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.     

Comparison of polyhydroxyalkanoates production by activated sludges from anaerobic and oxic zones of an enhanced biological phosphorus removal system: effect of sludge retention time

This study compared the PHAs production behavior of sludges from the anaerobic and oxic phases of an enhanced biological phosphorus removal (EBPR) system. This was accomplished by using the kinetics and stoichiometric coefficients obtained from aerobic batch tests to evaluate the performance of these two sludges. Experimental results indicated that the metabolic behavior of the sludges for PHAs production depend significantly on the operating sludge retention time (SRT) of the EBPR system. The oxic sludge with 5 days of SRT exhibited better PHAs production performance than anaerobic sludge. Conversely, the anaerobic sludge with 15 days of SRT had superior PHAs production capability compared to oxic sludge. These comparisons suggest that whether anaerobic or oxic sludge should be employed for PHAs production depends mainly on the operating SRT of the EBPR system.     

Long-term population dynamics and in situ physiology in activated sludge systems with enhanced biological phosphorus removal operated with and without nitrogen removal

Quantitative fluorescence in situ hybridization (FISH) and the combination of FISH with microautoradiography (MAR) were used in order to study the long-term population dynamics (2.5 years) and the in situ physiology in two parallel activated sludge pilot systems with enhanced biological phosphorus removal (EBPR). The two systems received the same influent wastewater, but were differently operated (with and without nitrogen removal, respectively). Both systems showed a significant P removal that increased when different substrates (phosphorus (P), acetate and glucose, respectively) were added to the influent wastewater. Rhodocyclus-related bacteria were present in both systems in significant numbers (ranging from 4 to 28%) throughout the whole period. This supports the hypothesis that these bacteria occur in significant numbers in different types of well-operating EBPR activated sludge processes. However, we observed a lower correlation (< 0.5) for the amount of Rhodocyclus-related bacteria to the P content in activated sludge than previous studies (> 0.9). The Actinobacteria were the only additional group of bacteria which showed a similar degree of correlation to the P content in activated sludge as the Rhodocyclus-related bacteria--but only for the system without nitrogen removal. Significant amounts (< or = 12%) of glycogen-accumulating bacteria (GAOs) were detected in the system with nitrogen removal (but not in the other system), but had no, in contrast to previous observations, apparent negative effect on the overall EBPR performance. FISH-MAR indicated that a significant part of the Betaproteobacteria (part of them identified as Rhodocyclus-related bacteria) as well as the Actinobacteria were able to take up 33Pi, [3H]-acetate and [3H]-glucose under anaerobic-aerobic conditions. The contribution of anoxic 33Pi uptake under alternating anaerobic-anoxic conditions was significantly lower. Interestingly, not all Rhodocyclus-related bacteria showed uptake of these three radioactive substrates. This may be due to differences in metabolic state, physiological potential or genotype, not detectable by the present probe set for Rhodocyclus-related bacteria. Comparison of the 33Pi, [3H]-acetate and [3H]-glucose uptake by activated sludge after different fixation and incubation procedures showed that a part of the observed 33Pi uptake may have been caused by a combination of a biological and chemical or biologically induced chemical P adsorption.     

The effects of hydraulic retention time and sludge retention time on the fate of di-(2-ethylhexyl) phthalate in a laboratory-scale anaerobic-anoxic-aerobic activated sludge system

A laboratory-scale anaerobic-anoxic-aerobic (AAA) activated sludge wastewater treatment system was employed to investigate the effects of hydraulic retention time (HRT) and sludge retention time (SRT) on the removal and fate of di-(2-ethylhexyl) phthalate (DEHP). In the range from 5 to 14h, HRT had no significant effect on DEHP removal. However, longer HRT increased DEHP accumulation in the system and DEHP retention in the waste sludge. When SRT was increased from 15 to 25d, DEHP removal efficiency stayed above 96%. Compared to the removal of only 88% at SRT of 10d, longer SRT enhanced DEHP degradation efficiency. The optimal HRT and SRT for both nutrients (nitrogen and phosphorus) and DEHP removal were 8h and 15d. At these retention times, about 71% of DEHP was degraded by the activated sludge process, 26% was accumulated in the system, 2% was released in the effluent, and 1% remained in the waste sludge. The anaerobic, anoxic and aerobic reactors were responsible for 15%, 19% and 62% of the overall DEHP removal, respectively.     

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 sludge retention time on tolerance of copper toxicity for polyphosphate accumulating organisms linked to polyhydroxyalkanoates metabolism and phosphate removal

This study explored the influence of sludge retention time (SRT) on tolerance of copper invasion for polyphosphate accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR). The experimental data showed the anaerobic polyhydroxyalkanoates (PHA) storage for the sludge at 10d SRT was less influenced by copper invasion than those at 5d and 15d SRTs. The reaction of PAOs aerobically taking up phosphate for the sludge at 5d or 15d SRT almost ceased at 2 mg Cu L⁻¹, whereas PAOs in the sludge at 10d SRT retained half of the ability to take up phosphate. Both the PHAs degradation and synthesis rates decreased with increasing copper concentration, regardless of the SRTs. However, the copper inhibition of the former was greater than that of the later.     

Determination of polyhydroxyalkanoates in activated sludge by ion chromatographic and enzymatic methods

Two new detection methods for the determination of poly-beta-hydroxybutyrate (PHB) and -valerate (PHV) are described. Both methods are based on depolymerization of PHB/PHV to 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Depolymerization was achieved by either propanolic or hydrolytic digestion. Propanolic digestion transformed commercial PHB/PHV stoichiometrically into 3HB/3HV and yielded apparently complete recoveries of bacterial PHB/PHV from activated sludge. Hydrolytic digestion was suitable only for PHB determination. For quantification of 3HB and 3HV directly from digested sludge, a method based on ion-exchange chromatography and conductivity detection was developed (IC-method). Alternatively, the total of 3HB and 3HV was quantified using a commercial enzymatic test kit and colorimetric detection (enzyme method). Both detection methods are easier to perform than previous methods and are suitable for complex matrices such as activated sludge. The IC-method is recommended for high sample throughputs or if distinction between PHB and PHV is essential. Enzymatic detection is recommended if a few samples per day have to be measured immediately or if an ion chromatograph is unavailable.     

Effect of low solids retention time and focused pulsed pre-treatment on anaerobic digestion of waste activated sludge

The interacting effects of Focused Pulsed (FP) treatment and solids retention time (SRT) were evaluated in laboratory-scale digesters operated at SRTs of 2-20 days. Anaerobic digestion and methanogenesis of waste activated sludge (WAS) were stable for SRT ? 5 days, but the effluent soluble organic compounds increased significantly for SRT = 2 days due to a combination of faster hydrolysis kinetics and washout of methanogens. FP treatment increased the CH₄ production rate and TCOD removal efficiency by up to 33% and 18%, respectively, at a SRT of 20 days. These effects were the result of an increase in the hydrolysis rate, since the concentrations of soluble components remained low for SRT ? 5 days. Alternately, FP pre-treatment of WAS allowed the same conversion of TCOD to CH₄ with a smaller SRT and digester size: e.g., 40% size savings with a CH₄ conversion of 0.23 g CH₄-COD/g COD_in.     

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.     

Batch production of polyhydroxyalkanoate by low-polyphosphate-content activated sludge at varying pH

Polyhydroxyalkanoate (PHA), a biodegradable plastic, can be produced from excess activated sludge by utilizing intracellular glycogen and polyphosphate as energy sources under growth-limiting conditions. Activated sludge of 2%, 6%, and 8% polyphosphate with similar glycogen content of 33% was investigated for batch PHA production by varying the pH values from 6 to 8. Acetate applied at 1000 mg COD/L was almost exhausted within 80 min of anaerobic stage. The remaining glycogen in the sludge was higher at a lower pH because of less energy used for acetate uptake. Highest PHA content of 51% was obtained from sludge with an 8% polyphosphate content at pH 8. PHA production occurred rapidly within the first 20 min, with a productivity rate of 2.19 g PHA/L-h. The results in this study indicate that PHA production by using activated sludge is a promising alternative to a typical pure culture approach.     

Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation

To reduce the production cost of polyhydroxyalkanoates (PHA) and disposal amount of excess sludge simultaneously, the feasibility of using fermentative volatile fatty acids (VFAs) as carbon sources to synthesize PHA by activated sludge was examined. At pH 11.0, 60 degrees C and fermentative time of 7d, the VFAs yield was 258.65 mgTOC/gVSS. To restrain cell growth during PHA production, the released phosphorus and residual ammonium in the fermentative VFAs was recovered by the formation of struvite precipitation. Acetic acid was the predominant composition of the fermentative VFAs. PHA accumulation in excess sludge occurred feeding by fermentative VFAs with aerobic dynamic feeding process. The maximum PHA content accounted for 56.5% of the dry cell. It can be concluded from this study that the VFAs generated from excess sludge fermentation were a suitable carbon source for PHA production by activated sludge.     

Active heterotrophic biomass and sludge retention time (SRT) as determining factors for biodegradation kinetics of pharmaceuticals in activated sludge

The present study investigates the biodegradation of pharmaceutically active compounds (PhACs) by active biomass in activated sludge. Active heterotrophs (X_bh) which are known to govern COD removal are suggested as a determining factor for biological PhAC removal as well. Biodegradation kinetics of five polar PhACs were determined in activated sludge of two wastewater treatment plants which differed in size, layout and sludge retention time (SRT).Results showed that active fractions of the total suspended solids (TSS) differed significantly between the two sludges, indicating that TSS does not reveal information about heterotrophic activity. Furthermore, PhAC removal was significantly faster in the presence of high numbers of heterotrophs and a low SRT. Pseudo first-order kinetics were modified to include X_bh and used to describe decreasing PhAC elimination with increasing SRT.     

Production of medium-chain-length polyhydroxyalkanoates by activated sludge enriched under periodic feeding with nonanoic acid

The potential use of activated sludge for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was investigated. The enrichment of bacterial populations capable of producing MCL-PHAs was achieved by periodic feeding with nonanoic acid in a sequencing batch reactor (SBR). Denaturing gradient gel electrophoresis analysis revealed Pseudomonas aeruginosa strains to be predominant in the bacterial community during the SBR process. The composition of PHA synthesized by the enriched biomass from nonanoic acid consisted of a large concentration (>89 mol%) of MCL monomer units and a small amount of short-chain-length monomer units. Under fed-batch fermentation with continuous feeding of nonanoic acid at a flow rate of 0.225 g/L/h and a C/N ratio of 40, a maximum PHA content of 48.6% dry cell weight and a conversion yield (Y_p/s) of 0.94 g/g were achieved. These results indicate that MCL-PHA production by activated sludge is a promising alternative to typical pure culture approaches.     

Influence of hydraulic retention time on anaerobic digestion of pretreated sludge

Continuous anaerobic digestion of waste activated sludge pretreated at low temperatures below 100°C increased methane generation by 30%. pH values of the digestion mixture increased, approximately from 0.3 to 0.55 by pretreatment, although its volatile fatty acids concentration was greater than the control. An abrupt increase in propionate : acetate ratio in digestion stage (e.g. from less than 1:1 to over 3.5 :1), provided a reliable indicator for impending failure.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

Ultrasonic sludge disintegration for enhanced methane production in anaerobic digestion: effects of sludge hydrolysis efficiency and hydraulic retention time

Hydrolysis of waste activated sludge (WAS) has been regarded as the rate limiting step of anaerobic sludge digestion. Therefore, in this study, the effect of ultrasound and hydraulic residence time during sludge hydrolysis was investigated with the goal of enhancing methane production from anaerobic digestion (AD). WAS was ultrasonically disintegrated for hydrolysis, and it was semi-continuously fed to an anaerobic digesters at various hydraulic retention times (HRTs). The results of these experiments showed that the solids and chemical oxygen demand (COD) removal efficiencies when using ultrasonically disintegrated sludge were higher during AD than the control sludge. The longer the HRT, the higher the removal efficiencies of solids and COD, while methane production increased with lower HRT. Sludge with 30% hydrolysis produced 7 × more methane production than the control sludge. The highest methane yields were 0.350 m(3)/kg volatile solids (VS)(add) and 0.301 m(3)/kg COD(con) for 16 and 30% hydrolyzed sludge, respectively. In addition, we found that excess ultrasound irradiation may inhibit AD since the 50% hydrolyzed sludge produced lower methane yields than 16 and 30% hydrolyzed sludge.     

Metabolic influence of lead on polyhydroxyalkanoates (PHA) production and phosphate uptake in activated sludge fed with glucose or acetic acid as carbon source

Sludge in a sequential batch reactor (SBR) system was used to investigate the effect of lead toxicity on metabolisms of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) communities fed with acetic acid or glucose as their sole carbon source, respectively. Results showed that the effect of lead on substrate utilization of both PAOs and GAOs was insignificant. However, lead substantially inhibited both of phosphate release and uptake of PAOs. In high concentration of acetic acid trials, an abnormal aerobic phosphate release was observed instead of phosphate uptake and the release rate increased with increasing lead concentration. Results also showed that PAOs could normally synthesize polyhydroxybutyrate (PHB) in the anaerobic phase even though lead concentration was 40 mg L⁻¹. However, they could not aerobically utilize PHB normally in the presence of lead. On the other hand, GAOs could not normally metabolize polyhydroxyvalerate (PHV) in both the anaerobic and aerobic phases.     

Long-term nitrogen deposition increases phosphorus limitation of bryophytes in an ombrotrophic bog

Here we investigate the effect of 4 years simulated atmospheric deposition of ammonium (NH₄) and nitrate (NO₃), applied alone or in combination with phosphorus and potassium (PK), on the surface phosphatase activities and nutrient acquisition behaviour of two species of moss (Sphagnum capillifolium and Hypnum jutlandicum) from an ombrotrophic peatland. Phosphatase activity was significantly enhanced by both the NH₄ and NO₃ treatments, particularly for Sphagnum, but the activity decreased when exposed to additions of PK. Regression analysis revealed that phosphatase activity on Sphagnum was positively related with tissue N and negatively related to tissue P concentrations. For Hypnum, a negative relationship between shoot P concentration and phosphatase activity was observed. Using a ³²P tracer, mosses removed from plots receiving PK in combination with NH₄ maintained their affinity for increased phosphorus uptake. These findings suggest that enhanced nutrient supply, even at modest doses, significantly alter the nutrient recycling behaviour of bryophytes.     

Effect of azithromycin on enhancement of methane production from waste activated sludge

In the methane production from waste activated sludge (WAS), complex bacterial interactions in WAS have been known as a major contribution to methane production. Therefore, the influence of bacterial community changes toward methane production from WAS was investigated by an application of antibiotics as a simple means for it. In this study, azithromycin (Azm) as an antibiotic was mainly used to observe the effect on microbial changes that influence methane production from WAS. The results showed that at the end of fermentation, Azm enhanced methane production about twofold compared to control. Azm fostered the growth of acid-producing bacterial communities, which synthesized more precursors for methane formation. DGGE result showed that the hydrolysis as well as acetogenesis stage was improved by the dominant of B1, B2 and B3 strains, which are Clostridium species. In the presence of Azm, the total population of archaeal group was increased, resulting in higher methane productivity achievement.