SEMINAR ON BIOCHEMICAL ROUTES :ANAEROBIC DIGESTIONTRANSESTERIFICATIONPHOTOFERMENTATION

1. SEMINAR ON
BIOCHEMICAL ROUTES :ANAEROBIC DIGESTION
TRANSESTERIFICATION
PHOTOFERMENTATION
By
ANURAG CHANDRA SHEKHAR
(M.TECH. FIRST YEAR )
ROLL No. 06
In The Expert Guidance Of
Dr. SONAL DIXIT
ASST. PROFESSOR(GUEST)
DEPT. ENVIROMENTAL SCIENCE

2. Anaerobic Digestion
Anaerobic digestion is the conversion of organic material in
the absence of oxygen to methane and carbon dioxide by a
microbial consortia.
CXHYOZ CO2 +CH4+ anaerobic bacterial
biomass

3. How Do We Get Methane?
Need Methanogens:
Either Hydrogen (Lithotrophic methanogens)
4H2 + CO2 CH4 + 2 H2O
Acetate (acetoclastic methanogens)
CH2COOH + H2O CH4 + CO2
But have a limited range of substrates can also include carbon
monoxide, formate, methanol.

4. The Anaerobic Digestion Microbial
Consortia
 The production of biogas is dependant on the successful
interaction of interdependent microbial species.
 The production of methane is a property of a (relatively)
limited number of micro-organism species, the
“methanogens”.
 There is a limited number of substrates that the “methanogens”
can use to produce methane.

5. 5
Anaerobic Digestion Process
Liquefaction
Liquefying
Bacteria
Acid Production
Liquefied
soluble organic
compounds
Insoluble
Compounds
(organic,
inorganic,
water)
Acid-Forming
Bacteria
Biogas
Production
Simple
organic
acids
Methane-Forming
Bacteria
Manure
BiogasBiogas
(Methane,
CO2, misc.)
Effluent
End
Products

6. The Stages in Anaerobic Digestion
 Hydrolysis
Long chain polymers broken down to smaller molecules.
 Acidogenesis
Production of hydrogen and volatile fatty acids.
 Acetogenesis
Alcohols, >C2 VFAs converted to acetate and hydrogen.
 Methanogenesis
Hydrogen and acetate converted to methane.

7. The Steps in Anaerobic Digestion
Hydrolysis
Acidogenesis
Acetogenesis
Methanogenesis
Taken from Guwy, (1996). Modified from Mosey, (1983)

8. Factors Effecting the Rate of
Hydrolysis
 Particle size of the waste, smaller is better.
 Accessibility of the substrate i.e. how easy is it for the
enzymes to attack the substrate.
 Problems with lipids .
 Residence time in the reactor.
 Chemical structure of substrate.
 Negative impact of lignin and hydrocarbons,
 Organic content of the substrate.

9. Acidogenesis
 The sugars, amino acids and longer chain fatty acids are
then fermented to acetate, propionate, butyrate, valerate,
ethanol, lactate, hydrogen, CO2, ammonia and sulphide by
the acidogenic bacteria.
 The proportion of the organic products of the acidogenic
bacteria is determined by the H2 concentration and pH.

10. Acetogenesis
 The acetogens (obligate hydrogen producing acetogenic
bacteria) convert the fermentation products which the
methanogens cannot use (alcohols, >C2 VFAs, aromatic
compounds) to the substrates which the methanogens can
utilise.
 The Gibbs free energies for the conversion of ethanol,
propionic and butyric to acetate and hydrogen are
energetically unfavourable i.e. positive at standard free
biochemical energy levels (pH 7.0, 1 atm.).

11. Why are Propionic and Butyric Acids
bad?
 Don`t smell very nice .
 Wasted energy as methanogens can`t use them.
 Very difficult to get rid of.
CH3CH2COOH + 2H2O CH3COOH + CO2+ 3H2
Propionic acid
NOT beneficial for the microorganisms as:
G0 =+ 76.1.9 KJ mol -1
Energy could be required to be put in to use the propionic acids.

12. Role of Interspecies Hydrogen
Transfer
 The H2 partial pressure must be maintained at between 10-6
-10-4
atmospheres for sufficient energy for growth to be obtained
from propionic and butyric acids.
 As H2 is continually being generated it must be continually be
removed for methanogenesis to occur.
 The removal of H2 is achieved by conversion to methane by
the hydrogen utilising methanogens.
 This symbiosis is known as interspecies hydrogen transfer and
is crucial to the success of the anaerobic digestion process.

13. Graphical representation of the hydrogen-dependant thermodynamic favourability of acetogenic
oxidations and inorganic respirations associated with the anaerobic degradation of waste organics.
(1) Propionic oxidation to acetic acid, (2) butyric oxidation to acetic, (3) ethanol to acetic, (4) lactic
to acetic, (5) acetogenic respiration of bicarbonate, (6) methanogenic respiration of bicarbonate, (7)
respiration of sulphate to sulphide, (8) respiration of sulphite to sulphide, (9) methanogenic
cleavage of acetic acid, (10) SRB-mediated cleavage of acetic acid. (From Harper and Pohland,
1985).
Methanogenic “Hydrogen” Niche

14. The Methanogens
 A group of gram –ve bacteria.
 Belong to the group of bacteria termed the Archaea or
archaebacteria.
 Are strict anaerobes they evolved billions (3.5 billion) years
ago when the earth had very low or zero levels of oxygen.
 Utilise simple inorganic substrates such as H2, CO2 or simple
organic substrates such as acetate, formate and methanol.

15. Image source: Dr L. Hulshoff and Professor van Lier; Sub-department of Environmental Technology, Wageningen
University, Netherlands
The Archaea
Scanning electron micrograph of archaea from the
genus Methanosaeta. Members of the genus
Methanosaeta are acetotrophic, i.e. they produce
methane from acetate.
Scanning electron micrograph of a
cocci-shaped archaea from the genus
Methanosarcina. Members of the
genus Methanosarcina can use all 3
routes to methane.

16. Specific Growth Conditions
These micro-organisms have been difficult to culture in vitro as in
nature they are dependant on other bacteria to provide a suitable
environment for their growth.
The fermentative bacteria provide a environment with :
Low redox potential (-330 mV).
Extremely low in O2 .
Suitable range of substrates .

17. Analysing The Consortia
 Traditional microbiology
 “petri” dish technology (some problems here)
 Enzymatic profiles
 Molecular Biology Techniques
 PCR/RT PCR
 DGGE
 FISH
 Genomics and Proteomics
 Microarrays

18. Limited Range of Substrates
Hydrogen (Lithotrophic methanogens)
4H2 + CO2 CH4 + 2 H2O
Very beneficial for the microorganisms as G0 =-138.9 KJ mol -1
is
gained.
But only 30% of methane produced via this route.

19. Cont…
Acetate (acetoclastic methanogens)
CH2COOH + H2O CH4 + CO2
Not so beneficial for the microorganisms as only G0 = - 32
KJ mol -1
is gained.
But 70% of methane produced via this route.

20. Other Anaerobic Processes
 Unfortunately other gases are produced as well as methane
and CO2.
 These include hydrogen sulphide (H2S) and ammonia (NH3).
 Hydrogen Sulphide is produced by a competing group of
bacteria to the methanogens called the sulphate reducing
bacteria.

21. What Effects the % and amount of
Methane in the Biogas
 Relative solubility of the gases.
 Is alkalinity being destroyed?
 Biodegradability of substrate.
 Chemical composition of biodegradable substrate.
– Buswell equation

22. Sulphate Reducing Bacteria (SRBs)
Are a problem for methanogenesis as?
 Compete with methanogens for their source of energy
(hydrogen and acetate).
 Can become inhibitory (cellular toxin) at dissolved levels of
50 mg/l.
 More of the dissolved form at lower pH.

23. Competitive Nature of SRBs
4H2 + SO4
2-
H2S + 2 H2O
Very beneficial for the micro-organisms as G0 =-154 KJ mol
-1
is gained.
CH2COOH + SO4
2-
H2S + 2HCO3
-
Not so beneficial for the micro-organisms as only G0 = - 43 KJ mol
-1
is gained BUT more than the methanogens

24. The bacterial consortium requires a number of
factors to be controlled to maintain performance.
These include:
 Temperature
 pH (related to buffering capacity)
 Essential Nutrients
 Avoid toxic compounds
 Sufficient residence time to reproduce

25. The Effect of Temperature
 Three temperature optima have been reported for the
anaerobic digestion process phsycrophilic (around 15o
C),
mesophilic (around 35o
C) and thermophilic (around 55o
C)
temperatures.
 Methanogenesis has been found to occur upto 75o
C but the
optimum temperature is thought to be 55-60o
C.
 The advantage of the higher temperature ranges is that the
process will proceed at a faster rate than the lower temperature
ranges as stated by the Arrhenius equation.

26. The Effect of pH
 Different groups in the anaerobic consortia can cope with
different pH levels.
 Methanogens prefer pH 6-5- 7.5
 Acidogens also prefer pH 6.5-7.5 BUT can cope with pH 5.2
 Therefore if pH is lowered methane production will slow and
consumption of VFA reduce while VFA production continues. Spiral
of Decline.
 Effects methane production but also distribution of volatile
fatty acids.

27. Bicarbonate Alkalinity and pH
 pH Scale is the log10 of the H+
concentration.
 Bicarbonate alkalinity is a measure of the H+
buffering
capacity of the reactor. Measured in mg/l CaCO3
 If you have a alkalinity buffer then difference is between walking
a tight rope and a plank when operating the digester.
 If alkalinity is decreasing may be a sign of trouble before pH
drops below critical level.

28. Nutrient Requirements
The anaerobic consortia need nutrients to grow
their cells and drive their enzymes and
metabolic processes.
Can be divided up into macro and micro
nutrients.
Need to be “available”

29. Macro nutrients
 Relatively large quantities
C/N/P ratios
Also need sulphur C N P S
500-1000:15-20: 5 : 3
Micronutrients (Trace Elements)
 Needed in much lower quantities (g per m3
)
 Required for enzyme activity
 Ni, Fe, Co, Se, Mg etc.

30. Toxic or Inhibitory Compounds
 Divided into two groups:
Too much of a good thing
Ammonia
Sulphate
Ca/Na/K
Should not be here at all
Cleaning compounds
Antimicrobials
Solvents
Heavy metals

31. 31
Environmental Benefits
 Reduces odor from land
application.
 Protects water resources.
 Reduces pathogens.
 Weed seed reduction.
 Fly control after digestion.
 Greenhouse gas reduction.
 Help in bio gas generation &
WWT.

32. Conclusions
 The anaerobic digestion process depends on the effective
working of a complex interaction of microorganisms.
 It has been working successfully for 3.5 billion years.
 However there a wide number of ways which we can make it
not work very well.
 It can be breaking down and still seem to be working.

33. Transesterification
 Transesterification is based on the chemical reaction of
triglycerides with Methanol to form methyl esters and glycerin
in the presence of an alkaline catalyst .
 Methanol & Catalyst are injected into Light layer from
esterification and virgin oil and reacted in the Bio-Conversion
Processor (BCP).
 Crude Biodiesel and Crude Glycerin are separated using
Centrifuge after completion of the transesterification reaction .
 Crude Biodiesel and Crude Glycerin are refined using
distillation columns.
 Use of Ultrasonication Conversion can speed up this process
significantly.

34. Transesterification RXN
O O
|| ||
CH2 - O - C - R1 CH3 - O - C - R1
|
| O O CH2 - OH
| || || |
CH - O - C - R2 + 3 CH3OH => CH3 - O - C - R2 + CH - OH
| (KOH) |
| O O CH2 - OH
| || ||
CH2 - O - C - R3 CH3 - O - C - R3
Triglyceride methanol mixture of fatty esters glycerin

35. Triglyceride Sources
 Rendered animal fats: beef tallow, lard
 Vegetable oils: soybean, canola, palm, etc.
 Chicken fat
 Rendered greases: yellow grease (multiple sources)
 Recovered materials: brown grease, soapstock, etc.

36. Feedstock
Oil
Methanol
Catalyst
Raw
Biodiesel
Fuel-Grade
Biodiesel
Water
Wash
Wate
r
Glycerol
(with methanol)
Glycerin
Transesterification
Drie
r
Reactor
Washer
Separator

37. Standard Recipe
100 lb oil + 21.71 lb methanol
→ 100.45 lb biodiesel + 10.40 lb glycerol + 10.86 lb
XS methanol
Plus 1 lb of NaOH catalyst

38. Competing Reactions
Free fatty acids are a potential contaminant of
oils and fats.
O
||
HO - C - R
Carboxylic Acid (R is a carbon chain)
O
||
HO - C - (CH2)7 CH=CH(CH2)7CH3
Oleic Acid

39.
O
|| + KOH
HO - C - (CH2
)7
CH=CH(CH2
)7
CH3
Oleic Acid Potassium
Hydroxide
O
||
→ K+ -
O - C - (CH2
)7
CH=CH(CH2
)7
CH3
+ H2
O
Potassium oleate (soap) Water
Fatty acids react with alkali catalyst to form
soap.

40. Water is also a problem
Water hydrolyzes fats to form free fatty acids,
which then form soap.
O
||
CH2 - O - C - R1 CH3 - OH
| |
| O | O O
| || | || ||
CH - O - C - R2 + H2O >>> CH3 - O - C - R2 + HO - C-R1
| |
| O | O
| || | ||
CH2 - O - C - R3 CH3 - O - C - R3
Triglyceride Water Diglyceride Fatty acid

41. Soap
 Soaps can gel at ambient temperature causing the the entire
product mixture to form a semi-solid mass.
 Soaps can cause problems with glycerol separation and
washing.

42. Process Issues
 Feedstock requirements
 Reaction time
 Continuous vs. batch processing
 Processing low quality feed stocks
 Product quality
 Developing process options

43. Feedstock Used in Biodiesel
Production
 Triglygeride or fats and oils (e.g. 100 kg soybean oil) –
vegetable oils, animal fats, greases, soapstock, etc.
 Primary alcohol (e.g. 10 kg methanol) – methanol or ethanol
(44% more ethanol is required for reaction)
 Catalyst (e.g. 0.3–1.0 kg sodium hydroxide)
 Neutralizer (e.g. 0.25 kg sulfuric or hydrochloric acid)

44. Reaction time
 Transesterification reaction will proceed at ambient (70°F)
temperatures but needs 4-8 hours to reach completion.
 Reaction time can be shortened to 2-4 hours at 105°F and 1-2
hours at 140°F.
 Higher temperatures will decrease reaction times but require
pressure vessels because methanol boils at 148°F (65°C).
 High shear mixing and use of cosolvents have been proposed
to accelerate reaction.

45. Batch vs Continuous Flow
 Batch is better suited to smaller plants (<1 million gallons/yr).
 Batch does not require 24/7 operation.
 Batch provides greater flexibility to tune process to feedstock
variations.
 Continuous allows use of high-volume separation systems
(centrifuges) which greatly increase throughput.
 Hybrid systems are possible.

46. Hybrid Batch/Continuous
Base Catalyzed Process
Ester
TG
Alcohol
TG
Alcohol
Catalyst
Ester
Biodiesel
Mixer
Glycerol
Decanter
CSTR 1 CSTR 2 Glycerol
Alcohol
Alcohol Water
Water
Dryer
Wash Water
Acid
Crude
Glycerol

47. Processing Lower Quality
Feedstock’s
Biodiesel feedstock’s vary in the amount of free fatty acids they
contain:
• Refined vegetable oils < 0.05%
• Crude soybean oil 0.3-0.7%
• Restaurant waste grease 2-7%
• Animal fat 5-30%
• Trap grease 75-100%
Price decreases as FFAs increase but processing demands
increase, also.
Not suitable for high FFA feeds because of soap formation.

48. Preferred method for High FFA
feeds: Acid catalysis followed by
base catalysis
 Use acid catalysis for conversion of FFAs to methyl esters,
until FFA < 0.5%.
Acid esterification of FFA is fast (1 hour) but acid-
catalyzed transesterification is slow (2 days at 60°C).
Water formation by
 FFA + methanol ==> methyl ester + water
 can be a problem.
 Then, add additional methanol and base catalyst to
transesterify the triglycerides.

49. Acid Catalyzed FFA Pretreat System
Hi-FFA TG Esters
to
Acid base-
process
Alcohol
Acid Reactor
Neutralize
Separate
Water
Methanol
recovery

50. Product Quality
 Product quality is important – modern diesel engines are very
sensitive to fuel.
 It is not biodiesel until it meets ASTM D6751.
 Critical properties are total glycerol (completeness of reaction)
and acid value (fuel deterioration). Reaction must be >98%
complete.

51. Developing Process Options
 Schemes for accelerating the reaction
Supercritical methanol
High shear mixing
Co solvents (Biox)
 Solid (heterogeneous) catalysts
Catalyst reuse
Easier glycerol clean-up

52. Environmental Impacts of Biodiesel
 Large Reductions in:
CO2
SOx
Particulates
Odor
 Slight Increase in:
NOx

53. Conclusions
 Biodiesel is an alternative fuel for diesel engines that can be
made from virtually any oil or fat feedstock.
 The technology choice is a function of desired capacity,
feedstock type and quality, alcohol recovery, and catalyst
recovery.
 Maintaining product quality is essential for the growth of the
biodiesel industry.

54. Photobiological
 This method involves using sunlight, a biological component,
catalysts and an engineered system.
 Specific organisms, algae and bacteria, produce hydrogen as a
byproduct of their metabolic processes.
 These organisms generally live in water and therefore are
biologically splitting the water into its component elements.
 Currently, this technology is still in the research and
development stage and the theoretical sunlight conversion
efficiencies have been estimated up to 24%.

55. Materials Requirements for
Photobiological Hydrogen Production
 Biological H2 production is expected to be one of many processes that
contribute to the ultimate supply of H2.
 But it is useful to consider that in an area of less than 5,000 square miles
(about 0.12 % of the U.S. land area) of bioreactor footprint in the desert
southwest, photobiological processes could in principle produce enough H2
to displace all of the gasoline currently used in the U.S. (236 million
vehicles).
 The underlying assumptions are that H2 could be produced from water at 10
% efficiency (the maximum theoretical efficiency of an algal H2 production
system is about 13 %) and that fuel cell-powered vehicles could get 60
miles/kg of H2.

56. DESCRIPTION OF THE PROCESS
 The photobiological production of H2 is a property of only three classes of organisms
photosynthetic bacteria, cyanobacteria, and green algae.
 These organisms use their photosynthetic apparatus to absorb sunlight and convert it into
chemical energy.
 Water (green algae and cyanobacteria) or an organic/inorganic acid (photosynthetic bacteria) is
the electron donor.
 This review will focus on oxygenic organisms such as green algae and some cyanobacteria,
which produce hydrogen directly from water, without an intermediary biomass accumulation
stage.
 This process is considered to have highest potential sunlight conversion efficiency to H2, which
as mentioned above, could be on the order of 10 to 13 %.
 To accomplish this, green algae and cyanobacteria can utilize the normal components of
photosynthesis to split water into O2, protons, and electrons, deriving the requisite energy from
sunlight.
 However, instead of using the protons and electrons to reduce CO2 and storing the energy as
starch molecules (the normal function of photosynthesis), these organisms can recombine the
protons and electrons and evolve H2 gas under anaerobic conditions (figure 5.1) using a reaction
catalyzed by an induced hydrogenate.

58. Conti..
 The enzymes responsible for H2 production are
metallocatalysts that belong to the classes of [NiFe]- (in
cyanobacteria) or [NiFe]- (in green algae) hydrogenases.
 Although sustained and continuous photobiological H2
production has been achieved with green algae, the
establishment and maintenance of culture anaerobiosis is
currently a sine qua non for the process.
 This requirement results from the following biological
realities.

59. Cont…
 The hydrogenase genes in green algae and in some cyanobacteria are not
expressed (the genes are not turned on) in the presence of O2, and a
variable period of anaerobiosis is required to induce gene transcription.
 The expression and function of the genes that catalyze the assembly of the
catalytic metallocluster of the algal [FeFe]-hydrogenases require
anaerobiosis, while in most cyanobacteria, the [NiFe]-hydrogenase genes
are expressed and the protein assembled in the presence of O2; in this case,
the proteins are assembled in an inactive form, but can be activated quickly
when exposed to anaerobic conditions.
 The activity of the catalytic metallocluster of the hydrogenases is inhibited
reversibly (in cyanobacteria) or irreversibly (in green algae) by the
presence of O2.

60. Reactor Materials
 Engineering design of full-scale photobioreactors and the
balance of the facility for photobiological hydrogen
production has not been considered beyond very general
concepts.
 Consequently, there has been little effort to identify
construction material and establish boundaries for specifying
materials performance and properties.

62. Major Benefits Of The Method
 High theoretical conversion yields.
 Lack of oxygen involving , which causes problem of oxygen
in activation of different biological systems.
 Ability to use wide spectrum of light.
 Ability to consume organic substrates derivable from waste
and then , for their potential to be used in association with
wastewater treatment.

63. Conclusions
The photo biological hydrogen production processes are mostly
operated at ambient temperature and pressure, but less energy
intensive. These process are not only environment friendly, but
also they lead to opening of new avenues for the utilization of
renewable energy resources , which are inexhaustible. They can
also use various waste material , which facilitate waste
recycling.