FEMS Microbiology Ecology 13 (1994) 295-302
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
295
FEMSEC 00509
The effect of incubation temperature on steady-state
concentrations of hydrogen and volatile fatty acids
during anaerobic degradation in slurries
from wetland sediments
Peter Westermann
*
Department of General Microbiology, Universityof Copenhagen, S¢lvgade 83H, DK-1307 Copenhagen K, Denmark
(Received 26 March 1993; revision received 24 November 1993; accepted 26 November 1993)
Abstract: Increasing the incubation temperature of two swamp slurries from 2°C to 37°C resulted in a 8- to 18-fold increase in the
H 2 partial pressure. The concentration of volatile fatty acids remained fairly constant except for butyrate, which decreased with
increasing temperature. Calculation of Gibbs free energies of syntrophic degradation of butyrate and propionate, and of
methanogenesis from acetate and H 2 revealed that these reactions were exergonic after the slurries had stabilized at the incubation
temperatures. The changes in H 2 partial pressure and butyrate concentration with temperature were found important to render
the processes exergonic within the tested temperature range.
Key words: Methanogenesis; Syntrophism; Thermodynamics; Acetate; Propionate; Butyrate
Introduction
Flooded soils and fresh-water wetlands of the
Northern hemisphere are considered to be some
of the most important sources of atmospheric
methane, thereby contributing to the global accumulation of infra-red absorbing trace gases in the
atmosphere [1,2].
In these ecosystems, where the concentrations
of inorganic electron acceptors other than CO2
are low, methanogenesis is controlled by physicochemical and biological processes among which
* Corresponding author.
SSDI 0 1 6 8 - 6 4 9 6 ( 9 3 ) E 0 0 6 9 - T
substrate availability and seasonal variations in
temperature are regarded as some of the most
important [3,4].
During metabolism of organic matter in anaerobic methanogenic ecosystems, volatile fatty acids
such as acetate, propionate, and butyrate are the
major reduced compounds produced by exergonic
fermentation processes. Propionate and butyrate
are metabolized to acetate and H 2 by syntrophic
bacteria in processes which are endergonic under
standard conditions. If the partial pressure of H2,
however, is kept sufficiently low by e.g. CO2-reducing methanogenic bacteria, the processes are
exergonic, i.e. have a negative Gibbs free energy
(AG'), [5]. Acetate is metabolized by methano-
296
genic bacteria or oxidized syntrophically (manuscript in preparation).
The capability of anaerobic bacteria to scavenge substrates from an anaerobic environment
has been shown to be dependent upon the energy
yield obtained from metabolism of the particular
compound [6]. Syntrophic metabolism is limited
by a certain substrate threshold concentration on
one side, and an inhibitory product concentration
on the other, beyond which the energy expenses
utilizing the substrate equals or exceeds the energy gained from the metabolism (i.e. where the
sum of Gibbs free energies of involved processes
> 0). For the metabolism of volatile fatty acids
such as propionate and butyrate, this implies a
certain interval where the H 2 concentration is
sufficiently low to allow syntrophic oxidation and
sufficiently high to allow methanogenesis from
H 2 [5]. Besides concentrations of substrates and
products, the AG' of a reaction is also dependent
upon temperature as described by the Nernst
equation. The standard Gibbs free energy (AG°),
however, also depends on temperature according
to the following Eq. [7]: AG O= AH ° - T × AS °
where AH ° and AS ° are the changes in enthalpy
and entropy under standard conditions respectively. Temperature corrections for AG O have
been shown important for incubation temperatures deviating from standard conditions (25°C)
to achieve correspondance between measured and
calculated threshold concentrations of hydrogen
in defined mono- and cocultures [8,9]. The energetic consequence of this temperature dependency is, that the energy yield of processes which
are endergonic under standard conditions decrease with decreasing temperature while processes which are exergonic under standard conditions increase their energy yield with decreasing
temperature. In syntrophic relationships, the oxidation process therefore becomes less energetically favourable with decreasing temperature,
while the hydrogen scavenging process becomes
more favourable, unless the concentrations of
participating compounds are altered proportional
to the temperature change. In stable natural
ecosystems such as sediments and permanent
swamps, where the size and composition of the
bacterial populations are adjusted to the prevail-
ing conditions and substrates, an approximation
between in situ and threshold concentrations of
non-recalcitrant substrates could be expected.
The consequence of this assumption should either be a change in the energy yield of t h e
degradation processes or a change in the concentrations of intermediate s u b s t r a t e s / p r o d u c t s
when the temperature varies throughout the year.
The purpose of this study was to investigate
this substrate - product - temperature relationship in slurries from an organic-rich swamp associated with an eutrophic lake and a less productive minerotrophic swamp.
Materials and Methods
Sampling and slurry preparation
Sediments were collected with corers or in
N2-flushed jars from two permanently waterlogged swamps in Denmark: (1) a permanently
waterlogged alder swamp in the Dyrehaven forest, where the primary carbon supply consists of
alder leaves and twigs; and (2) an old canal connected to the eutrophic lake Bagsv~erd, filled with
waterlogged algal detritus and leaves, but with no
standing water.
Upon return to the laboratory, the alder swamp
sediment was blended with anoxic porewater from
the swamp (1:1), while the canal sediment, due
to its higher water content, was blended without
dilution. The slurries were distributed into 50-ml
Nz-flushed serum vials (25 ml in each). The vials
were capped with butyl rubber stoppers, crimped
with aluminum seals, and then incubated at 2, 10,
15, 20, 30, and 37°C under static conditions in the
dark.
,
Analytical methods
Hydrogen was measured in the headspace by a
m e r c u r y / m e r c u r y vapor reduction gas analyzer
(Trace Analytical). To maintain a near-atmospheric pressure in the vials, overpressure was
occasionally relieved by inserting a needle through
the stopper during the first 2 weeks of incubation,
where the major activity occurred. Before analysis, the vials were shaken to equilibrate dissolved
H 2 with the gas phase. Methane was analyzed by
297
gas chromatography as previously described [10].
Bicarbonate concentrations at the end of the
experiments were calculated by inserting measured p H values and dissolved CO 2 concentrations into the Henderson-Hasselbach equation
after correcting the p K a of carbonic acid for
t e m p e r a t u r e influence on the equilibrium constant [7]. Dissolved CO 2 was calculated after correcting measured headspace CO 2 concentrations
for t e m p e r a t u r e / s o l u b i l i t y [11].
To measure concentrations of volatile fatty
acids in the low/~mol to high nmol range, samples were concentrated before gas chromatographic analysis. Diethyl ether extractions from
acidified samples as described by Larsson et al.
[12] and Balba and Nedwell [13] were tried without success, as the trace amounts of volatile fatty
acids present in commercially available ether,
even after washing with 1 N N a O H , interfered
with the very low acid concentrations in the slurries. Instead the p H was raised to 11 with N a O H
in supernatant decanted from centrifuged slurry
samples. T h e samples were dried at 95°C and
redissolved in 0.1 to 0.2 ml of 3 N H a P O 4 corresponding to a concentration factor of 25 to 50. 1
/zl was injected into the gas chromatograph. The
concentration of each acid was calculated from
standards carried through the same concentration
procedures. Analyses were performed on a Hewlett-Packard gas chromatograph 5890 equipped
with a flame ionization detector and a Nukol 15
m × 530 /zm capillary column (Supelco); oven
t e m p e r a t u r e 120°C; injector and detector 180°C,
carrier gas N 2. To avoid precipitation of salts in
the analytical column, a 1 m empty 530 /zm
capillary column was mounted as a preeolumn.
Thermodynamic calculations
The effect of temperature, substrate and product concentrations on the free energy available to
the bacteria carrying out butyrate, propionate,
acetate, and H E metabolism at in situ concentrations was calculated by substituting AG o in the
Nernst equation with AG O= A H ° - T × AS ° to
compensate for temperature. For methanogenesis
from H 2 this leads to:
A G ' = ( A H ° - AS ° × T) + R T
× In [H214 × [HCO~-] × [H +]
(1)
For methanogenesis from acetate:
a G ' = ( a l l ° - a S ° × T) + R T
( [CH.] X [HCO3] )
× In
[CH3COO_ ]
(2)
For syntrophic propionate degradation:
ziG' = (ziH 0 _ ASo × T) + RT
× In
( [CH3COO- ] x [HCO3 ] × [H+ ] × [H2]3 )
[CH3CHzCOO_ ]
(3)
and for syntrophic butyrate degradation:
A G ' = ( A H ° - AS ° X T) + R T
[CHaCOO-]
X In
X [H + ] × [HE] 2
[ C H a C H E C H 2COO_ ]
(4)
Table 1
Values for standard a Gibbs free energy, standard reaction enthalpies and entropies of methanogenesis from H2, acetate, and
butyrate and propionate oxidation b
Process
AG°
LI/mol
ZIH°
Ll/mol
AS°
Ll/mol. °C
4H 2 +CO 2 -o CH 4 +2H2 O
CH3COO- +H20 ~ CH 4 +HCO 3
CH3CH2CH2COO- + 2H20 -o 2CH 3COO- + H + + 2H 2
CH3CH2COO- +3H20 ~ CH3COO- +HCO 3 + H + +3H 2
- 130.7
-31.0
+ 88.2
+ 116.4
-253.0
+5.0
+ 135.6
+ 192.4
-0.410
+0.121
+ 0.159
+0.255
a Standard conditions are 1 M, 1 arm, pH = 0 and T = 25°C.
b Values were calculated from energies, enthalpies, and entropies of formation [7,9].
298
where AH ° is the standard reaction enthalpy,
AS ° is the entropy change of the reaction. T is
the t e m p e r a t u r e in Kelvin, and R is the gas
constant (0.008315 kJ X ° K - t x m o l - 1). Reaction
enthalpies, entropies and standard free Gibbs
energies used for the calculations are shown in
Table 1.
25
~'o 2o
X
E
Results
The basic C H 4 and CO 2 production during
the incubation of the two swamp slurries are
shown in Fig. 1. The highest methane production
was found in the canal sediment. Due to the
rough mechanical treatment of the slurries, the
concentrations of volatile fatty acids and H 2 were
initially high. During the incubation period, the
concentrations, however, fell to a steady state
level which remained virtually constant (less than
1% change per month). To allow the concentra-
0,6
0.5
/
/
.~0,4
0
T
"0
t-0.3
0
O~/1/"
0,2
0.1
"O
I
s
10
20
30
40
Temperature °C
Fig. 2. Hydrogen concentrations in swamp slurries at different
incubation temperatures, measured after 88 days of incubation. n alder swamp, zx, canal swamp.
0.7 ,
~
N
.. "
~
/
/
/
/
/
. ///1~
/
.II"
IF"
I"'"
10
29
30
40
Temperature °C
Fig. 1. Carbon dioxide and methane partial pressures in alder
swamp and canal slurries, measured after 88 days of incubation. 13, methane and II, carbon dioxide, alder swamp slurry.
o, methane and e, carbon dioxide, canal swamp slurry.
tion of intermediates to reach steady state, measurements were therefore carried out after 50-90
days of incubation. A pre-incubation of this length
was previously shown to be sufficient to ensure
depletion of the small amounts of inorganic electron acceptors such as SO 2 - and N O 3 present in
the swamps [10]. The p H in the slurries decreased
slightly with increasing t e m p e r a t u r e (from 7.04 to
6.59 in the alder slurries, and from 6.82 to 6.45 in
the canal slurries).
In Fig. 2, the H 2 partial pressures at the
different incubation temperatures are shown for
the alder swamp and canal slurries. The H 2 partial pressures increased with increasing temperature in both swamps and were almost similar in
the two slurries, despite the higher methanogenic
activity in the canal slurry.
In Fig. 3 the concentrations of acetate, propionate, and butyrate in the two swamp slurries are
shown as a function of temperature. Acetate and
propionate concentrations were slightly higher in
the alder swamp slurries than in the canal slurry,
299
remained fairly constant independently of temperature and ecosystem.
10o
-~-
"~
Discussion
~ ~°
-6
E
Z
"" ,q - G
~'~"~"=: ~ k . . " "
0,3
0,1
i
I
10
i
I
~
20
I
30
,
40
Temperature °C
Fig. 3, Concentrations of volatile fatty acids in aider swamp
and canal slurries at different incubation temperatures measured after 76 days of incubation, o, acetate; II, propionate;
A, butyrate, aider swamp slurry. 12, acetate; zx, propionate;
o, butyrate, canal swamp slurry.
while butyrate concentrations were almost similar. No increase in the concentrations of volatile
fatty acids with increasing temperature was, however, observed. Butyrate concentrations even decreased a factor 4 upon a temperature increase
from 2 to 37°C.
The effect of temperature on Gibbs free energy at the measured substrate and product concentrations in the two swamp slurries is shown in
Fig. 4. From a thermodynamic point of view, all
processes involved in the mineralization of propionate and butyrate to CH 4 and CO 2 were energetically possible at the different incubation temperatures. Propionate oxidation and methanogenesis from H 2 / C O 2 were the least energy yielding
processes. Despite the relatively large variations
in substrate/product concentrations and energy
yield of the part processes of propionate and
butyrate mineralization to CO 2 and CH4, the
total energy yield of the mineralization processes
To my knowledge, this is the first study of the
effect of temperature on the concentration of
volatile fatty acids and H 2 in natural environments. Conrad and Wetter have previously shown
that the H e threshold concentration in axenic
cultures of methanogenic bacteria decreased with
decreasing temperature [9]. Lee and Zinder [8]
also predicted lower H 2 partial pressures in syntrophic cocultures with decreasing temperature
but have not so far verified this experimentally.
The decrease in energy of syntrophic oxidation
with decreasing temperature can either lead to an
arrest in the metabolism of the syntrophic substrates or a maintainance of the oxidation activity
if the concentrations of regulating intermediates
are proportionally changed with changing temperature. The constancy of the total energy yield
of butyrate and propionate mineralization with
changing temperature demonstrates that these
processes can occur at low temperatures as previously confirmed for the alder swamp [14]. The
energy yield of propionate oxidation and methanogenesis from H 2 / C O z is more sensitive to
variations in the H a partial pressure than to
variations in concentrations of volatile fatty acids
(Eq. 1 and 3). As the H 2 partial pressure further
varies considerably more (8-18 times) than the
concentrations of volatile fatty acids, the H 2 partial pressure must be considered responsible for
the energetic compensation for temperature variations. The role of H 2 is supported by the fact
that propionate oxidation at 10°C is not possible
if the H a partial pressure measured at 37°C is
inserted in the Nernst equation ( z i G ' = +2.39
k J / m o l in canal swamp slurries and + 3.58 k J /
mol in alder swamp slurries). Similarly, methanogenesis from H 2 / C O 2 is not possible at 37°C if
the H a partial pressure measured at 10°C is used
GaG ' = +9.28 k J / m o l in canal swamp slurries
and + 9.12 in alder swamp slurries). The increase
in butyrate concentrations with decreasing temperature also contributes to a stable energy yield
300
of butyrate oxidation, but with less importance
than H 2, since H 2 is squared in the Nernst equation (Eq. 4).
Conrad et al. [15] measured H 2 partial pressures in lake sediments and sewage sludge which
were too high to allow syntrophic degradation of
propionate and butyrate. Based upon these observations they hypothesized the existence of two
pools of H 2 in these ecosystems: one pool inside
microniches where interspecies H 2 transfer is
made possible by rapid methanogenic consumption of H 2, and one common pool where H 2
partial pressures exceed the level necessesary to
substantiate energetically favourable oxidation of
propionate and butyrate. The pool sizes measured in the present study result in that both
syntrophic oxidation of propionate and butyrate
and methanogenesis from H 2 and acetate are
exergonic. Although thermodynamically possible,
none of the reactions, however, produce sufficient energy for the phosphorylation of ADP to
ATP with a AG' of +30.54 kJ/mol, correspond-
ing to approx. 70 kJ/mol when compensating for
an energy conservation efficiency of 42%. Assuming that the smallest amount of energy which can
be conserved corresponds to 1 / 4 of the phosphorylation energy (17.5 kJ/mol), only butyrate oxidation and methanogenesis from acetate are biologically possible. A further reduction of H 2 partial pressures within matrix structures could make
propionate oxidation biologically possible. This
would, however, lead to a further decrease in
AG' of methanogenesis from H 2.
The 5- to 10-times higher methanogenic activity in the canal swamp slurries was not reflected
in the concentrations of H2 and volatile fatty
acids, which generally varied less than a factor 2
between the two ecosystems. A possible explanation for this is, that the bacterial populations in
sediments which are not exposed to wash-out
adjust their size to the prevailing substrate availability. If this is a general property in stable
sediments, the need for juxtapositional arrangements as described by Conrad et al. [15] should
-100
- 1 O0
-.--A...........
-50
k- .....
A
•
....
•
O
-
4
-50
..•__
•
•
.........
-II
t6- .......
__
-30
-30
0
O
E
• "--
•
E
0-~
~,~- - 2 0
_ ~ 0
.O ......... O .....................
-
O ........
~ ~
O ............. O
" 0 ~ ~ ...0_ . . . .
-,•_ . . . . .
0
~0
~
-
- -20
{5
<~
•
-10
-10
2~--"
®
®
-5
I
10
~
I
~
20
Temperature °C
p
30
~
-5
40
i
J
10
J
I
I
20
30
40
Temperature °G
Fig. 4. Effects of incubation temperature on Gibbs free energy in canal (a) and alder swamp (b) slurries. 13, methanogenesis from
H2; e, methanogenesis from acetate; zx, propionate oxidation; ©, butyrate oxidation; i , propionate mineralization to CH 4 and
CO2; A, butyrate mineralization to CH 4 and CO 2.
301
only b e n e c e s s a r y in d i g e s t e r s a n d in a s s o c i a t i o n
with s u b s t r a t e h o t spots w h e r e t h e t h e H 2 - c o n s u m i n g m e t h a n o g e n i c p o p u l a t i o n is e i t h e r w a s h e d
o u t o r for o t h e r r e a s o n s u n a b l e to k e e p p a c e with
t h e H 2-producing p o p u l a t i o n s .
C o m p a r e d to a n a e r o b i c f r e s h w a t e r ecosystems,
a c e t a t e , p r o p i o n a t e , a n d b u t y r a t e w e r e p r e s e n t in
a similar c o n c e n t r a t i o n r a n g e in t h e two s w a m p
slurries ( 3 2 - 1 5 1 / z M a c e t a t e , 0 . 3 - 9 0 / z M p r o p i o n a t e , 0 . 2 - 2 / z M b u t y r a t e [15,16]). T h e H 2 p a r tial p r e s s u r e was, however, m a r k e d l y lower in t h e
c a n a l a n d a l d e r s w a m p slurries t h a n r e p o r t e d for
f r e s h w a t e r s e d i m e n t s ( 1 1 - 4 8 × 10 - 6 a t m [15,17]).
Dolfing introduced the concept of a microbial
" z e r o - s u m society" in which t h e i n c r e a s e in energy available to o n e o r g a n i s m following t h e
c h a n g e in t h e c o n c e n t r a t i o n o f an i n t e r m e d i a t e is
c o u n t e r b a l a n c e d by t h e d e c r e a s e in e n e r g y availa b l e to t h e p r o d u c e r o f this i n t e r m e d i a t e [18]
p r e s u p p o s i n g t h a t t h e p r o c e s s e s r e m a i n exergonic. T h e results o f t h e p r e s e n t study illustrate
this z e r o - s u m society c o n c e p t in t h a t t h e e n e r g y
yields o f t h e c o m p l e t e m i n e r a l i z a t i o n o f p r o p i o n a t e a n d b u t y r a t e r e m a i n fairly c o n s t a n t with
c h a n g i n g t e m p e r a t u r e , d e s p i t e t h e fluctuations in
t h e e n e r g y y i e l d o f t h e p a r t processes. F u r t h e r m o r e , s t a b i l i z a t i o n o f t h e e n e r g y yield with changing t e m p e r a t u r e can b e a d d e d to t h e roles o f H 2
as a c e n t r a l r e g u l a t i n g i n t e r m e d i a t e in s y n t r o p h i c
metabolism.
Acknowledgements
K a r i n V e s t b e r g is a c k n o w l e d g e d for e x c e l l e n t
t e c h n i c a l assistance. P a r t s o f this study w e r e supp o r t e d by T h e D a n i s h N a t u r a l Science R e s e a r c h
C o u n c i l (11-7701) a n d by T h e D a n i s h C e n t e r for
M i c r o b i a l Ecology.
References
1 Aselmann, I. and Crutzen, P.J. (1989) Global distribution
of natural freshwater wetlands and rice paddies, their net
productivity and possible methane emissions. J. Atmos.
Chem. 8, 307-358.
2 Tyler, S.C. (1991) The global methane budget. In: Microbial Production and Consumption of Greenhouse Gases:
Methane, Nitrogen Oxides, and Halomethanes (Rogers,
J.E. and Whitman, W.B., Eds.), pp. 7-39. ASM, Washington D.C.
3 Conrad, R., Schiitz, H. and Babbel, M. (1987) Temperature limitations of hydrogen turnover and methanogenesis
in anoxic paddy soil. FEMS Microbiol. Ecol. 45, 281-289.
4 Westermann, P. and Ahring, B.K. (1987) Dynamics of
methane production, sulfate reduction and denitrification
in a permanently waterlogged alder swamp. Appl, Environ. Microbiol. 53, 2554-2559.
5 Dolfing, J. (1988) Acetogenesis. In: Biology of Anaerobic
Microorganisms (Zehnder, A.J.B., Ed.), pp. 417-469. John
Wiley and Sons, New York.
6 Cord-Ruwisch, R., Seitz, H.-J. and Conrad, R. (1988) The
capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential
of the terminal electron acceptor. Arch. Microbiol. 149,
350-357.
7 Stumm, W. and Morgan, J.J. (1981) Aquatic Chemistry.
2nd Edn. Wiley Interscience, New York.
8 Lee, M.J. and Zinder, S.H. (1988) Hydrogen partial pressures in a thermophilic acetate-oxidizing methanogenic
coculture. Appl. Environ. Microbiol. 54, 1457-1461.
9 Conrad, R. and Wetter, B. (1990) Influence of temperature on energetics of hydrogen metabolism in homoacetogenic, methanogenic, and other anaerobic bacteria. Arch.
Microbiol. 155, 94-98.
10 Westermann, P. and Ahring, B.K. (1985) Terminal anaerobic carbon mineralization in a permanently waterlogged
alder swamp. In: Microbial Communities in Soil (Jensen,
V., Kj¢ller, A. and Scrensen, L.H., Eds.), pp. 305-314.
Elsevier Press, Amsterdam.
11 Wilhelm, E., Battino, R. and Wilcock, R.J. (1977) Lowpressure solubility of gases in liquid water. Chem. Rev. 77,
219-262.
12 Larsson, L., S~tllstr6m-Baum, S. and de la Cochetiere-collinet, M.-F. (1984) A two-step extraction procedure for
concentrating acidic organic volatiles in aqueous solution
prior to gas chromatographic head-space analysis, as exemplified by short-chain fatty acids produced by Bacillus
cereus. J. Microbiol Meth. 2, 9-14.
13 Balba, T. and Nedwell, D.B. (1982) Microbial metabolism
of acetate, propionate, and butyrate in anoxic sediment
from the Colne Point Saltmarsh, Essex, U.K.J. Gen. Microbiol. 128, 1415-1422.
14 Westermann, P. (1990) The effect of temperature on butyrate degradation. In: Microbiology and Biochemistry of
Strict Anaerobes Involved in Interspecies Hydrogen
Transfer (Belaich, J.-P. Bruschi, M. and Garcia, J.-L.,
Eds.), pp. 489-491. Plenum Press, New York.
15 Conrad, R., Schink, B. and Phelps, T.J. (1986) Thermodynamics of H2-consuming and H2-producing metabolic reactions in diverse methanogenic environments under in
situ conditions. FEMS Microbiol. Ecol. 38, 353-360.
16 Lovley, D.R. and Klug, M.J. (1982) Intermediary metabo-
302
lism of organic matter in the sediments of a eutrophic
lake. Appl. Environ. Microbiol. 43, 552-560.
17 Conrad, R., Phelps, T.J. and Zeikus, J.G. (1985) Gas
metabolism evidence in support of the juxtaposition of
hydrogen-producing and methanogenic bacteria in sewage
sludge and lake sediments. Appl. Environ. Microhiol. 50,
595-601.
18 Dolfing, J. (1992) The energetic consequences of hydrogen
gradients in methanogenie ecosystems. FEMS Microbiol.
Ecol. 101, 183-187.