Three‐dimensional visualization of mixed species biofilm formation together with its substratum

Biofilms, such as dental plaque, are aggregates of microorganisms attached to a surface. Thus, visualization of biofilms together with their attached substrata is important in order to understand details of the interaction between them. However, so far there is limited availability of such techniques. Here, non‐invasive visualization of biofilm formation with its attached substratum by applying the previously reported technique of continuous‐optimizing confocal reflection microscopy (COCRM) is reported. The process of development of oral biofilm together with its substratum was sequentially visualized with COCRM. This study describes a convenient method for visualizing biofilm and its attached surface.     

Mycobacterium marinum biofilm formation reveals cording morphology

Abstract The emergence of the nontuberculosis mycobacteria (NTM) as clinically relevant pathogens has warranted the study of these ubiquitous organisms in the context of their likely environmental niche, the biofilm. We assayed the NTM bacterium Mycobacterium marinum strain 1218R, a fish outbreak isolate, for biofilm formation on different surfaces over time using three different methods. Using the MBEC system, biofilm development occurred continually over the 14-day culture period reaching a mature or stable biofilm state after 7 days postinoculation. Quantification of M. marinum biofilm formation on high-density polyethylene (HDPE), polycarbonate (PC) and silicon (Si) coupons over a 14-day period was evaluated using a continuous flow reactor system. M. marinum developed biofilms on all of the surfaces tested. However, substantially more biofilm accumulated on the silicon than on the other substrates (Si>HDPE>PC) under the same growth conditions indicating that silicon was the most effective substratum studied for the generation of M. marinum biofilms and suggesting a correlation between surface hydrophobicity and attachment. Finally, confocal laser scanning microscopy was used to visualize M. marinum biofilm development in situ over time and revealed an unusual biofilm ultrastructure. Large cell clusters attached to the surface grew in parallel sinuous arrays of cells that formed large cords.     

Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors

In this article, the conditions for aerobic biofilm formation on suspended particles, the dynamics of biofilm formation, and the biomass production during the start-up of a Biofilm Airlift Suspension reactor (BAS reactor) have been studied. The dynamics of biofilm formation during start up in the biofilm airlift suspension reactor follows three consecutive stages: bare carrier, microcolonies or patchy biofilms on the carrier, and biofilms completely covering the carrier. The effect of hydraulic retention time and of substrate loading rate on the formation of biofilms were investigated. To obtain in a BAS reactor a high biomass concentration and predominantly continuous biofilms, which completely surround the carrier, the hydraulic retention time must be shorter than the inverse of the maximum growth rate of the suspended bacteria. At longer hydraulic retention times, a low amount of attached biomass can be present on the carrier material as patchy biofilms. During the start-up at short hydraulic retention times the bare carrier concentration decreases, the amount of biomass per biofilm particle remains constant, and biomass increase in the reactor is due to increasing numbers of biofilm particles. The substrate surface loading rate has effect only on the amount of biomass on the biofilm particle. A higher surface load leads to a thicker biofilm.A strong nonlinear increase of the concentration of attached biomass in time was observed. This can be explained by a decreased abrasion of the biofilm particles due to the decreasing concentration of bare carriers. The detachment rate per biofilm area during the start-up is independent of the substrate loading rate, but depends strongly upon the bare carrier concentration.The Pirt-maintenance concept is applicable to BAS reactors. Surplus biomass production is diminished at high biomass concentrations. The average maximal yield of biomass on substrate during the experiments presented in this article was 0.44 +/- 0.08 C-mol/C-mol, the maintenance value 0.019 +/- 0.012 C-mol/(C-mol h). The lowest actual biomass yield measured in this study was 0.15 C-mol/C-mol. (c) 1994 John Wiley & Sons, Inc.     

Biofilm reactors: an experimental and modeling study of wastewater denitrification in fluidized-bed reactors of activated carbon particles

A fluidized-bed biofilm reactor using activated carbon particles of 1.69 mm diameter as the support for biomass growth and molasses as the carbon source is used for wastewater denitrification.The start-up of the reactor was successfully achieved in 1 week by using a liquor from garden soil leaching as the inoculum and a superficial velocity u(0) = 5u(mf). Typical biofilm thickness is 800 mum; therefore covered activated carbon particles have 3.3 mm in diameter.Reactor hydrodynamics was studied by tracer (KCl solution) experiments. The analysis based on residence time distribution theory involved a model with axial dispersion flow and tracer diffusion with linear adsorption inside the biofilm. Peclet numbers higher than 100 were found, allowing the plug flow assumption for the reactor model.Experimental profiles of nitrate and nitrite species were explained by a kinetic model of two consecutive zero-order reactions coupled with substrate diffusion inside the biofilm. Under the operating conditons used thick biofilms were obtained working in a diffusion-controlled regime.Comparison is made with results obtained in the same reactor with sand particles as the support for biomass growth. Activated carbon as the support has the following advantages: good adsorptive characteristics, homogeneous biofilm thickness along the reactor, and easy restart-up of the reactor. (c) 1992 John Wiley & Sons, Inc.     

Single-species microbial biofilm screening for industrial applications

While natural microbial biofilms often consist of multiple species, single-species biofilms are of great interest to biotechnology. The current study evaluates biofilm formation for common industrial and laboratory microorganisms. A total of 68 species of biosafety level one bacteria and yeasts from over 40 different genera and five phyla were screened by growing them in microtiter plates and estimating attached biomass by crystal violet staining. Most organisms showed biofilm formation on surfaces of polystyrene within 24 h. By changing a few simple conditions such as substratum characteristics, inoculum and nutrient availability, 66 strains (97%) demonstrated biofilm formation under at least one of the experimental conditions and over half of these strains were classified as strong biofilm formers, potentially suitable as catalysts in biofilm applications. Many non-motile bacteria were also strong biofilm formers. Biofilm morphologies were visualized for selected strains. A model organism, Zymomonas mobilis, easily established itself as a biofilm on various reactor packing materials, including stainless steel.     

Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Modelling epilithic biofilms combining hydrodynamics,invertebrate grazing and algal traits

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Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

Methanotrophic attached-film reactor development and biofilm characteristics

The feasibility of using methanotrophs in an attached-film, fluidized-bed (MAFFB) reactor system has been under investigation since 1987. Mixed culture, methane-utilizing attached biofilms were developed on diatomaceous earth particles and on granular activated carbon. The required feed gases, methane and oxygen, were supplied to the attached biofilm in disolved form using separate gas-liquid aeration columns. Biofilm growth was steady despite low influent dissolved methane concentrations (1 to 3 mg/L). A breeder MAFFB operated consistently for 4.1 years with attached biofilm concentrations as high as 51.7 g VS/L static-bed with minimal biomass wasting and with minimal buffer and nutrient inputs. The maximum biomass concentration observed was 75.6 g VS/L static-bed in a MAFFB reactor treating trichloroethene. Biofilm thickness reached 160 mum with typical values of 70 mum under methane and oxygen growht-rate-limited conditions. Biofilm densities of 120 to 190 g VS/L film were observed. Growth rates varied from <0.01/d to 0.17/d. Greater than 90% of the biomass concentration in the bed was attached, and effluent total suspended solids ranged from 5 to 74 mg/L, with an average of 24 mg/L over 27 runs in four MAFFB systems at upflow velocities of 11.4 to 25 m/h. Heterotrophic attached-film methanotrophs appear to be stable and useful for applications in toxics treatment, and other product manipulations. (c) 1992 John Wiley & Sons, Inc.     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, 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.     

Current and future trends for biofilm reactors for fermentation processes

Biofilms in the environment can both cause detrimental and beneficial effects. However, their use in bioreactors provides many advantages including lesser tendencies to develop membrane fouling and lower required capital costs, their higher biomass density and operation stability, contribution to resistance of microorganisms, etc. Biofilm formation occurs naturally by the attachment of microbial cells to the support without use of any chemicals agent in biofilm reactors. Biofilm reactors have been studied and commercially used for waste water treatment and bench and pilot-scale production of value-added products in the past decades. It is important to understand the fundamentals of biofilm formation, physical and chemical properties of a biofilm matrix to run the biofilm reactor at optimum conditions. This review includes the principles of biofilm formation; properties of a biofilm matrix and their roles in the biofilm formation; factors that improve the biofilm formation, such as support materials; advantages and disadvantages of biofilm reactors; and industrial applications of biofilm reactors.     

Liquid flow in heterogeneous biofilms

Liquid flow was studied in aerobic biofilms, consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. Fluorescein microinjection was used as a qualitative technique to determine the presence of flow in cell clusters and voids. Flow velocity profiles were determined by tracking fluorescent latex spheres using confocal microscopy. Liquid was flowing through the voids and was stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, whereas in cell clusters diffusion is the dominant factor. The flow velocity in the biofilm depended on the average flow velocity of the bulk liquid. The velocity profiles in biofilms were linear and the velocity was zero at the substratum surface. The velocity gradients within biofilms were 50% of that near walls without biofilm coverage. The influence of the biofilm roughness on the flow velocity profiles was similar to that caused by rigid roughness elements. (c) 1994 John Wiley & Sons, Inc.     

Modeling and simulation of competition between two microorganisms for a single inhibitory substrate in a biofilm reactor

A simple biofilm model was developed to simulate the competition between two microorganisms for a common inhibitory substrate. The following assumptions were made for the simulations: (1) the biofilm has a uniform thickness and is composed of 5 segments, (2) growth of two microorganisms A and B which utilize the common substrate is expressed by the Haldane kinetics with a spatial limitation term and is independent of the other microorganism in the biofilm reactor, and (3) diffusion of the substrate, movement of the microorganisms, and continuous loss of the biomass by shearing are expressed by Fick's Law-type equations. The qualitative behavior of the biofilm reactor is characterized by five regions, I-V, depending on the operation conditions, the substrate concentration in feed, and the dilution rate. In region I, both microorganisms are washed out of the biofilm reactor. In region II, microorganism B is washed out, and in region III, microorganism A is washed out of the biofilm. In region IV, both microorganisms coexist with one another. In region V, both microorganisms coexist with a sustained oscillatory behavior. Convergence to regions I-V depends on the initial conditions. In regions II-V, washout of either or both microorganisms is also observed with initial conditions too far away.     

Patterns of Biofilm Succession on a Sheltered Rocky Shore in Hong Kong

Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment. Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers. In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong. In the succession, bacteria were followed by diatoms and cyanobacteria. Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter). Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period. The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer. Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.     

A novel planar flow cell for studies of biofilm heterogeneity and flow-biofilm interactions

Biofilms are microbial communities growing on surfaces, and are ubiquitous in nature, in bioreactors, and in human infection. Coupling between physical, chemical, and biological processes is known to regulate the development of biofilms; however, current experimental systems do not provide sufficient control of environmental conditions to enable detailed investigations of these complex interactions. We developed a novel planar flow cell that supports biofilm growth under complex two-dimensional fluid flow conditions. This device provides precise control of flow conditions and can be used to create well-defined physical and chemical gradients that significantly affect biofilm heterogeneity. Moreover, the top and bottom of the flow chamber are transparent, so biofilm growth and flow conditions are fully observable using non-invasive confocal microscopy and high-resolution video imaging. To demonstrate the capability of the device, we observed the growth of Pseudomonas aeruginosa biofilms under imposed flow gradients. We found a positive relationship between patterns of fluid velocity and biofilm biomass due to faster microbial growth under conditions of greater local nutrient influx, but this relationship eventually reversed because high hydrodynamic shear leads to the detachment of cells from the surface. These results reveal that flow gradients play a critical role in the development of biofilm communities. By providing new capability for observing biofilm growth, solute and particle transport, and net chemical transformations under user-specified environmental gradients, this new planar flow cell system has broad utility for studies of environmental biotechnology and basic biofilm microbiology, as well as applications in bioreactor design, environmental engineering, biogeochemistry, geomicrobiology, and biomedical research.     

Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications

Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.     

Nitrification using polyvinyl alcohol-immobilized nitrifying biofilm on an O2-enriching membrane

A combination of cell immobilization and membrane aeration approaches was used in a biological reactor to treat NH₄ ⁺ in wastewater. Nitrifying microorganisms, immobilized by polyvinyl-alcohol (PVA) and attached to the surface of a silicone membrane tube, were used to develop a novel reactor for nitrification. The immobilized biofilm had a rubber-like elasticity and resisted shear stress over 5 months of operation. The reactor removed 95% of ammonium, added at 1.97 g N m^–2 d^–1, with O_–2 enriching the membrane.     

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.