Download Extraction and distribution of free amino acids and

OCEANOLOGICA ACTA 1981 -VOL. 4 - No 4
~
- - - - -
Extraction and distribution
of free amino acids
and animonium
in sedimént interstitial waters
from the Limfjord, Déhmark
~
Amino acids
Ammonium
Sediment
Porewaters
HPLC
Ami no-acides
Ammonium
Sédiment
Eaux interstitielles
HPLC
N. O. G. Jy;rgensen a, P. Lindroth b, K. Mopperc *
Institute of Ecology and Genetics, University of Aarhus, Ny Munkegade, DK-8000
Aarhus C, Denmark.
b Department of Analytical and Marine Chemistry, Chalmers University ofTechnology
·
and University of Goteborg, S-412 96 Goteborg, Sweden.
c College of Marine Studies, University of Delaware, Lewes, Delaware 19958, USA.
a
• to whom correspondance should be adressed.
Received 11/6/80, in revised form 16/3/81, accepted 10/5/81.
ABSTRACT
Dissolved free amino acids and ammonium were extracted by centrifugation from four
diverse sediments from a Danish fjord. Analyses were performed by precolumn fluorigenic
labelling followed by HPLC separation and on-line fluorescence detection. The most
abundant amino acids found were glutamic acid, serine, glycine, alanine and leucine.
Glutamic acid and possibly ~-aminoglutaric acid were especially important in the anoxie
sediments. At sorne of the localities, a close resemblance of the amino acid spectra in the
overlying sea water to that of the sediment interstitial water was observed, which supports
the idea that free amino acids can diffuse into bottom sea water. The concentrations of
sediment interstial amino acids were found to decrease in the extracts with an increase in
duration of centrifugation. In addition, the amino acid spectra of the ex tracts changed to
varying degrees with increasing centrifugation time. These results are discussed in terms
of: (1) rapid and selective microbial degradation during sediment storage and extraction;
(2) stress induced excretion of intracellular amino acids, and (3) physiochemical
processes. It was tentatively concluded that physiochemical processes were dominant.
Oceanol. Acta, 1981, 4, 4, 465-474.
RÉSUMÉ
Extraction et répartition des acides aminés libres
et de l'ammonium dans les eaux interstitielles
des sédiments du Limfjord, Danemark.
Les amino-acides libres dissous et l'ammonium sont extraits par centrifugation de quatre
sédiments différents provenant d'un fjord danois. Les analyses comportent un marquage
préalable par formation de dérivés fluorescents suivi d'une séparation par HPLC et
détection des composés fluorescents sur la courbe d'élution. Les amino-acides les plus
abondants sont l'acide glutamique, la sérine, la glycine, l'alanine et la leucine. L'acide
glutamique et, peut-être, l'acide ~-amino glutarique, apparaissent particulièrement
importants dans les sédiments anoxiques. Dans quelques sites, on observe une forte
analogie entre le spectre des amino-acides de l'eau sus-jacente et celui de l'eau interstitielle
du sédiment, ce qui conforte l'idée que les amino-acides libres peuvent diffuser dans l'eau
du fond. Les concentrations en amino-acides des extraits interstitiels décroissent lorsque
augmente la durée de la centrifugation; de plus,le spectre des amino-acides évolue de façon
variable avec le temps de centrifugation. Ces résultats sont discutés en fonction : (1) d'une
·dégradation microbienne rapide lors de la conservation du sédiment et de l'extraction;
(2) d'une excrétion d'amino-acides intra-cellulaires causée par les phénomènes de stress;
et (3) des processus physico-chimiques. On tente, en conclusion, de montrer que les
processus physico-chimiques sont dominants.
Oceanol. Acta., 1981, 4, 4, 465-474.
0399-1784/1981/465/$ 5.00/ (!:) Gauthier-Villars
465
N. O. G. J!l'>RGENSEN. P. LINDROTH. K. MOPPER
INTRODUCTION
Free amino acids in sediment interstitial water usually
constitute less than 1% of the total "dissolved"
extractable nitrogen (e.g., Kemp, Mudrochova, 1973).
However, free amino acids are probably biologically
important in marine sediments, as turnover times from a
few minutes (Christensen, Blackburn, 1980) to severa!
hours (Hanson, Gardner, 1978) are reported. Thus,
detailed studies of these compounds should provide
important insights into nitrogen cycling in sediments.
However, the interpretation of dissolved free amino acid
data from sediment interstitial water analyses can be
rather difficult since these compounds, besides being free
components in interstitial water (Jl!lrgensen et al., 1980),
are also present in other sedimentary pools. e. g.: 1) as
exchangeable amino acids associated with sediment
particles (Christensen, Blackburn, 1980); 2) as irreversibly or strongly bound amino acids in particulate matter
(e.g., clay minerais) (Cheng, 1975; Rosenfeld, 1979a);
3) as biologically resistant organic matter, e. g., in humic
substances (Rashid, 1972); and 4) as intracellular amino
acids in sediment-inhabiting organisms such as bacteria
(Stanley, Brown, 1976), algae (Fowden, 1962), and
invertebrates (Gilles, 1975).
(estuary, Denmark; Jl!lrgensen ù al., 1980); and 180. 17 000 nM and 220-3 200 nM (west Atlantic sediment;
Henrichs, Farrington, 1979).
·
In order to determine to . what extent the applied
extraction procedure influences the concentration and
relative composition offree amino acids, we investigated
a centrifugation technique for two types of sediment.
Centrifugation was chosen, since, as opposed to other
methods such as nitrogen pressure filtration, the pore
water extraction efflciency could be readily varied and
studied by changing either the intensity or duration of
centrifugation. In the present investigation amino acids
were analyzed in interstitial water extracted as a function
duration of centrifugation. In addition, using what was
judged to be the optimal centrifugation time, interstitial
water from cores from four diverse sedimentary
environments were examined in terms of quantitative and
qualitative differences in the spectra of free amino acids
and ammonium.
MATERIALS AND METHODS
Sampling
Samples were taken during October, 1979 from four sites
in the eastern part of the Limfjord, Denmark (Fig. 1):
l) Rpnbjerg: exposed coast with coarse, sandy clastic
sediment; 2) Aggersborg: large intertidal flat with mixed
clastic sediment; 3) Aggersund: partly sheltered coast
with mixed clastic sediment; and 4) Lendrup: sheltered
lagoon with argillaceous clastic sediment; high organic
content. The water depth did not exceed 30 cm at any of
the sampling stations. Physical and chemical data for the
four sediments are given in Table 1. The redox potentials
of the sediments were not measured, however, due to the
high iron contents, the depths of the redox transition
Iayers could be reliably determined by visual examination. The pH range in the Limfjord sediments was
measured to be 7.9-8.3.
At each sampling location 3-5 replicate sediment cores
were taken. The cores were obtained with 200 mm long,
46 mm i. d. acrylic tubes. The tubes were previously
cleaned with a commercial detergent followed by copions
rinsings withfresh double distilled water. After sampling,
the bottom of the cores were immediately sealed with
rubber stoppers. Sea water samples (directly overlying
·the sediments) were taken in 100 ml glass botties which
has been previously combusted at 450°C to destroy
This complex distribution of amino acids in marine
sediments suggests that the term "free" is actually an
operational definition. Thus, if a gentle extraction
procedure is applied, only interstitial water from the
larger pores is obtained. On the other hand, if a harsh
procedure is used, the concentration of "free" amino
acids may be overestimated, as living organisms and
the structure of the sediment particles may be affected.
Varions extraction procedures of amino acids and total
primary amines in marine sediments are reported in the
literature:
a) pressure filtration with nitrogen gas (Stephens, 1975;
Jorgensen, 1979);
b) centrifugation of sediment sam pies (Hanson, Gardner,
1978; Jorgensen et al., 1980);
c) hydraulic squeezing (Henrichs, Farrington, 1979);
d) dialysis membranes placed directly into the sediment
(North, 1975);
e) filtration of sediment in situ through aeration stones
(Clark et al., 1972);
1) extraction with 80% acidifled ethanol (Starikova,
.Korzhikova, 1968);
g) extraction with water (Brinkhurst et al., 1971 );
h) extraction with ammonium acetate solutions (Whelan,
1977);
l) direct derivatization with dansyl-Cl of amino acids in
sediment-buffer slurries (Litchf1eld et al., 1974).
Results obtained from these extractions are difflcult to
compare due to the considerable differences in
methodologies and the expected natural variations in the
·concentrations of free amino acids. This is demonstrated
by methods (b) and (c) above where concentrations of
free alanine and aspartic acid, respective! y, were reported
as: 2-550 nM and 1-70 nM (salt-marsh in Georgia, USA;
Hanson, Gardner, 1978); 50-1900 nM and 90-1000 nM
Figure 1
Map of sampling locations in the Ûmj]ord, Denmark.
466
EXTRACTION AND DISTRIBUTION OF DFAA IN POREWATERS
Table 1
Samp/e background i'!formation.
Medium
grain
size
(!lm)
Locality
Rmnbjerg:
Seawater
0-2cm
4-6cm
10-12cm
Aggersborg:
Seawater
0-2cm }
4-6cm
10-12 cm
Aggersund:
Seawater
0-2cm }
4-6cm
10-12cm
Lendrup:
Seawater
0-2 cm }
4-6cm
10-12cm
}
f
367
171
158
134
Silt (")
(%)
CaC0 3
(%)
Porosity
(%,v/v)
Water content
%of total
[ml (b)] extracted
by centrif.
Org.
content
(%, w/w)
44
43
31
0.65
0.68
0.49
9
11
36
49
10
9
41
59
9.7
16.5
2.1
3.4
0.7
12.1
10.0
9.1
2.3
n
NH.i
htM)
(d)
(')
N03
(!lM)
NOï
(!lM)
0.3
{ 0.54
0.47
0.15
36.5
30.0
27.3
3.3
'0.18
( 0.18
0.18
47.3
51.0
39.7
15.7
17.0
13.2
23
15
17
1.79
1.34
0.98
34
121
104
171
34
128
109
224
1.7
3.8
1.7
6.9
{ 0.98
1.25
2.52
52.2
38.8
49.7
17.4
12.9
16.5
32
25
8
2.45
1.08
1.43
8
45
112
108
9
51
119
106
33.1
4.0
0.9
0.3
1.0
1.0 }
0.6
0.4
j 1.63
17.8
20.7
17.4
25
11
5
3.69
2.95
2.82
39
149
241
344
50
185
301
293
54.8
4.1
2.6
0.1
6.6
53.7
62.4
52.2
7.2
14.5
( 1.52
1.01
2.7}
0.5
1.7
1.3}
1.3
Oxicanoxie
zone (f)
(cm)
6-8
2.5
1.7
f
2.0
0.7}
0.6
0.6
1.5
(") Particle <63j!m. (b) Determined by drying at 105"C; sam pie volume 33.2 ml sediment.(') % of total water content extracted by centrifugation
(Fig. 6). (4) According to Solorzano (1969). (')Chromatographie values. (1 ) Visually determined.
contaminants. Ali cores and water samples were brought
to the laboratory within 1 hour and stored at the in situ
temperature (6.5°C). During the storage period (up to
24 hours) anoxie conditions were maintained in the
sediment cores by the in situ microbial processes.
Sediment cores were eut into 2 cm slices from the desired
· depth, placed in centrifugation containers (33.2 ml
capacity) (Blackburn, 1979) which were supplied with
thick, precombusted glass f1ber fllters (nominal pore
size -0.3 }lm, Gelman Type A, Gelman Sciences, Inc.,
USA) and centrifuged (Model Sigma 2; Sigma, FRG) at
a force of 20g . The volume of the separated interstitial
water was measured and samples for amino acid analysis
and inorganic nitrogen analyses were taken. The amino
acid analyses were performed on the pore water extracts
within 5 minutes after centrifugation. Th us, conservation
of the extracts was unnecessary.
the colorimetrie methods are: ammonium, ±0.5 }lM;
nitrite and nitrate, ±0.05 }lM.
Ail amino acid analyses were performed on the same day
of collection and, in the case of the sea water samples,
within 2 hours of collection. Sediment cores could be
stored for at least 24 hours at in situ temperatures with
no appreciable change in either arnino acid composition
or concentration of the extracted pore waters, which is in
agreement with Henrichs (1980). From these results it
was concluded that the addition of a bacteriocide to the
sediments was unnecessary and perhaps undesirable. The
sea water samples were analyzed unflltered in order to
minimize the possibility of contamination. In addition,
ali amino acid analyses were conducted without prior
desalting or concentration since these steps (together
with prolonged sample storage) cao give rise to
signiticant errors due to adsorption !osses, contamination and wall-induced hydrolyses (Garrasi et al., 1979;
Dawson, Mopper, 1978; Dawson, Liebezeit, 1980). A
recently developed HPLC technique was employed
(Lindroth, Mopper, 1979). Briefly, amino acids (contaioing a primary amine group) and ammonium- in the
samples were reacted directly with o-phthaldialdehyde
(Merck, FRG) and mercaptoethanol (Serva, FRG) to
form highly fluorescent isoindole derivatives. The
reaction time was 1 minute at a pH of 9.5 and room
temperature. Usually 100 }.tl of the derivatized sample
(corresponding to about 95 }.tl of original sample) was
injected directly into the liquid chromatograph (Altex
Scientitic, USA). The amino acid derivatives were
separated on a reversed phase column with a methanolphosphate butTer gradient. The derivatives were then
detected fluorimetrically (FS 970 L. C. Fluorimeter,
Schoeffel Instruments, USA). The detection limit is 50lOO x 10- 15 mol per injected amino acid. The precision,
reproducibility and accuracy of the method are very high.
(Lindroth, Mopper, 1979; · Hamberger et al., 1980).
Parallel sediment cores were used for grain size analysis
(wet sieving), porosity (vol/vol %), water content as
determined by drying in an oven at 105°C, and organic
content (loss on combustion of dried sediment at 500°C)
[note that CaC0 3 in the sediment was determined from
the Ca++ content after acidification, using atomic
absorption spectroscopy (Perkin Elmer 2380, Perkin
Elmer, USA)].
Analytical methods
Ammonium was measured colorimetrically according to
Solorzano (1969) as weil as chromatographicaily
(Lindroth, Mopper, 1979). The agreement between these
two independent methods is reasonable for most samples
(Table 1). Nitrite and nitrate were determined according
to Wood et al. (1967) using a Chemlab Autoanalyser
(Chemlab Instruments Ltd., England). The precisions of
467
N. O. G. JQ)RGENSEN. P. LINDROTH. K. MOPPER
Figure 2
INJ
l
Upper: calibration standard mixture; each peak 40 pmol.
Notation: Asp, aspartic acid; Glu, glutamic acid; ~-glu,
~-aminoglutaric acid; Asn, asparagine; Ser, serine; Gin,
glutamine; His, histidine; Thr, threonine; Gly, glycine;
Arg, arginine; Tau, taurine; Jyr, tyrosine; Afa, alanine;
ô-abu, 1>-aminobutyric acid; ex-abu, cx-aminobutyric acid;
Trp, tryptophane; Met, methionine; Val, valine; Phe,
phenylalanine; Ile, isoleucine; Leu, leucine; Orn, ornithine;
Lys, lysine. Lower: pore u:ater }rom anoxie Agyersund
sediment (10-12 cm); ~-aminoglutaric acid tentative/y
identijred (see text).
15
20
25
10
0
Tl ME (min)
Typical chromatograms of a standard mixture and an
interstitial water sample are shown in Figure 2. Most
amino acids were identifted by spiking samples with
authentic compounds. A few unknown amine peaks
were, however, encountered and the major unknown
peak, labelled "X", elu ting after leucine, was quantifted
assuming a response factor equal to serine (Lindroth,
Mopper, 1979).
A relatively large tryptophane peak was observed in
sorne of the chromatograms but the shape of this peak
differed from the "standard" tryptophane peak (Fig. 2),
and we suspect that this peak contained additional
primary amines. Tryptophane is therefore not included in
the results. Neither is ornithine and lysine, although they
occurred in most samples, but their peaks were difftcult to
quantify since they were present in low concentrations
relative to other amino acids and co-eluted with
interfering unidentifred compounds.
the initial compos1t10n (as determined directly after
centrifugation). Only a minor increase in the acidic
amino acids could be discerned in preserved samples. The
results on an absolute basis are given in Table 2. From
this table it is evident that freezing (slow or quick) is the
preferred form of preservation.
Table 2
Effect of preservation on the concentrations of amino acids and
ammonium in a pore water sample.
Method
Free amino
acids (!lM)
Ammonium
(J.lM)
Untreated
Toluene
Liquid Nitrogen
-20°C
2.36
2.95
2.48
2.21
28.2
37.0
29.0
27.0
RESULTS
Preservation of interstitial water samples
In the present study no preservation method was
employed because analyses were performed immediately
after centrifugation. In other studies,-immediate analyses
may not always be possible, therefore, the effect ofthree
preservation methods on the free amino acid composition of a pore water sample was examined: 1) addition of
toluene (20 ,.LI per ml pore water); 2) slow freezing at
- 20°C; and 3) quick freezing in liquid nitrogen
{followed by storage at - 20°C). After 24 hours the
amino acid compositions were determined. On a mole
percent basis, the compositions very closely resembled
Extraction of amino acids as..a fonction of centrifugation
ti me
In a previous study (J~rgensen et al., 1980) it was
suggested that at high centrifugai forces, >400 g, free
amino acids are extracted from organisms in sediment (or
that the organisms excrete amino acids in response to
high mechanical stress), especially surface sediments,
. where the greatest biological activity is found. Thus, in
order to minimize both mechanical disturbance of the
sediment and stress to the sediment-inhabiting organisms
during centrifugation, preliminary experi,ments were
468
EXTRACTION AND DISTRIBUTION OF DFAA IN POREWATERS
...........
'-
0-2CM
A0t&JERO
0-2CM
which simply reflects the higher water contents in the
upper layers (Table 1). In addition, with increasing
centrifugation time (Fig. 3), water appears to be Jess
extractable from the fme-grained, organic-rich Aggersund sediment than from the sandy, organic-poor
R0nbjerg sediment. Th us, on! y 8% of the total water
content (determined by desiccation at 105°C) was
centrifuged from lower Aggersund sediment (Fig. 4 ).
These results indicate that the interstitial water in the
fme-grained sediment is more strongly sorbed than in the
sandy sediment. However, it is possible that progressive
clogging of the glass fr ber ftlter with clay and fme silt may
have caused sorne underestimation of extractable water
from the frne-grained sediment.
RONBJERG
The concentrations of free amino acids in the interstitial
water extracts of all four samples demonstrated
signiftcant decreases with increasing centrifugation time
(Fig. 3). These decreases are evident even in the
integrated (or potential) concentrations (Fig. 4). For
example, for the upper R0nbjerg sediment, a concentration of 16 J.1M is obtained after 1 minute centrifugation,
whereas on! y 1/8 this concentration is obtained after a
total of 12 minutes (Fig. 4).
Like the amino acids, the concentrations of ammonium
in the collected fractions for all four samples were also
influenced by the centrifugation time, however, no clearly
defrned trend could be discerned (Fig. 3). The integrated
(or potential) concentrations, Figure 4, indicate that the
concentration of ammonium is less affected by the
duration of centrifugation than the free amino acids.
The relative molar compositions of free amino acids in
pore water of the two sediments in the individual
Figure 3
Actual concentrations of free amino acids and ammonium, and amounts
of pore water in the individual collected fractions extracted from 33.2 ml
of R;nbjerg and Aggersund sediments as a function of centrifugation
time (20g) and depth in the sediment.
performed to determine the lowest usa ble rotation speed
at which pore water could be separated from the four
sediment types. This speed was found to be 500 rpm
(20 g ). Sediment sam pies from R0nbjerg and Aggersund
were run at increasing centrifugation times at 500 rpm, to
investigate how the duration of the centrifugation
affected the concentrations of extracted amino acids and
ammonium. These two sediments were chosen since they
differ signiftcantly in texture, porosity, and content of
water and organic matter (Table 1).
The sediment was centrifuged ftrst for 1 minute at
500 rpm and the water obtained was removed and
analyzed. The same sediment was then centrifuged
without delay for additional periods of 1, 5 and
5 minutes (for a total of 12 minutes) and after each period
the new water obtained was collected and immediately
analyzed.
For clarity, the results are graphically depicted in two
ways. In Figure 3 the actual concentrations offree amino
acids and ammonium and amounts ofwater obtained for
the individual collected fractions (1, + 1, + 5 and
+ 5 minutes) are shown. In, Figure 4, the integrated (or
potential) concentrations and cumulative amounts of
water are given. Thus Figure 4 indicates the concentrations which would have been measured at a given
centrifugation time, if prior fractions had not been
removed.
Figure 4
lntegrated concentrations of free amino acids and ammonium, and
cumulative amounts of pore water from Figure 3. The percentages of the
total water contents extracted by centrifugation were: Aggersund (02cm) 34%, (10-12cm) 8%; R!Pnbjerg (0-2cm) 44%, (10-12cm) 31 %·
A number of trends are apparent in these results (Fig. 3
and 4 ). Grea ter amounts of water are extracted from the
upper layers of the sediments than in the lower layers,
469
N. O. G. J0RGENSEN. P. LINDROTH. K. MOPPER
that the concentrations of amino acids changed most
rapidly in the extracts during the frrst few minutes of
centrifugation. The concentrations in the later extracts
for aU samples were relatively low and generally more
uniform. Thus, in order to provide a more consistent
basis for comparison, a 12 minutes centrifugation period
was selected. The results are depicted in Figures 6 and 7.
The concentration of amino acids in the R~nbjerg
sediment decreased with increasing depth white the
opposite trend was observed in the Aggersborg sediment.
In Aggersund and Lendrup sediments, a concentration
maximum occurred in the 4-6 cm layer. Similar
concentration maxima have been previously observed in
sediments (J~rgensen, 1979) and are probably related to
enhanced microbial activity, such as sulfate reduction, at
and below the oxic-anoxic zone of the sediment
(J~rgensen, 1977; Wollast, 1977; Kepkay, Nevitsky,
1980).
ln all four sediments, the concentration of ammonium
generally increased with depth. The highest concentrations occurred in the reducing Lendrup sediment which
also had the highest content of organic matter.
Correspondingly, the lowest ammonium concentration
was observed in the sandy, organic-poor, oxygenated
R~nbjerg sediment.
The amino acid composition of the overlying sea waters
and extracted pore waters from the four localities is
shown in Figure 7. In the R~nbjerg sediment the 0-2 cm
pore water was dominated by serine; the 4-6 cm and 1012 cm layers by glutamine and alanine. The coïncidence
of a high proportion of serine in both the upper sediment
and the overlying sea water (Fig. 7) suggests that amino
acids diffuse from this coarse sediment.
In Aggersborg, the amino acids in the sea water differed
markedly from the amino acids in the pore water. Thus,
glycine and leucine dominatt~d in the sea water, white
alanine, glutamic acid and taurine were most abundant
in the pore water of the upper sediment layer. In the 46 cm and 10-12 cm layers, glutamic acid was the most
important amino acid.
ln Aggersund, glutamic acid dominated in all sediment
layers and in the overlying sea water and, therefore,
AOOERSUND 1D-12 CM
1 "'"
+1 MIN
0
n
A!&NBJERQ 0-2 CW
Rc&NBJERO 1D-12 CM
~~
~-'
~~!~
-~"
•
.
+1 MON
20
:~dl--:cn--rn-n
Figure 5
Relative molar compositions of free amino acids in the individual pore
water fractions from Figure 3; Abu, a+y-aminobutyric acid, X,
unknown peak in Figure 2.
collected fractions are given in Figure 5. In the R~nbjerg
sediment, only minor changes in the spectrum took place.
Serine was the dominant amino acid in the 0-2 cm layer.
In the later fractions, leucine and alanine increased
slightly.ln the 10-12 cm layer, sorne moderate changes in
the composition occurred. Initially, glutamic acid and
serine were most abundant, but in the later fractions,
glutamine, alanine, leucine and the unknown amine "X"
become more important.
Pore water from Aggersund was dominated by glutamic
acid. In the upper sediment, glutamic acid and to a lesser
extent glutamine predominated in the fust extractions,
but in the la ter extractions the relative abundance of the
neutra! amino acids serine, glycine and leucine increased.
In the 10-12 cm layer, the glutamic acid dominance was
distinct in the frrst two centrifugations, then, after further
centrifugation, leucine and the unknown amine "X"
· increased in abundance as in the other sediment.
Comparison of free amino acid spectra in four different
sediments
Variations in concentration and composition of free
amino acids were investigated in four sediment pore
waters and overlying sea waters from the Limfjord.
Sediment cores were collected at R~nbjerg, Aggersborg,
Aggersund and Lendrup as before. A centrifugation
speed of 500 rpm (20 g) and a duration of 12 minutes
were chosen. The basis for choosing the 12 minutes
centrifugation is shown in Figure 3, where it can be seen
Figure 6
Concentrations of free amino acids and ammonium, and amounts of
co/lected pore water in 33.2 ml of fresh sediment as a function of depth in
four sediments. Centrifugation for 12minutes at 20g.
470
EXTRACTION AND DISTRIBUTION OF DFAA IN POREWATERS
Rapid and selective microbial degradation during the
extraction procedure
llfrtpftUP BQ !TOM SEAWA n.B
Mole"
;~fJl C1-=-n
~NJ)RUP
Mole '!lo
+
nHl--,___,.,
-
R0NBJERG PORE ~ATJ.!!
v0--.2 '"CM
2100 t~
.AAt:2.a.M
==
Experimental evidence does not favor this explanation,
as sediment cores stored for 24 hours or longer at in situ
temperatures showed no major changes in either amino
.1cid composition or concentration of the extracted pore
waters. This stability supports the assumption that free
amino acids exist in dynamic equilibria between
production and assimilation, as turnover times of free
amino acids in marine sediments from minutes to hours
are reported (Hanson, Gardner, 1978; Christensen,
Blackburn, 1980). Alternatively, microbial degradation
of the extracted amino acids might have occurred white
and after centrifuging. During the extraction, however,
most bacteria are expected to be retained within the
sediment or on the glass ftber ftlters. Furthermore, the
amino acid analyses were performed immediately after
each extraction, therefore, we consider Joss from
microbial degradation during extraction to be negligible.
PORE WATER
;~r~O-OM ·~'2··~
;~fdk =·-cr ·~.,~
~Cn~·on
CM
n
·~ ,~0
J~!a:~â~~1!!:ff~~-
a
Mole'!lo
40
~ERSUND
BOTTOM SEAWATER
30
20
10
AGGERSBOR~
PORE WATER
0-2 CM
~f-ü-cn.ll
0
Rapid emptying of intracellular pools of free amino acids of
organisms due to stress during centrifugation
00
••
30
20
$
4-6 CM
Mol o J L ; : ' !
1G-12CM
Io
2
1
c
Three major groups of organisms are quantitatively
important in the investigated sediment: bacteria,
microalgae, and invertebrates. Glutamic acid has been
found to be the dominant free intracellular amino acid in
marine bacteria (Stanley, Brown, 1976; Henrichs, 1980).
In Figures 5 A, B, and Dit is seen that this amino acid is
also dominant in the initial extracts, suggesting that the
bacteria leak glutamic acid during the centrifugation.
Calculations of the actual pool size of intracellular
bacterial amino acids based on data from Stanley and
Brown (1976) and the number of bacteria in Limfjord
sediments (Lopez, Levinton, 1979) show, however, that
emptying of the total free amino a cid pools of the bacteria
would only increase the interstitial amino acid
concentrations about 25 nM in the present sediment
sam pies. Therefore, we conclude that bacterial intracellular pools are negligible sources of amino acids during the
actual extraction. However, this does not preclude that
bacteria are not important sources for interstitial free
amino acids on a long-term basis.
AAt: 11.0 .....
n
d
Figure 7
Relative molar compositions of free amino acids in pore waters from
sediments in Figure 6 and from the respective overlying sea waters.
indicates that diffusion of amino acids from the sediment
may be an active process at this locality.
In Lendrup, a high concentration of glutamié acid was
observed in the sea water. The amino acid spectrum of the
sea water, however, was not similar to that of the pore
water in the upper sediment layer, where glycine and
leucine prevailed. However, upper oxic sediment layers
at several of the locations tended to have much grea ter
variability in their amino acid spectra than the
corresponding lower anoxie layers. Thus, the spectrum
found in the upper Lendrup sediment may not be typical
of this location. In fact, the coïncidence of a high
proportion of glutamic acid in the 4-6 cm layer of the
sediment and the overlying water suggests that even in
this locality diffusion may be occurring. The 4-6 cm layer
is located just under oxic/anoxic zone (Table 1) and is
characterized by the highest concentration of free amino
acids (Fig. 7). Figure 7 also shows that with increasing
depth in the sediment, the relative amount of leucine
increases markedly.
DISCUSSION
Microalgae, e. g., dia toms, are restricted to the upper few
millimetres of sediment and thus probably do not
influence the pore water in deeper sediments. In R0nbjerg
sediment higher amino acid concentrations were
extracted from the 0-2cm layer than from the 10-12cm
layer (Fig. 3 and 4), whereas the opposite trend was
found in Aggersund. These inconsistent fmdings do not
demonstrate if microalgae excrete free amino acids as a
result of centrifugai stress. We have not been able to fmd
data on free amino acid concentrations in marine
microalgae in the literature.
The study on the effect of centrifugation time revealed
marked decreases in the free amino acid concentrations
and changes in the relative compositions in the pore
water extracts with increasing centrifugation time
Fig. 3-5). At !east three alternatives exist for explaining
these observed trends.
The invertebrate fauna in the investigated Limfjord
sediments consist mainly of molluscs and polychaetes.
The free amino acid pools of these animais ali contain
taurine as a major or the major amino acid, together with
glycine and alanine (Awapara, 1962; J~rgensen,
Kristensen, 1980). The occurrence of taurine in sorne of
471
N. O. G. JillRGENSEN. P. UNDROTH. K. MOPPER
the porewater samples, e. g. Figure 7 D, suggests that the
extracted amino acids partially originated from living
and dead invertebrates. With increasing centrifugation
(Fig. 5) the relative concentration of taurine does not
change signiftcantly indicating that induced excretion of
free invertebrate amino acids is probably not an
important source of amino acids during the extraction. In
addition, taurine was found in ali bottom sea water
samples (Fig. 7), further indicating that eventualleakage
of amino acids due to induced stress of the infauna
animais need not be responsible for the presence of
taurine in the sediment extracts.
We fmd that stress induced excretion of intracellular
amino acids from bacteria, microalgae, and invertebrates
is of min or importance in the present study, as the applied
centrifugation force, 20 g, is probably too mild to cause a
signif1cant amino acid excretion. If the sediment is rich in
infauna, the present extraction procedure might
overestimate the actual pore water amino acid
concentrations, especially if high centrifugai forces are
used (J9lrgensen et al., 1980).
tration actually increased with increasing centrifugation
time. This increase may be related to high local
concentrations of ammonium in micropores which are
only emptied after prolonged centrifugation.
Physiochemical factors may also be involved in the
changes observed in the amino acid spectra with
increasing centrifugation (Fig. 5). Within any sediment
various and probably numerous sorption sites exist for
organic molecules (Weiss, 1969; Thompson, Eglington,
1978; Tanoue, Handa, 1979; Christensen, Blackburn,
1980). The different chemistries of the amino acids will
result in varying degrees of sorption onto (or into) these
sites. Basic amino àcids, such as ornithine and lysine, are
very strongly adsorbed by cation exchange sites (Weiss,
1969). In addition basic amino acids preferentially
condense with sugars to form melanoidin-type
complexes, which are also strongly adsorped by clays
(Hedges, 1978). Once adsorbed, clays may catalyze the
deamination of basic amino acids (Weiss, 1969). Neutra!
and especially acidic amino acids are excluded from
adsorption by ion exchange at the pH of porewater
(pH,..., 8). Amino acids containing mercaptide linkages,
cysteine and cystine, form insoluble complexes with
transitions metals (Saxby, 1973) and are, thereby,
preferentially excluded from the porewater phase. Amino
acids ,with large hydrophobie portions, such as leucine
and ornithine, are excluded from the aqueous phase by
hydrophobie bonding onto the sedimentary organic
matter (Weiss, 1969; Khan, Schnitzer, 1972). The results
in Figure 5 are consistent with these processes. Thus,
acidic amino acids, which tend to be excluded from ion
exchange sorption sites, are enriched in the ftrst
centrifuged fractions. Amino acids selectively sorbed by
hydrophobie interactions, such as leucine and the
unknown amine "X" (Fig. 2 ), are released after
prolonged centrifugation and are preferentially found in
the later fractions. Irreversibly adsorbed amino acids,
such as ornithine (basic and hydrophobie) and cystine
(mercaptide-containing) are either not detected or
present at low concentrations in the porewater extracts.
At this point it is not clear to what extent physiochemical
processes or induced excretion of intraceUular amino
acids have influenced the results obtained in Figures 3-5.
Both may be occurring and reinforcing each other.
Insights into this problem may be gained by examination
of the amino acid porewater data of four different
sediments (Fig. 6, Table 1).
Physiochemical processes
The combined effect of a number of physiochemical
processes may also produce the amino acid trends
observed in Figures 3-5. When a solution containing
electrolytes is passed through a column packed with a
porous solid, the initial eluting aliquots are signiftcantly
more concentrated in the electrolytes than the original
solution. This process is known as electrolyte (or ion)
exclusion and is largely due to the enhanced structuring
of water at the solid-solution interface (Horne, 1969).
During centrifugation this process may explain the high
amino acid concentrations of the f1rst emergent ex tracts,
Figure 3. In addition to electrolyte exclusion, the change
of the surface-to-volume ratio of the sediment during
centrifugation may also signiftcantly affect the amino
acid concentrations of the individual extracts. Free
amino acids in pore waters exist in dynamic equilibrium
with exchangeable amino acids. Adsorption/desorption
experiments using C-14 and H-3 labelled alanine and
Limfjord sediments (Christensen, Blackburn, 1980)
indicate that the pool of free alanine is about 2 500 fold
smaller than the alanine pool adsorbed in the particulate
phases of the sediment. Compaction during centrifugation decreases the surface-to-volume ratio of the
sediment which, in turn, decreases the accessibility of the
large adsorbed amino acid pool to exchange reactions
with the pore waters. The result is that the amino acid
concentrations ofthe ex tracts progressive! y decrease with
increasing compaction, Figure 3.
The concentrations of free amino acids varied
considerably from sediment and even with depth in any
one sediment (Fig. 6). However, within the organic-rich,
ftne-grained sediments (Aggersborg, Aggersund and
Lendrup) a roughly inverse correlation was observed
between the free amino acid concentrations (Fig. 6) and
the content of organic matter (Table 1). In general,
microbial biomass in surface sediments is proportional to
the organic content (Moore, 1969; Dale, 1974). This is
also found in Limfjord sediments (E. Perry, pers. comm.,
1981). Thus, if one wants to invoke induced excretion of
intracellular amino acids to explain the amino acid
results in Figures 3 and 4, one would expect to ftnd the
highest concentrations of amino acids in the ex tracts of
Ammonium, on the other band, may be adsorbed to a
much Iesser degree than amino acids (Müller, 1977;
Rosenfeld, 1979 a), with the ratio of adsorbed to
dissolved ammonium being only 1-2:1 (Rosenfeld,
1979 b). Th us, the compaction effect discussed above
would not be expected to signiflcantly affect the
ammonium concentrations of the extracts, as shown in
· Figures 3 and 4. In fact, for the fmegrained, anoxie
Aggersund sediment, 10-12cm, the ammonium concen472
EXTRACTION AND DISTRIBUTION OF DFAA IN POREWATERS
sediments containing the highest microbial biomass (i.e.,
highest organic content). This result was not obtained
(Fig. 6 and Table 1), therefore, we tentative! y conclude
that physiochemical processes are main! y responsible for
the amino acid trends in Figures 3-5.
The molar compositions of free amino acids demonstrated signiftcant variations between localities and also with
depth, Figure 7. This is not surprising since numerous
biological, chemical and physical factors undoubtedly
influence the concentration and composition at any given
time. However, the most abundant amino acids, i.e.,
glutamic acid, serine, glycine, alanine and leucine are also
reported to be abundant in pore waters in other
sediments (Brinkhurst et al., 1971; Kemp, Mudrochova,
1973; Michaelis et al., 1976; Henrichs, Farrington, 1979;
J~rgensen et al., 1980). Henrichs and Farrington (1979)
reported high concentrations of the nonprotein amino
acid, ~-aminoglutaric acid, in pore waters from various
marine sediments. This amino acid occurs in severa!
marine algae (Fattorusso, Piattelli, 1980) and marine
bacteria (Henrichs, 1980). With the present HPLC
technique this amino acid elutes in the vicinity of
glutamic acid (C. Lee, pers. comm., 1980). in severa! of
the samples a large unknown peak was encountered
immediately after glutamic acid, Figure 2. Although this
peak is probably ~-aminoglutaric acid (C. Lee, pers.
comm., 1980), positive identification awaits comparison
to the authentic substance.
The resemblance of the amino acid spectra in the
overly}ng water to that of the sediment pore water at
sorne of the sampling locations (Fig. 7) suggests that
amino acids (as well as other low molecular weight
organic compounds) diffuse into the overlying sea water.
This transport may be quantitatively important in
shallow estuaries (J~rgensen et al., 1980).
Ammonium in the sediment porewater may be partially
derived from free amino acids, especially basic amino
·acids, by processes such as clay(Weiss, 1969) and enzyme
catalyzed deamination reactions. However, the pool of
free amino acids is most certain! y not a major source for
ammonium in pore waters, as no clear relationship
between the concentration of free amino acids and the
concentration
of
ammonium
was
observed
(Fig. 3, 4, 6). Ammonium is probably derived Iargely
from microbial remineralization of sedimentary proteineous materials (Blackburn, 1979), which are present in
substantially higher concentrations than free amino
acids. The lower concentrations of ammonium in the
upper layers compared to the lower layers of the
sediments, Figures 4 and 6, are probably due in part to
microbially mediated nitrification processes (Wollast,
1977) as evidenced by the generally higher nitrate
concentrations in the upper layers (Table 1).
Perhaps the most important conclusion to be drawn from
the present investigation is that the extraction technique
used for trace organics and ammonium, whether it be
centrifugation, nitrogen pressure filtration or hydraulic
squeezing, etc., can signiflcantly influence the results,
both quantitatively and qualitatively, which is in
agreement with Farke and Rieman (1980). Thus, a
thorough study of the potential effects of the extraction
technique on the results must ftrSt be Ùndertaken before
the results of sediment pore water analyses can be
meaningfully interpreted.
Acknowledgements
We are much obliged to Dr. T. H. Blackburn for !etting
us use his self-constructed centrifugation containers and
to Ors. D. Dyrssen, J. W. Farrington, S. M. Henrichs, C.
Lee and two anonymous reviewers for reading and
criticizing the manuscript. We also wish to thank Mrs. A.
Jensen for ber technical assistance in the chemical and
physical analysis of the sediment samples, and Mr. B.
Gustafsson for drafting the ftgures. This work . was
supported by The Danish ,Natural Science Research
Council, Grant No. 511-15884 (chromatographie equipment) and by grants from The Swedish National Science
Research Council (K 2549-028 and K 2549-029 ).
REFERENCES
Awapara J., 1962. Free amino acids in invertebrates: a comparative
study of their distribution and metabolism, in: A mina acid pools, edited
by J. T. Holden, Elsevier, London, 158-175.
Blackburn T. H., 1979. Method for measuring rates ofNH1' turnover in
anoxie marine sediments, using a 15 N-NH1' dilution technique, App/.
Microbiol., 37,760-765.
Brinkhurst R. O., Chua K. E., Batoosingh E., 1971. The free amino acids
in the sediments of Toronto Harbor, Limnol. Oceanogr., 16, 555-559.
Cheng C.-N., 1975. Extraction and desalting amino acids from soils and
sediments: evaluation of methods, Sail Biol. Biochem., 7, 319-322.
Christensen D., Blackburn T. H., 1980. Turnover of 14C-alanine in
anoxie marine sediments, Mar. Biol., 58, 97-103.
Clark M. E., Jackson G. A., North W. J., 1972. Dissolved free amino
acids in southern California coastal waters, Limnol. Oceanogr., 17,749758.
Dale N. G., 1974. Bacteria in intertidal sediments: factors related to
their distribution, Limnol. Oceanogr., 19, 509-518.
Dawson R., Mopper K., 1978. A note on the !osses of monosaccharides,
amino sugars and amino acids from extracts during concentration
procedures, Anal. Biochem., 84, 186-190.
Dawson R., Liebezeit G., 1981. The analytical methods for the
characterisation of organics in sea water, in: Marine organic chemistry,
· edited byE. K. Duursma and R. Dawson, Elsevier, in press.
Farke H., Rieman F., 1980. Dissolved organic carbon in littoral
sediments: concentrations and available amounts demonstrated by the
percolation method, Veroff.lnst. Meeresforsch. Bremerh., 18, 235-244.
Fattorusso E., Piattelli M., 1980. Amino acids from marine algae, in:
Marine natural products, vol. III, edited by P.J. Scheuer, Academie
Press, New York, 95-140.
473 '
N. O. G. J\ZIRGENSEN. P. LINOROTH. K. MOPPER
Fowden L., 1962. Amino acids and proteins, in: Physiology and
biochemistry of algae, edited by R. A. Lewin, Academie Press, New
York, 189-209.
·•
·
Garrasi C., Degens E. T., Mopper K., 1979. The free amino acid
composition of seawater obtained without desalting and preconcentration, Mar. Chem., 8, 71·75.
Giles R., 1975. Mechanism of ion and osmoregulation, in: Marine
ecology, Vol. 2, Part 1, edited by O. Kinne, John Wiley and Sons,
London, 259-348.
Hamberger A., Jacobson 1., Lindroth P., Mopper K., Nystriim B.,
Sandberg M., Molin S.-O., Svanberg U., 1980. Neuron-glia interactions
in the biosynthesis and release of transmitter amino acids, in: Amino
acid transmitters, edited by P. Mandel and F. V. Defeudis, Ra ven Press.
Hanson R. B., Gardner W. S., 1978. Uptake and metabolism of two
ami no acids by anaerobie microorganisms jn four diverse salt-marsh
soils, Mar. Biol., 46, 101-107.
Hedges J. 1., 1978. The formation and clay mineral reactions of
melanoidins, Geochim. Cosmochim.Acta, 42, 69-76.
Henrichs S. M., 1980. Biogeochemistry of dissolved free amino acids in
marine sediments, Ph. D. Thesis, Massachusetts I nstitute of Technology
and Woods Hale Oceanographie Institution, WHOI Report No. 80-39.
Henrichs S. M., Farrington J. W., 1979. Amino acids in interstitia1
waters of marine sediments, Nature, 279, 319-322.
Horne R. A., 1969. Marine chemistry, Wiley-Interscience, New York,
362-369.
J-'rgensen B. B., 1977. The sulphur cycle of a coastal marine sediment
(Limfjorden, Denmark), Limnol. Oceanogr., 22, 814-832.
Jorgensen N. O. G., 1979. Annual variation of dissolved free primary
amines in estuarine water and sediment, Oecologia (Berl.), 40,207-217.
Jorgensen N. O. G., Kristensen E., 1980. U ptake of amino acids by three
species ofNereis (Annelida: Polychaeta). 1. Transport kinetics and net
uptake from natural concentrations, Mar. Eco!. Prog. Ser., 3, 329-340.
Jllrgensen N. O. G., Mopper K., Lindroth P., 1980. Occurrence, origin,
and assimilation of free amino acids in an estuarine environment,
Ophelia, Suppl., 1, 179-192.
Kemp A. L. W., Mudrochova A., 1973. The distribution and nature of
amino acids and other nitrogen-containing compounds in Lake
Ontario surface sediments, Geochim. Cosmochim. Acta, 37,2191-2206.
Kepkay P. E., Novitsky J. A., 1980. Microbial control of organic carbon
in marine sediments: coupled chemoautotrophy and heterotrophy,
Mar. Biol., 55, 261-266.
Khan S. U., Schnitzer M., 1972. The retention of hydrophobie organic
compounds by humic acid, Geochim. Cosmochim. Acta, 36, 745-754.
Lindroth P., Mopper K., 1979. High performance 1iquid chromatographie determination of subpicomole amounts of amino acids by
precolumn fluorescence derivatization with o-phthaldialdehyde, Anal.
Chem., 51, 1667-1674.
Litchfield C. D., Munro! A. L. S., Massie L. C., Floodgate G. D., 1974.
Biochemistry and microbio1ogy of sorne Irish Sea sediments: 1. Amino
acid analyses, Mar. Biol., 26, 249-260.
/
474
Lopez G. R., Levinton J. S., 1979. The availability ofmicroorganisms
attached to sediment particles as food for Hydorbia ventrosa Montagu
(Gastropoda: Prosobranchia), Oecologia (Berl.), 32, 263-275.
Michaelis W., Mopper K., Garrasi C., Degens E. T., 1976. Organische
Substanzen im Sediment und Wasser der Hamburger Alster, Mitt.
Geol.-Palaont. Inst. Univ. Hamburg, Sonderbund Alster, 173-188.
Moore L. R., 1969. Geomicrobiology and geomicrobiological attack on
sedimented organic matter, in: Organic geochemistry, edited by G.
Eg1ington and M. T. J. Murphy, Springer-Verlag, Berlin, 265-303.
Müller P.J., 1977. C/N ratios hi Pacifie deep-sea sediments: effect of
inorganic ammonium and organic nitrogen compounds sorbed by
clays, Geochim. Cosmochim. Acta, 41, 765-776.
North B. B., 1975. Primary amines in Califomia coastal waters:
utilization by phytoplankton, Limnol. Oceanogr., 20, 20-27.
Rashid M. A., 1972. Amino acids associated with marine sediments and
humic compounds and their role in so1ubility and complexing of metals,
24th Int. Geol. Congr., 1972, Section 10, 346-353.
Rosenfeld J. K., 1979a. Amino acid diagenesis and adsorption in
nearshore anoxie sediments, Limnol. Oceanogr., 24, 1014-1021.
Rosenfeld J. K., 1979 b. Ammonium adsorption in nearshore anoxie
sediments, Limnol. Oceanogr., 24, 356-364.
Saxby J. D., 1973. Diagenesis of metal-organic complexes in sediments:
formation of metal sulphides from cystine complexes, Chem. Geai., 12,
241-248.
Solorzano L.,1969. Determination of ammonia in natural waters by the
phenolhypochlorite method, Limnol. Oceanogr., 14, 799-810.
Stanley S. 0., Brown C. M., 1976. lnorganic nitrogen metabolism in
marine bacteria: the intracellular free amino acid pools of a marine
pseudo-minad, Mar. Biol., 38, 101-109.
Starikova N. D., Korzbikova R. 1., 1968. Amino acids in the Black Sea,
Oceanology, 9, 509-518.
Stephens G. C., 1975. Uptake of naturally occurring primary amines by
marine annelids, Biol. Bull. Mar. Biol. Lab., Woods Hole, 397-407.
Tanoue E., Handa N., 1979. Differentiai sorption of organic matter by
various sized sediment particles in recent sediment from the Bering Sea,
J. Oceanogr. Soc. Jpn., 35, 199-208.
Thompson S., Eglington G., 1978. The fractionation of a recent sediment
fol_organic geochemical analysis, Geochim. Cosmochim. Acta, 42, 199201l.
Weiss A., 1969. Organic derivatives of clay minerais, zeolites, and
related minerais, in: Organic geochemistry, edited by G. Eglington and
M. 1. J. Murphy, Springer-Verlag, Berlin, 737-781.
Whelan J. K., 1977. Amino acids in a surface sediment core of the
Atlantic abyssal plain, Geochim. Cosmochim. Acta, 41, 803-810.
Wollast R., 1977. Factors affecting the composition of sediment pore
waters, Proceedings of the 2nd International Symposium on WaterSediment Interactions, Strasbourg, France, 1977.
Wood E. D., Armstrong F. A. J ., Richards F. A., 1967. Determination of
nitrate in sea water by cadmium-copper reduction to nitrite, J. Mar.
Biol. UK, 47, 23-31.