Measurement and mathematical modelling of competition between fast- and slow-growing ordinary heterotrophic organisms in low and high substrate-loaded systems

Ordinary heterotrophic organisms (OHO) of an activated sludge wastewater treatment system showed an atypical growth behaviour when they are inoculated to batch aerobic growth tests with a high substrate-loaded condition. For example, the OHO maximum specific growth rates on readily biodegradable substrates (μ _H) increased with a high ratio of substrate concentration to OHO active biomass concentration (So/Xo), although they were assumed to be constant in a conventional microbial growth kinetic model with a single OHO population group. We, therefore, set a hypothesis in that the change of OHO maximum specific growth rates in the batch test condition is caused by turnover of fast-growing OHO population against slow-growing OHO population. And, a competitive microbial growth kinetic model of the fast- and slow-growing OHO population groups was developed and validated with model-data fitting analysis for the batch test results. The competitive microbial growth kinetic model of process selection, rather than that of kinetic selection, was capable of simulating microbial growth kinetics in high substrate-loaded dynamic systems (i.e., batch tests) and low substrate-loaded steady-state systems (i.e., continuously operated wastewater treatment systems), better than the conventional non-competitive growth kinetic model.     

Transient response of aerobic and anoxic activated sludge activities to sudden substrate concentration changes

The state-of-the-art understanding of activated sludge processes as summarized in activated sludge models (ASMs) predicts an instantaneous increase in the biomass activity (which is measured, e.g., by the corresponding respiration rate OUR, NUR, etc.) under sudden substrate concentration changes. Experimental data (e.g., short-term batch respiration experiments under aerobic or anoxic conditions) collected for the calibration of the dynamic models (ASMs) often exhibit a transient phenomenon while attaining maximum activity, which cannot be explained by the current understanding of the activated sludge process. That transient phenomenon exhibits itself immediately upon addition of a substrate source to an endogenously respiring activated sludge sample and it usually takes a few minutes until the activated sludge reaches its maximum possible rate under given environmental conditions. This discrepancy between the state-of-the-art model and the experimental data is addressed in detail in this investigation. It is shown that the discrepancy is not caused by an error in the experimental set-up/data but it is rather due to model inadequacy. Among the hypotheses proposed, it appears that this transient response of the activated sludge most likely results from the sequence of intracellular reactions involved in substrate degradation by the activated sludge. Results from studies performed elsewhere with pure cultures (S. cerevisae and E. coli) support the hypothesis. The transient phenomenon can be described by a dynamic metabolic network model or by a simple first-order model, as adopted in this study. The transient phenomenon occurring in short-term batch respiration experiments is shown to interfere severely with parameter estimation if not modeled properly (2.8%, 11.5%, and 16.8% relative errors [average of three experiments] on Y(H), micro(maxH), and K(S), respectively). Proper modeling of this transient phenomenon whose time constant is on the order of minutes (1 to 3 min) is expected to contribute fundamentally to a better understanding and modeling of Orbal, carousel, and SBR-type treatment plants with fast-alternating process conditions, although such studies are beyond the scope of this report.     

Stoichiometry and kinetics of poly-beta-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.     

Critical review of activated sludge modeling: State of process knowledge,modeling concepts,and limitations

This work critically reviews modeling concepts for standard activated sludge wastewater treatment processes (e.g., hydrolysis, growth and decay of organisms, etc.) for some of the most commonly used models. Based on a short overview on the theoretical biochemistry knowledge this review should help model users to better understand (i) the model concepts used; (ii) the differences between models, and (iii) the limits of the models. The seven analyzed models are: (1) ASM1; (2) ASM2d; (3) ASM3; (4) ASM3 + BioP; (5) ASM2d + TUD; (6) Barker & Dold model; and (7) UCTPHO+. Nine standard processes are distinguished and discussed in the present work: hydrolysis; fermentation; ordinary heterotrophic organisms (OHO) growth; autotrophic nitrifying organisms (ANO) growth; OHO & ANO decay; poly‐hydroxyalkanoates (PHA) storage; polyphosphate (polyP) storage; phosphorus accumulating organisms PAO) growth; and PAO decay. For a structured comparison, a new schematic representation of these processes is proposed. Each process is represented as a reaction with consumed components on the left of the figure and produced components on the right. Standardized icons, based on shapes and color codes, enable the representation of the stoichiometric modeling concepts and kinetics. This representation allows highlighting the conceptual differences of the models, and the level of simplification between the concepts and the theoretical knowledge. The model selection depending on their theoretical limitations and the main research needs to increase the model quality are finally discussed. Biotechnol. Bioeng. 2013; 110: 24–46. © 2012 Wiley Periodicals, Inc.     

Effect of nitrogen limitation on enrichment of activated sludge for PHA production

Polyhydroxyalkanoates (PHA) are good candidates to plastics because of their material properties similar to conventional plastics and complete biodegradability. The use of activated sludge can be a cheaper alternative to pure cultures for PHA production. In this study, effect of nitrogen limitation during acclimatization period of biomass on production of polyhydroxyalkanoate was investigated. Activated sludge was selected in two sequencing batch reactors operated with and without nitrogen limitation. Batch tests were performed to examine polymer productions of activated sludges acclimatized to different nitrogen regimes. Responses of biomass to different organic loading rates, organic acids, and carbon to nitrogen (C/N) ratios were studied by determining specific polymer storage rate, polymer storage yield, and sludge polymer content of biomasses. Results obtained from batch experiments showed that concentrations of polymer accumulated by two different sludges increased directly with initial substrate concentration. Observed highest polymer yields for the biomasses enriched with and without nitrogen deficiency were 0.69 g COD PHA g(-1) COD S and 0.51 g COD PHA g(-1) COD S, and corresponding polymer contents of biomasses were 43.3% (g COD PHA g(-1) COD X) and 38.3% (g COD PHA g(-1) COD X), respectively. Polymer yields for both biomasses decreased with substrate shift however, biomass enriched with nitrogen deficiency adapted well to acetate-propionate mixture. The results presented in this study showed that polymer storage ability of biomass was improved more under dynamic conditions with nitrogen deficiency when compared to that without nitrogen deficiency. Limiting ammonia availability during batch experiments also caused higher polymer production by suppressing growth, as well as during enrichment of biomass.     

Aerobic phosphorus release linked to acetate uptake in bio-P sludge: process modeling using oxygen uptake rate

The main processes involved in enhanced biological phosphorus removal (EBPR) under anaerobic and subsequently aerobic conditions are widely described in the literature. Polyphosphate accumulating organisms (PAO) are the organisms responsible for this process. However, the mechanisms of PAO are not fully established yet under conditions that differ from the classical anaerobic/aerobic conditions. In this work, we made a comparison between the behavior of PAO under classical EBPR conditions and its behavior when consuming substrate under only aerobic conditions. In addition, oxygen uptake rate (OUR) was measured in the set of experiments under aerobic conditions to improve the characterization of the process. A kinetic and stoichiometric model based on Activated Sludge Model No.2 (ASM2) and including glycogen economy (AnOx model), calibrated for classical anaerobic/aerobic conditions, was not able to describe the experimental data since it underestimated the acetate consumption, the PHB storage, and the OUR. Two different hypotheses for describing the experimental measurements were proposed and modeled. Both hypotheses considered that PAO, under aerobic conditions, uptake acetate coupled to PHB storage, glycogen degradation, and phosphorus release as in anaerobic conditions. Moreover, the first hypothesis (PAO-hypothesis) considered that PAO were able to store acetate as PHB linked to oxygen consumption and the second one (OHO hypothesis) considered that this storage was due to ordinary heterotrophic organisms (OHO). Both hypotheses were evaluated by simulation extending the AnOx model with additional equations. The main differences observed were the predictions for PHB degradation during the famine phase and the OUR profile during both feast and famine phases. The OHO hypothesis described the experimental profiles more accurately than the PAO hypothesis.     

Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off-gas CO2 measurements

A method for detailed investigation of aerobic carbon degradation processes by microorganisms is presented. The method relies on an integrated use of the respirometric, titrimetric, and off-gas CO(2) measurements. The oxygen uptake rate (OUR), hydrogen ion production rate (HPR), and the carbon dioxide transfer rate (CTR) resulting from the biological as well as physicochemical processes, coupled with a metabolic model characterizing both the growth and carbon storage processes, enables the comprehensive study of the carbon degradation processes. The method allows the formation of carbon storage products and the biomass growth rates to be estimated without requiring any off-line biomass or liquid-phase measurements, although the practical identifiability of the system could be improved with additional measurements. Furthermore, the combined yield for biomass growth and carbon storage is identifiable, along with the affinity constant with respect to the carbon substrate. However, the individual yields for growth and carbon storage are not identifiable without further knowledge about the metabolic pathways employed by the microorganisms in the carbon conversion. This is true even when more process variables are measured. The method is applied to the aerobic carbon substrate degradation by a full-scale sludge using acetate as an example carbon source. The sludge was able to quickly take up the substrate and store it as poly-beta-hydroxybutyrate (PHB). The PHB formation rate was a few times faster than the biomass growth rate, which was confirmed by off-line liquid- and solid-phase analysis. The estimated combined yield for biomass growth and carbon storage compared closely to that determined from the theoretical yields reported in literature based on thermodynamics. This suggests that the theoretical yields may be used as default parameters for modeling purposes.     

Model-based analysis on growth of activated sludge in a sequencing batch reactor

A mathematical model is developed to describe the growth of multiple microbial species such as heterotrophs and autotrophs in activated sludge system. Performance of a lab-scale sequencing batch reactor involving storage process is used to evaluate the model. Results show that the model is appropriate for predicting the fate of major model components, i.e., chemical oxygen demand, storage polymers (X _STO), volatile suspended solid (VSS), ammonia, and oxygen uptake rate (OUR). The influence of sludge retention time (SRT) on reactor performance is analyzed by model simulation. The biomass components require different time periods from one to four times of SRT to reach steady state. At an SRT of 20 days, the active bacteria (autotrophs and heterotrophs) constitute about 57% of the VSS; the remaining biomass is not active. The model established demonstrates its capacity of simulating the reactor performance and getting insight in autotrophic and heterotrophic growth in complex activated sludge systems.     

Respirometric evaluation and modeling of glucose utilization by Escherichia coli under aerobic and mesophilic cultivation conditions

The study presents a mechanistic model for the evaluation of glucose utilization by Escherichia coli under aerobic and mesophilic growth conditions. In the first step, the experimental data was derived from batch respirometric experiments conducted at 37 degrees C, using two different initial substrate to microorganism (S(0)/X(0)) ratios of 15.0 and 1.3 mgCOD/mgSS. Acetate generation, glycogen formation and oxygen uptake rate profile were monitored together with glucose uptake and biomass increase throughout the experiments. The oxygen uptake rate (OUR) exhibited a typical profile accounting for growth on glucose, acetate and glycogen. No acetate formation (overflow) was detected at low initial S(0)/X(0) ratio. In the second step, the effect of culture history developed under long-term growth limiting conditions on the kinetics of glucose utilization by the same culture was evaluated in a sequencing batch reactor (SBR). The system was operated at cyclic steady state with a constant mean cell residence time of 5 days. The kinetic response of E.coli culture was followed by similar measurements within a complete cycle. Model calibration for the SBR system showed that E. coli culture regulated its growth metabolism by decreasing the maximum growth rate (lower microH) together with an increase of substrate affinity (lower K(S)) as compared to uncontrolled growth conditions. The continuous low rate operation of SBR system induced a significant biochemical substrate storage capability as glycogen in parallel to growth, which persisted throughout the operation. The acetate overflow was observed again as an important mechanism to be accounted for in the evaluation of process kinetics.     

Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding

This article describes a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. Enrichment was obtained through the selective pressure established by feeding the carbon source in a periodic mode (feast and famine regime) in a sequencing batch reactor. A concentrated mixture of acetic, lactic, and propionic acids (overall concentration of 8.5 gCOD L(-1)) was fed every 2 h at 1 day(-1) overall dilution rate. Even at such high organic load (8.5 gCOD L(-1) day(-1)), the selective pressure due to periodic feeding was effective in obtaining a biomass with a storage ability much higher than activated sludges. The immediate biomass response to substrate excess (as determined thorough short-term batch tests) was characterized by a storage rate and yield of 649 mgPHA (as COD) g biomass (as COD)(-1) h(-1) and 0.45 mgPHA (as COD) mg removed substrates (as COD(-1)), respectively. When the substrate excess was present for more than 2 h (long-term batch tests), the storage rate and yield decreased, whereas growth rate and yield significantly increased due to biomass adaptation. A maximum polymer fraction in the biomass was therefore obtained at about 50% (on COD basis). As for the PHA composition, the copolymer poly(beta-hydroxybutyrate/beta-hydroxyvalerate) with 31% of hydroxyvalerate monomer was produced from the substrate mixture. Comparison of the tests with individual and mixed substrates seemed to indicate that, on removing the substrate mixture for copolymer production, propionic acid was fully utilized to produce propionylCoA, whereas the acetylCoA was fully provided by acetic and lactic acid.     

Evaluation on factors influencing the heterotrophic growth on the soluble microbial products of autotrophs

In this work, the heterotrophic growth on the microbial products of autotrophs and the effecting factors were evaluated with both experimental and modeling approaches. Fluorescence in situ hybridization (FISH) analysis illustrated that ammonia oxidizers (AOB), nitrite oxidizers (NOB), and heterotrophs accounted for about 65%, 20%, and 15% of the total bacteria, respectively. The mathematical evaluation of experimental data reported in literature indicated that heterotrophic growth in nitrifying biofilm (30–50%) and granules (30%) was significantly higher than that of nitrifying sludge (15%). It was found that low influent ammonium resulted in a lower availability of soluble microbial products (SMP) and a slower heterotrophic growth, but high ammonium (>150 mg N L⁻¹) feeding would lead to purely AOB dominated sludge with high biomass‐associated products contained effluent, although the absolute heterotrophic growth increased. Meanwhile, the total active biomass concentration increased gradually with the increasing solids retention time, whereas the factions of active AOB, NOB, and heterotrophs varied a lot at different solids retention times. This work could be useful for better understanding of the autotrophic wastewater treatment systems. Biotechnol. Bioeng. 2011; 108:804–812. © 2010 Wiley Periodicals, Inc.     

Studies on the in situ physiology of Thiothrix spp. present in activated sludge

The in situ physiology of the filamentous sulphur bacterium Thiothrix spp. was investigated in an industrial wastewater treatment plant with severe bulking problems as a result of overgrowth of Thiothrix. Identification and enumeration using fluorescence in situ hybridization (FISH) with species-specific 16S and 23S rRNA probes revealed that 5–10% of the bacteria in the activated sludge were Thiothrix spp. By using a combination of FISH and microautoradiography it was possible to study the in situ physiology of probe-defined Thiothrix filaments under different environmental conditions. The Thiothrix filaments were very versatile and showed incorporation of radiolabelled acetate and/or bicarbonate under heterotrophic, mixotrophic and chemolithoautotrophic conditions. The Thiothrix filaments were active under anaerobic conditions (with or without nitrate) in which intracellular sulphur globules were formed from thiosulphate and acetate was taken up. Thiothrix -specific substrate uptake rates and growth rates in activated sludge samples were determined under different conditions. Doubling times of 6–9 h under mixotrophic conditions and 15–30 h under autotrophic conditions were estimated. The key properties that Thiothrix might be employing to outcompete other microorganisms in activated sludge were probably related to the mixotrophic growth potential with strong stimulation of acetate uptake by thiosulphate, as well as stimulation of bicarbonate incorporation by acetate in the presence of thiosulphate.     

Acute impact of tetracycline and erythromycin on the storage mechanism of polyhydroxyalkanoates

The study investigated acute impact of tetracycline and erythromycin on substrate storage under aerobic conditions. A fill and draw reactor fed with peptone mixture was maintained at steady-state at a sludge age of 10 days; the acclimated biomass was used in a series of batch runs. The first run served as control reactor with organic substrate alone and the others were started with antibiotic doses of 50 mg/L and 200 mg/L for assessing intracellular storage. Parallel batch reactors were also conducted for recording oxygen uptake rate profiles. Both antibiotics enhanced substrate storage, leading to higher levels of polyhydroxyalkanoates incorporated into biomass, but they impaired its internal utilization for microbial growth. The observed decrease in oxygen consumption under the acute effect of antibiotics could partially be related to substrate storage – except for 50 mg/L of erythromycin dosing – suggesting an additional substrate binding mechanism by antibiotics, leading to residual biodegradable substrate.     

A metabolic model of the biological phosphorus removal process: II. Validation during start-up conditions

A metabolic model of the biological phosphorus removal process has been developed and validated previously for complex conversions during the process under anaerobic and aerobic conditions at different growth rates in sequencing batch reactors in steady state. For additional validation of the metabolic model, the model was applied to the dynamic conditions which occur during the start-up phase of the biological P removal in the presence and absence of non-polyP heterotrophic microorganisms. In a laboratory scale sequencing batch reactor, experiments were performed to examine the enrichment of the population with polyphosphate organisms during the start-up and the subsequent shift from non-polyP, heterotrophic organisms to polyP organisms in the sludge. The effect of different influent loading patterns for acetate and phosphate was studied. In these experiments, the maximal growth rate of the polyP organisms and the behavior of the internal storage compounds could be derived. The metabolic model was capable of describing the experimental results, without the need to adjust the kinetic or stoichiometric parameters obtained under steady state conditions. (c) 1995 John Wiley & Sons, Inc.     

Development of mechanistically based model for simulating soluble microbial products generation in an aerated/non-aerated SBR

Soluble microbial products (SMPs) are considered as the main organic components in wastewater treatment plant effluent from biological wastewater treatment systems. To investigate and explore SMP metabolism pathway for further treatment and control, two innovative mechanistically based activated sludge models were developed by extension of activated sludge model no.3 (ASM3). One was the model by combining SMP formation and degradation (ASM3-SMP model) processes with ASM3, and the other by combining both SMP and simultaneous substrate storage and growth (SSSG) mechanisms with ASM3 (SSSG-ASM3-SMP model). The detailed schematic modification and process supplements were introduced for comprehensively understanding all the mechanisms involved in the activated sludge process. The evaluations of these two models were demonstrated by a laboratory-scale sequencing batch reactor (SBR) operated under aerated/non-aerated conditions. The simulated and measured results indicated that SMP comprised about 83% of total soluble chemical oxygen demand (SCOD) in which biomass-associated products (BAPs) were predominant compared with utilization-associated products (UAPs). It also elucidated that there should be a minimum SMP value as the reactive time increases continuously and this conclusion could be used to optimize effluent SCOD in activated sludge processes. The comparative results among ASM3, ASM3-SMP and SSSG-ASM3-SMP models and the experimental measurements (SCOD, ammonia and nitrate nitrogen) showed clearly the best agreement with SSSG-ASM3-SMP simulation values (R = 0.993), strongly suggesting that both SMP formation and degradation and SSSG mechanisms are necessary in biologically activated sludge modeling for municipal wastewater treatment.     

Evaluation of growth yield of Spirulina (Arthrospira) sp. in photoautotrophic, heterotrophic and mixotrophic cultures

In microbial cultures, both cellular growth rate and yield (defined as the degree of substrate conversion into the biomass) are important. Although effect of culture conditions on growth kinetics has been well documented for various microbial strains, there is almost no literature concerning the effect of environmental conditions on growth equilibrium, expressed as biomass yield coefficients from substrate. The present paper discusses the effect of culture conditions: irradiance (physical substrate) and glucose concentration (chemical substrate) on biomass yield coefficients from two chemical substrates: glucose and nitrate-nitrogen in photoautotrophic, heterotrophic and mixotrophic culture of blue-green alga Spirulina (Arthrospira) sp. The efficiency of substrates incorporation into the biomass can be precisely determined only if the elemental composition of the biomass is known. The experimental results showed that culture conditions had a substantial influence on biomass yield coefficients (biomass yield from glucose and nitrate-nitrogen). It was found that, the increase of irradiance favoured increase of biomass yield coefficient from both, glucose and nitrate-nitrogen. However, in the case of yield from nitrogen in mixotrophic culture, the effect was opposite. The effect of glucose concentration was different: the higher the initial glucose concentration, the lower the biomass yield coefficients from chemical substrates.