L. Borrás

Metabolic shift of polyphosphate-accumulating organisms with different levels of polyphosphate storage

Previous studies have shown that polyphosphate-accumulating organisms (PAOs) are able to behave as glycogen-accumulating organisms (GAOs) under different conditions. In this study we investigated the behavior of a culture enriched with Accumulibacter at different levels of polyphosphate (poly-P) storage. The results of stoichiometric ratios Gly(degraded)/HAc(uptake), PHB(synthesized)/HAc(uptake), PHV(synthesized)/HAc(uptake) and P(release)/HAc(uptake) confirmed a metabolic shift from PAO metabolism to GAO metabolism: PAOs with high poly-P content used the poly-P to obtain adenosine tri-phosphate (ATP), and glycogen (Gly) to obtain nicotinamide adenine dinucleotide (NADH) and some ATP. In a test where poly-P depletion was imposed on the culture, all the acetate (HAc) added in each cycle was transformed into polyhydroxyalkanoate (PHA) despite the decrease of poly-P inside the cells. This led to an increase of the Gly(degraded)/HAc(uptake) ratio that resulted from a shift towards the glycolytic pathway in order to compensate for the lack of ATP formed from poly-P hydrolysis. The shift from PAO to GAO metabolism was also reflected in the change in the PHA composition as the poly-P availability decreased, suggesting that polyhydroxyvalerate (PHV) is obtained due to the consumption of excess reducing equivalents to balance the internal NADH, similarly to GAO metabolism. Fluorescence in situ hybridization analysis showed a significant PAO population change from Type I to Type II Accumulibacter as the poly-P availability decreased in short term experiments. This work suggests that poly-P storage levels and GAO-like metabolism are important factors affecting the competition between different PAO Types in enhanced biological phosphorus removal systems.

Using SOM and PCA for analysing and interpreting data from a P-removal SBR

This paper focuses on the application of Kohonen self-organizing maps (SOM) and principal component analysis (PCA) to thoroughly analyse and interpret multidimensional data from a biological process. The process is aimed at enhanced biological phosphorus removal (EBPR) from wastewater. In this work, SOM and PCA are firstly applied to the data set in order to identify and analyse the relationships among the variables in the process. Afterwards, K-means algorithm is used to find out how the observations can be grouped, on the basis of their similarity, in different classes. Finally, the information obtained using these intelligent tools is used for process interpretation and diagnosis. In the data set analysed, both techniques yielded similar results regarding the relationships among the variables and the clustering of the observations (i.e., the same groups of observations were identified) and, therefore, identical process interpretation could be made. The cluster analysis allowed relating the observations to process behaviour, clearly distinguishing start-up, desirable and poor process conditions. The results demonstrate that the applied techniques are highly effective to compress multidimensional data sets and to extract relevant information from the process, making the interpretation and diagnosis much easier and evident.

Interactions between calcium precipitation and the polyphosphate-accumulating bacteria metabolism

A sequencing batch reactor that is operated for biological phosphorus removal has been operated under different influent calcium concentrations to study the precipitation process and the possible effects of phosphorus precipitation in the biological phosphorus removal process. Four experiments were carried out under different influent calcium concentrations ranging from 10 to 90 g Ca m(-3). The experimental results and the equilibrium study, which are based on the saturation index calculation, confirm that the process controlling the calcium behaviour is the calcium phosphate precipitation. This precipitation takes place at two stages: initially, precipitation of the amorphous calcium phosphate, and later crystallization of hydroxyapatite. Also the accumulation of phosphorus precipitated was observed when the influent calcium concentration was increased. In all the experiments, the influent wastewater ratio P/COD was kept constant. It has been observed that, at high calcium concentration, the ratio between phosphate release and acetate uptake (P(rel)/Ac(uptake)) decreases. Changes in the polyphosphate-accumulating organism (PAO) population and in the glycogen-accumulating organism (GAO) population during the experimental period were ruled out by means of fluorescence in situ hybridization. These results could suggest that PAO are able to change their metabolic pathways based on external conditions, such as influent calcium concentration. The accumulation of phosphorus precipitated as calcium phosphate at high influent calcium concentration throughout the experimental period confirmed that phosphate precipitation is a process that can affect the PAO metabolism.

Anaerobic central metabolic pathways active during polyhydroxyalkanoate production in uncultured cluster 1 Defluviicoccus enriched in activated sludge communities

A glycogen nonpolyphosphate-accumulating organism (GAO) enrichment culture dominated by the Alphaproteobacteria cluster 1 Defluviicoccus was investigated to determine the metabolic pathways involved in the anaerobic formation of polyhydroxyalkanoates, carbon storage polymers important for the proliferation of microorganisms in enhanced biological phosphorus removal processes. FISH-microautoradiography and post-FISH fluorescent chemical staining confirmed acetate assimilation as polyhydroxyalkanoates in cluster 1 Defluviicoccus under anaerobic conditions. Chemical inhibition of glycolysis using iodoacetate, and of isocitrate lyase by 3-nitropropionate and itaconate, indicated that carbon is likely to be channelled through both glycolysis and the glyoxylate cycle in cluster 1 Defluviicoccus. The effect of metabolic inhibitors of aconitase (monofluoroacetate) and succinate dehydrogenase (malonate) suggested that aconitase, but not succinate dehydrogenase, was active, providing further support for the role of the glyoxylate cycle in these GAOs. Metabolic inhibition of fumarate reductase using oxantel decreased polyhydroxyalkanoate production. This indicated reduction of fumarate to succinate and the operation of the reductive branch of the tricarboxylic acid cycle, which is possibly important in the production of the polyhydroxyvalerate component of polyhydroxyalkanoates observed in cluster 1 Defluviicoccus enrichment cultures. These findings were integrated with previous metabolic models for GAOs and enabled an anaerobic central metabolic pathway model for polyhydroxyalkanoate formation in cluster 1 Defluviicoccus to be proposed.

Identification and quantification of microbial populations in activated sludge and anaerobic digestion processes

Eight different phenotypes were studied in an activated sludge process (AeR) and anaerobic digester (AnD) in a full-scale wastewater treatment plant by means of fluorescent in situ hybridization (FISH) and automated FISH quantification software. The phenotypes were ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, denitrifying bacteria, phosphate-accumulating organisms (PAO), glycogen-accumulating organisms (GAO), sulphate-reducing bacteria (SRB), methanotrophic bacteria and methanogenic archaea. Some findings were unexpected: (a) Presence of PAO, GAO and denitrifiers in the AeR possibly due to unexpected environmental conditions caused by oxygen deficiencies or its ability to survive aerobically; (b) presence of SRB in the AeR due to high sulphate content of wastewater intake and possibly also due to digested sludge being recycled back into the primary clarifier; (c) presence of methanogenic archaea in the AeR, which can be explained by the recirculation of digested sludge and its ability to survive periods of high oxygen levels; (d) presence of denitrifying bacteria in the AnD which cannot be fully explained because the nitrate level in the AnD was not measured. However, other authors reported the existence of denitrifiers in environments where nitrate or oxygen was not present suggesting that denitrifiers can survive in nitrate-free anaerobic environments by carrying out low-level fermentation; (e) the results of this paper are relevant because of the focus on the identification of nearly all the significant bacterial and archaeal groups of microorganisms with a known phenotype involved in the biological wastewater treatment. GRAPHICAL ABSTRACT

Reliable method for assessing the COD mass balance of a submerged anaerobic membrane bioreactor (SAMBR) treating sulphate-rich municipal wastewater.

The anaerobic treatment of sulphate-rich wastewater causes sulphate reducing bacteria (SRB) and methanogenic archaea (MA) to compete for the available substrate. The outcome is lower methane yield coefficient and, therefore, a reduction in the energy recovery potential of the anaerobic treatment. Moreover, in order to assess the overall chemical oxygen demand (COD) balance, it is necessary to determine how much dissolved CH(4) is lost in the effluent. The aim of this study is to develop a detailed and reliable method for assessing the COD mass balance and, thereby, to establish a more precise methane yield coefficient for anaerobic systems treating sulphate-rich wastewaters. A submerged anaerobic membrane bioreactor (SAMBR) treating sulphate-rich municipal wastewater was operated at 33 °C for an experimental period of 90 d, resulting in a high COD removal (approximately 84%) with a methane-enriched biogas of 54 ± 15% v/v. The novelty of the proposed methodology is to take into account the sulphide oxidation during COD determination, the COD removed only by MA and the dissolved CH(4) lost with the effluent. The obtained biomethanation yield (333 L CH(4) kg(-1) COD(REM MA)) is close to the theoretical value, which confirms the reliability of the proposed method.

Modelling the metabolic shift of polyphosphate-accumulating organisms

Enhanced biological phosphorus removal (EBPR) is one of the most important methods of phosphorus removal in municipal wastewater treatment plants, having been described by different modelling approaches. In this process, the PAOs (polyphosphate accumulating organisms) and GAOs (glycogen accumulating organisms) compete for volatile fatty acids uptake under anaerobic conditions. Recent studies have revealed that the metabolic pathways used by PAOs in order to obtain the energy and the reducing power needed for polyhydroxyalkanoates synthesis could change depending on the amount of polyphosphate stored in the cells. The model presented in this paper extends beyond previously developed metabolic models by including the ability of PAO to change their metabolic pathways according to the content of poly-P available. The processes of the PAO metabolic model were adapted to new formulations enabling the change from P-driven VFA uptake to glycogen-driven VFA uptake using the same process equations. The stoichiometric parameters were changed from a typical PAO coefficient to a typical GAO coefficient depending on the internal poly-P with Monod-type expressions. The model was calibrated and validated with seven experiments under different internal poly-P concentrations, showing the ability to correctly represent the PAO metabolic shift at low poly-P concentrations. The sensitivity and error analysis showed that the model is robust and has the ability to describe satisfactorily the change from one metabolic pathway to the other one, thereby encompassing a wider range of process conditions found in EBPR plants.

Calcium phosphate precipitation in a SBR operated for EBPR: interactions with the biological process

The aim of this paper is to study the precipitation process in a sequencing batch reactor (SBR) operated for EBPR (enhanced biological phosphorus removal) and the possible effects of this phosphorus precipitation in the biological process. Four experiments were carried out under different influent calcium concentration. The experimental results and the equilibrium study, based on the Saturation Index calculation, confirm that the process controlling the calcium behaviour in a SBR operated for EBPR is the calcium phosphate precipitation. This precipitation takes place at two stages initially precipitation of the ACP and later crystallization of HAP. Also the accumulation of phosphorus precipitated was observed when the influent Ca concentration was increased. In all the experiments the influent wastewater ratio P/COD was kept constant. It has been observed that at high Ca concentration the amount of poly-P granules decrease, decreasing the ratio between phosphate release and acetate uptake (P(rel)/Ac(uptake)). Changes on PAO and GAO populations during the experimental period were ruled out by means of methilene blue stains for poly-P detection. These results confirmed the phosphate precipitation as a process that can affect to the PAO metabolism and the EBPR performance.

Influence of food waste addition over microbial communities in an Anaerobic Membrane Bioreactor plant treating urban wastewater

Notorious changes in microbial communities were observed during and after the joint treatment of wastewater with Food Waste (FW) in an Anaerobic Membrane Bioreactor (AnMBR) plant. The microbial population was analysed by high-throughput sequencing of the 16S rRNA gene and dominance of Chloroflexi, Firmicutes, Synergistetes and Proteobacteria phyla was found. The relative abundance of these potential hydrolytic phyla increased as a higher fraction of FW was jointly treated. Moreover, whereas Specific Methanogenic Activity (SMA) rose from 10 to 51 mL CH4 g-1 VS, Methanosarcinales order increased from 34.0% over 80.0% of total Archaea, being Methanosaeta the dominant genus. The effect of FW over AnMBR biomass was observed during the whole experience, as methane production rose from 49.2 to 144.5 L CH4 · kg-1 influent COD. Furthermore, biomethanization potential was increased over 82% after the experience. AnMBR technology allows the established microbial community to remain in the bioreactor even after the addition of FW, improving the anaerobic digestion of urban wastewater.

Effect of long residence time and high temperature over anaerobic biodegradation of Scenedesmus microalgae grown in wastewater

Anaerobic digestion of indigenous Scenedesmus spp. microalgae was studied in continuous lab-scale anaerobic reactors at different temperatures (35 °C and 55 °C), and sludge retention time - SRT (50 and 70 days). Mesophilic digestion was performed in a continuous stirred-tank reactor (CSTR) and in an anaerobic membrane bioreactor (AnMBR). Mesophilic CSTR operated at 50 days SRT only achieved 11.9% of anaerobic biodegradability whereas in the AnMBR at 70 days SRT and 50 days HRT reached 39.5%, which is even higher than the biodegradability achieved in the thermophilic CSTR at 50 days SRT (30.4%). Microbial analysis revealed a high abundance of cellulose-degraders in both reactors, AnMBR (mainly composed of 9.4% Bacteroidetes, 10.1% Chloroflexi, 8.0% Firmicutes and 13.2% Thermotogae) and thermophilic CSTR (dominated by 23.8% Chloroflexi and 12.9% Firmicutes). However, higher microbial diversity was found in the AnMBR compared to the thermophilic CSTR which is related to the SRT. since high SRT promoted low growth-rate microorganisms, increasing the hydrolytic potential of the system. These results present the membrane technology as a promising approach to revalue microalgal biomass, suggesting that microalgae biodegradability and consequently the methane production could be improved operating at higher SRT.