Biological N and P removal in
activated sludge processes
Sara Hallin
Department of Microbiology, SLU
©Sara Hallin
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).
Traditionell ASP
Organiskt material bryts ner av mikroorganismer i luftningsbassängen.
Slammet (biomassa och organiskt material)avskiljs från det renade
vattnet i sedimenteringsbassängen.
.
Microbiological reactions in the N cycle
Denitrification
N2
ATMOSPHERE
Nitrogen fixation
SOIL/WATER
N2O
Organically bound nitrogen
org-NH2
NO
Assimilation
Mineralization/
Ammonification
NH4+
Assimilation
NO2-
NO2-
NO3-
Nitrification
Dissimilatory nitrate reduction to ammonium
Kväverening
NH 4+
NH 2 OH
NO 2-
NO 3 -
NO 2-
NO
N 2O
N2
Nitrification - a two step oxidation process
Ammonia oxidation
NH3
NH2OH
Nitrite oxidation
NO2-
NO3-
Regulation of nitrous oxide emissions
NO3-
N removal
NO2-
NO
N2O
N2
Nitrification
Denitrification
Redox
-
Glucose
Glykolysis
2 Pyruvate
8 NADH
NADH
NO2
O22
O
2 GTP
TCA cycle
2 FADH
NH4+
2 ATP
2 NADH
6 CO2
ATP
FADH
ATP
-
ATP
NO3-
H20
+
NO2 N2
NO
N2O
Ammonia oxidation:
NH3 + 1,5O2
NO2- + H+ + H2O
Nitrite oxidation: NO2- + ½ O2
NO3-
Periplams
OH-
Metabolism
Carbon fixation
Cell constituents
Growth
• Lots of ATP needed!
• NADPH required!
Energetic constraints
1. ATP and NADH (reducing power) requirements in for
C-fixation in Calvin cycle
2. NADH formed by reverse e- flow:
O2
e2e-
Cyt c
e-
Cyt c
NAD+
Ammonia
diversity in soil
Denitrifieroxidizer
Diversity
Bacteria
Eukarya
Archaea
Ammonia
oxidizers
Ammonia oxidizing archaea and
bacteria (AOA and AOB), nitrite
oxidizing bacteria (NOB)
The organisms
NH3 oxidizers
Proteobacteria:
Nitrosomonas
Nitrosococcus
Nitrosospira
Thaumarchaeota:
Nitrosopumilis…
NO2- oxidizers
Bacteria:
Nitrobacter
Nitrospira
Screening of a 1215 Mb soil metagenomic library
amoAamoA
NH3
NH2OH
Treusch et al. 2005
Env Microbiol 7, 1985-1995
16S
NO2-
High-affinity ammonia oxidation by AOA
AOA: Nitrosopumilis maritimus (●)
AOB: Nitrosomonas spp; Nitrosospira spp.(●)
nitrification in ocean water ()
nitrification in soils ()
lowest Km for ammonium assimilation ( ).
(Martens-Habbena et al. Nature, 2009)
Kväverening
NH 4+
NH 2 OH
NO 2-
NO 3 -
NO 2-
NO
N 2O
N2
Microbiological reactions in the N cycle
Denitrification
N2
ATMOSPHERE
Nitrogen fixation
SOIL/WATER
N2O
Organically bound nitrogen
org-NH2
NO
Assimilation
Mineralization/
Ammonification
NH4+
Assimilation
NO2-
NO2-
NO3-
Nitrification
Dissimilatory nitrate reduction to ammonium
Denitrification
Denitrification pathway
Pathway
-/
NO3
+ O2 - =
NO2
Denitrification: anaerobic
respiration
Organic compound
ATP
CO2
Carbon flow
Electron flow
Biosynthesis
NO3-, (NO2-, N2O)
Denitrification
Cytoplasma
NO2-
NO3-
nar
NADH2 NAD+
2e-
2ee-
nor
2e-
nir
NO3-
Periplasma
H+ Proton motive
force
nos
NO N2O
NO2- NO
N2O N2
Denitrifier
Denitrifierdiversity
Diversity
Bacteria
Eukarya
Archaea
Denitrifiers
Nitrifikation
Denitrifikation
Redox
-
Glucose
Glykolysis
2 Pyruvate
8 NADH
NADH
NO2
O
O22
2 GTP
TCA cycle
2 FADH
NH4+
2 ATP
2 NADH
6 CO2
ATP
FADH
ATP
-
ATP
NO3-
H20
+
NO2 N2
NO
N2O
Nitrifierande bakterier
Denitrifierande bakterier
•Nitrifierare finns i mark och
vatten
•Denitrifierare finns nästan
överallt
•Bara några få arter
•Många bakteriesläkten
•Nitrifikation är två
energigivande processer som
utförs av två olika grupper av
bakterier
•Denitrifikation är en alternativ
andningsprocess i frånvaro av
syre
•Nitrifierare växer långsamt
•Denitrifierare är växer oftast
snabbt
Effekt av extern kolkälla
på kvävereningen
2. Denitrifikastionskapacitet:
1. Kvävereningsgrad (%):
a
b
Denitrification rate
(mg N O-N g -1 VSS h -1 )
80
E
60
40
2
Nitrogen reduction
(%)
100
20
R
0
0
10
20
30
Time
(days)
Tid
(dagar)
40
50
60
15
E
10
5
R
0
0
10
20
30
40
50
Time(dagar)
(days)
Tid
R = Fördenitrifikation utan extern kolkälla
E = Fördenitrifikation med etanoltillsats
60
Intermittent dosering av etanol
i en fördenitrifikationsprocess
Hasselblad & Hallin. 1998. Wat. Sci.Technol.
Kväverening
NH 4+
NH 2 OH
NO 2-
NO 3 -
NO 2-
NO
N 2O
N2
N2O producing processes and NO3 leaching
N2 O
N2O
NO
NH 4+
NH 2 OH
NO 2-
NO 3 -
NO3-
NO 2-
NO
N 2O
N2
DIET
AGRICULTURE
ENVIRONMENT
World greenhouse gas emissions by sector
Regulation of N2O emissions
NO3-
NO2-
NO
N2O
N2
Microbes without nosZ (N2O reductase gene)
1/3 of denitrifier genomes lack nosZ
(Jones et al. 2008 Molec Biol Evol)
Manipulation of soil denitrifier community
showed direct causality link between the
community composition and potential N2O
emissions.
N2O/(N2O+ N2)
0,8
0,6
0,4
0,2
(Philippot et al. 2011 Global Change Biol.)
0
Ratio of N2O-producers
500
500
1500
1500
2500
2500
N2O/(N2O+N2)
g-1
Potental denitrification
ng N
dry soil h-1
Importance
of on
rootthe
derived
carbon commmunity
Cattle
impact
denitrifying
10
30
Proportion of denitrifiers genetically capable
to reduce N2O (% nosZ/16S rDNA)
Denitrifier genetically capable to reduce N2O (nosZ)
N
W
E
3.102
1.1
2
1.10
1.4
2.102
nosZ
NO3-
NO2-
NO
NO
NNO
2O
N
NO
2
Environmental Microbiology (2009) 11(6), 1518-1526
Mapping field-scale spatial patterns of size and activity
of the denitrifier community
S
Low cattle
impact
1. 104
2.104
4
3.10
0.5
4.10
0.8 4
Gene copy
ng-1 DNA
Total bacteria (16S rRNA)
Gene copy
ng-1 DNA
50
70
40 m
High cattle
impact
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
Medium cattle
impact
(From L. Philippot, INRA)
Regulation of N2O emissions
NO3-
NO2-
NO
N2O
N2
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
Short chain
fatty acids
Energy
Phosphate
Energy consumption for uptake
of soluble organics.
ATP and PO43- is released.
AEROBIC
CO2+H2O
O2
Energy
Phosphate
Energy is conserved as polyphosphate granules. Uptake of
PO43-.
Consumption of stored
products (PHB).
PHB synthesis & degradation
Acetic acid
Acetyl-CoA
Acetoacetyl-CoA
Acetoacetate
ß-hydroxybutyrate
ß-hyroxybutyryl-CoA
Poly-ß-hyroxybutyrate
(PHB)
Biological phosphorus removal
Recirculation of NO3-
Organics
Energy
PO43-
AEROBIC REACTOR
Denitrification
ANAEROBIC REACTOR
CO2+H2O Energy
PO43O2
SEDIMENTATION
N-removal in wetlands
Constructed wetlands
Diffusion through aerenchyma
CH4
O2
O2
CO2 CH4
N2O
N2
Water
O2
CO2
O2
O2 +NH3
NO3NO3-
NO3-
SO42Reduction zone
CO2
Reduction zone
N2
Acetate
Root exudates
CH4
H2+CO2
Aerobic zone
O2 + NH3
Anaerobic zone
NO3Reduction zone
N2O
µg N/g DW/h
Plants affect denitrification
Ruiz et al., 2009, FEMS Microbiol. Ecol.
Sediment
Low rates
Rhizospehere
Wetland plants effects
DGGE of nosZ
High rates
• Typha and Fragmites select nosZ communities
• Seasonal differences
Ruiz et al., 2009, FEMS Microbiol. Ecol.
Conclusions and outlook
• 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?
Ekeby wetland in Eskilstuna
Ekeby Constructed Wetland
Total area: 36 ha
Flow: ~45000m3/day
Water-flow paths
Kjellin et al., 2007, Wat. Res.
N-removal in mining impacted waters