© Sara Hallin

Biological N and P removal in activated sludge processes

Sara Hallin Department of Microbiology, SLU

Metabolism

Oxidation:

KOLFÖRENING KOLDIOXID + ELEKTRONER + VÄTEJONER

Reduktion:

SYRE + ELEKTRONER + VÄTEJONER VATTEN

Fullständig reaktion:

KOLFÖRENING + SYRE KOLDIOXID + VATTEN

BIOKEMISKT BUNDEN ENERGI

Fermentation:

GLUKOS ETANOL + KOLDIOXID

BIOKEMISKT BUNDEN ENERGI

Fermentation av socker till etanol och koldioxid. En del av kolet isockret har oxiderats till koldioxid medan en del har reducerats tilletanol (vanlig sprit).

Organiskt material bryts ner av mikroorganismer i luftningsbassängen. Slammet (biomassa och organiskt material)avskiljs från det renade vattnet i sedimenteringsbassängen. .

Traditionell ASP

NH4+ NO2

- NO3-

N2O NO NO2

-

Organically bound nitrogen org-NH2

N2

Nitrogen fixation

Nitrification

Assimilation

Assimilation Mineralization/ Ammonification

ATMOSPHERE

SOIL/WATER

Denitrification

Dissimilatory nitrate reduction to ammonium

Microbiological reactions in the N cycle

Nitrification in water treatment

Nitrification lowers BOD load in recipient Nitrification coupled to denitrification lowers N load in recipient Nitrogen removal systems: - natural wetlands - constructed wetlands - wastewater treatment plants Process problems with inhibition: Microbial sensors for detecting nitrification inhibitors

Nitrification

Nitrosomonas

Nitrification involves several reactions and two different bacteria: 1. Dissociation of ammonium 2. Oxidation of ammonia Ammonia monooxygenase (AMO): Integral membrane protein Can oxidize methane Cometabolize halogenated compounds 3. Oxidation of hydroxylamine Hydroxylamine oxidoreductase (HAO): Soluble, periplasmic protein 4. Oxidation of nitrite

Ammonia oxidation: NH3 + 1,5O2 NO2

- + H+ + H2O

Periplams

OH-

Nitrite oxidation: NO2- + ½ O2 NO3

-

Carbon metabolism

Cell constituents Growth

Energetic constraints...

1. ATP and NADPH (reducing power) requirements in Calvin cycle

2. NAD(P)H formed by reverse e- flow:

Cyt c 2e- Cyt c

O2

NAD(P)+ e-

e-

Am

mo

nia

oxi

diz

ing

arc

ha

ea

an

d

ba

cte

ria

(A

OA

an

d A

OB

) The organisms

NH3 oxidizers Proteobacteria: Nitrosomonas Nitrosococcus Nitrosospira Thaumarchaeota: Nitrosopumilis…

NO2- oxidizers

Bacteria: Nitrobacter Nitrospira

NH4+ NO2

- NO3-

N2O NO NO2

-

Organically bound nitrogen org-NH2

N2

Nitrogen fixation

Nitrification

Assimilation

Assimilation Mineralization/ Ammonification

ATMOSPHERE

SOIL/WATER

Denitrification

Dissimilatory nitrate reduction to ammonium

Microbiological reactions in the N cycle

Denitrifikation är en andningsprocess som där nitrat omvandlas till kvävgas i flera komplicerade steg inom en och samma bakteriecell:

nitrat → nitrit → kväveoxid → lustgas → kvävgas

(NO3- → NO2

- → NO → N2O → N2)

Denitrification Pathway

O2 + = NO3

-/ NO2

-

Denitrification pathway

Denitrification: anaerobic respiration

Organic compound CO2

Biosynthesis

Carbon flow

NO3-, (NO2

-, N2O)

Electron flow ATP

Denitrification

Cytoplasma

Periplasma

NO3-

NO2- NO3

-

H+

NADH2 NAD+

Proton motive force

2e- 2e- 2e-

NO2- NO

NO N2O

N2O N2

e- nar nor

nir nos

Denitrifier Diversity

Denitrifiers

Bacteria Eukarya Archaea

Denitrifier diversity

O2

NH4+

H20 O2

ATP

Nitrifikation

NO2-

Redox

+

Glucose

2 ATP 2 NADH Glykolysis

2 Pyruvate

2 GTP

6 CO2

TCA cycle 8 NADH

2 FADH

ATP NADH

FADH ATP

NO3- NO2

- NO N2O N2

- Denitrifikation

NO 2 NO N 2 O N 2 NO 2 NO N 2 O N 2 NO 3 NH 4 NH 2 OH NO 2 NO - 3 NH NH 2 OH NO 2 - - +

Kvävrening

Nitrifierande bakterier Denitrifierande bakterier •Nitrifierare finns i mark och vatten •Bara några få arter •Nitrifikation är två energigivande processer som utförs av två olika grupper av bakterier •Nitrifierare växer långsamt

•Denitrifierare finns nästan överallt •Många bakteriesläkten •Denitrifikation är en alternativ andningsprocess i frånvaro av syre •Denitrifierare är växer oftast snabbt

Effekt av extern kolkälla på kvävereningen

0

20

40

60

80

100

0 10 20 30 40 50 60

R

E

0

5

10

15

0 10 20 30 40 50 60

R

E

1. Kvävereningsgrad (%): 2. Denitrifikastionskapacitet:

Tid (dagar) Tid (dagar)

R = Fördenitrifikation utan extern kolkälla E = Fördenitrifikation med etanoltillsats

Intermittent dosering av etanol i en fördenitrifikationsprocess

Hasselblad & Hallin. 1998. Wat. Sci.Technol.

NO 2 NO N 2 O N 2 NO 2 NO N 2 O N 2 NO 3 NH 4 NH 2 OH NO 2 NO - 3 NH NH 2 OH NO 2 - - +

Kvävrening

D I E T A G R I C U L T U R E E N V I R O N M E N T

1/3 of denitrifier genomes lack nosZ (Jones et al. 2008 Molec Biol Evol)

0

0,2

0,4

0,6

0,8

Ratio of N2O-producers

N2O

/(N

2O+

N2)

Manipulation of soil denitrifier community showed direct causality link between the community composition and potential N2O emissions. (Philippot et al. 2011 Global Change Biol.)

Some organisms only have nosZ and are potential N2O sinks. (Graf et al. in prep.)

Microber med och utan nosZ (N2O reduktase gen)

World greenhouse gas emissions by sector

Biological phosphorus removal

ANAEROBIC AEROBIC

Energy Phosphate

Short chain fatty acids

Energy Phosphate O2

CO2+H2O

Energy consumption for uptake of soluble organics. ATP and PO43- is released.

Energy is conserved as poly- phosphate granules. Uptake of PO4

3-. Consumption of stored products (PHB).

PHB synthesis & degradation

Acetoacetate ß-hyroxybutyryl-CoA

ß-hydroxybutyrate

Acetoacetyl-CoA

Poly-ß-hyroxybutyrate (PHB)

Acetic acid Acetyl-CoA

Biological phosphorus removal

PO43- O2

CO2+H2O Energy Energy PO4

3- Organics

Den

itri

fica

tion

ANAEROBIC REACTOR AEROBIC REACTOR SEDIMENTATION

Recirculation of NO3-

N-removal in wetlands

CH4 O2

Diffusion through aerenchyma

Water

O2

CO2

CO2 CH4

O2 +NH3 NO3-

N2OO2

CH4

H2+CO2

Acetate

Root exudates

N2

CO2Reduction zone

NO3- Aerobic zoneN2O N2

Anaerobic zone

NO3-

Reduction zoneSO4

2-

Reduction zone

NO3-

O2

O2 + NH3

CH4 O2

Diffusion through aerenchyma

Water

O2

CO2

CO2 CH4

O2 +NH3 NO3-

N2OO2

CH4

H2+CO2

Acetate

Root exudates

N2

CO2Reduction zone

NO3- Aerobic zoneN2O N2

Anaerobic zone

NO3-

Reduction zoneSO4

2-

Reduction zone

NO3-

O2

O2 + NH3

Constructed wetlands

µg

N/g

DW

/h

Ruiz et al., 2009, FEMS Microbiol. Ecol.

Plants affect denitrification

We

tla

nd

pla

nts

eff

ec

ts

Rhiz

ospe

here

Se

dim

ent

Low rates

High rates

Ruiz et al., 2009, FEMS Microbiol. Ecol.

DGGE of nosZ

• Typha and Fragmites select nosZ communities • Seasonal differences

• Typha and Phragmites select nosZ communities • Typha and Phragmites increase denitrification activity • Seasonal differences Is increase in rhizosphere enough for increased capacity of wetland? • What about the abundance of denitrifiers?

Conclusions and outlook

Ekeby Constructed Wetland

Ekeby wetland in Eskilstuna

Total area: 36 ha

Flow: ~45000m3/day

Wat

er-

flo

w p

ath

s

Kjellin et al., 2007, Wat. Res.

11.113.04103.2 NR CTPDA −−×=Multiple regressions: D

en

itri

fic

atio

n r

ate

s

Kjellin et al., 2007, Wat. Res.

N-removal in mining impacted waters