Bess Ward and Bess Ward and Amal JayakumarAmal JayakumarPrinceton UniversityPrinceton University

Diversity and Dynamics ofDiversity and Dynamics of DenitrifyingDenitrifyingBacteria in the Arabian SeaBacteria in the Arabian Sea

from Analysis of Nitrite from Analysis of Nitrite Reductase Reductase GenesGenes

Regulation of Denitrification

Secondary NO2- maximum?

N2O maxima?Cu limitation?Microbial biomass maximum?Denitrification vs. anammox?

Microbial Ncycle

0

5

10

15

20

25

30

0 50 100 150

Time (hr)

Nitra

te a

nd N

itrite

(M

)

0

50

100

150

200

250

Nitro

us O

xid

e (

nM

)

Nitrite

Nitrate

Nitrous Oxide

0

5

10

15

20

25

30

0 50 100 150

Time (hr)

Nitra

te a

nd N

itrite

(M

)

0

50

100

150

200

250

Nitro

us O

xid

e (

nM

)

Nitrite

Nitrate

Nitrous Oxide

ETNP Incubations without added carbon

•No denitrification observed in any of the treatments

Control +Copper

Bag incubations

Treatments:

Organic C

Cu, Fe

NO3

NO2

cells

Carrie Tuit

Denitrification in theETNP is C limited

1

2

17

Arabian Sea Cruise Track

ETNP

AS

ETSP 1985

Spinrad et al., 1989

Indicators ofdenitrification:

Most intensedenitrificationat SNM

Stage of Denitrification

Paracoccus denitrificans, AAB34245 ( )

Paracoccus pantotrophus, P72181 ( )

Roseobacter denitrificans, CAA12214 ( )

AS WC Cluster IV

CAS WC Cluster II

AS WC Cluster III

Baltic WC, AAY87345

CAS WC Cluster I

AS WC Cluster II

CAS WC Cluster III

Baltic WC, AAY87379

AS WC Cluster I

Magnetospirillum magneticum AMB-1, YP-423528 ( )

Candidatus Kuenenia stuttgartiensis, CAJ74898(Planctomycetacia)

PNW S pB16, CAB76786

PNW S wA20, CAB76794

AS WC Cluster V

Azospirillum brasilense, CAA12215 ( )

Thauera linaloolentis, AAL86934 ( )

Pseudomonas stutzeri JM300, AAA02944 ( )

Alcaligenes faecalis, CAA12216 ( )

Thauera terpenica, AAL86935 ( )

Acidovorax sp. 2FB7, AAL86942 ( )

Ralstonia eutropha H16, CAA62740 ( )

Pseudomonas aeruginosa, AAG03908 ( )

Pseudomonas fluorescens, AAG34381( )

CAS WC Cluster XI

AS WC Cluster VII

Azoarcus tolulyticus, AAL86941, ( )

PNW S pB20, CAB76787

Halomonas variabilis B9-12, CAB76795, ( )

ETSP WC, CAH18784

Marinobacter sp. C10-1, CAB76806, ( )

Marinobacter aquaeolei VT8, ZP_00816485, ( )

AS WC Cluster VI0.05 changes

100

100

100

99

100

88

72

55

49

49

100

100

100

76

79

74

99

100

100

99

100

100

100

Arabian SeanirS phylogeny

Neisseria gonorrhoeae AniA YP-208345 ( )

Shewanella sp. PV4, ZP-00839224 ( )

Nitrosococcus oceani ATCC19707 YP-343556 ( )

AS WC Cluster I

AS WC Cluster II

Nitrosomonas sp. NO3W, AAK53359 ( )

Coastal Aquifer bcD5 S, AAZ83881

Baltic S 30, ABD36682

Soil Ag8-47, ABC02598

AS WC Cluster III

Baltic S 19, ABD36613

Roseovarius sp. 217, EAQ23437 ( )

AS WC Cluster IV

AS WC Cluster V

Coastal Aquifer hbED2 S, AAZ83953

Ochrobactrum sp. 4FB14, AAL82512 ( )

Alcaligenes faecalis, BAA02440 ( )

Ochrobactrum sp. 3CB5 AAL82510 ( )

AS WC Cluster VI

Sludge KRP71, ABA54016

Mesorhizobium sp. 4FB11, AAL82513 ( )

Rhodobacterium bacterium HTCC2654 ZP-01012407 ( )

Baltic S 08, ABD36606

AS WC Cluster VII

AS WC Cluster VIII

Bradyrhizobium japonicum USDA110, BAC52354 ( )

Nitrosomonas sp. TA-921i-NH4, AAK53363 ( )

Waste water M1K30, BAD00133

Rhodopseudomonas palustris CGA009, NP-949481 ( )

Pseudomonas mendocina AAL82514 ( )

Alcaligenes sp. STC1 BAB21510 ( )

Enterococcus sp. R-24626, CAJ76706 (Lactobacillales)

AS WC Cluster IX

Sludge KA27, BAE43797

0.05 changes

100

100

100

79

100

92

90

65

100

100

54

76

55

55

50

9951

95

100

100

52

Arabian SeanirK phylogeny

• Diversity is highest whendenitrification is in the initialstage

• As denitrification intensifies,diversity decreases

0

5

10

15

20

25

1 2 3 4 5 6 7 8

Time

Nu

mb

er o

f U

niq

ue

OTU

s

nirS

Define OTUs fromsequences at 10%identity cutoff

Initial Advanced

208-K03-C4208-K03-G2

208-K03-E5208-K03-D5208-K03-D8

208-K03-A7208-K03-B6208-K03-A46

208-K03-H7208-K03-F9208-K03-E10208-K03-B1208-K03-B4

208-K03-D10208-K03-A12208-K03-C1208-K03-G6

208-K03-B9208-K03-E1

208-K03-A4208-K03-C9

208-K03-G12208-K03-H3

208-K03-E8208-K03-D11208-K03-E12208-K03-H5

208-K03-G4208-K03-F11208-K03-F3208-K03-F2208-K03-F1208-K03-E4208-K03-D9208-K03-B12208-K03-B11208-K03-B7208-K03-A9208-K03-B3

208-K03-F10208-K03-F6

208-K03-C2208-K03-C11

208-K03-F12208-K03-A5208-K03-D3

208-K03-T2-E5208-K03-T2-C6

208-K03-T1-A1208-K03-H11

208-K03-H9208-K03-C8208-K03-H2

208-K03-G3208-K03-C5208-K03-D4208-K03-B2

208-K03-C6208-K03-G5

208-K03-F8208-K03-B8

208-K03-H1208-K03-H8

208-K03-D6208-K03-A8

208-K03-G10208-K03-G1

Roseovarius Sp. 217Arabian Sea Isolate-209-2-6

Coastal Aquifer AAZ83857208-K03-T2-G2208-K03-T1-B1208-K03-T1-E1208-K03-T1-G3208-K03-T1-G5

208-K03-T1-A4208-K03-T2-B2208-K03-T1-B3208-K03-T2-F5

208-K03-T2-D5208-K03-T1-E2208-K03-T1-F5208-K03-T1-D5208-K03-T1-E3208-K03-T1-H3208-K03-T2-B4208-K03-T2-C4208-K03-T1-A6

208-K03-T2-H4208-K03-T2-C2208-K03-T2-D1208-K03-T2-B5208-K03-T2-D6208-K03-T2-H1208-K03-T2-F1208-K03-G9208-K03-T2-A3208-K03-T2-E1208-K03-T2-E6208-K03-T2-H5208-K03-T2-E2208-K03-T2-H3

208-K03-D12208-K03-T2-F3

208-K03-T2-E3208-K03-T2-F2208-K03-E7208-K03-T2-C5208-K03-T2-H2208-K03-T2-E4208-K03-T1-H5208-K03-T1-F2208-K03-T1-E5208-K03-T1-C5208-K03-T1-A5208-K03-T1-G4208-K03-T1-F6208-K03-T1-F3208-K03-T1-E4208-K03-T1-D3208-K03-T1-C6208-K03-T1-H1208-K03-T1-F4208-K03-T1-D4208-K03-T1-C4208-K03-T2-A4208-K03-T2-D4208-K03-T1-B6208-K03-T1-B4

208-K03-T2-G3208-K03-T2-F4

208-K03-T1-C2208-K03-T1-G1208-K03-T1-E6208-K03-T1-D1

208-K03-T2-D3208-K03-T1-G6

208-K03-B10208-K03-H4

208-K03-T1-H4208-K03-D2208-K03-T1-D2

208-K03-T2-C3208-K03-T1-A3208-K03-T2-B6208-K03-T2-C1208-K03-T1-B2

208-K03-T2-G5208-K03-T2-G4208-K03-T2-F6208-K03-T2-D2208-K03-T2-B3208-K03-T2-B1208-K03-T2-A5208-K03-F5208-K03-T1-H2208-K03-T1-G2208-K03-T1-D6208-K03-T1-C3208-K03-T1-C1

208-K03-T1-F1208-K03-T1-B5

208 K03 T2 G6

100

1008551

100

6065

100

94

100100

99

60

100

10098

100

100

Neisseira gonorrhoeae M97926

0.01 changes

~156 aa

_ T1

T2

Coastal (station 17)Arabian Sea Incubation3 time points (2.5 days)

T0

• community shifts towardsdominance

nirK

G. D. O Mullan

G. D. O Mullan

•Low initial diversity

•As denitrificationintensifies --> bloom anddominance

•Highest denit ratesoccur during bloom dueto a few phylotypesnirS

nirK

Kuypers et al. 2003

Indicators of anammox

Dalsgaard et al. 2003

Dalsgaard et al. 2003

Yes -- anammox

No -- denitrification

Bloom induction

ETNP

AS

What was the difference betweenthe ETNP and AS?

Episodic organic C supply

Here s what I think

Anammox: autotrophs, slow steadyconstant backgroundDenitrification: heterotrophs, episodic,bloom dynamics, pulses account formost N2 fluxDirect link to particle flux, anaerobicremineralizationDenitrification supports anammox

Regulation of Denitrification

Secondary NO2- maximum?

N2O maxima?Controlled by O2?Accurate low level [O2] !!!

0 200 400 600 800

Time (h)

0

50

100

150

200

250

0 200 400 600 800

Time (h)

hi Cu

hi Fe, lo Cu

hi Fe, lo Cu

N2O

, NO

2 (μ

M)

P. stutzeri NO2- and N2O reduction

are Cu-sensitive

Julie Granger

0

50

100

150

200

250

0 50 100 150 200 250

Time (h)

NO

2 (μ

M)

WLB Nitrite Reduction is Cu-sensitive

lo Cu

hi Cu

Julie Granger

Dep

th (

m)

Arabian Sea (22o30’N, 60o40’E)

0 0.5 1 1.5 2 2.5

0 2 4 6 8

Sulfide (nM)

Cu (nM)

0

500

1000

1500

2000

2500

3000

35000 10 20 30 40 50 60 70 80

0 50 100 150 200 250

p (

)

N2O (nM)

O2 (μM)

Cu: Saager etal., 1992

O2, N2O: Lawand Owens,1990

S-: Thebergeet al., 1997

Julie Granger

O2

N2O

Cu

S=

Station 17

0

5

10

15

20

25

30

0 50 100 150 200

Time

Nitra

te (

M)

0

5

10

15

20

25

30

0 50 100 150 200

Time

Nitri

te (

M)

0.E+00

1.E+05

2.E+05

3.E+05

4.E+05

5.E+05

0 50 100 150 200

Time

ce

lls/m

l

Copper

Control

TTMo

0

100

200

300

400

500

600

0 50 100 150 200

Time

Nitro

us O

xid

e(n

M)

Time (hr)Time (hr)

Arabian Sea nutrient profiles

0

100

200

300

400

500

600

700

800

0 100 200 300

Oxygen ( M)

Depth

(m

)

Station 1

Station 2

Station 17

0.0 2.0 4.0 6.0

Nitrite ( M)0 20 40 60

Nitrate ( M)

Naqvi et al. unpublished

16S rRNA T-RFLP

K03 bag incubation

T0

T1

T2

Arabian Sea 1996

Open Ocean OMZ in the Arabian Sea

Station 2

0

5

10

15

20

25

30

0 100 200 300 400

Time (hr)

Nitra

te (

M)

0

5

10

15

20

25

30

0 100 200 300 400

Time (hr)

Nitrite

(M

)

•Very large amounts of N2O ( M) in the

TTMo treatments

•Longer lag seen in second sample from

Go-Flo

•Cu(I) samples taken from TTMo and

Control

•Cu(II) in control equivalent to Go-Flo

samples0.E+00

2.E+05

4.E+05

6.E+05

8.E+05

0 100 200 300 400

Time (hr)

Cells

/ml

TTMo1 TTMo2

Control3 Control4

Cu5 Cu6

Time (hr)

0

1000

2000

3000

4000

5000

0 100 200 300 400

Time (hr)

Nitro

us O

xid

e(n

M)

Time (hr)

• Diversity is highest whendenitrification is in the initialstage

• As denitrification intensifies,diversity decreases

0

5

10

15

20

25

1 2 3 4 5 6 7 8

Time

Nu

mb

er o

f U

niq

ue

OTU

s

nirS

Examine diversity ofdenitrifiers during theprogression ofdenitrification processInitial Advanced

Water Column denitrification zones in the

Arabian Sea

Jayakumar et al., 2004

Jayakumar et al., 2004

V400

V483

G840, G857

Stn17 0.1 M NO2- 23.3 M NO3

-

Stn1 5.2 M NO2- 18.4 M NO3

-

Stn1 8 days, 13.3 M NO2- 4.3 M NO3

-

Stn17, 9 days, 14.6 M NO2- 7.9 M NO3

-

Rarefaction Analysis of nirK clonesRFLP and sequence data

Amal Jayakumar

Denitrification

Nitritereductasegenes

Water Column denitrification zones in the

Arabian Sea

Open Ocean Arabian Sea

0 10 20 30 40Nitrate (uM),

-1600

-1200

-800

-400

0

Depth

(m

)

0 50 100 150 200 250

DO (uM)

Station 1 (17N 68E)

Nitrite (uM)0 10 20 30

Nitrate (uM),

-200

-160

-120

-80

-40

0

Depth

(m

)

0 50 100 150 200 250

DO (uM)

Station 17 (18.3N 57.5E)

Nitrite (uM)

NO-3 NO-

3

NO-2

NO-2

Denitrification Rate

(mmol N m-3

y-1

)

0 5 10 20 40 600

50

100

Aug. 98

Sept. 99

Depth

(m)

150

200

250

300

350

Jan. 98

April 96

Sept. 99

Dec. 98

open ocean

coastal

0

5

10

15

20

25

30

0 50 100 150 200 250

Time (hr)

Nitra

te (

M)

0

5

10

15

20

25

30

0 50 100 150 200 250

Time (hr)

Nitrite

(M

)Station 1

0.E+00

2.E+05

4.E+05

6.E+05

8.E+05

0 50 100 150 200 250

Time (hr)

Cells

/ml

2 TTMo

3 TTMo

4 Control

6 Control

7 Cu

8 Cu

13 C+TTMo

14 C+TTMo

17 C

18 C

19 C+Cu

20 C+Cu

0

200

400

600

800

1000

1200

0 50 100 150 200 250

Time (hr)

Nitro

us O

xid

e(n

M)

Time (hr)Time (hr)

NO3-

NO2-

N2O

NO

N2

NH4+

PON,DON

Fe

Cu

MoFe

Fe

Cu

Cu

Fe

Fe

MoV

Fe

NH2OH

Fe

Fe

Open Ocean Arabian Sea

0 10 20 30 40

Nitrate (uM),

1600

1200

800

400

0D

epth

(m

)0 50 100 150 200 250

DO (uM)

Station 1 (17N 68E)

Nitrite (uM)

NO-3

NO-2

Arabian Sea 2004