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.

Serum antibodies to oral anaerobic bacteria in patients with rheumatoid arthritis

BACKGROUND: This study was conducted to determine the component that causes the disease in rheumatoid arthritis (RA), which shows great resemblance to periodontitis in a pathologic context. MATERIALS AND METHODS: Within this study, the pathogen-specific IgG levels formed against Porphyromonas gingivalis FDC 381, Prevotella melaninogenica ATCC 25845, Actinobacillus actinomycetemcomitans Y4, Bacteroides forsythus ATCC 43047, and Prevotella intermedia 25611 oral bacteria were researched from the blood serum samples of 30 RA patients and 20 healthy controls with the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The IgG levels of P gingivalis, P intermedia, P melaninogenica, and B forsythus were found to be significantly higher in RA patients when compared with those of the controls. Of the other bacteria antibodies, A actinomycetemcomitans was not found at greater levels in RA serum samples in comparison with the healthy samples. CONCLUSION: The antibodies formed against P gingivalis, P intermedia, P melaninogenica, and B forsythus could be important to the etiopathogenesis of RA.

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation' Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO2- uptake than NH4 uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO2- from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal. (c) 2006 Elsevier Inc. 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.

Comparison of cellulose solubilisation rates in rumen and landfill leachate inoculated reactors

The aim of this study was to conduct a number of controlled digestions to obtain easily comparable cellulose solubilisation rates and to compare these rates to those found in the literature to see which operational differences were significant in affecting cellulose degradation during anaerobic digestion. The results suggested that differences in volumetric cellulose solubilisation rates were not indicative of the true performance of cellulose digestion systems. When cellulose solubilisation rates were normalised by the mass of cellulose in the reactor at each time step, the comparison of the rates became more meaningful. Cellulose solubilisation was surface area limited. Therefore, changes in the loading rate of cellulose to the reactor altered the volumetric solubilisation rate without changing the mass normalised rate. Comparison of mass normalised solubilisation rates from this study and the literature demonstrated that differences in reactor configuration and operational conditions did not significantly impact on the solubilisation rate whereas the difference in composition of the microbial communities showed a marked effect. This work highlights the importance of using appropriately normalised data when making comparisons between systems with differing operational conditions. (c) 2005 Elsevier Ltd. All rights reserved.

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.

Efficient and stable nitritation and denitritation of ammonium-rich sludge dewatering liquor using an SBR with continuous loading

Separate treatment of dewatering liquor from anaerobic sludge digestion significantly reduces the nitrogen load of the main stream and improves overall nitrogen elimination. Such ammonium-rich wastewater is particularly suited to be treated by high rate processes which achieve a rapid elimination of nitrogen with a minimal COD requirement. Processes whereby ammonium is oxidised to nitrite only (nitritation) followed by denitritation with carbon addition can achieve this. Nitrogen removal by nitritation/denitritation was optimised using a novel SBR operation with continuous dewatering liquor addition. Efficient and robust nitrogen elimination was obtained at a total hydraulic retention time of 1 day via the nitrite pathway. Around 85-90% nitrogen removal was achieved at an ammonium loading rate of 1.2 g NH4+-N m(-3) d(-1). Ethanol was used as electron donor for denitritation at a ratio of 2.2gCODg(-1) N removed. Conventional nitritation/denitritation with rapid addition of the dewatering liquor at the beginning of the cycle often resulted in considerable nitric oxide (NO) accumulation during the anoxic phase possibly leading to unstable denitritation. Some NO production was still observed in the novel continuous mode, but denitritation was never seriously affected. Thus, process stability can be increased and the high specific reaction rates as well as the continuous feeding result in decreased reactor size for full-scale operation. (c) 2006 Elsevier Ltd. All rights reserved.

Modelling biological processes under anaerobic conditions through integrating titrimetric and off-gas measurements - applied to EBPR systems

An innovative method for modelling biological processes under anaerobic conditions is presented and discussed. The method is based on titrimetric and off-gas measurements. Titrimetric data is recorded as the addition rate of hydroxyl ions or protons that is required to maintain pH in a bioreactor at a constant level. An off-gas analysis arrangement measures, among other things, the transfer rate of carbon dioxide. The integration of these signals results in a continuous signal which is solely related to the biological reactions. When coupled with a mathematical model of the biological reactions, the signal allows a detailed characterisation of these reactions, which would otherwise be difficult to achieve. Two applications of the method to the enhanced biological phosphorus removal processes are presented and discussed to demonstrate the principle and effectiveness of the method.

Multidimensional modelling to investigate interspecies hydrogen transfer in anaerobic biofilms

Anaerobic digestion is a multistep process, mediated by a functionally and phylogenetically diverse microbial population. One of the crucial steps is oxidation of organic acids, with electron transfer via hydrogen or formate from acetogenic bacteria to methanogens. This syntrophic microbiological process is strongly restricted by a thermodynamic limitation on the allowable hydrogen or formate concentration. In order to study this process in more detail, we developed an individual-based biofilm model which enables to describe the processes at a microbial resolution. The biochemical model is the ADM1, implemented in a multidimensional domain. With this model, we evaluated three important issues for the syntrophic relationship: (i) is there a fundamental difference in using hydrogen or formate as electron carrier? (ii) Does a thermodynamic-based inhibition function produced substantially different results from an empirical function? and; (iii) Does the physical colocation of acetogens and methanogens follow directly from a general model. Hydrogen or formate as electron carrier had no substantial impact on model results. Standard inhibition functions or thermodynamic inhibition function gave similar results at larger substrate field grid sizes (> 10 mu m), but at smaller grid sizes, the thermodynamic-based function reduced the number of cells with long interspecies distances (> 2.5 mu m). Therefore, a very fine grid resolution is needed to reflect differences between the thermodynamic function, and a more generic inhibition form. The co-location of syntrophic bacteria was well predicted without a need to assume a microbiological based mechanism (e.g., through chemotaxis) of biofilm formation.

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 (N2O) 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. NO accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as NO. After mixing SNDPR sludge with other denitrifying sludge, N2O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N2O 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. (C) 2005 Elsevier B.V. All rights reserved.

Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90% identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic-aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6.6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33% of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55% of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

Spatial patterns of aerobic and anaerobic mineralization rates and oxygen penetration dynamics in coral reef sediments

Oxygen consumption rates (OCR), aerobic mineralization and sulfate reduction rates (SRR) were studied in the permeable carbonate reef sediments of Heron Reef, Australia. We selected 4 stations with different hydrodynamic regimes for this study. In situ oxygen penetration into the sediments was measured with an autonomous microsensor profiler. Areal OCR were quantified from the measured oxygen penetration depth and volumetric OCR. Oxygen penetration and dynamics (median penetration depths at the 4 stations ranged between 0.3 and 2.2 cm), OCR (median 57 to 196 mmol C m(-2) d(-1)), aerobic mineralization (median 24 to 176 mmol C m(-2) d(-1)) and SRR (median 9 to 42 mmol C m(-2) d(-1)) were highly variable between sites. The supply of oxygen by pore water advection was a major cause for high mineralization rates by stimulating aerobic mineralization at all sites. However, estimated bottom water filtration rates could not explain the differences in volumetric OCR and SRR between the 4 stations. This suggests that local mineralization rates are additionally controlled by factors other than current driven pore water advection, e.g. by the distribution of the benthic fauna or by local differences in labile organic carbon supply from sources such as benthic photosynthesis. Carbon mineralization rates were among the highest reported for coral reef sediments, stressing the role of these sediments in the functioning of the reef ecosystem.

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".