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Biotechnology

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Immobilization of microbial cells, usually by entrapment in a polymer gel matrix, ... BIOTECHNOLOGY INDUSTRIAL AND APPLIED MICROBIOLOGY Author: SYSTEM – PowerPoint PPT presentation

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Title: Biotechnology


1
Biotechnology
2
  • 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.

3
  • 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.

4
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.
5
  • 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.

6
  • 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.

7
  • 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

8
  • 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.

9
  • (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.

10
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.

11
  • 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.

12
  • 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.

13
  • 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 metal-chelating
    agent in, for example, detergent products.

14
  • 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.

15
  • 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.

16
Streptokinase is administered by intravenous or
intra-arterial infusion in the treatment of
thrombo-embolic disorders.
17
  • 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.

18
  • 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.

19
  • -In lab animals administration of
    neuraminidase-treated tumour cells was found to
    be effective against a variety of mouse
    leukaemias.

20
  • 4 ß-Lactamases
  • - ß-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 ß-lactams in
    blood or urine samples in patients undergo
    therapy with these drugs.

21
- One other important therapeutic application is
the rescue of patients presenting symptoms of a
severe allergic reaction following administration
of a ß-lactamase - sensitive penicillin.
22
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.

23
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.
24
  • 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.

25
  • 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.

26
  • - 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.

27
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.

28
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.
29
  • 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.

30
  • 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).

31
  • 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.

32
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.
33
  • 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.

34
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.
35
  • 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.

36
  • 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.

37
  • Chiral inversion
  • Several clinically used drugs, e.g. salbutamol (a
    ß-adrenoceptor agonist), propranolol (a
    ß-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.

38
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.

39
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.
40
  • 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.

41
  • 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.

42
  • 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.

43
  • 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.

44
  • 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.

45
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-

46
  • 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.

47
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.
48
  • 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 .

49
  • 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.

50
  • 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.

51
  • 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.

52
  • 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.

53
  • 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.

54
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.
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