16. MICROBIOLOGY AND BIOCHEMISTRY OF BIOGAS PRODUCTION.ppt

1. MICROBIOLOGY AND BIOCHEMISTRY OF BIOMETHANATION OF BIOGAS PRODUCTION

2. • Anaerobic digestion is a series of processes in which microorganisms break down biodegradable material in the absence of oxygen, used for industrial or domestic purposes to manage waste and/or to release energy which is also called as anaerobic digestion or biogasification • Ecosystem - several group of microorganisms works • Converts complex organic matter products such as methane, carbon dioxide, hydrogen sulfide, water vapour and ammonia Biomethanation 2

3. STAGES • Hydrolysis • Acidogenesis • Acetogenesis • Methanogenesis

5. • Subdivided in to various metabolic pathways with the participation of several microbial groups Biomethanation Process 1. Hydrolysis 2. Acidogenesis 3. Acetogenesis 4. Methanogenesis 5

6. • Solubilisation of insoluble particles and biological decomposition of organic polymers to monomers or dimers • Usually carried out by extracellular enzymes • Hydrolysis of a complex, insoluble substrate depends on different parameters such as; Particle size, pH, production of enzymes and diffusion and adsorption of enzymes to particles • Optimum pH = 6.5-7.5 Hydrolysis 6

7. Hydrolysis 7

8. ANAEROBIC BREAKDOWN OF COMPLEX ORGANIC MATTER In the first stage of hydrolysis, or liquefaction, hydrolytic bacteria convert the complex polymers to their respective monomers. For example, celluloses are converted to sugars or alcohols, proteins to peptides or amino acids and lipids to fatty acids. This is carried out by several hydrolytic enzymes (cellulases, amylases, lipases, proteases) secreted by microbes

9. • Decompose complex organic polymers (proteins, polysaccharides and lipids) into soluble organic monomers (sugars, organic acids, amino acids, etc) dS/dt = -Kh * S S – Concentration of insoluble substrate Kh – first order hydrolysis rate constant t - time Hydrolysis 9

10. Substrate Hydrolysis rate day-1 Carbohydrates 0.025 – 0.200 Cellulose 0.040 – 0.130 Proteins 0.015 – 0.075 Lipids 0.005 – 0.010 Hydrolysis 10

11. Soluble compounds can pass through the cell walls of the fermentative bacteria Hydrolysis 11

12. • Cellulose is hydrolysed by cellulase (mixture of endo- cellulases, exocellulases and cellobiases) Hydrolysis : Cellulose 12

13. • Protein - polypeptides - peptides - amino acids • Proteinases (Peptidases + Proteases) Hydrolysis : Proteins 13

14. • Most lipids in waste (water) are present as triacylglycerides Hydrolysis : Fats/Lipids 14

15. Substrate to be degraded Enzyme Product Bacterium Cellulose Cellulase Glucose Cellulomonas Proteins Protease Amino acids Bacteroides, Butyrivibrio, Clostridium, Fusobacterium, Selenomonas, and Streptococcus Fats/lipids Lipases LCFA Clostridia Micrococci Hydrolytic Bacteria 15

16. • Dissolved organic matter is biodegraded mainly to volatile fatty acids (VFAs) and alcohols by a heterogeneous microbial population • Organisms responsible for this process is acid formers (facultative anaerobes) Acidogenesis 16

17. Fermentative bacteria convert soluble organic to • Volatile fatty acids (acetatic, propionic, butyric) • Hydrogen (H2) • Carbon dioxide (CO2) • Pyruvic acid (CH3COCOOH) • Ethanol (CH3CH2OH) • Lactic acid (CH3CH(OH)COOH) 17 Acidogenesis

18. • Acidogenesis is the most rapid step • Fermentative bacteria can work at pH 4 to 5 Glucose to acetate* (ΔG -206 KJ) C6H12O6 + 2 H2O 2 CH3COOH + 2 CO2 + 4 H2 18 Acidogenesis

19. Glucose to acetate and propionate* 3 C6H12O6 + 2 H2 2 CH3COOH + 4 CH3CH2COOH + 2 H2 Glucose to ethanol* (ΔG -226 KJ) C6H12O6 2 CH3CH2OH + 2 CO2 Glucose to lactate* (ΔG -198 KJ) C6H12O6 2 CHCH(OH)COOH + 2 H2 Acidogenesis 19

20. 20 Acidogenesis

21. • Aeromonas • Bacteroides • Bifidobacteria • Citrobacter • Clostridium • Enterobacter • Erwinia • Escherchia • Klebsiella Acidogenic Bacteria • Lactobacillus • Pasteurella • Propionobacterium • Proteus • Providencia • Salmonella • Serratia • Shigella 21

22. • Conversion of fermentation products into acetic acid, CO2, and H2 • Mainly from propionic acid, butyric acid and ethanol • Sensitive to physical and chemical conditions (temperature, pH, hydrogen partial pressure) • Syntrophic Acetogenesis: Anaerobic oxidation of propionate and butyrate to acetate and H2 • Homoacetogenesis: Production of acetate as a sole end product from H2 and CO2 22 Acetogenesis

23. Propionate to acetate* Δ G0 = + 76.1 kJ/mole CH3CH2COOH + 2 H2O CH3COOH + 3 H2 + CO2 Butyrate to acetate* Δ G0 = + 48.1 kJ/mole CH3CH2CH2COOH + 2 H2O 2 CH3COOH + 2 H2 Palmitate to acetate* Δ G0 = + 9.6 kJ/mole CH3(CH2)14CH2COOH + 14 H2O 8 CH3COOH + 14 H2 23 Syntrophic Acetogenesis

24. 4H2+ 2CO2 CH3COOH + 2H2O • _______________________________________________________________________________________________________________________________-- ----- _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ • The syntrophic acetogenesis results in low energy yield (highly endergonic and do not occur naturally and thermodynamically) • When it is combined with the hydrogenotrophic methanogenesis, reduce the amount of H2, the reaction becomes feasible 24 Homoacetogenesis

25. • Hydrogen partial pressure extremely low (10 -5 atm) • Achieved through interspecies hydrogen transfer 25 Acetogenesis

26. Impact of pH2 on thermodynamics 26

27. Equations ΔG0,' (kJ/reaction) 1. Proton-reducing (H2-producing) acetogenic bacteria A. CH3CH2CH2COO- + 2H2O 2 CH3COO- + 2H2 + H+ +48.1 B. CH3CH2COO- + 3H2O CH3COO- + HCO3 - + H+ + 3H2 +76.1 2. H2-using methanogens and desulfovibrios C. 4H2 + HCO3 - + H + CH4 + 3 H2O -135.6 D. 4H2 + S04 2- + H+ HS- + 4 H2O -151.9 3. Co-culture of 1 and 2 A + C 2 CH3CH2CH2COO- + HCO3 - + H2O 4 CH3COO- + H+ + CH4 -39.4 A + D 2 CH3CH2CH2COO- + S04 2- 4 CH3COO- + H+ + HS- -55.7 B + C 4 CH3CH2COO- + 12H2 4 CH3COO- + HCO3 - + H+ + 3 CH4 -102.4 B + D 4 CH3CH2COO- + 3 S04 2" 4 CH3COO- + 4 HCO3 - + H+ + 3 HS- -151.3 Free energy changes for reactions 27

28. • Obligate H2 producing Microorganism Syntrophobacter wolinii Sytrophomonos wolfei Obligate Bacteria 28

29. • Methanogenesis is the final stage of the AD process • Methanogens microorganisms are obligate anaerobes, which can convert few substrates to methane – Acetate – Methanol – Formate – CO2/H2 – Methylamines 29 Methanogenesis

30. • Hydrogenotrophic methanogenesis Hydrogen to Methane • Acetoclastic methanogenesis Acetate to Methane • Methanogenesis is extremely sensitive to temperature, loading rate and pH fluctuations and inhibited by a number of organic and inorganic compounds 30 Methanogenesis

31. • Hydrogenotrophic archaea reduce the CO2 using hydrogen as electron donor CO2 + 4 H2 CH4 + 2 H2O • Higher growth rate and less sensitive organisms • About a 30% of the biogas is produced by this reaction 31 Hydrogenotrophic Methanogenesis

32. • Acetoclastic archaea decarboxilate acetate to form CH4 CH3COOH CH4 + CO2 • Slowest growth rate and most sensitive organisms • About a 70% of the biogas is produced by this reaction 32 Acetoclastic Methanogenesis

33. Genera Species Methanobacterium M. formicicum M. bryantii M. thermoautotroohicum Methanobrevribacter M. ruminantium M. arboriphilus M. smithii Methanococcus M. vannielli M. Voltae Methanogenic Bacteria

34. Genera Species Methanomicrobium M. mebile Methanogenium M. cariaci M. marisnigri Methanospirillum M. bungater Methanosarcina M. barkeri Methanogenic Bacteria (contd..)

35. 35 Carbon flow in anaerobic dogestion

36. • Oxidation of Reduced compounds, Acetate and Hydrogen to carbon dioxide by sulphate-reducing bacteria 36 Other Microorganism Reactions 1. Hydrolytic bacteria 2. Fermentative bacteria 3. Acetogenic bacteria 4. Methanogenic bacteria 5. Sulfate reducing bacteria

37. • The presence of inorganic electron acceptors (SO4 2) will decrease the methane production • Bacteria that convert sulphate (SO4 2-) to hydrogen sulphide (H2S), which mainly oxidize acetic acid or H2 Sulphate to hydrogen sulphite (ΔG -48 KJ) SO4 2- + CH3COOH H2S + 2 HCO3 – Sulphate to hydrogen sulphite (ΔG -10 KJ) SO4 2- + 4 H2 + 2 H+ H2S + 4 H2O 37 Sulfate reducing bacteria reaction

38. Competition between SRB and methanogens Eg: Desulfovibrio desulfuricans 38 Sulfate reducing bacteria reaction

39. Problems related with the presence of hydrogen sulphide in the digester • Is toxic for methanogenic bacteria • Lower methane yields > worse energy balance • Worse quality biogas • Need for biogas treatment • Odour problem • Corrosion problems, maintenance cost are increased 39 Sulfate reducing bacteria reaction

40. Bacterial reproduction • Bacteria can reproduce by binary fission (original cell becomes two new organisms) • Time required for each division, which is termed the generation time, can vary from days to less than 20 min • Example: if the generation time is 30 min, one bacterium would yield 16,777,216 (i.e 224) bacteria after a period of 12h

41. • Growth rate Increase of bacterial cell number per unit time • Generation The interval of formation of daughter cells from parental cell • Generation time Time required to form two daughter cells from a single cell Bacterial Growth in AD

42. Bacterial Growth Phases

43. Lag Phase • Represents the time required to acclimate to their new environment before significant cell division and biomass production occur • During this period, the cell increases its size • Synthesis new protoplasm, enzymes, co enzymes etc • The cells may be acclimating to changes in salinity, pH, or temperature • The cells are metabolically active Bacterial Growth Phases

44. Log Phase/Exponential growth phase • Cells are multiplying at their maximum rate • Generation time will be of constant • Cells will be sensitive to chemical inhibitors • Ideal stage to study the metabolic activity of the bacterium • The only factor that affects the rate of exponential growth is temperature Bacterial Growth Phases

45. Stationary phase • Cell division rate and cell death rate is uniform • Death due to low nutrients content, pH change, toxic wastes, reduced oxygen • Cells are very small and having low number of ribosomes • Some cases, cell do not die and at the same time do not multiply • Some bacteria produce antibiotics at this stage as secondary metabolites for competitiveness Bacterial Growth Phases

46. Death phase • The substrate has been depleted so that no growth is occurring and the change in biomass concentration is due to cell death • Exponential decline in the biomass concentration is often observed as an approximate constant fraction of the biomass remaining that is lost each day Bacterial Growth Phases

47. g biomass produced Biomass yield Y = ----------------------------------------- g substrate utilized (i.e. consumed) Example: For anaerobic degradation of volatile fatty acids (VFAs) to produce methane, the yield is expressed as g biomass / g VFAs used 47 Bacterial Growth & Biomass Yield

48. • Biomass yield is based on a measurable parameter reflecting the overall organic compound consumption, such as COD or BOD • Yield would be g biomass / g COD removed or g biomass / g BOD removed 48 Bacterial Growth & Biomass Yield

49. Theoretical Yield • By means of exact stoichiometric realtionship • Performing COD mass balance • COD utilized = COD cells + COD of oxidized substrate • Yield can be expressed as g cells / g COD used • Oxygen consumed = COD utilized – COD cells • Oxygen requirement can be expressed as g O2 / g COD used 49 Estimation of Biomass Yield

50. • Involves the application of thermodynamic principles to biological reactions • Chemical reactions – energy - can be described thermodynamically by a change in the free energy G°, known as the Gibbs free energy • Change in energy is termed as ∆G° 50 Estimation of Biomass Yield from Bioenergetics

51. Key steps in bioenergetics analysis are: • Identify the electron donor (substrate oxidized) and electron acceptor • Determine the energy produced from the bacteria oxidation reduction reaction • Determine the amount of energy needed for converting the growth carbon source into cell matter, and • Calculate the cell yield based on a balance between energy produced and energy needed for cell yield 51 Estimation of Biomass Yield

52. • Govern the oxidation (i.e., utilization) of substrate and the production of biomass which contributes to the total suspended solids concentration in a biological reaction 52 Microbial Growth Kinetics

53. • Cell growth generally involves o Raspiratory (electron transport phosphorylation) or Fermentative (substrate level phosphorylation) o Conversion of susbtrate to products (release energy in form of adenosine 5-triphosphate (ATP) o Energy used for both synthesis of new cell and maintenance of old ones 53 Microbial Growth Kinetics

54. • The ratio of ATP mass produced per substrate mass consumed is called ATP yield factor Similarly • Biomass Yield factor = ∆X / ∆S • Product Yield factor = ∆P / ∆S X – Amount of biomass P – Amount of product S – Amount of substrate 54 Microbial Growth Kinetics

55. • AD gives low biomass yield factor compared to aerobic • Due to low energy (ATP) yield of anaerobic metabolism • Anaerobic biomass yield factor is between 0.05 to 0.2 g of biomass produced per g of substrate consumed • The yield factors can be determined from stoichiometry of biochemical reactions 55 Microbial Growth Kinetics of AD

56. Rate of growth is described by the “Monod’s “ equation. µ = specific growth rate, day-1 µm = maximum specific growth rate, day-1 S = concentration of growth – limiting substrate, mg/l Ks = half saturation constant (equal to the limiting substrate concentration at half the maximum growth rate), mg/l 56           S K S s m   Monod’s Equation

57. • At S = Ks, µ = ½ µmax • dS/dt = substrate utilization rate • Y = yield coefficient 57 Substrate availability & bacterial growth Y X dt dS   

58. • Rate of consumption, dS/dt = rsu • Michaelis-Menten expression • Consequence of enzyme-substrate reaction kinetics 58 Rate of utilisation of soluble substrates

59. • When the substrate is being used at its maximum rate, the bacteria are also growing at their maximum rate µm = kY k = • µm = maximum specific bacterial growth rate, day-1 • k = maximum specific substrate utilization rate, day-1 • Y = true yield coefficient, g/g • Substrate utilisation rate 59 Y µm Rate of utilisation of soluble substrates

60. Microbial growth • rg also related to rsu by Y • Endogenous decay: Cell material consumed for maintenance energy Cell death • rd = kdX 60

61. Microbial growth Substitute rsu = - Specific growth rate µ = rg / X ) ( . S K Y XS µ s m  61

62. Rate of oxygen uptake The rate of oxygen uptake is related stoichiometrically to the organic utilization rate and growth rate ro = - rsu – 1.42rg ro = oxygen uptake rate, g O2/m3.d rsu = rate of substrate utilization, g bs COD/m3.d 1.42 = the COD of cell tissue, g bs COD/g VSS rg = rate of biomass growth, g VSS/m3.d 62

63. Effect of temperature The temperature dependence of the biological reaction-rate constants – very important in assessing the overall efficiency kT = k20(T-20) kT = reaction-rate coefficient at temperature T, oC k20 = reaction-rate coefficient at 20oC  = temperature-activity coefficient T = temperature, oC Values for  in biological systems can vary from 1.02 to 1.25 63

16. MICROBIOLOGY AND BIOCHEMISTRY OF BIOGAS PRODUCTION.ppt

16. MICROBIOLOGY AND BIOCHEMISTRY OF BIOGAS PRODUCTION.ppt

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16. MICROBIOLOGY AND BIOCHEMISTRY OF BIOGAS PRODUCTION.ppt