Evaluating trends in biofilm density using the UMCCA model

We present a series of modeling cases that illustrate the trends described by the unique features of the unified multiple-component cellular automaton (UMCCA) model for a heterogeneous, two-dimensional biofilm. The outputs of the UMCCA model show five general trends. (1) The concentration profiles for the two soluble microbial products are opposite the profile for original substrate. (2) The top of the biofilm is dominated by active biomass and EPS, while the bottom is dominated by residual inert biomass. Within the top layers, active biomass has a much higher concentration than EPS. (3) The top of all biofilm is quite "fluffy," while the bottom is dense. (4) The peak of the composite density does not correspond to the peak of active biomass. (5) All biomass types show considerable local heterogeneity. The series of cases also indicate what conditions lead to particular characteristics observed in some biofilms. Biofilm clusters are promoted by substrate limitation, a high detachment rate, or strong consolidation. A high biofilm density is associated with an old biofilm, which is favored by a low substrate concentration, a high detachment rate, and strong consolidation. Old biofilms also can develop low-density pockets near the substratum, a possible cause of sloughing. Local heterogeneity is generally related to the same factors that cause a high density. We also solved the UMCCA model for conditions similar to the experiments of Bishop et al. (Water Sci. Technol. 31(1) (1995) 143), who measured the total biomass density in layers from the substratum. The model outputs captured all the major trends in the experimental data: the overall thickness and density of biofilms increase with time, and the total biomass density is 5-10 times greater near the substratum than near the top of the biofilm. Furthermore, the model indicates that the residual inert biomass becomes denser toward the substratum, a trend observed experimentally; the UMCCA model suggests that this trend is due to the combined effects of consolidation and inert biomass having a larger maximum density.     

Evaluating operating conditions for outcompeting nitrite oxidizers and maintaining partial nitrification in biofilm systems using biofilm modeling and Monte Carlo filtering

In practice, partial nitrification to nitrite in biofilms has been achieved with a range of different operating conditions, but mechanisms resulting in reliable partial nitrification in biofilms are not well understood. In this study, mathematical biofilm modeling combined with Monte Carlo filtering was used to evaluate operating conditions that (1) lead to outcompetition of nitrite oxidizers from the biofilm, and (2) allow to maintain partial nitrification during long-term operation. Competition for oxygen was found to be the main mechanism for displacing nitrite oxidizers from the biofilm, and preventing re-growth of nitrite oxidizers in the long-term. To maintain partial nitrification in the model, a larger oxygen affinity (i.e., smaller half saturation constant) for ammonium oxidizers compared to nitrite oxidizers was required, while the difference in maximum growth rate was not important for competition under steady state conditions. Thus, mechanisms for washout of nitrite oxidizing bacteria from biofilms are different from suspended cultures where the difference in maximum growth rate is a key mechanism. Inhibition of nitrite oxidizers by free ammonia was not required to outcompete nitrite oxidizers from the biofilm, and to maintain partial nitrification to nitrite. But inhibition by free ammonia resulted in faster washout of nitrite oxidizers.     

Evaluation on the microbial interactions of anaerobic ammonium oxidizers and heterotrophs in Anammox biofilm

Anaerobic ammonium oxidation (Anammox) is a cost-effective new process to treat high-strength nitrogenous wastewater. In this work, the microbial interactions of anaerobic ammonium oxidizers and heterotrophs through the exchange of soluble microbial products (SMP) in Anammox biofilm and the affecting factors were evaluated with both experimental and modeling approaches. Fluorescent in situ hybridization (FISH) analysis illustrated that Anammox bacteria and heterotrophs accounted for 77% and 23% of the total bacteria, respectively, even without addition of an external carbon source. Experimental results showed the heterotrophs could grow both on SMP and decay released substrate from the metabolism of the Anammox bacteria. However, heterotrophic growth in Anammox biofilm (23%) was significantly lower than that of nitrifying biofilm (30-50%). The model predictions matched well with the experimental observations of the bacterial distribution, as well as the nitrogenous transformations in batch and continuous experiments. The modeling results showed that low nitrogen surface loading resulted in a lower availability of SMP leading to low heterotrophic growth in Anammox biofilm, but high nitrogen surface loading would lead to relative stable biomass fractions although the absolute heterotrophic growth increased. Meanwhile, increasing biofilm thickness increased heterotrophic growth but has little influence on the relative biomass fractions.     

Treatment of a colored groundwater by ozone-biofiltration: pilot studies and modeling interpretation

Pilot studies investigated the fates of color, dissolved organic carbon (DOC), and biodegradable organic matter (BOM) by the tandem of ozone plus biofiltration for treating a source water having significant color (50 cu) and DOC (3.2 mg/l). Transferred ozone doses were from 1.0 to 1.8 g O3/g C. Rapid biofilters used sand, anthracite, or granular activated carbon as media with empty-bed contact time (EBCT) up to 9 min. The pilot studies demonstrated that ozonation plus biofiltration removed most color and substantial DOC, and increasing the transferred ozone dose enhanced the removals. For the highest ozone dose, removals were as high as 90% for color and 38% for DOC. While most of the color removal took place during ozonation, most DOC removal occurred in the biofilters, particularly when the ozone dose was high. Compared to sand and anthracite biofilters, the GAC biofilter gave the best performance for color and DOC removal, but some of this enhanced performance was caused by adsorption, since the GAC was virgin at the beginning of the pilot studies. Backwashing events had no noticeable impact of the performance of the biofilters. The Transient-State, Multiple-Species Biofilm Model (TSMSBM) was used to interpret the experimental results. Model simulations show that soluble microbial products, which comprised a significant part of the effluent BOM, offset the removal of original BOM, a factor that kept the removal of DOC relatively constant over the range of EBCTs of 3.5-9 min. Although improved biofilm retention, represented by a small detachment rate, allowed more total biofilm accumulation and greater removal of original BOM, it also caused more release of soluble microbial products and the build up of inert biomass in the biofilm. Backwashing had little impact on biofilter performance, because it did not remove more than 25% of the biofilm under any condition simulated.     

Influence of growth history on sloughing and erosion from biofilms

The development of biofilms is determined by the balance of growth and detachment. But while the growth of biofilms is well studied, the influence of growth history and detachment on biofilm development is not. Here we report on laboratory scale experiments where heterotrophic biofilms were grown in a tubular reactor. Biofilm detachment was categorized based on particle size as erosion or sloughing. Erosion results in small particles and was approximated by the effluent suspended solids while sloughing was determined from the larger pieces of biomass that settled in a mixing tank. It was found that for all experiments, overall detachment was a combination of erosion and sloughing where erosion had a slightly larger contribution to the overall solids removal. However, sloughing had a significant influence on the biofilm morphology. Once the smooth biofilm surface was disturbed by a sloughing event (e.g., initiated through increasing liquid shear in the reactor), the biofilm became unstable resulting in spontaneous sloughing during subsequent operation. We propose that experimental investigations should consider sloughing events as an integral part of biofilm development rather than a disturbance.     

Effects of substrate loading rate on biofilm structure

The effects of substrate surface loading rate on biofilm growth and structure were investigated by chemical, biochemical and microscopic methods. Three tubular reactors were operated at equal C:N ratio of 0.1, with substrate loading rates of 1.2, 0.6 and 0.3g-C/m(2)/day. Substrate loading positively influenced the biofilm growth rate. Denser biofilms with lower porosities were formed at higher substrate loading. Slowly growing biofilms having porous structures were found to have higher specific activities. Nitrification was suppressed under the higher substrate loading conditions even at the equal C:N ratio of 0.1, thus proving that the spatial competition between nitrifiers and heterotrophs as one limiting criteria for stable nitrification. The spatial organization of the ammonia oxidizers was biofilm structure related. The strain variability of ammonia oxidizers was substrate loading dependent. These findings suggest that substrate loading is a key parameter in determining biofilm structure and function.     

Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations

Biofilm modeling is often considered as a complex mathematical subject. This paper evaluates simple equations to describe the basic processes in a biofilm system with the main aim to show several interesting applications. To avoid mathematical complexity the simulations are carried out in a simple spreadsheet. Frequently, only the solution for zero-order reaction kinetics of the reaction-diffusion equation is used (better known as half-order kinetics). A weighted average of the analytical solutions for zero- and first-order reactions is proposed as basic and useful model to describe steady-state (in biofilm composition) biofilm reactors. This approach is compared with several modeling approaches, such as the simple solution for zero-order reaction and more complex ones (i) direct numerical solution for the diffusion equations, (ii) 1-D AQUASIM and (iii) 2-D modeling. The systems evaluated are single and multiple species biofilms. It is shown that for describing conversions in biofilm reactors, the zero-order solution is generally sufficient; however, for design purposes large deviations of the correct solution can occur. Additionally, the role of diffusion in flocculated and granular sludge systems is discussed. The relation between the measured (apparent) substrate affinity constant and diffusion processes is outlined.     

Visualisation of transient processes in biofilms by optical coherence tomography

Biofilms occur in natural and engineered water systems. In technical processes, biofouling lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for the removal of organic and inorganic pollutants. For improvement of antifouling strategies and for process optimisation in wastewater treatment plants, analytical techniques for online monitoring of biofilms are needed. Optical coherence tomography (OCT) is a non-invasive optical tomography technique, which is increasingly applied in medical diagnostics. It reveals photon-reflecting structures in tissue with lateral and axial resolution in the range of 10 microm. In this paper, we demonstrate the capabilities of OCT for the monitoring of biofilm structures and their detachment. OCT is able to reveal spatially resolved structural information on biofilm without staining. A main focus of this work is set on the ability of OCT to monitor transient processes with temporal resolution in a second to minute scale. These key features of OCT allow online monitoring of biofilm growth and detachment in a flow channel. Three-dimensional (3D) imaging quality, spatial resolution, and temporally resolved profiling of biofilms are demonstrated. The results give rise to the hope that OCT may evolve to a standard tool for monitoring of biofilm density.     

Effect of shear stress and growth conditions on detachment and physical properties of biofilms

Detachment is one of the major processes determining the physical structure and microbial functionalities of biofilms. To predict detachment, it is necessary to take the mechanical properties of the biofilm and the effect of both hydrodynamic and growth conditions into account. In this work, experiments were conducted with biofilms developed under various shear stresses and with various substrate natures. In addition, two cases were considered in order to differentiate between the effect of hydrodynamic factors and growth factors: the biofilms were directly grown under the targeted shear stress (τ) condition or they were precultivated under very low shear stress (0.01 Pa) and then exposed to high shear stress in the range of 0.1-13 Pa. An exponential and asymptotic decrease of the biofilm thickness and mass with increasing τ was observed in both cases. On contrary density, expressed as the biofilm dry mass on a known substratum divided by the average thickness increased with τ. Denitrifying biofilms always showed greater thickness and density than oxic biofilms. These results showed the presence of a compact basal layer that resisted shear stresses as high as 13 Pa whatever the culture conditions. Above this basal layer, the cohesion was lower and depended on the shear stress applied during biofilm development. The application of shear stress to the biofilms resulted in both detachment and compression, but detachment prevailed for the upper part of the biofilms and compression prevailed for the basal layers. A model of biofilm structure underlying the stratified character of this aggregate is given in terms of density and cohesion.     

Non-steady state modeling of extracellular polymeric substances,soluble microbial products,and active and inert biomass

We present a modeling approach that quantifies the unified theory presented in the companion paper. In this approach, we use mathematical modeling to quantify the relationships among three solid species--bacteria, extracellular polymeric substances (EPS), and inert residual biomass-two soluble microbial products (SMP), original substrate, and an electron acceptor. According to the model, donor electrons are used for the synthesis of biomass, EPS, and utilization-associated products. Residual inert biomass and biomass-associated products are produced from the decay of active biomass and the hydrolysis of EPS, respectively. The model includes mass balance equations that consistently describe the flow of electrons among the components. It is solved with a set of parameters appropriate to the experimental study of Hsieh et al. (Biotech. Bioeng. 44 (1994) 219). Model outputs capture all trends observed in steady-state CSTR experiments and transient batch experiments. This agreement supports that the unified theory correctly captures the interconnections among SMP, EPS, and active and inert biomass.     

限制基质条件下生物膜特性的研究

在氮作为限制基质的情况下,采用库爱特-泰勒反应器分别培养异养菌生物膜和异养/自养硝化菌混合生物膜。当生物膜系统达到稳定后,通过提高水力剪切力使生物膜发生脱落,以研究生物膜内层基本特性及活性变化。结果表明,随着水力强度的增加则生物膜发生逐步脱落,脱落前后两种生物膜的特性及微生物活性均发生了较大变化。在两种生物膜内,靠近载膜片的生物膜比靠近液相的生物膜具有更强的粘结力,能抵抗高达10 Pa的水力剪切力,且生物活性较高。对于自养硝化菌生物膜,在生物膜发生脱落后,残余生物膜对氨氮的表面去除速率几乎保持不变,甚至对氨氮的比去除速率还略有增加,说明自养硝化菌可能主要分布在生物膜的内层。     

Phosphorus uptake into algal biofilms in a lowland chalk river

This paper examines the growth and uptake of phosphorus into algal biofilms in the River Kennet, a lowland chalk (Cretaceous-age) stream in southern England. Algal biofilms were grown on artificial plastic substrates (templates) placed (i) on the riverbed and (ii) within the mid-water column. Experiments were set up to examine differences in growth rates of newly colonising biofilms compared with biofilms left to accumulate for periods of up to 6 months. Rates of algal biofilm production were measured by the chlorophyll a concentration that had accumulated per cm² over the number of days that the biofilm template had been immersed in the river water. An algal biofilm bloom occurred in early spring, prior to peak suspended chlorophyll a concentrations within the water column. Biofilm samples collected in February and March had the highest chlorophyll a and total phosphorus concentrations. The biofilm bloom corresponded with increased solar radiation and declining river flow conditions. Periodic increases in soluble reactive phosphorus concentrations in the overlying river water did not correspond with any significant increase in biofilm production. These results suggests that light, rather than phosphorus is a key factor for biofilm growth in the River Kennet. Higher rates of chlorophyll a development in mid-water column biofilms may be linked to greater light exposure; however, maximum total-P concentrations were similar for both bed and water column biofilms. Newly colonising biofilms exhibited higher chlorophyll a and total-P concentrations than biofilms left to accumulate over longer terms, suggesting that fresh substrate availability promotes high rates of biofilm growth. Both ‘condensed and organic’ P (stored in biomass) and ‘inorganic’ (mineral) P fractions within the biofilms were present in varying proportions, although the early spring biofilm bloom resulted in maximum proportions and absolute concentrations of ‘condensed and organic’ P. Calcite was the only crystalline mineral detected within the biofilms. Ratios of Ca:inorganic P are largely consistent with the presence of CaCO₃–P co-precipitates, although one very low value suggested that there may also be additional sources of inorganic P, possibly P adsorbed to clays or organics within the biofilm. However, poor linkages between CaCO₃ and inorganic P concentrations suggest that, although the inorganic P fraction within the biofilm may be derived largely from CaCO₃–P co-precipitation, the subsequent processes controlling overall CaCO₃ and inorganic P concentrations in the biofilm are complex.     

Cohesiveness and hydrodynamic properties of young drinking water biofilms

Drinking water biofilms are complex microbial systems mainly composed of clusters of different size and age. Atomic force microscopy (AFM) measurements were performed on 4, 8 and 12 weeks old biofilms in order to quantify the mechanical detachment shear stress of the clusters, to estimate the biofilm entanglement rate ξ. This AFM approach showed that the removal of the clusters occurred generally for mechanical shear stress of about 100 kPa only for clusters volumes greater than 200 μm³. This value appears 1000 times higher than hydrodynamic shear stress technically available meaning that the cleaning of pipe surfaces by water flushing remains always incomplete. To predict hydrodynamic detachment of biofilm clusters, a theoretical model has been developed regarding the averaging of elastic and viscous stresses in the cluster and by including the entanglement rate ξ. The results highlighted a slight increase of the detachment shear stress with age and also the dependence between the posting of clusters and their volume. Indeed, the experimental values of ξ allow predicting biofilm hydrodynamic detachment with same order of magnitude than was what reported in the literature. The apparent discrepancy between the mechanical and the hydrodynamic detachment is mainly due to the fact that AFM mechanical experiments are related to the clusters local properties whereas hydrodynamic measurements reflected the global properties of the whole biofilm.     

Estimating biomass yield coefficients for autotrophic ammonia and nitrite oxidation from batch respirograms

Kinetic characterization of biological processes via batch respirometry requires an accurate estimate of the biomass yield coefficient because it provides the stoichiometric link between biomass synthesis, substrate consumption and oxygen uptake. Expressions for biomass yield coefficients describing autotrophic ammonia and nitrite oxidation were derived from a mechanistically based electron balanced equation. We demonstrate that applying the conventional expression used to calculate the heterotrophic biomass yield results in erroneous estimates for the autotrophic biomass yield. Yield coefficients for autotrophic NH4(+)-N to NO2(-)-N oxidation and NH4(+)-N to NO3(-)-N oxidation were overestimated by 27 to 36%. Due to correlation between the maximum specific growth rate and the biomass yield, the error in yield values propagated in 30 to 40% overestimates of the maximum specific growth rate coefficient for NH4(+)-N oxidation determined from batch respirograms. Therefore, it is essential to employ the correct expression to estimate the autotrophic biomass yield coefficient from batch respirograms due its inadvertent impact on subsequent parameter estimation.     

Demonstration of mass transfer and pH effects in a nitrifying biofilm

A bench-scale nitrifying trickling filter (surface AREA = 0.5 m²) was developed to permit evaluation of diffusion of oxygen within a biofilm, the pH dependence of ammonium oxidation and external mass transfer. In addition, a biofilm model was developed and verified for homogeneous nitrifying biofilms of varied thickness and for thin nitrifying biofilms covered by heterotrophic biofilms. The model uses literature values for the pH dependence of Monod coefficients for Nitrosomonas and Nitrobacter.

The diffusion coefficient of oxygen in the biofilm was found to be 40–80% of the value in pure water. Due to mass transfer resistance, the biomass ·sees” a lower pH than is measured in the water film passing over it. The surface uptake rate of ammonia is used as an indicator of pH gradients within the biofilm system. With the help of oxygen limitation experiments, the location of nitrifying biomass within mixed biofilms (heterotrophic, autotrophic) can be determined.

The biofilm model predicts ammonium uptake rate of a trickling filter as a function of the bicarbonate concentration in the water film.     

Modeling the development of biofilm density including active bacteria, inert biomass, and extracellular polymeric substances

We present the unified multi-component cellular automaton (UMCCA) model, which predicts quantitatively the development of the biofilm's composite density for three biofilm components: active bacteria, inert or dead biomass, and extracellular polymeric substances. The model also describes the concentrations of three soluble organic components (soluble substrate and two types of soluble microbial products) and oxygen. The UMCCA model is a hybrid discrete-differential mathematical model and introduces the novel feature of biofilm consolidation. Our hypothesis is that the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time. Each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass. The UMCCA model also adds a cellular automaton algorithm that identifies the path of least resistance and directly moves excess biomass along that path, thereby ensuring that the excess biomass is distributed efficiently. A companion paper illustrates the trends that the UMCCA model is able to represent and shows a comparison with experimental results.     

Acetate removal in sewer biofilms under aerobic conditions

Removal of acetate has been investigated in sewer biofilms by continuous-flow biofilm reactor studies simulating the conditions in a gravity sewer. Non-steady-state conditions are prevailing in sewers, due to periodic variations in substrate concentrations. In order to simulate two extreme situations in a gravity sewer, biofilms defined as high-loaded and low-loaded, respectively, were grown by continuously feeding wastewater to the reactors with and without supplementary addition of acetate. During short-term experiments with high acetate concentrations (1-2 h), surface removal rates of acetate and dissolved oxygen (DO) and observed yield coefficients were determined, as well as the influence of DO concentration on acetate removal rates. The low-loaded biofilms showed very high acetate removal rates in short-term experiments at high acetate concentrations. The DO uptake rates were low, resulting in an average observed yield coefficient of 0.79 g biomass produced per gram acetate (as chemical oxygen demand, COD) consumed. This indicated a luxury uptake by the cells probably for storage inside the cells or for production of extracellular polymeric substances. The high-loaded biofilms showed lower acetate removal rates during the short-term experiments, with an average yield coefficient of 0.49 g biomass produced per gram acetate (as COD) consumed. The level of the acetate removal rates seemed to be related to the structure of the biofilm. The highest acetate removal rates were found for the low-loaded biofilm, where the biofilm was very hairy with 'streamers" with a length of 8-9 mm. At low acetate removal rates (high-loaded biofilm), the "streamer" lengths were only 3-5 mm. The surface removal rates for acetate and DO seemed to follow 1/2 order approximations to biofilm kinetics. For a DO of 0.8 and 6.0 g/m3, the limiting acetate concentrations were about 34 and 20 g-COD/m3, respectively. Under real gravity sewer conditions, the typical concentration ranges for acetate and DO are at levels where any of them may be rate-limiting for microbial acetate removal.     

Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes

The action of the cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated to control biofilms (aged 7d) formed by Pseudomonas fluorescens on stainless-steel slides, using flow cells reactors, under turbulent and laminar flow. The effect of CTAB was also investigated using planktonic cells in the presence and absence of BSA, by measuring the cellular respiratory activity and the ATP released. The action of CTAB on biofilms was assessed by means of cellular respiratory activity and variation of biofilm mass, immediately and 3, 7 and 12h after the application of CTAB. The physical stability of the biofilm was also assessed using a rotating device, where the effect of the surfactant on the biofilm stability was evaluated through the variation of the mass remaining on the surface. CTAB significantly reduced the activity of the planktonic cells probably due to the rupture of the cells. This effect was significantly reduced in the presence of BSA. Planktonic cells were more easily inactivated than bacteria in biofilms. Biofilms formed under laminar flow were more susceptible than those formed under turbulent flow, but in both cases total inactivation was not achieved. Biofilm recovery was observed, in terms of respiratory activity, in almost all the cases studied. CTAB application by itself did not promote the detachment of biofilms. The physical stability tests showed that the synergistic action of the surfactant and the application of high shear stress to the biofilm increase its detachment.     

Species association increases biofilm resistance to chemical and mechanical treatments

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re_A). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re_A were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each other's survival to antimicrobials and the biofilm shear stress stability.