Biological N and P removal in activated sludge processes · Biological N and P removal in activated...

© 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 i

sockret har oxiderats till koldioxid medan en del har reducerats till

etanol (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

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 - - +

Kväverening

Nitrification - a two step oxidation process

NH3 NO2- NH2OH

Nitrite oxidation Ammonia oxidation

NO3-

N2O N2 NO3- NO2

- NO

N removal

Regulation of nitrous oxide emissions

O2

NH4+

H20 O2

ATP

Nitrification

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

- Denitrification

Ammonia oxidation: NH3 + 1,5O2 NO2

- + H+ + H2O

Periplams

OH-

Nitrite oxidation: NO2- + ½ O2 NO3

-

Metabolism

Cell constituents

Growth

Carbon fixation

• Lots of ATP needed!

• NADPH required!

1. ATP and NADH (reducing power) requirements in for

C-fixation in Calvin cycle

2. NADH formed by reverse e- flow:

Cyt c 2e- Cyt c

NAD+

O2 e-

e-

Energetic constraints

Denitrifier Diversity

Ammonia oxidizers

Bacteria Eukarya Archaea

Ammonia oxidizer diversity in soil

Am

mo

nia

ox

idiz

ing

arc

ha

ea

an

d

ba

cte

ria

(A

OA

an

d A

OB

), n

itri

te

ox

idiz

ing

ba

cte

ria

(N

OB

) The organisms

NH3 oxidizers Proteobacteria: Nitrosomonas Nitrosococcus Nitrosospira Thaumarchaeota: Nitrosopumilis…

NO2- oxidizers

Bacteria: Nitrobacter Nitrospira

amoA amoA

NH NH3 NH2OH NO2

-

Screening of a 1215 Mb soil metagenomic library

Treusch et al. 2005

Env Microbiol 7, 1985-1995

16S

AOA: Nitrosopumilis maritimus (●)

AOB: Nitrosomonas spp; Nitrosospira spp.(●) nitrification in ocean water () nitrification in soils ()

lowest Km for ammonium assimilation (

).

High-affinity ammonia oxidation by AOA

(Martens-Habbena et al. Nature, 2009)

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 - - +

Kväverening

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

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

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

Nitro

ge

n r

edu

ction

(%)

Time (days)

R

E

a

0

5

10

15

0 10 20 30 40 50 60

Denitri

ficati

on r

ate

(mg

N2O

-N g

-1 V

SS

h-1

)

Time (days)

R

E

b

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 - - +

Kväverening

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 - - +

N2O producing processes and NO3 leaching

NO

N 2 N O

NO3-

N 2 N 2 O

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

World greenhouse gas emissions by sector

N2O N2 NO3- NO2

- NO

Regulation of N2O emissions

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

+ N

2)

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

Microbes without nosZ (N2O reductase gene)

40 m

ng N

g-

1 dry

soi

l h

-1

Importance of root derived carbon Cattle impact on the denitrifying commmunity

N E

S W

Low cattle impact

Medium cattle impact

High cattle impact

10

30

50

70

N2O/(N2O+N2)

1.102

2.102

3.102

Denitrifier genetically capable to reduce N2O (nosZ)

Gene

cop

y ng

-1 D

NA

Total bacteria (16S rRNA) 1. 104

2.104

3.104

4.104 Gene

cop

y ng

-1 D

NA

0.5 0.8

1.1 1.4

Proportion of denitrifiers genetically capable to reduce N2O (% nosZ/16S rDNA)

500

1500

2500

500

1500

2500

Potental denitrification

Environmental Microbiology (2009) 11(6), 1518-1526

Mapping field-scale spatial patterns of size and activity

of the denitrifier community

Laurent Philippot,1,2,* Jiri Ćuhel,3 Nicolas P A Saby,4

Dominique Chèneby,1,2 Alicia Chroňáková, 3 David Bru, 1,2

Dominique Arrouays4, Fabrice Martin Laurent1,2 and Miloslav Śimek3

nosZ

NO N2 NO3- NO2

- NO N2O NO NO

(From L. Philippot, INRA)

N2O N2 NO3- NO2

- NO

Regulation of N2O emissions

Microbes that only have nosZ (N2O reductase gene)

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

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

Deni

trif

icat

ion

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-

N2O

O2

CH4

H2+CO2

Acetate

Root exudates

N2

CO2

Reduction zone

NO3- Aerobic zone

N2O N2

Anaerobic zone

NO3-

Reduction zone

SO42-

Reduction zone

NO3-

O2

O2 + NH3

CH4 O2

Diffusion through aerenchyma

Water

O2

CO2

CO2 CH4

O2 +NH3 NO3-

N2O

O2

CH4

H2+CO2

Acetate

Root exudates

N2

CO2

Reduction zone

NO3- Aerobic zone

N2O N2

Anaerobic zone

NO3-

Reduction zone

SO42-

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

her

e

Sedim

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

Wa

ter-

flo

w p

ath

s

Kjellin et al., 2007, Wat. Res.

N-removal in mining impacted waters