J. J. Heijnen

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

Abstract 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.

Microbiology and biochemistry of the enhanced biological phosphate removal process

Abstract This paper reviews microbiological and biochemical aspects of the enhanced biological phosphate removal (EBPR) process. The discussion includes: microorganisms responsible for EBPR, isolation of polyphosphate accumulating organisms (PAOs), microbial diversity of the EBPR sludge, biochemical metabolisms of PAOs, energy budget in PAOs metabolism, denitrification by PAOs, glycogen accumulating non-poly-P organisms (GAOs), etc. Since pure cultures which possess complete characteristics of PAOs have not been isolated yet, the biochemical mechanism cannot be definitively described. The criteria to obtain a pure culture isolate are proposed. Based on the review, essential characteristics of PAOs are summarized in a table and directions for future research are identified.

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Wastewater treatment with particulate biofilm reactors

The review presented in this paper focuses on applications of particulate biofilm reactors (e.g. Upflow Sludge Blanket, Biofilm Fluidized Bed, Expanded Granular Sludge Blanket, Biofilm Airlift Suspension, Internal Circulation reactors). Several full-scale applications for municipal and industrial wastewater treatment are presented and illustrated, and their most important design and operation aspects (e.g. biofilm formation, hydrodynamics, mass transfer, mixing) are analysed and discussed. It is clear from the review that this technology can be considered a grown up technology for which good design and scale-up guidelines are available.

Phosphorus and nitrogen removal with minimal cod requirement by integration of denitrifying dephosphatation and nitrification in a two-sludge system

Abstract Denitrifying dephosphatation enables the removal of phosphorus and nitrogen with minimal use of COD, minimal oxygen consumption and minimal surplus sludge production. Moreover it would make aeration only necessary for nitrification. Therefore we have studied an anaerobic-anoxic (A2) sequencing batch reactor (SBR) coupled to a nitrification SBR. Denitrifying phosphorus removing bacteria (DPB) and nitrifiers were completely separated in two sludges in these two SBRs. The nitrified supernatant was recirculated from the nitrification SBR to the A2 SBR where nitrate was utilized by DPB as an electron acceptor for phosphorus removal. The technical feasibility for simultaneous phosphorus and nitrogen removal in the proposed two-sludge system was evaluated. The benefits of two-sludge systems over single-sludge systems were also discussed. It could be concluded that the separation of the nitrification step leads to an optimal process design for the application of denitrifying dephosphatation. The two-sludge system showed stable phosphorus and nitrogen removal, and enabled the removal of 15 mg-P/1 and 105 mg N/1 at the expense of only 400 mg-COD/1 acetic acid. Stoichiometric calculations showed that, in the two-sludge system the required COD can be up to 50% less than for conventional aerobic phosphorus and nitrogen removal systems. Moreover oxygen requirements and sludge production can be decreased in significant amounts of about 30 and 50%, respectively.

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.

Stoichiometry and kinetics of poly-β-hydroxybutyrate metabolism in aerobic, slow growing, activated sludge cultures

This paper discusses the poly-beta-hydroxybutyrate (PHB) metabolism in aerobic, slow growing, activated sludge cultures, based on experimental data and on a metabolic model. The dynamic conditions which occur in activated sludge processes were simulated in a 2-L sequencing batch reactor (SBR) by subjecting a mixed microbial population to successive periods of external substrate availability (feast period) and no external substrate availability (famine period). Under these conditions intracellular storage and consumption of PHB was observed. It appeared that in the feast period, 66% to almost 100% of the substrate consumed is used for storage of PHB, the remainder is used for growth and maintenance processes. Furthermore, it appeared that at high sludge retention time (SRT) the growth rate in the feast and famine periods was the same. With decreasing SRT the growth rate in the feast period increased relative to the growth rate in the famine period. Acetate consumption and PHB production in the feast period both proceeded with a zero-order rate in acetate and PHB concentration respectively. PHB consumption in the famine period could best be described kinetically with a nth-order degradation equation in PHB concentration. The obtained results are discussed in the context of the general activated sludge models.

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.

Poly-β-hydroxybutyrate metabolism in dynamically fed mixed microbial cultures

The kinetics of production and degradation of poly-beta-hydroxybutyrate (PHB) by a mixed activated sludge culture growing on acetate was studied in a sequencing batch reactor (SBR). Occasionally a very high amount of acetate was added to the steady state system in order to obtain high PHB concentrations in the cells (fPHB). This made it possible to follow PHB production and degradation over a wide range of fPHB-data (between 0 and 0.8 Cmol/Cmol). The results were compared with data available in literature and with equations derived by metabolic modeling. This led to some remarkable observations. For the feast period, the ratio q(feast)PHB/-q(feast)Ac (specific PHB production rate over specific acetate uptake rate) was used to indicate which fraction of the substrate is stored. Experimentally and theoretically it was shown that this ratio has a constant value for dynamically fed systems operated at a sludge retention time (SRT) > 2d. This value is 0.6 Cmol/Cmol under aerobic conditions and 0.4-0.5 Cmol/Cmol under anoxic conditions, irrespective of the specific growth rate of the biomass and the specific acetate uptake rate in the feast period. Degradation of internal stored PHB could be described with a first order degradation rate with respect to the PHB content of the cells. Degradation of PHB appeared to be independent of the type of electron acceptor present in the system and independent of the SRT of the system. The kinetic descriptions can be used to predict PHB production and consumption in general in dynamic fed wastewater treatment systems, and they provide some trends for modeling purposes.

A simple method to determine the enantiomeric ratio in enantioselective biocatalysis

The enantiomeric ratio (E) is commonly used to characterize the enantioselectivity in enzyme-catalyzed kinetic resolution. In this paper this parameter is directly derived from the enantiomeric excess of substrate and product. This is formally more correct than using Chens equation after calculating the degree of conversion from both ee values using the relation of Sih and Wu. New expressions and useful graphs have been generated for reversible and irreversible uni-uni reactions. The theoretical predictions have been verified experimentally for various reactions. Values for E and the thermodynamic equilibrium constant, KEQ, were obtained for a (DL)-dehalogenase-catalyzed dehalogenation, a hydrolysis reaction by porcine pancreatic lipase, and for C. Cylindracea lipase-catalyzed esterification and transesterification. In view of the current developments in the field of chiral analysis, this method is an easily available tool in the quantitative treatment of enzyme-catalyzed resolution of enantiomers.