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METHANOTROPHS: MMO=methane monooxygenase
+ NAD+ reducing Hase
CH4
NADH+H+
O2
H2O
NAD+
O2
H2O
Xox
Xred
sMMOpMMO
CH3OH
NAD+ reduction• when CH4 is the
carbon cource
• bioremediation
Must be active in the presence of O2
Methanotrophs in the environment
O2
CH4
Sediment CH4
Methanogens
CO2
Water
Aerobic / Anaerobic interface
CO2 CH4
Methane oxidation
Methane oxidation by the pMMO complex
DQH2
Cu
DQ
O2
CH4
CH3OH
H2O
26 kDa
45 kDa
25 kDa
618DaCu Cu Cu
618DaCu Cu Cu
618DaCu Cu Cu
618DaCu Cu Cu
The sMMO enzyme complex of Methylococcus capsulatus (Bath)
CH OH + H O2H+ + CH + O
C Protein C
3 2
NADH
NAD + 2H+ +
2e -54kDa
54kDa
42kDa
42kDa
17kDa
17kDa
17kDa
45kDa
4 2
H
FeFe
H
FeFe
Fe Fe
S
S
Protein A
Protein B
Biotechnological potential of methanotrophs
• Bioremediation: Degradation of chlorinated hydrocarbons
• Bioconversion: Methanol production
Chemical methanol synthesis
50-100atm
230-280 oC
1-25
atm700-900 oC
CH4+H2O CO+H2 CH3OH
methanotrophic bacteria
CH4+H2 +O2
Biological alternative
Atmospheric pressure(1 atm)
25-70 oC
CH3OH + H2O
CH4CH 3OH
NA
DH
+H
+
NA
D+
HCOOH CO 2
MDH
RuMP pathway
Serine pathway
Type II Methanotrophs
Type I Methanotrophs
FADH FDH
pMMO
H2CO
Xre
d
Xox
Utilization of methane
NA
DH
+H
+
NA
D+
sMMO
O2
O2
H2O
H2O
H2 driven MMO activity
20
40
60
80
100
120
160
0% 10% 20% 30% 40% 50%A
kti
vitá
s
H2 concnetration in headspace
140
40
80120
160
200240
280320
360
0% 10% 20% 30% 40% 50% H2 concentration in headspace
Act
ivit
y
pMMO sMMO
Hydrogen driven MMO activities exhibit oxygen and heat tolerance
0,00
20,00
40,00
60,00
80,00
100,00
120,00
50% H2
Act
ivit
y (n
mol
es p
ropo
x fo
rmed
/ m
in*m
g dr
y ce
ll)
45°C57°C
ActivitiesOxygenconcentration(%)
pMMO sMMO
0 10 65 34 2410 86 4015 123 62
Hydrogenase activities in M. capsulatus (Bath)
• Membrane bound: methylene blue reducing uptake activity
Activities are expressed in nmol H2 min-1 (mg membrane protein)-1
Strain Activity Reduced substratesNADH NADPH BV+ MV+ MBEvolution0 0 1.2 4.0 0Electron acceptorNAD+ NADP+ BV2+ MV2+ MB2+
M.c.wildtype
Uptake
0 0 3.7 0.4 84.3
(%)
hupS
hupS
hupL hupE hupC hupD
hupL hupC hupD hupE
hupS hupL hupC hupD
Methylococcus capsulatus (Bath)
Thiocapsa roseopersicina
Rhizobium leguminosarum
88/75 88/78 60/44 68/47 63/43
86/74 89/81 69/48 67/48 (%)
hupSLECD genes of M. capsulatus (Bath)
Hup type hydrogenase genes in methanotrophs
Met
hyl
omic
robi
um
alb
um
Met
hyl
omon
as m
eth
anic
a
Met
hyl
obac
ter
lute
us
Met
hyl
ocal
dum
sze
gedi
ense
Met
hyl
ocys
tis
parv
us
Mar
ker
Mar
ker
Met
hyl
ococ
cus
caps
ula
ts
Met
hyl
ocys
tis
sp. M
Met
hyl
ocal
dum
sp.
L
K5
Site directed mutagenesis of M. capsulatus Hup hydrogenase
pJQ501SK
KmR‘hupL
OriV
OriTsacB
GmR
hupS hupL hupE hupDhupC
H2 production in methanotrophs
11 46 64
444
0
100
200
300
400
500
H2
evol
utio
n /
OD
540
Nitrogenase repressed Nitrogen fixing
In vivo hydrogen evolutionwild
hupSL
Summary: Characteristics of the MBH
MBH SHLocalisation: membrane cytoplasm
Expression: Constitutive ConstitutiveGene family: [Ni-Fe] type ???H2 evolution from: viologens (benzyl,
methyl)NADH,viologensmethylene blue
H2 uptake with: viologensmethylene blue
viologensNAD+
Function: H2-uptake under N2-aserepressed and N2-fixingcondition
Not known yet
NAD-dependent hydrogenase activity in M. capsulatus soluble fraction
Activities are expressed as nmol H2 min-1 (mg soluble protein)-1.
Reduced substratesActivity StrainNADH NADPH BV+ MV+ MB
M.c. wild type 2.9 0 6.0 11.3 0.6H2-producingM.c.hup-mutant 2.4 0 4.7 15.3 0.8
Electron acceptorNAD+ NADP+ BV2+ MV2+ MB2+
M.c. wild type 25.5 0 9.5 1.7 17.5
H2-uptake
M.c.hup-mutant 23.5 0 21.3 4.8 0
MBH SHLocalisation: membrane cytoplasm
Expression: Constitutive ConstitutiveGene family: [Ni-Fe]-type ???H2 evolution from: viologens (benzyl,
methyl)NADH,viologensmethylene blue
H2 uptake with: viologensmethylene blue
viologensNAD+
Function: H2-uptake under N2-aserepressed and N2-fixingcondition
Not known yet
Conclusion
Two distinct hydrogenase present in M. capsulatus (Bath)
POLYMERS
AcetateH2
Acetate, FormiateSuccinate
Acetate, CO2
Propionate
AcetateH2
+ CO2 + CO2
CH4
METHANOGENESIS
1 2 3 4 5 6 7 8 9 10 11 12 1305
1015202530354045G
áz te
rmel
és
Hónapok
Biogáz termelés sertés hígtrágyából
Beoltás
Biogas production from pig manure
Inoculation
Months
Gas
pro
du
ctio
n
BIOGAS field experiments
Waste Volume(m3)
Biogas production(%)
Cattle manure 0.01 180
Pig manure 0.01 250Pig manure 1 220Pig manure 15 200Pig manure 250 220Pig manure 10,000 180
Household solid 300 140Waste water sludge 0.01 170Waste water sludge 2,500 160
A national Széchenyi project
Renewable energy from
wasteK+F capacity
Regional conditions
Industrial needs
The project structure
Biogas
Termophilic fermenter
Pig slurry Energy plants
Power plant
ElectricityLocal use and transportation
Natural gas residue
Nanocomposit gas storage
Waste heat
Termoelectric devices
Hazardous waste
Chlorinated hydrocarbons• inert• anaerobic degradaation: vinyl chloride
Sulfonated aromatic compounds• bactericide
Nitrate Keratine
• food processing industry
Hazardous waste
Chlorinated hydrocarbons• inert• anaerobic degradation product: vinyl chloride
Sulfonated aromatic compounds• bactericide
Nitrate Keratine
• food processing industry
Hazardous waste
Chlorinated hydrocarbons• inert• anaerobic degradation product: vinyl choride
Sulfonated aromatic compounds• bactericide
Nitrate Keratin
• food processing industry
Denitrification-2
NO3CO2 + N2
cellulose fiber
Pseudomonas denitrificans
Acetivibrio cellulolyticus
immobilizing matrix
Hazardous waste
Chlorinated hydrocarbons• inert• anaerobic degradation product: vinyl chloride
Sulfonated aromatic compounds• bactericide
Nitrate Keratine
• food processing industry