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.     

rRNA and poly-beta-hydroxybutyrate dynamics in bioreactors subjected to feast and famine cycles

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-beta-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.     

rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.     

Substrate uptake tests and quantitative FISH show differences in kinetic growth of bulking and non-bulking activated sludge

The competition between filaments and floc formers in activated sludge has been historically described using kinetic selection. However, recent studies have suggested that bacterial storage may also be an important factor in microbial selection, since the dynamic nature of substrate flows into wastewater treatment plants elicit transient responses from microorganisms. Respirometry-based kinetic selection should thus be reevaluated by considering cell storage, and a more reliable method should be developed to include bacterial storage in the analysis of growth of filaments and floc formers in activated sludge. In this study, we applied substrate uptake tests combined with metabolic modeling to determine the growth rates, yields and maintenance coefficients of bulking and non-bulking activated sludge developed in lab scale reactors under feast and famine conditions. The results of quantitative fluorescence in situ hybridization (FISH) showed that the filaments Eikelboom Type 1851, Type 021N, and Thiothrix nivea were dominant in bulking sludge, comprising 42.0 % of mixed liquor volatile suspended solids (MLVSS), with 61.6% of the total filament length extending from flocs into bulk solution. Only low levels of Type 1851 filament length (4.9% of MLVSS) occurred in non-bulking sludge, 83.0% of which grew inside the flocs. The kinetic parameters determined from the substrate uptake tests were consistent with those from respirometry and showed that filamentous bulking sludge had lower growth rates and maintenance coefficients than non-bulking sludge. These results provide support for growth kinetic differences in explaining the competitive strategy of filamentous bacteria.     

Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts

Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S-) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction. Vibrio spp. and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S-, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant gamma-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable gamma-proteobacteria could thus be described as a "feast and famine" existence involving different activation levels of substrate concentration.     

Denitrification with endogenous carbon source at low C/N and its effect on P(3HB) accumulation

The objective of the work reported here was to determine whether the ratio of COD/Nox has an impact on poly-beta-hydroxybutyrate (PHB) metabolism in activated sludge. Furthermore, it was tested if the ratio influenced the percentage use of organic compounds present in wastewater, for endogenous respiration, oxidation, accumulation and denitrification. Gas flow rate in SBR reactor was controlled by thermal mass flow controller (TMFC). Constant amount of air entering sequencing batch reactor was automatically adjusted to stable set-point 2mg O2 L(-1). It means that DO concentration in the reactor could change with oxygen uptake. During the filling period and part of the reaction time DO was nearly zero. Feast period of the external substrate availability and famine period of little amount or no external carbon availability were determined. At 23 h of the reaction time, and COD/Nox ratio 8, denitrification took place only during feast period. What was interesting, poly-beta-hydroxybutyrate degradation was observed in the feast period as well. However, at 11h of the reaction time and COD/Nox ratio 37, denitrification occurred in feast and famine period. In the feast period PHB was accumulated and in the famine period was used as the endogenous carbon source. COD consumption to reduce 1mg N-nitrate was ranging from 1.15 to 6.26 depending on carbon source and increased when exogenous and endogenous carbon were used by activated sludge. The increase in PHB content from 0.25 to 0.43 Cmol/Cmol resulted in a double increase in the amount of nitrogen removed due to denitrification was observed.     

Effect of magnetic field on the accumulation of polyhydroxyalkanoates (PHAs) by microorganism in activated sludge

The effect of static magnetic field on polyhydroxyalkanoates (PHAs) syntheses by activated sludge under aerobic dynamic feeding (ADF) was evaluated in sequence batch reactors (SBR), with magnetic field intensities of 42, 21, 11 and 7 millitesla (mT) exposure in the feast, feast-famine and famine periods, respectively, and one control group without magnetic field exposure. Under each level of magnetic field intensity, the effect of magnetic field exposed in the famine period to PHAs syntheses was most significant in comparison with that in the feast or feast-famine period. Maximal hydroxybutyrate (HB) and (HV) yield occurred at 21 and 11 mT, respectively, and the minimal yield occurred at 42 mT during exposure in the famine period. The maximum biodegradable rate constant of PHA was noted at 11 mT during exposure in the famine period.     

Plug flow simulating and completely mixed reactors with a premixing tank,in the control of filamentous bulking

Summary A sequence of a substrate rich and substrate starvation phase (feast and famine strategy) is created in both compartmentalized reactors and reactors with premixing tanks. This condition is known to be essential in the control of bulking. With a synthetic waste water containing glucose a reactor with 12 compartments was effective in the control of filamentous bulking. With a dairy industrial waste water, containing a slowly biodegradable COD-fraction, this reactor could not suppress the growth of filamentous bacteria. With dairy and brewery waste water, reactors with premixing tanks were used to create a more pronounced exogenous (substrate rich) phase. Using three or more premixing tanks filamentous bulking could be controlled. During the exogenous phase the floc-forming microorganisms, having a higher substrate uptake rate, can take up the largest amount of substrate and can survive better during the endogenous phase.Substrate concentration and respiration rate profiles were studied and the concentration of the reserve materials was monitored.For each type of waste water the sludge loading and the fraction of readily available COD will determine the system's design.     

Characteristics of aerobic granular sludge in a sequencing batch reactor with variable aeration

Aerobic granules can be used for the treatment of industrial or municipal wastewater, but high aeration rate is required for the stable operation of the granular sludge system. Therefore, the aim of this research was to reduce aeration rate greatly to decrease the energy consumption for the technology of aerobic granules. Based on the characteristics of sequencing batch reactor with distinct feast and famine periods, aeration rate was reduced from 1.66 to 0.55 cm s⁻¹ in the famine period after granules were formed. It was found that the settleability of aerobic granules in reactor R1 with reduced aeration was the same as that of aerobic granules in reactor R2 with constant aeration rate of 1.66 cm s⁻¹. However, the outer morphology of aerobic granules gradually changed from round shape to long shape, and minor population showed certain shift after aeration rate was reduced in the famine period. Since good settleability is the most essential feature of aerobic granules, it can be said that reducing aeration rate in famine period did not influence the stable operation of aerobic granular sludge system. Furthermore, the experimental results indicated that aeration rate in feast period was much more important to the stable operation of aerobic granules than that in famine period.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

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.     

Growth and storage processes in aerobic granules grown on soybean wastewater

Aerobic granules in a sequencing batch reactor (SBR) are subjected to alternative feast and famine conditions, and are able to take up carbon substrate in wastewater rapidly and to store it as intracellular storage products when the substrate is in excess. This phenomenon could not be described by the widely used activated sludge model No.3 (ASM3). In this work, taking adsorption process, microbial maintenance, and substrate diffusion into account, the simultaneous growth and storage processes occurring in an aerobic-granule-based SBR are investigated with experimental and modeling approaches. A new model is established and successfully validated with the experimental results of an SBR fed with soybean-processing wastewater. Simulation results show that our approach is appropriate for elucidating the fates of major model components. Comparison between ASM3 and the model established in this work demonstrates that the latter is better to describe the substrate removal mechanisms and simultaneous growth and storage processes in aerobic granules.     

Effect of the applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactor

This article studies the operation of a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) at different applied organic load rates (OLRs). The process is based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. A mixture of acetic, lactic, and propionic acids at different concentrations (in the range 8.5-31.25 gCOD/L) was fed every 2 h in a sequencing batch reactor (SBR). The resulting applied OLR was in the range 8.5-31.25 gCOD/L/day. Even though, as expected, the increase in the OLR caused an increase in biomass concentration (up to about 8.7 g COD/L), it also caused a relevant decrease of maximal polymer production rate. This decrease in polymer production rate was related to the different extent of "feast and famine" conditions, as function of the applied OLR and of the start-up conditions. As a consequence the best performance of the process was obtained at an intermediate OLR (20 gCOD/L/day) where both biomass productivity and PHA storage were high enough. However, at this high OLR the process was unstable and sudden decrease of performance was also observed. The sludge characterized by the highest PHA storage response was investigated by 16S rDNA clone library. The clone library contained sequences mostly from PHA producers (e.g., Alcaligenes and Comamonas genera); however many genera and among them, one of the dominant (Thauera), were never described before in relation to PHA storage response.     

Determination of the decay rate of nitrifying bacteria

The growth and decay of nitrifying organisms determines the amount of nitrifying bacteria in activated sludge systems. The growth rate of the nitrifying organisms is reasonable, well defined, and studied, while the decay rate is still rather uncertain. Experiments in previous studies were over periods up to 14 days and obtained results were not confirmed. Contradicting decay rates of nitrifiers in different bacterial communities is reported. No differentiation between ammonia and nitrite oxidizers was made. Therefore, in this studyper day the decay rate of the nitrifying organisms was studied. The starvation condition (aerobic, anoxic, or anaerobic), temperature, type of bacterial community, and the presence of higher organisms are the main aspects that were investigated. A simple and reliable method (adapted from previous studies) for determining the decay rate of nitrifying organisms under different starvation conditions and different temperatures was developed. The test procedure has been used for determining the decay rate of ammonium and nitrite oxidizing bacteria in an enriched nitrifying culture and in activated sludge. The test was successfully applied at starvation periods up to 30 days. The decay rate of the enriched culture of nitrifiers was very low compared to values for nitrifiers in activated sludge. The decay rate of the nitrifiers in activated sludge was found to be to 0.2, 0.1, and 0.06 per day for aerobic, anoxic, and anaerobic conditions, respectively. The decay rate of ammonia oxidizers and nitrite oxidizers was the same at the corresponding conditions.     

Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater

Production of polyhydroxyalkanoates (PHAs) in activated sludge treating wastewater represents an economical and environmental promising alternative to pure culture fermentations. A process for production of PHA from a paper mill wastewater was examined at laboratory scale. The three stage process examined consisted of acidogenic fermentation to convert wastewater organic matter to volatile fatty acids (VFAs), an activated sludge system operating under feast/famine conditions to enrich for PHA producing organisms and accumulation of PHA in batch experiments. After fermentation of the wastewater, 74% of the soluble COD was present as VFA (acetate, propionate, butyrate and valerate) and the resulting PHA after batch accumulation consisted of 31-47 mol% hydroxybutyrate and 53-69 mol% hydroxyvalerate. The maximum PHA content achieved was 48% of the sludge dry weight and the three stage process exhibited a potential to produce 0.11 kg of PHA per kg of influent COD treated.     

Influence of 2,4-dinitrophenol on the characteristics of activated sludge in batch reactors

The response of activated sludge characteristics to the presence of 2,4-dinitrophenol (dNP) in batch cultures was investigated in this study. The sludge yield slightly decreased with an increase in dNP concentration. At 10 mg l(-1), or lower, dNP significantly reduced sludge yield and relative specific growth rates (mu/mu0), but didn't substantially affect its relative specific chemical oxygen demand removal rate (q/q0). Presence of dNP at 1-20 mg l(-1) increased the specific oxygen uptake rate of activated sludge, and slightly changed its hydrophobicity. An analysis on inhibition indicated that the reduction in sludge yield in the presence of dNP was mainly attributed to the significant decreased sludge growth, rather than the reduced substrate degradation.     

Succession of Pelagic Marine Bacteria during Enrichment: a Close Look at Cultivation-Induced Shifts

Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S−) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction. Vibrio spp. and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S−, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant γ-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable γ-proteobacteria could thus be described as a “feast and famine” existence involving different activation levels of substrate concentration.     

Comparison of maximum cell yield and maintenance coefficients in axenic cultures and activated sludge communities

Comparison of the equations that describe the relationship between the maximum cell yield coefficient, the maintenance coefficient, and the specific growth rate at steady-state conditions revealed that the equations used for axenic cultures are congruent with those commonly used for mixed-culture system such as activated sludge. A unified basis was proposed. The expression of the yield and maintenance coefficients in carbon units according to the unified basis permitted one to evaluate literature data on both axenic and mixed-culture systems. From this it appears that the maximum cell yield ranges from 0.50–0.80 (mg biomass carbon formed/mg substrate carbon used) for both axenic and mixed systems. However, the maintenance coefficient (mg substrate C/mg biomass C·hr) for the axenic cultures was between 0.010 and 0.100, but for activated sludge communities it was between 0.001 and 0.010. Microorganisms were isolated from sludge communities with these apparently low maintenance requirements and grown axenilly. Their maintenance coefficients but not their maximum yield coefficients decreased with decreasing specific growth rates. The consequences of this finding with regard to species selection in mixed-culture systems and the concept of cellular maintenance requirement are discussed.