ATP Production

Ruminal fermentation is an exergonic process the converts

carbohydrates and other substrates to partially oxidized
fermentation end-products, and some of the change in free-energy
is trapped as ATP.

This ATP is then used to drive anabolic reactions (bacterial

biomass formation) and maintain vital cell functions.
NH3

Substrates Amino
Acids

Catabolism q ATP Anabolism µ

Products Cells

© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

1
Introductory textbooks of biology often describe ATP as the ‘energy currency’ of
the cell. The idea that ATP is an 'energy rich' compound is a valuable teaching
tool, but it is not entirely correct.

In 1982, Nichols corrected this misconception. "It is frequently, and

misleadingly, supposed that the phosphate anhydride bonds of ATP are 'high
energy' bonds which are capable of storing energy and driving reactions in
otherwise unfavorable directions.”

“The extent to which the observed mass action ratio is displaced from
equilibrium is the factor that defines the capacity of the reactants to do work,
rather than the attribute of a single component."

ATP ---> ADP + Pi (pH 7.0, 10 mM Mg++)

Keq = [ADP] [Pi] ÷ [ATP] = 105 M

If cellular [ADP] and [Pi] = 10-2 M, [ATP] would be 10-9 M

However, bacteria normally have 10-2 M ATP (not 105 M),

so the equilibrium is displaced nearly 7 orders
2
 Hexose Catabolism
CO2

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The Embden-Meyerhof-Parnas (EMP) Many aerobes have the Entner-

pathway is the most common pathway of Doudoroff (ED) pathway.
hexose metabolism.
3
 Pentose Catabolism

Pentoses can be metabolized by the pentose pathway or via the enzyme phosphoketolase.

The ATP yield of the pentose cycle is greater than the pathway involving phosphoketolase.

Labeling studies indicated that 75% of the xylan was fermented by the pentose pathway
while 25% would have been fermented via phosphoketolase.

4
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.
 Pyruvate Catabolism

Pyruvate Lactate
Some bacteria convert pyruvate that
COOH NADH NAD COOH
arising from the EMP pathway to
C=O H-C-OH
lactate, but most of the pyruvate is
CH 3 Lactate CH3
converted to acetyl CoA. Dehydrogenase

Clostridia and selenomonads have a Pyruvate Acetyl CoA

pyruvate-ferredoxin oxidoreductase COOH FD FDH S-CoA
that produces acetyl CoA reduced C=O C=O + CO 2
ferredoxin and CO2 rather than Pyruvate
CH3 Ferredoxin CH 3
formate. Oxidoreductase
COOH S-CoA
E. coli, S. bovis and butyrivibrios C=O C=O + HCOOH
have pyruvate-formate lyases that Pyruvate
CH 3 Formate CH Formate
3
produce acetyl CoA and formate. Lyase
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

5
 Acetate S-CoA
C=O Acetyl CoA
Acetyl CoA arising from pyruvate
CH3
metabolism can be converted to
Pi Phospho-
acetate.
trans-
acetylase
If the bacteria have phosphotrans-
P
acetylase, energy of the CoA bond is
conserved as a phosphate ester C=O Acetyl P
(acetyl phosphate). CH3 +CoASH
ADP
Acetate
Acetyl phosphate can then be ATP Kinase
converted to acetate by acetate
kinase, and ATP is produced. COOH
Acetate
CH 3
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

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 Butyrate
Alternatively, two acetyl CoA molecules can be
condensed to produce acetyl-acetyl CoA.

The acetyl-acetyl CoA can then be converted to

butyryl CoA by a reversal of -oxidation, and this
scheme allows the bacteria to dispose of excess
reducing equivalents (e.g., NADH or NADPH).

Butyrivibrios are the most important butyrate-

producing bacteria in the rumen, but they have two
different schemes of butyrate production.

Some species convert butyryl CoA to butyryl

phosphate and use a butyrate kinase to obtain more
ATP directly (see arrows in red).

Other species lack butyrate kinase and use a

butyryl CoA acetyl CoA transferase. Because
acetate is ultimately converted to additional acetyl
CoA, phosphotrans-acetylase and acetate kinase
drive additional ATP formation (see arrows in
yellow). 7
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.
 Propionate and Succinate

There were no obvious sites of substrate

level phosphorylation, and the
carboxylation and formation of succinyl
CoA could have caused a loss of ATP.

However:

1) OAA synthesis is catalyzed by a biotin-

dependent, transcarboxylation reaction
that conserved the energy of succinyl CoA
decarboxylation (red box).

1) Propionyl CoA transferase conserves the

free energy to drive the synthesis of
succinyl CoA (purple box)

2) cytochrome-linked fumarate reductase (P.

ruminicola) produces ATP via electron
transport (green box).

8
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.
 Glucose L-Lactate
Propionate via Direct Pathway
The 2 pathways of propionate metabolism
can be differentiated by 14C labeling. D-Lactate Lactyl CoA

ETF
When [2-14C] glucose is metabolized by the
EMP pathway, the label is found in the ETF-H
ADP + Pi
number two position of pyruvate. ?
------- Pyruvate ATP

If pyruvate (or PEP) is metabolized by the FDH ETF-H

'randomizing pathway,' the intermediates CO
2 FD ETF
(fumarate and succinate) are symmetrical
molecules, and the label is seen in the Acetyl CoA Acrylyl CoA
second and third positions of propionate. CoA

When [2-14C] glucose is catabolized by the Acetyl Phosphate Propionyl CoA

direct reductive pathway, the label only ATP

shows up in the second carbon of ADP + Pi CoA

propionate.
Acetate Propionate

9
 Propionate
Butyrate

Valerate
Ethanol
Lactate
Acetate
Enzyme
Higher volatile fatty acids
(valeric, caproic, etc.) are
formed by the condensation of Glucokinase -1 -1 -1 -1 -1 -1
acetyl-CoA and/or propionyl- Phosphofructokinase -1 -1 -1 -1 -1 -1
CoA. Glycertate kinase 2 2 2 2 2 2
Pyruvate kinase 2 2 2 2 2 2
Acetate kinase - 2 - - - -
Fumarate reductase - - 2 - - -
Basic Principle:
Butyrate kinase - - - 1 - -
Total (~ P) 2 4 4 3 2 -
Bacteria will sacrifice ATP to
dispose of reducing equivalents G-3-P dehydrogenase 2 2 2 2 2 2
Lactate dehydrogenase -2 - - - - -
and vice versa!!!!!
Pyruvate oxidoreductase - 2 - 2 2 1
Alcohol dehydrogenase - - - - -4 -
Malate dehydrogenase - - -2 - - -1
Fumarate reductase - - -2 - - -1
-OH butyrate dehydrogenase - - - -1 - -
Butyryl CoA dehydrogenase - - - -1 - -
-OH valerate dehydrogenase - - - - - -1
Valeryl CoA dehydrogenase - - - - - 10-1
Total (2H) 0 4 -2 2 0 -1
 Na+ Na+
Histidine Histidine NH 3

Na+ Na+
Glutamine Glutamine
NH 3
Amino acid fermentations yield little Na+ Na+
Glutamate
ATP, and obligate amino acid Glutamate  Ketoglutarate

fermenting ruminal bacteria methods if Glutaconyl CoA

Na+ Na+
conserving ATP needed to take up Crotonyl CoA Acetate + Butyrate
+ CO
amino acids. 2
ADP
+ Pi ATP

C. aminophilum
This can be done by decarboxylases
Out In
that extrude sodium, product/sodium
Arginine Arginine
efflux or by the use of facilitated ADP+Pi
Na+ Na+
diffusion. ATP

Na+ Na+ 2NH 3

Ornithine Ornithine 2NH
3
ADP+Pi

Arginine Arginine ATP

C. sticklandii
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002. 11
 NH CO2
3
Valine  Ketoisovalerate Isobutyrate
Branched chain amino acids 2[H] 2[H]
can be converted to branched NH CO2
3
chain VFA that are then Leucine  Ketoisocaproate Isovalerate
utilized by cellulolytics. 2[H] 2[H]
NH CO2
3
Isoleucine  Ketomethylvalerate 2-Methylbutyrate
The reducing equivalents must
2[H] 2[H]
then be utilized by
methanogens. 4[H] 2H 2 CH4
CO2

© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

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 Out In
Until the 1990's, the energetics of CO + MF + H 2
+ 2
methane production were not well Na
Formyl-MF
understood.
2H 2
4e-
The initial steps of CH4 formation 4 H+
have low or even positive free Methylene-H MPT
4
2 H+ +CoMSH
energy changes, and only the last Na
+ 2e-

step, methanol reduction has a CH -S-CoM

3
free energy change (-26.9 kcal) +H 4MPT
sufficient to drive ATP formation. 4 H+

2e- 2 H+
However, the free energy change CH 4 + CoM-SH
ATP
of the overall process is very
H + or Na+
negative (-32.4 kcal), and ATP can ADP + Pi
+
Na
be produced by a chemiosmotic
mechanism. H+

© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

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 Redox, Fermentation Balances,
and Interspecies Hydrogen Transfer
Compound Formula Redox
If oxygen is not available as a
terminal electron acceptor, Valerate C5H10O2 -3
oxidations must be Methane CH4 -2
Ethanol C2H6O -2
simultaneously coupled with
Butyrate C4H8O2 -2
reductions. Propionate C3H6O2 -1
Acetate C2H4O2 0
Oxidation-reduction state (redox) Water H20 0
is determined arbitrarily by Glucose C6H12O6 0
Lactate C3H6O3 0
assigning each [H] in the Formate CH2O2 +1
compound a value of -1/2 and Carbon dioxide CO2 +2
each [O] a value of +1. Succinate C4H6O4 +1
Tricarballylate C6H8O6 +2
Aconitate C6H10O6 +3
As a consequence, highly reduced Citrate C6H8O7 +3
compounds have negative values
while highly oxidized compounds Cells C4.44 H8.88 O2.35 -1.88
have positive values.
© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

14
Redox is illustrated by the
conversion of glucose to lactate.
Glucose Lactate
Glucose (C6H12O6) and lactate
(C3H6O3) have the same overall H-C-OH CH
3
oxidation-reduction state,
H-C-OH H-C-OH
Where does the energy come HO-C-H O COOH
from?
H-C-OH COOH
The molecules have a different H-C H-C-OH
distribution of redox.
CH 2OH CH 3
All of the carbon atoms in
C H O C H O
glucose have a neutral 6 12 6 3 6 3
oxidation-reduction state. © Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

The methyl carbon of lactate is Redox = 0 Redox = 0

highly reduced while the
carboxyl carbon is highly
oxidized.
15
 Selenomonas ruminantium

Glucose -----> Propionate + Acetate + Formate + Water

C6H12O6 -------> 1.0 C3H6O2 + 1.0 C2H4O2 + 1.0 CH2O2 + 1.0 H2O

6 C --------> 3C + 2 C + 1C
12 H ------> 6H + 4H+2H+ 2H
6 O ---------> 2O +1O+2O+1O

1(0) --------> 1(-1) + 1(0) + 1 (+1) + 1(0)

4 ATP----> 2 from EMP, 1 from fumarate reductase, 1 from acetate kinase

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 Hydrogenase Activity
Non-methanogenic bacteria have
membrane bound hydrogenases to
produce H2 from reduced electron
carriers, but this process can be
thermodynamically unfavorable.

Hydrogenases linked to NADH can be

inhibited by even small amounts of
hydrogen.

Methanogens scavenge H2 and keep

the partial pressure of H2 low enough
©
so the NADH-linked hydrogenases Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.

can operate.

17
 Interspecies Hydrogen Transfer
When R. albus is grown in pure culture, the hydrogenase is unable to oxidize
NADH. Alcohol production provides an alternative method of reducing
equivalent disposal, but the ATP yield is lower.

Pure Culture With a Methanogen

© Rumen Microbiology and Its Role In Ruminant Nutrition. 2002. © Rumen Microbiology and Its Role In Ruminant Nutrition. 2002.
18
 Crossfeeding
Cellulose Proteins
Pure cultures of ruminal bacteria
produce products not detected in
ruminal fluid.
Peptides,
The absence of some products (e.g., NH3 Amino Acids
ethanol) could be explained by the
inability of the pure cultures to
transfer H2 to methanogens (last slide).
Celluloytics
In other cases, the end-product of one HAB
species is an essential nutrient for
another.

Amino acid fermentation is generally

considered a wasteful process, but it Branched Chain VFA
produces branched chain volatile fatty
acids and ammonia. Both of these © James B . Russell. 2005.

products are needed by cellulolytic

bacteria.
19
 Crossfeeding
Starch, Sugar, Cellulose
The end-product of one species
Cellulose
is a substrate for another.
Starch, Sugar
Succinate is taken up and
converted to propionate by Celluloltyics
Selenomonas ruminantium. Succinate
Succinate
Producers
Producers Amylolytics
Lactate can be used by other
bacteria such as Megasphaera
elsdenii and Selenomonas Cellodextrins
ruminantium. Succinate
Lactate

Cellulolytics secrete cello-

dextrins but this carbohydrate
can be used by non- Propionate Non-
Lactate Users
cellulolytics. Producers Cellulolytics

© James B . Russell. 2005. 20

 Crossfeeding
Starch
Extracellular enzymes can
provide other bacteria with the
product.
Maltose Sugar
For example, bacteria that Fermenters
produce an amylase and
degrade starch release maltose
to bacteria that cannot degrade Peptides,
Amylolytics HAB
starch. Amino
Acids
Bacteria that are proteolytic,
release peptides and amino
acids to bacteria that do not Proteins Proteolytic
have proteinases including Bacteria
(HAB) hyper-ammonia © James B . Russell. 2005.

producing bacteria.

21
Starch & Sugar Cellulose Proteins Starch, Sugar,
Cellulose
Lactate H2

Sugar Peptides,
Maltose Amino Acids
Fermenters Succinate
NH3
Producers
NH3

Peptides, Cellulolytics
Amylolytics Amino HAB
Succinate
Acids

Lactate H2
Propionate
NH3 Cellodextrins Branched Chain VFA Producers

Lactate Users H2 Methanogens CH4

© James B . Russell. 2005.

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