Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of "Candidatus Competibacter phosphatis", a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by "Candidatus Accumulibacter phosphatis", a known PAO, and did not contain Competibacter. In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter. Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems.     

Microbial selection on enhanced biological phosphorus removal systems fed exclusively with glucose

The microbial selection on an enhanced biological phosphorus removal (EBPR) system was investigated in a laboratory-scale sequencing batch reactor fed exclusively with glucose as the carbon source. Fluorescence In Situ Hybridization analysis was performed to target two polyphosphate accumulating organisms (PAOs) (i.e., Candidatus Accumulibacter phosphatis and Microlunatus phosphovorus) and two glycogen accumulating organisms (GAOs) (i.e., Candidatus Competibacter phosphatis and Micropruina glycogenica). The results show that glucose might not select for Candidatus Accumulibacter phosphatis. However, Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica might be selected. The highest percent relative abundance (% RA) of Candidatus Accumulibacter phosphatis was about 42%; this occurred at the beginning of the experimental period when phosphorus removal was efficient. However, the % RA of these bacteria decreased, reaching below 4% at the end of the run. The maximum % RA of Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica was about 21, 37, 17%, respectively. It appears that a higher glucose concentration might be detrimental for Microlunatus phosphovorus and Micropruina glycogenica. Results also indicate a dominance of GAOs over PAOs when EBPR systems are fed with glucose. It is possible that the GAOs outcompete the PAOs at low pH values; it has been reported that at low pH, GAOs use glycogen as the energy source to uptake glucose. As a result, P-removal deteriorated. Therefore, glucose is not a strong candidate as a carbon source to supplement EBPR systems that do not contain sufficient volatile fatty acids.     

Comparison of acetate and propionate uptake by polyphosphate accumulating organisms and glycogen accumulating organisms

Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, "Candidatus Accumulibacter phosphatis" (a known PAO) and "Candidatus Competibacter phosphatis" (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses.     

Kinetic model of a granular sludge SBR: influences on nutrient removal

A mathematical model was developed that can be used to describe an aerobic granular sludge reactor, fed with a defined influent, capable of simultaneously removing COD, nitrogen and phosphate in one sequencing batch reactor (SBR). The model described the experimental data from this complex system sufficiently. The effect of process parameters on the nutrient removal rates could therefore be reliably evaluated. The influence of oxygen concentration, temperature, granule diameter, sludge loading rate, and cycle configuration were analyzed. Oxygen penetration depth in combination with the position of the autotrophic biomass played a crucial role in the conversion rates of the different components and thus on overall nutrient removal efficiencies. The ratio between aerobic and anoxic volume in the granule strongly determines the N-removal efficiency as it was shown by model simulations with varying oxygen concentration, temperature, and granule size. The optimum granule diameter for maximum N- and P-removal in the standard case operating conditions (DO 2 mg L(-1), 20 degrees C) was found between 1.2 and 1.4 mm and the optimum COD loading rate was 1.9 kg COD m(-3) day(-1). When all ammonia is oxidized, oxygen diffuses to the core of the granule inhibiting the denitrification process. In order to optimize the process, anoxic phases can be implemented in the SBR-cycle configuration, leading to a more efficient overall N-removal. Phosphate removal efficiency mainly depends on the sludge age; if the SRT exceeds 30 days not enough biomass is removed from the system to keep effluent phosphate concentrations low.     

Net P-removal deterioration in enriched PAO sludge subjected to permanent aerobic conditions

Recently, some research in the field of enhanced biological phosphorus removal (EBPR) has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. This can occur, for example, in a full scale wastewater treatment plant during heavy rainfall periods when the anaerobic hydraulic retention time is temporarily shortened. To study this situation that could induce EBPR failure, the operation of a sequencing batch reactor (SBR) working under alternating anaerobic-aerobic conditions with an enriched EBPR population (50% Candidatus Accumulibacter phosphatis and less than 1% Candidatus Competibacter phosphatis) was shifted to strict aerobic operation. Seven cycle studies were performed during the 11 days of aerobic operation. Net P-removal was observed in this aerobic SBR during the first 4 days of operation but the system could not achieve net-P removal after this period, although the microbial composition, in terms of percentage of Accumulibacter and Competibacter, did not change significantly. The observed changes in the different compounds analysed (phosphorus, acetate, glycogen and PHB) as well as in the OUR profile indicate that metabolic changes are produced for the adaptation of PAO to aerobic conditions.     

Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge

Aerobic granular sludge technology offers a possibility to design compact wastewater treatment plants based on simultaneous chemical oxygen demand (COD), nitrogen and phosphate removal in one sequencing batch reactor. In earlier studies, it was shown that aerobic granules, cultivated with an aerobic pulse-feeding pattern, were not stable at low dissolved oxygen concentrations. Selection for slow-growing organisms such as phosphate-accumulating organisms (PAO) was shown to be a measure for improved granule stability, particularly at low oxygen concentrations. Moreover, this allows long feeding periods needed for economically feasible full-scale applications. Simultaneous nutrient removal was possible, because of heterotrophic growth inside the granules (denitrifying PAO). At low oxygen saturation (20%) high removal efficiencies were obtained; 100% COD removal, 94% phosphate (P-) removal and 94% total nitrogen (N-) removal (with 100% ammonium removal). Experimental results strongly suggest that P-removal occurs partly by (biologically induced) precipitation. Monitoring the laboratory scale reactors for a long period showed that N-removal efficiency highly depends on the diameter of the granules.     

Experimental evaluation of decrease in the activities of polyphosphate/glycogen‐accumulating organisms due to cell death and activity decay in activated sludge

Decrease in bacterial activity (biomass decay) in activated sludge can result from cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The goal of this study was to experimentally differentiate between cell death and activity decay as the cause of decrease in bacterial activity. By means of measuring maximal anaerobic phosphate release rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in situ hybridization (FISH), the decay rates and death rates of polyphosphate‐accumulating organisms (PAOs) in a biological nutrient removal (BNR) system and a laboratory phosphate removing sequencing batch reactor (SBR) system were determined, respectively, under famine conditions. In addition, the decay rate and death rate of glycogen‐accumulating organisms (GAOs) in a SBR system with an enrichment culture of GAOs were also measured under famine conditions. Hereto the maximal anaerobic volatile fatty acid uptake rates, live/dead staining, and FISH were used. The experiments revealed that in the BNR and enriched PAO‐SBR systems, activity decay contributed 58% and 80% to the decreased activities of PAOs, and that cell death was responsible for 42% and 20% of decreases in their respective activities. In the enriched GAOs system, activity decay constituted a proportion of 74% of the decreased activity of GAOs, and cell death only accounted for 26% of the decrease of their activity. Biotechnol. Bioeng. 2010; 106: 399–407. © 2010 Wiley Periodicals, Inc.     

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70 degrees C) conditions in series with a methane reactor operated at mesophilic (35 degrees C), thermophilic (55 degrees C), and hyperthermophilic (65 degrees C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22-46% at 70 degrees C, while it was 21-29% at 55 degrees C. The average protein solubilization was 44% at 70 degrees C. The double bond fatty acid removal ratio at 70 degrees C was much higher than at 55 degrees C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70 degrees C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55 degrees C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70 degrees C and methanogenesis at 55 degrees C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.     

Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal

The recently described process of simultaneous nitrification, denitrification and phosphorus removal (SNDPR) has a great potential to save capital and operating costs for wastewater treatment plants. However, the presence of glycogen-accumulating organisms (GAOs) and the accumulation of nitrous oxide (N(2)O) can severely compromise the advantages of this process. In this study, these two issues were investigated using a lab-scale sequencing batch reactor performing SNDPR over a 5-month period. The reactor was highly enriched in polyphosphate-accumulating organisms (PAOs) and GAOs representing around 70% of the total microbial community. PAOs were the dominant population at all times and their abundance increased, while GAOs population decreased over the study period. Anoxic batch tests demonstrated that GAOs rather than denitrifying PAOs were responsible for denitrification. N(2)O accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as N(2)O. After mixing SNDPR sludge with other denitrifying sludge, N(2)O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N(2)O accumulation observed in the SNDPR reactor is an artefact of the low microbial diversity facilitated by the use of synthetic wastewater with only a single carbon source.     

Factors influencing the density of aerobic granular sludge

In the present study, the factors influencing density of granular sludge particles were evaluated. Granules consist of microbes, precipitates and of extracellular polymeric substance. The volume fractions of the bacterial layers were experimentally estimated by fluorescent in situ hybridisation staining. The volume fraction occupied by precipitates was determined by computed tomography scanning. PHREEQC was used to estimate potential formation of precipitates to determine a density of the inorganic fraction. Densities of bacteria were investigated by Percoll density centrifugation. The volume fractions were then coupled with the corresponding densities and the total density of a granule was calculated. The sensitivity of the density of the entire granule on the corresponding settling velocity was evaluated by changing the volume fractions of precipitates or bacteria in a settling model. Results from granules originating from a Nereda reactor for simultaneous phosphate COD and nitrogen removal revealed that phosphate-accumulating organisms (PAOs) had a higher density than glycogen-accumulating organisms leading to significantly higher settling velocities for PAO-dominated granules explaining earlier observations of the segregation of the granular sludge bed inside reactors. The model showed that a small increase in the volume fraction of precipitates (1–5 %) strongly increased the granular density and thereby the settling velocity. For nitritation–anammox granular sludge, mainly granular diameter and not density differences are causing a segregation of the biomass in the bed.     

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.     

Nutrient removal in an A2O-MBR reactor with sludge reduction

In the present study, an advanced sewage treatment process has been developed by incorporating excess sludge reduction and phosphorous recovery in an A2O-MBR process. The A2O-MBR reactor was operated at a flux of 17 LMH over a period of 210 days. The designed flux was increased stepwise over a period of two weeks. The reactor was operated at two different MLSS range. Thermo chemical digestion of sludge was carried out at a fixed pH (11) and temperature (75 °C) for 25% COD solubilisation. The released phosphorous was recovered by precipitation process and the organics was sent back to anoxic tank. The sludge digestion did not have any impact on COD and TP removal efficiency of the reactor. During the 210 days of reactor operation, the MBR maintained relatively constant transmembrane pressure. The results based on the study indicated that the proposed process configuration has potential to reduce the excess sludge production as well as it didn’t detoriated the treated water quality.     

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

Potential of biogas recirculation to enhance biomass accumulation on supporting media

Two lab-scale anaerobic hybrid reactors (AHR) were operated to investigate the effect of recirculated biogas on the development of biomass on supporting media during the start-up. The reactor comprised of two distinct zones; sludge bed on the bottom and packed bed using nylon fiber as the media on the upper half of the reactor. Both reactors were continuously fed with cassava starch wastewater. The organic loading rate (OLR) was increased from 0.3 to 5.5 g COD/L/day by gradually decreasing the hydraulic retention time (HRT) from 37 to 3.5 days in two months. The biogas at 2.6 L/L/day was recirculated merely in the first month of the operation in order to allow the attached biomass to grow according to the organic matters present in the reactor at the final stage of the start up. Chemical oxygen demand (COD) removal efficiency of over 80% was achieved throughout the study. The result demonstrated a better COD removal efficiency for the reactor with biogas recirculation, especially at low HRTs. The amounts of biomass accumulated on the media in both reactors were slightly different with 11.9 gVSS found on the one with biogas recirculation compared to 9.8 gVSS on the other. In addition, 16.3% increase of the sludge bed was achieved with biogas recirculation as opposed to 9% in the control one. The attached biomass activity test indicated a greater amount and more favorable ratio of the methanogenic bacterial group on the media with the recirculation correlating well to a relatively higher methane content in biogas. As a result, the recirculation of biogas has a potential of improving the characteristics of the AHR especially in terms of biomass accumulation.     

Fine-scale population structure of Accumulibacter phosphatis in enhanced biological phosphorus removal sludge

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting G1PAO, G2PAO, and G3PAO groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non- Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (G1NPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the G4PAO group of Accumulibacter phosphatis, which suggests that G1NPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.     

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor

A novel bioreactor containing self-flocculated anaerobic granular sludge was developed for high-performance hydrogen production from sucrose-based synthetic wastewater. The reactor achieved an optimal volumetric hydrogen production rate of approximately 7.3 L/h/L (7,150 mmol/d/L) and a maximal hydrogen yield of 3.03 mol H2/mol sucrose when it was operated at a hydraulic retention time (HRT) of 0.5 h with an influent sucrose concentration of 20 g COD/L. The gas-phase hydrogen content and substrate conversion also exceeded 40 and 90%, respectively, under optimal conditions. Packing of a small quantity of carrier matrices on the bottom of the upflow reactor significantly stimulated sludge granulation that can be accomplished within 100 h. Among the four carriers examined, spherical activated carbon was the most effective inducer for granular sludge formation. The carrier-induced granular sludge bed (CIGSB) bioreactor was started up with a low HRT of 4-8 h (corresponding to an organic loading rate of 2.5-5 g COD/h/L) and enabled stable operations at an extremely low HRT (up to 0.5 h) without washout of biomass. The granular sludge was rapidly formed in CIGSB supported with activated carbon and reached a maximal concentration of 26 g/L at HRT = 0.5 h. The ability to maintain high biomass concentration at low HRT (i.e., high organic loading rate) highlights the key factor for the remarkable hydrogen production efficiency of the CIGSB processes.     

Effect of long-term cobalt deprivation on methanol degradation in a methanogenic granular sludge bioreactor

The effect of the trace metal cobalt on the conversion of methanol in an upflow anaerobic sludge bed (UASB) reactor was investigated by studying the effect of cobalt deprivation from the influent on the reactor efficiency and the sludge characteristics. A UASB reactor (30 degrees C; pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT). The loading rate was increased stepwise from 2.6 g chemical oxygen demand (COD) x L reactor(-1) x d(-1) to 7.8 g COD x L reactor(-1) x d(-1). Cobalt deprivation had a strong impact on the methanogenic activity of the sludge. In batch tests, the methanogenic activity of the sludge with methanol as the substrate increased 5.3 (day 28) and 2.1 (day 257) times by addition of 840 nM of cobalt. The sludge had an apparent K(m) for cobalt of 948 nM after 28 days of operation and 442 nM at the end of the run. Cobalt deprivation during 54 days of operation led to a methanol conversion efficiency of only 55%. Continuous addition of cobalt (330 nM) for 33 days improved the methanol removal efficiency to 100%. In this period of cobalt dosing, the cobalt concentration in the sludge increased 2.7 times up to 32 microg x g TSS(-1). Upon omission of the cobalt addition, cobalt washed-out at a stable rate of 0.1 microg x g VSS(-1) x d(-1). At the end of the run, the cobalt concentration of the sludge was similar to that of the seed sludge.     

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.