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 when CH 4 is the carbon cource bioremediation w Must be active in the presence of O 2

Methanotrophs in the environment O2O2 CH 4 Sediment CH 4 Methanogens CO 2 Water Aerobic / Anaerobic interface CO 2 CH 4 Methane oxidation

Methane oxidation by the pMMO complex DQH 2 Cu DQ O2O2 CH 4 CH 3 OH H2OH2O 26 kDa 45 kDa 25 kDa 618Da Cu 618Da Cu 618Da Cu 618Da Cu

The sMMO enzyme complex of Methylococcus capsulatus (Bath) CHOH + HO 2H + + CH+ O C Protein C       32 NADH NAD + 2H + + 2e - 54kDa 42kDa 17kDa 45kDa 42 H  Fe H  S S Protein A Protein B

Biotechnological potential of methanotrophs Bioremediation: Degradation of chlorinated hydrocarbons Bioconversion: Methanol production

Chemical methanol synthesis atm o C atm o C CH 4 +H 2 O  CO+H 2  CH 3 OH methanotrophic bacteria CH 4 +H 2 +O 2  Biological alternative Atmospheric pressure (1 atm) o C  CH 3 OH + H 2 O

CH 4 CH 3 OH NADH+H + NAD + HCOOHCO 2 MDH RuMP pathway Serine pathway Type II Methanotrophs Type I Methanotrophs FADH FDH pMMO H 2 CO X red X ox Utilization of methane NADH+H + NAD + sMMO O2O2 O2O2 H2OH2O H2OH2O

H 2 driven MMO activity %10%20%30%40%50% H 2 concentration in headspace Activity 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% H 2 Activity (nmoles propox formed / min*mg dry cell) 45°C 57°C

Hydrogenase activities in M. capsulatus (Bath) Membrane bound: methylene blue reducing uptake activity Activities are expressed in nmol H 2 min -1 (mg membrane protein) -1

(%) hupS hupLhupEhupChupD hupLhupChupDhupE hupShupLhupC hupD Methylococcus capsulatus (Bath) Thiocapsa roseopersicina Rhizobium leguminosarum 88/7588/7860/4468/4763/43 86/7489/8169/4867/48 (%) hupSLECD genes of M. capsulatus (Bath)

Hup type hydrogenase genes in methanotrophs Methylomicrobium album Methylomonas methanica Methylobacter luteus Methylocaldum szegediense Methylocystis parvus Marker Methylococcus capsulats Methylocystis sp. M Methylocaldum sp. LK5

Site directed mutagenesis of M. capsulatus Hup hydrogenase pJQ501SK Km R ‘hupL OriV OriT sacB Gm R hupS hupL hupEhupDhupC

H 2 production in methanotrophs H 2 evolution / OD 540 Nitrogenase repressedNitrogen fixing In vivo hydrogen evolution wild  hupSL

Summary: Characteristics of the MBH

NAD-dependent hydrogenase activity in M. capsulatus soluble fraction Activities are expressed as nmol H 2 min -1 (mg soluble protein) -1.

Conclusion Two distinct hydrogenase present in M. capsulatus (Bath)

BIOGAS

POLYMERS Acetate H 2 Acetate, Formiate Succinate Acetate, CO 2 Propionate Acetate H 2 + CO 2 CH 4 METHANOGENESIS

Biogas production from pig manure Inoculation Months Gas production

BIOGAS field experiments

A national Széchenyi project Renewable energy from waste K+F capacity Regional conditions Industrial needs

The project structure Biogas Termophilic fermenter Pig slurryEnergy plants Power plant Electricity Local use and transportation Natural gas residue Nanocomposit gas storage Waste heat Termoelectric devices

An integrated energy production system

BIOREMEDIATION

Hazardous waste w Chlorinated hydrocarbons inert anaerobic degradaation: vinyl chloride w Sulfonated aromatic compounds bactericide w Nitrate w Keratine food processing industry

Hazardous waste w Chlorinated hydrocarbons inert anaerobic degradation product: vinyl chloride w Sulfonated aromatic compounds bactericide w Nitrate w Keratine food processing industry

Hazardous waste w Chlorinated hydrocarbons inert anaerobic degradation product: vinyl choride w Sulfonated aromatic compounds bactericide w Nitrate w Keratin food processing industry

Denitrification Interspecies hydrogen transfer

Denitrification-2 NO 3 CO 2 + N 2 cellulose fiber Pseudomonas denitrificans Acetivibrio cellulolyticus immobilizing matrix

Hazardous waste w Chlorinated hydrocarbons inert anaerobic degradation product: vinyl chloride w Sulfonated aromatic compounds bactericide w Nitrate w Keratine food processing industry

Feather

KERATIN: pig hair