ANAEROBIC DEGRADATION:Anaerobic degradation is defined as the biological process that produce a gas mixture (called biogas) that contains methane (CH4) and carbon dioxide (CO2) as its primary constituents, through the concerted action of a mixed microbial population under conditions of oxygen deficiency. Biological methane production was first noticed by Volta in 1776, who described the release of methane from a swamp. Anaerobic digestion is most widely used and one of the oldest methods for sewage sludge stabilization. It was first used for high-solids municipal wastewater treatment toward the end of the nineteenth century by Louis H. Mouras, who designed and constructed sewage sludge digesters in Vesoul, France. Complete Aerobic digestion of glucose to carbon-dioxide yields up to 38 mole ATP/mole glucose while Anaerobic fermentation to mixed organic acids yields 2-4 mole ATP/mole glucose. Microorganisms involved in degradation: Acid - forming bacteria : Clostridium sp , Corynebacterium sp , Lactobacillus sp ,Actinomycetes sp, Staphylococcus sp,Peptococcus anaerobus, Escherichia coli, Pseudomonas,Bifidobacterium, Propionibacterium, Enterobacteriaceae . Methanogenic bacteria: Methanobacterium formicium,Methanobacterium bryantii, Methanobacterium thermoautotrophicum,Methanosarcina barkeri, Methanobrevibacte ruminantiurn,Methanobrevibacter smithii ,Methanobrevibacter arboriphilus, Methanococcus vannielii , Methanococcus thermolithotrophicus, Methanobacterium cariaci, Methanobacillus omelianskii. Stages of Anaerobic biodegradation Hydrolysis, Acidogenesis, Acetogenesis and Methanogenesis Anaerobic Degradation of Carbohydrates: The anaerobic degradation of cellulose, can be divided into hydrolytic, fermentative, acetogenic and methanogenic phases. The hydrolysis of carbohydrates proceeds favourably at a slightly acidic pH. Hemicellulose and pectin are hydrolyzed 10 times faster than lignin-encrusted cellulose. In the methane reactor, beta-oxidation of fatty acids,especially of propionate or n-butyrate, is the rate limiting step. Anaerobic degradation of Proteins: Hydrolysis of precipitated or soluble protein is catalyzed by several types of proteases that cleave membrane-permeable amino acids, dipeptides, or oligopeptides. The hydrolysis of proteins requires a neutral or weakly alkaline pH. For complete degradadtion of amino acids in an anaerobic system , a syntrophic relationship of amino acids-fermenting anaerobic bacteria with methanogens or sulfate reducers is required. Anaerobic degradation of Neutral fats and Lipids: Glycerol and saturated and unsaturated fatty acids(palmitic acid,linolic acid,stearic acid etc.) are formed from neutral fats. The long chain of fatty acids are degraded by acetogenic bacteria by beta-oxidation to acetate and molecular hydrogen. If acetate and molecular hydrogen accumulate, the anaerobic digestion process is inhibited. Very low H2 partial pressure is mainatained by hydrogen-utilizing methanogens .

1. MECHANISM OF ANAEROBIC
BIODEGRADATION

2. Contents
Overview of Anaerobic biodegradation
Microorganisms involved in biodegradation
Microbiology of Anaerobic Degradation
Hydrolytic and Acidogenic Bacteria
H 2-Producing Acetogenic Bacteria
Methanogenic Bacteria
Anaerobic treatment of wastes
Anaerobic metabolism of organic matter
Stages of anaerobic biodegradation
Mechanism of anaerobic biodegradation
Comparison between Anaerobic and Aerobic degradation
Advantages and Disadvantages
Applications

3. ANAEROBIC DEGRADATION
• Anaerobic degradation is defined as the biological
process that produce a gas mixture (called biogas) that
contains methane (CH4) and carbon dioxide (CO2) as
its primary constituents, through the concerted action
of a mixed microbial population under conditions of
oxygen deficiency.
• Biological methane production was first noticed by
Volta in 1776, who described the release of methane
from a swamp.
• Anaerobic digestion is most widely used and one of
the oldest methods for sewage sludge stabilization.

4. • It was first used for high-solids
municipal wastewater treatment toward
the end of the nineteenth century by
Louis H. Mouras, who designed and
constructed sewage sludge digesters in
Vesoul, France.
• Complete Aerobic digestion of glucose
to carbon-dioxide yields up to 38 mole
ATP/mole glucose while Anaerobic
fermentation to mixed organic acids
yields 2-4 mole ATP/mole glucose.

5. Microorganisms involved in degradation
• Acid - forming bacteria : Clostridium sp , Corynebacterium sp , Lactobacillus sp
,Actinomycetes sp, Staphylococcus sp,Peptococcus anaerobus, Escherichia coli,
Pseudomonas,Bifidobacterium, Propionibacterium, Enterobacteriaceae .
• Methanogenic bacteria: Methanobacterium formicium,Methanobacterium bryantii,
Methanobacterium thermoautotrophicum,Methanosarcina barkeri, Methanobrevibacte
ruminantiurn,Methanobrevibacter smithii ,Methanobrevibacter arboriphilus,
Methanococcus vannielii , Methanococcus thermolithotrophicus, Methanobacterium
cariaci, Methanobacillus omelianskii.

6. Microbiology of Anaerobic Degradation
• The methane fermentation is classified into two groups
of bacteria or stages of degradation, the acid forming
bacteria and methane forming bacteria.
• The acid-forming stage involves the acidogenic
bacteria which hydrolyze polymers and convert the
products to organic acids, alcohols, carbon dioxide and
hydrogen.
• The methane-forming stage involve the
methanogenic bacteria which catabolized these
compounds to the final products methane and Carbon
dioxide.
• However, methanogenic bacteria are not able to
catabolize alcohols other than methanol or organic
acids other than acetate and formate that is why at least
three groups of bacteria decompose organic material
into methane and carbon dioxide.
Fig 1: The three stages of the methane fermentation.
Percentages represent the flow of electrons
from organic compounds to methane.

7. Hydrolytic and Acidogenic Bacteria
Fig. 2. Bacterial fermentation products of pyruvate.
Pyruvate is formed by the catabolism of carbohydrates
• All the polymeric organic material such as
polysaccharides, fats and proteins are hydrolyzed by
extracellular enzymes, excreted by several bacteria.
Most of the biopolymers are easily degradable, the
cellulose of highly lignified plant material (straw,
wood, etc.) has been shown to be very resistant to
hydrolysis.
• The sugars and amino acids formed are then taken
up by the bacteria and fermented mainly to acetate,
propionate, butyrate, H2, CO2,lactate, valerate,
ethanol, ammonia and sulfide.
• Succinate produced by many bacteria is
decarboxylated by others to yield propionate and
Carbon dioxide.

8. • The concentration of Hydrogen plays a
central role in controlling the proportions
of the various products from acidogenic
bacteria.
• As shown in fig; the free energy change for
the oxidation of NADH2 to NAD and H 2
is negative at a partial pressure of H 2
below 10 -3 atm.
• Thus in mixed cultures, where p H2 is kept
low by the action of H 2 -utilizing
methanogenic bacteria, the fermentative
metabolism is shifted to the production of
more hydrogen and less reduced organic
compounds.
• when the partial pressure of H2 is
maintained at a very low level, most of the
carbohydrate is converted into acetate, CO2
and H 2 and without major production of
other fatty acids.

9. H 2-Producing Acetogenic Bacteria
Fig. 4. Effect of hydrogen partial pressure on the free
energy of conversion of ethanol, propionate,acetate and
hydrogen during methane fermentation.
• Syntrophobacter wolinii, degrades propionate
to acetate, CO2 and H2 only in coculture with
an H 2-utilizing organism.
• In the absence of sulfate members of the genus
Desulfovibrio it can oxidize ethanol and lactate
when combined with methanogenic bacteria.

10. Methanogenic Bacteria
• Several new coenzymes and factors have
been discovered in methanogens.
Coenzyme M (HS--CH 2--CH 2--SO 3H), is
involved in the last step of methanogenesis.
• Coenzyme F 420, a 5-deazaisoalloxacine
derivative, acts as an electron carrier.
• Factor F430, the prosthetic group of methyl
CoM reductase, is a nickel tetrapyrrole;
therefore the growth of methanogens
depends on nickel.

11. • Most methanogenic bacteria grow on CO 2 and
H2 as sole carbon and energy sources.
• Labelling data and enzymatic studies indicate
that autotrophic CO2 fixation in these bacteria
does not proceed via the Calvin cycle.
• It was found that acetyl-CoA is the key
intermediate of CO2 assimilation in
Methanobacterium thermoautotrophicum.
• CO2 is fixed via the carboxylation of acetyl-
CoA of pyruvate, of phospho-enolpyruvate to
oxalacetate and of succinyl CoA to a-
ketogluterate.

12. Table 1: Characteristics of methanogenic species in pure Culture

13. Anaerobic treatment of wastes
• Sulfate-reducing bacteria can be used for anaerobic biodegradation of organic matter or
for the precipitation/immobilization of heavy metals of sulfate containing hazardous
wastes.
• Anaerobic biodegradation of organic matter and detoxication of hazardous wastes can be
significantly enhanced as a result of precipitation of toxic organics,phenols, or cyanide by
Fe(II).
• Nitrate-respiring bacteria can be used in denitrification i.e. the reduction of nitrate to
gaseous nitrogen. Nitrate can be added to the hazardous waste to initiate the
biodegradation of different types of organic substances.
• For example: polycyclic aromatic hydrocarbons.
• Anaerobic fermenting bacteria (e.g. from genus Clostridium) perform two important
functions in the biodegradation of hazardous organics: a) they hydrolyze different natural
polymers and b) ferment monomers with production of alcohols,organic acid and carbon
doxide.

14. Contd...
• Many hazardous substances, for e.g. chlorinated solvents, phenols,ethyleneglycol and
polyethylene glycols can be degraded by anaerobic microorganisms.
• Different biotechnological systems perform anaerobic biotreatment of wastewater :
biotreatment by suspended microorganisms, anaerobic biofiltration, and biotreatment in
upflow anaerobic sludge blanket (UASB) reactors.
• A combined anaerobic and aerobic biotreatment can be more effective than aerobic or
anaerobic treatment alone. The simplest approach for this type of treatment is the use of
aerated stabilization ponds,aerated and non-aerated lagoons and natural and artificial
wetland systems.

15. Anaerobic metabolism of organic matter
• In general terms,under completely anaerobic conditions,organic compounds are converted
according to the overall reaction:
• For example, carbohydrates (n=1, a=2, b=1) gives a 50-50 mixture of carbon dioxide and
methane.
• From this simplified reaction , it becomes apparernt that the constitution of the generated
biogas will depend on the redox state of the organic carbon.
• Therefore,hydrocarbons generate equal amounts of methane and carbon dioxide,methanol
and lipids generate biogas rich in methane,oxalic acid will produce biogas low in methane
and urea will produce no methane .

16. Fig : Basic digestion pathways of anaerobic
digestion showing main substrates. The major
intermediates carbon dioxide and water are not
shown.

17. Stages of Anaerobic biodegradation
Hydrolysis of Particulate Substrates:
• Organisms cannot take up non-soluble and particulate substrates that are too large to pass through the cell
membrane. Therefore, extra-cellular enzymes are released that cleave polymers into smaller substrate
molecules. This process is commonly referred to as hydrolysis.
Hydrolysis
Acidogenesis
Syntrophic acetogenesis
Acetoclastic methanogenesis

18. Hydrolysis of particulate Carbohydrates
Fig. showing that Cellulose is a regular linear
polymer of D-glucose with β-1,4 glucosidic
bonds. • Depending on operating conditions degradation of carbohydrate based
particulates may be rate limiting compared to methanogenesis.
• Cellulose is taken as an example substrate here. Degradation of
cellulose from the fiber to glucose requires four major enzyme assisted
steps as shown below:
Cellulose
Cello-
oligosacchrides
Cellobiose Glucose
Fig: Hydrolysis of cellulose fibres
• For example, Clostridium Thermocellum, one of the major
cellulolytic anaerobic bacteria in thermophilic environments produce
a cellulase matrix that catalyses the complete hydrolysis.
• Cellulases have a pH optimum of about 4-6 but since the optimal pH
of each of the steps may be different, it is difficult to determine a
single optimum.

19. Hydrolysis of particulate Proteins
• Globular proteins are rapidly hydrolysable
while fibrous proteins are difficult.
• There are three main groups of proteases:
serine, metallo and acid proteases which have
alkaline (8-11), neutral (6-8) and acidic (4-6)
pH optimums respectively.
• The triggers for enzyme production vary
widely. Some clostridia secrete in growth
phase, stationary phase, and under stress.
• Enzyme production may be suppressed when
readably biodegradable substrates such as
glucose or amino-acids are supplied.

20. Hydrolysis of Fats and Oils
• Hydrolysis of oils is normally more rapid than fats due to the higher level of
emulsification and hence higher specific surface area.
• Hydrolysis of Triglycerides:
• Hydrolysis is catalysed by long-chain fatty acid ester hydrolases, called lipases.
• There are three main products from the hydrolysis of fats. These are non-fatty acid
products (mainly glycerol), unsaturated fatty acids, and saturated fatty acids.
• Lipase production can be stimulated by the presence of both triglycerides and by fatty
acids.
• Activity of lipases increases greatly when the concentration of triglycerides reaches
saturation and forms a second phase. The lipases are adsorbed at the interface. Because
there is an adsorption mechanism, combined reaction and adsorption rate may be
dependent on surface area of the insoluble triglycerides.

21. • Bacterial lipases can be divided into three main types;
Non-specific lipases, 1,3-
specific lipases, and fatty acid specific lipases.
• Non-specific lipases can hydrolyse any fatty acid
triglyceride regardless of structure, acting at any of the
fatty acids. These can completely hydrolyse the ester
bonds acting equally at all alcyl sites.
• 1,3-specific lipases can only act at the outside bonds of
the triglycerides, yielding 1,2-diacylglycerols and 2-
monoacylglycerols. These glyceride esters are unstable
and undergo acyl migration to 1,3-diacylglycerol and
1-monoacylglycerol. These can be degraded further by
the 1,3-specific lipase to glycerol and free fatty acids.
• Fatty acid specific lipases catalyse the removal of a
specific fatty acid, removing cis-∆9-monounsaturated
fatty acids. Other fatty acids are degraded very slowly,
especially those containing an additional double bond
between ∆1 and ∆9.

22. Mechanics of Hydrolysis
• There are three main mechanical
pathways for release of enzymes and
hydrolysis.
• (a) The organisms secrete enzymes to the
bulk liquid where it adsorbs onto a
particle or reacts with a soluble substrate.
• (b) The organism attaches to the particle,
secretes enzymes into the vicinity of the
particle. The organism benefits from the
soluble substrates being released.
• (c) The organism has an attached enzyme
which may double as a transport receptor
to the interior of the cell. This method
requires the organism to adsorb onto the
surface of the particle.

23. Acidogenesis/Fermentation
• Acidogenesis is the first energy yielding step.
Because LCFA(long chain fatty acids) require
an external electron acceptor for oxidation,
degradation of LCFA is covered in
OHPA(obligate hydrogen producing
acetogenesis) (Acetogenesis). The main
substrates for acidogenesis are soluble
saccharides and proteins.
 Acidogenesis of Soluble Carbohydrates:
For example, production of propionate only
(equation (d) below) is not normally observed
as it is coupled with an oxidation reaction such
as (f) to give (a).

24. References to production of propionate only, even in pure cultures have not been seen
though the key intermediate to propionate, succinate is produced by the organism
Fibrobacter Succinogenes.
Acidogenesis of lactate:
The products of lactate fermentation are the similar to that of glucose (though with
adjusted energy yields) and therefore regulation and relative concentrations of products
may be similar.
Regulation of products by environmental conditions:

25.  Amino Acids: Amino acids can be degraded in two main ways:
(a) As a Stickland oxidation-reduction paired fermentation.
(b) As a single amino acid with an external electron acceptor.
 Stickland reactions: Stickland reactions require one amino acid to act as an electron
donor
(oxidation) and the other to act as an electron acceptor (reduction).
The products of the oxidation step are always NH3, CO2 and a carboxylic acid with one
carbon less than the original chain as well as ATP. The reduction step results in a
carboxylic acid with the same number of total carbon atoms as the original amino acid and
NH3.
The coupled oxidation/reduction reaction for alanine and glycine in Clostridium
sporogenes is shown below:

26. Histidine is the only amino acid not able to be digested via Stickland reactions.
 Products of amino acid fermentations:
Stickland reactions normally produce volatile fatty acids up to valerate (C5) from non-
aromatic amino acids. Aromatic amino acids produce aromatic intermediates such as
phenol, cresol and indole derivatives found accumulated significantly as when hydrogen
concentrations were high.

27.  Other Fermentation Processes:
1. Fermentation of glycerol
2. Hydrogenation of unsaturated fatty acids.
2.
1

28. Obligate Hydrogen Producing Acetogenesis (OHPA) and
Hydrogenotrophic Methanogenesis
• Obligate hydrogen producing acetogenesis is acetate producing reactions that can only
oxidise the substrate while reducing hydrogen ions to hydrogen or bicarbonate to
formate.
• All organic fatty acids and ethanol are degraded by OPHA.
• Substrates have been grouped into C4+ fatty acids and propionate by differences in
pathways.
Oxidation of C4+ fatty acids:

31. Methanogenesis
• Methanogens are obligate anaerobic microorganisms that may be found in natural
environment such as the rumen,the interior part of the stem of certain trees and in
freshwater sediments.
• Methane has also been found to be released from high-salt environments as well as
high-temperature environments (e.g. Methanothermus fervidus isolated from thermal
springs has an optimal growth temperrature of 83 degree C).
• Recently, it was believed that all methanogens can generate methane from hydrogen
and carbon dioxide.
• However, it was shown that although most methanogenic species have this ability,
there are some that use acetic acid as a substrate and thus they have been divided into
two groups: (a) acetotrophs such as Methanothrix soehngenii, Methanosacrina TM-1,
Methanosacrina acetivorans and (b) obligate methylotrophs such as Methanolobus
tindarus,Methanococcus halophlus etc, which metabolize only methanol,
methylamines and dimethyl sulfide.

32. Contd...
• Among hydrogen-utilizing methanogens, there are quite a few species that metabolize
formic acid (e.g. Methanobacterium thermolithotrophicus, Methanobacterium
formicicum) and carbon monoxide (e.g. Methanobacterium thermoautotrophicum).

33. Anaerobic Degradation of Carbohydrates
• The anaerobic degradation of cellulose,
can be divided into hydrolytic,
fermentative, acetogenic and
methanogenic phases.
• The hydrolysis of carbohydrates
proceeds favourably at a slightly acidic
pH.
• Hemicellulose and pectin are
hydrolyzed 10 times faster than lignin-
encrusted cellulose.
• In the methane reactor, beta-oxidation
of fatty acids,especially of propionate
or n-butyrate, is the rate limiting step.

34. Anaerobic degradation of Proteins
• Hydrolysis of precipitated or soluble
protein is catalyzed by several types of
proteases that cleave membrane-
permeable amino acids, dipeptides, or
oligopeptides.
• The hydrolysis of proteins requires a
neutral or weakly alkaline pH.
• For complete degradadtion of amino
acids in an anaerobic system , a
syntrophic relationship of amino acids-
fermenting anaerobic bacteria with
methanogens or sulfate reducers is
required.

35. Anaerobic degradation of Neutral fats and Lipids
• Glycerol and saturated and unsaturated
fatty acids(palmitic acid,linolic
acid,stearic acid etc.) are formed from
neutral fats.
• The long chain of fatty acids are
degraded by acetogenic bacteria by
beta-oxidation to acetate and
molecular hydrogen.
• If acetate and molecular hydrogen
accumulate, the anaerobic digestion
process is inhibited.
• Very low H2 partial pressure is
mainatained by hydrogen-utilizing
methanogens or sulfate reducers.

36. Degradation of Herbicides
• Diuron is a systemic herbicide derived from urea,
relatively persistent in soil.
• Half-lives from 90-180 days.
• It shows slight acute toxicity.
• It is a likely carcinogen
• Diuron has a very slow rate of natural hydrolysis
in a neutral solution at 25o C.
• However, when hydrolysis occurs the degradation
in water solution is an irreversible reaction giving
3,4-DCA as the only product.

37. Contd...

38. Comparison between Anaerobic and Aerobic
Biodegradation
Aerobic degradation
• Most rapid and fast degradation.
• No pungent gas produced.
• More expensive
• Large disposable waste generated.
• Microbes are Xanthomonas,
Comamonas.
Anaerobic degradation
• Time consuming and slow.
• Pungent gas produced.
• Less expensive
• Less waste is generated
• Clostridia, Eubacteria etc.

39. Energy flow comparison

40. Factors Aerobic Process Anaerobic Process
Reactors Aerated lagoons, oxidation
ditches,stabilization ponds
,trickling filters, and biological
discs.
UASB, anaerobic filter, upflow packed bed
reactor, CSTR, down flow fixed-film reactor,
buoyant filter bioreactor.
Reactor size Aerated lagoons, oxidation
ditches,stabilization ponds
,trickling filters, and biological
discs requires larger land area but
SBR needs comparatively lower
area.
smaller reactor size is required.
Effluent quality excellent effluent quality in terms
of COD, BOD and nutrient removal
is achieved.
effluent quality in terms of COD is fair but
further treatment is required. nutrient removal
is poor.
Energy high energy is required the process produce energy in the form of
methane
Biomass yield in comparison to anaerobic
process 6-8 times greater biomass
is produced.
lower biomass is produced
Loading rate maximum 9000 g COD/m3 d is
required in literature.
31 KgCOD/m3 d is required. This is the reason
for smaller reactor volume and lesser area.

41. Advantages
• The advantages of anaerobic digestion as a wastewater treatment method over its
aerobic oxidation counterpart are :
Stabilization of high organic strength wastes
Generation of reduced amounts of sludge
Reduced nutrients (N and P) requirements
Low energy consumption
Biogas production (which may be used as an energy source)
Anaerobic microorganisms can be maintained for extended periods without
feeding
The generated stabilized biosolids can be suitable as a soil amendment.

42. Disadvantages
The disadvantages of anaerobic digestion as a wastewater treatment method over
its aerobic oxidation counterpart are :
The senstivity of methanogens to a variety of toxic compounds
The control problems frequently exhibited by anaerobic bioprocesses
The relatively long startup times required for anaerobic digesters (often 8-12
weeks).
Some form of post treatment is usually necessary.

43. APPLICATIONS
• The anaerobic processes is usually used for the treatment of significantly concentrated
wastewater along with objectives of producing biofuels.
• Anaerobic processes may not be apt for municipal wastewaters with lower
concentrations of biodegradable COD, lower temperature, high effluent quality needs,
and nutrient removal requirements.
• For industrial wastewaters with much higher biodegradable COD concentrations and
evolved temperature, anaerobic processes are considered more economical.
• Even if anaerobic processes are resulting in effluents having higher COD
concentrations that desired,it may still be advantageous to use anaerobic system as first
stage of treatment , which could be than followed by second stage aerobic or advanced
treatment steps to get the desired quality of effluent.

44. Contd...
• Types of industries whose effluent can be treated using Anaerobic Processes
Slaughterhouses and cold
storage facilities
Alcohol production Potato processing
Breweries Starch production Coffee processing
Leather factories Yeast production Fruit processing
Dairies Soft drink production Fish processing
Sugar refineries Wine production Vegetable processing

45. REFERENCES
• Environmental Biotechnology by Lawrence K.Wang, Volodymyr lvanov,Joo-Hwa Tay
and Yung-Tse Hung
• Environmental Microbiology by Pradipta K. Mohapatra
• Introduction to environmental biotechnology by A.K chatterji
• Anaerobic Wastewater Treatment by Institute for Biotechnology der
Kernforschungsanlage Jülich, D-5170, Jülich, FRG H. Sahm