1. Methylotrophy
• Catabolic utilization of Methane (CH4) and many other C1 compounds
• Methylotrophs are organisms that use organic compounds that lack
C—C bonds as electron donors and carbon sources
• Methanotrophy is part of methylotrophy
• Methanotrophic organisms utilize methane
• Methane is found extensively in nature.
• It is produced in anoxic environments by methanogenic Archaea and is
a major gas of anoxic muds, marshes, anoxic zones of lakes, the rumen,
and the mammalian intestinal tract.
• Methane is the major constituent of “natural gas” widely used as a
heating and industrial fuel, and is also present in many coal formations.
2. • Methanotrophs assimilate either all or one-half of their carbon
(depending on the pathway used) at the oxidation state of formaldehyde
(CH2O).
• This affords a major energy savings compared with the carbon
assimilation of autotrophs, which also assimilate C1 units, but exclusively
from CO2 rather than organic compounds.
3. • Aerobic oxidation of Ch4 is carried out by enzyme MMO (Methane
monooxygenase)
• monooxygenases catalyze the incorporation of oxygen atoms from O2
into carbon compounds (and into some nitrogen compounds)
• NADH is probably the electron donor for the MMO
Reactions and bioenergetics
4. Oxidation of methane by methanotrophic bacteria. CH4 is oxidized to CH3OH by the enzyme methane
monooxygenase (MMO). A proton motive force is established from electron flow in the membrane, and this fuels
ATPase. Note how carbon for biosynthesis comes from CH2O. Although not depicted as such, MMO is actually a
membrane-associated enzyme and methanol dehydrogenase is periplasmic. FP, flavoprotein; cyt, cytochrome; Q,
quinone; MP, methanopterin.
5. • In the MMO reaction, an atom of oxygen is introduced into CH4, and
CH3OH and H2O are the products. Reducing power for the first step
comes from later oxidative steps in the pathway.
• CH3OH is oxidized by a periplasmic dehydrogenase, yielding formaldehyde
and NADH.
• Once CH2O is formed, it is oxidized to CO2 by either of two different
pathways.
1- pathway using enzymes that contain the THF, a coenzyme widely
involved in C1 transformations.
2- pathway employing the coenzyme methanopterin
• However, regardless of the CH2O oxidation pathway employed, e- from
the oxidation of CH2O enter the ETC, generating a PMF from which ATP is
synthesized
6. C1 Assimilation into Cell Material
• two distinct pathways for C1 incorporation into cell material exist.
1- serine pathway
2-ribulose monophosphate pathway
7. 1- Serine pathway
• utilized by type II methanotrophs,
• In this pathway, a two-carbon unit, acetyl-CoA, is synthesized from
one molecule of CH2O (produced from the oxidation of CH3OH, and one
molecule of CO2.
• requires reducing power and energy in the form of two molecules each of
NADH and ATP, respectively, for each acetyl-CoA synthesized.
• employs a number of enzymes of the citric acid cycle and one enzyme,
serine transhydroxymethylase, unique to the pathway
8. The serine pathway for the
assimilation of C1 units
into cell material by methylotrophic
bacteria.
The product of the pathway,
acetyl-CoA, is used as the starting
point for making new cell material.
The key enzyme of the pathway is
serine transhydroxymethylase.
9. 2-ribulose monophosphate pathway
• used by type I methanotrophs,
• pathway is more efficient than the serine pathway because all of the
carbon for cell material is derived from CH2O.
• And, because CH2O is at the same oxidation level as cell material, no
reducing power is needed.
• requires one molecule of ATP for each molecule of glyceraldehyde 3-
phosphate (G-3-P) synthesized
• Two G-3-Ps can be converted into glucose by the glycolytic pathway.
• Due to lower energy requirements of this pathway, the cell yield (grams of
cells produced per mole of CH4 oxidized) of type I methanotrophs is higher
than for type II methanotrophs.
10. The ribulose monophosphate
pathway for assimilation
of C1 units by methylotrophic
bacteria.
Three molecules of CH2O
are needed to complete the
cycle, with the net result
being one molecule
of glyceraldehyde 3-
phosphate.
The key enzyme of this
pathway is hexulose
P-synthase.
The sugar rearrangements
require enzymes of the
pentose
phosphate pathway
11. Methanotrophs
• Methanotrophs oxidize methane and a few other one-carbon compounds as
electron donors in energy metabolism and as carbon sources.
• grow aerobically and are widespread in soils and waters.
• exhibit diverse morphologies but are related in terms of their phylogeny and
ecology.
• In marine sediments and a few other anoxic environments, methane is
oxidized
• under strictly anoxic conditions by a consortium of methanogenic Archaea
and sulfate-reducing bacteria.
12. • Methane-oxidizing bacteria are virtually unique among bacteria in
possessing relatively large amounts of sterols.
• Sterols are rigid planar molecules found in the cytoplasmic and other
membranes of eukaryotes but are absent from most bacteria.
• Sterols may be an essential part of the complex internal membrane
system for methane oxidation.
• The only other group of bacteria in which sterols are widely
distributed is in the cell wall–less mycoplasmas
13. Classification of methanotrophs
• These bacteria were initially distinguished on the basis of morphology
• and formation of resting stages. However, now they are
• classified into two major groups based on their internal cell structure,
phylogeny, and carbon assimilation pathway
14. Type I methanotrophs assimilate one-carbon compounds via the ribulose monophosphate cycle and are
phylogenetically Gammaproteobacteria.
By contrast, type II methanotrophs assimilate C1 intermediates via the serine pathway and are phylogenetically
Alphaproteobacteria.
15. • Both groups of methanotrophs contain extensive internal membrane systems
for methane oxidation.
• Membranes in type I methanotrophs are arranged as bundles of disc-shaped
vesicles distributed throughout the cell.
• Type II species possess paired membranes running along the periphery of the
cell.
• The key enzyme methane monooxygenase is located in these membranes.
16. Ecology
• Methanotrophs are widespread in aquatic and terrestrial environments, being
found wherever stable sources of CH4 are present.
• Methane produced in the anoxic regions of lakes rises through the water
column, and methanotrophs are often concentrated in a narrow band at the
zone where CH4 and O2 meet.
• Methane-oxidizing bacteria therefore play an important role in the carbon cycle,
converting CH4 derived from anoxic decomposition back into cell material and
CO2.
17. • Methanotrophic bacteria and certain marine mussels and sponges have
developed symbiotic relationships.
• Some marine mussels live in the vicinity of hydrocarbon seeps on the seafloor,
places where CH4 is released in substantial amounts.
• Isolated mussel gill tissues consume CH4 at high rates in the presence of O2.
• In these tissues, coccoid-shaped bacteria are present in high numbers.
• The symbionts reside in vacuoles within animal cells near the gill surface,
which probably ensures an effective gas exchange with seawater,
• Assimilated CH4 is distributed throughout the animal by the excretion of
organic compounds by the methanotrophs.
18. Isolation
• All that is needed to enrich methanotrophs is a mineral salts medium
containing a headspace about 50% each of CH4 and air.
• Colonies appearing on the plates are typically of two types:
1- nonmethanotrophic chemoorganotrophs growing on traces of organic matter
in the medium, which appear in 1–2 days, and methanotrophs, which appear
after about a week.
2- The colonies of some methanotrophs are pink from the presence of various
carotenoid pigments and high levels of cytochromes in their membranes, and
this feature can assist in identifying these organisms on plates.
19. Methanotrophs and Ammonia-Oxidizing Bacteria
• Besides CH4, methanotrophs can also oxidize ammonia (NH3), and ammonia-
oxidizing bacteria can also oxidize CH4; however, neither group can actually
grow using the other group’s substrate as electron donor.
• It has been hypothesized that methanotrophic bacteria evolved from ammonia-
oxidizing bacteria via selection for the conversion of an ammonia
monooxygenase into a methane monooxygenase.
• The fact that methanotrophs and nitrifiers have similar internal membrane
systems and are phylogenetically closely related supports this theory.
• However, it has also been found that methanotrophic bacteria contain some of
the same genes and make some of the same proteins as methanogenic Archaea,
and so the evolution of methanotrophy is still very much an open question
