Waste Water Treatment Technology Oxygen supply Major investment

Waste Water Treatment Technology Oxygen supply Major investment (1 M$/y per treatment plant) Fine bubble diffusers Nitrogen Removal How? : Aerobic Nitrification NH 3 + O 2 NO 3 Anaerobic Denitrification NO 3 + organics N 2 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 O 2) dichromate as the oxidant BOD 5 = biochemical oxygen demand(mg/Lof O 2 in 5 days microbial O 2 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 3

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 4

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 (Fe 3+ > Fe 2+) Sulfate reduction to sulfide (H 2 S) (poison, oxygen scavenger Solubilisation of iron and phosphate (ferric phosphate poorly soluble) Further supply of nutrients cycle back to beginning O 2 depletion, sulfide and ammonia buildup Upwards shift of chemocline > Killing of aerobic organisms Further sedimentation 5 Collapse of ecosystem

Simplified Principle of of Activated Sludge COD, NH 4+, phosphate to ocean Activated Sludge (O 2 + 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: NH 4+ conversion to N 2 ? How? 6

What is Nitrification? Microbial oxidation of reduced nitrogen compounds (generally NH 4+). Autotrophic ammonium oxidising bacteria (AOB) (Nitrosomonas, Nitrosospira etc. ): NH 4+ + 1. 5 O 2 NO 2 + H 2 O + 2 H+ Autotrophic nitrite oxidisers (Nitrobacter, Nitrospira etc. ) NO 2 + 0. 5 O 2 NO 3 Aerobic conversion of NH 4+ to NO 3 + removes some of the oxygen demand (COD) + removes NH 4+ toxicity ot 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 3 + 2 H+ + 2 e NO 2 + H 2 O (nitrate reductase) NO 2 + 2 H+ + e NO + H 2 O 2 NO + 2 H+ + 2 e N 2 O + H 2 O (nitrite reductase) (nitric oxide reductase) N 2 O + 2 H+ + 2 e N 2 + H 2 O (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 CO 2) • 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 CO 2 autotrophic

The Nitrogen cycle Ox State 3 2 1 0 +1 +2 +3 +4 +5 CNH 2 NH 4+ N 2 NO NO 2 NO 3

The Nitrogen cycle Ox State 3 2 1 0 +1 +2 +3 +4 +5 CNH 2 NH 4+ Dotted lines are assimiliative paths N 2 NO NO 2 NO 3

The Nitrogen cycle Ox State 3 2 1 0 +1 +2 +3 +4 +5 CNH 2 NH 4+ Nitrogen fixation: Atmospheric N 2 reduction to ammonium and amino acids. N 2 Syntrophic Rhizobia types, free living bacteria and cyanobacteria. NO 2 Reactions serves assimilation. NO 3 NO

The Nitrogen cycle Ox State 3 2 1 0 +1 +2 +3 +4 +5 CNH 2 NH 4+ N 2 NO NO 2 NO 3

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

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

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

How to accomplish overall N removal? Nitrification typically occurs during the aerobic treatment of wastewater: COD + O 2 Ammonium + O 2 CO 2 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 Aerobic Treatment Anaerobic Treatment Recycled sludge Effluent

How to accomplish overall N removal? • N removal by the anaerobic step requires an electron donor to reduce NO 3 to N 2. • This electron donor is organic material. • Solution A: Add organic material to the anaerobic treatment step. • Example: Methanol • Problems: costs, contamination • Alternative solutions? NH 4+ COD N 2 CO 2 Clarifier NO 3 CO 2 Aerobic Treatment Anaerobic Treatment Recycled biomass (sludge) Effluent

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? NH 4+ COD N 2 CO 2 Clarifier NO 3 CO 2 Aerobic Treatment Anaerobic Treatment Recycled biomass (sludge) Effluent

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. NH 4+ COD N 2 CO 2 Clarifier NO 3 CO 2 Aerobic Treatment Anaerobic Treatment Recycled biomass (sludge) Effluent

Idea for SND • Q: How to allow anaerobic denitrification at the same time as aerobic nitrification? • A: Intelligent oxygen control, not straightforward: • Aerobic: COD + O 2 CO 2 • Ammonium + O 2 Nitrate • Anaerobic: CO 2 COD + Nitrate N 2 + • COD should be e donor for nitrate reduction, not oxygen reduction. • Oxygen supply will burn COD faster than ammonium • No COD No denitrification NO 3 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 + NH 4+ + O 2 NO 3 + residual COD • Anoxic: Residual COD + NO 3 N 2 • There is always substantial COD + O 2 CO 2 wastage. Effective N removal is limited Which phase is anaerorobic, which lines are COD, NO 3 - and NH 4+ ?

Alternating Aeration in Batch Systems • Aerobic: COD + NH 4+ + O 2 NO 3 + residual COD • Anoxic: Residual COD + NO 3 N 2 • There is always substantial COD + O 2 CO 2 wastage. Effective N removal is limited COD anoxic NH 3 NO 3 aerobic

Alternating Aeration in Batch Systems • Aerobic: COD + NH 4+ + O 2 NO 3 + residual COD • Anoxic: Residual COD + NO 3 N 2 • There is always substantial COD + O 2 CO 2 wastage. Effective N removal is limited COD NH 3 NO 3 anoxic COD oxidation with NO 3 aerobic COD and NH 3 oxidation

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. 28

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

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

Oxygen dependency of SND Under oxidation Over oxidation Underoxidation: NH 3 build up Over oxidation: NO 3 build up Rate To match Nitrif. and Denitri. : Nitrif. Denitri. SND DO (mg/L) Flux of reducing power (NH 3, 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?

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 p. H fluctuations (costs, performance loss) • Save further O 2 and COD by SND via nitrite • Simplified operation 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 costs for p. H 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 33

SND pathway NH 3 NH 2 OH O 2 N 2 O NO 2 - NO 3 - NO 2 - N 2 If nitrification and denitrification can occur simultaneously there is a possibility of by passing nitrate formation and nitrate reduction SND via nitrite. COD Has the advantage of oxygen savings and COD savings.

DO Effect on Nitrification and Denitrification Rate SND via NO 2 can operate more easily than SND via NO 3 as oxygen has a stronger inhibition effect on nitrate reduction than nitrite reduction Nitrification NO 3 NO 2 reduction DO (mg/L) If SND proceeds via nitrite, then: how much savings are generated?

Under oxidation Over oxidation Nitrif. Rate [N] in outflow Nitrif. NH 3 Denitri. 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, N 2 O emission, O 2 minimisation

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

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 NH 3 • no COD left for denitrification Advantages of bacterial Storage of COD 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. 41 PHB

Why Storage Driven Denitrification? Denitrification needs organic reducing power: • Either sufficient COD or PHB storage • Problem with COD: degrades quicker than NH 3 • no COD left for denitrification Advantages of bacterial Storage of COD 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. 42 PHB

BOD storage as PHB needs ATP 2 Acetate 2 Co. A 4 ATP 2 Acetyl Co. A (16 e ) Mechanisms for ATP generation: • O 2 respiration • Nitrate respiration • Glycogen fermentation • Poly P hydrolysis PHB (18 e ) 1 NADH Bio (2 e ) mass 2 Co. A TCA cycle 8 NADH (16 e ) Glycogen, P complicated NO 3 too low. Aerobic bioselector? H 2 O ETC 2 CO 2 24 ATP Our results: Storage under some O 2 supply O 2 PHB

Expected Benefit of Storing Reducing Power Inside the Floc N 2 COD O 2 NO 2 - PHB anoxic NH 3 • PHB physically separated from O 2 • Selective availability of O 2 to AOB. • PHB may be more readily oxidised by nitrate or nitrite being formed by the aerobic reaction aerobic CO 2 PHB

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

4 Aerobic Anoxic 8 3 6 PHB 2 4 NO 3 1 0 SOUR (mg. O 2/g/h) Carb. comp. (Cm. M) Nitrog comp. (m. M) 10 0 50 40 30 20 10 0 0 50 100 Time (min) 250 2 0 300 350 Three phases could be observed • 1 st : COD PHB • 2 nd : PHB driven SND (60%) • OUR indicates NH 3 depletion • 3 rd : wastage of reducing power • 69 % N removal, no reducing power left NH 3 OUR 50 150 200 Time (min) • Needed: Automatic stopping of aeration when ammonia is oxidised to prevent PHB oxidation with oxygen • Could be detected from OUR monitoring

Effect of auto aeration cut off on PHB levels and N removal 4 Aerobic Anoxic 8 3 6 PHB 2 4 NO 3 1 2 010 4 Anoxic Aerobic Carb. comp. (m. M) Nitrog. comp. (m. M) 0 Carb. comp. (Cm. M) Nitrog comp. (m. M) 10 Settle 8 3 6 2 4 1 2 0 0 0 50 100 150 200 250 Time (mins) 300 350 Aim: Avoid wastage of reducing power by: auto aeration cut off Outcomes: • More PHB preserved • N rem 69 86% • Less air • Shorter treatment

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. 4 Anoxic Aerobic Carb. comp. (m. M) Nitrog. comp. (m. M) 10 Settle 8 3 6 2 4 1 2 0 0 0 50 100 150 200 250 Time (mins) 300 350

Use of negative derivative of OUR to detect ammonium depletion Ammonium depletion d(SOUR)/dt (mg/g/h 2 ) 2 1 Effect of aeration cut off on next cycle? 0 50 100 150 Time (mins) 200

Longer term effects of PHB buildup (not examinable) 70 PHB analysis and SPOUR monitoring show: 60 PHB can be build up over several cycles SOUR (mg/L) 50 Cycle 12 40 improved SND explains biomass “adaptation” 8 30 20 5 no need for emptying cells Cycle 1 one over aerated cycle can NH 3 – OUR 10 loose all “savings” from prev. cycl. review end of aeration DO high? 0 0 50 Time (min) 150

PHB build up over 12 cycles PHB analysis and SPOUR monitoring show: 5 PHB (m. M) 4 PHB can be build up over several cycles 3 2 enabling more reducing power and better denitrification 1 0 0 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 1. 6 With close to complete N removal: no point for front denitrification phase DO required for COD storage 1. 4 Conc (m. M) 1. 2 1 0. 8 NH 4+ 0. 6 0. 4 NO 3 NO 2 0. 2 0 0 50 100 150 200 Time (min)

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 BIO 301 - Leonie Hughes

Multiple sludge approach to WWT Stage 1 storage of influent COD Influent wastewater BIOFILM Heterotrophic denitrifiers Acetate and Ammonia Effluent wastewater Ammonia Acetate BIO 301 - Leonie Hughes PHB

Multiple sludge approach to WWT Stage 2 oxidation of ammonia BIOFILM Heterotrophic denitrifiers Influent wastewater BIOFILM or SBR Autotrophic nitrifiers Ammonia Nitrate Ammonia BIO 301 - Leonie Hughes Effluent wastewater Nitrate

Multiple sludge approach to WWT Stage 3 reduction of nitrate Effluent wastewater BIOFILM Heterotrophic denitrifiers Influent wastewater Nitrate Nitrogen gas PHB + Nitrate BIO 301 - Leonie Hughes BIOFILM or SBR Autotrophic nitrifiers

Commercialisation of PHB • Enhanced bacterial food source for use in aquaculture • Biopol - biological alternative to petrochemical plastics BIO 301 - 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 BIO 301 - Leonie Hughes

History of Biopol • ICI/Zenica published the first patents in the 1980 s 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 BIO 301 - 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 BIO 301 - 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? BIO 301 - 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 (N 2 O) Production During SND The Environmental Impact of N 2 O • Nitrous oxide is a greenhouse gas • global warming potential 250 times greater than CO 2 • Estimated N 2 O 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 (N 2 O) Production During SND • N 2 O is an intermediate of denitrification • Produced from the reduction of NO 2 (nitrite reductase) • N 2 O is reduced to N 2 (nitrous oxide reductase) • Nitrous oxide reductase is highly oxygen sensitive • Oxygen, even at very low levels (0. 02 mg O 2/L), will stop the enzyme working and cause N 2 O to be emitted

Nitrous Oxide (N 2 O) Production During SND • N 2 O 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 N 2 O 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.

urface aeratio n of activate d sludge

Bulking sludge due to Filamentous Bacteria (S. natans)

Anaerobic Ammonium Oxidation (Anammox) ation of ammonium to dinitrogen gas (N 2) with nitrite as the electron acceptor by autotrophic Discovered at the Kluyver Laboratory, Delft, The Netherlands in 1995. vered to be oxidised in the absence of oxygen by a rare species of bacteria Planctomycetes NH 4+ + NO 2 N 2 + 2 H 2 O ( Go’ = 357 k. J mol 1) rectly to dinitrogen gas, without the need for the multi step process of aerobic nitrification an Foaming sludge due to Nocardia