FEMS Microbiology Ecology 38 (1986) 11.-17
Published by Elsevier
11
FEC 00052
Mass spectrometry as en ecological tool for in situ measurement
of dissolved gases in sediment systems
(Denitrification; methanogenesis; anaerobic sediment)
D. Lloyd, K a t h r y n J.P. D a v i e s a n d L y n n e B o d d y
Department of Microhioloyo', Unit~ersio, College, Newport Road. Cardiff. CF2 1 TA. Wales. U.K.
Received 4 November 1985
Revision received and accepted 2 January 1986
1. S U M M A R Y
2. I N T R O D U C T I O N
The use of membrane-inlet quadrupole mass
spectrometry, as a method for quantitative monitoring of dissolved gases in natural or semi-natural
environments, is described. Its advantages over
other methods lie in the fact that it provides an
accuratc, sensitive means for non-invasive, continuous analysis of several dissolved gases simultaneously. The potential of mass spectrometry as an
ecological tool is illustrated by representative resuits from measurements made on undisturbed
and experimentally amended estuarine and freshwater sediments.
Dissolved gas profiles from the surface to a
depth of 10 cm in the estuarine sediment showed
that the dissolved oxygen decreased gradually until at 10 cm it was undetectable ( < 0.25 /.tM);
dinitrogen reached a maximum at 6 cm, where
oxygen was 20 ~M. In a fresh-water sediment,
methane reached 1.5 mM at 10 cm depth. N O t
was also detected; quantitation of carbon dioxide
nccessit~,tes a correction for the contribution of
NO~. Manipulation of conditions (gas phase,
nitrogen and carbon sources) permitted ecological
modelling.
A variety of methods is available for the quantification of dissolved gases in laboratory cultures,
although many of these require removal of samples, or show low accuracy a n d / o r poor reliability
[1-5]. These problems are magnified and further
deficiencies in methodology become evident when
such techniques are applied to natural biological
systems. Further, if more than one gas is to be
measured, several different techniques are usually
needed. Membrane-inlet quadrupole mass spectrometry, however, provides an accurate, sensitive
means for non-invasive, continuous quantitative
analysis of several gases simultaneously in the
liquid phase [1,3,6,7], and should thus have great
potential as an ecological tool. It has already been
used successfully in liquid culture studies in the
laboratory on numerous occasions, e.g., [8-10], in
model ecosystems [11] and in situ in the rumen of
sheep [12].
The theory and use of mass spectrometry have
been described in detail elsewhere [3,13,14]. Briefly,
the mass spectrometer (MS) ionises neutral atoms
and molecules of introduced gas samples, and
these ions can then be analysed according to their
0168-6496/86/$03.50 ¢~ 1986 Federation of European Microbiological Societies
12
mass to charge ratio ( m / z ) . Each atom and molecule has a characteristic peak, or rather group of
peaks, in the mass spectrum. The position of the
peak ( m / z ) is characteristic of the component,
and the height of the peak is proportional to its
concentration. Permanent gases which are of particular ecological interest are usually measured at
the following m / z values: H~, 2; CO, 12, N:, 28,
CH~, 15, nitrogen oxides, 3 0 : 0 2 , 32: H2S, 34;
CO,, 44. However, it should be realised that ions
originating from different sources may sometimes
interfere (see [15]). For example, when 02 is measured at m / z = 32 there are two potential sources
of interference, from H2S or methanol. This problena is easily surmountable by determining the
contribution of these other ions: H , S being 44%
of its largest peak at talc = 34, and methanol
being 67% of its largest peak at 31. It is also useful
to measure Ar (at m / z = 40 no other gases interfere) as this can be used as an internal standard
(e.g., used to correct for slow drift in sensitivity
over extended periods) or for producing anaerobic
conditions.
In this paper we illustrate the potential of mass
spectrometry for measurement of a number of
gases simultaneously in sediment systems in the
laboratory.
3. M A T E R I A L S A N D M E T H O D S
3.1. Sampling site and procedures
Sediments were obtained from Penarth mud
flats, Cardiff, National Grid (N.G.) ref. ST181735,
and from the Melingriffith feeder stream 10 m
from the river Taft, N.G. ref. ST131808. The
former is an estuarine site, polluted by sewage,
which experiences extreme tidal rise and fall, exposing large areas of mud flats at low tide. Cores
of sediment were taken from a region several
metres seaward of a zone that was stabilised by
vegetation ( Aster tripolurn, Salicornia sp., Spartina
sp., Suaeda maritima). Some large organic debris
of plant origin was present in the samples. The
Taft feeder stream is an industrially polluted, shallow ( < 1 m), slow-flowing fresh-water stream. Both
sediments contained numerous polychaete and
nematode worms of various species.
Sediment cores were collected intact from
Penarth mud flats using a Perspex coring tube (10
cm i.d., 30 cm length), with a removable stainless
stcel screw lid having an hollow perpendicular
tube handle. The Perspex tube was forced into the
sediment, and a rubber bung was then inserted
into the hollow tube to prevent the sample from
falling out on withdrawal from the sediment. The
lower end of the Perspex tube was sealed b',
inserting a large rubber bung immediately' following withdrawal from the sediment. The intact scdiment sample plus any superficial liquid was transported, in a vertical position, back to the laboratory where it was kept with its lower 10-20 cm
immersed in sea water at room temperature
(18--20°C), for the duration of the expcriments.
The fresh-water sediment cores were collected in
the same way, but were of 3 cm diameter, and
their lower ends were kept tightly stoppercd during measurements.
3.2. Instrumentation
Dissolved gascs were monitored by inserting a
probc, attached to a mass spectrometer, to the
required location in a sediment core. The probe
consisted of 'Simplifix' stainless steel capillary
tubing (1.56 mm o.d., 0.5 mm i.d., 140 cm length)
which was sealed at the apex distal to the mass
spectrometer, and had an entry port for gases 0.4
cm from the tip. The entry port was a 50 /~m
diameter hole drilled through the tube wall, and
covercd with a sleeve of silicone rubber (0.8 mm
i.d., 1.7 mm o.d.).
Quadrupole mass spectrometers were from
Spectrum Scientific Ltd., Radnor Park Estate,
C o n g l e t o n , Cheshire, C W 2 4 4 X R , U.K.
(Dataquad), or from VG Systems, Ashton Way,
Middlewich, Cheshire, C W I 0 0HS (SX 200 with
DPP 16). Both systems used turbomolecular pumps
(40 I • s ~, Pfeifer Vacuumtechnik Wetzlar, Asslar,
F.R.G.), and the backing pumps were rotary
pumps from Edwards High Vacuum. Filaments
were of tungsten (in the Dataquad) and Thorium
(in thc SX200): with a vacuum of 10 6 tort,
filament life can be > 5000 h. Digital-analogue
conversion of signal voltages in 8 channels provided the capability for quasi-simultaneous rnonitoring of 8 different mass numbers (channel-scan-
13
ning rate 1 s t); data presentation was as a histogram (on oscilloscope display) and Y-t chart recorder traces, while RS 232 compatibility enabled
computer storage and data handling. Response
times ( t l / 2 values for all gases 1.3-1.4 min) were
measured, and calibrations established, by mixing
measured volumes of saturated solutions of gases
with 10 ml of argon-saturated sea water or stream
water, as appropriate, at 20°C while monitoring
dissolved gases. Stirring was stopped almost immediately so as to present the probe with a stationary film of liquid (similar to conditions during
sediment measurements). From tables, solubilities
of gases at 22°C are as follows (all in p,M): O,.
1330: N 2, 677; N20, 26700: H 2, 790; CH a, 1470:
CO 2, 36 700 [ 10].
3.3. Experimentation
A number of sediments were examined and
general procedures are outlined below. However,
details of treatments varied slightly from one experiment to another and precise details are given,
in the figures, only for those illustrative results
described in this paper.
In a preliminary experiment the potential for
denitrification was assessed using homogenised
sediment. Samples (4.5 ml) were injected into the
reaction vessel [1,3] of an MS SX200 (50 ~ m
Teflon membrane) at 20°C, along with 10 mM
N a N O 3 and 10 mM Na succinate. Initially, measurements were made under an atmosphere of Ar
and then O~ was added so as to sequentially
increase its concentration by injecting 5, 10 or 20
kPa 02 into the mobile gas phase: m / z values 28,
30 and 32 were monitored continuously over the
several hours duration of the experiment.
In subsequent experiments intact sediment cores
were used. Depth profiles of dissolved gases were
obtained by lowering or raising the probe, fitted
with silicone membrane and attached to the MS,
in steps of 5 or 10 mm. The probe remained at
each depth for at least 20 rain. Dissolved gases
were monitored continuously and included m / z =
2, 15, 28, 30, 32, 34 and 44, depending on the
experiment.
In some cores, following characterisation of
dissolved gas profiles, 0.5 ml of I M N a N O 3
initrogen source), a n d / o r Na succinate (carbon
source), a n d / o r Na acetate (carbon source) was
added and changes in dissolved gases monitored
for 24 h. These additions were made at the surface
and gases monitored either at 0.5 cm below the
sediment surface or at 6.5 cm depth. In these
experiments the gas phase above the sediment was
either air, 100% 02 or 100~ Ar.
4. RESULTS
4.1. Denitrification in homogenised samples
Both dinitrogen and N()~ (characterised as
100~ N20 by gas chromatography on a CTR
Column (Alltech, Type 8700, Carnforth U.K.) with
tle as carrier gas and a katharometer detector)
were detected in a number of samples under an
atmosphere of Ar and their concentrations increased when 02 was added. In a typical experiment (Fig. 1) addition of 5 kPa O~ (40 /~M 02 )
after 20 rain made little difference to the rate of
production of these gases, but following additions
of 10 kPa (100 FM) 02 after 60 min N 2 and N , O
production was twice that attained under argon.
Following addition of 20 kPa (230 p,M) O, after
70 rain, even greater production occurred; the
maximum concentrations for N 2 and N,O were
rcspectively 4.5 #M and 600 ~tM.
NO x
....
;JM
I0~ 'raM
5
pM
I¢:
b
20
4
.' /
-, .3,1
a 2()(~
i
i
i o ? ~ i,'):)
2~
•
_
~i.-
.
. . . . . . .
.... :-L
20
J
:...:. :_::: _..:'
........
..6
40
6"0
..0
BO
Fig. 1. Effect of O, on N 2 and b,'O, p r o d u c t i o n at 200( ` in a
4.5 ml homogenized s e d i m e n t sample, It:. which 10 m M N a N O a
a n d 10 m M N a succinate ~.ere a d d e d at the start of the
experiment. N 2 ( . . . . . .
), NO~ ( . . . . . ) and 0 2 ( . . . . . . ) were
m o n i t o r e d over the 110 min d u r a t i o n of the e x p e r i m e n t , initially the gas phase was argon. 0 2 was mixed into the m o b i l e
gas phase as i n d i c a t e d by the arrows.
14
;%1
i"
t.
• 101( (],
'
-
I:~cc
,,,M 400 [" "•
I
'
tIM
"2CJO
~,oc-.
,,
,.~
o.
~
o
-10)
Sedlme,~l
W~ller
..4
mM
:[)~i
i ,)
bottom
lop
oe:
INL,.
IJM
pM
L
I
I
:H,_,'
~JM
O' h
" #
:~-: ~r-~ ~ 0
,I{). .*'- . . . . . . . . .
1 :(:tCI!
20, •
.5 ;.JM
Depth
.1UO
:'°(:I "\
: i:111.
Fig. 2. P r o f i l e o f dissolved gases obtained by insertion of the
m a s s spectrometer probe through an estuarine sediment core.
The freshly-obtained core was not supplemented with nitrogen
o r c a r b o n sources and the gas phase was air. (a) O , ( O ) . N ,
(A) a n d C O ? ( 0 ) . ( b ) ('1-14 ( @ ) a n d 112 (tl).
c
"
100i
~O0
i
' i
,'} . . . . . . . . .
...........
i b
o
i
:CH~
),M
gas
4. 2. Dissoh'ed
profiles
Typical dissolved gas profiles for the estuarine
and freshwater stream sediments, incubated at
room temperature under an atmosphere of air, are
illustrated respectively in Figs. 2 and 3. m / z values. 2, 15, 28, 30, 32, 34 and 44 were monitored
simultaneously in the estuarine sediment, although
only data for concentration of dissolved H 2. C H 4,
N,, O, and CO~ are presented, as NO, and H2S
were not detectable at any depth. The sensitivity
of the probe for detecting H 2 S is not as high as
for the other gases monitored: this is because H , S
is adsorbed strongly onto the stainless steel surface
of the capillary'. At the time of sampling it was
however noted that some sulphate reduction is
associated with this sediment as evidenced by
smell and the occurrence of black sulphide deposits. Both dissolved 02 and N: concentrations
decreased rapidly with depth being respectively 70
/.tM (equivalent to 25 and 14ql of air
saturation) at 1 cm beneath the surface. Dissolved
O, concentration continued to decrease with increasing depth, and was undetcctable at 10 cm
depth. Dissolved N, concentration, however, declined to a minimum of 7 # M at 2.4 cm depth, and
then increased reaching a maximum of 360 p.M
between 5-.7.5 cm depth; subsequently it declined
to 35 #M by about 8.5 cm depth. Dissolved CO,
was maximal (14 x air-saturated) at the surface of
the sediment: it decreased to less than half this
concentration at 6 cm depth and then slowly
and 80
';;OO [
:(:()e'
I
mM
I
90Q }
i
600 '
~. '91 • 1 0 0 0
--
4i 500
i
300
"
!H.,i
I
!!o
o!7, ..° ,
pM
:~o01
iN(hi
PM
lo
200
1(70
.,
0J
o
-'
'
is
•b
'
so
A
16
o
Oepltl ~.Cm'
Fig. 3. P r o f i l e o f dissolved gases obtained by insertion of the
mass spectrometer probe through an untreated freshwater sediment core (water depth 4 c m ) . (a) 0 2 ( O ) and N 2 (zx); ( b l ( ' O 2
10) and ('1t a ((:)): i t ) H 2 (11) a n d N O ~ (C)).
increased again at greater depths. Methane reached 3 ~M at 6.5 cm depth: hydrogen was detectable at every point below 0.75 cm depth.
tt 2, N 2, CH 4, NO x, 02 and CO 2 were quantified with depth in the fresh-water stream sediment
(Fig. 3), and show marked differences from the
estuarine sediment. 02 concentration decreased
15
rapidly over the first few cm beneath the water
surface, but remained at approximately 50 p.M at
all levels in the sediment. N 2 concentration, however, remained at a constant 360 # M through the
water and then declined rapidly to 200 p.M by 8.5
cm depth. Both C O 2 and CH4 were present at low
levels in the aerobic 4 cm of water but increased in
the sediment, respectively to 1000 mM and 1500
p.M. Presumably anaerobic microenvironments
provide methanogenic niches in the predominantly
aerobic sediment. Concentration of H 2 and NO~
both increased with decreasing depth, although
neither reached 20 p.M. C o m p a r e d with the C O 2
concentrations that of NO~ is so low (50-fold less)
that corrections for the major contribution of N O ,
at m / z = 44 (148% of that at m / z = 30) arc not
necessary. Figures for dissolved H e represent an
over-estimate as a proportion of the signal at
m / z -- 2 arises from water which freely penetrates
the silicone rubber membrane. However, this
b a c k g r o u n d contribution is similar at all depths.
N2, and an increase in C() 2 after a lag of 70 min.
These changes were partially reversible.
5. D I S C U S S I O N
Manipulation of sediment systems, by altering
carbon and nutrient sources and external atmosphere, to investigate ecological processes is illustrated in Fig. 4. Here neither nitrate nor carbon
source were initially limiting (Na succinate, Na
acetate and N a N O 3 were added at the surface)
and external gaseous regime was either 100%
oxygen or 100% argon. On altering the atmosphere
above the sediment from argon to 100% O 2, dissolved O2 at 5 m m beneath the surface increased,
with a c o n c o m i t a n t increase of NO~, decrease of
This report indicates the potential usefulness of
m e m b r a n e inlet mass spectrometry in ecological
investigations requiring dissolved gas measurements. Thus, the method described enables key
products of denitrification to be measured directly
rather than the more usual assays of nitrate and
nitrite disappearance [15,16]. For methanogenic
systems both the product and H 2 [17,18], the
rate-controlling intermediate [19-23], can be
simultaneously monitored. "l-he high sensitivity,
rapid response of the system (limited by the membrane rather than the instrument itself), and the
inherent stability of the measuring system (drift
< 1% per week) have been detailed elsewhere [14]
and are all characteristics which make this method
superior to those currently employed (e.g., electrode methods for dissolved 02). More importantly, no sensors arc available for measurement of other gases in solution, except for N,O,
C O , and H 2, all of which can be measured using
rather unsatisfactory and unstable electrode systems [1]. Furthermore the method is virtually
non-invasive as insertion of the steel capillary
causes minimal displacement of sediment layering
and the gas c o n s u m p t i o n by the measuring device
is insignificant.
It is likely that this methodology will aid considcrably the unravelling of complex ecological
IN,]':
N~O.
4.3. Effects of external atmosphere in dissoh,ed gases
~-
)JMI
2 0 0 -'
Ar
02
IC&N
I source
100 .- ;
i
- ~.
...........
4
t
~.
.-/ ~
..
0
1
I. . . . .
2
A,
f
._-.~I
-~
i
~ ' . - I~
'
l_
3
4
5
-:1..
6
- 2
'
I
.:1
//'~
-% ~
i
i'-- - -iT . . . .
0
%
~- ~
..........
.
mM
1
I
~150
[0.~
}JM
]~o0
I
~ 50
\
..........
I_
O.
J 0
..:
7
T i m e (h)
Fig. 4. Effect of addition of carbon and nitrogen sources (0.5 M each of Na succinate. Na acetate and NaNO.~) and of changing gas
phase from argon to 02 on dissolved gases at 0.5 cm depth in an estuarine sediment core. Initially the gas phase was argon. Substrate
additions and step changes in the gas phase wee as indicated by arrows. Symbols as in Fig. 1.
16
processes. For example, here we havc shown, in
homogenised samples and at 0.5 cm below the
surface of intact cores, that dinitrogen and NO,
are not only produced under 100% argon, but also
under high O, concentration. This contrasts
markedly with the widespread view that bacterial
dcnitrification is a predominantly anaerobic protess. We have however, on the basis of a number
of laboratory experiments, put forward the
suggestion that aerobic denitrification may be as
widespread and ecologically important as its
anaerobic counterpart (D. Lloyd. L. Boddy and
K.J.P. Davies. submitted). In the homogenised
sample concentrations of dinitrogen and N(), both
increased with increasing O 2 concentration,
whereas in the intact sediment, although concentration of dissolved N()~ increased, that of N,
decreased. Similar effects of O, on denitrification
have been observed with non-prolifcrating suspensions of various bactcria: the former occurring
with
P,~ettdomonas aerugmosa a n d Propionibacterium thoenii, and the latter with Paracoccus
denitr~fican,s, Pseudomona.s" stutzeri and a number
of Pscudomonads obtained from this same
estuarine site (D. Lloyd. L. Boddy and K.J.P.
I)avies, submitted).
Several other results differ from what is generally thought to be the case. The profiles obtained
here for (): indicate penetration to depths of over
8 cm from the surface. This contrasts markedly.
with measurements made using micro-electrodes
in other nutritionally enriched sediments, where
(), is not usually detectable below about 3 mm
from the surface [24,25]. In the present study'.
there was, however, considerable disturbance o f
vertical stratification brought about by animal
activity. The latter made numerous burrows which
provided aeration channels. In the ease of the
fresh-water sediment, its loosely packed nature
facilitated casv mechanical disturbance, by' water
flow and burrowing activity of worms, thus allowing rapid gaseous exchange by,' mixing.
Additional work is required to chtcidate further
unusual features rcported herc. For instance, we
raise questions relating to the control of denitrification by O,, the microheterogeneity of methanogenie floes, and the relationship between metham>
genesis and sulphate reduction in estuarine sedinlents.
Improvements in methodology currently under
investigation in our laboratory include (1) reduction of capillary' size to o.d. 0.1 mm, (2) increased
spatial resolution of measurements using a computer-controlled micromanipulator for controlled
probe insertion: (3) dcvclopment of a multi-probc
facility cnabling simultaneous monitoring of
several depths and inclusion of a reference probe
in an environment of known constant gas composition.
The portability and rugged construction of the
mass spectrometer makes in situ environmental
use feasible: this has already been achieved for
instance for pollution monitoring [26] and routine
usc in hospitals [27,281.
Evaluation of possible discrepancies between
more conventional monitoring systems and the
mass spectrometer should obviously be performed.
These arc likely' to further reveal the limitations of
each approach.
AC KN OW L E D G EM ENTS
This work was supported by a Research Studentship to K.J.P.D. from the Natural Environmental Research Council, U.K. The authors wish
to acknowledge the expert advice of Dr. J.C. Fry,
and to thank all of those who helped with sampling.
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nleasurt:'nlcnt of dissolved gases ill biochemical systems
'.~.ith the quadrupole mass spectrometer, m Methods of
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[2] Lloyd. I). (1985) Simultaneous dissolved O, and redox
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17
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[20] Scott, R.l.. Williatns, T.N. and Lloyd. D. (1983) ()xygen
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Appl. Microbiol. Biotcchnol. 18, 236 241.
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