METHANOTROPHS: MMO=methane monooxygenase + NAD + reducing Hase CH 4 NADH+H + O2O2 H2OH2O NAD + O2O2 H2OH2O X ox X red sMMO pMMO CH 3 OH w NAD + reduction.

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)

BIOGAS

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

An integrated energy production system

BIOREMEDIATION

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

Interspecies hydrogen transfer

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

Feather

KERATIN: pig hair