Many microorganisms affect anaerobic digestion, including acetic acid-forming bacteria (acetogens) and methane-forming archaea (methanogens). These organisms promote a number of chemical processes in converting the biomass to biogas.

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
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7. Hydrolysis
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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
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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
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11. Soluble compounds can pass through
the cell walls of the fermentative bacteria
Hydrolysis
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12. • Cellulose is hydrolysed by cellulase (mixture of endo-
cellulases, exocellulases and cellobiases)
Hydrolysis : Cellulose
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13. • Protein - polypeptides - peptides - amino acids
• Proteinases (Peptidases + Proteases)
Hydrolysis : Proteins
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14. • Most lipids in waste (water) are present as triacylglycerides
Hydrolysis : Fats/Lipids
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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
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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
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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)
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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
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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
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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
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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
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Syntrophic Acetogenesis

24. 4H2+ 2CO2 CH3COOH + 2H2O
• _______________________________________________________________________________________________________________________________-- -----
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• 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
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Homoacetogenesis

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

26. Impact of pH2 on thermodynamics
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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
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28. • Obligate H2 producing Microorganism
Syntrophobacter wolinii
Sytrophomonos wolfei
Obligate Bacteria
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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
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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
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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
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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
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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
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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
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Sulfate reducing bacteria reaction

38. Competition between SRB and methanogens
Eg: Desulfovibrio desulfuricans
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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
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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
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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
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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
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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°
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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
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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
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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
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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
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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
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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
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









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
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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
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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
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61. Microbial growth
Substitute rsu = -
Specific growth rate µ = rg / X
)
(
.
S
K
Y
XS
µ
s
m

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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
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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
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