The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

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.     

Simultaneous nitrification and denitrification using stored substrate (PHB) as the electron donor in an SBR

The potential for PHB (poly-beta-hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2-L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L(-1). However, the increased percentage of SND at a low DO concentration was compromised by a 2-times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L(-1) was optimal for both SND efficiency (61%) and rate (4.4 mmol N x Cmol x(-1) x h(-1)). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH(4) (+) and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first-order kinetics (df(PHB)/dt = -0.19 x f(PHB)), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen into the floc, due to the corresponding increased SOUR. Process control techniques to accumulate higher internal PHB concentrations to improve PHB-driven SND are discussed.     

N-removal in a granular sludge sequencing batch airlift reactor

The removal of N-compounds in the sequencing batch airlift reactor (SBAR) containing granular sludge was studied under conditions of decreased dissolved oxygen (DO). A simulation model was developed to describe and evaluate the effects of several process conditions in the SBAR on N-removal performance. The model described the experimental data reasonable well. It has been shown that nitrification, denitrification, and removal of chemical oxygen demand (COD) can occur simultaneously in a granular sludge SBR. It has also been shown that the exact location of the autotrophic biomass influences the net N-removal. The distribution of the autotrophic biomass is influenced by the DO in the reactor. The optimal DO value is expected to be around 40% air saturation. It was shown that storage and subsequent degradation of poly-beta-hydroxybutyrate (PHB) benefit the denitrification. In particular, PHB is stored in bacteria situated in deeper layers of the granules below where the autotrophic activity occurs, serves as a C-source for denitrification.     

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.     

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.     

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.     

Simultaneous removal of carbon and nitrate in an airlift bioreactor

This paper presents the integrated removal of carbon (measured as chemical oxygen demand i.e. COD) and NO(x)-N by sequentially adapted sludge, studied in an airlift reactor (ALR). Simultaneous removal of COD and nitrate occurs by denitrification (anoxic) and oxidation (aerobic). Aerobic (riser) and anoxic (remaining part) conditions prevail in different parts of the reactor. Studies were carried out in a 42 L ALR operated at low aeration rate to maintain anoxic and aerobic conditions as required for denitrification and COD removal, respectively. The sludge was adapted sequentially to increasing levels of NO(x)-N and COD over a period of 45 days. Nitrate removal efficiency of the sludge increased due to adaptation and degraded 900 ppm NO(3)-N completely in 2h (initially the sludge could not degrade 100 ppm NO(3)-N). The performance of the adapted sludge was tested for the degradation of synthetic waste with COD/N loadings in the range of 4-10. The reduction of COD was significantly faster in the presence of NO(x)-N and was attributed to the availability of oxygen from NO(x)-N and distinct conditions in the reactor. This hypothesis was justified by the material balance of COD.     

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.     

A comparative study of methanol as a supplementary carbon source for enhancing denitrification in primary and secondary anoxic zones

A comparative study on the use of methanol as a supplementary carbon source to enhance denitrification in primary and secondary anoxic zones is reported. Three lab-scale sequencing batch reactors (SBR) were operated to achieve nitrogen and carbon removal from domestic wastewater. Methanol was added to the primary anoxic period of the first SBR, and to the secondary anoxic period of the second SBR. No methanol was added to the third SBR, which served as a control. The extent of improvement on the denitrification performance was found to be dependent on the reactor configuration. Addition to the secondary anoxic period is more effective when very low effluent nitrate levels are to be achieved and hence requires a relatively large amount of methanol. Adding a small amount of methanol to the secondary anoxic period may cause nitrite accumulation, which does not improve overall nitrogen removal. In the latter case, methanol should be added to the primary anoxic period. The addition of methanol can also improve biological phosphorus removal by creating anaerobic conditions and increasing the availability of organic carbon in wastewater for polyphosphate accumulating organisms. This potentially provides a cost-effective approach to phosphorus removal from wastewater with a low carbon content. New fluorescence in situ hybridisation (FISH) probes targeting methanol-utilising denitrifiers were designed using stable isotope probing. Microbial structure analysis of the sludges using the new and existing FISH probes clearly showed that the addition of methanol stimulated the growth of specific methanol-utilizing denitrifiers, which improved the capability of sludge to use methanol and ethanol for denitrification, but reduced its capability to use wastewater COD for denitrification. Unlike acetate, long-term application of methanol has no negative impact on the settling properties of the sludge.     

活性污泥积累PHB (Poly-β-hydroxybutyrate)及其菌群多样性分析

【目的】通过调整活性污泥在驯化过程中碳、氮、磷比例以及供氧模式, 以提高其积累PHB的能力。应用PCR-DGGE定期对驯化期间菌群动态进行分析。【方法】以乙酸钠为碳源, 在SBR (Sequencing batch reactor) 内以厌氧/好氧 (A/O) 交替的驯化模式, 逐级提高碳源浓度, 限制氮源浓度, 人为创造营养不均衡条件来逐步提高活性污泥积累PHB的能力。【结果】当碳浓度逐步提高, COD升高至1 200 mg/L, COD/N/P为1 200/9.6/30时, 活性污泥中PHB积累量达到最大, 占细胞干重的64.2%。【结论】驯化过程中逐步提高COD负荷, 并增加COD/N的值有利于PHB积累, 利用苏丹黑和Albert法染色显示菌群内产PHB菌占很大比例, 透射电镜显示菌体胞内含有大量白色PHB颗粒。经DGGE菌群动态分析, 发现驯化过程中菌群种类出现较为明显的演替, 而在一个反应周期(6 h)内菌种数量也有一定改变。驯化获得的高产PHB的菌群中含Acinetobacter、Bacillus、Bacteroidetes、Chryseobacteria 及proteobacteria等5个属的微生物, 菌群多样性较为丰富。     

Bacterial Flocculation and Production of Poly-β-Hydroxybutyrate

Experiments with a number of bacteria isolated from activated sludge have shown that flocculation is independent of the presence of poly-beta-hydroxybutyrate (PHB) in the cells. Several strains gave flocculent growth without any PHB detectable. Other strains, producing PHB in varying amounts, utilized this compound as an endogenous substrate, and after its disappearance the floc structure remained unchanged. The PHB content of various samples of activated sludge was found to be negligible.     

Carbon source recovery from waste activated sludge by alkaline hydrolysis and gamma-ray irradiation for biological denitrification

The recovery of an organic carbon source from a waste activated sludge by using alkaline hydrolysis and radiation treatment was studied, and the feasibility of the solubilized sludge carbon source for a biological denitrification was also investigated. The effects of an alkaline treatment and gamma-ray irradiation on a biodegradability enhancement of the sludge were also studied. A modified continuous bioreactor for a denitrification (MLE reactor) was operated by using a synthetic wastewater for 47 days. Alkaline treatment of pH 10 and gamma-ray irradiation of 20 kGy were found to be the optimum carbon source recovery conditions. COD removal of 84% and T-N removal of 51% could be obtained by using the solubilized sludge carbon source through the MLE denitrification process. It can be concluded that the carbon source recovered from the waste activated sludge was successfully employed as an alternative carbon source for a biological denitrification.     

Feast/famine growth environments and activated sludge population selection

The effect of feast/famine growth conditions on activated sludge cultures indicates that nonfilamentous cultures can be selected by providing proper substrate gradients and extended periods of endogenous metablism. Reactor operating strategies providing intermittently high substrate concentrations result in cultures characterized by high peak substrate and oxygen uptake activities, rapid settling rates, and high resistance to starvation. Sludge settleability can be manipulated using controlled variations in growth environment with corresponding changes noted in sludge activity. In combination with the low net growth rates associated with activated sludge systems, feast/famine environments would logically convey a selection advantage to microbes capable of readily assimilating substrate materials and maintaining viability during extended starvation periods.     

Variable aeration in sequencing batch reactor with aerobic granular sludge

This study investigated the effects of reduced aeration in famine period on the performance of sequencing batch reactor (SBR) with aerobic granular sludge. Aerobic granules were first cultivated in two SBRs (R1 and R2) with acetate as sole carbon source. From operation day 27, aeration rate in R1 was reduced from 1.66 to 0.55 cm s(-1) from 110 min to the end of each cycle and further reduced from 30 min to the end of each cycle from day 63. R2 as a control was operated with a constant aeration rate of 1.66 cm s(-1) in the whole cycle during the entire experimental period. Results showed that changing trends of SVI, concentration, average size and VSS/SS of biomass with time in R1 and R2 were similar although different aeration modes were adopted. At steady state, SVI of aerobic granules and biomass concentration maintained at about 40 ml g(-1) and 6 g l(-1), respectively. Average size of granules was about 750 microm in R1 while 550 microm in R2. This is the first study to demonstrate that aerobic granular sludge could be stable at reduced aeration rate in famine period during more than 3-month operation. Such an operation strategy with reduced aeration rate will lead to a significant reduction of energy consumption, which makes the aerobic granular sludge technology more competitive over conventional activated sludge process. Furthermore, the stability of aerobic granular system with variable aeration further indicates that the difference of physiology and kinetics of aerobic granule in feast and famine periods results in the different requirements of oxygen and shear stress for the stability of granules, which will deepen the understanding of mechanism of aerobic granulation in sequencing batch reactor.     

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.     

Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.     

Synthesis and degradation of poly-β-hydroxybutyrate in a sequencing batch biofilm reactor

The aim of this work was the study of poly-β-hydroxybutyrate (PHB) formation and degradation in a sequencing batch biofilm reactor (SBBR). The SBBR was operated in cycles comprising three individual phases: mixed fill, aeration and draw. A synthetic substrate solution with acetate and ammonium was used.PHB was formed during the aeration phase immediately after acetate depletion, and was subsequently consumed for biomass growth, owing to the high oxygen concentration in the reactor. It was observed a combination of suspended and biofilm growth in the SBBR with predominance of the fixed form of biomass (506 Cmmol and 2102 Cmmol, respectively). Maximum PHB fraction of suspended biomass (0.13 Cmol/Cmol) was considerably higher than that of biofilm (0.01 Cmol/Cmol). This may possibly be explained by a combination of two factors: lower mass transfer limitation of acetate and higher fraction of heterotrophs in suspended biomass compared to the ones of biofilm.     

Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.