N removal Denitrification and Nitrification How is Den and Nit used for N removal? What are the critical process conditions What is the effect of Oxygen on both? To what extent are Den and Nit exclusive? Can they both happen in the same reactor? Can they happen in the same reactor at the same time ? How? How can process conditions be optimised to achieve simultaneous nitrif, denitrif.? Eutrophication explain

Examinable concepts on Wast Water Treatment (WWT) Eutrophication, why is dissolved nitrogen harmful? Biological WWT, biomass recycle, flocculation Nitrification, denitrification Nitrification followed by denitrification, why does it not work? Alternating nitrification denitrification Simultaneous N and D (SND) SND via nitrite Storage capacity as PHB of most bacteria Parallel nitrification denitrification Anammox

Eutrophication COD BOD nutrients Biomass recycle Flocs (Stokes law) SRT> HRT Intermittent high COD supply High feed COD/biomass ratio Either plug flow or SBR Reactor types Batch Chemostat SBR Plugflow Fedbatch

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: Waste Water Treatment Technology

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

6 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

7 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

8 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

9 Simplified Principle of of 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? Activated Sludge (O 2 + X) Clarifyer 100:1 Biomass Recycle (Return Activated Sludge) COD, NH4+, phosphate to ocean Excess sludge

What is Nitrification? Microbial oxidation of reduced nitrogen compounds (generally NH 4 + ). Autotrophic ammonium oxidising bacteria (AOB) (Nitrosomonas, Nitrosospira etc.): NH O 2  NO H 2 O + 2 H + Autotrophic nitrite oxidisers (Nitrobacter, Nitrospira etc.) NO O 2  NO 3 - Aerobic conversion of NH4+ to NO3 + removes NH4+ toxicity to fish and odor from wastewater - does not accomplish nutrient removal

What is denitrification? Microbial reduction of oxidised nitrogen compounds (generally NO 3 - ). 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)

Review of Terms: Ana-Cata 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 NH N NO +3 NO NO3-

The Nitrogen cycle Ox State -3 CNH2 NH N NO +3 NO NO3- Dotted lines are assimiliative paths

The Nitrogen cycle Ox State -3 CNH2 NH N NO +3 NO 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 NH N NO +3 NO NO3-

The Nitrogen cycle Ox State -3 CNH2 NH N NO +3 NO 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 NH N NO +3 NO 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 NH N NO +3 NO 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? Recycled sludge Aerobic Treatment Anaerobic Treatment Clarifier Effluent 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?

How to accomplish overall N- removal? Recycled biomass (sludge) Aerobic Treatment Anaerobic Treatment Clarifier Effluent 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? NH4+ COD NO3- CO2 N2 CO2

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

How to accomplish overall N- removal? Recycled biomass (sludge) Aerobic Treatment Anaerobic Treatment Clarifier Effluent 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. NH4+ COD NO3- CO2 N2 CO2

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

Return Activated Sludge Air Line Influent Effluent Waste Sludge Clarifier Plug flow allows alternating aerobic / anaerobic conditions without time schedule 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 NH 3 NO 3 - aerobicanoxic

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 NH 3 NO 3 - aerobic COD and NH3 oxidation anoxic COD oxidation with NO3-

31 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. What is SND (Simultaneous Nitrification and Denitrification) ?

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

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

Oxygen dependency of SND 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? Over- oxidation Under- oxidation Nitrif. DO (mg/L) Rate Denitri. SND

35 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 O 2 and COD by SND via nitrite Simplified operation Why Simultaneous nitrification and denitrification(SND) ?

SND pathway 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. NO 2 - NO 3 - N2N2 NH 3 COD O2O2 NH 2 OH N2ON2O O.S

Nitrification DO Effect on Nitrification and Denitrification DO (mg/L) Rate NO 2 - reduction NO 3 - 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?

Nitrif. DO (mg/L) Rate Denitri. Over- oxidation Under- oxidation Nitrif. DO (mg/L) Denitri. NH3 [N] in outflow 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 ) 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

43 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: 1.Oxidises slower  lasts longer  important for SBR 2.Reducing power inside the floc rather than outside 3.Reducing power can be settled and build up. Why Storage Driven Denitrification? PHB

Influent Effluent Waste Sludge Cycle Fill Aeration Settle Decant Use of Sequencing Batch Reactor (SBR) for a) Biomass Retention via internal biomass feedback b) floc formation by oxposing biomass to a sudden high inflow of biomass Biomass Retention in WWTP

45 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: 1.Oxidises slower  lasts longer  important for SBR 2.Reducing power inside the floc rather than outside 3.Reducing power can be settled and build up. Why Storage Driven Denitrification? PHB

BOD storage as PHB needs ATP 2 Acetate TCA cycle 2 Acetyl-CoA (16 e - ) 2 CoA 8 NADH (16 e - ) Bio- mass PHB (18 e - ) ETC 2 CoA 4 ATP 24 ATP 2 CO 2 O2O2 H2OH2O 1 NADH (2 e - ) 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? PHB

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 COD NH 3 O2O2 NO 2 - PHB anoxic N2N2 aerobic Expected Benefit of Storing Reducing Power Inside the Floc CO 2 PHB

AB CD Increasing PHB (dark) buildup in bacterial biomass (red) during early phase of SBR PHB

Three phases could be observed 1 st : COD  PHB 2 nd : PHB driven SND (60%) OUR indicates NH3 depletion 3 rd : wastage of reducing power Nitrog-comp. (mM) Carb. comp. (CmM) Anoxic Aerobic NO Time (min) Time (min) SOUR (mgO2/g/h) NH 3 OUR PHB 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

Aim: Avoid wastage of reducing power by: auto-aeration cut- off Outcomes: More PHB preserved N-rem 69  86% Less air Shorter treatment Effect of auto-aeration cut-off on PHB levels and N-removal Nitrog-comp. (mM) Carb. comp. (CmM) Anoxic Aerobic Time (mins) Nitrog. comp. (mM) Carb. comp.(mM) Aerobic Anoxic Settle PHB NO3-

Time (mins) Nitrog. comp. (mM) 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.

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

Longer term effects of PHB buildup (not examinable) Time (min) SOUR (mg/L) NH 3 – OUR Cycle Cycle 12 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?

cycle PHB (mM) 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

PHB driven SND performance after 12 cycles of controlled PHB build-up Time (min) Conc (mM) NO3- NO2- NH4 + With close to complete N-removal: no point for front denitrification phase  DO required for COD storage

56 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 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.

57 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.

Nitrogen removal by separating nitrifiers from denitrifiers Annamox (anaerobic ammonium oxidation)

Possible ways of N removal Alternating aerobic and anoxic conditions SND SND via nitrite SBR (COD to PHB, preserving reducing power Two biomass systems (PND) Anammox –E-donor NH3 –E-acceptor NO2- –Product N2 –NO2- can come from CANON (not examinable)

60 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-  N2 + 2 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.

61 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.