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.     

Influence of biomass production and detachment forces on biofilm structures in a biofilm airlift suspension reactor

The influence of process conditions (substrate loading rate and detachment force) on the structure of biofilms grown on basalt particles in a Biofilm Airlift Suspension (BAS) reactor was studied. The structure of the biofilms (density, surface shape, and thickness) and microbial characteristics (biomass yield) were investigated at substrate loading rates of 5, 10, 15, and 20 kg COD/m3. day with basalt concentrations of 60 g/L, 150 g/L, and 250 g/L. The basalt concentration determines the number of biofilm particles in steady state, which is the main determining factor for the biofilm detachment in these systems. In total, 12 experimental runs were performed. A high biofilm density (up to 67 g/L) and a high biomass concentration was observed at high detachment forces. The higher biomass content is associated with a lower biomass substrate loading rate and therefore with a lower biomass yield (from 0.4 down to 0.12 gbiomass/gacetate). Contrary to general beliefs, the observed biomass detachment decreased with increasing detachment force. In addition, smoother (fewer protuberances), denser and thinner compact biofilms were obtained when the biomass surface production rate decreased and/or the detachment force increased. These observations confirmed a hypothesis, postulated earlier by Van Loosdrecht et al. (1995b), that the balance between biofilm substrate surface loading (proportional to biomass surface production rate, when biomass yield is constant) and detachment force determines the biofilm structure. When detachment forces are relatively high only a patchy biofilm will develop, whereas at low detachment forces, the biofilm becomes highly heterogeneous with many pores and protuberances. With the right balance, smooth, dense and stable biofilms can be obtained. Copyright 1998 John Wiley & Sons, Inc.     

Formation of nitrifying biofilms on small suspended particles in airlift reactors

For a stable and reliable operation of a BAS-reactor a high, active biomass concentration is required with mainly biofilm-covered carriers. The effect of reactor conditions on the formation of nitrifying biofilms in BAS-reactors was investigated in this article. A start-up strategy to obtain predominantly biofilm-covered carriers, based on the balancing of detachment and a biomass production per carrier surface area, proved tp be very successful. The amount of biomass and the fraction of covered carrier were high and development of nitrification activity was fast, leading to a volumetric conversion of 5 kg(N) . m(-3) . d(-1) at a hydraulic retention time of 1h. A 1-week, continuous inoculation with suspended purely nitrifying microorganisms resulted in a swift start-up compared with batch addition of a small number of biofilms with some nitrification activity. The development of nitrifying biofilms was very similar to the formation of heterotrophic biofilms. In contrast to heterotrophic bio-films, the diameter of nitrifying biofilms increased during start-up. The detachment rate from nitrifying biofilms decreased with lower concentrations of bare carrier, in a fashion comparable with heterotrophic biofilms, but the nitrifying biofilms were much more robust and resistant. Standard diffusion theory combined with reaction kinetics are capable of predicting the activity and conversion of biofilms on small suspended particles. (c) 1995 John Wiley & Sons Inc.     

Application of two component biodegradable carriers in a particle-fixed biofilm airlift suspension reactor: development and structure of biofilms

Two component biodegradable carriers for biofilm airlift suspension (BAS) reactors were investigated with respect to development of biofilm structure and oxygen transport inside the biofilm. The carriers were composed of PHB (polyhydroxybutyrate), which is easily degradable and PCL (caprolactone), which is less easily degradable by heterotrophic microorganisms. Cryosectioning combined with classical light microscopy and CLSM was used to identify the surface structure of the carrier material over a period of 250 days of biofilm cultivation in an airlift reactor. Pores of 50 to several hundred micrometers depth are formed due to the preferred degradation of PHB. Furthermore, microelectrode studies show the transport mechanism for different types of biofilm structures, which were generated under different substrate conditions. At high loading rates, the growth of a rather loosely structured biofilm with high penetration depths of oxygen was found. Strong changes of substrate concentration during fed-batch mode operation of the reactor enhance the growth of filamentous biofilms on the carriers. Mass transport in the outer regions of such biofilms was mainly driven by advection.     

Biofilm development in laboratory methanogenic fluidized bed reactors

Biofilm development on sand with different heterogeneous inocula was studied in laboratory-scale methanogenic fluidized bed reactors. Both the course of biofilm formation during reactor start-up and the bacterial composition of newly developed biofilms at steady-state were found to be similar irrespective of the type of inoculum applied. Biofilm formation proceeded according to a fixed pattern that could be subdivided in three consecutive phases, designated as the lag phase, biofilm production phase, and steady-state phase. Methanogenic activity and biomass content of the fluidized bed granules were found to be accurate parameters of the course of biofilm formation. More indirect parameters monitored did not give unambiguous results in all instances. The composition of the newly developed biomass as assessed on the basis of potential methanogenic activities on different substrates and of the concentration of specific methanogenic cofactors was consistent with electron microscopic observations.     

Dynamics of biofilm detachment in biofilm airlift suspension reactors

The dynamic change in the overall detachment rate of spherical biofilms in a biofilm airlift suspension reactor was measured after a downshift of the substrate loading rate to zero while all other conditions remained constant. In contrast to the expectations, the overall detachment rate decreased rapidly to a nearly stable level. Correlations available from literature were not able to describe this phenomenon. Concepts were formulated which can describe the observations from this study. Research under dynamic conditions and careful monitoring of the biofilm surface area and biofilm morphology are necessary to elucidate and discriminate biofilm detachment mechanisms. (c) 1995 John Wiley & Sons, Inc.     

Quantification of viable biomass in biofilm reactors by extractable lipid phosphate

 Lipid phosphate, which is a measure of viable biomass, was determined using biofilm samples from three different laboratory-scale reactors. The analysis procedure proposed in the literature was modified and tested for suitability in experiments with biofilm reactors. The microbial contents of the biofilms studied are compared in three types of reactor. Received: 2 November 1994/Accepted: 23 January 1995     

Advanced start-up of anaerobic attached film expanded bed reactor by pre-aeration of biofilm carrier

The start-up and performance of the anaerobic attached film expanded bed (AAFEB) reactor with pre-aeration of carrier were investigated. The carriers of the reactors had been aerated for 10 days before they were put into the AAFEB reactors. The results indicated that the reactors advance the start-up by 15 days, and maintain higher efficiency when they were subjected to organic and hydraulic loading shock, but during steady-state operation, the reactors did not show better performance than the control reactors without pre-aeration of carrier. The thicker biofilm and higher biomass concentration of the reactors with pre-aeration were observed during the start-up period, but the difference between two types of reactors tapered with the time course, and at the steady-state operation, the difference between two types of reactors on these two parameters was not obvious. Maximum specific methane or acids production rates, dehydrogenase activity and coenzyme F(420) content were continuously higher than those of the control reactors. After running 30 days, filamentous bacteria dominated in the reactors with pre-aeration, whereas the cocci were predominant species in the control reactors. It was suggested that the action of the biofilm is strongly dependent on the biofilm thickness or the biomass concentration in normal circumstances, but under adverse circumstances, such as organic or hydraulic loading shock, the characteristics and activity of the anaerobic granular sludge play key roles on the reactor performance. These results clearly indicated that pre-aeration of carrier favor to enhance the start-up and performance of AAFEB reactor.     

Detachment forces and their influence on the structure and metabolic behaviour of biofilms

The biofilm reactor has been frequently applied in wastewater treatment. The formation, structure and metabolic activity of the biofilms are closely associated with the detachment forces in the reactors. This paper reviews the essential role of detachment forces in the biofilm process. A more compact, stable and denser biofilm can be formed at a relatively higher detachment force. The detachment force has significant influence on the structure, mass transfer, production of exopolysaccharides, metabolic and genetic properties of the biofilm. In an engineering sense, the detachment forces can be manipulated, as a control parameter, to produce a more stable and compact biofilm for use in wastewater treatment. The molecular and genetic mechanisms responsible for the detachment force-associated phenomena are not yet fully understood.     

A general description of detachment for multidimensional modelling of biofilms

A general method for describing biomass detachment in multidimensional biofilm modelling is introduced. Biomass losses from processes acting on the entire surface of the biofilm, such as erosion, are modelled using a continuous detachment speed function F(det). Discrete detachment events, i.e. sloughing, are implicitly derived from simulations. The method is flexible to allow F(det) to take several forms, including expressions dependent on any state variables such as the local biofilm density. This methodology for biomass detachment was integrated with multidimensional (2D and 3D) particle-based multispecies biofilm models by using a novel application of the level set method. Application of the method is illustrated by trends in the dynamics of biofilms structure and activity derived from simulations performed on a simple model considering uniform biomass (case study I) and a model discriminating biomass composition in heterotrophic active mass, extracellular polymeric substances (EPS) and inert mass (case study II). Results from case study I demonstrate the effect of applied detachment forces as a fundamental factor influencing steady-state biofilm activity and structure. Trends from experimental observations reported in literature were correctly described. For example, simulation results indicated that biomass sloughing is reduced when erosion forces are increased. Case study II illustrates the application of the detachment methodology to systems with non-uniform biomass composition. Simulations carried out at different bulk concentrations of substrate show changes in biofilm structure (in terms of shape, density and spatial distribution of biomass components) and activity (in terms of oxygen and substrate consumption) as a consequence of either oxygen-limited or substrate-limited growth.     

Influence of dissolved oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor

The biofilm airlift suspension (BAS) reactor can treat wastewater at a high volumetric loading rate combined with a low sludge loading. Two BAS reactors were operated, with an ammonium load of 5 kg N/(m(3) d), in order to study the influence of biomass and oxygen concentration on the nitrification process. After start-up the nitrifying biomass in the reactors gradually increased up to 30 g VSS/L. Due to this increased biomass concentration the gas-liquid mass transfer coefficient was negatively influenced. The resulting gradual decrease in dissolved oxygen concentration (over a 2-month period) was associated with a concomitantly nitrite build-up. Short term experiments showed a similar relation between dissolved oxygen concentration (DO) and nitrite accumulation. It was possible to obtain full ammonium conversion with approximately 50% nitrate and 50% nitrite in the effluent. The facts that (i) nitrite build up occurred only when DO dropped, (ii) the nitrite formation was stable over long periods, and (iii) fully depending on DO levels in short term experiments, led to the conclusion that it was not affected by microbial adaptations but associated with intrinsic characteristics of the microbial growth system. A simple biofilm model based on the often reported difference of oxygen affinity between ammonium and nitrite oxydizers was capable of adequately describing the phenomena.Measurements of biomass density and concentration are critical for the interpretation of the results, but highly sensitive to sampling procedures. Therefore we have developed an independent method, based on the residence time of Dextran Blue, to check the experimental methods. There was a good agreement between procedures.The relation between biomass concentration, oxygen mass transfer rate and nitrification in a BAS reactor is discussed. (c) 1997 John Wiley & Sons, Inc.     

Control of heterotrophic layer formation on nitrifying biofilms in a biofilm airlift suspension reactor

A Biofilm Airlift Suspension (BAS) reactor was operated with nitrifying biofilm growth and heterotrophic suspended growth, simultaneously converting ammonium and acetate. Growth of heterotrophs in suspension decreases the diffusion limitation for the nitrifiers, and enlarges the nitrifying capacity of a biofilm reactor. Neither nitrifiers nor heterotrophs suffer from additional oxygen diffusion limitation when the heterotrophs grow in suspension. Control of the location of heterotrophic growth, either in suspension or in biofilms over the nitrifying biofilms, was possible by manipulation of the hydraulic retention time. A time delay for formation and disappearance of the heterotrophic biofilms of 10 to 15 days was observed. Surprisingly, it was found that in the presence of the heterotrophic layers the maximum specific activity on ammonia of the nitrifying biofilms increased. The reason for the increase in activity is unknown. The effect of heterotrophic biofilm formation on oxygen diffusion limitation for the nitrifiers is discussed. Some phenomena compensating the increased mass transfer resistance due to the growth of a heterotrophic layer are also presented. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 397-405, 1997.     

Detachment of biomass from suspended nongrowing spherical biofilms in airlift reactors

In three-phase internal loop airlift reactors, the detachment of biomass from suspended biofilm pellets in the presence of bare carrier particles was investigated under nongrowth conditions. The detachment rate was dominated by collisions between bare carrier particles and biofilm pellets. The concentration of bare carrier particles and the carrier roughness strongly influenced the detachment rate. A change in flow regime from bubbling to slug flow considerably increased the detachment rate. Otherwise, the superficial gas velocity did not directly affect the detachment rate. The influence of particle size was not clear. The bottom clearance did not affect the detachment rate within the tested range. Other aspects of reactor geometry might be important. The main detachment processes were abrasion and breakage of biofilm pellets. During the detachment process, two phases could be distinguished. In the first phase the detachment was relatively high, and both breakage and abrasion of biofilm pellets occurred. During the second phase, breakage dominated and the detachment rate was lower. The two-phase behavior is explained by differences in strength between the inner and outer biofilm layers, possibly caused by variations in local growth rates during biofilm formation. Differences in growth history might also explain the various detachment rates observed with different biofilm batches. (c) 1995 John Wiley & Sons, Inc.     

A modeling study of anaerobic biofilm systems: II. Reactor modeling

A rigorous steady-state model of anaerobic biofilm reactors taking into account acid-base and gas-phase equilibria in the reactor in conjunction with detailed chemical equilibria and mass transfer in acetate-utilizing methanogenic biofilms is presented. The performances of ideal completely stirred tank reactors (CSTRs) and plug-flow reactors, as well as reactors with nonideal hydraulic conditions, are simulated. Decreasing the surface loading rate increases the acetate removal efficiency, while decreasing the influent pH and increasing the buffering capacity improves the removal efficiency only if the bulk pH of the reactor shifts toward more optimal values between 6.8 to 7.0. The reactor can have negative or positive removal efficiencies depending on the start-up conditions. The respiration coefficient plays a critical role in determining the minimum influent pH required for reactor recovery after failure. Having multiple CSTRs-in-series generally increases the overall removal efficiency for the influent conditions investigated. Monitoring of the influent feed quality is critical for plug-flow reactors, becasue failure of the initial sections of the reactor may cause a cascading effect that may lead to a rapid reactor failure. (c) 1995 John Wiley & Sons, Inc.     

Early stages in biofilm development in methanogenic fluidized-bed reactors

Summary Biofilm development in methanogenic fluidized-bed reactors with sand as the carrier was studied on a laboratory scale. The microorganisms present in consecutive layers of the biofilm of mature sludge granules were preliminarily characterized on the basis of their morphology, element composition and adhesion capacity and were compared to bacteria which take part in the initial colonization of sand. The early phase of biofilm development was monitored with reactors receiving waste-waters containing different mixtures of volatile fatty acids and inoculated with fluidized-bed reactor effluent for different lengths of time. The results obtained indicate that facultative anaerobic bacteria abundantly present in the outermost biofilm layers of mature sludge granules are probably the main primary colonizers of the sand. Methanothrix spp. or other methanogens were rarely observed among the primary colonizers. The course of biofilm formation was comparable under the various start-up conditions employed including variations in waste-water composition, inoculation and anaerobicity. However, omission of waste-water and thus of substrate resulted in rapid wash-out of the attached biomass. Offprint requests to: W. Heinen     

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.     

Startup of anaerobic downflow stationary fixed film (DSFF) reactors

One of the serious problems limiting the application of full-scale anaerobic fixed film processes is reactor startup. To better understand startup, studies with downflow stationary fixed film (DSFF) reactors were conducted to characterize the effects of influent concentration, support material, and surface-to-volume ratio on biofilm development and overall reactor performance. Materials with roughened surfaces gave the best startup performance and as expected increased surface area in the reactors led to more rapid increases in loading rates and higher ultimate loadings. Soluble influent COD concentrations between 5 x 10(3) and 2 x 10(4) mg/L influenced the rate of biofilm development. Lower COD concentrations resulted in faster development of the biofilm, even though ultimate loadings were not necessarily achieved as rapidly as in reactors fed higher strength wastes. No decrease in specific activity of the biofilms in each reactor was observed as the thickness of the biofilms increased to their maximum value at the ultimate loadings. The operation of reactors fed lower strength wastes was more stable than reactors receiving higher strength feeds at comparable loadings. Biofilm yield and activity, COD removals, suspended growth and activity, and other system parameters are discussed.     

Biofilm development during the start-up period of anaerobic biofilm reactors: the biofilm Archaea community is highly dependent on the support material

To evaluate the impact of the nature of the support material on its colonization by a methanogenic consortium, four substrata made of different materials: polyvinyl chloride, 2 polyethylene and polypropylene were tested during the start-up of lab-scale fixed-film reactors. The reactor performances were evaluated and compared together with the analysis of the biofilms. Biofilm growth was quantified and the structure of bacterial and archaeal communities were characterized by molecular fingerprinting profiles (capillary electrophoresis-single strand conformation polymorphism). The composition of the inoculum was shown to have a major impact on the bacterial composition of the biofilm, whatever the nature of the support material or the organic loading rate applied to the reactors during the start-up period. In contrast, the biofilm archaeal populations were independent of the inoculum used but highly dependent on the support material. Supports favouring Archaea colonization, the limiting factor in the overall process, should be preferred.     

A statistical analysis of the effect of substrate utilization and shear stress on the kinetics of biofilm detachment

One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment.Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed.A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono- and mixed-population biofilms. (c) John Wiley & Sons, Inc.