Objective

To explain the process of ATP generation in methanogenic bacteria showing how the
products of metabolism of other rumen micro-organisms are used as growth substrates.
Methanogens are autotrophic archebacteria that use anaerobic respiration for ATP synthesis.
Methanogens use CO2 taken up from their growth environment as the carbon substrate for
growth. They use some CO2 as the ultimate oxidizing agent of an electron transport chain
which, by a chemiosmotic mechanism, maintains a transmembrane electrochemical ion
gradient which powers ATP production.
The reducing agent that drives the electron transport chain is hydrogen also taken up from the
growth environment. This hydrogen is the waste end product of the metabolism of other,
heterotrophic microorganisms. Methanogens use this hydrogen and this process maintains a
lowered hydrogen partial pressure in the reticulo-rumen. Some of the hydrogen producing
heterotrophic microorganisms show altered patterns of metabolism because of methanogen
usage of the hydrogen they produce. This process lies at the heart of some of the best
characterised syntrophic relations seen in the reticulo-rumen and and is referred to as
"interspecies hydrogen transfer"
This phenomenon is important because hydrogen utilization by the methanogens reduces the
hydrogen partial pressure of the reticulo-rumen and this alters the pattern of metabolism of
syntrophic hydrogen "donor" partner species. This is due to these organisms having
hydrogen-sensitive hydrogenases. Raising or lowering the partial pressure of hydrogen in their
growth environment determines their fermentation pathways and thus affects their ATP
production which affects their growth. As we shall see further on in this section when
studying the fermentation patterns of Ruminococcus flavifaciens a lowering of the partial
pressure of hydrogen by interspecies hydrogen transfer allows enhanced growth of the
syntrophic hydrogen "donor" species.
The equation shows the reduction of CO2 by H2 to produce methane. This redox reaction
sustains anaerobic respiration which allows the production of ATP.

The methane produced by reduction of the carbon dioxide is lost from the reticulo-rumen by
eructation. It is a waste of feed carbon because the rumen does not have methanotrophic
bacteria and the host ruminant can not utilize this gas. Methane lost in this way is one reason
why methanogens contribute to lowered food conversion efficiency of the host ruminant.
There is another way they cause lowered food conversion efficiency which we deal with in the
next section. It involves indirect effects the methanogens have on propionate production.
Remember however that these inter-relations are complex because as indicated briefly above
and as we deal with in detail later, co-culture with methanogens can improve the growth of
some species of heterotrophic microorganisms.

Effect of Methanogens
Methanogens affect the growth of some but not all hydrogen producing species of

microorganism in the reticulo-rumen.

The growth of Fibrobacter succinogenes for example is not affected by the presence of
methanogens. This is because hydrogenase activity in Fibrobacter is not sensitive to the
prevailing hydrogen tension. These microorganisms do not therefore live in syntrophic
relations with methanogens but methanogens do use the hydrogen they produce which is
available to them as a result of interspecies hydrogen transfer.
Ruminococcus species however do have their metabolic processes altered by the presence of
methanogens and they do live in syntrophic relations with the methane producing species
which use the hydrogen that they produce.
One species, R. albus, produces ethanol, H2, CO2 and acetate but in proportions that vary
according to the abundance of methanogens in the culture medium.
In the presence of vigorously growing methanogens the products of fermentation are biased
towards acetate and away from ethanol.
This change in product proportions occurs because the methanogens remove the H2 that R.
albus produces and the decreased hydrogen partial pressure that results causes R. albus to
alter the way NAD is regenerated so as to reduce ethanol production and increase acetate
production. Increased acetate production is accompanied by increased ATP production and it
is the extra ATP that confers a growth advantage to the hydrogen "donor" species. This is a
striking feature of the syntrophic relationship.
Fenchel and Finlay give another example of syntrophy involving R. albus where the partner
organism is Vibrio succinogenes; another species taking advantage of interspecies hydrogen
transfer. This Vibrio uses hydrogen waste from R. albus to reduce fumarate so forming
succinate and in so doing generates a transmembrane ion gradient by a chemiosmotic
process that supports ATP synthesis by anaerobic respiration.
Ruminococcus flavifaciens shows a similar change in fermentation products brought about by
co-cultured methanogens and we analyse this in detail below.
When grown in monoculture the products of R. flavifaciens are acetate, succinate, H2 and CO2,
but when grown with a vigorous co-culture of methanogens, the production of acetate (and
ATP) and hydrogen increase at the expense of succinate. If, in the laboratory, products of the
growth of R. flavifaciens growing alone and growing in co-culture with methanogen are
compared, the production of hydrogen and carbon dioxide both appear to decrease in the
presence of methanogens. They do not in fact decrease they are present at reduced
concentrations because they are used as growth substrates by the methanogens.
The growth advantage to the ruminant of having methanogens present in the reticulo-rumen is
that the various syntrophic species of bacteria grow better so there is an increase in bacterial
mass. Remember it is largely bacterial biomass that is digested in the upper small intestine of
adult ruminants.
The organisms that can switch to acetate production when the partial pressure of hydrogen
decreases grow better because the ATP yield per molecule of glucose fermented is greater.
This is revealed from a study of the diagrams below.
The first of the following three diagrams shows fermentation in R. flavifaciens with glucose
being oxidised to form pyruvate, yielding ATP, and with NAD regeneration being possible by

two sequences; one that generates succinate and the other generating hydrogen due to
activity of a hydrogenase.
The hydrogenase is however hydrogen sensitive, it is inhibited by hydrogen. This means that
this enzyme is of reduced functionality when the hydrogen produced by the hydrogen
insensitive hydrogenase that regenerates oxidized ferredoxin used as an oxidizing agent in
pyruvate conversion to acetyl CoA is not removed by co-cultured methanogens.

If the hydrogen sensitive hydrogenase is not working then NAD regeneration must be mainly
by the succinate producing pathway. This means that succinate production is higher when
methanogens are in lower abundance.
The next diagram shows the redox reactions demonstrating the chemistry of succinate
production.

The third diagram shows the production of acetate as the other fate of pyruvate.
When R. flavifaciens is not co-cultured with methanogens there is production of some acetate
and some succinate because the hydrogen sensitive hydrogenase has low activity and NAD
regeneration is mainly by the succinate producing pathway.
With methanogens present the partial pressure of hydrogen in the reticulo-rumen decreases
and the hydrogen sensitive hydrogenase is released from inhibition and so can regenerate
NAD. This then obviates the need for the succinate producing pathway to do this and so
acetate production increases as more pyruvate is available for this pathway.
In additon note that this pathway allows Ruminococcus to grow better because ATP
production increases along with acetate production.

The decrease in succinate and increase in acetate production by R. flavifaciens when grown in
co-culture with methanogens has profound consequences for feed utilization in the host
ruminant.
We are now at a point where understanding the relationship between succinate producing and
succinate utilizing microorganisms becomes important for understanding food conversion
efficiency of the host ruminant.
The decrease in succinate production by Ruminococcus growing in the presence of
methanogens means that species like Selenomonas and Veillonella have less substrate
available for making propionate. You will remember that in these organisms propionate
production from succinate was linked to chemiosmotic mechanisms for ATP production by
anaerobic respiration. Lower succinate production by Ruminococcus means lower growth
rates of Selenomonas and Veillonella and thus less propionate for the host ruminant.
Since the host ruminant would normally use propionate extensively for gluconeogenesis lower
propionate availability means that more amino acids must be used for gluconeogenesis and

this means that protein production is compromised with a resultant reduction in feed
conversion efficiency for the host ruminant.
This is one of the reasons that primary producers seek to control methanogen growth. This
subject will be discussed in the section on Agribusiness interventions.