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08BIOTECHNOLOG

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B. BIOLOGICAL FUELS

1The need for biological fuels2Raw materialsThese include wastes and crops;

wastes Dry Wastes Wet wastesCrops

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 the

more traditional batch culture methods.

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 Some farms now place

animal manure and 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 crops

of tomatoes, peppers and other vegetables.

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 acidsAmino 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 acidsExamples 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 metal-

chelating agent in, for example,

detergent products.

3 Iron-chelating agentsGrowth 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 Enzymes 1- 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 of thrombo-embolic

disorders.

2 - L-Asparaginase- L- 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 administered with the

intention of depleting tumor of asparagine

by converting it to aspartic acid and ammonia.

3 - Neuraminidase- Neuraminidase derived from Vibrio cholerae

has been used 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 increase in their

immunogenicity.

--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 important therapeutic

application is the rescue of patients

presenting symptoms of a severe allergic

reaction following administration of a β-

lactamase - sensitive penicillin.

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.

15- Extraction:A- Removal of 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 advances involving the

employment of M.O.s in 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), propranolol (a

β-adrenoceptor antiagonist) and the 2-

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 and amino acid bioassays

- The 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 and mutagen testing- A 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 mutagenesis in specially

constructed bacterial mutants derived from

Salmonella typhimurium.

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.

- It must be economical to produce, stable on storage and

preferably rapidly acting. Bacterial and viral pathogens

have so far shown the most promise.

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, such as Salmonella typhi

(typhoid), pathogenic Escherichia coli

(gastroenteritis) and Ascaris 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.