Combined application of 13C NMR spectroscopy and confocal laser scanning microscopy—Investigation on biofilm structure and physico-chemical properties

Long-term biofilm processes are influenced by the interplay of biofilm accumulation and detachment, which in turn depend partially on the biofilm structure and composition. In this study a combination of confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) spectroscopy was applied to analyze biofilm structure, composition and molecular mobility. Whereas CLSM delivers information about the structure of biofilms the NMR measurement provides detailed but not locally resolved information about the chemical composition of biofilm constituents. Heterotrophic mixed-species biofilms were cultivated in rotating annular reactors exposed to different flow conditions and glucose concentrations in order to obtain biofilms with diverse architectural structures. The growth state of the biofilms appeared to influence the composition of biofilm and detached biomass. The difference in the ¹³C NMR spectra between the differently structured biofilms or between biofilm and detached biomass was small, except for the still exponential growing biofilm supplied with the highest glucose concentration. More information was gained from the mobility of specific molecular groups within the biofilm biomass. Molecules within the biofilm biomass of the non-filamentous biofilms were more strongly bound than the molecules within the respective detached biomass. Glucose starvation resulted in a reduction in the biofilm molecular mobility. The opposite was observed in the filamentous biofilm. In this case, the molecular mobility in the biofilm increased after starvation and the molecules in the detached biomass were bound more strongly than in the respective biofilm biomass. It could be shown that the combination of CLSM and ¹³C NMR spectroscopy is a promising approach to analyze the interactions between biofilm architecture, composition or growth state and biofilm detachment.     

Sloughing and limited substrate conditions trigger filamentous growth in heterotrophic biofilms—Measurements in flow-through tube reactor

The aim of this study was to investigate the interaction between biofilm structure and sloughing in a flow-through tube reactor exposed to constant, limiting and non-limiting substrate conditions. Biofilm development and detachment were analysed by means of gravimetrical methods and confocal laser scanning microscopy (CLSM). This study revealed the impact of sloughing on biofilm structure. After six weeks of cultivation all biofilms were dominated by filamentous growth. In three out of four cultivations fungal networks developed after the first or second major sloughing event. In one biofilm, experiencing the highest substrate limitations, filamentous bacteria dominated the biofilm community prior to the first sloughing. Despite structural changes the overall biofilm substrate conversion rates remained rather constant. Several factors were identified, which possibly led to the first major sloughing event. For example, all biofilms had a density less than 40 kg m⁻³, a biofilm thickness above 80 μm, an increased surface roughness and presence of protozoa prior to sloughing. The observed fungal development may have several reasons: (1) small colonies dormant in the base biofilm adapted rapidly towards new conditions after sloughing, (2) spores attached after sloughing within the remaining base biofilm and (3) the absence of bacterial reseeding as a result of no recirculation of the bulk-fluid containing planktonic bacteria. Filamentous bacterial growth was due to the combination of limited substrate availability and high flow rates. These results can be significant for industrial systems where biofilm stability and sloughing as well as community composition are critical factors for process stability.     

Analytical effectiveness factor calculations concerning product-inhibited fermentations associated with biofilm growth and maintenance

An analytical reaction engineering model, recently presented by van Ede et al. (1992), which describes formation of inhibiting products associated with the growth of immobilized biofilms, is extended to describe simultaneous production associated with biofilm maintenance. The model accounts for both the metabolic rate controlling behaviour of an inhibitory product in the biofilm, and the effect of diffusion limitation in the transport of this product or a substrate, on the biofilm thickness. Simple criteria are presented to check the applicability of the model in the case of true Monod kinetics. As a potentially industrially important example, ethanol production by Zymomonas mobilis is treated.     

Biofilm Structure and Extracellular Polymeric Substances in Low and High Dissolved Oxygen Membrane Bioreactors

Abstract

This paper discusses the effect of dissolved oxygen (DO) on the biofilm structure in membrane bioreactor (MBR) and their consequence on membrane permeability and EPS. Two MBRs under high DO (6.0 mg/L, HDO) and low DO (<0.1 mg/L, LDO) were operated in parallel under same hydrodynamic conditions. The microbiological aspects in MBR systems were explored through a series of analysis techniques including PCR‐DGGE, gel filtration chromatography (GFC), confocal laser scanning microscope (CLSM), and image analysis.

The rate of membrane fouling for the LDO MBR was 5 times faster than that for the HDO MBR. The microbial communities between HDO and LDO MBR were quite different, which is likely to be the reason for different structures and permeabilities of the biofilms. The specific biofilm resistance in HDO MBR was lower to that in LDO MBR. This is attributed to relatively lower porosity and higher amount of EPS for the biofilm in LDO MBR. The distributions of cell and EPS were not uniform in the biofilms in both HDO and LDO MBR. The biofilm in LDO MBR contained larger amount of EPS than that in HDO MBR. The ratio of protein to polysaccharide was also higher for biofilm in HDO MBR than in LDO MBR.     

Transport of hydrophobic pollutants through biofilms in biofilters

Treatment of air pollutants in a biofilter requires that the compound be effectively transported from the gas phase to the organisms that reside in a biofilm that forms upon a packing material. It has been suggested that biofilms have different transport properties than water making hydrophobic pollutants have higher removal rates than predicted based on water's transport properties. The objectives of this study were to experimentally determine partition and diffusion coefficients of a model hydrophobic compound (α-pinene) through natural and artificial biofilms and to relate these to biofilm characteristics such as solids content and the substrate (VOC) being consumed during biofilm generation. This was done by setting up bench-scale biofilters to generate biofilm to use in partitioning and transport experiments. Batch partitioning experiments were conducted that indicated that α-pinene has a higher degree of partitioning into biofilm than into water due to the presence of solids (two orders of magnitude). A diffusion cell has also been designed and built to study the partitioning from air and diffusion of α-pinene through various artificial biofilms. The average diffusion coefficient of α-pinene through agar, which has the same partitioning properties as water, was found to be (S.D.: ). Diffusion cell experiments performed with α-pinene using inactivated biofilm, previously grown on methanol and α-pinene, immobilized in agar indicate that initially sorption takes place within the film but after this initial lag phase, the transport rate is not significantly different from agar indicating the ratio of the diffusion and partition coefficient of the mobile phase is the same. Therefore, at steady state we expect the transport rates of hydrophobic pollutants through biofilms to be the same as through water.     

Modeling mass transport and microbial activity in stratified biofilms

The most recent mathematical models of microbial activity in heterogeneous biofilms are based on cellular automata. The main weakness of these models is that to obtain numerical solutions the operator must specify the rules governing microbial cell behaviour in the biofilm, and these rules are difficult to establish experimentally. To avoid this difficulty, we have used an alternative approach, discretizing biofilms into layers, to include the effects of biofilm heterogeneity on biofilm activity. This procedure conceptually converts heterogeneous biofilms into a stack of stratified layers of various densities, activities, and diffusivities, and can include some effects of biofilm heterogeneity, e.g vertical distribution of biofilm density, activity, and effective diffusivity. We present this model and selected examples of computational procedures illustrating it. We found that the activity of homogeneous biofilms can be lower, higher, or equal to the activity of stratified biofilms; since homogeneous biofilms do not exist, their properties have to be assumed. As expected, the model predicts that the growth-limiting nutrient penetrates deeper into stratified biofilms than it does into homogeneous biofilms.     

Biofilm growth pattern in honeycomb monolith packings: Effect of shear rate and substrate transport limitations

Potential application of monolith reactors in a biological process was investigated experimentally. A possible problem when using monolith reactors in biological applications is clogging due to biofilm formation. An interesting phenomenon is the pattern in which biofilms develop inside the monolith channels. Rather unexpectedly at a first glance, it was repeatedly observed that biofilm formation started in the middle of a side of the square-section monolith channels, instead of colonizing first the low-shear areas in the corners. To explain this biofilm formation pattern, a two-dimensional mechanistic model based on substrate diffusion and consumption accompanied by microbial growth and detachment was developed in this study. Simulation results suggest that the unexpected biofilm patterns are generated by the balance between biofilm growth and biofilm detachment due to shear stress induced erosion. In the early stages, the biofilm growth in the corners is strongly limited by the external resistance to substrate transfer. As time passes and the biofilm grows in thickness, mechanical forces due to passing gas bubbles will lead to a more regular biofilm shape, including the channel corners.     

A transient mathematical model for maximum respiration activity and oxygen diffusion coefficient estimation in non-steady-state biofilms

A transient mathematical model was established in order to evaluate oxygen diffusivity in non-steady-state biofilms. A submerged fixed bed biofilm system with efficient medium recirculation was investigated for p-toluenesulphonic acid degradation by Comamonas testosteroni T-2 in a multi-species biofilm. Static mixer elements (Sulzer Chemtech Ltd, Switzerland) were used as a support matrix for biofilm formation. Biofilm respiration was estimated using the dynamic gassing-out oxygen uptake method. Based on the dissolved oxygen concentration profiles, the oxygen diffusion coefficient and the maximum respiration activity were calculated. The values of the dissolved oxygen diffusion coefficient varied with biolfilm development and values reported here (2×10⁻¹⁰–1.2×10⁻⁹ m² s⁻¹) are in good agreement with literature data. Calculated oxygen consumption rates fit well with values obtained in respirometry tests with washed out biofilms. The knowledge of diffusivity changes in biofilms is particularly important for removal capacity estimation and appropriate reactor design. © 1998 Society of Chemical Industry     

用于生化膜Monod模型的新Thiele模数

A new Thiele＇s modulus, φF, was developed to provide a gradual transition between zero and the first order of kinetics, and to accurately calculate the mass transfer flux and the effectiveness factor for the Monod biofilm. Values of the effectiveness factor, calculated using the new Thiele＇s modulus, were compared with those obtained from numerical solutions and from other published moduli and empirical formulae. The comparison indi- cated that the new Thiele＇s modulus was the best modulus for the Monod biofilm model. In addition, another Thiele＇s modulus, φG, was developed for a Monod biofilm, covered with an external water layer. The overall effectiveness factor can also be calculated by using both moduli φF and φG. The criteria that were proposed for identifica- tion were based on the values of φF and φG, the limiting processes for biomass growth, and substrate conversion. Developed from φF, a new parameter ψ was related uniquely to such features as the depth and shallowness of the generalized substrate concentration profiles inside a Monod biofilm. Criteria were developed to identify the types of concentration distribution inside a Monod biofilm. These methods were used to estimate the substrate flux and the concentration distribution of the biofilms defined in the first benchmark problem （BM1）, by a task group of the International Water Association on Biofilm Modeling.     

Transport of oxygen, sodium chloride, and sodium nitrate in biofilms

Diffusion coefficients of sodium chloride, sodium nitrate and oxygen were determined for heterotrophic biofilms. The biofilms were cultivated under different hydrodynamic and substrate loading conditions in tubular reactors resulting in biofilm densities between 3 and dry mass. Quantifying solute diffusion in the biofilm for these biofilms allowed to specifically evaluate the influence of biofilm density on diffusion while keeping other factors such as the type of substrate, the inoculum, and the reactor type constant. Two methods were used to measure diffusion coefficients. The two-chamber method was used to quantify the diffusion of sodium chloride and sodium nitrate. The diffusion coefficients for oxygen were measured based on oxygen concentration profiles in the biofilm measured using microelectrodes. The ratio between the diffusion coefficient in biofilm and water (f_D=D_F/D_W) was found to be lower than 1 in the majority of experiments. A clear correlation between f_D and biofilm density was found where f_D decreased with increasing biofilm density. For mean biofilm densities in the range of 10- can be approximated between 0.5 and 1. For larger densities of 20 or can be approximated as 0.8 or 0.4, respectively. For densities higher than is below 0.6. Advective transport mechanisms that would have resulted in f_D>1 can be neglected in the biofilms cultivated.     

Modelling and simulation of anaerobic stratified biofilm for methane production and prediction of multiple steady states

The present work deals with modelling of anaerobic metabolism in a stratified biofilm consisting of two distinct layers of microorganisms in a fixed film reactor. The model is applied to steady state conditions and is based on substrate utilization kinetics and mass transport. The model assumes that the substrate is first metabolized by the outer acidogenic layer and the subsequent products are metabolized by the inner layer of methanogens to produce finally methane and carbon dioxide. The model considers slab geometry of the biofilm, Monod kinetics for growth rate of acidogens and the inhibition effect of volatile fatty acids on growth rate of methanogens. The diffusion equations are solved simultaneously using a modified form of the technique of orthogonal collocation on finite elements to obtain concentration profiles and the rate of methane production per unit volume of the biofilm is determined. Computer simulations are carried out to study the effect of various parameters on the rate of methane production and to investigate the existence of multiple steady states. The simulation shows that for a range of parameters values three steady states exist. It is shown that the upper steady state with highest rate of methane production may lead to unsatisfactory reactor performance.     

Evaluation of anti-microfouling activity of marine paints by microscopical techniques

The use of two microscopical techniques, SEM and CLSM, was investigated to facilitate the observation of adhered microfouling on antifouling paints. Bacterial and diatom colonisation was quantified on antifouling paints and compared with the standard test, ASTM D 3623. The results indicate that there is a likely correlation between the microscopic study and the antifouling activity. The study revealed the interest of CLSM for the evaluation of antifouling paints and reports on the efficacy of CLSM to study the initial microfouling layer. This method can yield important data relating to biofilm morphology, particularly film thickness and biomass measurements. This non-invasive technique enabled us to obtain qualitative and quantitative data about the biofilm formation during the first weeks of immersion in seawater.     

固碳产甲烷微生物阴极能质传输特性数值模拟

固碳产甲烷微生物电合成系统以附着其电极表面的生物膜为催化剂，可以在处理废水的同时将CO₂转化为甲烷，极具应用前景。微生物阴极是该系统的核心部件之一，其表面生物膜内的能质传输特性极大地影响系统性能。针对微生物阴极能质传输特性尚不明确的问题，推导了微生物阴极电极反应动力学方程（Nernst-Monod方程），构建了耦合生化/电化学反应的物质传输理论模型，研究了不同阴极电势、生物膜电导率以及孔隙率对阴极生物膜内电荷及物质传输的影响规律。研究结果表明当阴极电势高于-0.5 V (vs SHE)时，随阴极电势的降低生物膜内电流密度增大，底物浓度降低；但当阴极电势降低至-0.5 V(vs SHE)后，生物膜消耗电子还原底物的能力几乎达到饱和；低电导率（<10^-3 S/m）会导致生物膜内形成明显的电势差，使得底物利用速率降低，严重影响微生物阴极的性能；生物膜孔隙率控制在0.4时，微生物阴极可达到最佳电流密度。     

Quantifying selected growth parameters of Leptothrix discophora SP-6 in biofilms from oxygen concentration profiles

It is a dubious but common practice to use growth parameters measured in suspended cultures to predict substrate concentration profiles in biofilms. To obtain biofilm biokinetic parameters that apply to biofilms, a reliable method is needed that allows the computation of biokinetic parameters from substrate concentration profiles measured directly in biofilms. We have developed such a method and demonstrated its utility by evaluating biokinetic parameters from oxygen concentration profiles measured in biofilms of Leptothrix discophora SP-6 grown on a membrane, which was placed on top of an agar plate by fitting the data to Monod or Tessier growth kinetics, including maintenance substrate consumptions. We found that the Monod model represented the growth of L. discophora SP-6 biofilms marginally better than the Tessier model. The Monod half saturation coefficient was .     

Mathematical modeling of granular activated carbon (GAC) biofiltration system

In this study, a mathematical model of a fixed bed Granular Activated Carbon (GAC) biofiltration system was developed to predict the organic removal efficiency of the filter. The model consists of bulk transportation, adsorption, utilization, and biodegradation of organics. The variation of the specific surface area due to biofilm growth and the effect of filter backwash were also included in the model. The intrapellet diffusion and the diffusion of substrate in the biofilm were described by linear driving force approximation (LDFA) method. Biodegradation of organics was described by Monod kinetics. Sips adsorption isotherm was used to analyze the initial adsorption equilibrium of the system. The model showed that the organic removal efficiency of the biofilter greatly depends on the parameters related to the biological activities such as the maximum rate of substrate utilization (k_max) and biomass yield (Y) coefficients. Parameters such as suspended cell concentration (X_s) and decay constant (K_d) had little effects on the model simulation results. The filter backwash also had no significant impact on the performance of the biofilter.     

Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa

Biofilms are communities of microorganisms embedded in extracellular polymeric substances (EPS) matrix. Bacteria in biofilms demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to immune system and antibiotics that render biofilm a source of chronic and persistent infections. A deeper understanding of biofilms will ultimately provide insights into the development of alternative treatment for biofilm infections. The opportunistic pathogen Pseudomonas aeruginosa, a model bacterium for biofilm research, is notorious for its ability to cause chronic infections by its high level of drug resistance involving the formation of biofilms. In this review, we summarize recent advances in biofilm formation, focusing on the biofilm matrix and its regulation in P. aeruginosa, aiming to provide resources for the understanding and control of bacterial biofilms.     

A Combination of the Natural Molecules Gallic Acid and Carvacrol Eradicates P. aeruginosa and S. aureus Mature Biofilms

Wound infection, especially the development of bacterial biofilms, delays wound healing and is a major public health concern. Bacteria in biofilms are more tolerant to antimicrobial agents, and new treatments to eradicate mature biofilms are needed. Combining antimicrobial molecules with different mechanisms of action is an attractive strategy to tackle the heterogeneous nature of microbial communities in biofilms. This study focused on three molecules of natural origin: gallic acid (G), carvacrol (K) and curcumin (Q). Their abilities, individually or in combination, to eradicate biofilms were quantified on mono- and dual-species mature biofilms of Pseudomonas aeruginosa and Staphylococcus aureus, the strains most commonly found in infected wounds. G presented biofilm eradicating activity on P. aeruginosa, whereas K had biofilm eradicating activity on S. aureus and P. aeruginosa. Q had no potent biofilm eradicating activity. The combination of G and K increased the effects previously observed on P. aeruginosa biofilm and led to complete eradication of S. aureus biofilm. This combination was also efficient in eradicating a dual-species biofilm of S. aureus and P. aeruginosa. This work demonstrates that K and G used in combination have a strong and synergistic eradicating activity on both mono- and dual-species mature biofilms of S. aureus and P. aeruginosa and may therefore represent an efficient alternative for the treatment of biofilms in wounds.     

Modelling and analysis of membrane‐attached biofilms

The theory of diffusion and reaction has been applied to describe mass transfer and reaction phenomena in membrane‐attached biofilms (MABs) growing in extractive membrane bioreactors (EMBs), and to establish the rate‐limiting mechanisms in these systems. The model formulated accounts for substrate counter‐diffusion and two‐limiting‐substrate reaction within the biofilm. Model simulations are compared to experimental data obtained in a lab‐scale EMB and a simple case study is considered to show how MABs affect performance of EMBs. It is found that the organic substrate flux across the membrane is strongly affected by MABs, which constitute an additional resistance to mass transfer and in most cases reduce the flux across the membrane. As a result of the investigation, it is concluded that the decrease of flux in the presence of MABs, observed experimentally and predicted theoretically, is dominated by the resistance to organic substrate transfer caused by the biofilm.     

In vitro Streptococcus mutans biofilm formation on surfaces of chlorhexidine-containing dentin bonding systems

This in vitro study evaluated the influence of chlorhexidine diacetate (CDA) when blended within dentin bonding systems (DBSs) on Streptococcus mutans (S. mutans) biofilm formation.One commercially available 0.2% wt CDA-containing DBS (Peak Universal Bond) and five experimental 0.2% wt CDA-containing DBS formulations (experimental Adper Scotchbond 1XT plus experimental resins, R₂, R₃, R₄, R₅) were assessed vs their no-CDA containing counterparts. Twenty-eight DBSs disks were prepared for each group (6.4 mm×1.0 mm) and cured for 80 s at 800 mW/cm² in a nitrogen atmosphere. A modified Drip-Flow Reactor was used to grow S. mutans biofilms on specimen surfaces for 24 h and adherent, viable biomass was evaluated using a tetrazolium salt assay (MTT). Two specimens from each of the tested materials were processed with LIVE/DEAD stain and observed using laser confocal microscopy (CLSM) while two disks from each group were examined by using scanning electron microscopy (SEM).MTT assay, CLSM and SEM observations showed that CDA addition decreased, increased or did not change S. mutans biofilm formation. The lowest biofilm formation was obtained with Peak Universal Bond and R₅ (with and without CDA).It may be concluded that the chemical composition of DBSs determines their ability to promote or hamper biofilm formation. Therefore, CDA addition may be helpful in modulating biofilm formation provided that DBS formulation is tuned and optimized.     

Microbial composition and structure of a multispecies biofilm from a trickle‐bed reactor used for the removal of volatile aromatic hydrocarbons from a waste gas

The microbial composition and structure of a multispecies biofilm of a laboratory‐scale trickle‐bed bioreactor for the treatment of waste gas was examined. The model pollutant was a volatile organic compound‐mixture of polyalkylated benzenes called Solvesso 100^®. Fluorescent in‐situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) were applied. Two new Solvesso 100^®‐degrading Pseudomonas sp strains were isolated from the multispecies biofilm. Corresponding isolate‐specific oligonucleotide probes were designed and applied successfully. A major finding was that the fraction of Solvesso 100^®‐degrading bacteria in the biofilm was low (about 3–6% during long‐term operation). The majority of the active cells were saprophytes which utilized intermediates and cell lysis products. The measured fraction of extracellular polymeric substances of the mature biofilm was 89–93% of the total biomass. The CLSM examinations of a 3‐days‐old approx 10 µm thick biofilm revealed highly heterogeneous structures with distinguished three‐dimensional matrix‐enclosed microcolony bodies spread across the substratum surface. The 28‐days‐old 80–960 µm thick biofilm exhibited voids, cell‐free channels, and pores of variable sizes. In both cases, an even distribution of active cells and pollutant‐degrading bacteria throughout the biofilm cross‐section as well as through the biofilm depth was observed. Copyright © 2003 Society of Chemical Industry