Transcript biotechnology (3) _m..

2007-2008
BIOTECHNOLOGY
B.
BIOLOGICAL FUELS
1 The need for biological fuels
2 Raw materials
These include wastes and crops;
wastes
Dry Wastes
Wet wastes
Crops
In the future, crops may be grown specially for
energy production, perhaps on land unsuitable
for growing foodstuffs. Sugar cane is already
being grown in Brazil for this purpose.
 A.
ETHANOL PRODUCTION
1- Substrates include sugar cane, cassava roots,
cellulose waste and corn.
Cassava roots contain starch which must be
hydrolysed to sugars, and cellulose waste, such as
timber and straw, needs quite complex pre-treatment
with ligno-cellulase enzymes or chemicals.
2- At present, alcohol production is similar to the
traditional process but much research is taking
place.
It is hoped that more efficient, genetically engineered
M.O.s will be developed and that newer fermentor
designs and immobilized enzyme technology will
improve efficiency.
3- Distillation costs can be reduced by using a
cheap fuel, and bagasse (the waste from sugar cane)
has proved to be an economical fuel for raising
steam for the process by combustion.
4- A range of M.O.s have been used in the production
of ethanol, using many different carbohydrates as
substrate.
Traditionally, ethanol production has relied upon the
use of yeasts, mostly Saccharomyces species.
5- Zygomonas mobilis has been used in South
America for many years in the production of tequila,
and in Indonesia and Africa to make palm wine.
However, its use in the western world is quite
new. Recent research into Zygomonas has
shown that it is more efficient than yeasts in
converting sugar to ethanol.
6- A technique has been developed to produce
ethanol using Zygomonas in a continuous
culture
process,
rather
than
traditional batch culture methods.
the
more
6.4
The production of methane
(1) Sewage
(2) Urban waste, landfill gas
(3) Biogas fermentors
However, while this is a useful small-scale
process, it is unlikely to be commercially
avaible on a large scale because:
methane can be produced far more cheaply
from coal at present;
natural gas is cheaper than microbially
produced methane.
There are many natural sources of methane
Gas is expensive to store, transport and
distribute at present.
It is expensive and difficult to liquefy.
(4) Agricultural wastes
animal
manure
and
Some farms now place
other
crop
residues
into
anaerobic digestion tanks.
Here, the waste is fermented by M.O.s and the
methane produced is collected, liquefied and used to
power farm machinery.
In some cases it may be used to fire boilers, which
heat
glasshouses
and
produce
early
tomatoes, peppers and other vegetables.
crops
of
C. PHARMACEUTICALS PRODUCED BY M.O.S:
1.
Dextrans
Dextrans are polysaccharides produced by
lactic acid bacteria,
in particular members of
the genus Leuconostoc (e.g. L. dextranicus
and mesenteroides) following growth on
sucrose.
 2-
Vitamins, amino acids and organic acids
1.
Vitamins
Vitamin B2 (riboflavin) is a constituent of yeast
extract and incorporated into many vitamin
preparations.
Vitamin B2 deficiency is characterized by symptoms
which include an inflamed tongue, dermatitis and
a sensation of burning in the feet.
2.
Amino acids
Amino acids find applications as ingredients
of
infusion
solutions
for
parenteral
nutrition and individually for treatment of
specific conditions.
They are obtained either by fermentation
processes similar to those used for antibiotics
or in cell-free extracts employing enzymes
isolated from bacteria.
3. Organic acids
Examples of organic acids (citric, lactic,
gluconic) produced by M.O.s.
Citric and lactic acids also have widespread
uses in the food and drink and plastics
industries, respectively.
Gluconic acid is also used as a metalchelating agent in, for example, detergent
products.
3
Iron-chelating agents
Growth of many M.O.s in iron-deficient growth
media results in the secretion of low molecular
weight
iron-chelating
agents
called
siderophores, which are usually phenolate or
hydroxamate compounds.
-The therapeutic potential of these compounds
has generated considerable interest in recent
years.
4
 1-
Enzymes
Streptokinase and streptodornase
Mammalian blood will clot spontaneously if
allowed to stand: however, on further standing,
this clot may dissolve as a result of the action of
a proteolytic enzyme called plasmin.
Plasmin is normally present as its inactive
precursor, plasminogen.
Streptokinase
is
administered
by
intravenous or intra-arterial infusion in
the
treatment
disorders.
of
thrombo-embolic
2
-L-
- L-Asparaginase
Asparaginase, an enzyme derived from E. coli or
Erwinia carotovora, has been employed in cancer
chemotherapy where its selectivity depends upon
the essential requirement of some tumors for the
amino acid L-asparagine .
- Normal tissues do to require this amino acid and
thus
the
enzyme
is
intention of depleting
administered
with
the
tumor of asparagine by
converting it to aspartic acid and ammonia.
3
- Neuraminidase
-Neuraminidase
been
used
derived from Vibrio cholerae has
experimentally
to
increase
the
immunogenicity of tumour cells.
-It is capable of removing N-acetylneuraminic
(sialic) acid residues from the outer surface of
certain
tumor
cells,
thereby
exposing
new
antigens which may be tumor specific together
with
a
concomitant
immunogenicity.
increase
in
their
--In
lab
animals
administration
of
neuraminidase-treated tumour cells was
found to be effective against a variety of
mouse leukaemias.
4
-
β-Lactamases
β-Latamase
enzymes,
whilst
being
a
considerable nuisance because of their ability
to confer bact. resistance by inactivating
penicillins and cephalosporins are useful
in the sterility testing of certain antibiotics
and, prior to culture, in inactivating various
β-lactams in blood or urine samples in
patients undergo therapy with these drugs.
-
One
other
application
is
important
therapeutic
the
of
rescue
patients
presenting symptoms of a severe allergic
reaction following administration of a
lactamase - sensitive penicillin.
β-

3- APPLICATIONS OF M.O.S IN THE PARTIAL
SYNTHESIS OF PHARMACEUTICALS:
3.1
Production of antibiotics
Alexander Fleming's accidental discovery of
penicillin in 1929 is well known.
He found the mould Penicillium notatum
contaminating a Petri dish of pathogenic
bacteria and inhibiting their growth.
He isolated penicillin but it was not until the
Second World War that it was successfully
produced on a large scale.
At first, it was grown in static liquid culture in
flasks, shallow pans and bottles, but this process
was inefficient and it was not possible to
produce enough penicillin to meet demand.
Two
theories have been proposed to
explain antibiotic production.
1- Antibiotics are secondary metabolites, so
they may be produced to keep enzyme
systems operative when the microbe has run
out of nutrients and cell division is no
longer possible.
Normally, when the substrate has been used
up, the enzymes of that particular pathway
would be broken down.
-Then,
if a new nutrient supply was found, there
would be a delay while the necessary enzymes
were produced.
-It
has been suggested that making a secondary
metabolite keeps the enzymes active, so that
the microbe can quickly take advantage of any
new food supply.
-2-
Some scientists think antibiotic production is
for ridding of the cell toxic metabolic waste.
-
-
Although
not
toxic
to
the
organism
producing them, these substances could still
be highly toxic to other M.O.s.
-If
the toxin phenylacetic acid is added to a
culture of Penicillium, penicillin production is
increased. This observation supports this
theory.
- It is of course, possible that both theories are
correct since they are not contradictory.

THE INDUSTRIAL PRODUCTION OF
ANTIBIOTICS;
 PENICILLIN
PRODUCTION
1- M.O. the organism used for production of penicillin
was Penicillium notatum, but the mostly common
used is P. chrysogenus .
2- Inoculum Preparation; a pure inoculum in
sufficient
volume
and
in
the
fast
growing
(logarithmic) phase so that a high population
density is soon obtained.
3- The fermenter; A typical fermenter is closed,
vertical, cylinderical, stainless steel vessel with
convexly dished ends and 25 - 250 m3 capacity.
The height is usually two to three times its
diameter.
4- Oxygen supply; Penicillin fermentation need
oxygen, which is supplied as filtered sterilised air
from a compressor.
5-
Temperature
control;
The
production
of
penicillin G is very sensitive to temperature, the
tolerance being less than 1 C.
Heat is generated both by the metabolism of
nutrients and by the power dissipated in stirring,
and has to by removed by controlled cooling.
6- Defoaming agents; The fermenter system stirred
vigorously and aerated usually foam, so provision
has to made for adding defoaming agents.
7- Instrumentation; The vessel is fitted with
several probes to detect foaming, temperature,
pH, O2-tension and exhaust gas.
8- Media; Not all the nutrients required during
fermentation are initially provided in the culture
medium.
Provision is therefore made to add these while the
fermentation is in progress. The media used is corn
steep liquor (CSL).
9- Transfer and sampling systems; Appropriate
pipework is provided to transfer the inoculum to
the vessel, to allow taken routine sample and to
transfer the final content to the extraction
plant.
10- The optimum temperature and pH for growth
are not those for penicillin production they must
be changed during the process.
11- The production phase begin with the
addition of phenylacetic acid (PAA).
12- PAA supplies the side chain of penicillin G.
13- PAA is toxic for the M.O so it must be
supplied
in
small
quantities
without
approaching the toxic level.
14- Termination; The harvest is carried out
shortly after the first signs of faltering in the
efficiency of conversion of the most costly raw
material to penicillin.
15A-
Removal
of
Extraction:
the
cell;
penicillin
G
is
extracellular the first step is to remove the cells
by filtration.
B- Isolation of penicillin G; Penicillin G is very
unstable, so it must be quickly extracted by
organic solvent (amyl acetate) from the acidified
aqueous solution.
C- Treatment of crude extract; first formation of
an appropriate salt, charcoal treatment to remove
pyrogens and sterilization by using dry heat.
Interferons are antiviral chemicals, which also
have some tumour inhibiting properties.
These used to be extracted from human fibroblast
cells, but yields were minute.
Recombinant DNA methods have now been used to
synthesize interferons using a suitable bacterium,
such as Escherichia coli. Some other anti-tumour
pharmaceuticals are also made microbiologically.
An example is bleomycin, a glycopeptide, made by
Streptomyces verticillus. This drug has the ability
to disrupt the DNA and RNA of tumour cells.

Steroid biotransformation
Since steroid hormones can only be obtained in
small
quantities
directly
from
mammals,
attempts were made to synthesize them from
plant sterols which can be obtained cheaply and
economically in large quantities.
However,
all
adrenocortical
steroids
are
characterized by the presence of an oxygen at
position 11 in the steroid nucleus.
-More
recent
employment
of
advances
M.O.s
in
involving
the
biotransformation
reactions utilize immobilized cells (both living
and dead).
-
Immobilization of microbial cells, usually by
entrapment in a polymer gel matrix, has several
important advantages.
Chiral
inversion
Several clinically used drugs, e.g. salbutamol (a
β-adrenoceptor
agonist),
adrenoceptor
antiagonist)
propranolol
and
(a
the
β2-
arylpropionic acids (NSAIDs) are employed in
the racemic form.
-
It
has
thus
been suggested that the
enantiomerically pure S(+) form could be
administered clinically
to
give a reduced
dosage and possible less toxicity.

4-
USE OF M.O.S AND THEIR PRODUCTS
IN ASSAYS
Microbiological
assays
In microbiological assays the response of a growing
population of M.O.s to the antimicrobial agent is
measured.
The usual methods involve agar diffusion assays, in
which the drug diffuses into agar seeded with a
susceptible microbial population and produces a zone
of growth inhibition.
In the commonest form of microbiological
assay used today, samples to be assayed are
applied in some form of reservoir (porcelain
cup, paper disc or well) to a thin lay of agar
seeded with indicator organism.
The drug diffuses into the medium and after
incubation a zone of growth inhibition forms, in
this case as a circle around the reservoir.
 Vitamin
-The
and amino acid bioassays
principle of microb. bioassays for growth factors
such as vitamins and amino acids is quite simple.
-Unlike
antibiotic assays which are based on studies
of growth inhibition, these assays are based on growth
exhibition.
- All that is required is a culture medium which is
nutritionally adequate for the test M.O. in all
essential growth factors except the one being assayed.
-If
a range of limiting concentrations of the test
substance is added, the growth of the test M.O. will be
proportional to the amount added.
Carcinogen
-A
and mutagen testing
carcinogen is a substance which causes living
tissues to become carcinomatous (to produce a
malignant epithelial tumor).
-A
mutagen is a chemical (or physical) agent which
induces mutation in a human (or other) cell.
The Ames test
The Ames test is used to screen a wide variety
of chemicals for potential carcinogenicity or as
potential cancer chemotherapeutic agents.
-The
test enables a large No. of compounds to be
screened rapidly by examining their ability to
induce
bacterial
mutagenesis
mutants
typhimurium.
in
specially
derived
from
constructed
Salmonella
 Use
of microbial enzymes in sterility testing
- Sterile pharmaceutical preparations must be tested
for the presence of fungal and bacterial contamination
before use.
-If
the preparation contains an antibiotic, it must be
removed or inactivated where membrane filtration is
the usual recommended method.
-
However, this technique has certain disadvantages.
Accidental contamination is a problem, as is the
retention of the antibiotic on the filter and its
subsequent liberation into the nutrient medium.
6
Insecticides
- Like animals, insects are susceptible to infections
which may be caused by viruses, fungi bacteria or
protozoa.
- The use of M.O.s to spread diseases to particular
insect pests offers an attractive method of bio-control,
particularly in view of the ever-increasing incidence of
resistance to chemical insecticides.
- However, any M.O. used in this way must be highly
virulent, specific for the target pest but non-
pathogenic to animals, man or plants.

MICROBIAL DEGRADATION
- Biodegradation and biodeterioration
The use of M.O.s to break down substances is usually
called biodegradation.
However, M.O.s often break down substances in a
way that is not beneficial to humans, for example in
causing food spoilage.
This activity is generally called biodeterioration.
Sewage
Sewage is composed of the following:-
a- Human waste made up of human excreta
mixed with waste household water.
This contains many M.O.s including potential
pathogens.
A major pollutant from waste household water
is detergent, which causes persistent foam and
has high levels of phosphates.
b- Industrial wastes which are variable in
nature, depending on the industry.
Some can be very toxic to M.O.s and must
undergo pretreatment so that they do not kill
or inhibit the M.O.s which degrade the
sewage.
Many industries are required to treat their
own sewage, either wholly or partially.
c- Road drainage consists of rain water
together with grit and other debris which
enters the sewers from roadside gutters.
Sewage
treatment
Sewage is treated in two or three stages as
follows.
Primary
treatment.
Materials which will settle out are removed.
The sedimented solids pass on to a digester for
further treatment, while the liquid (effluent)
continues into the secondary treatment stage .
Secondary treatment.
Aerobic M.O.s are used to break down most of the
organic matter in the effluent. Any sludge produce in
this process is passed on to anaerobic digesters.
Tertiary treatment
This involves chemical and biological treatment which
renders the sewage effluent fit for drinking. However,
this is a very expensive treatment, so it is only carried
out when absolutely necessary.
There are two main reasons for treating sewage.
Firstly, sewage can contain pathogens which
cause
diseases,
(typhoid),
such
as
pathogenic
(gastroenteritis)
and
Salmonella
Escherichia
Ascaris
typhi
coli
lumbricoides
(roundworm).
Secondly, by treating sewage, pollution of the
environment can be avoided.
Microbial
Mining
- Some bacteria are useful in extracting
metals from low-grade ores.
- This is because they are chemoautotrophic
which means they derive their energy from
inorganic chemicals.
- Bacteria of the genus Thiobacillus are used
commercially to extract copper and uranium
from otherwise uneconomic reserves.
-Cobalt,
lead and nickel may also be extracted
in this way in the near future.
The extraction process may require extremes of
environmental conditions, such as heat and
pH.
Genetic engineering techniques are being used
to confer acid- and heat resistance on these
M.O.s.
Problems
of biologically active
biotechnology products:
Vaccines and antibiotics are obvious examples of
biologically active products, and care must be
taken to prevent their indiscriminate dispersal.
Contaminants in otherwise safe processes may
produce toxic molecules that could become
incorporated into final products, leading to food
poisoning.
Allergenic reactions to produce formulations
must also be guarded against.
Overuse of antibiotics in agriculture could lead
to carry-over into human foods, resulting in
possible development of antibiotic resistance in
human disease organisms.
Many countries now restrict the use of antibiotics
in agriculture.