the free pore space, biomass accumulation increased by a factor of ～3 over 40 h. The effect is shown to be retained when allowing for biomass detachment or erosion as a consequence of adjacent velocity shear. We conclude that biofilm permeability should be included in biofilm models and that further experimental work is required to better describe the link between biofilm permeability and local microstructure. Biotechnol. Bioeng. 2012; 109:1031–1042. © 2011 Wiley Periodicals, Inc.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Assessment of biofilm cell removal and killing and biocide efficacy using the microtiter plate test

Biofilm formation on surfaces has serious economic and environmental implications. Growth of biofilm within a water distribution system can lead to problems such as biocorrosion and biofouling accumulation. To prevent and control these occurrences, it is necessary to use suitable biocides to remove the biofilm and kill biofilm cells. In this study, the genera Actinobacillus, Branhamella, Bacillus, Micrococcus and Acinetobacter were isolated from biofilms formed on brass coupons exposed to a cooling water system. It was shown by the microtiter plate test that a mixed culture of the isolates and a single culture of Acinetobacter sp(2) produced high levels of biofilm formation. A microwell plate technique was applied for assessment of the ability of various biocides to remove and kill mixed-culture biofilm cells and Acinetobacter sp(2), the latter as a single-species biofilm with a high rate of biofilm production. The results showed that the mixed-culture biofilm cells had more resistance to removal and killing by some biocides, such as hydrogen peroxide and sulfathiazole, than the single-species biofilm cells (Acinetobacter sp(2)). Oxidising biocides, such as sodium hypochlorite and hydrogen peroxide, demonstrated a higher potential for biofilm removal and killing compared with non-oxidising biocides (sulfathiazole and glutaraldehyde).     

Biofilms and Planktonic Cells of Pseudomonas aeruginosa Have Similar Resistance to Killing by Antimicrobials

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.     

Non-toluene-associated respiration in a Pseudomonas putida 54G biofilm grown on toluene in a flat-plate vapor-phase bioreactor

Non-toluene-associated respiration (NTAR) within a Pseudomonas putida 54G biofilm growing on toluene as sole external carbon source was evaluated using oxygen microelectrodes in a flat-plate vapor-phase biological reactor. Two fluorescent probes, 2,4-diamidino-2-phenylindole and 5-cyano-2,3-ditolyltetrazolium chloride, were used to evaluate the number of total and respiring cells respectively within the biofilm. Biofilm samples were also analyzed for viable and toluene-culturable cells by spread-plating on non-selective and selective media respectively. Fractions of viable stressed, respiring and non-respiring cells within the biofilm were evaluated. The NTAR rate was positively correlated with the fraction of viable stressed and non-respiring cells within the biofilm, which suggested the capability of some cells to grow at the expense of leakage and lysis products coming from injured and dead cells. This effect was more pronounced at higher toluene concentration. Results suggest that NTAR should be incorporated into mathematical models of biofilm reactors degrading volatile organic carbon compounds. Received: 4 January 1997 / Received revision: 20 March 1997 / Accepted: 27 March 1997     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

Evaluation of a model biofilm for the ranking of biocide performance against sulphate-reducing bacteria

A model artificial biofilm was developed and evaluated for ranking the performance of biocides for application in oil production pipelines. The biofilm consisted of an alginate gel matrix into which were incorporated bacteria, scrapings from the inner surfaces of oil production pipelines and some crude oil.
The viability and sulphide-respiration rates of sulphate-reducing bacteria (SRB) within freshly-prepared artificial biofilm remained largely unchanged during a 2-week storage period. Furthermore, storage of the model biofilm did not alter the susceptibility of the incorporated SRB to a biocide. These findings showed that artificial biofilm may be produced in advance of a biocide assessment study and stored for at least 2 weeks over the course of the study without the model system undergoing changes which affected the relative performance of the biocides assessed. Artificial biofilms were found to be more resistant to biocides than planktonic bacteria and the addition of oil pipeline scrapings and crude oil to the artificial biofilm was found to increase further the resistance of biofilm to biocides.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

A novel approach to investigate biofilm accumulation and bacterial transport in porous matrices

Knowledge of bacterial transport through, and biofilm growth in, porous media is vitally important in numerous natural and engineered environments. Despite this, porous media systems are generally oversimplified and the local complexity of cell transport, biofilm formation and the effect of biofilm accumulation on flow patterns is lost. In this study, cells of the sulphate-reducing bacterium, Desulfovibrio sp. EX265, accumulated primarily on the leading faces of obstructions and developed into biofilm, which grew to narrow and block pore throats (at a rate of 12 micro m h(-1) in one instance). This pore blocking corresponded to a decrease in permeability from 9.9 to 4.9 Darcy. Biofilm processes were observed in detail and quantitative data were used to describe the rate of biofilm accumulation temporally and spatially. Accumulation in the inlet zone of the micromodel was 10% higher than in the outlet zone and a mean biofilm height of 28.4 micro m was measured in a micromodel with an average pore height of 34.9 microm. Backflow (flow reversal) of fluid was implemented on micromodels blocked with biofilm growth. Although biofilm surface area cover did immediately decrease (approximately 5%), the biofilm quickly re-established and permeability was not significantly affected (9.4 Darcy). These results demonstrate that the glass micromodel used here is an effective tool for in situ analysis and quantification of bacteria in porous media.     

The influence of nonconjugative Escherichia coli plasmids on biofilm formation and resistance

Aims: This work describes the effects of the presence of nonconjugative plasmids in Escherichia coli cells forming biofilms on a flow cell system under turbulent conditions. Methods and Results: The pET28 and pUC8 plasmids were separately used to transform E. coli JM109(DE3). Biofilm formation, removal and antimicrobial susceptibility to the cationic biocide benzyldimethyldodecylammonium chloride (BDMDAC) were assessed. Transformed cells formed thicker biofilms with higher cell densities, and the metabolic activity was higher whereas nontransformed cells had higher viabilities. Biocide treatment was not efficient for biofilm removal but was effective for cell killing. Biofilms formed by nontransformed cells were less affected by the treatment. Conclusions: Cell transformation with the tested plasmids has significant impacts on biofilm formation, cell viability, metabolic activity and resistance to biocide treatment. Our results show that in biofilm studies involving deletion/complementation experiments, a control with the strain carrying a plasmid devoid of the gene under investigation must be included so that the real effects of the genetic manipulation are not biased by the presence of the plasmid backbone. Significance and Impact of the Study: This is the first report where the presence of nonconjugative plasmids is assessed in flow conditions analysing biofilm formation, removal and antimicrobial susceptibility of high cell‐density biofilms.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 adapted to 2-phenoxyethanol shows cross-resistance to dissimilar biocides and increased susceptibility to antibiotics

The growth adaptability to increasing concentration of the biocide 2-phenoxyethanol (PE) was determined in Pseudomonas aeruginosa PAO1 (P.a.) as part of efforts to understand and control the biocide tolerance and its effect on cross-resistance to other biocides and resistance to antibiotics. After repeated subculturing in media containing increasing sub-minimum-inhibitory PE concentration, P.a. exhibited an adaptive resistance indicated by two-fold increase in MIC at the 10th passage. The resistance was stable and remained after passaging the strain in further 7 successive passages in PE-free growth media. The strain showed cross-resistance towards dissimilar biocides and displayed increased susceptibility to antibiotics, which was not influenced by the presence of the efflux inhibitor ‘carbonyl cyanide m-chlorophenyl hydrazone’. Outer membranes of adapted strain showed altered protein profile when examined by SDS-PAGE.     

Modeling of Biocide Action Against Biofilm

We consider the mathematical model of dynamic antimicrobial action against bacterial biofilms. A mixture model is used in which the biofilm consisting of live and dead bacteria is modeled as one fluid component, while the solvent containing biocide is modeled as the other, and each component is represented by its volume fraction. The whole system is assumed to be an incompressible fluid and the velocity is governed by the Navier-Stokes equation. Biocide kills the live bacteria and its transport is governed by an advection-reaction-diffusion equation. Certain biocide also weakens the mechanical cohesiveness of the biofilm and results in biofilm removal under the shear stress of the external flow. Spatial and temporal patterns of antimicrobial action of three different biocides are considered and numerical simulation results by finite difference method are presented.     

Identification of biofilm proteins in non-typeable Haemophilus Influenzae

Background  

Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is "slimy" and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.