Ana Lanham

Optimisation of glycogen quantification in mixed microbial cultures

This study addressed the key factors affecting the extraction and quantification of glycogen from floccular and granular mixed microbial cultures collected from activated sludge, nutrient removal systems and photosynthetic consortiums: acid concentration, hydrolysis time and concentration of biomass in the hydrolysis. Response surface modelling indicated that 0.9 M HCl and a biomass concentration of 1 mg mL(-1) were optimal conditions for performing acid hydrolysis. Floccular samples only needed a 2-h hydrolysis time whereas granular samples required as much as 5 h. An intermediate 3 h yielded an error of 10% compared to the results obtained with the hydrolysis times specifically tailored to the type of biomass and can thus be recommended as a practical compromise.

Metabolic versatility in full-scale wastewater treatment plants performing enhanced biological phosphorus removal

This study analysed the enhanced biological phosphorus removal (EBPR) microbial community and metabolic performance of five full-scale EBPR systems by using fluorescence in situ hybridisation combined with off-line batch tests fed with acetate under anaerobic-aerobic conditions. The phosphorus accumulating organisms (PAOs) in all systems were stable and showed little variability between each plant, while glycogen accumulating organisms (GAOs) were present in two of the plants. The metabolic activity of each sludge showed the frequent involvement of the anaerobic tricarboxylic acid cycle (TCA) in PAO metabolism for the anaerobic generation of reducing equivalents, in addition to the more frequently reported glycolysis pathway. Metabolic variability in the use of the two pathways was also observed, between different systems and in the same system over time. The metabolic dynamics was linked to the availability of glycogen, where a higher utilisation of the glycolysis pathway was observed in the two systems employing side-stream hydrolysis, and the TCA cycle was more active in the A(2)O systems. Full-scale plants that showed higher glycolysis activity also exhibited superior P removal performance, suggesting that promotion of the glycolysis pathway over the TCA cycle could be beneficial towards the optimisation of EBPR systems.

Long-term operation of a reactor enriched in Accumulibacter clade I DPAOs: Performance with nitrate, nitrite and oxygen

The microbiology of denitrifying enhanced biological phosphorus removal systems has been a subject of much debate. The question has centred on the affinities of different types of Candidatus Accumulibacter PAOs, type I and type II, towards different electron acceptors such as oxygen, nitrate and nitrite. This study used a propionate anaerobic/anoxic/aerobic lab-scale sequencing batch reactor where a microbial culture was successfully enriched in Accumulibacter type I organisms (approx. 90%). The culture was able to take up phosphorus using nitrate, nitrite and oxygen as electron acceptors, although experiments with oxygen led to the fastest P removal rate. The phosphorus uptake to nitrogen consumed ratio (P/N ratio), when using both nitrate and nitrite, was shown to be affected by pH in the range of 7-8.2, achieving higher values for lower pH values (7.0-7.5). The effect of pH on P removal seems to follow a similar trend for both nitrate and nitrite. To our knowledge, this is the first study where the impact of pH in the phosphate removal stoichiometry using the three most significant electron acceptors is shown for such a high enrichment in Accumulibacter type I.

Metabolic modelling of full-scale enhanced biological phosphorus removal sludge.

This study investigates, for the first time, the application of metabolic models incorporating polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) towards describing the biochemical transformations of full-scale enhanced biological phosphorus removal (EBPR) activated sludge from wastewater treatment plants (WWTPs). For this purpose, it was required to modify previous metabolic models applied to lab-scale systems by incorporating the anaerobic utilisation of the TCA cycle and the aerobic maintenance processes based on sequential utilisation of polyhydroxyalkanoates, followed by glycogen and polyphosphate. The abundance of the PAO and GAO populations quantified by fluorescence in situ hybridisation served as the initial conditions of each biomass fraction, whereby the models were able to describe accurately the experimental data. The kinetic rates were found to change among the four different WWTPs studied or even in the same plant during different seasons, either suggesting the presence of additional PAO or GAO organisms, or varying microbial activities for the same organisms. Nevertheless, these variations in kinetic rates were largely found to be proportional to the difference in acetate uptake rate, suggesting a viable means of calibrating the metabolic model. The application of the metabolic model to full-scale sludge also revealed that different Accumulibacter clades likely possess different acetate uptake mechanisms, as a correlation was observed between the energetic requirement for acetate transport across the cell membrane with the diversity of Accumulibacter present. Using the model as a predictive tool, it was shown that lower acetate concentrations in the feed as well as longer aerobic retention times favour the dominance of the TCA metabolism over glycolysis, which could explain why the anaerobic TCA pathway seems to be more relevant in full-scale WWTPs than in lab-scale systems.

Kinetic and metabolic aspects of Defluviicoccus vanus-related organisms as competitors in EBPR systems

A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Defluviicoccus vanus-related organisms presenting a Glycogen Accumulating Organisms (GAO) phenotype. Initial batch tests were performed using anaerobic/aerobic conditions to assess the capacity of different carbon sources utilization frequently abundant in wastewater: acetate, propionate, butyrate, valerate and glucose. Acetate and propionate were totally consumed in the anaerobic phase as well as butyrate and valerate, though these last ones with a very low consumption rate. All substrates were converted to polyhydroxyalkanoates (PHA). Glucose had a very slight anaerobic consumption but failed to disclose a typical GAO phenotype. In aerobic conditions, again all carbon sources were readily consumed except for glucose, with acetate and propionate having the higher consumption rates. Therefore, glucose seems not be used by this type of organisms. Acetate and propionate consumption rates indicated that these GAOs could reveal good competition advantages in EBPR systems where these carbon sources are available, especially propionate. Volatile Fatty Acid (VFA) uptake in aerobic phase and consequential PHA production indicate these organisms as possible candidates for PHA production.

Ethylenediamine-N,N’-diglutaric acid (EDDG) as a promising biodegradable chelator: Quantification, complexation and biodegradation

[S,S]-ethylenediamine-N,N’-diglutaric acid (EDDG) has been gaining interest in the industrial sector as a promising chelator. In this study, the effective metal complexing capacity of EDDG over a wide pH range was modelled and its biodegradability assessed. Results showed that EDDG could effectively bind to several metallic ions in a wide pH range and was completely biodegraded after approximately 15 days by un-acclimatized sludge. To confirm its biodegradability, an accurate quantification method based on the combination of liquid chromatography and tandem quadrupole mass spectrometry (LC-MS/MS) was developed. Good linearity of the detector response was found for EDDG at concentrations ranging from 0,15 to 1,2 mg/L.