E M S MicrobiologyEcology 101 (1992) 183-187
0 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00
Published by Elsevier
183
FEMSEC 00402
The energetic consequences of hydrogen gradients
in methanogenic ecosystems
Jan Dolfing
Department of Biochemistry, University of Groningen, Groningen, Netherlands
Received 11 February 1992
Revision received 7 May 1992
Accepted 14 May 1992
Key words: Interspecies hydrogen transfer; Methanogenesis; Waste water treatment; Zero-sum concept
1. SUMMARY
In the dense microbial aggregates usually found
in methanogenic waste water treatment systems,
hydrogen has to diffuse from producers to consumers at considerable rates. The ensuing hydrogen gradients dissipate part of the potential energy that would otherwise be available to the
hydrogen-consuming organisms. The present paper evaluates the energetic consequences of this
phenomenon.
2. INTRODUCTION
Mineralization of organic compounds in
methanogenic ecosystems is a joint effort, carried
out sequentially by a series of organisms [l].The
energy available during the degradation process,
Correspondence to: J. Dolfing, Department of Biochemistry,
University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands.
therefore, has to be divided up between the participating organisms, while the organisms live in
what can be labelled a zero-sum society: an increase in the concentration of an intermediate
increases the specific amount of energy available
to the consumers of this intermediate, but simultaneously results in a decrease of the same magnitude in the specific amount of energy available
to the producer of this intermediate [2].
The recent interest in diffusion gradients in
high-rate anaerobic waste water treatment systems [3,4] sheds new light on the zero-sum concept outlined above. It has been pointed out that
the high fluxes that occur in dense microbial
biolayers are sustained by steep concentration
gradients between producers and consumers of
the central intermediate in methanogenic ecosystems, hydrogen. This implies that the hydrogen
consumers in such consortia ‘see’ lower hydrogen
concentrations than the producers [3,4]. Ultimately this leads to the conclusion that the organisms do not harness all of the available energy,
since part of it is dissipated in these hydrogen
gradients. The present paper evaluates the energetic consequences of this phenomenon.
184
3. MATERIALS AND METHODS
The reduction of CO, to methane by hydrogenotrophic methanogens can be described by
the following reaction:
CH, + 2H,O + OH(1)
4H2 + HCO;
This equation served as the basis to calculate the
Gibbs free energy changes associated with the
energy metabolism of hydrogenotrophic methanogens. The calculations were made after Thauer et al. [5]. The Gibbs free energy change of
hydrogenotrophic methanogenesis was taken to
be - 135.6 kl/reaction under standard conditions [51. This value is valid for pH = 7 with
hydrogen and methane in the gaseous state at a
partial pressure of 1 atmosphere, and bicarbonate
dissolved at a concentration of 1.0 M. The values
for the Gibbs free energy change of hydrogenotrophic methanogenesis under non-standard conditions were calculated by using equation 2:
AG' = - 135.6 + 5.7 ~O~[CH,][H,]-~[HCO;]-'
kJ/mol CH,
(2)
where the concentrations of hydrogen and
methane are expressed in atm while [HCO;] is in
M IS]. To allow the direct use of hydrogen concentrations the Henry coefficient published by
Wilhelm et al. [6] was used. These authors state
that 1 atm H, is in equilbrium with 600 p M H,.
Thus, Eqn. 2 changes into
AG'= -209.1 + 5.7 ~o~[CH,][H~]-~[HCO;]-'
(3)
kJ/mol CH,
where the concentration of methane is expressed
in atm while [HCO;] and [H21 are in M.
4. RESULTS AND DISCUSSION
To evaluate the implications of energy dissipation via hydrogen gradients in methanogenic aggregates, two questions must be answered: (i)
how much energy is lost in these gradients, and
(ii) how much energy would be available to the
hydrogen consumers in the absence of such gradi-
Table 1
Energy dissipation in hydrogen gradients and its implications
for the amount of Gibbs free energy available to hydrogenotrophic methanogens
Concentration
gradient
H, (nM)
Energy
dissipation
(kl/molH2)
100-,90 10-9
+8
-80
+ 70
+1
-6
-60
0.26
0.55
0.88
1.26
1.72
2.27
2.98
3.98
5.71
-50
-40
-30
-20
-10
+5
+4
+3
-2
+1
Relative energy loss (%) at
1M)nM
10nM
2
5
8
12
16
21
27
31
53
5
11
17
24
33
44
58
77
111
Calculations were made for methanogenesis at 25T, pH
PcH, = 0.6 atm and [HCO,] = 50 mM.
7,
ents. The answers to both questions are based on
Eqn 2. Typical conditions for methanogenic
ecosystems are pH = 7, [HCO;] = 50 mM, and
PcH,= 0.6 atm. For these conditions Eqn. 2 can
be written as:
AG'= 2.2-22.8 log[H,] kJ/mol CH4
(4)
or
AG' = 0.55-5.7 log[H,] kJ/mol H,
(5)
with the hydrogen concentration in nM.
Two points with respect to the amount of
energy potentially available to hydrogenotrophic
methanogens in anaerobic consortia can be derived from Eqn. 5, which states that the amount
of available free energy is a linear function of the
logarithm of the hydrogen concentration: (i) the
amount of available Gibbs free energy (expressed
per mol of H, consumed) decreases by 5.7 kl for
every 10-fold drop in the H 2 concentration, and
(ii) the decrease in the amount of available free
energy depends on the percentual value of the
hydrogen gradient. This is illustrated in Table 1.
The amount of potential energy dissipated in
gradients with a concentration drop of e.g. 30% is
0.88 kT per mol of H, irrespective whether this is
a drop from 100 to 70 nM or from 10 to 7 nM.
The importance of a loss of 0.88 kl per mol of H,
converted is however about two times larger for
185
Table 2
Energy dissipation in hydrogen gradients and its implications
for the amount of Gibbs free energy available to hydrogenotrophic methanogens
Concentration
gradient
H, (nM)
Energy
dissipation
(kl/mol H,)
Relative energy loss
(%I at
100nM
10nM
100-99
-98
+97
+96
-95
10+9
+8
+7
+6
+5
0.02
0.05
100nM
0.2
0.5
-94
-93
4
4 3
0.15
0.18
0.26
0.55
0.88
1.26
1.72
2.27
492
491
~
+
+
2
+1
0.08
0.10
0.13
0.21
0.23
2.98
3.98
5.71
10nM
5
0.9
1.2
11
17
24
33
1.4
44
1.7
1.9
2.2
58
77
111
0.7
~~
Calculations were made for methanogenesis at 25”C, pH = 7,
pCH,
= 0.6 atm. and [HCO;] = 50 mM.
organisms that operate at a H, concentration of
10 nM than for organisms operating at 100 nM.
The effect of the diffusion gradient on the
mass transfer to the methanogen should also be
considered. The mass transfer is a function of the
gradient, so a gradient of 10 nM to 7 nM at the
methanogen’s surface would deliver a certain flux
of H,, and a gradient of 100 nM to 97 nM would
provide the same flux of H,. This comparison is
elaborated in Table 2. The calculations (Table 2)
show that a gradient of 3 nM dissipates only 0.7%
of the potential energy available to the methanogen if the gradient is from 100 nM to 97 nM,
versus 17% if the gradient is from 10 nM to 7
nM.
Goodwin and co-workers [41 recently reported
for a lactate degrading consortium a H, concentration of 210 nM at the surface of the lactate-degrading organisms vs. a H, concentration of 170
nM at the surface of the hydrogenotrophic
methanogens. This gradient of 40 nM encompasses a drop in the H, concentration of about
20%. According to Eqn. 5, 12.2 W was available
to the methanogens per mol of hydrogen at a H,
concentration of 170 nM, vs. 12.7 kJ per mol of
hydrogen at a H, concentration of 210 nM. Thus,
this hydrogen gradient of 20% costs the
methanogens 0.5 kJ per mol of hydrogen, i.e.
about 4% of the potential energy available at the
surface of the H,-producers.
The measured concentrations of H, in
methanogenic environments cluster between 2
and 200 nM [4,7-lo]. The example elaborated
here was at the upper limit of this range where
the potential thermodynamic effects are comparatively smaller than at lower H, concentrations.
At present this is the only quantified example of
a concentration gradient in dense methanogenic
consortia. A full evaluation of the direct energetic consequences of hydrogen concentration
gradients for hydrogenotrophic methanogens has
to wait until more data are available, but the
calculations presented here indicate that such
consequences cannot in advance be dismissed as
negligible.
In addition to directly influencing the energetics of hydrogenotrophic methanogens via an effect on the ‘caloric value’ of H,, hydrogen gradients also influence the energetics of the
methanogens via their effect on the kinetics of
these organisms. H concentrations in the range
of 2 to 200 nM are well below the apparent K ,
values of hydrogenotrophic methanogens [4,11]
and by consequence the hydrogen consumption
rate is a linear function of the H, concentration.
Taken together this implies that e.g. a 20% decrease in the H, concentration in the range of 10
to 100 nM results in a 25-31% decrease in the
energy flux through the methanogens.
In the last few years the question has been
raised whether reducing equivalents in methanogenic environments are transferred in the form of
hydrogen or of formate [3,12]. Sometimes formate
rather than hydrogen is the intermediate for reducing equivalents between juxtaposed bacteria
[12]. To explain this phenomenon it has been
argued that formate production would be thermodynamically favoured at high bicarbonate and
hydrogen concentrations [121. The zero-sum concept [2], however, implies that there is no energetic advantage for the consortium as a whole in
either pathway. The energy gain to the H,/formate producer would be the energy loss to the
HJformate consumer, and vice versa. The advantage to a syntrophic consortium of using formate rather than H, as interspecies electron car-
,
186
rier therefore seems to be the three-fold higher
diffusion coefficient of formate [3]. The other
mechanism to allow high substrate fluxes in
methanogenic consortia is of course the formation of close physical associations between ‘electron-producing’ and ‘electron-consuming’ bacteria, as this will minimize the development of
electron gradients. These are the very conditions
which high-rate waste water treatment systems
impose on the resident microflora [13-151. This is
done by offering a substratum on which biofilms
are formed or by selecting for the formation and
maintenance of well-settling dense micobial aggregates. In such environments the distances between the bacteria are in the order of 0.05-1 p m
[ 16,171. These short cell-cell distances allow for
high hydrogen fluxes at relatively low hydrogen
concentrations.
An important point in this context is how the
H producers and consumers are positioned relative to each other in such biofilms. The optimal
distance between the various physiological groups
in microbial aggregates is determined by the diffusion constant and the kinetic characteristics of
the H, producing and the H,-consuming organisms, including the affinity of the organisms for
H, at low substrate concentrations, the existence
of threshold concentrations for H, uptake, and
inhibition of the H , producers by elevated H ,
concentrations. Both H, producers and H, consumers are diverse microbial groups [2,18,19]. The
group of H, producers is generally subdivided
into acidogens and acetogens, with both subgroups having their own kinetic and physiological
responses to variations in the hydrogen concentration [2,18]. The group of the H, consumers is
also quite diverse. Here we can distinguish between methanogens, acetogens, suffate reducers,
and organisms that use chlorinated compounds as
electron acceptors [2,20,21]. While variations of
the kinetic properties occur within these subgroups, there is a tendency that the range of
concentrations at which these organisms function
is determined by the redox potential of the terminal electron acceptor [22]. At identical hydrogen
concentrations, for example, the amount of free
energy that becomes available per mol of hydrogen consumed is higher for sulfate reducers than
,
for methanogens [21. This enables sulfate reducers to function at lower hydrogen concentrations
than methanogens. This suggests that the effect
of a hydrogen gradient will significantly influence
the energetics of the hydrogen scavenging
catabolic reaction of sulfate reducing bacteria,
but since the thermodynamics of hydrogenotrophic sulfate reduction are more favorable t h a n
the thermodynamics of methanogenesis, it is not
possible to predict a priori whether the optimal
distance between hydrogen producers and hydrogen consumers in sulfate-reducing environments
will be different from that in methanogenic environments.
A factor of prime importance in this context is
also the ratio between hydrogen producers and
hydrogen consumers in microbial aggregates. ]t
will be interesting to see how this ratio is influenced by the types of substrates and electron
acceptors used, and how these factors influence
the organization and the occurrence of hydrogen
gradients in microbial aggregates. Only little information on the microanatomy of such systems is
currently available [231, but gas metabolism and
microscopic evidence suggests that juxtapositioning of hydrogen producing and methanogenic
bacteria occurs in many environments without
significant microcolony formation [10,24]. The
calculations presented here indicate that energy
dissipation via hydrogen gradients is one of the
factors that must be taken into consideration
during the interpretation of such information.
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