the ppt contain information about environmental biotechnology to reduce pollution

1. Dr. Syed M. Usman Shah Kazmi

2. 2

3. 3
Recalcitrant Compounds
1. They are difficult to recognize as substrates
2. They are highly stable, chemically and biologically inert
3. They are insoluble in water/adsorbed to soil
4. They are highly toxic
5. Their large molecular weight prevents entry into microbial cells
Recalcitrant compounds like polychlorinated biphenyls, synthetic polymers, oil mixtures,
pesticides, steroids, antibiotics and azo dyes may be recalcitrant due to:

4. 4
Enzymatic and Non-Enzymatic Biotransformation
o Enzymatic elimination is the biotransformation process occurring due to various enzymes present
in the body. Enzymatic are further divided into microsomal and non-microsomal.
o Microsomal biotransformation is caused by enzymes present within the lipophilic membranes
of smooth endoplasmic reticulum of liver. e.g. monooxygenases (catalyzes the incorporation of
a hydroxyl group into a variety of substrates) and glucuronyl transferase (changes hormones,
medicines, and toxins) are important microsomal enzymes.
o Non-Microsomal biotransformation involves the enzymes which are present within the plasma,
cytoplasm and mitochondria. e.g. Alcohol dehydrogenase responsible for metabolism of
ethanol into acetaldehyde, xanthine oxidase converting hypoxanthine into xanthine etc.

5. 5
o Non-enzymic: It is spontaneous and non-catalyzed type of biotransformation. It is used for highly
active, unstable compounds taking place slowly at physiological pH.
o e.g Chlorazepate converted into Dimethyl diazepam (Clorazepate is used to treat anxiety disorders)
o Mustine HCl converted into Ethyleneimonium (Mustine Hcl is used for the treatment of lung cancer)
o Hexamine converted into Formaldehyde (Hexamine used to formulate antiseptic agent for livestock)

6. 6
Rate of Transformation
o The scope and rate of all transformations of recalcitrant compounds depends on:
o Chemical structure and concentration of the recalcitrants
o Type and number of microorganisms capable of degrading or transforming the recalcitrants
o Environment conditions

7. 7
Biological Catalysts used for Microbial Transformation Reactions
 Growing cells
 Resting cells
 Killed Cells
 Immobilized cells
 Immobilized enzymes

8. 8
Comparison of Microbial Transformation with Plant or Animal Cells
o Microbial cells (bacteria, filamentous fungi, algae, yeast) are preferred more as compared to animal
cells or plant cells due to the following reasons:
 Surface-volume ratio
 Growth Rate
 Sterility
 Metabolism Rate

9. 9
Comparison with chemical synthesis
o Substrate specificity: Only one specific reaction step is normally catalyzed by an enzyme. e.g.
o Enzyme lactase can only hydrolyze the β-1, 4- glycosidic bond of lactose to yield
galactose and glucose. Similarly, Maltase can only act on the α-1, 4- glycosidic linkage of two
glucose molecules in maltose.
o Site specificity: If several functional groups of one type are present in the molecule, only one
specific position may be affected. It is possible to obtain conversions at centers that are chemically
unreactive. e.g.
o Progesterone to 11alpha hydroxyprogesterone by Rhizopus nigricans
o Reaction conditions: Several reactions can be combined, either in one fermentation step using an
organism with suitable enzyme systems, or by step-wise conversions. e.g.
o Conversion of steroids

10. 10
o Number of reaction steps: The number of process reaction steps are much less. The total chemical
transformation of one steroid to another may require many steps, and the process may be costly and
provide only low yields because of certain difficult steps in the process. e.g.
o The biosynthesis of Androsterones via chemical route involves several steps as compared to the
steroidal transformation (catalyzed by microorganism) where the multi step reaction is reduced to
a single step reaction.
o Microbial transformations is gaining importance and extensively utilized to generate metabolites in
bulk amounts with more specificity.

11. 11
Types of Microbial
Transformation
Reactions
1. Oxidation
2. Reduction
3. Hydrolysis
4. Phosphorylation
6. Formation of new C=C and C≡C bonds
5. Isomerization
7. Amination and Deamination

12. 12
1. OXIDATION
– Hydroxylation at C-5 occurs by the presence of B. subtilis
– Conversion efficiency = 100%

13. 13
– Dehydrogenation (60% conversion efficiency)

14. 14
– Oxidation of aliphatic side chains with the formation of aldehydes, ketones or carboxyl
functions (80% conversion efficiency)
– Oxidative splitting of aromatic rings (70% conversion efficiency)

15. 15
2. REDUCTION
– Reduction of carbonyl functions ( 50% conversion efficiency)
– Reduction of NO2

16. 16
3. HYDROLYSIS
– Hydrolysis of carboxylic acid esters
– Hydrolysis of N- derivatives

17. 17
4. PHOSPHORYLATION
– Phosphorylation of streptomycin
Phosphoryl group

18. 18
5. ISOMERIZATION
– Isomerization catalyzed by maleate isomerase
Pseudomonas putida

19. 19
6. Deamination
Action of bacterial (E. coli, Bacillus mycoides)
enzymes (L-amino acid oxidase)

20. 20
Biotransformation of Drugs
Drugs may be:
 Excreted unchanged (highly polar drugs e.g., Aminoglycosides, Methotrexate, Neostigmine)
 Metabolized by enzymes ( Microsomal, Cytoplasmic, Mitochondrial)
 Spontaneously changed (Spontaneous molecular rearrangement)

21. 21
Drug Biotransformation Sites
o Liver: Meperidine, pentazocine,
morphine, nitroglycerine, lidocaine,
propranolol, paracetamol, prazosin.
o GIT: Insulin, catecholamines,
clonazepam, chlorpromazine,
tyramine, salbutamol
o Lung: Prostanoids
o Blood Plasma: Suxamethonium

22. 22
Drug Metabolism
o The body typically deals with a foreign compound by making it more water-soluble, to increase the
rate of its excretion through the urine.
o Drugs can undergo one of four potential biotransformations:
i. Active Drug to Inactive Metabolite: e.g paracetamol to glucuronide, chloramphenicol to
chloramphenicol glucuronate etc.
ii. Active Drug to Active Metabolite: e.g morphine to morphino-6-glucuronide
iii. Inactive Drug to Active Metabolite: e.g levodopa to dopamine, carbidopa to decarboxylase
inhibitors
iv. Active Drug to Toxic Metabolite (biotoxification): e.g Isoniazid to acetyl-isoniazid

23. 23
The Pathways of Drug Metabolism
o Phase І reaction
o Includes oxidative, reductive and hydrolytic reactions.
o In these types of reactions, a polar group is either introduced or unmasked, so the drug molecule becomes
more water-soluble and can be excreted.
o Reactions are non-synthetic in nature and in general produce a more water-soluble and less active
metabolites.
o The majority of metabolites are generated by a common hydroxylating enzyme system known as
Cytochrome P450.
o Phase II reaction
o These reactions involve covalent attachment of small hydrophilic endogenous molecule such as glucuronic
acid, sulfate, or glycine to form water-soluble compounds, that are more hydrophilic.
o This is also known as a conjugation reaction.
o The final compounds have a larger molecular weight.

24. 24
Microbial Transformation of Steroids
o Hormones of adrenal cortex (cortisone, cortisol, corticosterone)
o Progestational hormone (progesterone)
o Androgens or male sex hormones (testosterone, dihydrotestosterone)
oEstrogens or female sex hormones (estradiol, estrone, etc.)
o Steroidal transformation is carried out by:
– Oxidation
– Reduction
– Hydrolysis
– Isomerization
– Aminations
– Esterification

25. 25
Progesterone is an endogenous steroid
and progestogen sex hormone involved
in the menstrual cycle, pregnancy, and
embryogenesis of humans and other
species.
Transformation of Progesterone by different fungi

26. 26
Transformation of Glycerol and Prostaglandins
Dihydroxyacetone from glycerol:
Prostaglandins:
These prostaglandins can be produced
from unsaturated fatty acids by microbial
transformation with pathogenic fungi
such as Cryptococcus neoformans.
Gluconobacter melanogenus

27. 27
Transformation of D- Glucose to L-Ascorbic acid (vitamin C)
The process for the production of L-
ascorbic acid is called Reichstein-
Grussner synthesis. This process was
devised by Nobel Prize winner
Reichstein and his colleagues in
1933. This L-ascorbic acid is also
used as an antioxidant in food
industries.
Gluconobacter sp.
Erwinia sp.
Corynebacterium sp.

28. 28
Transformation of Pollutants
o Both aerobic and anaerobic bacterial genera found have been associated with the
biotransformation of a wide range of xenobiotic chemicals. Bacillus, Pseudomonas, Escherichia,
Rhodococcus, Gordonia, Moraxella, Micrococcus are members of the aerobic genera
o while the anaerobic types includes Methanospirillum, Pelatomaculum, Syntrophobacter,
Desulfotomaculum, Syntrophus, Desulfovibrio and Methanosaeta. Mycobacterium vaccae have
been demonstrating the capabilities to catabolize acetone, cyclohexane, styrene, benzene,
ethylbenzene, propylbenzene, dioxane, and 1,2-dichloroethylene.
o Anaerobic methanogens (Methanospirillum hungatei, Methanosaeta concilii, Syntrophobacter
fumaroxidens) are mainly involved in the degradation of Phthalate (plasticizers) compound.

29. 29
The microbes degrade oil or other organic contaminants like Polycyclic
Aromatic Hydrocarbons (PAHs) either by aerobic biodegradation or
anaerobic biodegradation.
Aerobic metabolism
Microbes use O2 in their
metabolism to degrade
contaminants
Anaerobic metabolism
Microbes substitute another
chemical for O2 to degrade
contaminants.
Nitrate, iron, sulfate, carbon
dioxide, uranium, technicium,
perchlorate

30. 30
Microbial Transformation of Dalapon to Pyruvate
Herbicide dalapon (a chlorinated fatty acid) is converted by Arthrobacter species into pyruvate.
Metabolism: Recalcitrant compounds can serve as substrates for microbial
growth and energy production.

31. 31
Azo Dyes
o Azo compounds are compounds bearing the functional group R–N=N–R′, in which R and R′
can be either aryl or alkyl.
o Azo compounds have vivid colors, especially reds, oranges, and yellows. Therefore, they are
used as dyes, and are commonly known as azo dyes.
o Azo compound degradation is done by three key enzymes
1. Azo reductase
2. Peroxidase
3. Laccase
o These enzymes are collected from bacterial species like Bacillus subtilis, Pseudomonas
aeruginosa and Psuedomonas putida.
o Some strains of Acetobacter and Klebsiella have also been able to bio-fix carcinogenic azo
compounds.
General formula of
azo compounds

32. 32
Research Aspects of Microbial Transformation
1. Organism
2. Sterilization
3. Aeration and stirring
4. Diffusion
5. Elimination of side reactions
6. Product isolation
7. Large-scale biotransformation

33. 33
Conversion and Interference
1) Conversion of uncommon substrates
2) Interference with metabolic pathways
3) Interference with biotransformation
4) Interference with biosynthesis

34. 34
Current Trends of Biodegradation
o Modifications have to be made using genetic engineering to develop such a strain of microbe that
can degrade a specific type of pollutant all by itself.
o Such processes include plasmid transfer by conjugation, transduction and transformation,
recombinant technology etc. By doing so microbial degradation can be made more efficient, cheap
and easy to use.
o The modification of molecular mechanisms involves:
o mutation
o transfer of genes through plasmids e.g. TOL plasmids, pAC21, pAC25