denitrification in the oxygen minimum layer of the eastern tropical pacific ocean
TRANSCRIPT
Deep-Sea Research, 1968, Vol. 15, pp. 157 to 164. Pergamon Press. Printed in Great Britain.
Denitrification in the oxygen minimum layer of the eastern tropical Pacific Ocean*
JOHN J. GOERING t
(Received 29 November 1967)
Abstract--Molecular nitrogen and nitrite are produced concurrently from nitrate-15N added to the oxygen-deficient and nitrite-rich water below the thermocline in the tropical eastern Pacific Ocean. Molecular nitrogen originates by reduction of nitrate (denitrification) and not from the oxidation of ammonia by nitrate or nitrite. The ratio of molecular nitrogen produced to nitrate-nitrogen lost varied from 0.1 to 0'8. Denitrification decreased by 58% with an increase in oxygen saturation from 0"4 to 3"5 %.
I N T R O D U C T I O N
THE PRESENCE of oxygen-deficient water below the thermocline in the tropical eastern Pacific Ocean is well documented (SVERDRUP, JOHNSON and FLEMING, 1942; RICHARDS, 1957; BRANDHORST, 1959; WYRTKI, 1967). The region near the continental shelf of Mexico, the site of this study, contains low oxygen water from just under the thermo- cline to depths of about 1200 m. The most pronounced 02 deficiencies, less than 0-25 ml/l., generally occur between about 150 and 800 m.
The low oxygen waters of the tropical eastern Pacific often contain considerable quantities of nitrite. In addition to a primary nitrite maximum associated with the thermocline a deeper secondary maximum exists, where, under quasi-anoxic conditions, nitrite is presumably produced by reduction of nitrate (BRANDHORST, 1959). THOMAS (1966) discovered a significant decrease in nitrate in the region of the secondary nitrite maximum, and further suggested that gaseous nitrogen compounds are formed because the decrease in nitrate appeared to be greater than the increase in nitrite. The primary maximum is usually attributed to oxidation of ammonia or the produc- tion of extraceUular nitrite by phytoplankton (VACCARO and RVTrmR, 1960).
GOERING and V. A. DUGDALE (1966) and GOERING and R. C. DUGDALE (1966) using a more direct approach showed that nitrate-15N was converted to N2 in anoxic lake water and in sea water containing little or no 02. No evidence of denitrification was found in water collected below the thermocline off the coast of Peru, which has low 02 and high nitrite concentrations, suggesting that in these waters nitrate reduction does not proceed at significant rates beyond nitrite if nitrite results from reduction rather than from oxidation of ammonia.
The purpose of this paper is to present data recently obtained on rates of denitri- fication (measured by 15N-tracer techniques) in the oxygen-deficient layer of the Pacific Ocean near the west coast of Mexico. These data demonstrate that nitrate-15N added to this water is converted to N2. In some experiments nitrite and N2 were produced simultaneously.
*Contribution No. 477 from the Institute of Marine Science, University of Alaska, College. i'Institute of Marine Science, University of Alaska, College.
157
158 JOHN J. GOERING
METHODS
The 15N method used to measure denitrification rates has been described previously (GOERING and V. A. DUGDALE, 1966). Nitrate-lSN is added to the water contained in glass bottles and, after appropriate incubation, the dissolved N2 is analyzed for increase in 15N by mass spectrometry.
A Bendix Time-of-Flight Model 17-210 Mass Spectrometer was used to determine 30N2/28N2 + 29N2 ratios. The precision of this instrument is about 0.01 atom ~ for replicate samples containing the natural abundance oflSN (0.370 at. ~). The fractions of N2 evolved during incubation are thus in error by about + 0.0001.
The water for this study was collected off the west coast of Mexico (the area from 140°W to 11 l°W and from 16°N to 24°N) with Teflon-lined Nansen bottles on cruise 14 of the R.V. Te Vega. Samples for determination of" dissolved 02 and for rates of denitrification were drawn immediately after retrieval of the sampling bottles. Follow- ing the addition of nitrate-15N [ 133.3/zg at/1. of 15N as KNOs (95.7 at. ~ 15N)] the 15N enriched samples were incubated in the dark at 20 ° or 22°C for 186 or 336 hr. After incubation the samples were preserved with 7 mg/1. HgCI2 until analyzed for nitrogen isotopes, nitrate, and nitrite.
Temperature, salinity (by inductive salinometer), nitrate, and nitrite were deter- mined for each sampling depth. Nitrate and nitrite concentrations were determined promptly after sample collection. Nitrate was measured by the method of GRASSHOFF (1964); nitrite by the Griess method outlined in STRICKLAND and PARSONS (1965); and O2 by the Winkler method as modified by CARPENTER (1965).
RESULTS
The concentrations of nitrite, nitrate, 02, fractions of total N2 evolved from nitrate-15N during incubation, temperature, and salinity are plotted against depth for R.V. Te Vega Sta. 655 (Fig. 1) and Sta. 664 (Fig. 2). At both stations a decrease in nitrate is evident in the region of the secondary nitrite maximum. The fractions of N2 evolved at Sta. 655 indicate a sharp zone of denitrification with maximum rates occurring at or just above the nitrite maximum. At Sta. 664 the amounts of N2 evolved did not vary greatly with depth but the depths sampled for determination of denitrification rates were below the nitrite maximum. At Sta. 655 rates approach zero at shallower depths than at Sta. 664.
At the above stations the secondary nitrite maximum occurred in the upper part of the oxygen minimum layer and was associated with a layer of maximum salinity characteristic of equatorial Pacific water (sec WOOSTER, 1967). Apparently micro- organisms capable of using nitrate as a terminal electron acceptor in the absence o1" sufficient oxygen find the low oxygen water associated with the layer of maximum salinity a suitable environment. Denitrification was not detected at five locations where low nitrite concentrations (< 0.1/~g at NO-2-N/1.) were present throughout the oxygen-deficient layer (< 0.25 ral O2/10, suggesting that organisms capable of denitrifying are not omnipresent in deep ocean water.
In Table 1 denitrification rates were obtained by multiplying the fraction of total N2 evolved by the weight of N2 dissolved; the latter were taken from the N2 solubility values of RAKESTRAW and EMMEL (1938), saturation with N2 being assumed at a chlor- inky of 19~o at in situ temperatures.
Denitritication in the oxygen minimum layer of the eastern tropical Pacific Ocean 159
o NO]" -N Io 2o 3o 4okl at./I ter ) NO~" - N ~ ~ ~ 4 ug at./liter
.O N2 evolved o.s~lo ") 1~1o-] 1.s~1o -s / h r
1c4 ~ ~
201
5 e ~ ~" - ' - ' ' " ~ " " O] N
i:tao \ 8O
1
i T " 4 e ~ Us n 24 2=
o,
Fig. 1. Distribution of fractions of total N2 evolved during incubation (196 hr at 22°C) from nitrate-ZSN, nitrite, nitrate, 02, temperature, and salinity with depth at R.V. Te Vega Sta. 655.
The denitrification and nitrite data indicate that nitrite and N2 are produced concurrently• This is particularly evident in water from 347 m at Sta. 655 and 392 m at Sta. 664. Considerable quantities of nitrite were produced along with 15N2, nitrite apparently representing an intermediate step in the reduction of nitrate.
The amount of nitrate-N lost which was converted to N2-N varied greatly, from about 8 to 80 ~o at Sta. 655 and from 26 to 68 ~ at Sta. 664. Presumably reduction of nitrate to nitrite, ammonia or organic nitrogen (particulate and dissolved) accounts for the additional loss.
Fractions of N2 gas produced by denitrification in identical water containing different initial O2 concentrations are given in Table 2. The O2 concentrations in the
NO;- N to 2p 3,o NOi-N ~ ~ :~
~ evolved o.s,~lo ~ t~lQ"
201
301 ~.-.
:E 400 O E ~ N
~soo
60G
700
80¢
9O(
1oo(~
40 lag a t . / l l t e r 4 I~g at./liter
t,s~ 1o') / h r
S~ . 34.0 .2 .4 ..6 . I 35.0 T" 4 I~ 1~2 ~ 2o ~ ~4;
L
/ / / /
f / T * !
• !
!
Fig. 2. Distribution of fractions of total N2 evolved during incubation (336 hr at 20°C) from nitrate-15N, nitrite, nitrate, 02, temperature, and salinity with depth at R.V. Te Vega Sta. 664.
Tabl
e 1.
F
ract
ions
of
tota
l N
2 ev
olve
d fr
om n
itra
teA
~N,
rate
s o
f de
nitr
ifica
tion,
an
d co
ncen
trat
ions
o
f ni
trat
e an
d ni
trit
e be
fore
an
d af
ter
incu
batio
n in
wat
er c
olle
cted
fro
m v
ario
us d
epth
s at
tw
o R
.V.
Te V
ega
stat
ions
in
the
nort
heas
tern
tr
opic
al P
acifi
c.
Con
cent
ratio
ns
expr
esse
d as
/zg
at./l
. (e
xcep
t 02
is
ml/1
.), f
ract
ions
in/
hr,
and
rate
s in
/zg
N/(
1. ×
hr)
.
N2
Evo
lved
In
cuba
tion
In
itia
l co
ncen
trat
ions
D
epth
St
atio
n (m
) F
ract
ion
Rat
e hr
T
emp
(°C
) 0
2
NO
a-N
N
O-2
-N
Fin
al c
once
ntra
tion
s
NO
-a-N
*
NO
-2-N
655
149
0.00
004
0-5
186
22
0" 1
5 28
"7
0.49
94
.5
1" 1
9 (1
4 ° 5
5'N
, 19
9 0.
0004
8 6"
7 18
6 22
0"
18
29"4
0.
01
11.2
0-
08
100
° 0
4'W
) 25
0 0.
0007
5 10
"6
186
22
0" 1
2 24
"8
1.36
0"
1
0.10
29
8 0.
0004
0 5"
6 18
6 22
0'
13
21 "6
1.
21
ut
0.06
34
7 0.
0000
2 0"
3 18
6 22
0-
05
23-6
0-
83
109.
1 6.
80
664
294
0.00
032
4'6
33
6 20
0
' 13
28"0
0.
94
u 0.
04
(19
° 0
2'N
, 31
9 0.
0004
3 6
'2
336
20
0"14
2
9'0
0.
95
u 0.
03
105
° 4
1'W
) 34
3 0
'00
03
5
5"2
336
20
0"18
30
-9
0"74
u
0"03
36
8 0"
0002
4 3"
6 33
6 20
0-
08
32"0
0-
56
u 0"
02
392
0-00
012
1 "8
336
20
0"13
28
"7
0-42
0"
8 15
'50
417
0-00
033
5.0
336
20
0" 1
0 29
" 1
0-56
u
0"05
"133
'3 t
zg a
t./l
iter
of
t~N
as
KN
Oa
(95'
7 at
. ~
15N
) w
as a
dd
ed t
o e
ach
sam
ple
bef
ore
sta
rt o
f in
cub
atio
n,
"ha
=
un
det
ecta
ble
.
U,
z ~3
Denitrification in the oxygen minimum layer of the eastern tropical Pacific Ocean 161
Table 2. Fractions of total N2 evolved and amounts of nitrogen produced from nitrate during incubation (96 hr at 20°C) of 350 m water (R.V. Te Vega Sta. 670, 19 ° 26'N, 100 ° 12'W) containing different initial oxygen concentrations. Fractions calculated using the three equations given by HAUCK, MELSTED and YANKWICH (1958). Fractions
expressed as/96 hr and rates in t~g N/(300 ml × 96 hr).
03 roll1. Fraction Mean fraction Rate
0.02 - 0"0074 0.0097 0.0079 28.9 0.0065
0.19 0"0047 0.0049 0.0048 16.9 0.0047
experimental systems were controlled by mixing water (in an atmosphere of CO2), which had Oz scrubbed from it by sparging with N2, with water that was 02 saturated at 19°C by bubbling O2 through it. The O2 solubility value was taken from GREEN and CARRITT (1967) for a salinity of 34-75~o, the approximate in situ salinity. Differ- ences in amounts of N2 present in each system were taken into account for computation of denitrification rates, which were obtained assuming saturation with N2 at a chlorinity of 19 per mill and 19°C. Supersaturation with O2 may have resulted in a slight under- estimate of the O2 present but the results show that denitrification in sea water is adversely affected by small changes in the bulk concentration of 02. At 0.02 ml O2 (about 0.47o saturation with 02) denitrification was about 1.7 times greater than at 0.19 ml O3 (3.570 saturation). Unfortunately the experiment containing no Oz was lost, so no rate for anoxic conditions is available. Although O2 concentrations would be expected to change during incubation, due to cell utilization, the system presumably did not become anoxic as small amounts of O2 were present (determined by mass spec- trometry) in the extracted gases. The 0.19 ml O2 sample had about twice as much O2 as the 0.02 ml O3 sample. Evidence is presented in the discussion which indicates that sample contamination with atmospheric N2 was minor, thus the O2 probably did not arise by contamination with atmospheric 02. O2 gradients may have occurred within the experimental systems due to differences in the distribution of cells utilizing 02, therefore, denitrification may have occurred at O2 tensions somewhat different from the bulk O2 concentration.
DISCUSSION
Data presented above show that nitrite and N2 are produced from nitrate in the low oxygen and nitrite-rich water below the thermocline in the northeastern tropical Pacific Ocean. The process results in a loss of combined nitrogen available to micro- organisms unless recaptured by nitrogen fixation. Further investigations are needed to characterize the exact conditions under which the reaction occurs so that accurate estimates of the total significance of this nitrate loss in the world oceans may be asses- sed. Large areas of the Indian and Pacific Oceans appear to have conditions favorable for denitrification.
The fractions of total Nz evolved from nitrate were computed using the three expressions proposed by HAUCK, MELS~D and YANKWICH (1958) which assume that
162 JOHN J. GOERING
bacteria act only as a biological catalyst in the denitrification process; that is, all of the Nz produced in the experimental system comes from nitrate. The equations represent the mole fractions of the various isotopic species (14N2,14N15N, 15N2) in the dissolved Nz, the quantities of each being functions of the nitrogen isotope fractions characteristic of the system initially and of the processes which alter the original state of the system. A study of soil denitrification gave similar values for fractions of N2 evolved (variability from the mean comparable to that obtained in this study) indicating the N2 produced during denitrification comes largely from nitrate. WDLER and DELWICHE (1954) using nitrate-15N likewise found that all the Nz evolved by mixed denitrifying cultures had its origin in nitrate.
The similar values for fractions of N2 production in these sea water experiments (Tables 2 and 3) indicate that nitrate was the source of the N2 produced during incuba- tion. Significant amounts of N2 were not produced by the" Van Slyke " reaction (the purely chemical reaction of nitrite with ammonium ion), or by the oxidation of am- monia at the expense of nitrate (RICHARDS, 1965), nor was contamination with atmos- pheric N2 prior to mass spectrometry significant.
Table 3. Fractions of total N2 produced from nitrate during incubation (186hr at 22°C) at R.V. Te Vega Sta. 655 (14 ° 55'W, 100 ° 04'W) in the northeastern tropical Pacific. Fractions calculated using the three equations given by HAUCK, MELSTED and
YANKWICH (1958).
Depth (m) Fraction Mean fraction
149 0'0062 0.0080 0-0065 0'0054
199 0-0891 0.0934 0'0899 0'0873
250 0.1376 0.1442 0'1390 0'1353
298 0"0735 0'0745 0"0737 0"0732
347 0.0044 0.0038 0"0043 0'0047
The gases extracted from the nitrate-15N experiments were passed through a liquid- nitrogen trap to remove oxides of nitrogen, allowing only N2 to be analyzed for nitro- gen isotopes, since experiments with lake water showed N2 to be the only significant product of denitrification (GoERING and V. A. DUCDALZ, 1966). The pH of sea water favors production of N2. With a mixed flora, WIJLER and DELWICHE (1954) found that above pH 7.3 any NzO produced is readily reabsorbed and further denitrified to Nz, whereas at lower pH values there was little reabsorption of N20. The evolution of NO is significant only at pH values below 7.
Because of the lengthy incubation periods employed, the denitrification rates measured are probably not realistic in terms of the process in the natural environment. Nitrate and nitrite would soon be depleted in the oxygen minimum layer if denitrifi- cation occurred at these rates. High rates are probably due to large increases, during
Dertitrification in the oxygen minimum layer of the eastern tropical Pacific Ocean 163
incubation, of bacterial cells capable of denitrifying. A rapid proliferation of plank- tonic bacteria occurs when sea water is stored for just a few days in glass bottles (ZosBLL and ANDERSON, 1936), presumably the result of providing a surface for attach- ment, a site for concentration of dissolved organic matter by adsorption, and a barrier against diffusion of bacterial enzymes away from the cell. However, the 15N experi- ments do indicate that adequate organic matter is present in these waters to support vigorous denitrification at existing 02 tensions providing the water is put into a glass container.
The denitrification rates at some depths (Table 1) are considerably higher than those measured in previous studies (GoERING and V. A. DUGDALE, 1966; GOERING and R. C. DUODALE, 1966), in which rates of about 3.5/zg N/(I. × hr) were produced from nitrate in the water overlying the sediment and 0.6/~g N/(1. x hr) in water collected from 1 m below the ice in a subarctic lake, which becomes anoxic for part of the winter. Similar rates were obtained in water from a stagnant basin in an island bay in the equatorial Pacific Ocean. These differences between studies or with depth may be due to different numbers of microorganisms or different species, or both. Differences may also result from the higher incubation temperatures (20 ° and 22°C versus 18°C) used in the present study or a lag period in the reduction of nitrate by cells which had not originated in water containing nitrate (see review by NASON and TAKAHASHI, 1958). Nitrate and nitrite were undetectable or present in low concentra- tions in the anoxic waters used in the earlier studies, presumably depletion having resulted from denitrification.
Substantial amounts of nitrate reduction to products other than N2 occurred in these eastern tropical Pacific low 02 waters. BREZOrqK and LEE (1966) found a ratio of N2 formed to NO-a-N loss of 0.6 in their study of the sources of N2 in fermentation gases. GOEmNG and V. A. DUGDALE (1966) reported that in lake water reduction of nitrate to ammonia was small (2-18 ~) compared to reduction to N2, but reduction to particulate and dissolved organic nitrogen was not examined. Additional study is required to establish the importance of nitrate reduction to ammonia and to organic nitrogen in oxygen deficient marine waters.
The effect of 02 on sea water denitrifiers needs further study. 02 is known to be a potent inhibitor of the process by virtue of its effective competition with nitrate as an electron acceptor in the energy metabolism of cells (DELWJCHE, 1956). The amount of dissolved 02 which limits denitrification is uncertain, but it is thought to be a few tenths ml/1. and may be dependent on the concentration of nitrate (MCKINNEY and CONWAY, 1957). SKERMAN and MACRAE (1957) found no reduction of nitrate by Pseudomonas dentrificans at dissolved O2 concentrations above 0.2 mg/l. Perhaps with low 02 concentrations (few tenths ml/1.) localized anoxic zones exist which are the active sites of denitrification. To obtain useful information denitrification must be studied in an experimental system where O2 concentrations are precisely known and can be varied in all areas of the system at all times.
GOERING and R. C. DUGDALE (1966) did not detect denitrification in the low oxygen and high nitrite water off the coast of Peru. They did not investigate a depth profile and perhaps the active region of denitrification was missed, or reduction proceeded only to nitrite. ZOBELL (1946) states that fewer than 5 ~ of the marine bacterial species can reduce either nitrate or nitrite to N2 or ammonia, whereas about half can reduce nitrate to nitrite in the presence of sufficient organic matter. An alternate explanation
164 J'om~ J. GOERING
is that ni t r i te results p r imar i ly f rom a m m o n i a oxidat ion. Fur the r work is needed to de termine i f mic roorgan isms capable o f a m m o n i a ox ida t ion o r ni t ra te reduct ion exist and funct ion under condi t ions prevai l ing in these waters.
Acknowledgements--This study was supported in part by National Science Foundation Grants GB-5469 and GB-5532. The author is grateful to W. BARIBAULT, W. A. FORD, C. LEVENSON, .1. NORTON, W. SAMUEL, G. SILBERSTEIN and D. WALLEN for technical assistance.
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