METHANOTROPHS: MMO=methane monooxygenase + NAD+ reducing HaseNAD+ reductionwhen CH4 is the carbon courcebioremediation

Must be active in the presence of O2

Methanotrophs in the environment

Methane oxidation by the pMMO complex

The sMMO enzyme complex of Methylococcus capsulatus (Bath)

Biotechnological potential of methanotrophs

Bioremediation: Degradation of chlorinated hydrocarbons

Bioconversion: Methanol production

Chemical methanol synthesis

CH4CH3OHNADH+H+NAD+HCOOHCO2MDHRuMP pathwaySerine pathway Type II MethanotrophsType I MethanotrophsFADHFDHpMMOH2COXredXoxUtilization of methaneNADH+H+NAD+sMMOO2O2H2OH2O

H2 driven MMO activity140sMMO

Hydrogen driven MMO activities exhibit oxygen and heat tolerance

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 substrates

M.c.wild

type

Evolution

NADH

NADPH

BV+

MV+

MB

0

0

1.2

4.0

0

Uptake

Electron acceptor

NAD+

NADP+

BV2+

MV2+

MB2+

0

0

3.7

0.4

84.3

(%)hupSLECD genes of M. capsulatus (Bath)

Hup type hydrogenase genes in methanotrophsMethylomicrobium albumMethylomonas methanicaMethylobacter luteusMethylocaldum szegedienseMethylocystis parvusMarkerMarkerMethylococcus capsulats Methylocystis sp. MMethylocaldum sp. LK5

H2 production in methanotrophs

Summary: Characteristics of the MBH

MBH

SH

Localisation:

membrane

cytoplasm

Expression:

Constitutive

Constitutive

Gene family:

[Ni-Fe] type

???

H2 evolution from:

viologens (benzyl, methyl)

NADH,

viologens

methylene blue

H2 uptake with:

viologens

methylene blue

viologens

NAD+

Function:

H2-uptake under N2-ase repressed and N2-fixing condition

Not known yet

NAD-dependent hydrogenase activity in M. capsulatus soluble fractionActivities are expressed as nmol H2 min-1 (mg soluble protein)-1.

Activity

Strain

Reduced substrates

NADH

NADPH

BV+

MV+

MB

H2-producing

M.c. wild type

2.9

0

6.0

11.3

0.6

M.c.hup-mutant

2.4

0

4.7

15.3

0.8

H2-uptake

Electron acceptor

NAD+

NADP+

BV2+

MV2+

MB2+

M.c. wild type

25.5

0

9.5

1.7

17.5

M.c.hup-mutant

23.5

0

21.3

4.8

0

ConclusionTwo distinct hydrogenase present in M. capsulatus (Bath)

MBH

SH

Localisation:

membrane

cytoplasm

Expression:

Constitutive

Constitutive

Gene family:

[Ni-Fe]-type

???

H2 evolution from:

viologens (benzyl, methyl)

NADH,

viologens

methylene blue

H2 uptake with:

viologens

methylene blue

viologens

NAD+

Function:

H2-uptake under N2-ase repressed and N2-fixing condition

Not known yet

BIOGAS

METHANOGENESIS

Biogas production from pig manureInoculationMonthsGas production

Chart1

0.20.2

2.90.5

6.21

9.41.5

12.52.5

15.73.8

17.55.1

228.1

2611.3

3214.2

3816.8

4219.3

4521.7

Beolts

Hnapok

Gz termels

Biogz termels serts hgtrgybl

Biogaz1

Munka1

0.20.2

0.52.9

16.2

1.59.4

2.512.5

3.815.7

5.117.5

8.122

11.326

14.232

16.838

19.342

21.745

Munka1

Munka2

Beolts

Hnapok

Gz termels

Biogz termels serts hgtrgybl

Munka3

BIOGAS field experiments

Waste

Volume

(m3)

Biogas production

(%)

Cattle manure

0.01

180

Pig manure

0.01

250

Pig manure

1

220

Pig manure

15

200

Pig manure

250

220

Pig manure

10,000

180

Household solid

300

140

Waste water sludge

0.01

170

Waste water sludge

2,500

160

A national Szchenyi project Renewable energy from wasteK+F capacityRegional conditionsIndustrial needs

The project structure

An integrated energy production system

BIOREMEDIATION

Hazardous wasteChlorinated hydrocarbonsinertanaerobic degradaation: vinyl chlorideSulfonated aromatic compoundsbactericideNitrateKeratinefood processing industry

Hazardous wasteChlorinated hydrocarbonsinertanaerobic degradation product: vinyl chlorideSulfonated aromatic compoundsbactericideNitrateKeratinefood processing industry

Hazardous wasteChlorinated hydrocarbonsinertanaerobic degradation product: vinyl chorideSulfonated aromatic compoundsbactericideNitrateKeratinfood processing industry

DenitrificationInterspecies hydrogen transfer

Denitrification-2CO2 + N2cellulose fiberPseudomonas denitrificansAcetivibrio cellulolyticusimmobilizing matrix

Hazardous wasteChlorinated hydrocarbonsinertanaerobic degradation product: vinyl chlorideSulfonated aromatic compoundsbactericideNitrateKeratinefood processing industry

Feather

KERATIN: pig hair