2007-2008BIOTECHNOLOGY

B. BIOLOGICAL FUELS1The 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 PRODUCTION1- 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 biotransformationSince 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 inversionSeveral 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 assaysIn 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 biodeteriorationThe 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.SewageSewage 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 treatmentSewage 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.