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Partial nitrification combined with Anammox in a single reactor (the CANON process) is an energy-efficient N-removal technology that could substantially lower the N-load of a WWTP by separate treatment of nitrogen-rich side streams, preventing the need for extensive expansion and reducing the total energy requirement. This study looks at the enrichment of Anammox from activated sludge and its application in the CANON process on lab-scale. The aim was to identify the critical process control parameters necessary for successful operation of CANON. An Anammox culture capable of removing 0.6 kg N/m3/d was enriched in 14 weeks in a sequencing batch reactor. Nitrifying biomass was inoculated into the Anammox reactor (10% v/v) together with limited oxygen supply (< 8 mL/min) to initiate the CANON process in continuous culture. The small flocs formed by the biomass (< 1000 μm) were sensitive to low O2 concentrations (< 0.1 mg/L) which prevented simultaneous nitrification and Anammox. Operation with 20 min aerobiosis and 30 min anaerobiosis was necessary to achieve sustained, completely autotrophic N-removal for an extended period at a rate of 0.08 kg N/m3/d. Essential process control parameters for stable CANON operation were the nitrite concentration, oxygen concentration, pH and the temperature.

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH 4+ (g biomass) -1 h-1 (0.58 kg total-N m-3 day-1). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus 'Brocadia anammoxidans' strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m-3 day-1) for an extended period of time.

Management of the aeration length in a sequencing batch reactor (SBR) can improve N-removal by minimising the amount of organic substrate that is oxidised aerobically. This study investigates the long-term effect of aeration control on N-removal via simultaneous nitrification and denitrification (SND) by a mixed culture in a 2 L acetate-fed SBR, using PHB as the electron donor for denitrification. The reactor was operated continuously with automated termination of the aerobic phase after ammonium depletion, using the specific oxygen uptake rate (SOUR) as the control parameter. This resulted in an increase of the organic loading rate (OLR) from 0.33 to 0.59 g BOD g-1 d -1. Over the first 12 cycles of operation, the PHB content of the biomass increased three-fold and resulted in a progressively increasing SOUR, which allowed an increased amount of nitrogen removal via SND from 34% to 52%. After one month of continuous operation with controlled aeration, the settling efficiency of the biomass had significantly improved (SVI 70 mL g-1 X). Long-term oxygen management resulted in biomass with a higher capacity for N-removal via SND and improved settling characteristics. Our results may help to explain long-term historical effects of N-removal capabilities in WWTPs and assist design engineers in choosing an appropriate aeration length and OLR.

For economic and efficient nitrogen removal from wastewater treatment plants via simultaneous nitrification and denitrification the nitrification process should stop at the level of nitrite such that nitrite rather than nitrate becomes the substrate for denitrification. This study aims to contribute to the understanding of the conditions that are necessary to improve nitrite reduction over nitrite oxidation. Laboratory sequencing batch reactors (SBRs) were operated with synthetic wastewater containing acetate as COD and ammonium as the nitrogen source. Computer controlled operation of the reactors allowed reproducible simultaneous nitrification and denitrification (SND). The oxygen supply was kept precisely at a low level of 0.5 mgL-1 and bacterial PHB was the only electron donor available for denitrification. During SND little nitrite or nitrate accumulated (< 20% total N), indicating that the reducing processes were almost as fast as the production of nitrite and nitrate f rom nitrification. Nitrite spiking tests were performed to investigate the fate of nitrite under different oxidation (0.1-1.5 mgL-1 of dissolved oxygen) and reduction conditions. High levels of reducing power were provided by allowing the cells to build up to 2.5 mM of PHB. Nitrite added was preferentially oxidised to nitrate rather than reduced even when dissolved oxygen was low and reducing power (PHB) was excessively high. However, the presence of ammonium enabled significant reduction of nitrite under low oxygen conditions. This is consistent with previous observations in SBR where aerobic nitrite and nitrate reduction occurred only as long as ammonium was present. As soon as ammonium was depleted, the rate of denitrification decreased significantly. The significance of the observed strongly stimulating effect of ammonium on nitrite reduction under SND conditions is discussed and potential consequences for SBR operation are suggested.

This study builds on previous experience of maximising the formation of COD as poly-hydroxybutyrate (PHB) and now describes a feedback technique of preserving the use of PHB for denitrification resulting in enhanced nitrogen removal rather than allowing its wasteful oxidation by oxygen. The feedback technique uses on-line SOUR monitoring for detecting the end-point of nitrification and controlling the aerobic phase length accordingly. The laboratory SBR was operated such that all organic substrate (acetate) was rapidly converted to PHB, which then served as the electron donor for nitrogen removal via simultaneous nitrification and denitrification (SND) during the aerobic phase (up to 70% SND). During SBR cycling with a fixed aeration length (240 minutes), PHB was unnecessarily oxidised after ammonium depletion, resulting in little denitrification and poor total nitrogen removal (69%). However, when the aerobic phase length was controlled via the SOUR, up to 1.8 CmM PHB (58 mg L -1 COD) could be preserved, enabling improved total nitrogen removal (86%). The drop in the SOUR after ammonium depletion was a reproducible event that could be detected even when using raw wastewater and fresh activated sludge. The SOUR-control technique holds promise to build up PHB over a number of SBR cycles. While advanced oxygen-control is used for improved N-removal in several existing WWTPs, this study investigates the importance of oxygen control with relevance to PHB driven SND in sequencing batch reactors.

Het IWA-rapport 'Sequencing batch reactor technology, scientific and technical report no. 10' is een deskundig georiënteerd werk, dat een helder overzicht geeft van de technologie achter het SBR -proces. Het bevat zowel onderzoeksresultaten op kleine en grote schaal als praktijkervaringen. Het rapport is bedoeld voor ontwerpers en bedrijfsvoerders van SBR-installaties én onderzoekers op het gebied van de SBR-technologie. De aandacht richt zich met name op de toepassing van een variabel volume en discontinu bedreven actiefslibsystemen

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-β-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (kLa 6 to 16 h-1, 0 mg L-1 DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (kLa 30, 51 h-1, DO >0.9 mg L-1). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (YPHB/Ac 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (YPHB/Ac 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L-1 PHB) accounted for an additional denitrification potential of up to 18 mg L-1 nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB

The potential for PHB (poly-β-hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2-L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L-1. However, the increased percentage of SND at a low DO concentration was compromised by a 2-times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L-1 was optimal for both SND efficiency (61%) and rate (4.4 mmol N. Cmol X-1 · h-1). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH4+ and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first-order kinetics (dfPHB/dt = -0.19 · fPHB), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen

Shake flask and stirred tank bioleaching experiments showed that the dissolution of chalcopyrite is inhibited by ferric ion concentrations as low as 200 mg L-1 and redox potentials >420 mV (vs. Ag/AgCl). Chemical leaching of chalcopyrite (4% suspension, surface area 2.3 m2 g-1) was enhanced fourfold in the presence of 0.1 M ferrous sulphate compared with 0.1 M ferric sulphate. A computer-controlled reactor was designed to function as a "potentiostat"-bioreactor by arresting the air supply to the reactor when the redox potential in solution was greater than a designated setpoint. Leaching at a low, constant redox potential (380 mV vs. Ag/AgCl) achieved final copper recoveries of 52%-61%, which was twice that achieved with a continuous supply of oxygen (<30% extraction). The bacterial populations were observed to continue growing under oxygen limitation but in a controlled manner that was found to improve chalcopyrite dissolution. As the control mechanism is easily established and is likely to decrease production cost, the use of this technology may find application in industry.

A series of bacterial and chemical leaching experiments were conducted to clarify contradictory reports in the literature regarding the role of bacteria in the bioleaching of chalcopyrite. Tests containing a high bacterial concentration showed inhibited leaching, even lower than non-inoculated controls. However, when bacterial cells were washed before inoculation, it was apparent that it was not the bacterial cells but rather the chemical species introduced with them that influenced the leaching rate. In addition, the results of comparative tests with 0.1 M ferrous sulphate or ferric sulphate showed that copper was leached from the ore 2.7 times faster in leach solutions containing ferrous ion, suggesting that ferric ions inhibit chalcopyrite dissolution. The results indicated that the chalcopyrite dissolution rate is strongly dependent on the reduction potential (Eh) in solution, and that this parameter is far more influential than the number or activity of bacterial cells. These results imply that the role of bacteria may only be stimulatory when the prevailing electrochemical conditions are also favourable.