Okafor N. Modern Industrial Microbiology and Biotechnology
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" Modern Industrial Microbiology and Biotechnology
Chaplin, M.F., Bucke, C. 1990. Enzyme Technology. Cambridge University Press. New York,
USA.
Cheetam, S.J. 2004. Bioprocesses for the Manufacture of Ingredients for Food and Cosmetics.
Advances in Biochemical Engineering/Biotechnology, 86, 83–158.
Desai, M.A. 2000. Downstream Processing of Proteins. Humana Press Totowa, New Jersey, USA.
Fogarty, M., Kelly, C.T. (eds) 1990. Microbial Enzymes and Biotechnology. 2
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Imanaka, T. 1994. Improvement of Useful Enzymes by Protein Engineering. In: Rombinant
Microbes for Industrial and Agricultural Applications. Y. Murooka, T. Imanaka, (eds). Marcel
and Dekker. New York, USA. pp. 449-465.
Kennedy, J.F. 1995. Principles of Immobilization of Enzymes. In: Handbook of Enzyme
Biotechnology. A Wiseman, (ed) 3
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Bioprocessing. Advances in Biochemical Engineering/Biotechnology. 68, 61–132.
Pasechnik, V.A. 1995. Practical Aspects of Large-scale Protein Purification. In: Handbook of
Enzyme Boiotecnology. A Wiseman, (ed) 3
rd
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enzyme naringinase. Biotechnology Advances, 18, 207-217.
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Biochemical Engineering/Biotechnology, 86, 159–190.
Schugerl, K. 2000. Recovery of Proteins and Microorganisms from Cultivativation Media by
Foam Flotation. Advances in Biochemical Engineering/Biotechnology. 68, 191-233.
Tanaka, A, Tosa, T, Kobayashi, T, (1993). Industrial Applications of Immobilized Biocatalysts
New York: Dekker.
Mining Microbiology: Ore Leaching (Bioleaching) by Microorganisms "
23.1 BIOLEACHING
The term bioleaching refers to the conversion of an insoluble metal (generally a metal
sulfide, e.g., CuS, NiS, ZnS) into a soluble form (usually the metal sulfate, e.g., CuSO4,
NiSO4, ZnSO4). When this happens, the metal is extracted into water; this process is
called bioleaching. As these processes are oxidations, this process may also be termed
bio-oxidation. However, the term bio-oxidation is usually used to refer to processes in
which the recovery of a metal is enhanced by microbial decomposition of the mineral, but
the metal being recovered is not solubilized. An example is the recovery of gold from
arsenopyrite ores where the gold remains in the mineral after bio-oxidation and is
extracted by cyanide in a subsequent step. The term bioleaching is clearly inappropriate
when referring to gold recovery (although arsenic, iron, and sulfur are bioleached from
the mineral). Biomining is a general term that may be used to refer to both processes.
Bacterial leaching of metals is a process in which the ores of the metals, usually their
sulphides are solubilized by bacterial action. Basically the process is a chemical
oxidation following the equation
Microorganisms
MS + 2O
2
MSO4
where M is a bivalent metal. The solution collected after the solubilization is processed to
recover the metal. In the case of insoluble sulfides such as is the case with lead sulfide, the
fact of insolubility may be used to separate it from the dissolved metals.
Bacterial leaching has been practiced by man over many centuries without any
understanding of the microbiological basis of the process. It was used for mining copper
by the Romans in Wales, in Rio Tinto, Spain in the 18th century and in the USA in this
century. Indeed about 12% of copper produced in the USA is obtained by bacterial
leaching of low grade ores.
Mining Microbiology: Ore
Leaching (Bioleaching) by
Microorganisms
23
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" Modern Industrial Microbiology and Biotechnology
23.2 COMMERCIAL LEACHING METHODS
There are two types of processes for commercial microbially-assisited metal recovery: the
irrigation-type and the stirred tank type.
23.2.1 Irrigation-Type Processes
The irrigation-type processes can be grouped into three: the dump, the heap and the in-
situ methods. The most widely used methods are dump and heap leaching. The metal
most commonly bioleached metal in the irrigation methods is copper.
23.2.1.1 Dump leaching
In this method large quantities of low-grade ore are placed in valleys with impermeable
grounds. The dumps are shaped by bull-dozers into cones which may be as high as
600 ft with 600 ft diameter at the base. Acid solutions usually H
2
SO4, known as leach
solutions, are then sprayed into, or flooded over, the dumps or injected into it through
steel pipes. The acid provides the low pH required by the microorganisms whose
activities are responsible for dissolving the ores. Liquid collected at the bottom of the
dump contains dissolved metal which is recovered after processing.
In some processes the dumps are subjected to preconditioning, irrigation, rest, and
conditioning, each of which may extend for a year. The irrigation is done with an iron-
and sulfate-rich recycyled waste-water from which the copper has been removed.
Microorganisms growing in the dump bring about the reactions which cause the
insoluble copper sulfides to become soluble copper sulfate. The copper sulfate is collected
from the bottom of the dump and the copper is recovered by solvent extraction and
electrolysis. One of the best-known dump leaching sites is the Kennecot Copper mine in
Bingham Canyon, Utah, USA; another is Bala Ley plant in Coledo, Chile.
23.2.1.2 Heap leaching
Heap leaching is very similar to dump leaching, except that steps are taken to make the
process more efficient through ores of smaller particles and a smaller scale of operation.
The ores are crushed to make them finer, and then staked in much smaller heaps of about
6 to 50 ft high. Aeration pipes may be included to permit forced aeration so as to speed up
the process. To further speed up the reaction inorganic ammonium salts and phosphates
may be added. The heaps are placed in mounds on drainage pads or in concrete tanks
with false bottoms through which liquid collects in a dump. Tanks have capacities of
about 12,000 tons. On account of the steps taken to accelerate the process the operation is
complete in months rather than in years. An example of a large modern heap plant is to be
found in Quabrada Blanca in northern Chile.
Heap bioleaching may also be used to mine gold in low grade gold ores. In this case the
ore is initially flooded with acid-ferric iron. Thereafter, it is treated with recycled heap
effluent. After the ore is sufficiently broken down, the ore is mixed with lime and the gold
extracted with cyanide.
23.2.1.3 In-situ Mining
In in-situ irrigation bioleaching the ore is not brought to the surface and the ore is
extracted in situ. In some instances such as in the copper mine at San Manuel, Arizona,
Mining Microbiology: Ore Leaching (Bioleaching) by Microorganisms " !
injection wells are used to introduce acidified leaching fluids into the mineral deposit. It
percolates and collects in disused mines or a specially prepared catchment location. The
geology of the location must have a suitable impermeable layer. In spite of this leaching,
fluid is lost in this process.
The mining of uranium is generally done in situ in the mine. The uranium ore (uranite)
is not attacked by acid producing bacteria. However it is solubilized when it is oxidized
from the tetravalent form which is insoluble, to the soluble hexavalent form by chemical
oxidants such as ferric iron, MnO
2
, H
2
O
2
chlorates or nitric acid. In uranium leaching
mediated by bacteria, the role of the bacteria is to produce ferric iron which then oxidizes
the uranite to make it soluble.
23.2.2 Stirred Tank Processes
Stirred tank bioreactors are highly aerated tanks much like the fermentors already
discussed except that the sterility maintained in fermentors used, for example for
antibiotics, is not necessary in these bioreactors. They are expensive to construct and
maintain and hence they are used only for high value minerals such as gold.
They are usually arranged in series and function as continuous fermentors. The tanks
in the first stage are usually arranged in parallel so that the fermentation broth is retained
long enough for the cell numbers to reach a steady state without being washed out. The
mineral ore is suspended in water to which fertilizer grade (NH
4
)
2
SO
4
and KH
2
PO
4
are
added.
The pH is adjusted 1.5–2.0. The bioreactor is vigorously aerated using air spargers
and baffles. The released heat is removed by a jacket of cold water. Systems using aerated
bioreactors use pretreatments for the recovery of gold, especially where the gold is finely
divided in a mixture of pyrite and arsenopyrite (i.e. iron ores mixed with arsenic). Such
ores are known as recalcitrant ores. Normally gold is recovered from rich ores by
treatment with cyanide. However, because gold forms only a small proportion of
recalcitrant aesenopyrite ores, the ores are pretreated in the aerated bioreactors. During
the pretreatment which lasts for about four days microbes oxidize the arsenopyrite which
is decomposed into iron and sulfate releasing the gold, and is then treated with cyanide.
Stirred bioreactors are used to pretreat gold ores in many countries around the world
including Australia, Brazil, Ghana, and Peru. The Ghana plant in Sensu is perhaps the
largest fermentation in the world with 24 tanks each with a capacity of 1 million liters.
Aerated bioreactors are used for other minerals in France and Uganda (cobalt) and
South Africa (nickel).
23.3 MICROBIOLOGY OF THE LEACHING PROCESS
The primary biomining microorganisms involved in ore leaching have several properties
in common:
(i) They are Gram-negative specialized chemolitho-authotrophic able to use ferrous
iron and reduced inorganic sulphur compounds or both as electron donors;
(ii) They are able to fix carbon with energy derived from the oxidation of inorganic
compounds such as ferrous iron, sulfur and sulfides according to the following
equations:
" " Modern Industrial Microbiology and Biotechnology
4FeSO4 + 02 + 2H2SO4 ® 2Fe
2
(SO4) + 2H
2
O (1)
(S
8
+ 120
2
+ 8H
2
) ® 8H
2
SO4 (2)
H
2
S + 20
2
® H
2
SO4 (3)
(iii) They are acidophilic and will thrive under very acid conditions at pH ranges of 1.5
– 2.0.
(iv) Although they can use electron acceptors other than oxygen they grow best in
highly aerated solutions.
(v) Some are able to fix atmospheric nitrogen, when the oxygen supply is limited
(vi) Some are obligately autotrophic, while others are facultative autotrophs and able
to grow in the presence of organic matter.
(vii) The organisms are usually found naturally in waters in contact with exposed
sulphides or in mines.
The organisms involved are the following:
(a) Acidothiobacillus: Organisms belonging to this genus were previously classified as
Thiobacillus. Following 16S rRNA analysis they have been reclassified as
Acidothiobacillus to accommodate the very acidophilic members of the genus. These
include Acidothiobacillus ferrooxidans (formerly Thiobacillus ferooxidans) which is
the most intensively studied and until recently was thought to be the sole
microorganism involved. It is an obligate autotroph and obtains its energy for the
fixation of CO
2
from the oxidation of ferrous irons, sulfides, and other sulfur
compounds such as thiosulfate.
(b) Leptospirillum ferrooxidans: This grows only in soluble ferrous iron and not on
sulfur or mineral sulfides.
(c) Acidothiobacillus thiooxidans (formerly Thiobacillus thiooxidan): It cannot oxidize
iron, but grows on elemental sulfur and soluble compounds including those
generated in the leaching systems of T. ferrooxidans.
(d) Other bacteria: Thiobacillus organosporus is facultatively chemolithotrophic (i.e.
while it is autotrophic it will also grow when organic compounds are available to
it). It oxidizes sulfur, but not iron or sulfides. Mixed with other organisms e.g.
Leptospirriullum ferrooxidans it will degrade iron sulfides which neither can do
alone.
Unlike other industrial microbiology processes there is no conscious attempt to use
pure cultures. The highly acidophilic organisms create conditions which are unsuitable
for other organisms.
23.4 LEACHING OF SOME METAL SULFIDES
(i) Copper sulfides: One of the world’s main source of copper is the iron-copper sulfide,
chalcopyrite (CuFe
2
S
2
). Others are chalcocite (Cu
2
S) and covellite (CuS). The
reactions in which these minerals are leached by bacterial action are complex, but
the end equations may be given as follows:
Mining Microbiology: Ore Leaching (Bioleaching) by Microorganisms " #
Chalcopyrite
Bacteria
60CuFeS
2
+ 25SO
2
+ 90H
2
O 60CuSO
4
+ 20H Fe (SO
4
)
2
. 2Fe (OH)
3
+ 20H
2
SO4 (4)
Chalcocite
Bacteria
10 Cu
2
S + 10H
2
SO4 20CuSO4 + 10H
2
O (5)
(ii) Uranium extraction: Bacteria especially T. ferrooxidans, oxidize ferrous sulphate to
ferric sulphate (Equation I). The latter then reacts with uranium ores thus:
Uranite
UO
2
+ Fe
2
(SO4)
3
UO
2
SO4 + FeSO4 (6)
The tetravalent form in the ore is insoluble in the leach solution while the
oxidized hexavalent form is. There is thus a two-stage action which seems
especially appropriate as the uranyl ion is toxic to most strains of T. ferrooxidans.
(iii) Cobalt and nickel sulfides leaching: The major nickel bearing sulfide ore is usually
pentlandite which contains about 1% of nickel. The reaction for leaching with T.
ferrooxidans is given as follows:
Bacteria
(Ni, Fe)
9
S
8
+ 175/8 O
2
+ 3¼ H
2
SO4 4½ NiSO4
+ ¼ Fe
2
(SO4)
3
+ 3¼ H
20
(7)
(iv) Zinc and lead sulfide leaching: Zinc and lead sulfides respectively may be oxidized
by T. ferrooxidans according to the equations:
Bacteria
ZnS + 20
2
Zn SO4 (8)
Bacteria
PbS + 20
2
Pb SO4 (9)
23.5 ENVIRONMENTAL CONDITIONS AFFECTING
BACTERIAL LEACHING
The following factors affect the efficiency of leaching via bacterial action. Much of this
has been studied using T. ferrooxidans.
(i) Temperature: The optimum temperature for the bacterial solubilization of metals
lies between 25°C and 45°C for different strains of T. ferrooxidans. Above 55°C the
activity is mainly chemical. No minimum temperature has been established, but it
is believed that action stops at freezing.
(ii) pH: T. Ferrooxidans is acidophilic and has been studied at pH values ranging from
1 to 5. The optimum pH values for acting on many minerals lie between pH 2.3 and
2.5. Other organisms outside T. ferrooxidans appear to have about the same pH
requirements.
" $ Modern Industrial Microbiology and Biotechnology
(iii) Nutrient status of the leaching medium: Like any other bacterium, the iron-oxidizing
thiobacilli must have the appropriate nutrients in the leaching medium. The
energy source is, as has been stated, ferrous sulfate, but minerals containing iron or
sulfur may be utilized. The carbon is obtained from CO
2
of the air. It also needs
nitrogen, potassium, magnesium, phosphate, and calcium. Most of these are
available from the surrounding rocks; where the deficiency of one or more of them
is established, it must be replaced in the leaching solution for optimum
productivity. The thiobacilli are strict aerobes and oxygen deficiency leads to
limitations in leaching productivity.
(iv) Particle size: In general the finer the particle size of the ore the greater the extraction
of the leaching solution. Below a certain particle size however, especially in the
case of low grade ores, the ratio of the mineral to unwanted part of the ore increases
and productivity falls.
SUGGESTED READINGS
Kelly, D.P., Norris, P.R., Brierley, C.L. 1979: In: Microbial Technology: Current State, Future
Prospects. A.T., Bull, D.C. Ellwood, C. Ratledge, (eds). Cambridge University Press,
Cambridge. UK. pp. 263-308.
Lundgren, D.G. 1980. Ore Leaching by bacteria Ann. Rev. Microbiol. 24, 263-283.
Rawlings, D.E. 2002. Heavy Metal Mining Using Microbes Annual Review of Microbiology, 56,
65-91.
Zajic, J.E. 1969. Microbial Biogeo-chemistry Academic Press, New York, USA.
Production of Commodities
of Medical Importance
Section/
Modern Industrial
Microbiology and Biotechnology
As currently defined, antibiotics are chemicals produced by microorganisms and which
in low concentrations are capable of inhibiting the growth of, or killing, other
microorganisms. Anti-microbial substances are also produced by higher plants and
animals. Such substances are however excluded by this definition. Bacteriocins although
produced by microorganisms are also not included in this definition because they are not
only larger in molecular size than the usual antibiotics, but they are mainly protein in
nature; furthermore they affect mainly organisms related to the producing organism. In
comparison with bacteriocins, conventional antibiotics however are for more diverse in
their chemical nature and attack organisms distantly related to themselves. Most
importantly, while the information specifying the formation of ‘regular’ antibiotics is
carried on several genes, that needed for bacteriocins being single proteins need single
genes. It will be seen later that in the last few years this definition has been somewhat
broadened by some authors to include materials produced by living things – plants,
animals or microorganisms – which inhibit any cell activity.
Antibiotics may be wholly produced by fermentation. Nowadays, however, they are
increasingly produced by semi-synthetic processes, in which a product obtained by
fermentation is modified by the chemical introduction of side chains. Some wholly
chemically synthesized compounds are also used for the chemotherapy of infectious
diseases e.g. sulfonamides and quinolones. But these will not be considered since they
are not produced wholly or partially by fermentation. Some antibiotics e.g.
chloramphenicol were originally produced by fermentation, but are now more cheaply
produced by chemical means.
Thousands of antibiotics are known; and every year dozens are discovered. However,
only a small proportion of known antibiotics is used clinically, because the rest are too
toxic.
24.1 Classification and Nomenclature of Antibiotics
Several methods of antibiotic classification have been adopted by various authors. The
mode of action has been used, e.g. whether they act on the cell wall, or are protein
inhibitors, etc. Several mechanisms of action may operate simultaneously making such a
Production of Antibiotics
and Anti-Tumor Agents
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