Download some chemical and microbiological observations in the pacific

SOME CHEMICAL
AND MICROBIOLOGICAL
OBSERVATIONS
IN THE PACIFIC OCEAN
OFF THE HAWAIIAN
ISLANDS
K. Gundersen, C. W. Mountain, Diane Taylor,
R. Ohye, and J. Shen
Department
of Microbiology,
University
of Hawaii,
Honolulu
96822
ABSTRACT
Simultaneous
chemical and microbial
analyses of the water column were made at 33
stations off the leeward Hawaiian
Islands. The overall distribution
of oxygen, nitrate, and
pH was similar at all stations located more than 5 km offshore.
These parameters were
closely correlated and also correlated with nitrifying
and nitrate-reducing
activity in the
water column. The distribution
of nitrite and ammonium did not correlate with the oxygen
distribution.
A nitrite band was consistently found in the lower portion of the photic zone
and appeared to have originated
from reduction of nitrate rather than from oxidation
of
ammonium.
The distribution
of aerobic and anaerobic bacteria was regulated
by the
amount of available organic nutrients and not by the oxygen concentration.
INTRODUCTION
Little information is available regarding
the water chemistry and microbiology
off
the leeward Hawaiian Islands, Earlier microbiological work (Adair and Gundcrsen
1969a, b; Ohyc and Gundersen 1969)
was not accompanied by chemical-physical
analyses of the ocean environment, and we
were therefore not in a position to draw
any conclusions as to the ecological significance of our findings. We became convinced, then, that to understand microbial
processes as they occur in the sea it is
imperative to make simultaneously precise
analyses of biologically
important parameters of the water column and to relate
these to the processes studied; during subsequent cruises such parameters were routinely determined
along with microbial
analysis.
During
1969, we made 17 separate
cruises (Bug Scafari) off the leeward Hawaiian Islands, from Kawaihae Bay, Hawaii, in the east to Niihau in the west
(Fig, 1). Altogether 33 stations were investigated, 14 of which were on or beyond
the l,OOO-fathom contour. No station was
closer to land than 5 km. Station 17A was
1 Hawaii
No. 398.
LIMNOLOGY
Institute
AND
of
Geophysics
OCEANOGRAPHY
Contribution
revisited twice in early 1970 and some of
the observations made during these later
cruises have been included in this report.
Northeast winds, rarely exceeding 10 m
/set, were prevailing
during the cruises.
All cruises were made on the RV Teritzc.
We are indebted to Mr. L. I. Knowles and
to officers and crew of the RV Teritu for
their interest and cooperation during this
work.
METHODS
Sampling
Water samples for most chemical analysis, salinity, and pH were taken with a
set of twelve 13-liter PVC samplers (a
locally manufactured
modification
of the
Van Dorn sampler); samples for particulate organic matter and protein were taken
with Niskin sterile bag samplers (General
Oceanics, Inc. ) ; bacterial samples were
also obtained with these samplers and
with JZ sterile bottle samplers (Kahl Sci.
Instr. Corp. ) .
Samplings were made at 50-m intervals,
12 bottles to the cast; in the upper water
column, closer intervals were sampled in
several cases. The reported depths are
taken from the winch meter and corrected,
when rcquircd, for wire angle.
524
JULY
1972, V. 17(4)
ANALYSES
160”
--
159”
OF IIAWAIIAN
OCEAN
158”
WATIZRS
157”
156”
l55”W
L,I--
22”N
IOA
l
,’
I.-_.
HAWAIIAN ISLANDS -.. -- --.--.--+
FIG. 1.
Bug Seafari
cruises, 1969.
Physical-chemical
methods
The temperature of surface water, taken
in a bucket, was recorded to the nearest
0.5C. The temperature profile of the upper 275 m was determined
on every station with a standard bathythermograph.
Equipment was not available for temperature measurements below this depth.
Light penetration was measured with a
locally
manufactured
light transmission
metering system, having a range from 3001,000 nm and with a sensitivity from 107107,640 lux.
Samples for salinity
were stored in
tightly stoppcred glass bottles and analyzed in the labloratory with a Hytech
salinometer (model 6210).
pH and alkalinity were dcterrnined with
a Beckman pH meter according to standard procedures I(Strickland and Parsons
1968). Because temperature data were not
available from depths belomw the reach of
the bathythermograph,
we were not able
Position
of stations.
to make tempcraturc corrections for the
pH
readings
1
-~
.y so the. values
.
. reported are
Lower than the true in situ values.
Oxygen was determined routinely by two
independent
methods : polarographically
with a Beckman oxygen analyzer (model
777) and chemically by standard Winkler
titration.
The two methods showed good
correlation but only the values obtained
by titration are reported here.
Ammonium
was determined according
to the method of Solorzano (1969). Nitrite,
nitrate, and reactive phosphate were determined according to methods described by
Strickland and Parsoas ( 1968) .
Samples for particulate protein analysis
were filtered through 47-mm Millipore HA
filters (pore size, 0.45 p). The amount of
water varied with the origin of the samplc: 800 ml of surface water were filtered;
1 liter from between 25 and 500 m; 1.5
liters between 500 and 1,000 m; 2 liters
below 1,000 m. The filters were trcatcd
526
K.
GUNDERSEN
according to the Lowry-phenol method for
protein analysis ( Lowry et al. 1951). A
15-min alkaline hydrolysis was used, followed by the addition of Folin’s reagent.
The absorbance was read after exactly 30
min at 750 nm.
Water for particulate carbon (POC) and
nitrogen (PON) analyses was transferred
to e-liter polyethylene
bottles which had
previously been rinsed with distilled water
made particle-free
by filtration
through
0.22-p Millipore
filters.
The actual filtering was through 1.2-p Sclas Flotronic
silver membrane filters (45 mm) in a Millipore Pyrex filtering
assembly under a
vacuum of less than 550 mm of mercury.
One-liter quantities wcrc filtcrcd and the
filtering funnel washed with particulatcfree distilled
water bctwecn filtrations.
The filters were individually
stored in pctri dishes until analyzed in a CHN analyzer (Hewlett-Packard
F&M, model 185)
according to standard procedure.
Unless otherwise stated, all chemical
analyses were made on shipboard.
Microbiological
methods
Standard Milliporc
filtration
of asep titally sampled water was used to determine
bacterial distribution in the water column.
Samples of lo-100 ml were passed through
47-mm Milliporc IIA filters (porosity, 0.45
p) using a manifold and a vacuum of
about 700 mm of mercury. Four or six
filtrations were made of each sample and
the filters subsequently placed on marine
agar (Difco) plates. Half the plates were
incubated aerobically in the dark and the
other half incubated under hydrogen gas
in anaerobic jars (Torsion Balance Co.).
Plates were examined after 48-72 hr of
incubation at 28 rt 2C. Most aquatic bacteria occur in aggregates of variable numbers of individual cells and in close spatial
proximity
on the surfaces of particulate
matter (ZoBcll 1946; Scki 1970) and consequently give rise to considerably fewer
colonies than there were cells in the original sample, so we have preferred to report bacterial counts as “colony-forming
ET
AL.
units” ( c.f.u. ) , rather than as “number of
cells,” per unit volume.
Several different tcchniqucs wcrc used
in the investigation
of nitrification
and
nitrifying microorganisms in the water column but only a few will bc reported here;
a more cxtensivc report of methods used
in nitrogen transformation studies in these
waters was recently given by Mountain
( 1971). Enrichment
cultures were prcpared by injecting a few milliliters
of
( NH4)&04
or NaN02 solutions directly
into the polycthylcnc
sampling bags, cstimated to give final concentrations of about
1,000 pg-atom N/liter.
Exact detcrmination of initial
ammonium,
nitrite,
and
nitrate concentrations was made on subsamples withdrawn
immediately after cnrichmcnt. During the incubation period of
several weeks in the dark at 25 -L 2C, water was withdrawn
at intervals for chcmical and bacteriological
analysis.
The same tcchniquc used for nitrification was used for nitrate reduction.
Enrichments consisted of NaN03 (about 1,000
pug-atom N/liter) alone or in combination
with glycerol ( 0.1% ) .
All the initial microbiological
work was
done without delay on shipboard.
RESULTS
AND
DISCUSSION
The overall vertical distribution
of pH,
oxygen, nitrate, phosphate, protein, particulatc organic matter, and bacteria in the
water column was within the cxpccted
range for the pelagic distribution of these
parameters. A slight, but consistent oxygen maximum was found near 100 m and
a minimum, of about 1 mg Oa/liter, cxistcd bctwecn 700 and 900 m (Fig. 2A).
The salinity data agree well with data
given by Wyrtki ct al. (1967) and Scckcl
(1968) and show a sharp maximum near
100 m ( Fig. 2B), characteristic of North
Pacific Central Water. Below this water
mass and down to the lower depth of this
study (1,150 m), the low salinity North
Pacific Interrnediate Water makes up the
water column.
Whereas nitrate was practically
absent
from surface waters at all times, a fairly
ANALYSES
A.
IO 15 20
OF IIAWAIIAN
OCEAN
527
WATERS
7.50 175 8.00 8.25 pH
25 30°C T
c.f.u.bacteria/titer
\
b
‘Aerobic bacteria
Anaerobic
I: CO% 1.90
780
PH
R
-z
c.
20 25 .50°c
1.95
8.00
2.00 mM/li ter
8.20
T
15.0
20.0
25.O”C
T
0
66
cm0
2
4
6
8
IO ,uq PON-N/liter
0 20 40 60 80 IOOjkj POC- C/liter
0
1
10.0
I% \iqht penet
8.00 8.25 pH
I
bacteria
2
4
6
I
8 x 104c.f.u. bacteria/liter
I
I
I
IA-
p.m.
FIG. 2. Characteristics
of the water cohmm of three stations investigated
during the Bug Seafari
cruises off the leeward Hawaiian
Islands. A. Station 6B, off Oahu. pH uncorrected.
Bacterial counts
are reported as colony-forming
units (c.f.u.).
B. Characteristics
of the upper 300 m of station 17A,
off Oahu. C. Station 5B, a shallow nearshore station off Maui. The two casts were made at 0800 and
1600 hours.
528
K. GUNDERSEN
sharp nitrite band, amounting to 0.06-0.07
pug-atom N/liter,
was consistently found
between 100 and 200 m ( Fig. 2B). There
was, however, no nitrite band associated
with the oxygen minimum at about 800 m.
Ammonium was most abundant in the upper water column but rarely cxcceded 0.5
pg-atom N/liter.
Its distribution was usually irregular and did not always show a
clear peak ( Fig. 2B ) . In deeper water,
nitrite and ammonium were usually absent, but nitrate increased rapidly below
about 150 m reaching a high of about 40
pug-atom N/liter at about 800 m. The highest value measured was about 47 pg-atom
N/liter below 1,000 m.
Minor fluctuations in the chemical paramctcrs over the year apparently were not
seasonal variations.
The temperature of
the surface water varied about 5C betwecn winter and summer.
The island effect described by Doty and
Oguri (1956) was not reflected in our
chemical data. For example, there was
little diffcrcnce in the three stations 6A,
6B, and 6C, situated along a track perpcndicular to the Waianae coast of Oahu.
In the shallow waters off Maui (station
5B, Fig. 2C) and between Molokai and
Lanai (station 1OD) there was no significant vertical variation of any chemical paramcter and little, if any, numerical differcnce in data from a comparable depth
farther offshore. However, bacteria were
4-5 times more abundant in the shallow
ncarshore water than in the open ocean.
One parameter possibly reflecting a land
influence was the vertical distribution
of
particulate matter. The values for POC
and PON were more irregular and usually
also higher than at the windward station
Gollum (22” 10 N, 158” 00 W) investigated
by Gordon ( 1970). Since we used exactly
the same tcchniquc and equipment, the
data are comparable. The higher values
and the irregular distribution
on the leeward side of the islands may be due to
the dumping
into the ocean of large
amounts of solid wastes from Hawaiian
sugar mills; windrows of such materials
were frequently
observed
during
the
ET AL.
TABLE 1. Particulate
organic carbon (POC) and
nitrogen (PON), Bug Seafari cruises, 1969,
station 1 SD
Depth
(ml
O-5
50
100
150
250
300
400
450
500
550
600
650
700
750
800
900
950
1,000
1,050
1,100
POC
PON
( m/liter)
62.17
32.71
68.46
21.36
27.43
26.59
18.04
59.29
43.02
50.66
45.08
45.09
15.86
22.85
37.29
26.29
30.41
55.40
21.93
13.87
10.30
6.57
11.53
7.10
5.85
6.01
5.07
9.38
8.53
5.03
6.07
6.57
4.94
4.16
6.97
3.76
2.59
8.60
3.70
3.66
C:N
(w/w)
6.0
5.0
6.0
3.0
4.7
4.4
3.6
z-i
10:1
7.4
6.9
z*z
514
7.0
11.7
6.4
6.0
3.8
cruises, sometimes far out to sea. These
particles
continuously
settle throughout
the area and the prcsencc of such wastes
should easily be detected in the POC-PON
analysis. The high C : N ratios occasionally
found ( Table 1) would tend to indicate
a predominance of particulate matter of
plant origin such as cellulose and other
polysaccharides .
The C : N ratio of whole mixed plankton
is about 5.7 on a weight basis (Fleming
1940). Carbon-nitrogen
analyses made on
whole plankton collected in a 60-p-mesh
net at about 5-m depth during one of our
cruises gave C :N ratios between 4.65 and
5.20. Since the bulk of particulate organic
matter in the sea originates from plankton,
a high C : N ratio could also mean that a
fraction of the nitrogen-containing
components of dead plankton had already been
decomposed in the upper portions of the
water column, liberated
as ammonium,
and rapidly reassimilated by the phytoplankton, Our nitrification
studies support
this belief.
As was the case with POC and PON,
the distribution
of particulate protein and
ANALYSES
Nitrate
oo
g&lo900-
in
W~~WCO~WWI
5
10
15
0
0
%
E IOOO-
2,o
l
00
l
25
l e
0
IIAWAIIAN
jKptorn NO,-N/liter
30
35
4
:
l
l :.
0
P 1100 -
0
1200
I
I
I
-20
-15
-10
-5
0
Chanqe in nitrate durinq incubation
OF
I
5
l
I
I
IO
15
:
pq-atom NO,-N/liter
FIG. 3. Nitrifying
activity and nitrate reduction
demonstrated
by changes in nitrate concentration
of water samples during 30 days of incubation
at
room temperature
in the dark. *No
enrichment; O-enriched
with 1,000 pug-atom N/liter
as (NH4)2SOe.
The water samples were collected
and subsequently
incubated in presterilized
Niskin
polyethylene
bags.
bacteria showed considcrablc vertical variation. Most of the: protein was found in
the upper portions of the water column,
as expected, but occasionally substantial
values were found as deep as 500 m. Below this depth the values were always low.
A remarkably good correlation was found
bctwecn the distribution of particulate protein and bacteria when these paramctcrs
were determined simultaneously (Fig. 2A).
The filtration
method used for protein
analysis will, of course, include almost all
the bacterial protein of the sample, and
30-50% of the protein measured by this
method is of bacterial origin.
The pattern of vertical distribution
of
OCEAN
529
WATERS
bacteria and protein indicates the existcnce of narrow biotic strata in the water
column. We have found such bands on
several occasions, and they do not appear
to bc technical artifacts. Sorokin (1971)
described similar layers of plankton and
bacteria in tropical waters.
Nitrification
was only detectable below
200 m (Table 2, Fig. 3). Although ammonium was produced in samples of unenriched surface water incubated fo,r 30 days
in the dark, it was not nitrified
(Table
2). Apparently,
nitrifying
organisms are
either absent, or present in low numbers,
in surface waters; Carlucci and Strickland
(1968) estimated their numbers to be less
than one cell per liter in the open ocean.
A possible explanation for their insignificant role could bc the inability
of the
thermodynamically
disadvantaged nitrifiers
to compete with the phytoplankton
and
heterotrophic microorganisms for the small
amount of available ammonium.
In deeper water, however, the absence
of competing phytoplankton
and less o,rganic matter to support heterotrophic
growth ,would favor the nitrifying
organisms. Consistent with this point of view
is the nitrifying
capacity of most samples
taken from below about 200 m ( Fig. 3).
Bcforh the results of this experiment can
be intebrctcd,
it is necessary to realize
that two apparently
opposite biological
proccsscb, nitrification
and nitrate rcduction, both occur in the water. The factors
controllibg nitrification
are the availability
of ammonium ( as an energy source), car-
2. Changes in inorganic nitrogen (in ,ug-atom N/liter3
in unenriched
water samples from the
upper 300 m of the water column following
30 days of dark incubation
at 26C. Samples were taken
and incubated in sterile Niskin sampler bags. Bug Syfari
cruises, 1969, station 17A
TABLE
Initial
Depth
(111)
NH,+
NO,-
100
150
200
250
300
0.14
0.24
0.50
0.52
0.14
0.00
0.00
0.02
0.01
0.02
0.09
0.03
0.02
0.02
After 30 days
NO,-
NH,+
NO,-
0.42
0.50
0.36
1.78
4.80
9.60
16.00
4.82
6.67
9.27
10.01
2.98
1.35
1.25
0.11
0.09
0.13
0.19
0.20
0.45
0.58
Increase
WI-
NE&+
NO,-
NO,-
0.62
0.62
0.56
1.88
4.93
11.40
20.00
4.68
6.43
8.77
9.49
2.84
1.35
1.25
0.09
0.08
0.11
0.10
0.17
0.43
0.56
0.20
0.12
0.20
0.10
0.13
1.80
4.00
530
K. GUNDERSEN
bon dioxide (as a carbon source), and
oxygen
( as an electron acceptor ) , thus :
NH4’ + 02 + COz --) Cells + NOS-. (1)
Nitrate reduction, on the other hand, is
controlled by the availability
of organic
matter (as a carbon and energy source)
and nitrate (as an electron acceptor), thus:
Organic matter + NOJ- + Cells + NOz- (2)
(other forms of reduced nitrogen, e.g. NZ,
may bc the end product),
Facultative anaerobic bacteria which reduce nitrate arc
present at all depths in the sea and nitrite
production from nitrate, even in the prcsencc of oxygen, can easily bc dcmonstratcd in water samples enriched with
glycerol or other organic matter (Mountain 1971). Thus, with only small amounts
of ammonium available to the nitrificrs,
nitrate reduction will be the predominant
process and any nitrification
bccomcs
masked. On the other hand, if ammonium
is added and nitrifying organisms are prcsent, nitrification
becomes predominant and
will mask nitrate reduction,
In the unenriched water bags (Fig. 3,
open circles ), the preexisting nitrate was
rcduccd to nitrite whereas in the ammonium-enriched
bags ( solid circles ) additional nitrate was formed. Nitrite accumulated only as a result of nitrate reduction
and not as a result of oxidation of ammonium by nitrifiers.
If it can bc assumed
that nitrate reduction will occur whether
or not there is simultaneous nitrification,
the nitrifying capacity of the enriched watcr samples in Fig. 3 must bc considerably
greater than shown.
We had expected to find some corrclation bctwecn the distribution of oxygen in
the water column and the distribution
of
aerobic and anaerobic bacteria, but this
was not the cast. Although oxygen distribution is apparently corrclatcd with the
distribution
of aerobic bacteria, it is also
correlated with the distribution of bacteria
capable of growing in the complctc absence of oxygen, at least below the photic
zone (Fig. 2A). The finding that about
ET AL.
two-thirds of all heterotrophic marine bactcria isolated by standard techniques, irrespcctivc of what depth and oxygen level
they came from, will grow whether oxygen
is available or not (Ohyc and Gundersen
1969) seems to support the view that oxygcn within the range found in oceanic
waters dots not have a significant controlling cffcct on the distribution and biochemical activities of marine bacteria (ZoBcll
1940). Even the aerobic marinc nitrifying
bacterium Nitrosocystis oceanus will oxidize ammonium to nitrite at low oxygen
tensions ( Gundcrsen 1966; Carlucci and
McNally
1969). Th e very close correlation between the distribution
of protein
and of heterotrophic
bacteria (Fig. 2A)
supports the view that the distribution
of
nutrients plays a much more important
role in the distribution
of bacteria than
dots oxygen.
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ADAIR, F. W., AND K. GUNDERSEN.
Chemoautotrophic
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rinc environment.
345353.
1969h.
Chemoautotrophic
AND -.
-7
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1969.
Nitrification
by marine bacteria in low conLimcentrations
of substrate and oxygen.
nol. Oceanogr. 14: 736-739.
AND J, D. H. STRICKLAND. 1968.
The
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OCEAN
WATERS
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801.
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Oceanographic
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-.
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