Comparative analysis of biological phosphate removal (BPR) and non-BPR activated sludge bacterial communities with particular reference to Acinetobacter

The bacterial community of a biological phosphate removal (BPR) activated sludge process was studied and compared to that of a non-BPR process treating the same municipal waste water. Bacterial isolates from the BPR process, as characterized by whole cell fatty acids, belonged to more than twenty genera, with Micrococcus, Staphylococcus and Acidovorax scoring highest. Acinetobacter spp represented 4% of cultured bacteria, ≤3% as estimated by fluorescence in situ hybridization, and well under 10% on the basis of the proportion of ubiquinone Q9 in the sludge. The mole proportions of ubiquinones, Q8 : Q10 : Q9 in the sludge were maintained fairly stable at approximately 9:4:1. The spectra of the isolated strains and the proportions of ubiquinones in the processes (BPR vs non-BPR) were otherwise similar, but a significant number of isolates related to actinomycetes were obtained from the BPR sludge only. The BPR process did not enrich Acinetobacter. Pure cultures of Acinetobacter isolated from the sludge stained for polyphosphate, but Acinetobacter cells responding to the ACA probe in native sludge from the BPR process did not. Instead, the bulk of the polyphosphate in the BPR sludge was located in a distinct morphotype of large, coccoid, highly clustered cells. Received 3 August 1998/ Accepted in revised form 29 October 1998     

Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge

The coaggregation traits of two pairs of sewage sludge bacteria were tested and characterized. Oligotropha carboxidovorans S23 coaggregated with two strains of the genus Acinetobacter viz. Acinetobacter junii S33 (56%) and Acinetobacter johnsonii S35 (99%). Coaggregates of O. carboxidovorans S23 and A. junii S33 were small (20-40 microm), weak and susceptible to EDTA and a commercial protease (Actinase E). Actinase/periodate pretreatment of the partners prior to coaggregation revealed that interaction in this case was mediated by protein surface components. Coaggregates of O. carboxidovorans S23 and A. johnsonii S35 were large (above 100 microm), strong and not deflocculated by EDTA or Actinase E. Only periodate pretreatment of A. johnsonii S35 prevented this coaggregation indicating a role for a carbohydrate-containing moiety without the involvement of protein components. The potential mechanisms and strength of bacterial coaggregations seem to be pair dependent.     

Enhancement of the activated sludge process by activated carbon produced from surplus biological sludge

Surplus biological sludge from wastewater treatment operations was converted into activated carbon and then added to the aerated vessel of an activated sludge process treating phenol and glucose. The addition of activated carbon, either sludge-based or commercial, enhanced phenol removal from 58 to 98.7% and from 87 to 93% for COD with feed concentrations of 100 mg phenol l^–1 and 2500 mg COD l^–1. No differences were found between the activated sludge-activated carbon bench scale continuous reactors operating with either commercial or sludge-based activated carbon in spite of the higher adsorption capacity of the former.     

Polyphosphate production by strains of Acinetobacter

Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA3123. No correlation was found between the polyphosphate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.     

A general kinetic model for biological nutrient removal activated sludge systems: model evaluation

Hu et al. (2007) presented a general kinetic model for biological nutrient removal (BNR) activated sludge (AS) systems in general, but for external nitrification (EN) BNRAS (ENBNRAS) systems in particular. In this article, this model is evaluated against a large number of experimental data sets. In this evaluation, the model is first used to simulate a wide variety of conventional internal nitrification (IN) BNRAS systems to evaluate its predictions and also evaluate the model parameters suggested by Hu et al. (2007), and to calibrate those constants for which values are not available in the literature. Simulation results indicate that the model, with appropriately calibrated parameters, is capable of predicting COD removal, nitrification and denitrification and two types of biological excess phosphorus removal (BEPR), namely aerobic and anoxic/aerobic P uptake BEPR. The model is then used to simulate the ENBNRAS systems to evaluate its capacity of simulating the behaviour of this system. Simulation results show that the model is capable of simulating the behaviour of the ENBNRAS systems, including COD, nitrification, denitrification and BEPR, particularly anoxic P uptake BEPR, with the values of kinetic and stoichiometric parameters obtained in modelling conventional BNRAS systems, except for micro(NIT), K(MP), eta(PAO) and eta(H) which required calibration.     

Differentiation of polyphosphate and poly-β-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.     

RAPD-PCR typing of Acinetobacter isolates from activated sludge systems designed to remove phosphorus microbiologically

AIMS: This study investigated whether there were differences in RAPD fingerprints between already described genomic species of Acinetobacter and those from activated sludge systems. Whether plant-specific populations of acinetobacters exist was also examined. METHODS AND RESULTS: Fifty-two isolates of Acinetobacter from four biological phosphorus removal (EBPR) systems of different configurations, and the known genomic species, were characterized using RAPD-PCR, and fragments separated on agarose gels. Patterns were analysed using Gel Pro software and data analysed numerically. RAPD-PCR produced patterns suggesting that many environmental isolates differ from known genomic species. In two cases, strains from individual plants clustered closely enough together to imply that there may be plant-specific populations of acinetobacters. CONCLUSION: The data suggest that current understanding of the taxonomic status of Acinetobacter may need modifying to accommodate non-clinical isolates, as many of the clusters emerging after numerical analysis of RAPD-PCR fragments from activated sludge isolates were quite separate from the clusters containing the already described genomic species. Some evidence was also obtained from the clusters generated to support a view that particular populations of Acinetobacter may occur in individual activated sludge plants. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest that the current understanding of the systematics of Acinetobacter, based as it is almost exclusively on clinical isolates, may need drastic revision to accommodate environmental strains. They also suggest that a re-examination of the importance and role of Acinetobacter in the activated sludge process may be appropriate.     

A general kinetic model for biological nutrient removal activated sludge systems: model development

In this article, a kinetic model is developed and presented for biological nutrient removal (BNR) activated sludge (BNRAS) systems in general, but for external nitrification (EN) BNRAS (ENBNRAS) systems in particular. The model is based on the UCTPHO model, but includes some significant modifications, such as anoxic P uptake and associated denitrification by phosphorus accumulating organisms (PAOs). Some key features of the model are described and discussed before the model is presented. Model evaluation will be addressed in another article (Hu et al., 2007).     

A simple method to release polyphosphate from activated sludge for phosphorus reuse and recycling

In enhanced biological phosphorus removal processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP). It was discovered that nearly all of the polyP could be released from activated sludge simply by heating it at 70 degrees C for about 1 h. The chain length of released polyP ranged from 100 to 200 phosphate (P(i)) residues. The addition of CaCl(2) precipitated approximately 75% of the total phosphorus without pH adjustment. The formed precipitate contained more P and less Ca than typical natural phosphorite deposits. Hence, in combination with enhanced biological phosphorus removal, the present method has potential for the development of a simple process for recovering phosphorus in a reusable form from wastewater.     

Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. We investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. We observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. From these observations, we postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimisation of EBPR. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 507–515, 1999.     

Actinomycete diversity associated with foaming in activated sludge plants

Large numbers of mycolic acid-containing actinomycetes were isolated from foam and scum samples taken from three activated-sludge sewage-treatment plants using several selective isolation media. Organisms presumptively identified as gordonae formed the dominant population in all of the samples. A representative set of these strains have chemical properties consistent with their classification in the genusGordona. Forty-eight of theGordona strains were compared through 165 unit characters with the type strains of validly described species ofGordona. The resultant data were examined using the Jaccard and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. The numerical classification was only marginally affected by the statistics used or by test error, estimated as 3.92%. The isolates were assigned to five multi-membered and 28 single-membered clusters defined by the simple matching coefficient at the 89% similarity level. With few exceptions, the isolates were sharply separated from theGordona marker strains. Essentially the same classification was obtained when the test strains were examined using a Curie-point pyrolysis mass spectrometric procedure. It can be concluded that the gordonae form a heterogeneous taxonomic group, the members of which can be distinguished from representatives of validly described species ofGordona.     

Impact of enzymatic pre-hydrolysis on batch activated sludge systems dealing with oily wastewaters

Dairy wastewater containing different oil and grease contents was treated in batch activated sludge systems with and without (control) an enzymatic pre-hydrolysis stage [with 0.2% (w/v) of fermented babassu cake containing Penicillium restrictum lipases]. When the oil and grease concentration in the control bioreactor was increased (400, 600 and 800 mg l^–1), the COD removal efficiency fell (86%, 75% and 0%). However, in the reactor fed with pre-hydrolysed wastewater, COD removal efficiency was maintained (93%, 92% and 82%). At an oil and grease concentration of 800 mg l^–1, the control bioreactor presented final volatile suspended solids (VSS) values ten times greater (2225 mg l^–1) than those obtained for bioreactor fed with pre-hydrolysed wastewater (200 mg l^–1).     

Phylogeny and in situ identification of a novel gammaproteobacterium in activated sludge

Failure of a continuously aerated sequencing batch reactor (SBR) pilot plant-enhanced biological phosphorus removal (EBPR) process, designed to remove phosphorus from the clarified effluent from a conventional non-EBPR wastewater treatment plant, was associated with the dominance ( c . 50% of the biovolume) of gammaproteobacterial coccobacilli. Flow cytometry and subsequent clone library generation from an enriched population of these Gammaproteobacteria showed that their 16S rRNA genes were most similar to partial clone sequences obtained from an actively denitrifying SBR community, and from anaerobic : aerobic EBPR communities. Under the SBR operating conditions used here, these cells stained for poly-β-hydroxyalkanoates, but never polyphosphate. Applying FISH probes designed against them in combination with microautoradiography showed that they could also assimilate acetate 'aerobically'. FISH analyses of biomass samples from the full-scale treatment plant providing the pilot plant feed showed that they were present there in high numbers. However, they were not detected by FISH in laboratory-scale communities of the same aerated laboratory-scale EBPR process even when EBPR had failed, or from several full-scale EBPR plants or other activated sludge processes.     

Diversity and assembly patterns of activated sludge microbial communities: A review

Understanding diversity and assembly patterns of microbial communities in activated sludge (AS) is pivotal for addressing fundamental ecological questions and wastewater treatment engineering. Recent applications of molecular methods especially high-throughput sequencing (HTS) have led to the explosion of information about AS community diversity, including the identification of uncultured taxa, and characterization of low-abundance but environmentally important populations such as antibiotic resistant bacteria and pathogens. Those progresses have facilitated the leverage of ecological theories in describing AS community assembly. The lognormal species abundance curve has been applied to estimate AS microbial richness. Taxa-area and taxa-time relationships (TAR and TTR) have been observed for AS microbial communities. Core AS microbial communities have been identified. Meanwhile, the roles of both deterministic and stochastic processes in shaping AS community structures have been examined. Nonetheless, it remains challenging to define tempo-spatial scales for reliable identification of community turnover, and find tight links between AS microbial structure and wastewater treatment plant (WWTP) functions. To solve those issues, we expect that future research will focus on identifying active functional populations in AS using omics- methods integrated with stable-isotope probing (SIP) with the development of bioinformatics tools. Developing mathematic models to understand AS community structures and utilize information on AS community to predict the performance of WWTPs will also be vital for advancing knowledge of AS microbial ecology and environmental engineering.     

Growth inhibition of activated sludge by humus

Humus at 0.5 to 1 mg l–1 inhibited growth of activated sludge by about 55% in batch and long-term repeated batch cultures without decreasing sugar utilization. The growth inhibition was considerable when concentrations of substrates in the medium supplied per unit weight of activated sludge were low.     

Examination into the gamma irradiation of activated sludge

This study has shown that the treatment of activated sludge by gamma irradiation resulted in a deterioration in the filterability, a decrease in the size of the floc particles and an increase in the organic matter present in the sludge supernatant. A significant difference was found between the results obtained for filamentous and non-filamentous sludges in relation to the amount of soluble polysaccharide produced.     

In situ detection of protein-hydrolysing microorganisms in activated sludge

Protein hydrolysis plays an important role in the transformation of organic matter in activated sludge wastewater treatment plants, but no information is currently available regarding the identity and ecophysiology of protein-hydrolysing organisms (PHOs). In this study, fluorescence in situ enzyme staining with casein and bovine serum albumin conjugated with BODIPY dye was applied and optimized to label PHOs in activated sludge plants. A strong fluorescent labeling of the surface of microorganisms expressing protease activity was achieved. Metabolic inhibitors were applied to inhibit the metabolic activity to prevent uptake of the fluorescent hydrolysates by oligopeptide-consuming bacteria. In five full-scale, nutrient-removing activated sludge plants examined, the dominant PHOs were always different morphotypes of filamentous bacteria and the epiflora attached to many of these. The PHOs were identified by FISH using a range of available oligonucleotide probes. The filamentous PHOs belonged to the candidate phylum TM7, the phylum Chloroflexi and the class Betaproteobacteria. In total they comprised 1-5% of the bacterial biovolume. Most of the epiflora-PHOs hybridized with probe SAP-309 targeting Saprospiraceae in the phylum Bacteroidetes and accounted for 8-12% of the total bacterial biovolume in most plants and were thus an important and dominant part of the microbial communities.     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998