FEMS Microbiology Ecology 14 (1994) 33-44
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
33
FEMSEC 00518
Organic matter mineralization in an organic-rich
sediment: Experimental stimulation of sulfate
reduction by fish food pellets
M a r i a n n e H o l m e r * and Erik Kristensen
Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark
(Received 15 September 1993; revision received 23 December 1993; accepted 27 December 1993)
Abstract." The combined effects of organic matter additions and temperature on short chain fatty acid (SCFA) turnover, sulfate
reduction and nutrient accumulation were examined in an organic-rich fish farm sediment. Fish food pellets, which contribute
significantly to the organic matter loss from fish farms, were added to surface sediment at three loadings (2.8; 14.0; 28.0 mg ww g-1
ww sediment; equivalent to organic matter loadings measured during fish farming) and incubated for 30 days in anaerobic bags at
5°C and 15°C. SCFA accumulated to high levels (acetate up to 85 mM, propionate up to 17 raM, butyrate up to 25 mM) in
sediments amended with food pellets, and sulfate reduction was stimulated up to 30 times relative to unamended sediments.
Sulfate reducers appeared saturated with substrates (SCFA) even in the lowest additions. A low C / N ratio (0.4-1.8) of the major
mineralization products (TCO 2 and NH~-) indicated preferential nitrogen mineralization in amended sediment compared with the
total particulate pool ( C / N = 8.8-11.9) and added food pellets ( C / N = 8.4).
Key words: Organic stimulation; Sulfate reduction; Short chain fatty acid dynamics; Carbon and nitrogen mineralization
Introduction
An increased input of organic matter to coastal
sediments has significant impact on numerous
biogeochemical processes, e.g. sulfate reduction,
methanogenesis, ammonium and phosphate production, as well as on the turnover of small organic molecules such as short chain fatty acids
* Corresponding author. Present address: Institute of Life
Sciences and Chemistry, 17.2 Roskilde University, PO Box
260, DK-4000 Roskilde, Denmark. Tel: (46) 75 7711; Fax: (46)
75 7721; e-mail: [email protected].
SSDI 0 1 6 8 - 6 4 9 6 ( 9 3 ) E 0 0 7 8 - V
and amino acids [1-5]. Anaerobic mineralization
rates stimulated several orders of magnitude have
been found in sediments underlying marine fish
farms [6,7]. There is, however, a need for elucidating the factors controlling mineralization processes in such eutrophic sediments, since both the
quantity as well as the quality of the sedimentary
organic matter affects both reaction rates and
pathways of microbial processes [8-12].
Several studies have examined the relationship
between organic matter input and anaerobic mineralization in sediments [10-13], but all have been
conducted with loading rates much less than those
usually found in fish farm sediments. Sulfate re-
34
Jutland
55030'
9o30'
9o40'
Fig. 1. Location of sampling station in Kolding Fjord of the
east coast of Jutland, Denmark.
duction, the most important respiration process
in marine sediments is generally considered to be
controlled by the availability of substrates such as
short chain fatty acids (SCFA) [2,14-16]. Mineralization in fish farm sediments may not necessarily
be dependent on substrate and electron acceptor
availability alone, but also by other known (e.g.
pH, nutrients, sulfides) and, as yet unknown, factors. In anaerobic digesters and in pure cultures,
inhibition of sulfate reducing bacteria by substrates such as acetate or by products like dissolved sulfides has frequently been observed [17191.
In most marine sediments preferential mineralization of nitrogen relative to carbon generally
occurs [2,20,21]. Pore water analysis of fish farm
sediments have shown very low C / N ratios of the
mineralization products (TCO 2 and NH~-) compared to the particulate organic pools, indicating
that nitrogen also is mineralized faster than carbon in these sediments [7]. Carbon and nitrogen
mineralization appears not to be coupled. While
the former is driven primarily by respiration processes, the latter seems to be a result of fermentation processes [22].
The purpose of the present study was to determine the effect of organic matter additions on
anaerobic mineralization rates, with particular
emphasis on the factors controlling sulfate reduction. Results from two series of laboratory experiments with fish farm sediment are presented, and
used to evaluate the kinetics of anoxic mineralization. The role of substrate (short chain fatty
acids) availability for the sulfate respiration is
examined, with the focus on fermentation processes.
Materials and Methods
Sample collection and procedures
Two experiments were conducted with surface
sediment (0-5 cm) from a salmon farm located in
Kolding Fjord, Denmark (Fig. 1). In the first
experiment (Expt. A) sediment was collected with
a Van-Veen grab during active fish farming in
September 1991. Two stations were examined:
one underneath a net cage (Sta. 1) and one at a
control site 30 m away from the farming area
(Sta. 2). Water depth at both stations was 5 m.
The sediment from both stations appeared muddy
with particulate organic carbon (POC) content of
7.4%dw and 6.6%dw and particulate organic nitrogen (PON) content of 0.65%dw and 0.64%dw
at Sta. 1 and 2, respectively. After return to the
laboratory, the sediment was homogenized in a
glove bag and transferred into two parallel 1-1
gas-tight polyamide/polythene bags (Riloten) as
Table 1
Added amount of feed pellets (FP) in mg ww g - i ww sed,/xmol C cm -3 sed (Cad d) a n d / x m o l N cm 3 sed (Nadd). POC and PON
are the measured particulate pools (% dw) in the sediment for controls and after feed pellet additions in the amended sediments
given as mean ( ___range) of duplicate measurements
ID
FP
mg ww g - 1 ww
Cad d
/~mol C cm -3
Nadd
/zmol N cm -3
POC
% dw
Control
Low
Med
High
2.8
14.0
28.0
102
513
1023
12
61
122
6.7 +
6.7 +
8.1 +
12.8 +
PON
% dw
0.3
0.5
0.3
0.2
0.67 ++_0.05
0.72 + 0.07
0.95 +_0.05
1.69 + 0.04
35
described by Kruse [23], which were sealed by
melting. The bags were incubated at in situ temperature (15°C) for 14 days. Every second day the
bags were carefully mixed before subsamples were
taken (through a glass-fitting) for measurements
of pore water solutes.
In the second experiment (Expt. B) surface
sediment (0-5 cm) was sampled only at Sta. 1
during the spring of 1992, before fish farming had
begun. Homogenized food pellets (ECOLINE 19,
Dansk Orredfoder) were mixed into the sediment
at 3 concentrations (Table 1). The pellets contained 48.7%dw POC and 6.8%dw PON. Two
bags were incubated without food pellet addition
(control), 2 with low addition (Low), 2 with
medium addition (Med) and 2 with high addition
(High). The amount of added food pellets was
close to normal loading of POC and PON at the
farming site: the Low addition was 12 and 17%
higher than the background concentration of POC
and PON, respectively, at the control station in
spring (Sta. 2); Med addition was comparable to
the maximum POC measured at the fish farm
during farming (59% and 86% higher than POC
and PON in the control sediment). The High
addition resulted in 30% higher POC than ever
recorded at the farming site, but with a PON
content similar to the measured maximum of
1.55%dw. One bag of each treatment was incubated at 5°C and 15°C, respectively, for 30 days.
The mixtures were allowed to stabilize for 3 days,
before sampling was initiated. Subsequently, subsamples were taken at weekly intervals.
chlorid acid (pH = 2) and stored at 5°C until
analysis by HPLC anion chromatography. TCO 2
was determined within 12 h by flow injection
analysis [25]. Samples were kept in glass vials to
prevent loss of TCO 2, and interfering sulfides
were precipitated with zinc chloride. Samples for
NH~- and WOA were stored frozen until analysis. NH~- was determined using the standard autoanalyzer method according to Solarzano [26].
WOA were measured according to Bctte [27],
modified for seawater analysis. Separation was
performed using a Waters IC-PAK Ion-Exclusion
column with a Guard-Pak module and 1.00 mM
sulphuric acid as eluent followed by an Anion
MicroMembrane Suppressor (AMMS-ICE, Dionex Corp.) with 5.00 mM tetrabutylammonium
hydroxide as regenerant. The precision was better
than 5%. There were no requirements for pretreatments when the pore water was diluted twice
in eluent. Dissolved sulfides (EH2S) were determined by the method of Cline [28] on samples
precipitated with 0.5 M zinc acetate, pH was
measured on intact sediment by inserting a pH
glass-electrode (Radiometer) directly into subsampled sediment and allowing the reading to
stabilize.
Exchangeable NH~- was measured occasionally
during the time course in both experiments. Approximately 3 g of wet sediment was transferred
to 3 ml of 2 M potassium chloride and extracted
for 0.5-1 h at 5°C. After centrifugation (5 rain.,
3000 rpm) the supernatant was frozen until NH ~analysis as previously described.
Pore water solutes
Pore water solutes were obtained by squeezing
subsamples through combusted (300°C) G F / C
filters according to Reeburgh [24]. After discarding the first ml, pore water (5-10 ml) was sampled for sulfate (SO42-), total dissolved inorganic
carbon (TCO2), ammonium (NH~-), weak organic
acids (WOA; e.g. short-chain fatty acids (C1-C4),
lactate, succinate, glutarate), dissolved sulfide
(EH2S) and pH analysis. A test of the squeezing
method showed constant concentrations of all
components until approx. 25% of the pore water
remained in the sediment pellet.
Samples for sulfate were preserved in hydro-
Sediment characteristics
Water content was determined on subsamples
by drying (105°C) for 6 h. Density was obtained as
wet weight of a known volumen. Particulate organic carbon (POC) and nitrogen (PON) were
determined with a Carlo Erba Elemental Analyzer EA ll00A after the method of Kristensen
and Andersen [29].
Sulfate reduction
In Expt. A, and in the amended sediments of
Expt. B, sulfate reduction rates were determined
from the change in sulfate concentration with
time. In the Expt. B control sediments, where the
36
rate of SO 2- concentration change was slow, a
35S-radiotracer technique was used to improve
the precision on rate measurements. Control sediment was transferred directly from the bags into
3 cut-off syringes ( = 5 cm 3) sealed with butyl
stoppers. After injection of 50 t,l 3SS-SO42- (50
kBq), through the stoppers, the syringes were
incubated for 4 to 6 h. Incubations were terminated by transfer of the sediment into 1 M zinc
acetate (1:1 of volumen). Samples were stored
frozen until analysis. Separation of reduced sulfur
compounds were performed by the one-step distillation procedure of Fossing and J0rgensen [30].
sediments, with rates 9-30 times higher than the
controls (Table 2). The average SRR in the controis (determined by radiotracer technique) were
40.1 and 55.6 nmol cm -3 d -I at 5°C and 15°C,
respectively. There was no simple relationship
between SRR and the added OM, as SRR were
higher in the Low and Med than in the High
treatment (Fig. 2A). In the High treatment at
5°C, SRR was only 20% of Low and Med; a rate
corresponding to the control rate. SRR in High
at 15°C was 42% lower than the two other additions. Sulfate was depleted after 200-400 h in the
enriched 15°C incubations.
TCO 2 production
The initial concentration, and the production
rate of TCO 2 (Table 2) in Expt. A, were 75% and
84% higher respectively at Sta. 1 than at Sta. 2,
indicating a notable difference between fish farm
sediment and unaffected control sediment. In the
amended Expt. B sediments, T C O 2 accumulated
to high concentrations (15-25 raM) and the production was 4-12 times higher than in the Expt.
Results
Sulfate reduction
Sulfate concentration declined linearly in all
incubations, and in Expt. A the estimated sulfate
reduction rate (SRR) was more than twice as
high at Sta. 1 than at Sta. 2 (Table 2). In Expt. B,
sulfate was consumed rapidly in all the amended
Table 2
Rates of sulfate reduction (SRR), T C O 2 production (TCO 2) and net N H 4 production (APR) ( + exchangeable pool) in Expt. A and
Expt. B estimated by least squares regression analysis. Rates are presented as nmol c m - 3 d -1. Values for Expt. A are given as
m e a n and range of two bags. S R R in the Expt. B control bags rates are determined by tracer method, and given as mean + (S.E.) of
5 measurements. R 2 are the variation explained by each regression
SRR
Temp.
Expt. A
Sta. 1
Sta. 2
15
15
Expt. B
Control
Control
Low
Low
Med
Med
High
High
Low
Med
High
5
15
5
15
5
15
5
15
15
15
15
TCO 2
(nmol c m - 3 d -1)
80
33
(28)
(6)
40 (7)
55 (9)
306
1324 a
330
1436 a
63
825 c
a Initial time points < 162 h.
b Time points > 162 h.
c Time points < 405 h.
R2
0.68-0.89
0.51-0.54
0.99
0.999
0.998
0.9998
0.84
0,97
APR
(nmol c m - 3 d -1)
165
90
68
137
277
1251
697
1673
437
1624
256
- 632
-451
(26)
(16)
a
a
~
b
b
h
R2
0.85 -0.92
0.85-0.87
0.99
0.99
0.91
0.91
0.95
0.86
0.81
0.96
0.87
0.97
0.97
(nmol c m - 3 d
21(1)
12(1)
4
11
109
343
210
1185
592
873
1)
R2
0.98-0.99
0.96-0.97
0.99
0.99
0.94
0.96
0.98
0.93
0,99
0,98
37
B control sediments (Table 2). The rates were not
proportional to the OM additions, as they were
lower in High than in the Med treatments (Fig.
2B). At 15°C in Expt. B, the initial linear increase
of TCO 2 in the amended sediments only lasted
for 162 h, followed by either a reduced accumulation (Low) or a rapid loss (Med and High) coinciding with sulfate depletion and with a subse-
2.0
A
1.5
?
-6
E
o3
1.o
0.5
i
0
1
i
~ OI
B
1.5
o
E
0
1.0
O.5
i
O
2.0
I
i
I
i
I
i
I
c
In Expt. A the behaviour of NH~ was similar
to TCO2, but with a much slower accumulation
rate (Table 2). The rates of net NH~- production
(APR) were calculated by including the exchangeable NH~. The non-dimensional adsorption constant K was 1 + 0.2. APR was 70% higher
at Sta. 1 than at Sta. 2. In Expt. B the APR
accumulation rates were stimulated up to 120
times in the amended sediments compared to the
controls. APR generally increased with the
amount of added OM (Fig. 2C), except in the
High treatment at 15°C which attained 25% lower
rates compared to Med.
In the amended Expt. B sediments, a rapid
accumulation of dissolved sulfides (EHzS) was
observed after 100 h of incubation at 15°C and
after 400 h at 5°C (Fig. 3), reaching 5-8 mM
within the next 200 h (except High at 5°C). In all
treatments, ]~H2S decreased after reaching the
peak concentration. The behaviour of EHzS reflected the rate of sulfate reduction, as the accumulation of ZH2S in High at 5°C was significantly lower compared to the other amended
sediments. There were no measureable changes
of EH2S concentrations in Expt. A and in the
Expt. B control bags (data not shown).
pH
1.5
?
E
o
m
o
............ O
E
Ilk
Ammonium production
Dissolved sulfides
p
2.0
quent accumulation of methane (observed as
methane bubbles).
0.5
0=
o
2so
soo
7so
1o o o
C~ut [pmol C era-3]
Fig. 2. (A) Sulfate reduction rates (SRR); (B) TCO 2 production rates (TCOz); and (C) net ammonium production (APR)
(+exchangeable pool) in Expt. B as a function of added
organic carbon (Cad d) at 5°C (solid symbols) and 15°C (open
symbols). Each symbol represents one bag. At 15°C only
initial rates ( < 162 h) are plotted.
pH decreased with time in the Expt. B
amended sediments (data not shown), and was
lowest at 15°C in Med and High treatments. In
High, initial pH was 6.2 and final pH below 6
(5.7-5.8). In Low and Med pH started at 6.8-7.3
and decreased to 6.0-6.8 towards the end. There
were no significant pH changes in Expt. A and in
the Expt. B control sediments throughout the
incubation period (range: 7.0-7.6).
Weak organic acids (WOA)
WOA were below detection limit (< 2 tzM) in
Expt. A throughout the incubation period (data
not shown). Acetate was detected in the Expt. B
38
10.0
Exp B 5'C
Exp B 15°C
7.5
E
oL
5.0
• LOW
"7"
w
2.5
v HIGH
200
400
600
I
I
I
I
I
800
200
400
soo
eoo
TIME {hours)
TIME (hours)
Fig. 3. Accumulation of dissolved sulfides (~2H2S) in the amended Expt. B sediments. Each point represents the mean of duplicate
measurements (range < 7%).
control sediments initially (4-6 /xM), but decreased to values below detection limit after 90 h
(Fig. 4A, B). In the amended Expt. B sediments,
on the other hand, short chain fatty acids (SCFA)
accumulated to high concentrations as shown in
Fig. 4 for acetate (A,B), formate (C), propionate
(E,F) and butyrate (G,H). The highest concentrations of SCFA were observed at 15°C, with acetate as the most dominant SCFA reaching 85
mM in the High treatment at the end of the
experiment. No other WOA were measured above
detection limit. In general, pools of acetate almost doubled with temperature from 5°C to 15°C
for each OM addition. Initially, acetate increased
linearly in all incubations, but the rate generally
ceased towards the end. This was most pronounced at 15°C where acetate actually declined
after approx. 400 h in Low and Med. Acetate
production increased with OM addition at 5°C
from 582 nmol cm -3 d -1 in Low to 1049 and
2789 nmol cm -3 d-1 in Med and High, respectively (Fig. 5). At 15°C, however, rates only increased with OM addition from Low (1073 nmol
c m - 3 d -1) to Med 4165 nmol cm -3 d -t, and the
rate in High was 27% lower than in Med.
At 5°C, a formate peak appeared in the Med
(6 raM) at 200 h, and in High the concentration
increased irregularly to high levels (approx. 15-30
mM) during the experiment (Fig. 4C). Formate
was found initially in amended sediment at 15°C
(200 /zM-9 raM) (Fig. 4D), but decreased below
detection limit within 200 h.
The accumulation of propionate and butyrate
was essentially linear throughout the experiment,
except for Low at 5°C, where no butyrate and
very little propionate was detected (Fig. 4E-H).
The longer SCFA were most important at the
high loadings and at the high temperature, and
especially butyrate accumulated in High treatments up to 14 and 25 mM at 5°C and 15°C,
respectively.
Discussion
SCFA net production
This study demonstrates that the net production of short chain fatty acids (SCFA) from fish
food pellets decomposing in anoxic sediment was
remarkably high. Even at low organic additions
(equivalent to approx. 10% of the annual primary
production in this coastal area [31]) a significant
accumulation of acetate (18-20 mM), propionate
(0.5-3 mM) and butyrate (1.5 mM) occurred. The
Fig. 4. Accumulation of (A,B) acetate 5°C and 15°C; (C,D) formate 5°C and 15°C, (E,F) propionate 5°C and 15°C; and (G,It)
butyrate 5°C and 15°C for the amended sediments. Each symbol represents mean of duplicate measurements (range < 5%).
Regression lines are fitted by least squares method. Notice differences in scales.
39
~zo
A
12o
Exp B 5"C
100
~
* control
• LOW
Exp B 15"C
100
control
eMEO
BO
B
v HIGiH
D LOW
o MED
80
ILl
1-60
<
.
60
FLU
0 40
,9(
_._..-.V"....
..-V
.,.._°.,..,°o ................. 0..
40
........ ~ .........
.......IF........
20 ~
20
0 .
.
.
0
30
..........
~
.
.
200
.
0
400
600
800
3O
C
D
25
25
/
20
/
III
I-. ( 15
1P.......................'qP................. •
15
/"
10
u.
/
/
-
..e..
e" ............
5 '"
",..."V\\
200
400
600
800
0
20
20
400
20O
600
800
600
800
F
15
v
ul
F' < 10
Z
0
0
IZ:
5
0
200
30
400
600
0
800
30
G
200
400
H
25
25
~
20
ILl
I'-- 15
15
10
s
5
i
0 v
0
'
20O
~
~
r O - -
'
O~
4O0
TIME (hours]
60O
800
0
'=-
~
200
~
400
TIME (hours}
'
600
800
40
6.0
--..
o 15°C
•
T
E
•
5"C
40
O
2.0
g
ev-
0
'
i
250
i
500
h
750
1000
C,d d (~mol C cm -~]
Fig. 5. A c c u m u l a t i o n r a t e s of a c e t a t e (Racet) in Expt. B as a
function of a d d e d o r g a n i c c a r b o n (Cad d) at 5°C (solid) a n d
15°C (open). E a c h symbol r e p r e s e n t s o n e bag.
stimulation of sulfate reduction was extensive,
with rates up to 30 times higher than in unamended sediment (Table 2). Sulfate reduction
rates measured previously at the same fish farm
during summer [7] gave results equivalent to those
obtained here in the Low and Med treatments at
15°C. The particulate pools at the field site were
at that time corresponding to the Med and High
addition. Rates obtained in unamended sediments were similar to rates measured at the field
site indicating that the bag incubation is a reliable simulation of in situ measurements.
The weak relationship between sulfate reduction, TCO 2 and ammonium production and organic matter additions (Fig. 2) is in contrast to
other experimental studies of marine sediments
[8,10,13], where a linear relationship between organic loading and sediment metabolism generally
is found. The organic matter loading and the
lability of the food pellets used in this study is
higher compared to other studies, but realistic
related to the fish farm site. The stimulation
pattern in the present experiment indicated saturation kinetics for sulfate reduction (e.g.
Michaelis-Menten) with respect to substrate (e.g.
SCFA). Maximum rates were achieved at the
lowest loadings. Substrate saturation of SRR has
previously been found in sediment slurries with
K m of 0.3 mM for acetate [32], but has not been
shown for natural sediments. In these amended
sediments acetate concentrations were higher
than the K m value from slurries ( > 1 mM), and
the maximum rate of sulfate reduction (Vmax)
with respect to SCFA was apparently reached in
lowest additions indicating that Vmax may be
achieved at even smaller loadings.
The apparent inhibition of SRR at High additions relative to Low and Med suggested, that
SRB were affected by the accumulation of microbial metabolites. The growth of SRB may have
been influenced by low pH, since values around 6
dominated in the High treatments. This is lower
than both the optimum pH of SRB [19,33], and
pH of the other treatments (pH ~ 7). The toxicity of dissolved sulfides, which were present in
high concentrations, is inversely related to pH.
H2S(aq) is considered to be the most toxic form
of sulfide, inhibiting a wide range of organisms,
including bacteria [34]. At pH 6.2, which was the
initial value in the High treatments, the ratio
between the two major forms of dissolved sulfides
( H 2 S ( a q ) / H S - ) is 6.3, whereas in the low additions (pH = 7.3) most ~2HzS was in the form of
less toxic H S - ( H 2 S ( a q ) / H S - - - 0 . 5 ) . Inhibition
of SRB by ~ H 2 S has been observed in anaerobic
digesters at concentration levels of 2.0-3.5 mM
H2S(aq) [17,18,35]. Furthermore, a concomitant
inhibition by acetate and hydrogen sulfide has
been shown for pure cultures of Desulfovibrio
vulgaris [19]. Concentrations of 0.7-7 mM for
hydrogen sulfide and 20-80 mM for acetate as
found in the present study may cause significant
inhibition of SRB [19]. The very low sulfate reduction rate at the low temperature may indicate
a difference in population dynamics where the
growth of tolerant SRB is slow at the low temperature. The reduced sulfate reduction rates may
also be due to competition from the methanogens,
which has been found in high organic loaded
chemostats and mixed cultures in association with
H2-producing fatty acid oxidizing bacteria [33,36].
Methane production was observed in the High
treatments.
The extent of sulfate reduction was reflected
in the TCO 2 production, although the usual stoichiometry of 2 ( T C O 2 : S R R ) between the processes [37] was not found in all incubations (Table
3). In the sediments where sulfate reduction
41
Table 3
Ratios of TCO 2 production and sulfate reduction in Expt. A
and Expt. B. Rates are given in Table 2
Temp.
TCO 2 / S R R
Expt. A
Sta. 1
Sta. 2
15
15
2.1
2.7
Expt. B
Control
Control
Low
Low
Med
Med
High
High
5
15
5
15
5
15
5
15
1.7
2.5
0.9
0.9
2.1
1.1
6.9
2.0
a Initial time points < 162 h.
showed saturation, (Low and Med) ratios were
foremost lower, whereas they were higher in sediments with reduced SRR (High). A study similar
to the Low addition at 5°C showed a methane
production rate of approximately 30% of the SRR
(Holmer and Kristensen, manuscript submitted),
and methane bubbles were observed in the
amended sediments in this study indicating that
the deficit in TCO 2 was due to methanogenesis.
Methanogenesis was also present in the High
additions, but the ratios, especially in the High at
5°C, are probably affected by a production of
TCO 2 during fermentation processes [33,38]. The
decreasing concentration of acetate after sulfate
depletion at the high temperature indicate presence of methanogenesis eventually in association
with Hz-producing fatty acid oxidizing bacteria.
The ammonium production showed a pattern
like the sulfate reduction with increasing organic
loadings, except for the High loading at the low
temperature. Apparently the ammonium accumulation was linked more closely to the acetate
production instead of the SRR, indicating that
the production of ammonium was related to fermentation processes rather than directly to sulfate reduction, as previously suggested for other
sediments [39].
Stoichiometry of decomposition
The ratio between terminal organic carbon
mineralization and inorganic nitrogen production
can be examined from pore water concentrations
of the products (e.g. TCO 2 and NH~-) as described by Burdige [22]. C / N ratios obtained in
unamended active fish farm sediment from TCO z
and NH~ concentrations through time (ATCO2
Table 4
Stoichiometry of terminal mineralization products ( A T C O 2 / z l N H 4) calculated from regressions of TCO e concentration versus
(including exchangeable pool). The C/N ratio between acetate accumulation (2 C-atoms) and ammonium
production (Cacet/NH4) is calculated from regressions of acetate versus NH + concentration ( + exhangeable pool). E C / N is
calculated from the summed carbon pool (SCFA plus TCO2) versus the NH~- pool
NH~- concentration
Temp.
ATCO 2 / A N H 4
R2
Expt. A
Sta. 1
Sta. 2
15
15
4.03
3.67
0.85
0.91
Expt. B
Control
Control
Low
Low
Med
Med
High
High
5
15
5
15
5
15
5
15
7.75
6.46
1.76
1.82 a
1.80
0.71 a
0.37
0.93 a
0.88
0.75
0.96
0.99
0.75
0.57
a Initial time points < 162 h before sulfate depletion.
b Time points < 405 h.
c Time points < 596 h.
Cacet/NH 4
R2
EC/N
R2
5.00
1.42 c
5.00
3.48 a
1.64
4.40 b
0.87
0.80
0.99
0.88
0.84
0.999
6.61
4.95 c
10.46
5.07 a
7.93
6.63 b
0.86
0.80
0.99
0.87
0.84
0.999
42
Table 5
Total production of SCFA plus TCOe and NH~- (+exchangeable pool) in Expt. B (0-708 h). The decomposedpools
of organic carbon (Cdecom) and nitrogen (Ndecom) are given in
/zmol cm-3. SCFA plus TCO2 and NH~- production is presented as percentage of the initially added pool, %Cadd and
%Nadd respectively
Temp.
°C
Cdecom
/xmol C
cm
Low
Low
Med
Med
High
High
5
15
5
15
5
15
54
61
103
169
256
317
%Cad d
Ndecom
p~molN
3
cm
53
60
20
33
25
31
%Nadd
- 3
7.3
18.5
14.7
53.1
36.5
74.3
60
> 100
24
87
30
61
/z~NH~) were much lower (3.7-4.0) (Table 4)
than the C / N ratio of the particulate organic
pools ( P O C / P O N = 11.9-13.4), and substantiated the general contention of a preferential nitrogen mineralization in marine sediments
[7,20,22,40,41].
The A T C O z / z l N H ~ ratios obtained in unamended control sediments (6.5-7.8) were higher
than in the amended sediments (0.4-1.8) (Table
4), which showed a decreasing trend with increasing organic matter addition. The P O C / P O N ratio of the particulate organic pools only decreased from 11.7 in the controls to 8.8-10.8 in
the amended sediments. Unfortunately, total dissolved organic carbon (DOC) and nitrogen (DON)
were not determined here, but as SCFA accumulation plus TCO 2 production accounted for up to
60% of the initially added organic matter during
the experiment (Table 5), the SCFA may have
represented a large fraction of the total D O C
pool. Of the final dissolved carbon pool 65-77%,
82-96% and 96-97% at Low, Med and High,
respectively, was recovered as SCFA. A similar
study with Low addition at 5°C showed that D O N
initially attained a pool of approximately 20% of
the ammonium pool, but during 30 days of incubation the D O N pool decreased and finally attained only 2.5% of the ammonium pool [42]. By
combining the SCFA and T C O 2 pools into a
~ C / N ratio, values approaching the elemental
C / N ratio of the added food pellets ( P O C / P O N
= 8.4) were obtained in the amended sediments
(~2C/N = 5.0-10.5) (Table 4). The very low
A T C O z / A N H ~- ratios combined with the large
recovery of added nitrogen in inorganic form
implied a rapid nitrogen mineralization. The
higher ~2C/N ratios similar to the particulate
pools and the strong relationship between acetate
and NH~- accumulation suggested a coupling between the production of SCFA and NH~. One
route for N H 2 production is through D O N
(amino acids), where SCFA, e.g. acetate, is produced with a simultaneous release of NH~[21,22,42-44].
This study shows that loading of natural sediments with large quantities of organic matter of
high quality may result in a saturation of the
sulfate reduction with respect to substrate; i.e.
SCFA. The saturation levels are low compared to
the particulate pools found in sediments underlying marine fish farms [7], but at the field site
additional factors such as sediment-water interactions have to be considered during the decomposition of waste products. Sulfate reduction rates
corresponding to the maximum rates obtained in
this study have been found at the field site [7].
Pools of SCFA were, however, low during the
incubation of autumn fish farm sediment (Expt.
A), although the mineralization was twice as high
compared to a control sediment. At that time
sulfate reduction is probably controlled by the
supply of SCFA.
The temperature dependence of sulfate reduction is very strong, and rates were increased with
a factor of 4 by elevating the temperature 10°C.
Correspondingly, the apparent inhibition of sulfate reduction at high organic loadings was most
significant at the low temperature indicating the
importance of population dynamics in the terminal mineralization of organic matter. The mineralization of fish food pellets was rapid at both
incubation temperatures, and short chain fatty
acids are very important intermediates in the
carbon cycling. After 30 days of incubation, dissolved carbon was primarily recovered as SCFA,
whereas the nitrogen was found as ammonium,
indicating a rapid nitrogen mineralization.
43
Acknowledgement
Thanks for kind assistance during sampling to
the crew on the fish farm 'Fiskegruppen
Lilleb~elt'. M.H. was supported by a grant from
Odense University.
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