1. The Metabolism of Hydrogen Producing Bacteria Tõnu Malsub TTÜ, 2005

2. Subjects Introduction Metabolic pathways Research in our facility –Appr. 20 min.

3. Biological hydrogen production processes

6. Yields and production rates Extreme Thermophiles –Yield 83 – 100% (of the maximal theoretical value of 4 mol hydrogen/mol glucose) –Production rate low due to low densities Clostridia –Max 4 mol/mol, normally 2 mol/mol –23 mmol/(L*h) Enterobacter –Below 2 mol/mol wild strains, mutants 3 mol/mol –58 mmol/(L*h) - mutant Co- and Mixed cultures –2,6 mol/mol –50 mmol/(L*h) Our consortia, Residual Sludge as only substrate –Hydrogen: 0,01 mmol/(L*h) –Methane: 0,19 mmol/(L*h)

8. Substrates Residual Sludge from WWTP Brewery waste Milk industry waste Agro-industrial waste Yeast industry waste Paper industry waste Animal manure

9. Substrates Polysaccharides –Amylose –Amylopectin –Cellulose –Xylan Disaccharides –Sucrose –Lactose –Maltose –Cellobiose Monosaccharides –Glycose –Galactose –Fructose –Xylose Amino Acids –Alanine

10. Energia C 6 H 12 O 6 → 3CO 2 + 3CH 4 1 mooli CH 4 põletamisel saab 889,6 kJ 3 x 889,6 = 2668,8 kJ C 6 H 12 O 6 + 2H 2 O → 2CH 3 COOH + 4H 2 + 2CO 2 1 mooli H 2 põletamisel saab 284 kJ 4 x 284 = 1136 kJ Metaanist saab ~2,35 korda rohkem energiat

11. Calculated for Higher Heating Value (HHV) 1 mole of glucose yields either 12 moles of H 2 (3.4 MJ/mole) or 4 moles of H 2 and 2 moles of CH 4 gives (2,9 MJ/mole) or 3 moles of CH 4 (2.7 MJ/mole) or 2 moles of C 2 H 5 OH (2,0 MJ/mole)

15. End-products that can be formed Organic acids –Lactic acid –Formic acid –Acetic acid –Propionic acid –Butyric acid (Valeric acid) –Succinic acid –Fumaric acid –Malic acid –Capronic acid –Etc. Alcohols –Ethanol –Propanol –Butanol –2,3-Butanediol –Etc. Rest –Aceta-aldehyde –Diacetyl –Acetoin –Acetone –Etc.

16. 1Formic acidH-COOH 2Acetic acidCH3-COOH 3Oxalic acidHOOC-COOH 4Malonic acidHOOC-CH2-COOH 5Propionic acidCH3-CH2-COOH 6Pyruvic acidCH3-CO-COOH 7Lactic acidCH3-CH2O-COOH 8Butyric acidCH3-(CH2)2-COOH 9Isobutyric acid(CH3)2-CH-COOH 10Succinic acidHOOC-(CH2)2-COOH 11Fumaric acidHOOC-HC=CH-COOH 12Malic acidHOOC-CH2-CH2O-COOH 13Methylmalonic acid 14Oxalacetic acidHOOC-CO-CH2-COOH

17. 15alpha-Ketoglutaric acid HOOC-CO-(CH2)2- COOH 16Valeric acidCH3-(CH2)3-COOH 17Isovaleric acid(CH3)2-CH-CH2-COOH 18Caproic acidCH3-(CH2)4-COOH 192-Methylvaleric acid 204-Methylvaleric acid 21Heptanoic acidCH3-(CH2)5-COOH 22phenylacetic acid 23p-hydroxyphenylacetic acid 24Caprylic acidCH3-(CH2)6-COOH 253-p-phenylpropionic acid 263-(p-hydroxyphenyl)propionic acid 27Palmic acidCH3-(CH2)14-COOH 28Stearic acidCH3-(CH2)16-COOH

18. nrReaction∆Go'Comments 1Propionate + 2H2O > Acetate + 3H2 + CO2 24Propionate- + 12H2O > 4Acetate- + 4HCO3- + 4H+ + 12H2∆Go' = +304,6 kJ/mol reaction 3Propionate- + H+ + 3H2O > Acetate- + HCO3- + 2H+ + 3H2∆Go' = +76,1 kJ/mol 4Butyrate- + 2H2O > 2Acetate- + 2H2 52Butyrate- + 4H2O > 4Acetate- + H+ + 4H2∆Go' = +96,2 kJ/mol reaction 6Valerate- + 2H2O > Propionate- + Acetate- + 2H2 7Palmitate + 14H2O > 8Acetate + 14H2 8Glucose > 2CO2 +2H2 + butyrate- + H+∆Go' = -255 kJ/mol glucose*Biology of Prokaryotes 9Glucose > 2CO2 +4H2 + 2acetate- + 2H+∆Go' = -206 kJ/mol glucose 103Glucose > 2Acetate- + 2CO2 + 4Propionate- + 6H+∆Go' = -934 kJ/2mol acetatePropionibacterium 11Glycerol > Propionate- + H+ + H2O∆Go' = -149 kJ/molPropionibacterium 123Glycerol > 2Propane-3-diol + Acetate- + formate- + 2H+ + H2O 13Ethanol + H2O > Acetate- + H+ + 2H2∆Go' = +9,6 kJ/mol ethanolClostridium kluyveri 14Ethanol + Acetate- > Butyrate- + H2O∆Go' = -39 kJ/mol ethanolClostridium kluyveri

19. 152Ethanol + Acetate- > Caproate- + 2H2O∆Go' = -77 kJ/mol ethanolClostridium kluyveri 166Ethanol + 3Acetate- > 3butyrate- + Caproate- + H+ + 2H2 + 4H2O∆Go' = -183 kJ/mol H+Clostridium kluyveri 173Ethanol + 2Succinate2- > 2Butyrate- + 3Acetate- + H+ + H2O∆Go' = -165 kJ/mol H+Clostridium kluyveri 183Cystein + H2O > 3NH4+ + 3H2S + 2CO2 + 2Acetate- + Propionate-Clostridium propionicum 193Serine + 4H2O > 3NH4+ + 2CO2 +2Acetate- + Propionate-Clostridium propionicum 203Threonine + H2O > 3NH4+ + 2CO2 + 2Propionate- + Butyrate-Clostridium propionicum 21Threonine + H2O > (glycine + acetaaldehyde) > NH4+ + 2Acetate- + H+Clostridium pasteurianum 225Glutamate- + 6H2O +2H+ > 5NH4+ + 5CO2 + 6Acetate- + 2Butyrate- + H2∆Go' = -59 kJ/mol glutamateClostridium 234Formate- > CH4 + 3CO2 + 2H2OMethanosarcinales 24Acetate- + 4H2O > 2HCO3- + 4H2 + H+ 25Acetate- > CH4 + CO2Methanosarcinales 26CO2 > CH4Methanogens 274H2 + CO2 > CH4 + 2H2OMethanogens 28Pyruvate- + H2O > Acetate- + CO2 + H2 29Lactate- + H2O > Acetate- + CO2 + 2H2

22. Future plans Fermentations with Residual Sludge mixed with other wastes rich in carbohydrates Analyse microbial communities by using V3 region of 16S rDNA sequences using gel electrophoresis (DGGE) Determination chemical composition of residual sludge Fermentations on different temperatures 30 – 55 o C Monitor pH during fermentation Enrich fermentations with pure cultures and their combinations of E. coli, Enterobacter aerogenes, Clostridium acetobutylicum Explore the effect of extra nutrients (glucose, peptone)

23. Waste to Gold