Short-term effects of carbon source on the competition of polyphosphate accumulating organisms and glycogen accumulating organisms

The effectiveness of enhanced biological phosphorus removal (ESPR) systems is directly affected by the competition of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigated the short-term effects of carbon source on PAO and GAO performance. The tests were designed to clearly determine the impact of volatile fatty acid (VFA) composition on the performance of two types of biomass, one enriched for PAOs and the other for GAOs. The two populations were enriched in separate reactors using identical operating conditions and very similar influent compositions with acetate as the sole carbon source. The only difference was that a very tow level of phosphorus was present in the influent to the GAO reactor. The abundance of PAOs and GAOs was quantified using fluorescence in-situ hybridisation. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to utilise different carbon substrates. While both are able to take up acetate rapidly and completely, the GAOs are far slower at consuming propionate than the PAOs during short-term substrate changes. This provides a potentially highly valuable avenue to influence the competition between PAOs and GAOs. Other VFAs studied seem to be less usable in the short term by both PAOs and GAOs; as indicated by their much lower uptake rates.

Focused beam reflectance technique for in situ particle sizing in wastewater treatment settling tanks

Due to the complexities involved with measuring activated sludge floc size distributions, this parameter has largely been ignored by wastewater researchers and practitioners. One of the major reasons has been that instruments able to measure particle size distributions were complex, expensive and only provided off-line measurements. The Focused Beam Reflectance Method (FBRM) is one of the rare techniques able to measure the particle size distribution in situ. This paper introduces the technique for monitoring wastewater treatment systems and compares its performance with other sizing techniques. The issue of the optimal focal point is discussed, and similar conclusions as found in the literature for other particulate systems are drawn. The study also demonstrates the capabilities of the FBRM in evaluating the performance of settling tanks. Interestingly, the floc size distributions did not vary with position inside the settling tank flocculator. This was an unexpected finding, and seriously questioned the need for a flocculator in the settling tank. It is conjectured that the invariable size distributions were caused by the unique combination of high solids concentration, low shear and zeolite dosing. (C) 2004 Society of Chemical Industry.

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_TI038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to,study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [C-13]acetate was used in SIP to label the DNA of the denitrifiers. The [C-13]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the C-13 library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking Up [C-14] acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the waste-water industry to enhance denitrification.

Challenges for simultaneous nitrification, denitrification, and phosphorus removal in microbial aggregates: mass transfer limitation and nitrous oxide production

The microbial community composition and activity was investigated in aggregates from a lab-scale bioreactor, in which nitrification, denitrification and phosphorus removal occurred simultaneously. The biomass was highly enriched for polyphosphate accumulating organisms facilitating complete removal of phosphorus from the bulk liquid; however, some inorganic nitrogen still remained at the end of the reactor cycle. This was ascribed to incomplete coupling of nitrification and denitrification causing NO3- accumulation. After 2 h of aeration, denitrification was dependent on the activity of nitrifying bacteria facilitating the formation of anoxic zones in the aggregates; hence, denitrification could not occur without simultaneous nitrification towards the end of the reactor cycle. Nitrous oxide was identified as a product of denitrification, when based on stored PHA as carbon source. This observation is of critical importance to the outlook of applying PHA-driven denitrification in activated sludge processes. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis. (c) 2005 Wiley Periodicals, Inc.

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. (c) 2005 Wiley Periodicals, Inc.

Optimisation of poly-b-hydroxyalkanoate analysis using gas chromatography for enhanced biological phosphorus removal systems

Poly-beta-hydroxyalkanoate (PHA) is a polymer commonly used in carbon and energy storage for many different bacterial cells. Polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), store PHA anaerobically through metabolism of carbon substrates such as acetate and propionate. Although poly-beta-hydroxybutyrate (PHB)and poly-beta-hydroxyvalerate (PHV) are commonly quantified using a previously developed gas chromatography (GC) method, poly-beta-hydroxy-2-methyl valerate (PH2MV) is seldom quantified despite the fact that it has been shown to be a key PHA fraction produced when PAOs or GAOs metabolise propionate. This paper presents two GC-based methods modified for extraction and quantification of PHB, PHV and PH2MV from enhanced biological phosphorus removal (EBPR) systems. For the extraction Of PHB and PHV from acetate fed PAO and GAO cultures, a 3% sulfuric acid concentration and a 2-20 h digestion time is recommended, while a 10% sulfuric acid solution digested for 20 h is recommended for PHV and PH2MV analysis from propionate fed EBPR systems. (c) 2005 Elsevier B.V. All rights reserved.

Anaerobic and aerobic metabolism of glycogen-accumulating organisms selected with propionate as the sole carbon source

In the microbial competition observed in enhanced biological phosphorus removal (EBPR) systems, an undesirable group of micro-organisms known as glycogen-accumulating organisms (GAOs) compete for carbon in the anaerobic period with the desired polyphosphate-accumulating organisms (PAOs). Some studies have suggested that a propionate carbon source provides PAOs with a competitive advantage over GAOs in EBPR systems; however, the metabolism of GAOs with this carbon source has not been previously investigated. In this study, GAOs were enriched in a laboratory-scale bioreactor with propionate as the sole carbon source, in an effort to better understand their biochemical processes. Based on comprehensive solid-, liquid- and gas-phase chemical analytical data from the bioreactor, a metabolic model was proposed for the metabolism of propionate by GAOs. The model adequately described the anaerobic stoichiometry observed through chemical analysis, and can be a valuable tool for further investigation of the competition between PAOs and GAOs, and for the optimization of the EBPR process. A group of Alphaproteobacteria dominated the biomass (96% of Bacteria) from this bioreactor, while post-fluorescence in situ hybridization (FISH) chemical staining confirmed that these Alphaproteobacteria produced poly-beta-hydroxyalkanoates (PHAs) anaerobically and utilized them aerobically, demonstrating that they were putative GAOs. Some of the Alphaproteobacteria were related to Defluvicoccus vanus (16% of Bacteria), but the specific identity of many could not be determined by FISH. Further investigation into the identity of other GAOs is necessary.

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. (c) 2005 Elsevier B.V. All rights reserved.

Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, Candidatus Accumulibacter phosphatis. DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of non-pure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater. Keywords DNA extraction; EBPR; metagenomic library; 16S rRNA gene.

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3) d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L-1. Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosonlonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662. (c) 2005 Elsevier Ltd. All rights reserved.