Denitrification Nitrification Dinitrogen...

11-10-2010

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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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Carbon sourceDenitrification with separate sludge

Denitrification with combined sludge

D

D

Recycle

Return sludge

N

N


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


Others?

Glycogen-accumulating organisms (GAO)


Defluviicoccus

Others?


Microthrix sp.

Mycolata

Type 0041, 1701, 1851, 0092, 0803


Others

Other bacteria

Iron reducers


Fermenters


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


Others?

Glycogen-accumulatingorganisms (GAO)


Defluviicoccus

Others?


Microthrix sp.

Mycolata ??

Type 0041, 1701, 1851, 0092, 0803


Others

Other bacteria

Iron reducers


Fermenters ?


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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6

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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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


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


Type of sludge Carbon source Maximum-OUR

(mgO2/(gVSS*h))

Maximum-NUR

(mgNO3-N/(gVSS*h))

Källby, 2005-07-04


Acetate

Methanol

Acetate+ Methanol

17

6.6

18

4.5

1.3

5.8

Källby, 2005-10-03


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

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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?

Denitrification Nitrification Dinitrogen...

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