denitrification nitrification dinitrogen...
TRANSCRIPT
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Denitrification
Identity, physiology and ecology of denitrifiers in activated sludge
Jeppe Lund NielsenSection of Biotechnology
Aalborg University
The nitrogen cycle
NH 3
NO 2-
Nitrification
NO 3-
NO 2-
NON2O
N2
Biomass
N2Aerobic (O2 present)
Biomass
Dinitrogenfixation
Anaerobic (O2 absent)
The process
• NO3- + organic substrate → N2 (via NO2
- )
• Microorganisms often facultative aerobic
• No oxygen present (aerobic denitrification important?)
• Growth rate and yield ca. 70-80% of aerobic growth
• Consume some alkalinity (pH may increase)
• Some denitrifiers may also use Fe3+ as e-acceptor or ferment
Dissimilative reduction of nitrate(denitrification to N2)
NO3- NO2
- N2N2ONOnitrate dinitrogennitrous oxidenitric oxidenitrite
Gasous
Nitrate reductase
Nitrite reductase
Nitric oxide reductase
Nitrous oxide reductase
Acetylen inhibitorChange in oxidation state : +5 to 0
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Dissimilative reduction of nitrate in the environment
NO3- NO2
- N2N2ONO
NO3- NO2
-
NO2- N2N2ONO
NO3- NO2
-
NO2- N2N2ONO
Same organism:
Two different organisms:
Start and endproduct can vary from between denitrifiers and depend on the environment
Reduction of nitrate to ammonium (DNRA)
NO3- NO2
- NH4+
Change in oxidation state : +5 to -3
Mainly in very reduced environments– lack of electron acceptors (electron sink) – e.g. in digesters
Rule of thumb: permanent anoxic environments favors DNRA
ATP
Electron donors
NO3- + 1.8 CH3OH + H
+
0.065 CH5OH7O2N (biomass) + 0.47 N2 + 0.76 CO2 + 2.44 H2O
Electron donors: many different organic compounds (inexpensive), H2, reduced sulfur compounds, Fe(II), others
Consume some alkalinity (pH may increase)
Process design for denitrification
Simultaneous denitrification plant with varying aerobic and anoxic conditions
N
N
D
D
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Process design for denitrification
Carbon sourceDenitrification with separate sludge
Denitrification with combined sludge
D
D
Recycle
Return sludge
N
N
Process design for denitrification
Denitrification with alternating operation
D
N
D
N
N
N
N
N
Phase A Phase B
Phase C Phase D
Important questions - denitrification
• Who? • Carbon source Sufficient C/N ratio in wastewater Addition of external carbon (Methanol, glycol, …) Hydrolysis
• Rates Determine tank volumes
• Aerobic denitrification Is most likely not important in full-scale treatment plants
• …
Microorganisms involved?
Many have been cultured from activated sludge:
Proteobacteria (α, β, γ and ε)FirmicutesBacteroidetes
ArchaeaAquificae
ThermodesulfobacteriaThermotogae
Dictyoglomus
Deinococcus-ThermusChloroflexi
ThermomicrobiaCyanobacteria
Firmicutes
Actinobacteria
Acidobacteria
Nitrospira
Gemmatimonadetes
Planctomycetes
Chlamydia
Verrucomicrobia
ChrysiogenetesDeferribacteres
Bacteroidetes
Chlorobi
Fibrobacteres
Spirochaetes
Fusobacteria
Proteobacteriaα-, β-, δ-, γ-, ε-
Which are the important denitrifiers?
Currently known prokaryoticdenitrifiers represent85 bacterial and 5 archaeal genera
Mostly facultative anaerobes, NO Enterobacteria or obligate anaerobes
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Identifying active denitrifiers in sludge via mRNA
Incubations
Ac: 2mM Acetate
Aa: 2mM Amino acids
Ac+Aa: 2mM Acetate +
2mM Amino acids
Each with 0.4mM Nitrite
mRNA → cDNA
Reverse transcription
nirS, nirK, nosZPCR amplification
RNA extraction
nirS, nirK, nosZ
Process tanks
DGGE fingerprints
4 4 5 3 10 7 9 1 4# bands:
Isolation
Thauera terpenicaS2_AAH73
S3_AAH92S1_AAH100
S1_AAH97S1_AAH33
Thauera sp. S3_AAH114
S3_AAH84S2_AAH170
Simplicispira psychrophilaS1_AAH34
Dechloromonas aromaticaS1_AAH36Dechloromonas sp.
S1_AAH101S3_AAH195
Acidovorax delafieldiiS2_AAH104
Acidovorax sp. JS42S3_AAH116
S1_AAH99S3_AAH85
S1_AAH32S3-AAH115
Azoarcus sp.S2_AAH102
S2_AAH112S1_AAH98
S2_AAH103S2_AAH79
Thiobacillus denitrificansS2_AAH78
S2_AAH75S2_AAH81
S3_AAH90S2_AAH107
Brachymonas denitrificansS2_AAH111
Paracoccus sp.S1_AAH95
S1_AAH35
0.10
Thiobacillus
Thauera
Simplicispira
Dechloromonas
Acidovorax
Azoarcus
Brachymonas
Paracoccus
β
α
Proteobacteria
Expressed
nitrite reductase (NirS)
14
Identification of active denitrifiers (clone and isolate sequences) Dendrogram of 13 WWTPs based on qFISH
qFISH: 14 most dominant denitrifiers (determined by NirS and NirK)
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Dominant bacteria in full-scale N- and P-removing municipal wastewater treatment plants
Nitrifiers
Nitrosomonas sp.
Nitrospira sp.
Denitrifiers
Curvibacter sp.
Azoarcus sp.
Thauera sp.
Accumulibacter (PAO)
Others
Polyphosphate-accumulating organisms (PAO)
Accumulibacter
Actinobacteria-related
Others?
Glycogen-accumulating organisms (GAO)
GB-group (Competibacter)
Defluviicoccus
Others?
Filamentous bacteria
Microthrix sp.
Mycolata
Type 0041, 1701, 1851, 0092, 0803
Haliscomenobacter-like
Others
Other bacteria
Iron reducers
Sulfate reducers (Desulfovibrio sp.)
Fermenters
Hydrolysing bacteria (Microthrix, Saprospiraceae, Actinobacteria)
Dominant bacteria in full-scale N- and P-removing municipal wastewater treatment plants
Nitrifiers
Nitrosomonas sp.
Nitrospira sp.
Denitrifiers
Curvibacter sp.
Azoarcus sp.
Thauera sp.
Accumulibacter (PAO)
Others
Polyphosphate-accumulating organisms (PAO)
Accumulibacter
Actinobacteria-related
Others?
Glycogen-accumulating organisms (GAO)
GB-group (Competibacter)
Defluviicoccus
Others?
Filamentous bacteria
Microthrix sp.
Mycolata
Type 0041, 1701, 1851, 0092, 0803
Haliscomenobacter-like
Others
Other bacteria
Iron reducers
Sulfate reducers (Desulfovibrio sp.)
Fermenters
Hydrolysing bacteria (Microthrix, Saprospiraceae, Actinobacteria)
40-50%
10-15%
20-30%
5-8%
1-5%
1-3%
Nitrifiers
Nitrosomonas sp. ?
Nitrospira sp.?
Denitrifiers
Curvibacter sp. ?
Azoarcus sp. ?
Thauera sp. ?
Accumulibacter (PAO) ?
Others
Polyphosphate-accumulating organisms(PAO)
Accumulibacter
Actinobacteria-related
Others?
Glycogen-accumulatingorganisms (GAO)
GB-group (Competibacter)
Defluviicoccus
Others?
Filamentous bacteria
Microthrix sp.
Mycolata ??
Type 0041, 1701, 1851, 0092, 0803
Haliscomenobacter-like
Others
Other bacteria
Iron reducers
Sulfate reducers (Desulfovibrio sp.)
Fermenters ?
Hydrolysing bacteria (Microthrix, Saprospiraceae, Actinobacteria)
Ecophysiology - uptake of organic compounds: versatile versus specialized species The usual suspects
0
5
10
15
20
25
Curvibacter Thauera Azoarcus Rhodocyc. Zoogloea
Rel
ativ
e a
bu
nd
ance
(%)
Aalborg East WWTP
At least 3-4 abundant denitrifiers present
in all full-scale N and P-removal plants
investigated so far
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0
2
4
6
8
10
12
Curvibacter Zoogloea Azoarcus Thauera Accumulibacter
Per
cetn
age
of
EUB
mix
[%
]
Jan
May
Aug
Nov
Denitrifiers in EBPR plants (n=23)
At least 3-4 abundant denitrifiers present
in all full-scale N and P-removal plants
investigated so far
Non-EBPR plants(n=5)
Reduction of nitrate with Fe(III)
NO2- + Fe(II) N2 + Fe(III)
0
0.2
0.4
0.6
0.8
1
1 2 3 4 5 6 7 8
Time [hour]
Nit
rate
[m
M]
1
2
3
4
Ferr
ou
s I
ron
[m
M]
NO3-
Wastewater
Dewatering
Effluent
Return sludge
Settling
Process tanksPrimarysettling
Anaerobic digestion
Agriculture
Landfill
IncinerationReuse/recovery
SludgehandlingEnergy
(Methane)
Sidestream hydrolysis can increase the improve themicrobial processes in the process tank:
Two ways of improving the denitrification process:
Addition of easily degradable substrate
Anaerobichydrolysis
Sludge COD
VFA COD
Sludge COD
Reduction of Nitrate with different electron donors
Ryaverket WWTP, Gothenborg, Sweeden (24/4 2006)Methanol is regularly added to maintain sufficient denitrification
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Reduction of Nitrate with different electron donors
Type of sludge Carbon source Maximum-OUR
(mgO2/(gVSS*h))
Maximum-NUR
(mgNO3-N/(gVSS*h))
Källby, 2005-07-04
(No carbon dosing at the plant)
Acetate
Methanol
Acetate+ Methanol
17
6.6
18
4.5
1.3
5.8
Källby, 2005-10-03
(No carbon dosing at the plant)
Acetate
Ethanol
Acetate+ Ethanol
15
10
18
3.7
2.5
4.7
Ryaverket, Gothenborg, 2005-09-13
(Methanol dosing at the plant)
Acetate
Methanol
Acetate+ Methanol
45
72
84
13
15
14
Sjölunda, Malmö, 2006-04-28
(Methanol dosing at the plant )
Acetate
Methanol
Acetate+ Methanol
14
53
79
6.2
34
40
At least two populations exists, specialized on utilizing Acetate and Methanol, respectively
Reduction of Nitrate with different electron donors
Type of sludge Carbon source Maximum-OUR
(mgO2/(gVSS*h))
Maximum-NUR
(mgNO3-N/(gVSS*h))
Källby, 2005-07-04
(No carbon dosing at the plant)
Acetate
Methanol
Acetate+ Methanol
17
6.6
18
4.5
1.3
5.8
Källby, 2005-10-03
(No carbon dosing at the plant)
Acetate
Ethanol
Acetate+ Ethanol
15
10
18
3.7
2.5
4.7
Ryaverket, Gothenborg, 2005-09-13
(Ethanol dosing at the plant)
Acetate
Ethanol
Acetate+ Ethanol
45
72
84
13
15
14
Sjölunda, Malmö, 2006-04-28
(Methanol dosing at the plant )
Acetate
Methanol
Acetate+ Methanol
14
53
79
6.2
34
40
At least two populations exists, specialized on utilizing Acetate and Methanol, respectively
3H-Acetate
3H-Methanol
MAR FISH MAR-FISH
Generally higher silver graindensity, with Acetate show betterIncoorporation in biomass
Organisms Probe namePercentage of
Eubacteria [%]
MAR
Methanol/NO3
MAR
Acetate/NO3
Alphaproteobacteria ALF968 10.4 Neg Pos (72%)
Betaproteobacteria BET42a 53.8 Pos (36%) Pos (58%)
Curvibacter-related
bacteriaCurv997 0.9 Neg Neg
Rhodocyclus-related
polyphosphate
accumulating
organisms
PAOmix
PAO462, PAO651 and
PAO846
2.1 Neg Pos (53%)
Most Azoarcus Azo644 30.2 Pos (46%) Pos (85%)
Zoogloea ramigera ZRA 0.9 Neg Pos (89%)
Firmicutes LGCmix 2.7 Neg Neg
Azoarcus/Thauera AT1458 2.8 ND ND
Thauera spp. Thau646 1.5 ND ND
Gammaproteobacteria GAM42a 6.1 Neg Pos (69%)
Most
Deltaproteobacteria
and other Bacteria
SRBmix
SRB385 + SRB385Db13.3 ND ND
Phylum Chloroflexi CFX 1223 3.5 ND ND
Phylum Chloroflexi Gnsb941 4.0 Neg Neg
Archaea Arch915 8.4 ND Neg
Nonsense probe
(negative control)NonEUB338 Neg Neg
Identification of Nitrate reducers in Ryaverket WWTP
Very similar numbers were obtained with nitrite…
(Methanol dosing at the plant)
Nitrate and nitrite consumption in activated sludge
Morgan et al., 2008
Measured in situ by Unisense biosensor
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Substrate uptake by potential denitrifiersin full-scale N and P-removal plants (Aalborg East)
0
1
2
3
4
5
6
Incr
ease
in N
O3
-U
pta
ke R
ate
(mg
N/g
VSS
.h)
Fatty acids Amino acids
Suga
r
Alc
oh
ol Combined
substrates
Measured in situ by Unisense biosensor
Indication of specializedsubstrate consumers
Microautoradiography
Radioactive cells
Non-radioactive cells
Autoradiographic film
Silver grains
AalborgUniversity
Immersion oil
Cover slip
CLSM
Radio-labelledsubstrate [3H, 14C, 33P]
Electron acceptor[aerobic, anaerobic]
Incubation
Fixation
ExposureFilm emulsion Film development
Microscopicexamination
Washing step
Microautoradiography (MAR) Detection of dual substrate uptake by use of two isotopeswith different energies
14C-acetate
14C-acetate and 3H-propionate
3H-propionate
Filaments Single cells
3H: low energy
14C: high energy
3H + 14C:
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0
5
10
15
20
25
[1.0
7-1.
33[
[1.3
3-1.
60[
[1.6
0-1.
87[
[1.8
7-2.
13[
[2.1
3-2.
40[
[2.4
0-2.
67[
[2.6
7-2.
93[
[2.9
3-3.
20[
[3.2
0-3.
47[
[3.4
7-3.
73[
Quantitative MAR
Acetate [mM]
0.1 0.2 0.3 0.4 0.9 1.0
Ce
ll-sp
ecific
activity [x1
0-1
5 m
ol/ce
ll/h
]
0
1
2
3
4
5
Acetate (mM)
Up
take
rat
e (f
mo
l cel
l-1 h
-1)
Ks= approx. 2 µM
Apparent KsSubstrate uptake kinetics of
Meganema sp.in activated sludge
Activity distribution
Nielsen et al. 2003: Environ. Microbiol. 3:202-211Cell-specific activity [x10-15 mol/cell/h]
Information from MAR-FISH - single cells
Active/inactive
Heterotrophic, autotrophic, mixotrophic activity
Consumption of organic substrates
Uptake of micropollutants and other specific substances
Potential denitrifier, fermenter, sulfate reducer, methanotroph, ....
Luxury uptake of orthophosphate
Potential metabolic pathways
Growth kinetics
Substrate uptake rate
Substrate affinity (Km)
Effect of various factors on substrate uptake e.g. inhibiting substances, light, temperature etc.
....
Denitrification by uncultured bacteria– PAOs and GAOs
No NOx
Pre-incubationunlabelled substrate
3H or 14C-labeled substrate
3-6 hours 3 hours
MAR-positiveMAR-negative
No NOx
3 hours
Addition of nitrate/nitrite
Potential denitrification by probe-defined bacteria
Usual suspected denitrifiers:
Thauera sp. (Thau646)
Azoarcus (Azo644)
Rhodocyclus
(Accumulibacter) (PAOmix)
Zoogloea (ZRA)
Aquaspirillum (Aqs997)
others
Thauera sp.
Uptake of 3H-acetatewith nitrate as e-accep.
MAR
EUBmix
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Amino acids common
substrate for all probe-defined
species
Acetate common substrate for
at least 3 probe-defined
Substrate uptake by potential denitrifiersin full-scale N and P-removal plants
Substrate
(labeled)
Aqua-
spirillum
Thauera Azoarcus Accumli
bacter
Fatty
acids
Formate - - - -
Acetate - + + +
Propionate - + - +
Lactate - - - -
Pyruvate - + (+) +
Oleic acid - + - -
Sugars Glucose - (+) - -
Galactose - - - -
Mannose - - - -
Alcohols Ethanol - + + -
Amino
acids
Glutamate - - - +
Leucine - - - -
Amino acid
mix.+ + + +
Genera contain several
phenotypes
Are other important
deniitrifiers present?
Substrate uptake by potential denitrifiersin full-scale N and P-removal plants
Substrat
e
(labeled)
Aqua-
spirillum
Thauera Azoarcus Accumli-
bacter
Fatty
acids
Formate - - - -
Acetate - +(50%) +(20-30%) +(22-35%)
Propionate - +(30-40%) - +
Lactate - - - -
Pyruvate - +(10-20%) +(5-10%) +
Oleic acid - +(10%) - -
Sugars Glucose - +(10-20%) - -
Galactose - - - -
Mannose - - - -
Alcohols Ethanol - +(10-20%) +(20-30%) -
Amino
acids
Glutamate - - - +
Leucine - - - -
Amino
acid mix.+(20-30%) +(15-25%) +(50-60%) + (ND)
Density of silver grain formation
Oligonucleotide probe targeted
bacteria
Incubation with nitrite
(Nitrite-reducing conditions)
Incubation without nitrite
(Anaerobic conditions)
Chloroflexi ─ ─
Nitrospira ─ ─
Alphaproteobacteria + +
Betaproteobacteria + +
Aquaspirillum-related + ─
Azoarcus + ─/+
Thauera + ─
Rhodocyclus ++ +
Gammaproteobacteria + +
Deltaproteobacteria ─ ─
Firmicutes ++ ++
Actinobacteria ─/+ ─/+
Planctomycetes ─ ─
Bacteroidetes ─ ─
Assimilation of 14CO2 with a complex substrate mixture as electron donor and carbon source under nitrite-reducing and anaerobic conditions.
─ No or insignificant silver grain coverage:
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Denitrifiers in activated sludge
?
Others
Identifyable
denitrifiers
-genera
10 µm10 µm
Fluorescence in situhybridization (FISH)
Microautoradiography (MAR)3H-acetate + 3H-amino acids
Unidentified
denitrifiers
Multiphasic approach
- 16S rRNA phylogeny
- Stable isotope probing
- Quantitative FISH
- Quantitative MAR-FISH
- Transcriptomics
- Metatranscriptomics
41
Stable Isotope Probing:- α-, β-, γ- and ε- Proteobacteria- Acidobacteria- Firmicutes- Actinobacteria- Bacteroidetes
Denitrifiers in activated sludgecultivation-independent methods
Full denitrification – important denitrifiers:
Betaproteobacteria:
Family Comamonadadeae (Acidovorax, Comamonas)
Family Neisseraceae (Curvibacter)
Family Rhodocyclaceae (Dechloromonas, Azoarcus, Thauera, Zoogloea, Accumulibacter)
(Juretschko et al., 2002, Thomsen et al., 2004, Kong et al., 2004, Ginige et al., 2005/06, Hagmann et al., 2008, Morgan et al., 2008, Hansen and Nielsen, 2009, Nielsen et al., in prep)
Denitrification in activated sludge
• Relatively few species in full-scale plants can carry out full denitrification
• Many can probably carry out reduction of nitrate to nitrite
• Very specialized and more versatile groups/species are present
• Amino acids and acetate common substrate for most probe defined groups
• Aquaspirillum – high abundance physiology/substrates literally unknown
• We do not know what control the presence of different species
• Aerobic denitrification??
• Does species composition reflect denitrifying activity?