Determination of pollutant diffusion coefficients in naturally formed biofilms using a single tube extractive membrane bioreactor

A novel technique has been used to determine the effective diffusion coefficients for 1,1,2-trichloroethane (TCE), a nonreacting tracer, in biofilms growing on the external surface of a silicone rubber membrane tube during degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 and monochlorobenzene (MCB) by Pseudomonas JS150. Experiments were carried out in a single tube extractive membrane bioreactor (STEMB), whose configuration makes it possible to measure the transmembrane flux of substrates. A video imaging technique (VIT) was employed for in situ biofilm thickness measurement and recording. Diffusion coefficients of TCE in the biofilms and TCE mass transfer coefficients in the liquid films adjacent to the biofilms were determined simultaneously using a resistances-in-series diffusion model. It was found that the flux and overall mass transfer coefficient of TCE decrease with increasing biofilm thickness, showing the importance of biofilm diffusion on the mass transfer process. Similar fluxes were observed for the nonreacting tracer (TCE) and the reactive substrates (MCB or DCE), suggesting that membrane-attached biofilm systems can be rate controlled primarily by substrate diffusion. The TCE diffusion coefficient in the JS150 biofilm appeared to be dependent on biofilm thickness, decreasing markedly for biofilm thicknesses of >1 mm. The values of the TCE diffusion coefficients in the JS150 biofilms <1-mm thick are approximately twice those in water and fall to around 30% of the water value for biofilms >1-mm thick. The TCE diffusion coefficients in the GJ10 biofilms were apparently constant at about the water value. The change in the diffusion coefficient for the JS150 biofilms is attributed to the influence of eddy diffusion and convective flow on transport in the thinner (<1-mm thick) biofilms.     

Determination of diffusion coefficients in biofilms by confocal laser microscopy

Microbial exopolymer may hinder the diffusion of nutrients, antibiotics, and other materials to the cell surface. Studies of diffusion in biofilms have been limited to indirect measurements. This study demonstrated the use of fluorescein and size-fractionated fluor-conjugated dextrans in conjunction with scanning confocal laser microscopy to directly monitor and determine diffusion coefficients within biofilms. The monitoring approaches were simple and, when combined with computerized image collection, allowed assembly of a data set suitable for calculation of one-dimensional diffusion coefficients for biofilm regions. With these techniques, it was shown that regional variability in the mobility of the dextrans occurred within mixed-species biofilms. Some regions exhibited rapid diffusion of all test molecules, while adjacent regions were only penetrated by the lower-molecular-weight compounds. The effective diffusion coefficients (D(e)) determined in a mixed-species biofilm were a function of the molecular radius of the probe (i.e., fluorescein, D(e) = 7.7 x 10 cm s; 4,000 molecular weight, D(e) = 3.1 x 10 cm s; and 2,000,000 molecular weight, D(e) = 0.7 x 10 cm s). These results demonstrated that diffusion in the biofilm was hindered relative to diffusion in the bulk solution. The study indicated that in situ monitoring by scanning laser microscopy is a useful approach for determining the mobility of fluorescently labeled molecules in biofilms, allowing image acquisition, appropriate scales of study, both xy and xz monitoring, and calculation of D(e) values.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Biopolymer and water dynamics in microbial biofilm extracellular polymeric substance

Nuclear magnetic resonance (NMR) is a noninvasive and nondestructive tool able to access several observable quantities in biofilms such as chemical composition, diffusion, and macroscale structure and transport. Pulsed gradient spin echo (PGSE) NMR techniques were used to measure spectrally resolved biomacromolecular diffusion in biofilm biomass, extending previous research on spectrally resolved diffusion in biofilms. The dominant free water signal was nulled using an inversion recovery modification of the traditional PGSE technique in which the signal from free water is minimized in order to view the spectra of components such as the rotationally mobile carbohydrates, DNA, and proteins. Diffusion data for the major constituents obtained from each of these spectral peaks demonstrate that the biomass of the biofilm contains both a fast and slow diffusion component. The dependence of diffusion on antimicrobial and environmental challenges suggests the polymer molecular dynamics measured by NMR are a sensitive indicator of biofilm function.     

Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy

A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluorescein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) were measured in the cell clusters and interstitial voids of a heterogeneous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capacity decreased and the diffusion coefficient could be measured. The diffusivity of phycoerythrin in cell clusters was impeded by 41%, compared to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteristics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. (c) 1997 John Wiley & Sons, Inc.     

Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Magnetic Resonance Imaging of Mass Transport and Structure Inside a Phototrophic Biofilm

The aim of this study was to utilize magnetic resonance imaging (MRI) to image structural heterogeneity and mass transport inside a biofilm which was too thick for photon based imaging. MRI was used to map water diffusion and image the transport of the paramagnetically tagged macromolecule, Gd-DTPA, inside a 2.5 mm thick cyanobacterial biofilm. The structural heterogeneity of the biofilm was imaged at resolutions down to 22 × 22 μm, enabling the impact of biofilm architecture on the mass transport of both water and Gd-DTPA to be investigated. Higher density areas of the biofilm correlated with areas exhibiting lower relative water diffusion coefficients and slower transport of Gd-DTPA, highlighting the impact of biofilm structure on mass transport phenomena. This approach has potential for shedding light on heterogeneous mass transport of a range of molecular mass molecules in biofilms.     

Anisotropic nutrient transport in three-dimensional single species bacterial biofilms

The ability for a biofilm to grow and function is critically dependent on the nutrient availability, and this in turn is dependent on the structure of the biofilm. This relationship is therefore an important factor influencing biofilm maturation. Nutrient transport in bacterial biofilms is complex; however, mathematical models that describe the transport of particles within biofilms have made three simplifying assumptions: the effective diffusion coefficient (EDC) is constant, the EDC is that of water, and/or the EDC is isotropic. Using a Monte Carlo simulation, we determined the EDC, both parallel to and perpendicular to the substratum, within 131 real, single species, three-dimensional biofilms that were constructed from confocal laser scanning microscopy images. Our study showed that diffusion within bacterial biofilms was anisotropic and depth dependent. The heterogeneous distribution of bacteria varied between and within species, reducing the rate of diffusion of particles via steric hindrance. In biofilms with low porosity, the EDCs for nutrient transport perpendicular to the substratum were significantly lower than the EDCs for nutrient transport parallel to the substratum. Here, we propose a reaction-diffusion model to describe the nutrient concentration within a bacterial biofilm that accounts for the depth dependence of the EDC.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Adaptive responses to antimicrobial agents in biofilms

Bacterial biofilms demonstrate adaptive resistance in response to antimicrobial stress more effectively than corresponding planktonic populations. We propose here that, in biofilms, reaction-diffusion limited penetration may result in only low levels of antimicrobial exposure to deeper regions of the biofilm. Sheltered cells are then able to enter an adapted resistant state if the local time scale for adaptation is faster than that for disinfection. This mechanism is not available to a planktonic population. A mathematical model is presented to illustrate. Results indicate that, for a sufficiently thick biofilm, cells in the biofilm implement adaptive responses more effectively than do freely suspended cells. Effective disinfection requires applied biocide concentration that increases quadratically or exponentially with biofilm thickness.     

Magnetic resonance imaging of structure, diffusivity, and copper immobilization in a phototrophic biofilm

Magnetic resonance imaging (MRI) was used to spatially resolve structure, water diffusion, and copper transport and fate in a phototrophic biofilm [corrected]. MRI was able to resolve considerable structural heterogeneity, ranging from classical laminations approximately 500 mum thick to structures with no apparent ordering. Pulsed-field gradient (PFG) analysis spatially resolved water diffusion coefficients which exhibited relatively little or no attenuation (diffusion coefficients ranged from 1.7 x 10(-9) m(2) s(-1) to 2.2 x 10(-9) m(2) s(-1)). The biofilm was then reacted with a 10-mg liter(-1) Cu(2+) solution, and transverse relaxation time parameter maps [corrected].were used to spatially and temporally map copper immobilization within the biofilm. Significantly, a calibration protocol similar to that used in biomedical research successfully quantified copper concentrations throughout the biofilm. Variations in Cu concentrations were controlled by the biofilm structure. Copper immobilization was most rapid (approximately 5 mg Cu liter(-1) h(-1)) over the first 20 to 30 h and then much slower for the remaining 60 h of the experiment. The transport of metal within the biofilm is controlled by both diffusion and immobilization. This was explored using a Bartlett and Gardner model which examined both diffusion and adsorption through a hypothetical film exhibiting properties similar to those of the phototrophic biofilm. Higher adsorption constants (K) resulted in longer lag times until the onset of immobilization at depth but higher actual adsorption rates. MRI and reaction transport models are versatile tools which can significantly improve our understanding of heavy metal immobilization in naturally occurring biofilms.     

Diffusion of phenol through a biofilm grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor

Diffusion of phenol through a biofilm attached to activated carbon particles was investigated. The biofilm was grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor operating in a fed-batch mode. It was found that phenol did not adsorb on the biofilm and that the diffusion coefficient of phenol within the biofilm varied from 13 to 39% of its corresponding value in water. The diffusion coefficient of phenol within the biofilm was reduced by increasing the biofilm density. An extensive literature review of diffusion of substrates through biofilms indicated that this conclusion could be extended to biofilms grown on flat surfaces, rotating cylinders, and even bioflocs.     

Microelectrode measurements of local mass transport rates in heterogeneous biofilms

Microelectrodes were used to measure oxygen profiles and local mass transfer coefficient profiles in biofilm clusters and interstitial voids. Both profiles were measured at the same location in the biofilm. From the oxygen profile, the effective diffusive boundary layer thickness (DBL) was determined. The local mass transfer coefficient profiles provided information about the nature of mass transport near and within the biofilm. All profiles were measured at three different average flow velocities, 0.62, 1.53, and 2.60 cm sec-1, to determine the influence of flow velocity on mass transport. Convective mass transport was active near the biofilm/liquid interface and in the upper layers of the biofilm, independent of biofilm thickness and flow velocity. The DBL varied strongly between locations for the same flow velocities. Oxygen and local mass transfer coefficient profiles collected through a 70 micrometer thick cluster revealed that a cluster of that thickness did not present any significant mass transport resistance. In a 350 micrometer thick biofilm cluster, however, the local mass transfer coefficient decreased gradually to very low values near the substratum. This was hypothetically attributed to the decreasing effective diffusivity in deeper layers of biofilms. Interstitial voids between clusters did not seem to influence the local mass transfer coefficients significantly for flow velocities of 1.53 and 2.60 cm sec-1. At a flow velocity of 0.62 cm sec-1, interstitial voids visibly decreased the local mass transfer coefficient near the bottom.     

Spatial Physiological Heterogeneity in Pseudomonas aeruginosa Biofilm Is Determined by Oxygen Availability

The role of oxygen availability in determining the local physiological activity of Pseudomonas aeruginosa growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 μm thick with biofilms grown in air, 2 μm thick with biofilms grown in pure nitrogen, and 46 μm thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 μm. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within P. aeruginosa biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 μm of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.     

Channel structures in aerobic biofilms of fixed-film reactors treating contaminated groundwater.

Scanning electron microscopy, confocal scanning laser microscopy, and fatty acid methyl ester profiles were used to study the development, organization, and structure of aerobic multispecies biofilm communities in granular activated-carbon (GAC) fluidized-bed reactors treating petroleum-contaminated groundwaters. The sequential development of biofilm structure was studied in a laboratory reactor fed toluene-amended groundwater and colonized by the indigenous aquifer populations. During the early stages of colonization, microcolonies were observed primarily in crevices and other regions sheltered from hydraulic shear forces. Eventually, these microcolonies grew over the entire surface of the GAC. This growth led to the development of discrete discontinuous multilayer biofilm structures. Cell-free channel-like structures of variable sizes were observed to interconnect the surface film with the deep inner layers. These interconnections appeared to increase the biological surface area per unit volume ratio, which may facilitate transport of substrates into and waste products out of deep regions of the biofilm at rates greater than possible by diffusion alone. These architectural features were also observed in biofilms from four field-scale GAC reactors that were in commercial operation treating petroleum-contaminated groundwaters. These shared features suggest that formation of cell-free channel structures and their maintenance may be a general microbial strategy to deal with the problem of limiting diffusive transport in thick biofilms typical of fluidized-bed reactors.     

Antimicrobial efficacy of the alkaloid harmaline alone and in combination with chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic and biofilm cultures

Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures. Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro‐organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy. Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm. Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro. Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug‐resistance of S. aureus both in suspension and in biofilm.