Bess Ward and Amal Jayakumar Princeton University Diversity and
Transcript of Bess Ward and Amal Jayakumar Princeton University Diversity and
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