Paper No: 15 Environmental Microbiology & Biotechnology
Module: 6 Microbial Biotransformation
Development Team
Principal Investigator
Prof. R.K. Kohli
&
Prof. V.K. Garg &Prof.AshokDhawan
Co- Principal Investigator
Central University of Punjab, Bathinda
Dr Babita Khosla,
Paper Coordinator
MDU, Rohtak
Dr. Yogalakshmi K N and Dr. K N Sangeetha
Content Writer Central University of Punjab, Bathinda; Stem cell
research Centre, Stanley medical college
Content Reviewer Prof. VK Garg
Central University of Punjab, Bathinda
Anchor Institute Central University of Punjab 1
Environmental Microbiology & Biotechnology
Environmental
Sciences Microbial Biotransformation
Description of Module
Subject Name Environmental Sciences
Paper Name Environmental Microbiology & Biotechnology
Module Name/Title Microbial Biotransformation
Module Id EVS/EP-XV/6
To have basic knowledge in science and interest to learn about biotransformation involving
Pre-requisites
microorganisms
1. To familiarize the concept of biotransformation
2. To study about microbial biotransformation
3. To familiarize about the enzymes involved in microbial biotransformation
Objectives
4. To understand the merits and demerits associated with microbial biotransformation
5. To discuss the role of microbial biotransformation in transforming various compounds and
pollutants
Biotransformation, xenobiotics, microbial biotransformation, transformation of pollutants,
Keywords
transformation of steroids, pesticides, petroleum
Environmental Microbiology & Biotechnology
Environmental
Sciences Microbial Biotransformation
Module 6: Microbial Biotransformation
Objectives:
1. To familiarize the concept of biotransformation
2. To study about microbial biotransformation
3. To familiarize about the enzymes involved in microbial biotransformation
4. To understand the merits and demerits associated with microbial biotransformation
5. To discuss the role of microbial biotransformation in transforming various compounds and
pollutants
1. Biotransformation
Biotransformation is the structural modification of a chemical compound by organisms /enzyme systems
that lead to the formation of molecules with relatively greater polarity. The chemical process involved
in the modification of endogenous or exogenous chemicals into water soluble chemicals aid in the easy
elimination of the toxic/foreign chemicals from the body. Klaus Kieslich (1984) defined the term
biotransformation as “chemical reactions by microorganisms or enzymes”. To differentiate
biotransformation from bioconversion and biodegradation, the definition was later modified to include
two major points: the substrate used in biotransformation must be a foreign material and atleast one
product should hold the structure of the parent material (Lilly 1984). Natural Biotransformation
mechanisms are catalyzed by enzymes in the liver and other tissues. However, the natural transformation
process is slow, nonspecific and less productive. Biotransformation reactions catalysed by microbes are
called as microbial biotrasformation which was developed to acclimatize to environmental changes
making them useful in several biotechnological processes. The most significant aspect of
biotransformation is that it maintains the original carbon skeleton after obtaining the products.
Biotransformations can also be used to synthesize compounds or materials where synthetic approaches
are challenging. Moreover, microbial transformations are carried out in mild conditions, high regio- and
stereo-specificity. Biotransformation results in the generation of derivatives that are difficult to be
prepared by chemical or any other means.
Environmental Microbiology & Biotechnology
Environmental
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2. Microbial transformation
Microorganisms have been used with considerable success in biotechnological applications and in
chemical industries. They possess the capability to enzymatically modify a wide range of organic
compounds. Microbial biotransformation is the process of harnessing the microbial enzymes to catalyze
the conversion of organic compounds into structurally related products. In other words, microbial
biotransformation deals with microbial (enzymatic) conversion of a substrate into a product with limited
number (one or a few) of enzymatic reactions. This process is in contrast to fermentation process which
often is complex and involves large number of reactions.
Targeted microbial biotransformation have emerged only in the 19th century where the microbial
enzymes have been exploited and great advances have been made in the transformation of several
reactions that have industrial applications. For example, yeast mediated transformations have been
extensively and commonly used for the production of bread, dairy products and alcoholic beverages.
Microbial transformation offers many advantages over the conventional chemical methods. Many types
of chemical reactions such as oxidation, reduction, hydrolysis, condensation, isomerization, formation
of new C—C bonds, synthesis of chiral compounds and reversal of hydrolytic reactions occur in the
process of microbial biotransformation. Among these, oxidation, reduction, isomerization and
hydrolysis reactions are more commonly observed.
In the biotransformation reaction, the conversion time required is dependent on the type of reaction, the
substrate concentration and the microorganism used. A wide variety of biological catalysts are being
used for microbial biotransformation reactions. These include growing cells, resting cells, killed cells,
immobilized cells, cell-free extracts, enzymes and immobilized enzymes. In most biotransformation
reactions, the desired end products are extracellular and may be either in a soluble or suspended state.
The bio transformed product can be recovered by techniques such as precipitation by salts, extraction
with solvents, adsorption to ion-exchangers etc. The volatile products can be recovered by direct
distillation from the medium.
Environmental Microbiology & Biotechnology
Environmental
Sciences Microbial Biotransformation
Table 1 Illustrates the enzymes catalysing the microbial biotransformation reaction
Enzyme group Reaction types Potential products
Oxidoreductase Oxidation Alcohol, Epoxide, Lactone, Amino
Reduction Acid, Sulphoxide
Alcohol, Lactone
Transferase Hydroxy methyl transfer Hydroxyamino acid
Amino group transfer Amino acid, Amine
Hydrolase Ester hydrolysis Alcohol, Carboxylic acid,
Trans-esterification Carboxylic ester
Nitrile/amide hydrolysis Alcohol, Carboxylic acid,
Hydantoin hydrolysis Carboxylic ester
Alkylhalide hydrolysis Carboxylic acid
Amino acid
Haloalkanoic acid, Alcohol, Epoxide
Lyase C-C bond formation Amino acid, Acyloin, Cyanohydrin
C-O bond formation Alcohol, Amino acid
C-N bond formation Amino acid
Isomerase Lactone formation Lactone
(Source: Sachin Shah 2006)
Table 2 List of biotransformation reactions catalysed by microorganisms are detailed below
S.No. Type of reaction Example Commonly used
microorganism
1. Oxidation Tryptophan 5 hydroxytruptophan Bacillus subtitis
Naphthalene Salicylic acid Corynebacterium spp
2. Reduction Benzaldehyde Benzyl alcohol Saccharomyces
cerevisiae
Environmental Microbiology & Biotechnology
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Sciences Microbial Biotransformation
Nitropentachlorobenzol Streptomyces
Pentachloroaniline aureofaciens
3. Hydrolysis Anhydrotetracycline Tetracycline Streptomyces
aureofaciens
Menthyl laureate Menthol Mycobacterium phlei
4. Condensation Streptomycin Steptomycin Streptomyces griseus
phosphate
(Source: Sachin Shah 2006)
The White biotechnology involves the use of microbial biotransformation for generating products of
interest. Living cells such as bacteria, filamentous fungi, animals, plants, algae, yeast and actinomycetes
are used.
Microbial cells are ideal choice for biotransformation due to certain reasons like:
Surface-volume ratio: Microbial biotransformation has high surface-volume ratio.
Growth Rate: Higher growth rate of microbial cells reduces the time of biomass transformation.
Metabolism Rate: Higher rate of the metabolism in microbes leads to efficient transformation of
substrate.
Sterility: It is easier to maintain sterile conditions when microbes are used.
2.1 Advantages of microbial biotransformation
For centuries microbial biotransformation has proved to be an imperative tool in alleviating the
production of various chemicals used in food, pharmaceutical, agrochemical and other industries.
Microbes have been widely applied for steroid biotransformation to prepare specific derivatives,
the production of which is difficult by traditional synthetic methods.
In pharmaceutical research and development, biotransformation studies have been extensively
applied to investigate the metabolism of compounds using animal models.
Environmental Microbiology & Biotechnology
Environmental
Sciences Microbial Biotransformation
The microbial biotransformation phenomenon is commonly employed in comparing metabolic
pathways of drugs and scaling up the metabolites of interest discovered in these animal models
for further pharmacological and toxicological evaluation.
Biotransformation is good solution for environmental problems like degradation of xenobiotics
and petroleum hydrocarbons as they are real threat in recently.
It is a cost-effective method.
Microbial biotransformation aids in easy synthesis of complex molecules (steroids).
It has been widely used in the industrial production of testosterone and estrogen.
Transformations are carried out with very efficient biocatalyst (enzyme/ whole cells) and are
environmentally acceptable.
Biocatalyst operates under mild conditions of ambient temperatures 20 to 40 ºC, atmospheric
pressure and at or near neutral pH in the range 5.0 to 8.0.
As biocatalyst is compatible, several biotransformation reactions can be performed in one flask.
It is applicable for the sequential reactions using multi-enzyme system as the isolation of unstable
intermediates can be omitted.
They showed specific selectivity, chemoselectivity, regioselectivity and enantioselectivity.
Biocatalyst can carry out reactions which is not possible through conventional chemical
synthesis or not economically feasible by traditional chemical synthesis.
Biotransformations are producing a “nature-like”, biodegradable compounds which is “Green
Chemistry”. They are widely used in the transformation of various pollutants or a large variety
of compounds including hydrocarbons, pharmaceutical substances and metals.
2.2 Disadvantages:
In biotransformation, biocatalyst requires narrow operation parameters. If a reaction proceeds
slowly under given parameters, there is only a narrow scope of alteration as it leads to
deactivation of the enzyme/protein.
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Biocatalyst reaction shifting from an aqueous medium to an organic medium is most of the time
not feasible and reduces its activity. Biocatalyst displays its highest catalytic activity in aqueous
condition, which is the least desired solvent for most of organic compound.
Many biocatalytic reactions are prone to substrate or product inhibition, which cause the
enzymes / whole cells to cease to work at higher substrate or product concentrations.
Enzymes as a biocatalyst are provided by nature in one enantiomeric form and sensitive to
environmental extremes of temperature, pH etc.
Some biocatalysts may cause allergies.
2.3 Applications of Microbial Biotransformation
2.3.1 Transformation of steroids and sterols
Steroids are a class of natural products that is widely distributed throughout nature. They are present in
bile salts, adrenal-cortical and sex-hormones, insect molting hormones, sapogenins, alkaloids and some
antibiotics. All steroids have the same basic structure, a cyclopentanoperhydrophenanthrene which
consists of four fused rings. The steroid molecule has several asymmetric centres and it makes the total
synthesis of steroid very difficult. Testosterone was the first steroid to be produced by microbial
biotransformation from dehydroepiandrosterone by using Corynebacterium sp. The process of microbial
biotransformation of steroid increases its biological activity.
Example: Cortisone is an anti-inflammatory drug prescribed for rheumatold arthritis and skin diseases.
Structural modification of the cortisone molecule by incorporating a 1, 2 double bond in ring A to
produce prednisone markedly enhances its anti-inflammatory effect.
2.3.2 Transformation of Non-Steroid Compounds
Dihydroxyacetone is widely used in lotions and cosmetics. Gluconobacter melanogenus, an acetic acid
bacteria is used in the microbial transformation of glycerol from Dihydroxyacetone. Prostaglandins are
unsaturated fatty acids/hormones that have a lot of medical significance with wide application as
contraceptive, alleviating pain during childbirth, treatment of congenital heart failure and digestive
diseases. Prostaglandins can be produced from unsaturated fatty acids by microbial transformation by
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Cryptococcus neoformans, a pathogenic fungi. L-Ascorbic acid (vitamin C), an anti-oxidant with wide
application is synthesized by Reichstein-Grussner synthesis through microbial conversion carried out
by Acetobacter suboxydans.
2.3.3 Transformation of Antibiotics
The microbial transformation of existing antibiotics has been done with aim of developing new,
modified and improved antibiotics which contain many qualities like reduced toxicity, broad anti-
microbial spectrum, enhanced oral adsorption, less resistant/ allergic effects. In most cases, any
transformation step causes a partial or complete inactivation of antibiotic.
Indirect transformation: In controlled biosynthesis, altered antibiotics are produced in the presence of
inhibitors or modified precursors in the medium.
Direct transformation: Hydrolysis of the functional groups led to inactivation of the antibiotics.
2.3.4 Transformation of Pesticides
Microbial transformation is of interest not for the production of new active agents, but for the greatest
possible detoxification of the environment. Use of pesticides for the control of plant disease and pests
is necessary for improving the agricultural production. Pesticides are compounds with high stability and
are used for the vector control programs, but this stability has a negative effect on the environment.
Detoxification and removal of xenobiotics from ecosystem can be accomplished by enzymatic
conversions.
Metabolism: Xenobiotics can serve as substrates for microbial growth and energy production. Complete
breakdown of some substrates to carbon dioxide and water.
Cometabolism: Cometabolism normally causes slight modification of the molecules, which may result
in either a decrease or an increase in toxicity. Microorganisms involved in it do not obtain energy from
the transformation reaction and require another substrate for the growth. Complete breakdown of a
compound can be achieved through the combined action of different organisms. For example,
dehalogenation reactions are important cometabolism reactions which may make pesticide molecules
accessible for further breakdown. Compounds with complicated structure and high degree of
Environmental Microbiology & Biotechnology
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halogenation are not easily attacked by microorganisms eg. chlordecone, a hexachlorocyclopentadiene
derivative is a potent excellent insecticide that cannot be easily transformed.
2.3.5 Transformation of Pollutants
Recently, biotransformation of various pollutants using microorganisms has garnered attention to clean
up the polluted environment. The catabolic diversity of microbes have been utilized for the
bioremediation of a huge range of compounds including polyaromatic hydrocarbons (PAHs),
pharmaceutical substances, radionuclides, hydrocarbons (e.g. oil) and polychlorinated biphenyls
(PCBs). The main disturbing trend is the toxicity threat these pollutants pose to the public health.
Although most organisms have detoxifying abilities, microbes especially bacteria has an important part
to play in this process. High growth rate, metabolic diversity and horizontal gene transfer help them to
grow and adjust according to the ever changing environmental conditions. Both aerobic and anaerobic
bacterial genera are found to be associated with the biotransformation of a wide range of xenobiotic
chemicals. Example of aerobic bacteria are Bacillus, Pseudomonas, Escherichia, Rhodococcus,
Gordonia, Moraxella, Micrococcus 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. Pseudomonas and
Bacillus degrade PCB (Polychlorinated Biphenyls) very efficiently. Some strains of Pseudomonas,
Acetobacter and Klebsiella have also been able to bio-fix carcinogenic azo compounds. Pseudomonas
BCb12/1 and BCb12/3 have exceptional degradation capabilities of low ethoxylated NPnEO (Non
phenol Polyethoxylates). Anaerobic methanogens (Methanospirillum hungatei, Methanosaeta concilii,
Syntrophobacter fumaroxidens) are mainly involved in the degradation of Phthalate compound.
2.3.6 Petroleum Biotransformation
Major concerns these days is the hydrocarbon contamination resulting from the activities related to the
petrochemical industry release, the main source of soil and water pollution. Petroleum oil is carcinogenic
and neurotoxic for all living forms. Bioremediation using microbes is the most efficient method to
detoxify the pollutants because it leads to complete mineralization and is cost effective. Many aquatic
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and marine microflora, bacteria, yeast and fungi have been shown to play an important role in the
biodegradation of oil spills by breaking down oil contaminants into nontoxic forms.
Bacterial genera such as Rhodococcus, Pseudomonas, Arthrobacter, and Mycobacterium are active
degraders of alkylaromatic degradation of petroleum hydrocarbons. Bacterial genera, namely, Gordonia,
Brevibacterium, Corynebacterium sp., Flavobacterium sp., Pseudomonas fluorescens, P. aeruginosa,
Actinocorallia, Klebsiella, Rhizobium, Bacillus sp and Alcaligenes sp, Aeromicrobium, Dietzia,
Burkholderia and Mycobacterium and fungal genera namely Aspergillus, Penicillium, Talaromyces,
Amorphoteca, Neosartorya, Cephalosporium have been identified to play an important role in
bioremediation of oil spills.
2.4 Summary
At the end of this module we have learnt about
• Concept of biotransformation and microbial biotransformation
• Various enzymes involved in microbial biotransformation
• Merits and demerits of microbial biotransformation
• Microbial biotransformation of pesticides, pollutants, petrol, steroids and non-steroids and
antibiotics
References
1. Kieslich K (1984) Biotechnology. A Comprehensive treatise in 8 volumes. Rehm H-J and Reed G.
(eds.) Biotransformation 6: 1.
2. Lilly MD (1984) Advances in biotransformation processes. Trans Inst Chem Eng 72: 27-34
3. http://shodhganga.inflibnet.ac.in/bitstream/10603/2219/12/12_chapter%201.pdf
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