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

• 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 rec ycle• 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 NO3Anaerobic Denitrification NO3 + organics N2

Problems Nitrifiers grow slow and are sensitive and need oxygenDenitrifiers 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 scavengerSolubilisation of iron and phosphate (ferric phosphate poorly soluble)Further supply of nutrients cycle back to beginningO2 depletion, sulfide and ammonia buildupUpwards shift of chemocline --> Killing of aerobic organismsFurther sedimentationCollapse 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 (O2 + 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 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 NH4+ toxicity to 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: Ana-Cata

• Metabolic processes can be differentiated between:• Processes that make use of exergonic redox reactions, conserve the

energy of the reaction as ATPCatabolism or Dissimilation or Respirationtypically oxidative process (degradation or organics to CO2)

• Processes that drive endergonic reactions by using the ATP generated from Dissimilation

Anabolism or Assimilation or Biomass Synthesistypically reductive processes (synthesis of complex organics from

small building blocksIf the building block is CO2 autotrophic

Energy source Photo Chemo

Electrondonor

Organo Litho

C-source Hetero Auto

The Nitrogen cycleOxState

-3 CNH2 NH4+ -2-10 N2+1+2 NO+3 NO2-+4+5 NO3-


-3 CNH2 NH4+ -2-10 N2+1+2 NO+3 NO2-+4+5 NO3-

Dotted lines are assimiliative paths


-3 CNH2 NH4+ -2-10 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.


-3 CNH2 NH4+ -2-10 N2+1+2 NO+3 NO2-+4+5 NO3-


-3 CNH2 NH4+ -2-10 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.


-3 CNH2 NH4+ -2-10 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.


-3 CNH2 NH4+ -2-10 N2+1+2 NO+3 NO2-+4+5 NO3-

Denitrificationusing 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

AerobicTreatment

Anaerobic Treatment

Clarifier

Effluent

Nitrification typically occurs during the aerobic treatment of wastewater:

COD + O2 CO2Ammonium + 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?


Recycled biomass(sludge)

AerobicTreatment

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

N2CO2



AerobicTreatment

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

N2CO2



AerobicTreatment

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

N2CO2

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 SludgeAir 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+ ?



COD

NH3

NO3-

aerobicanoxic



COD

NH3

NO3-

aerobicCODand NH3oxidation

anoxicCOD oxidationwith 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 limitedby the substrate concentration (nitrate) but also by the oxygen concentrationdouble limitation\

Nitrif.

DO (mg/L)

Rat

e

3

33

3

33max NH

OO

O

NHNH

NHNHNH X

kS

S

kS

SSURSUR

Oxygen dependency of Denitrification

DO (mg/L)

Rat

e

Denitri.

Oxygen inhibition constant (ki)can be measured and used for modeling

Similar to half saturation constant

half inhibition constant

3

33

3

33max NO

OO

O

NONO

NONONH X

kiS

ki

kS

SSURSUR

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)

Rat

e

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 O2 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.

NO2-NO2

-

NO3-

N2

NH3

COD

O2

NH2OH

N2O

O.S-3-2-1012345

Nitrification

DO Effect on Nitrification and Denitrification

DO (mg/L)

Rat

e

NO2- reduction

NO3-

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)

Rat

e

Denitri.

Over-oxidation

Under-oxidation

Nitrif.

DO (mg/L)

Denitri.

NH3

[N]

in o

utflo

w

Conclusion: For best N-removal in the outflow of the treatment process, a low DO should be chosen

Laboratory Sequencing Batch Reactor

Tenix / Murdoch UniversitySND SBRpilot plant(Woodman Pt.03-12-24)

Labview control

Bioselector,

Online OURmonitoring,

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 SludgeCycle

Fill

Aeration

Settle

Decant

Use of Sequencing Batch Reactor (SBR) for a) Biomass Retention via internal biomass feedbackb) 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 CO2O2

H2O

1 NADH (2 e-)

Mechanisms for ATP generation:• O2 respiration• Nitrate respiration• Glycogen fermentation• Poly-P hydrolysis

Our results:Storage under

some O2 supplyGlycogen, 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

NH3

O2

NO2- PHB

anoxic

N2

aerobic

Expected Benefit of Storing Reducing Power Inside the Floc

CO2PHB

A B

C D

Increasing PHB (dark) buildup in bacterial biomass (red) during early

phase of SBRPHB

Three phases could be observed• 1st : COD PHB• 2nd : PHB driven SND

(60%)• OUR indicates NH3

depletion• 3rd : wastage of

reducing power

0

1

2

3

4

Nitr

og-c

omp.

(m

M)

0

2

4

6

8

10

Car

b. c

omp.

(C

mM

)

AnoxicAerobic

NO3-

0 50 100 250 300 350Time (min)

00 50 150 200

Time (min)

10

20

30

40

50

SO

UR

(m

gO2/

g/h)

NH3

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 powerby: auto-aeration cut-off

Outcomes:• More PHB preserved• N-rem 6986%• Less air• Shorter treatment

Effect of auto-aeration cut-off onPHB levels and N-removal

0

1

2

3

4

Nitr

og-c

omp.

(m

M)

0

2

4

6

8

10

Car

b. c

omp.

(C

mM

)

AnoxicAerobic

0

1

2

3

4

0 50 100 150 200 250 300 350

Time (mins)

Nitr

og. c

om

p. (

mM

)

0

2

4

6

8

10

Car

b. c

omp.

(mM

)

Aerobic Anoxic Settle

PHB

NO3-

0

1

2

3

4

0 50 100 150 200 250 300 350

Time (mins)

Nitr

og. c

om

p. (

mM

)

0

2

4

6

8

10

Car

b. c

omp.

(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.

0

1

2

50 100 150 200

Time (mins)

-d(S

OU

R)/

dt (

mg

/g/h

2 )

Ammonium

depletion

Use of negative derivative of OUR to detect ammonium depletion

Effect of aeration cut-offon next cycle?

Longer term effects of PHB buildup (not examinable)

0

10

20

30

40

50

60

70

0 50 150Time (min)

SO

UR

(m

g/L

)

NH3 –OUR

Cycle 1

5

8

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?

0

1

2

3

4

5

0 1 2 3 4 5 6 7 8 9 10 11 12 13cycle

PH

B (

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

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 50 100 150 200 Time (min)

Co

nc

(m

M)

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

• Annamox (anaerobic ammonium oxidation)

• E-donor NH3• E-acceptor NO2-• Product N2

Possible ways of N removal

• Altenating aerobic and anoxic conditions• SND• SND via nitrite• SBR (COD to PHB, preserving reducing

power• Two biomass systems• Anammox

BIO301 - Leonie Hughes

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


Multiple sludge approach to WWT

Stage 1 - storage of influent COD

BIOFILM Heterotrophic denitrifiers

Influent wastewater

Acetate and Ammonia

Effluent wastewater

Ammonia

Acetate PHB



Stage 2 - oxidation of ammonia

BIOFILM Heterotrophic

denitrifiers

Influent wastewater

Ammonia

Ammonia Nitrate

BIOFILM or SBR

Autotrophic nitrifiers

Effluent wastewater

Nitrate



Stage 3 - reduction of nitrate

BIOFILM Heterotrophic

denitrifiers

Effluent wastewater

Nitrogen gas PHB + Nitrate

BIOFILM or SBR

Autotrophic nitrifiers

Influent wastewater

Nitrate


Commercialisation of PHB

• Enhanced bacterial food source for use in aquaculture

• Biopol - biological alternative to petrochemical plastics


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


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


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


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?

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


• 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


• 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+ + NO 2- 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+ + NO 2- 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.

Documents

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