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METHANOTR OPHS : MMO=methane monooxygenase + N AD + reducing Hase

pMMO. sMMO. CH 4. O 2. O 2. X red. NADH+H +. X ox. NAD +. H 2 O. H 2 O. CH 3 OH. METHANOTR OPHS : MMO=methane monooxygenase + N AD + reducing Hase. NAD + reduction when CH 4 is the carbon cource bioremediation Must be active in the presence of O 2. O 2. Methane oxidation.

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METHANOTR OPHS : MMO=methane monooxygenase + N AD + reducing Hase

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  1. pMMO sMMO CH4 O2 O2 Xred NADH+H+ Xox NAD+ H2O H2O CH3OH METHANOTROPHS:MMO=methane monooxygenase + NAD+ reducing Hase • NAD+ reduction • when CH4 is the carbon cource • bioremediation • Must be active in the presence of O2

  2. O2 Methane oxidation Water CO2 CH4 Aerobic / Anaerobic interface Methanogens Sediment CO2 CH4 CH4 Methanotrophs in the environment

  3. 618Da Cu Cu Cu 618Da Cu Cu Cu CH4 DQH2 26 kDa O2 Cu H2O DQ CH3OH 45 kDa 25kDa 618Da Cu Cu Cu 618Da Cu Cu Cu Methane oxidation by the pMMO complex

  4. + 2H + CH + O CH OH + H O 4 2 3 2 Protein B 17kDa H a O g Fe Fe 17kDa 54kDa Protein A + + NAD + 2H - 42kDa 2e b b S NADH Fe Fe 42kDa S H a 45kDa O C Protein C Fe Fe g 54kDa 17kDa The sMMO enzyme complex of Methylococcus capsulatus (Bath)

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

  6. 1-25 atm 50-100 atm CH4+H2O CO+H2  CH3OH 700-900 oC 230-280 oC Biological alternative Atmospheric pressure (1 atm) 25-70 oC CH4+H2 +O2  CH3OH + H2O methanotrophic bacteria Chemical methanol synthesis

  7. Utilization of methane sMMO Type I Methanotrophs RuMP pathway H2O NADH+H+ NADH+H+ O2 NAD+ NAD+ FADH MDH FDH CH4 CH OH H2CO HCOOH CO 3 2 Xred Xox Serine pathway O2 H2O Type II Methanotrophs pMMO

  8. 360 160 320 280 120 240 100 Activity Aktivitás 200 pMMO 80 160 60 120 40 80 20 40 0% 10% 20% 30% 40% 50% 0% 10% 20% 30% 40% 50% H2 concentration in headspace H2 concnetration in headspace H2 driven MMO activity sMMO 140

  9. 120,00 100,00 80,00 45°C Activity (nmoles propox formed / min*mg dry cell) 60,00 57°C 40,00 20,00 0,00 50% H2 Hydrogen driven MMO activities exhibit oxygen and heat tolerance

  10. Hydrogenase activities in M. capsulatus (Bath) • Membrane bound: methylene blue reducing uptake activity Activities are expressed in nmol H2 min-1 (mgmembrane protein)-1

  11. Rhizobium leguminosarum hupS hupL hupC hupD hupE 88/75 88/78 60/44 68/47 63/43 Methylococcus capsulatus (Bath) hupS hupL hupE hupC hupD Thiocapsa roseopersicina hupS hupL hupC hupD (%) 86/74 89/81 69/48 67/48 hupSLECD genes of M. capsulatus (Bath) (%)

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

  13. Site directed mutagenesis of M. capsulatus Hup hydrogenase sacB OriT GmR pJQ501SK OriV ‘hupL KmR hupE hupC hupD hupS hupL

  14. wild In vivo hydrogen evolution  hupSL 444 500 400 H2 evolution / OD540 300 64 200 46 11 100 0 Nitrogenase repressed Nitrogen fixing H2 production in methanotrophs

  15. Summary: Characteristics of the MBH

  16. NAD-dependent hydrogenase activity in M. capsulatus soluble fraction Activities are expressed as nmol H2 min-1 (mgsoluble protein)-1.

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

  18. BIOGAS

  19. POLYMERS Acetate, Formiate Succinate Acetate, CO2 Propionate Acetate H2 Acetate H2 + CO2 + CO2 CH4 METHANOGENESIS

  20. Biogas production from pig manure Inoculation Gas production Months

  21. BIOGAS field experiments

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

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

  24. An integrated energy production system

  25. BIOREMEDIATION

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

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

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

  29. Denitrification Interspecies hydrogen transfer

  30. NO3 Denitrification-2 CO2 + N2 Pseudomonas denitrificans Acetivibrio cellulolyticus cellulose fiber immobilizing matrix

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

  32. Feather

  33. KERATIN: pig hair

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