A Modeling and Simulation Study of the Role of Suspended Microbial Populations in Nitrification in a Biofilm Reactor

Many biological wastewater treatment processes are based on bacterial biofilms, i.e. layered aggregates of microbial populations deposited on surfaces. Detachment and (re-)attachment leads to an exchange of biomass between the biofilm and the surrounding aqueous phase. Traditionally, mathematical models of biofilm processes do not take the contribution of the suspended, non-attached bacteria into account, implicitly assuming that these are negligible due to the relatively small amount of suspended biomass compared to biofilm biomass. In this paper, we present a model for a nitrifying biofilm reactor that explicitly includes both types of biomass. The model is derived by coupling a reactor mass balance for suspended populations and substrates with a full one-dimensional Wanner–Gujer type biofilm model. The complexity of this model, both with respect to mathematical structure and number of parameters, prevents a rigorous analysis of its dynamics, wherefore we study the model numerically. Our investigations show that suspended biomass needs to be considered explicitly in the model if the interests of the study are the details of the nitrification process and its intermediate steps and compounds. However, suspended biomass may be neglected if the primary interests are the overall reactor performance criteria, such as removal rates. Furthermore, it can be expected that changes in the biofilm area, attachment, detachment, and dilution rates are more likely to affect the variables primarily associated with the second step of nitrification, while the variables associated with the first step tend to be more robust.     

Growth characteristics and expression of iron-regulated outer-membrane proteins of chemostat-grown biofilm cells of Pseudomonas aeruginosa.

An in vitro chemostat system was used to study the growth and the expression of iron-regulated outer-membrane proteins (IROMPs) by biofilm cells of Pseudomonas aeruginosa cultivated under conditions of iron limitation. The population of the planktonic cells decreased when the dilution rate was increased. At a dilution rate of 0.05 h-1, the populations of planktonic cells of both mucoid and nonmucoid P. aeruginosa were 3 x 10(9) cells/mL. This value dropped to 5 x 10(6) cells/mL when the dilution rate was increased to 1.0 h-1. The reverse was observed for the biofilm cells. The number of biofilm cells colonising the silicone tubing increased when the dilution rate was increased. The number of biofilm cells of the mucoid strain at steady state was 2 x 10(8) cells/cm (length) when the dilution rate was fixed at 0.05 h-1. The figure increased to 8 x 10(9) cells/cm when the dilution rate was increased to 1.0 h-1. The population of biofilm cells of the nonmucoid strain was 9 x 10(7) cells/cm (length) when the dilution rate was 0.05 h-1. It increased to 2 x 10(9) cells/cm when the dilution rate was set at 1.0 h-1. The expression of IROMPs was induced in the biofilm cells of both mucoid and nonmucoid strains when the dilution rates were 0.05 and 0.2 h-1. IROMPs were reduced but still detectable at the dilution rate of 0.5 h-1. However, the expression of IROMPs was repressed when the dilution rate was increased to 1.0 h-1. The data suggest that the biofilm cells of P. aeruginosa switch on the expression of IROMPs to assist iron acquisition when the dilution rate used for the chemostat run is below 0.5 h-1. The high affinity iron uptake system is not required by the biofilm cells when the dilution rate is increased because the trace amount of iron present in the chemostat is sufficient for the growth of adherent biofilm cells.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

Performance of three phase fluidized bed reactor for quinoline degradation on various supports at steady state and dynamic conditions

Quinoline degradation by Comamonas acidovorans was investigated in a three phase fluidized bed reactor at dilution rates below and above the critical value (mu(max) = 0.42 h(-1)). Quinoline was used as the sole source of carbon, nitrogen, and energy. Two attachment carriers, polyurethane foam (Bayvitec(R)) and modified cellulose (Aquacel(R)), and a gel entrapment carrier (polyvinyl alcohol) were studied and compared with regard to their effectiveness to immobilize cells. Attachment and biofilm formation was best at higher dilution rates, regardless of carrier type used. Except for the maximum biomass concentration on the carrier, Y(V) (biomass per volume of solid particles), there was no significant difference in reactor performance between the investigated carriers under stationary conditions. The highest value for Y(V) was found for the gel entrapment carrier (Y(V) = 35 g L(-1)). In a long-term run (66 days), the gel entrapment carrier established a permanent biofilm on the surface of the gel beads after 900 h of cultivation time. Complete quinoline mineralization was achieved at a dilution rate of 2.0 h(-1), which is 4.7 times higher than the critical dilution rate. Identical substrate overloads were applied to the gel entrapment and the cellulose carrier by a step increase of the quinoline feed concentration at a dilution rate of 0.8 h(-1) (D approximately 2mu(max)). The cells survived the overload, but the accumulation of quinoline and quinoline degradation products and the degradation efficiency were different for the two systems during the overload, showing the influence of the carrier type on the dynamic performance and stability of the process. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 295-303, 1997.     

Activity of Pseudomonas aeruginosa in biofilms: Steady state

Aerobic glucose metabolism by Pseudomonas aeruginosa in steady-state biofilms at various substrate loading rates and reactor dilution rates was investigated. Variables monitored were substrate (glucose), biofilm cellular density, biofilm extracellular polymeric substance (EPS) density, and suspended cellular and EPS concentrations. A mathematical model developed to describe the system was compared to experimental data. Intrinsic yield and rate coefficients included in the model were obtained from suspended continuous culture studies of glucose metabolism by P. aeruginosa. Experimental data compared well with the mathematical model, suggesting that P. aeruginosa does not behave differently in steady-state biofilm cultures, where diffusional resistance is negligible, than in suspended cultures. This implies that kinetic and stoichiometric coefficients for P. aeruginosa derived in suspended continuous culture can be used to describe steady-state biofilm processes.     

Modeling of an aerobic biofilm reactor with double-limiting substrate kinetics: bifurcational and dynamical analysis

A mathematical model of an aerobic biofilm reactor is presented to investigate the bifurcational patterns and the dynamical behavior of the reactor as a function of different key operating parameters. Suspended cells and biofilm are assumed to grow according to double limiting kinetics with phenol inhibition (carbon source) and oxygen limitation. The model presented by Russo et al. is extended to embody key features of the phenomenology of the granular‐supported biofilm: biofilm growth and detachment, gas–liquid oxygen transport, phenol, and oxygen uptake by both suspended and immobilized cells, and substrate diffusion into the biofilm. Steady‐state conditions and stability, and local dynamic behavior have been characterized. The multiplicity of steady states and their stability depend on key operating parameter values (dilution rate, gas–liquid mass transfer coefficient, biofilm detachment rate, and inlet substrate concentration). Small changes in the operating conditions may be coupled with a drastic change of the steady‐state scenario with transcritical and saddle‐node bifurcations. The relevance of concentration profiles establishing within the biofilm is also addressed. When the oxygen level in the liquid phase is <10% of the saturation level, the biofilm undergoes oxygen starvation and the active biofilm fraction becomes independent of the dilution rate. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Deposition of bacterial cells onto glass and biofilm surfaces

Deposition rates of Pseudomonas putida and Hyphomicrobium ZV620 onto glass and biofilm surfaces were quantified. Both species deposited to glass at a much slower rate than to biofilm. A definite bias by depositing cells for biofilms of their own species was evident in the highest attachment rates observed in this study.     

Detachment of multi species biofilm in circulating fluidized bed bioreactor

In this study, the detachment rates of various microbial species from the aerobic and anoxic biofilms in a circulating fluidized bed bioreactor (CFBB) with two entirely separate aerobic and anoxic beds were investigated. Overall detachment rate coefficients for biomass, determined on the basis of volatile suspended solids (VSS), glucose and protein as well as for specific microbial groups, i.e., for nitrifiers, denitrifiers, and phosphorous accumulating organisms (PAOs), were established. Biomass detachment rates were found to increase with biomass attachment on carrier media in both beds. The detachment rate coefficients based on VSS were significantly affected by shear stress, whereas for protein, glucose and specific microbial groups, no significant effect of shear stress was observed. High detachment rates were observed for the more porous biofilm structure. The presence of nitrifiers in the anoxic biofilm and denitrifiers in the aerobic biofilm was established by the specific activity measurements. Detachment rates of PAOs in aerobic and anoxic biofilms were evaluated.     

The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.     

High shear enrichment improves the performance of the anodophilic microbial consortium in a microbial fuel cell

In many microbial bioreactors, high shear rates result in strong attachment of microbes and dense biofilms. In this study, high shear rates were applied to enrich an anodophilic microbial consortium in a microbial fuel cell (MFC). Enrichment at a shear rate of about 120 s^?1 resulted in the production of a current and power output two to three times higher than those in the case of low shear rates (around 0.3 s^?1). Biomass and biofilm analyses showed that the anodic biofilm from the MFC enriched under high shear rate conditions, in comparison with that under low shear rate conditions, had a doubled average thickness and the biomass density increased with a factor 5. The microbial community of the former, as analysed by DGGE, was significantly different from that of the latter. The results showed that enrichment by applying high shear rates in an MFC can result in a specific electrochemically active biofilm that is thicker and denser and attaches better, and hence has a better performance.     

Development of pure culture biofilms of P. putida on solid supports

Pseudomonas putida biofilms were developed on and biofilm accumulation rate data were obtained for the following two classes of support materials: charged surfaces and noncharged hydrophobic and hydrophilic surfaces. The effects of surface roughness and porosity on the rate of microbial attachment were also examined.Materials bearing a net positive or negative surface charge supported the greatest biofilm accumulation and the highest biofilm accumulation rate. Uncharged hydrophobic materials achieved the next greatest biofilm accumulation, averaging approximately 50% of the total biomass which was accumulated on the charged surface materials after 16 days. Uncharged hydrophilic materials supported very little biofilm development. In general, biofilm accumulation increased with decreased surface roughness. The effect of pore size on biofilm accumulation was not conclusive.The biofilm accumulation kinetics showed an exponential accumulation rate for the charged surfaces and an approximately linear accumulation rate for the hydrophobic materials. This difference in accumulation kinetics is consistent with proposed differences in the physicochemical mechanism governing attachment to these two types of surface materials.     

Growth models of the continuous bacterial leaching of iron pyrite by Thiobacillus ferrooxidans

The leaching of iron pyrite by Thiobacillus ferrooxidans was studied in a continuous stirred tank reactor at a variety of dilution rates (0.012-0.22 h(-1)), pyrite surface areas (18-194 m(2)/L), and inlet soluble substrate (Fe(2+)) concentrations (0-3000 ppm). The bacterial leaching rate was found to increase with increasing pyrite surface area, dilution rate, and inlet Fe(2+) concentration. The concentration of bacteria in solution was related to the concentration of bacteria attached to the pyrite surface by a Langmuir-type adsorption-desorption relation. Fitting the experimental data to this relation yielded a value for the area occupied per bacterium of 86 mum(2). This result is consistent with the concept of preferential bacterial attachment of certain sites on the solid. A bacterial growth model was developed that included both bacterial growth in solution and growth of bacteria attached to the pyrite surface. The specific growth rate of the attached bacteria was calculated from this model and was found to increase with increasing solid dilution rate and to decrease with increasing pyrite surface area and soluble substance concentration. An explanation of these results based on an active-inactive site mechanisms was also developed.     

Fluorescence-Based Quasicontinuous and In Situ Monitoring of Biofilm Formation Dynamics in Natural Marine Environments

Analyzing the dynamics of biofilm formation helps to deepen our understanding of surface colonization in natural environments. While methods for screening biofilm formation in the laboratory are well established, studies in marine environments have so far been based upon destructive analysis of individual samples and provide only discontinuous snapshots of biofilm establishment. In order to explore the development of biofilm over time and under various biotic and abiotic conditions, we applied a recently developed optical biofilm sensor to quasicontinuously analyze marine biofilm dynamics in situ. Using this technique in combination with microscope-assisted imaging, we investigated biofilm formation from its beginning to mature multispecies biofilms. In contrast to laboratory studies on biofilm formation, a smooth transition from initial attachment to colony formation and exponential growth could not be observed in the marine environment. Instead, initial attachment was followed by an adaptation phase of low growth and homogeneously distributed solitary bacterial cells. Moreover, we observed a diurnal variation of biofilm signal intensity, suggesting a transient state of biofilm formation of bacteria. Overall, the biofilm formation dynamics could be modeled by three consecutive development stages attributed to initial bacterial attachment, bacterial growth, and attachment and growth of unicellular eukaryotic microorganisms. Additional experiments showed that the presence of seaweed considerably shortened the adaptation phase in comparison with that on control surfaces but yielded similar growth rates. The outlined examples highlight the advantages of a quasicontinuous in situ detection that enabled, for the first time, the exploration of the initial attachment phase and the diurnal variation during biofilm formation in natural ecosystems.     

Degradation of quinoline by immobilized Comamonas acidovorans in a three-phase airlift reactor

Quinolie degradation by Comamonas acidovorans was studied in a continuously operated three-phase airlift reactor. Porous glass beads were applied as support matrix for cell imobilization by colonization. Under steady-state conditions (S approximately 0), cell attachment was poor at low dilution rates but imporved considerably with increasing dilution rate. Conversion of quinoline was investigated below and above the washout for suspended culture (D(crit) = mu(max) = 0.42 h(-1)). With immobilized cells the reactor could be operated at D > mu(max), and complete conversion of quinoline was achieved as long as the specific quinoline feed rate D*S(0)/X did not exceed the maximum specific degradation rate (r(S, max)). The biofilm thickness was about 100 mum, and its efficiency was about 54% compared to suspended organisms. If quinoline overloads were supplied to the reactor, quinoline, as overloads were supplied to the reactor, quinoline, as well as its pathway intermediates, appeared in the reactor and conversion was low. Hence, the immobilized microorganisms remained viable and active. They could survive quinoline overloads. If the quinoline feed rate was reduced agains, complete conversion was reestablished. (c) 1995 John Wiley & Sons, Inc.     

溶菌酶对微小小单胞菌生物膜的影响

目的测定溶菌酶对微小小单胞菌及其生物膜的抑菌作用,并测出最小抑菌浓度(MIC)、最小杀菌浓度(MBC)和抑菌率。方法采用对倍稀释的方法,测定溶菌酶对微小小单胞菌的MIC、MBC;在96孔板中体外建立微小小单胞菌生物膜模型,采用MTT法检测溶菌酶对微小小单胞菌生物膜的影响;在六孔板中建立生物膜模型,使用激光共聚焦显微镜(CLSM)观察不同浓度溶菌酶对微小小单胞菌生物膜作用后的变化。结果溶菌酶对微小小单胞菌的MIC为0.0195 mg/mL,MBC为0.3125 mg/mL;CLSM观察结果显示,溶菌酶对微小小单胞菌生物膜的抑制作用随着浓度的增加而增强。结论溶菌酶对微小小单胞菌及其生物膜的生长和活性均具有抑制作用。     

An experimental study of mass diffusion and reaction rate in an anaerobic biofilm

An experimental reactor consisting of two chambers, separated by a porous ceramic immobilization matrix, was constructed to measure the effective diffusivity of different compounds and the consumption rates of acetate in developing biofilms. In initial experiments, effective diffusivities for acetate, propionate, isopropanol, and lithium salt through the ceramic immobilization matrix in the absence of biofilm were determined to be 40% to 50% less than in water at infinite dilution. The effective diffusivity of the lithium salt was similar to that of acetate. The effective diffusivity of the lithium salt through biofilms of thickness in the range of 200 to 1200 mum was essentially constant with a value of approximately 7% of that in water at infinite dilution. Acetate consumption in the biofilm was linearly proportional to biofilm thickness up to a biofilm depth of 800 mum. Deviation from linearity appeared in biofilm thicknesses greater than 800 mum. Results of these experiments support previous reports that immobilized cell reactors have significantly higher bioconversion rates than suspended cell systems.     

Form and Function of Clostridium thermocellum Biofilms

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h⁻¹) 12-fold higher than the bacterium''s maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.     

Mathematical Model and Mechanisms for Biofilm Wastewater Treatment Systems

Summary Fundamental theoretical depiction is still lacking on biofilm wastewater treatment systems up to today. A mathematical model of biofilm wastewater treatment systems, taking account of suspended microorganisms and some factors influencing biofilm formation and stabilization, is developed in this paper. By theoretical and numerical analyses, the factors influencing biofilm formation and stabilization, such as the dilution rate, influent organic concentration, detachment and initial inoculum concentration etc, are discussed. Qualitative investigations were carried out and suggestions on industrial applications are then proposed. This paper not only plays an important role in understanding the physical mechanisms of biofilm dynamics, but also has far-reaching implications for industrial practices.