Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h−1, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research.

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Nitrous oxide emission during wastewater treatment.

The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge

Aerobic granulation in a sequencing batch reactor

Aerobic granular sludge technology offers a possibility to design compact wastewater treatment plants based on simultaneous chemical oxygen demand (COD), nitrogen and phosphate removal in one sequencing batch reactor. In earlier studies, it was shown that aerobic granules, cultivated with an aerobic pulse-feeding pattern, were not stable at low dissolved oxygen concentrations. Selection for slow-growing organisms such as phosphate-accumulating organisms (PAO) was shown to be a measure for improved granule stability, particularly at low oxygen concentrations. Moreover, this allows long feeding periods needed for economically feasible full-scale applications. Simultaneous nutrient removal was possible, because of heterotrophic growth inside the granules (denitrifying PAO). At low oxygen saturation (20%) high removal efficiencies were obtained; 100% COD removal, 94% phosphate (P-) removal and 94% total nitrogen (N-) removal (with 100% ammonium removal). Experimental results strongly suggest that P-removal occurs partly by (biologically induced) precipitation. Monitoring the laboratory scale reactors for a long period showed that N-removal efficiency highly depends on the diameter of the granules.

Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge

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/(m3 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. © 1997 John Wiley & Sons, Inc.

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

Bubble recirculation regimes in an internal-loop airlift 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.

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

Fluorescent microparticles were used as tracer beads to measure the dynamics of solids in spherical biofilms in a biofilm airlift suspension reactor Attachment to, release from, and penetration into the biofilms of the tracer beads were measured The coverage of the biofilm surface was low and the steady state particle concentration on the surface was dependent on the biofilm surface characteristics The measured attachment rate constant was identical in both experiments and appeared to be determined by the hydrodynamic conditions in the turbulent reactor The attachment rate was much faster than the release rate of the tracer beads and, therefore, the solidsretention time in the biofilm particle is not due to a simple reversible adsorption-desorption process The heterogeneity of the distribution oftracer beads on different sectors on the biofilm surface decreased duringthe attachment period Due to random detachment processes the heterogeneity of the tracer bead distribution increased during the release periodThe tracer beads quickly penetrated into the biofilm and became distributed throughout the active layer of the biofilm The observed penetration into biofilms, the nonuniform distribution on the biofilm surface, and the fast uptake and slow release of tracer beads cannot be described by a simple model based on a reversible adsorption-desorption mechanism, nor withexisting biofilm models These biofilm models, which balance growth and advection assuming a uniform biofilm with a homogeneous surface, are inadequate for the description of the observed solids retention time in biofilms Therefore, a new concept of biofilm dynamics is proposed, in which formation of cracks and fissures, which are rapidly filled with growing biomass, combined with nonuniform local detachment, explains the observed fast penetration into the biofilm of tracer beads, the long residence time, and the nonuniform distibution of fluorescent microparticles (c) 1994 John Wiley & Sons, Inc

Solids retention time in spherical biofilms in a biofilm airlift suspension reactor.

The influence of the bare basalt carrier concentration on nitrifying biofilm formation in biofilm airlift suspension (BAS) reactors was studied. The time needed to develop fully covered biofilm particles strongly increased with an increasing initial carrier concentration. This was caused by the increased shear and subsequent higher detachment. During startup the diameter of the biofilm particles increased, leading to a higher biomass concentration, larger biofilm surface area, and lower surface-specific substrate load. Eventually a “steady state” thickness is obtained, where surface-specific growth and detachment are equal. The biofilm density decreased with a decreasing bare basalt concentration. The steady state biomass concentration was not very much influenced by the initial carrier concentration. Up to 75% of the produced biomass was retained in the reactors. Maximum biomass retention was found when the initial bare basalt concentration was in between 10 and 30 g·L−1. The continuous detachment of biofilm fragments leads, upon growth of these small fragments, to the formation of granules (biofilms without a carrier). The granules have average density and size equal to those of the biofilm particles. The granules are formed, if the detached biofilm fragments are large enough to be retained by the three-phase separator on top of the BAS reactor. Since the superficial liquid velocity in the three-phase separator is scale dependent, granule formation will be influenced by reactor scale, hydraulic load, and dimensions of the three-phase separator.

W. A. J. van Benthum

Papers

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

Bubble recirculation regimes in an internal-loop airlift reactor

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

Solids retention time in spherical biofilms in a biofilm airlift suspension reactor.

Formation and Detachment of Biofilms and Granules in a Nitrifying Biofilm Airlift Suspension Reactor