1. 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:

2. 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

3. 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

4. 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

5. 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

6. 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?

7. What is Nitrification?
Microbial oxidation of reduced nitrogen compounds (generally NH4+).
Autotrophic ammonium oxidising bacteria (AOB) (Nitrosomonas, Nitrosospira etc.):
NH O2  NO2- + H2O + 2 H+
Autotrophic nitrite oxidisers (Nitrobacter, Nitrospira etc.)
NO 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

8. 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)
NO H+ + 2 e-  NO2- + H2O (nitrate reductase)
NO 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)

9. 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

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

12. 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

13. 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.

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

15. 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.

16. 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.

17. 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.

18. 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

19. 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)

20. 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)

21. 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)

22. 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.

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

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

25. 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+ ?

26. 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-

27. 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-

28. 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.

29. 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)

30. 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)

31. 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)

32. 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

33. 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

34. 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-

35. 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)

36. 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

37. Laboratory Sequencing Batch Reactor

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

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

41. 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

42. 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

43. 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

44. 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

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

46. 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)

47. Effect of auto-aeration cut-off on PHB levels and N-removal10 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

48. 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

49. Use of negative derivative of OUR to detect ammonium depletion1 2 50
100
150
200
Time (mins)
-d(SOUR)/dt (mg/g/h2 )
Ammonium
depletion
Effect of aeration cut-off
on next cycle?

50. 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

51. 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

52. 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

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

54. 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

55. 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

56. 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

57. 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

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

59. 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

60. 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

61. 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

62. 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

63. 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.

64. 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.

65. 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

66. 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

67. 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.

68. 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.

69. Surface aeration of activated sludge

70. Bulking sludge due to Filamentous Bacteria (S. natans)

71. 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

72. 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.

73. 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 = h-1), probably lives in nature at the oxic/anoxic interface.
Advent of molecular microbiological techniques, eg. molecular probing greater insight into natural habitats.

74. 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.);
NH O2  NO H+ + H2O
• The nitrite produced can be used by anammox;
NH NO­2-  N H2O
• Overall nitrogen removal by CANON:
1 NH O2  0.5 N 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.

75. 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.