Waste Water Treatment Technology Oxygen supply - Major investment (1 M$/y per treatment plant) Fine bubble diffusers Nitrogen Removal How? : Aerobic Nitrification NH3 + O2  NO3 Anaerobic Denitrification NO3 + organics  N2 Problems Nitrifiers grow slow and are sensitive and need oxygen Denitrifiers need organics but no oxygen Nitrification can be either sequential or simultaneous:

List Pollutants to be removed Suspended material (inorganic, bacteria, organic) Dissolved organics (COD,BOD) COD = chemical oxygen demand (mg/L of O2) dichromate as the oxidant BOD5 = biochemical oxygen demand(mg/Lof O2 in 5 days microbial O2 consumption over 5 days N P pathogens odor, colour ultimate aim: recycle of water for re-use

Why organic pollutant removal? Organic pollutants represent an oxygen demand (COD or BOD) Bacteria in the environment will degrade the pollutants and use oxygen. If oxgygen uptake > oxygen transfer  oxygen depletion .  Collapse of ecosystem

Why nutrient removal? Simplified Sequence of events of eutrophication Pristine aquatic ecosystems are typically limited by nutrients. Supply of nutrients (N or P)  photosynthetic biomass (primary and secondary).  More oxygen production and consumption  Sedimentation and decay of dead biomass  Depletion of oxygen in sediment/water column  Collapse of ecosystem

Why nutrient removal? comprehensive Sequence of events of eutrophication (needs understanding of anaerobic respirations) Pristine aquatic ecosystems are typically limited by nutrients. Supply of nutrients (N or P)  photosynthetic biomass (primary and secondary).  More oxygen production and consumption  Sedimentation and decay of dead biomass  Depletion of oxygen in sediment/water column Oversupply of e- donors Use of other electron acceptors (anaerobic respirations) Ferric iron reduction to ferrous iron (Fe3+ --> Fe2+) Sulfate reduction to sulfide (H2S) (poison, oxygen scavenger Solubilisation of iron and phosphate (ferric phosphate poorly soluble) Further supply of nutrients  cycle back to beginning O2 depletion, sulfide and ammonia buildup Upwards shift of chemocline --> Killing of aerobic organisms Further sedimentation Collapse of ecosystem

Simplified Principle of of Activated Sludge COD, NH4+, phosphate to ocean Activated Sludge (O2 + X) Clarifyer 100:1 Excess sludge Biomass Recycle (Return Activated Sludge) After primary treatment (gravity separation of insoluble solids) Secondary treatment: Oxidation of organic pollutants, (COD and BOD removal, partial N removal Needed: NH4+ conversion to N2 ? How?

What is Nitrification? Microbial oxidation of reduced nitrogen compounds (generally NH4+). Autotrophic ammonium oxidising bacteria (AOB) (Nitrosomonas, Nitrosospira etc.): NH4+ + 1.5 O2  NO2- + H2O + 2 H+ Autotrophic nitrite oxidisers (Nitrobacter, Nitrospira etc.) NO2- + 0.5 O2  NO3- Aerobic conversion of NH4+ to NO3 + removes some of the oxygen demand (COD) + removes NH4+ toxicity ot fish and odor from wastewater - does not accomplish nutrient removal

What is denitrification? Microbial reduction of oxidised nitrogen compounds (generally NO3-). Anoxic process using nitrate as an alternative electron acceptor to oxygen (anaerobic respiration) Catalysed by non- specialised factultative aerobic heterotrophic bacteria. A series of reduction steps leading to potential accumulation of intermediates Electron donor: organic substances (BOD, COD) NO3- + 2 H+ + 2 e-  NO2- + H2O (nitrate reductase) NO2- + 2 H+ + e-  NO + H2O (nitrite reductase) 2 NO + 2 H+ + 2 e-  N2O + H2O (nitric oxide reductase) N2O + 2 H+ + 2 e-  N2 + H2O (nitrous oxide reductase)

Review of Terms Metabolic processes can be differentiated between: Processes that make use of exergonic redox reactions, conserve the energy of the reaction as ATP Catabolism or Dissimilation or Respiration typically oxidative process (degradation or organics to CO2) Processes that drive endergonic reactions by using the ATP generated from Dissimilation Anabolism or Assimilation or Biomass Synthesis typically reductive processes (synthesis of complex organics from small building blocks If the building block is CO2  autotrophic

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3-

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3- Dotted lines are assimiliative paths

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3- Nitrogen fixation: Atmospheric N2 reduction to ammonium and amino acids. Syntrophic Rhizobia types, free living bacteria and cyanobacteria. Reactions serves assimilation.

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3-

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3- Nitrification step 1 Nitritification: Ammonium as the electron donor for aerobic respiration. Chemo-litho-autrophic. Nitrosomonas type species.

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3- Nitrification step 2 Nitratification: Nitrite as electron donor for aerobic oxidation to nitrate Chemo-litho-autrophic Nitrobacter type species.

The Nitrogen cycle Ox State -3 CNH2 NH4+ -2 -1 0 N2 +1 +2 NO +3 NO2- +4 +5 NO3- Denitrification using either nitrate (NO3-) or nitrite (NO2-) as the electron eacceptor for anaerobic respiration. Most COD can serve as electron donor. Non-specific bacteria replacing O2 with Nitrate as e- acceptor when oxygen is depleted.

How to accomplish overall N-removal? Nitrification typically occurs during the aerobic treatment of wastewater: COD + O2  CO2 Ammonium + O2  Nitrate In addition to the aerobic activated sludge treatment an anaerobic treatment step is included aiming at N-removal (tertiary treatment) Insufficient N removal is typically achieved. why? Clarifier Anaerobic Treatment Aerobic Treatment Effluent Recycled sludge

How to accomplish overall N-removal? N removal by the anaerobic step requires an electron donor to reduce NO3- to N2. This electron donor is organic material. Solution A: Add organic material to the anaerobic treatment step. Example: Methanol Problems: costs, contamination Alternative solutions? N2 CO2 NO3- CO2 NH4+ COD Clarifier Anaerobic Treatment Aerobic Treatment Effluent Recycled biomass (sludge)

How to accomplish overall N-removal? The obvious solution to successful N removal: Use the COD as electron donor for nitrification and denitrification How to allow anaerobic denitrification to occur in the presence of oxygen? N2 CO2 NO3- CO2 NH4+ COD Clarifier Anaerobic Treatment Aerobic Treatment Effluent Recycled biomass (sludge)

How to accomplish overall N-removal? Observations in the laboratory have shown that aerobic nitrification and anerobic denitrification can sometimes occur at the same time. This simultaneous nitrification and denitrification (SND) has been the focus of many R&D projects for improved N-removal. N2 CO2 NO3- CO2 NH4+ COD Clarifier Anaerobic Treatment Aerobic Treatment Effluent Recycled biomass (sludge)

Idea for SND Q: How to allow anaerobic denitrification at the same time as aerobic nitrification? A: Intelligent oxygen control, not straightforward: Aerobic: COD + O2  CO2 Ammonium + O2  Nitrate Anaerobic: COD + Nitrate N2 + CO2 COD should be e-donor for nitrate reduction, not oxygen reduction. Oxygen supply will burn COD faster than ammonium No COD  No denitrification  NO3- pollution Goal for improved N removal: Slow down aerobic COD oxidation, to leave electron donor for denitrif.

Ideas for SND 1: Alternating aeration 2: Limiting aeration 3: SBR technology: Slowing down COD oxidation by conversion to PHB Intelligent aeration control

Plug flow allows alternating aerobic / anaerobic conditions without time schedule Clarifier Influent Effluent Waste Sludge Return Activated Sludge Air Line Biomass Retention in WWTP

Alternating Aeration in Batch Systems Aerobic: COD + NH4+ + O2  NO3- + residual COD Anoxic: Residual COD + NO3-  N2 There is always substantial COD + O2  CO2 wastage. Effective N removal is limited Which phase is anaerorobic, which lines are COD, NO3- and NH4+ ?

Alternating Aeration in Batch Systems Aerobic: COD + NH4+ + O2  NO3- + residual COD Anoxic: Residual COD + NO3-  N2 There is always substantial COD + O2  CO2 wastage. Effective N removal is limited COD anoxic aerobic NH3 NO3-

Alternating Aeration in Batch Systems Aerobic: COD + NH4+ + O2  NO3- + residual COD Anoxic: Residual COD + NO3-  N2 There is always substantial COD + O2  CO2 wastage. Effective N removal is limited COD anoxic COD oxidation with NO3- aerobic COD and NH3 oxidation NH3 NO3-

What is SND (Simultaneous Nitrification and Denitrification) ? Compromise with DO to go so low that ammonium oxidation is still working and denitrification is enabled. Basically: Run nitrification and denitrification at same speed  sophisticated control needed.

Oxygen dependency of Nitrification Nitrification is not only limited by the substrate concentration (nitrate) but also by the oxygen concentration double limitation \ Rate Nitrif. DO (mg/L)

Oxygen dependency of Denitrification Oxygen inhibition constant (ki) can be measured and used for modeling Similar to half saturation constant half inhibition constant Rate Denitri. DO (mg/L)

Oxygen dependency of SND Under-oxidation Over-oxidation Underoxidation: NH3 build- up Over-oxidation: NO3- build-up To match Nitrif. and Denitri.: Flux of reducing power (NH3, COD) should match flux of oxidation power. But how? What is the magical DO level that enables max SND? How does the SND curve change with different loading rates, biomass levels and N:C levels? Rate Nitrif. Denitri. SND DO (mg/L)

Why Simulaltaneous nitrification and denitrification(SND) ? Minimise aeration costs by running at low DO Avoid external COD addition to (a) lower costs (b) encourage (AOB) rather than heterotrophs   adapt high N-removal performance sludge Avoid pH fluctuations (costs, performance loss) Save further O2 and COD by SND via nitrite Simplified operation

Why Simulaltaneous nitrification and denitrification(SND) ? Minimise aeration costs by running at low DO Avoid external COD addition to (a) lower costs (b) encourage (AOB) rather than heterotrophs   adapt high N-removal performance sludge Avoid costs for pH corrections (nitrification uses acid while denitrification produces acid (can you show this with stoichiometric equations?) Save further O2 and COD by SND via nitrite Simplified operation

SND pathway NH3 NH2OH N2 N2O NO2- NO2- NO3- O2 COD If nitrification and denitrification can occur simultaneously there is a possibility of by-passing nitrate formation and nitrate reduction  SND via nitrite. Has the advantage of oxygen savings and COD savings. NH3 NH2OH N2 O2 N2O COD NO2- NO2- NO3-

DO Effect on Nitrification and Denitrification SND via NO2- can operate more easily than SND via NO3- as oxygen has a stronger inhibition effect on nitrate reduction than nitrite reduction If SND proceeds via nitrite, then: how much savings are generated? Rate Nitrification NO2- reduction NO3- DO (mg/L)

Nitrif. Nitrif. Rate [N] in outflow Denitri. Denitri. DO (mg/L) Under-oxidation Over-oxidation Nitrif. Nitrif. NH3 Rate [N] in outflow Denitri. Denitri. DO (mg/L) DO (mg/L) Conclusion: For best N-removal in the outflow of the treatment process, a low DO should be chosen

Laboratory Sequencing Batch Reactor

Tenix / Murdoch University SND SBR pilot plant (Woodman Pt. 03-12-24) Labview control Bioselector, Online OUR monitoring, N2O emission, O2 minimisation

Return activated sludge ready to be contacted with incoming feed to allow “feast time” and enhance floc formation

Why Storage Driven Denitrification? Idea: Making use of bacteria’s behaviour of taking up organic substances for storage as PHB. Denitrification needs organic reducing power: Either sufficient COD or PHB storage Problem with COD: degrades quicker than NH3  no COD left for denitrification Advantages of bacterial Storage of COD as PHB as PHB: Oxidises slower  lasts longer  important for SBR Reducing power inside the floc rather than outside Reducing power can be settled and build up. PHB

Why Storage Driven Denitrification? Denitrification needs organic reducing power: Either sufficient COD or PHB storage Problem with COD: degrades quicker than NH3  no COD left for denitrification Advantages of bacterial Storage of COD as PHB as PHB: Oxidises slower  lasts longer  important for SBR Reducing power inside the floc rather than outside Reducing power can be settled and build up. PHB

BOD storage as PHB needs ATP Mechanisms for ATP generation: O2 respiration Nitrate respiration Glycogen fermentation Poly-P hydrolysis Our results: Storage under some O2 supply Glycogen, P complicated NO3- too low. Aerobic bioselector? 2 Acetate TCA cycle 2 Acetyl-CoA (16 e-) 2 CoA 8 NADH (16 e-) Bio-mass PHB (18 e-) ETC 4 ATP 24 ATP 2 CO2 O2 H2O 1 NADH (2 e-) PHB

Expected Benefit of Storing Reducing Power Inside the Floc PHB physically separated from O2 Selective availability of O2 to AOB. PHB may be more readily oxidised by nitrate or nitrite being formed by the aerobic reaction N2 COD O2 NO2- PHB anoxic NH3 aerobic CO2 PHB

A B C D Increasing PHB (dark) buildup in bacterial biomass (red) during early phase of SBR PHB

Three phases could be observed 1st : COD PHB 10 4 Three phases could be observed 1st : COD PHB 2nd : PHB driven SND (60%) OUR indicates NH3 depletion 3rd : wastage of reducing power Aerobic Anoxic 8 3 6 Nitrog-comp. (mM) PHB Carb. comp. (CmM) 2 4 NO3- 1 2 50 100 250 300 350 Time (min) 50 40 69 % N-removal, no reducing power left Needed: Automatic stopping of aeration when ammonia is oxidised to prevent PHB oxidation with oxygen Could be detected from OUR monitoring SOUR (mgO2/g/h) 30 20 10 NH3 OUR 50 150 200 Time (min)

Effect of auto-aeration cut-off on PHB levels and N-removal 10 4 Aerobic Aim: Avoid wastage of reducing power by: auto-aeration cut-off Outcomes: More PHB preserved N-rem 6986% Less air Shorter treatment Anoxic 8 3 6 Nitrog-comp. (mM) PHB Carb. comp. (CmM) 2 4 NO3- 1 2 1 2 3 4 50 100 150 200 250 300 350 Time (mins) Nitrog. comp. (mM) 6 8 10 Carb. comp.(mM) Aerobic Anoxic Settle

Special features of PHB hydrolysis kinetics PHB degradation kinetics is ~ first order: dependent on PHB, but independent of biomass However, ammonium oxidation is proportional to biomass: higher sludge concentrations should favour autotrophic over heterotrophic activity  helps SND. 1 2 3 4 50 100 150 200 250 300 350 Time (mins) Nitrog. comp. (mM) 6 8 10 Carb. comp.(mM) Aerobic Anoxic Settle

Use of negative derivative of OUR to detect ammonium depletion 1 2 50 100 150 200 Time (mins) -d(SOUR)/dt (mg/g/h2 ) Ammonium depletion Effect of aeration cut-off on next cycle?

Longer term effects of PHB buildup (not examinable) 70 PHB analysis and SPOUR monitoring show: PHB can be build up over several cycles improved SND explains biomass “adaptation” no need for emptying cells one over-aerated cycle can loose all “savings” from prev. cycl. review end of aeration DO high? 60 8 5 Cycle 12 50 40 SOUR (mg/L) 30 Cycle 1 20 NH3 – OUR 10 50 Time (min) 150

PHB build-up over 12 cycles PHB analysis and SPOUR monitoring show: PHB can be build up over several cycles  enabling more reducing power and better denitrification 5 4 3 PHB (mM) 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 cycle

PHB driven SND performance after 12 cycles of controlled PHB build-up 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 50 100 150 200 Time (min) Conc (mM) NO3- NO2- NH4+ With close to complete N-removal: no point for front denitrification phase  DO required for COD storage

Below this point for 2007 only Nitrogen removal by separating nitrifiers from denitrifiers

Biological nutrient removal As the main influent N species of wastewater is ammonia, nitrification must precede denitrification BUT if oxygen and organic carbon are present, heterotrophic organisms will consume the carbon This is a waste of both oxygen ($$) and carbon ($$) causing the cost of operation to increase If the influent COD can instead be stored internally by the heterotrophs for later use in denitrification, this would save on both oxygen and carbon BIO301 - Leonie Hughes

Stage 1 - storage of influent COD Multiple sludge approach to WWT Stage 1 - storage of influent COD BIOFILM Heterotrophic denitrifiers Influent wastewater Acetate and Ammonia Effluent wastewater Ammonia Acetate PHB BIO301 - Leonie Hughes

Stage 2 - oxidation of ammonia Multiple sludge approach to WWT Stage 2 - oxidation of ammonia BIOFILM or SBR Autotrophic nitrifiers BIOFILM Heterotrophic denitrifiers Influent wastewater Ammonia Effluent wastewater Nitrate Ammonia Nitrate BIO301 - Leonie Hughes

Stage 3 - reduction of nitrate Multiple sludge approach to WWT Stage 3 - reduction of nitrate BIOFILM or SBR Autotrophic nitrifiers BIOFILM Heterotrophic denitrifiers Effluent wastewater Influent wastewater Nitrate Nitrogen gas PHB + Nitrate BIO301 - Leonie Hughes

Commercialisation of PHB Enhanced bacterial food source for use in aquaculture Biopol - biological alternative to petrochemical plastics BIO301 - Leonie Hughes

The need for biodegradable plastics 6 billion plastic bags are used every year in Australia All plastic products make up 4% of all waste going to landfill Reduction in plastic going to landfill will make landfill lifespans longer BIO301 - Leonie Hughes

History of Biopol ICI/Zenica published the first patents in the 1980s for a complete production pathway of PHB with minimal cost extraction Biological fermentation method Shampoo bottle for Wella was highest profile product In 1996 Monsanto purchased the patents and shifted the focus to PHB production in genetically modified crops Continued public perception affecting commercialisation of GM crops contributed to the selling of the PHB patents to Metabolix Metabolix now have exclusive rights to manufacture, sell and use PHA related products regardless of origin BIO301 - Leonie Hughes

Wastewater - free source of PHB? One of the limitations of PHB production is the high cost compared to petrochemical based thermoplastics If we know that Activated sludge can make it and Wastewater can be used as the substrate Surely this may change the economics? Much research is focused on pursuing this BIO301 - Leonie Hughes

Wastewater - free source of PHB? Question: Consider that wastewater is a waste product that people are currently paid to remove If it becomes a resource, what would stop governments charging those who want it What if this counteracts the previous economic statement? BIO301 - Leonie Hughes

Phosphorous Removal Called “phosphorous accumulating organisms” (PAO’s) Require fluctuating conditions of aerobic and anaerobic conditions à SBR can provide perfect environment. The PAO’s have a pool of poly-inorganic phosphate (poly-Pi) inside the cell.

Phosphorous Removal Anaerobic conditions hydrolyse a phosphate bond to produce energy in order to import substrate (typically acetate) into the cell. Hydrolysed Pi released into the medium and PHA is produced Called the “P release phase”. Aerobic conditions the bacteria take up phosphorous to regenerate poly-Pi pool PHA as the energy source Called the “P uptake phase” Overall net reduction of phosphorus in the wastewater.

Nitrous Oxide (N2O) Production During SND The Environmental Impact of N2O Nitrous oxide is a greenhouse gas global warming potential 250 times greater than CO2 Estimated N2O responsible for 6% of global warming involved in the destruction of the ozone layer leading to an increase in the incidence of skin cancer and related health problems

Nitrous Oxide (N2O) Production During SND N2O is an intermediate of denitrification Produced from the reduction of NO2- (nitrite reductase) N2O is reduced to N2 (nitrous oxide reductase) Nitrous oxide reductase is highly oxygen sensitive Oxygen, even at very low levels (0.02 mg O2/L), will stop the enzyme working and cause N2O to be emitted

Nitrous Oxide (N2O) Production During SND N2O also produced by Autotrophic ammonium oxidising (nitrifying) bacteria, if the oxygen concentration is very low. In an SBR operated for SND both nitrifiers and denitrifiers in the flocs will be exposed to low dissolved oxygen concentrations Result: SBR's operated for SND have a greater tendency to emit N2O than traditionally wastewater treatment plants could be of environmental concern.

In a nutshell Nutrient rich wastewater released into waterways can lead to eutrophication. During nutrient removal of wastewater, aerobic and anaerobic processes need not be separated as traditionally thought. Under oxygen limitation, simultaneous nitrification (aerobic) and denitrification (anaerobic) can be achieved, due to anoxic zones inside the floc. Effective denitrification requires a carbon source. Control of aeration to DO < 1 can help conserve carbon for heterotrophic denitrification, improving denitrification. SND via nitrite provides savings in reduced oxygen and BOD consumption.

Surface aeration of activated sludge

Bulking sludge due to Filamentous Bacteria (S. natans)

Foaming sludge due to Nocardia Anaerobic Ammonium Oxidation (Anammox) The oxidation of ammonium to dinitrogen gas (N2) with nitrite as the electron acceptor by autotrophic bacteria. Discovered at the Kluyver Laboratory, Delft, The Netherlands in 1995. For the first time, ammonium was discovered to be oxidised in the absence of oxygen by a rare species of bacteria Planctomycetes, Candidatus Brocadia anammoxidans. NH4+ + NO­2-  N2 + 2 H2O (Go’ = -357 kJ mol-1) Ammonium can be oxidised directly to dinitrogen gas, without the need for the multi-step process of aerobic nitrification and heterotrophic denitrification. Anaerobic Ammonium Oxidation (Anammox) The oxidation of ammonium to dinitrogen gas (N2) with nitrite as the electron acceptor by autotrophic bacteria. Discovered at the Kluyver Laboratory, Delft, The Netherlands in 1995. For the first time, ammonium was discovered to be oxidised in the absence of oxygen by a rare species of bacteria Planctomycetes, Candidatus Brocadia anammoxidans. NH4+ + NO­2-  N2 + 2 H2O (Go’ = -357 kJ mol-1) Ammonium can be oxidised directly to dinitrogen gas, without the need for the multi-step process of aerobic nitrification and heterotrophic denitrification. Foaming sludge due to Nocardia

Anammox Accidental observations at WWTP showed some N-removal during aeratio Anaerobic Ammonium Oxidation (Anammox The oxidation of ammonium to dinitrogen gas (N2) with nitrite as the electron acceptor by autotrophic bacteria. Discovered at the Kluyver Laboratory, Delft, The Netherlands in 1995. For the first time, ammonium was discovered to be oxidised in the absence of oxygen by a rare species of bacteria Planctomycetes, Candidatus Brocadia anammoxidans. NH4+ + NO­2-  N2 + 2 H2O (Go’ = -357 kJ mol-1) Ammonium can be oxidised directly to dinitrogen gas, without the need for the multi-step process of aerobic nitrification and heterotrophic denitrification.

Anammox The electron donor is ammonium, the electron acceptor is nitrite. Ammonium (ox. state -3) gets oxidised to N2 (0), and nitrite (+3) is reduced to N2. Autotrophic  avoids the need for addition of a carbon source, which is sometimes a cost in conventional systems. All original attempts to isolate the responsible microorganism failed; organism grows extremely slowly (max = 0.003 h-1), probably lives in nature at the oxic/anoxic interface. Advent of molecular microbiological techniques, eg. molecular probing greater insight into natural habitats.

Cannon CANON (Completely Autotrophic Nitrogen removal Over Nitrite) Cooperation between aerobic and anaerobic ammonium oxidisers under oxygen limitation. Completely autotrophic  promising opportunity for wastewaters with a very low organic carbon content (eg. landfill leachates, aquaculture waste). • Ammonium is oxidised to nitrite by aerobic ammonium oxidisers (Nitrosomonas, Nitrosospira etc.); NH4+ + 1.5 O2  NO2- + 2 H+ + H2O • The nitrite produced can be used by anammox; NH4+ + 1.3 NO­2-  N2 + 2 H2O • Overall nitrogen removal by CANON: 1 NH4+ + 0.75 O2  0.5 N2 + 1.5 H2O + H+ Advantages of CANON system; low aeration costs (60% less than traditional systems), requires no addition of a carbon source (process is autotrophic) and the only end product is N2.

Conclusions Importance of Oxygen Limitation Without aeration control, the DO in the aeration tanks can reach up to 5 mg L-1 (close to saturation, 8 mg L-1). At high DO concentrations, DO penetrates into the flocs  the possibility of SND is decreased. Low oxygen partial pressures (< 1 mg L-1) favour SND and the CANON process. Sophisticated computer feedback control needed to run process effectively. Conclusions Aerobic and anaerobic processes need not necessarily be separated, as traditionally thought. Under oxygen limitation, simultaneous nitrification (aerobic) and denitrification (anaerobic) can be achieved, due to anoxic zones inside the floc. A new type of bacterium, of the order Planctomycetes can oxidise ammonium in the absence of oxygen, using nitrite as the electron acceptor (anammox). Under oxygen limitation, anammox bacteria can cooperate with aerobic ammonium oxidisers, and nitrogen can be removed directly to dinitrogen gas.