Mixed Population Biofilms Research Articles

Multi-channel microfluidic biosensor platform applied for online monitoring and screening of biofilm formation and activity.

Bacterial colonization of surfaces and interfaces has a major impact on various areas including biotechnology, medicine, food industries, and water technologies. In most of these areas biofilm development has a strong impact on hygiene situations, product quality, and process efficacies. In consequence, biofilm manipulation and prevention is a fundamental issue to avoid adverse impacts. For such scenario online, non-destructive biofilm monitoring systems become important in many technical and industrial applications. This study reports such a system in form of a microfluidic sensor platform based on the combination of electrical impedance spectroscopy and amperometric current measurement, which allows sensitive online measurement of biofilm formation and activity. A total number of 12 parallel fluidic channels enable real-time online screening of various biofilms formed by different Pseudomonas aeruginosa and Stenotrophomonas maltophilia strains and complex mixed population biofilms. Experiments using disinfectant and antibiofilm reagents demonstrate that the biofilm sensor is able to discriminate between inactivation/killing of bacteria and destabilization of biofilm structures. The impedance and amperometric sensor data demonstrated the high dynamics of biofilms as a consequence of distinct responses to chemical treatment strategies. Gene expression of flagellar and fimbrial genes of biofilms grown inside the microfluidic system supported the detected biofilm growth kinetics. Thus, the presented biosensor platform is a qualified tool for assessing biofilm formation in specific environments and for evaluating the effectiveness of antibiofilm treatment strategies.

Potential for Largemouth Bass Virus to Associate with and Gain Protection from Bacterial Biofilms

Quantitative PCR (qPCR) was used to investigate whether largemouth bass virus (LMBV) can exist within biofilms. Suspended LMBV was partitioned into either laboratory-grown Pseudomonas fluorescens biofilms or pond-grown, mixed-population biofilms. Biofilm-entrapped LMBV retained infectivity when tested on epithelioma papillosum cyprini tissue culture cells. The LMBV associated with P. fluorescens biofilms were resistant to disinfection by sodium hypochlorite and an iodine-based compound (betadine) but were susceptible to ethanol. Largemouth bass virus was not detected in biofilms or water from ponds that had previously contained LMBV-positive fish, suggesting either that the viral concentrations were below the detection limit of qPCR or that the fish represented the main LMBV reservoir. This study illustrates the potential for LMBV to associate with bacterial biofilms and thereby gain protection from some chemical disinfectants.

Survival Response of Bacteriovorax in Surface Biofilm Versus Suspension when Stressed by Extremes in Environmental Conditions

The Bacteriovorax, previously in the genus Bdellovibrio, are prokaryotes that prey upon many Gram-negative bacteria. They are ubiquitous in salt-water environments and have been reported to have a strong association with biofilms. The purpose of this study was to test the hypothesis that this association affords protection for the Bacteriovorax and enhances their survival in nature when exposed to extreme environmental conditions. Experiments were designed to compare their survival in biofilms versus in suspension when exposed to extremes in salinity and temperature. Natural mixed-population biofilms generated in moderate-salinity (16 per thousand) Patuxent River water and containing Bacteriovorax were exposed to drastic changes in salinity by placing in low-salinity (1 per thousand) river water and salt-free (no measurable salinity) distilled water for up to 14 days. In a separate trial, the biofilm was exposed to extremes in temperature, 5 degrees C and 35 degrees C, for up to 12 weeks in aquarium mesocosms. Simultaneously, suspensions of the Bacteriovorax were exposed to the same extremes in salinity and temperature as biofilms. The results revealed that the Bacteriovorax typically were able to survive for a week or longer while in association with biofilms than when in suspension. These results are consistent with observations from nature and establish that biofilms are important in the survival and ecology of the Bacteriovorax.

Effect of Acetate on Nitrite Oxidation in Mixed-Population Biofilms

While ammonia oxidation has been widely studied, nitrite oxidation is still not well understood. To study the effect of organics on nitrite oxidation, one control group and four treatment groups were designed with sodium acetate (C) to nitrite (N) ratios of 0, 0.44, 0.88, 4.41, and 8.82. Normal nitrite oxidization reactions were performed when C/N=0, but nitrite oxidization and partial denitrification occurred with low C/N ratios (0.44 or 0.88). At high C/N ratios (4.41 or 8.82), we mainly observed denitrification. In contrast to C/N=0, the nitrite oxidization rate was unaffected when C/N=0.44, but decreased with C/N = 0.88. The structure of bacterial communities varied significantly between autotrophic and mixotrophic conditions. Nitrobacter was hard to detect by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) while heterotrophs and especially denitrifiers were in the majority under mixotrophic conditions. Real-time PCR indicated that the Nitrobacter population decreased from 2.42 x 10(4) to 1.34 x 10(3) 16S rRNA gene copies/ng DNA, while the quantity of denitrifiers obviously increased from 0 to 2.51 x 10(4) nosZ gene copies/ng DNA with an increasing C/N ratio. The degree of denitrification differed between C/N=4.41 and C/N=8.82, as indicated by nitrite-N and nitrate-N curve analysis, as well as by the apparent bacterial community structure. Our findings provide critical insight regarding the relationship between organics and nitrification in biofilms.

Effects of marine microbial biofilms on the biocide release rate from antifouling paints—A model-based analysis

The antifouling (AF) paint model of Kiil et al. [S. Kiil, C.E. Weinell, M.S. Pedersen, K. Dam-Johansen, Analysis of self-polishing antifouling paints using rotary experiments and mathematical modelling, Ind. Eng. Chem. Res. 40 (2001) 3906–3920] and the simplified biofilm growth model of Gujer and Wanner [W. Gujer, O. Wanner, Modeling mixed population biofilms, in: W.G. Characklis, K.C. Marshall (Eds.), Biofilms, Wiley–Interscience, New York, 1990] are used to provide a reaction engineering-based insight to the effects of marine microbial slimes on biocide leaching and, to a minor extent, polishing behaviour of AF paints. It is concluded that the perturbation of the local sea water conditions (e.g. pH), as a consequence of the metabolic activity of the biofilm should not affect the net biocide leaching and binder reaction rates significantly. This results from the thin and poorly active biofilms which presumably grow onto the highly effective modern AF paints. According to simulations, the experimental decrease in the biocide leaching rate caused by biofilm growth must be mainly attributed to adsorption of the biocide by the exopolymeric substances secreted by the microorganisms. The effects of biofilms on the leaching of any generic active compound (e.g. natural antifoulants) are discussed in relation to their potential release mechanisms. The largest influence of biofilms is predicted for those active compounds that are released by a diffusion-controlled mechanism (typically tin-free algaecides).

Influences of Biofilm Structure and Antibiotic Resistance Mechanisms on Indirect Pathogenicity in a Model Polymicrobial Biofilm

Indirect pathogenicity (IP), the commensal protection of antibiotic-sensitive pathogens by resistant microorganisms of low intrinsic virulence, can prevent the eradication of polymicrobial infections. The contributions of antibiotic resistance mechanisms and biofilm structure to IP within polymicrobial biofilms were investigated using a model two-member consortium. Escherichia coli ATCC 33456 was transformed with vectors conferring either ampicillin or spectinomycin resistance, creating two distinct populations with different resistance mechanisms. Each strain alone or the consortium was grown as biofilms in flow cells and planktonically in chemostats. Comparisons in survival and activity were made on the basis of MICs and minimum biofilm preventative concentrations, a newly introduced descriptor. In ampicillin-containing medium, commensal interactions were evident during both modes of cultivation, but the sensitive strain experienced a greater benefit in the chemostat, indicating that the biofilm environment limited the commensal interaction between the Amp(r) and Spt(r) strains. In spectinomycin-containing medium, growth of the sensitive strain in chemostats and biofilms was not aided by the resistant strain. However, green fluorescent protein expression by the sensitive strain was greater in mixed-population biofilms (9% +/- 1%) than when the strain was grown alone (2% +/- 0%). No comparable benefit was evident during growth in the chemostat, indicating that the biofilm structure contributed to enhanced activity of the sensitive strain.

A bioremediation approach using natural transformation in pure-culture and mixed-population biofilms

Bacterial transformation by naked DNA is thought to contribute to gene transfer and microbial evolution within natural environments. In nature many microbial communities exist as complex assemblages known as biofilms where genetic exchange is facilitated. It may be possible to take advantage of natural transformation processes to modify the phenotypes of biofilm communities giving them specific and desirable functions. Work described here shows that biofilms composed of either pure cultures or mixed populations can be transformed with specific catabolic genes such that the communities acquire the ability to degrade a particular xenobiotic compound. Biofilms were transformed by plasmids bearing genes encoding green fluorescent protein (mut2) and/or atrazine chlorohydrolase (atzA). Confocal microscopy was used to quantify the number of transformants expressing mut2 in the biofilms. Degradation of atrazine by expressed atzA was quantified by tandem mass spectrometry. PCR analysis was performed to confirm the presence of atzA in transformed biofilms. These results indicate that it should be possible to use natural transformation to enhance bioremediation processes performed by biofilms.

Persistence of biofilm-associated Escherichia coli and Pseudomonas aeruginosa in groundwater and treated effluent in a laboratory model system.

This study was based on the hypothesis that groundwater-derived biofilms may provide a reservoir for coliform or pathogenic bacteria as has been observed in drinking water distribution systems. Escherichia coli, labelled with green fluorescent protein, was found to colonize all layers of mixed-population biofilms developed in association with indigenous groundwater micro-organisms in a laboratory-scale reactor. Biofilm-associated E. coli was removed at a slower rate from the reactor flasks than planktonic E. coli under a continuous flow regime. During flow-through of groundwater, planktonic E. coli removal was slower in flasks containing coverslips for enhanced biofilm development compared to a control flask without coverslips. Conversely, during flow-through of treated effluent, planktonic E. coli removal was faster in flasks with coverslips compared to without. Removal of attached E. coli was also fastest in the coverslip-containing flasks with effluent flow-through. This suggests that an increase in available nutrients may reduce E. coli survival potential due to either enhanced competition for nutrients or enhanced antagonism by the indigenous microbial population. Under identical conditions, GFP-labelled Pseudomonas aeruginosa was found to persist in the biofilms for longer than E. coli, most notably when exposed to flow-through of treated effluent. However, prolonged persistence of P. aeruginosa in the effluent could not be attributed to an association with the biofilms. This study has shown that under certain conditions the presence of mixed-population biofilms may limit the survival potential of enteric bacterial pathogens introduced into groundwater.

Binary and mixed population biofilms: time-lapse image analysis and disinfection with biocides.

Simultaneous binary population biofilm formation by a bacterium and filamentous fungus was demonstrated by time-lapse image analysis in a flow cell system. The accumulation of attached bacterial cells followed an S-shaped graph similar to batch culture bacterial growth, with continual attachment, detachment, rotation, and movement of bacteria over the surface. An extensive hyphal network formed on the surface of the flow cell, protruding into the bulk flow, which subsequently detached. Multiple species mixed fungal-bacterial model biofilms were tested for isothiazolone biocide susceptibility. Biofilms were less susceptible to biocide treatment than planktonic cells of the same organisms. Mixed species biofilms, particularly for the bacterial species, offered greater protection against the action of the biocide compared to single species biofilms. Microbial loss as a result of biocide activity was shown by reduced cell surface coverage in electron micrographs.

Binary and mixed population biofilms: Time-lapse image analysis and disinfection with biocides

Simultaneous binary population biofilm formation by a bacterium and filamentous fungus was demonstrated by time-lapse image analysis in a flow cell system. The accumulation of attached bacterial cells followed an S-shaped graph similar to batch culture bacterial growth, with continual attachment, detachment, rotation, and movement of bacteria over the surface. An extensive hyphal network formed on the surface of the flow cell, protruding into the bulk flow, which subsequently detached. Multiple species mixed fungal–bacterial model biofilms were tested for isothiazolone biocide susceptibility. Biofilms were less susceptible to biocide treatment than planktonic cells of the same organisms. Mixed species biofilms, particularly for the bacterial species, offered greater protection against the action of the biocide compared to single species biofilms. Microbial loss as a result of biocide activity was shown by reduced cell surface coverage in electron micrographs.

Chlorination of model drinking water biofilm: implications for growth and organic carbon removal

The influence of chlorine on biofilm in low organic carbon environments typical of drinking water or industrial process water was examined by comparing biomass and kinetic parameters for biofilm growth in a chlorinated reactor to those in a non-chlorinated control. Mixed-population heterotrophic biofilms were developed in rotating annular reactors under low concentration, carbon-limited conditions (<2 mg/L as carbon) using three substrate groups (amino acids, carbohydrates and humic substances). Reactors were operated in parallel under identical conditions with the exception that chlorine was added to one reactor at a dose sufficient to maintain a free chlorine residual of 0.09–0.15 mg/L in the effluent. The presence of free chlorine resulted in development of less biofilm biomass compared to the control for all substrates investigated. However, specific growth and organic carbon removal rates were on the average five times greater for chlorinated biofilm compared to the control. Observed yield values were less for chlorinated biofilm. Although chlorinated biofilm's specific organic carbon removal rate was high, the low observed yield indicated organic carbon was being utilized for purposes other than creating new cell biomass. The impacts of free chlorine on mixed-population biofilms in low-nutrient environments were different depending upon the available substrate. Biofilms grown using amino acids exhibited the least difference between control and chlorinated kinetic parameters; biofilm grown using carbohydrates had the greatest differences. These findings are particularly relevant to the fundamental kinetic parameters used in models of biofilm growth in piping systems that distribute chlorinated, low-carbon-concentration water.

Resistance of biofilms containing alginate‐producing bacteria to disintegration by an alginate degrading enzyme (Algl)

Pure culture biofilms of Pseudomonas aeruginosa (strains 8830 and ATCC 700829) and mixed population biofilms composed of Pseudomonas aeruginosa (ATCC 700829), Pseudomonas fluorescens (ATCC 700830), and Klebsiellapneumoniae (ATCC 700831) were treated with an alginate‐degrading enzyme (AlgL). The enzyme effectively depolymerized the mannuronic acid rich (92%), partially O‐acetylated bacterial alginate produced by P. aeruginosa (8830), both in dilute solution and in a gel‐like, concentrated state. However, both biofilms were unaffected by the presence of the enzyme. These findings suggest either that bacterial alginates do not contribute significantly to the cohesiveness of biofilms or that the alginate is protected from enzymatic degradation in biofilms.

Water binding in biofilms

The main component of biofilms is water, which can make up to over 90% of the wet weight. Biofilms are therefore considered as hydrogels. Water binding and mobility are crucial for diffusion processes in biofilms as well as for dewatering of sludges. In a FTIR-ATR study, the hydrogen-deuterium exchange in native drinking water mixed population biofilms was investigated non-destructively in a flow-through cell. The exchange process of water is characterized by two steps, the first completed after 49 seconds and the second after 21 minutes, indicating different water retention mechanisms. The process is fully completed after about 2 hours. If the biofilm is treated with chlorine (6 mg L−1 for 12h), exchange happens faster and no steps can be observed, indicating a change in the matrix with respect to water binding properties. It is concluded that the water in the EPS matrix is organized in a fine structure which influences the properties of the biofilm.

Water binding in biofilms

The main component of biofilms is water, which can make up to over 90% of the wet weight. Biofilms are therefore considered as hydrogels. Water binding and mobility are crucial for diffusion processes in biofihns as well as for dewatering of sludges. In a FTIR-ATR study, the hydrogen-deuterium exchange in native drinking water mixed population biofilms was investigated non-destructively in a flow-through cell. The exchange process of water is characterized by two steps, the first completed after 49 seconds and the second after 21 minutes, indicating different water retention mechanisms. The process is fully completed after about 2 hours. If the biofilm is treated with chlorine (6 mg −1 for 12h), exchange happens faster and no steps can be observed, indicating a change in the matrix with respect to water binding properties. It is concluded that the water in the EPS matrix is organized in a fine structure which influences the properties of the biofilm

Measurement of local effective diffusivity in heterogeneous biofilms

We have developed a novel technique to measure local effective diffusivity distribution in heterogeneous biofilms. Mobile microelectrodes (tip diameter 10 μm) and the limiting current technique were employed to measure the effective diffusivity of electroactive species introduced to natural and artificial biofilms. We calibrated the microelectrodes in artificial biofilms of known effective diffusivity and known density. In mixed population biofilms, local effective diffusivity varied from one location to another and decreased toward the bottom of the biofilm. We related local effective diffusivity to local biofilm density using an empirical equation. Surface-averaged biomass density depended on liquid flow velocity at which the biofilms were grown. The higher the flow velocity, the denser were the biofilms. Our technique permits fast evaluation of local effective diffusivity and biofilm density in heterogeneous biofilms.

Significance of biofilm structure on transport of inert particulates into biofilms

Evolutional changes in interior structures of mixed population biofilms grown on domestic wastewater were quantitatively analyzed using a cryosectioning technique and an image analysis. Meanwhile, transport of particulates into the biofilms was also experimentally investigated using fluorescent microbeads as tracers to relate the biofilm structure and particulate transport into the biofilm. Microscopic observation of the cryomicrotomy biofilm sections indicated the biofilms were very porous and consisted of interwinded filamentous biomass acting as a framework of the biofilm. A honeycomb structure was often found, which would make the biofilm more resistant to water flow. There were micropores with the diameter of about 10 μm microcolony aggregates attached to filamentous biomass and macropores with the diameter of 20–200 μm in the biomass matrix. These pores did not clog during two months of cultivation. Areal porosity was about 30% in the bottom biofilm and more than 90% in the surface. Significant difference in transport efficiency was not observed for various sizes of microbeads due to the presence of macropores. Therefore, even 10 μm tracer beads could quickly traverse throughout a biofilm 640 μm thick via water channels or macropores and then penetrated into the micropores. Convective transport from the bulk to the bottom biofilm, rather than molecular diffusion, was responsible for this rapid transport. Based on experimental results, it can be concluded that the biofilm structure seems to be well designed to maximize the transport efficiency of substrates and products and the strength of biofilm structure.

Oscillation characteristics of biofilm streamers in turbulent flowing water as related to drag and pressure drop.

Mixed population biofilms consisting of Pseudomonas aeruginosa, P. fluorescens, and Klebsiella pneumoniae were grown in a flow cell under turbulent conditions with a water flow velocity of 18 cm/s (Reynolds number, Re, =1192). After 7 days the biofilms were patchy and consisted of cell clusters and streamers (filamentous structures attached to the downstream edge of the clusters) separated by interstitial channels. The cell clusters ranged in size from 25 to 750 microm in diameter. The largest clusters were approximately 85 microm thick. The streamers, which were up to 3 mm long, oscillated laterally in the flow. The motion of the streamers was recorded at various flow velocities up to 50.5 cm/s (Re 3351) using confocal scanning laser microscopy. The resulting time traces were evaluated by image analysis and fast Fourier transform analysis (FFT). The amplitude of the motion increased with flow velocity in a sigmoidal shaped curve, reaching a plateau at an average fluid flow velocity of approximately 25 cm/s (Re 1656). The motion of the streamers was possibly limited by the flexibility of the biofilm material. FFT indicated that the frequency of oscillation was directly proportional to the average flow velocity (u(ave)) below 9.5 cm/s (Re 629). At u(ave) greater than 9.5 cm/s, oscillation frequencies were above our measurable frequency range (0.12-6.7 Hz). The oscillation frequency was related to the flow velocity by the Strouhal relationship, suggesting that the oscillations were possibly caused by vortex shedding from the upstream biofilm clusters. A loss coefficient (k) was used to assess the influence of biofilm accumulation on pressure drop. The k across the flow cell colonized with biofilm was 2.2 times greater than the k across a clean flow cell.

Sulfate reduction and sulfide oxidation in aerobic mixed population biofilms

The microzonation of O2 respiration, H2S oxidation and SO42− reduction in aerobic biofilms grown on rotating disk reactors of a sewage treatment plant was studied by measuring concentration profiles with microelectrodes for O2, S2− and pH. The vertical distribution of sulfate-reducing bacteria (SRB) in the biofilms was also determined by the conventional culture-dependent MPN method and fluorescently labeled 16S rRNA-targeted oligonucleotide probes for SRB. The SRB probe stained cells were distributed throughout the biofilm with a distinct higher fluorescence intensity in the middle part of the biofilm. This result corresponded well with O2 and S2− concentration gradients measured by microelectrodes, showing sulfate reducing activity was largely restricted to a narrow anaerobic zone located in the middle of the biofilm. Measurements of accumulation of reduced sulfur compounds (FeS, FeS2 and SO) in the biofilm indicated that the H2S produced by SRB became oxidized by O2 and other oxidants, probably ferric/ferrous hydrates, and precipitated as FeS and SO just above the sulfate reduction zone.

Measurement of local effective diffusivity in heterogeneous biofilms

We have developed a novel technique to measure local effective diffusivity distribution in heterogeneous biofilms. Mobile microelectrodes (tip diameter 10 µm) and the limiting current technique were employed to measure the effective diffusivity of electroactive species introduced to natural and artificial biofilms. We calibrated the microelectrodes in artificial biofilms of known effective diffusivity and known density. In mixed population biofilms, local effective diffusivity varied from one location to another and decreased toward the bottom of the biofilm. We related local effective diffusivity to local biofilm density using an empirical equation. Surface-averaged biomass density depended on liquid flow velocity at which the biofilms were grown. The higher the flow velocity, the denser were the biofilms. Our technique permits fast evaluation of local effective diffusivity and biofilm density in heterogeneous biofilms.

Sulfate reduction and sulfide oxidation in aerobic mixed population biofilms

The microzonation of O2 respiration, H2S oxidation and S042- reduction in aerobic biofilms grown on rotating disk reactors of a sewage treatment plant was studied by measuring concentration profiles with microelectrodes for O2, S2- and pH. The vertical distribution of sulfate-reducing bacteria (SRB) in the hiolilms was also determined by the conventional culture-dependent MPN method and fluorescently labeled 16S rRNA-targeted oligonucleotide probes for SRB. The SRB probe stained cells were distributed throughout the biofilm with a distinct higher fluorescence intensity to the middle part of the biofilm. This result corresponded well with O2 and S2- concentration gradients measured by microelectrodes, showing sulfate reducing activity was largely restricted to a narrow anaerobic zone located in the middle of the biofilm. Measurements of accumulation of reduced sulfur compounds (FeS, FeS2 and SO) in the biofilm indicated that the H2S produced by SRB became oxidized by O2 and other oxidants, probably ferric/ferrous hydrates, and precipitated as FeS and SO just above the sulfate reduction zone.