Microbial Technology

Faculty of Mineral Resources Technology
Department of Minerals Engineering
Lecture Material on
MR 379 Microbial Technology
Compiled by:
Assoc Prof Grace Ofori-Sarpong
August 2018
UNIVERSITY OF MINES AND TECHNOLOGY, TARKWA UMaT
Lecture Outline
 Learning Outcomes
 Introduction to Microbial Technology/Biotechnology
 Basic Microbiology
 Role of Microorganisms in the Environment
Algae, Fungi, Viruses and Bacteria
 Microbial Growth
 Microorganisms in Water, Food, Waste, and soil
 Bioremediation of the environment
 Acid mine/rock drainage
 Biogeochemical cycles
 Application of Microbial Technology in Minerals Engineering
 Genetic Engineering
 References
Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 2
1
Learning Outcomes
 At the end of the semester’s lecture on Microbial
Technology, the student should be able to:
 Define biotechnology and identify the various areas;
 Define microorganisms, and differentiate among the various
microorganisms; algae, protozoa, fungi, bacteria, archaea,
viruses;
 List the major roles of microorganisms in the environment;
 Describe the conditions for optimum growth of microorganisms;
 Explain the application of industrial microbiology and
biotechnology in minerals engineering; bioprocessing,
biodegradation, etc.; and
 Design an experiment on application of microbial technology in
minerals engineering.
 Describe the genetic manipulation of microorganisms for
specific applications;
INTRODUCTION
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Microbial Technology/Biotechnology
 There have been several definitions of Microbial
Technology/Biotechnology including the following:
 The use of microorganisms, typically grown on a large scale, to
yield products or carry out chemical transformation.
 The aspect of technology concerned with the application of
living organisms to meet the needs and ends of man.
 Any technique that uses living organisms or substances from
those organisms to make or modify a product, improve plants
or animals, or develop microorganisms for specific uses.
 Any technological application that uses biological systems,
living organisms, or derivatives thereof, to make or modify
products or processes for specific use.
Microbial Technology/Biotechnology
 Microbial Technology/Biotechnology has undergone
transformations over the years:
 Ancient Biotechnology - early history as related to food
and shelter.
 Classical Biotechnology – relating to fermentation/food
production and medicine.
 Modern Biotechnology – relating to manipulation of
genetic information in organism as in genetic
engineering.
 The application of Microbial Technology/Biotechnology
involves several fields including, biochemistry,
microbiology, computer science, genetics, molecular
biology, engineering.
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Benefits of Microbial Technology
 Microbial Technology makes it possible to study the large-
scale and profit-motivated production of microorganisms or
their products for direct use, or as inputs in the
manufacture of other goods.
 It offers an efficient, environmentally friendly and cost-
effective remedy for many chemical processes.
 The principles are applied in many areas including
 food processing, municipal composting, medicine, and
industrial leaching processes aimed at metal recovery.
 production of yeasts as animal feed, for use in bread-
making; ethanol for use in alcoholic beverages, or in the
manufacture of perfumes, pharmaceuticals, etc.
 production of antibiotics like penicillin, dairy product like
yoghurt; and amino acids among others.
Microbiology
 Study of microorganisms, and their beneficial and

adverse effects on man and the environment.
 Can be grouped broadly into:
 Medical microbiology
 Food microbiology
 Industrial microbiology
Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout

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Microbiology
 Medical microbiology
 Pathogens (Tuberculosis, Buruli ulcer)
 Antibiotics
 Food microbiology
 Dairy products
 Bread making
 Wine making
 Industrial microbiology
 Mineral leaching
 Environmental clean-up

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 a) Microbial diversity and versatility

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 Basic concepts of microorganisms

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 Physiology
 Metabolism
 Influence on the environment
 b) Biofilms

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Biogeochemical Cycles
 Sulphur, carbon, nitrogen cycles

 Movement of elements or compounds through
living organisms and the non-living environment
 The role of microorganisms in the cycle
 Human alteration of the cycles
Bioprocessing of Minerals
 a) Biooxidation of minerals
 Bacterial oxidation of metal sulphides and its
application in precious metal extraction
 Fungal biotransformation of carbonaceous matter
and its application in precious metal extraction
 b) Bioleaching of minerals
 Bacterial leaching of copper, zinc and uranium ores
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Acid Mine Drainage and Bioremediation
 a) Acid mine drainage and mitigation

 Microbial influence on acid generation
 Effects of acid mine drainage on the environment
 Acid-base accounting
 Mitigation of acid mine drainage
 b) Bioremediation
 Biosorption of heavy/toxic metals in wastewater
 Remediation of contaminated soils
 c) Microbial degradation of cyanide
Basic
Microbiology
Ass
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Microorganisms
 Microbes (microorganisms) are tiny organisms that are
too small to be seen individually by the naked eye and
must be viewed with the help of a microscope;
Archaea Bacteria, fungi, algae, virus and protozoa.
 Eukaryotic microbes include yeast, algae and
protozoans.
 Prokaryotes are single celled microorganisms which
include the domains Bacteria and Archaea.
 Microorganisms are efficient factories for the
production of several substances and development of
many processes.
 Major ones of industrial interest are bacteria, fungi
and archaea.
Assignment One - Microorganisms

 Form six groups and select at random one of the
microoganisms:
 Archaea,
 Bacteria,
 Fungi,
 Algae,
 Virus, and
 Protozoa.
 Research about what they are, their beneficial use and
adverse effects to mankind and the environment.
 Prepare not more than 10 slides for presentation on
Monday, 10th September, 2018
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Protozoa
 Protozoans have animal-like characteristics, and thus
classified as the animal portion of the kingdom protista
under the eukaryotes
 They are typically heterotrophic, and come in different
shapes, and can be free-living or parasitic.
 They improve the fertility of soil by maintaining a
youthful balance in soil bacteria.
Algae
 Algae are aquatic organisms that are mostly unicellular though
some are multicellular.
 They occur usually in fresh water, salt water or moist ground,
with examples being pond scum, seaweeds (phytoplankton)
and algal blooms in lakes.
 Algae uses photosynthesis to capture sunlight energy and
carbon dioxide to produce oxygen and carbohydrates.
 It grows so quickly and can be used as food for humans (sea
weed) and fish.
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Achaea
 Archaea share properties of both eukaryotes and
prokaryotes.
 They are similar to bacteria in size and simplicity of
structure but radically different in molecular organization.
 They usually live in extreme environments (halophiles,
thermophiles) and have unique metabolic properties.
 Archaea provide the major routes for ammonia oxidation
through nitrite to nitrate for plants and other animals.
Fungi
 Fungi are eukaryotic organisms, usually multicellular,
examples of which are yeasts, moulds and mushrooms.
 They are heterotrophs, with great importance in nutrient
cycling in an ecosystem.
 Fungi provide essential nutrients for plants growth and serve
as food for human beings and other organisms.
 Fungi are used in the production of some drugs and food.
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Virus
 A virus is an infective agent that typically consists of a nucleic
acid molecule in a protein coat, and it is too small to be seen
by light microscopy.
 A virus can only reproduce within the living cells of a host.
Thus it invades living cells and uses the chemical machinery to
keep itself alive and to replicate itself. Examples are hepatitis
B virus, HIV virus, Herpes simplex virus, influenza virus.
Bacteria
 Bacteria are a large group of microorganisms that are
single-celled prokaryotes that are found in every
environment, inside and/or outside other organisms.
 Bacteria degrade dead animals and plants and recycles
valuable nutrients in soil, bioremediate the environment
by cleaning up harmful pollutants.
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Microorganisms
 Bacteria were the first life forms on earth and have existed for
over 3.5 billion years.
 Bacteria are estimated to comprise over 50% of the earth's living
matter.
 They live in our mouths, on our skin and in our digestive
tract.
 There are approximately 10 times as many bacterial cells in
the human body, than our own cells.
 Bacteria are adapted to living in some of the harshest
environments on the planet:
 polar ice caps, deserts, boiling hot springs and under
extraordinary high pressure in deep-sea vents miles under the
ocean's surface.
 Less than 1% of all bacterial species have been identified,
cultured and studied, so we can only imagine the contribution
of microbes to biotechnology in the future.
Properties of useful microbes

 Produces spores or can be easily inoculated
 Grows rapidly on a large scale in inexpensive medium
 Should not be pathogenic
 Amenable to genetic manipulation
 Produces desired product quickly
 Microbial cells
 Enzymes
 Antibiotics, steroids, alkaloids
 Food additives
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Composition of Dry Microbial Cell
 A wet cell contains 70% of water

Genera and Species

 Escherichia coli
 Streptomyces setonii
 Acidithiobacillus thiooxidans
 Leptospirillum ferrooxidans
 Mycobacterium ulcerans
 Phanerochaete chrysosporium
 Pennicillium citrinum
 Trametes versicolor
 Saccharomyces cerevisiae (baker's yeast)
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Classification of Microorganisms
 Shapes/morphology
 pH
 Temperature
 Oxygen requirement
 Nutrition/Food
 Energy requirement
 Osmotic Pressure
 Moisture requirement
Morphological Classification
 Coccus
 Spirillus
 Bacillus
 Filamentous
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Morphological Classification
 Bacteria vary in size and shape
 Most common shapes
 Cocci – spherical cells
 Bacilli – rod-shaped cells
 Spiral – corkscrew-shaped cells
 Filamentous - threadlike
Bacillus
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Observation of Morphologies
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Observation of Colonies
Staphylococcus aureus
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pH Classification
 Acidophiles (0.0-5.4)
 acidic growth medium (pH 1-5)
 microbes that can live in the stomach
 Neutrophiles (5.4-8.5)
 neutral growth medium (pH 6-8)
 most microorganisms
 Alkalinophiles (8-11.5)
 alkaline growth medium(pH 8.5-10.5)
 found in intestine
Temperature classification
 Psychrophilic (-10 to 20 0 C)
 Psychrotrophic (10 to 25 0 C)
 Cryophilic (< 20 0 C)
 Mesophilic (20 – 45 0 C)
 Thermophilic (40 to 75 0 C)
 Hyperthermophilic/Thermoduric (65 to 120 0 C)
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Oxygen Requirements
AEROBES
 use molecular O2 for life and reproduction
 Obligate aerobes
 require an atmosphere that contains O2 similar to
room air (20-21% O2),
 Eg. Mycobacterium
 Microaerophiles
 require O2 lower than room air (5% O2)
 Eg. Neisseria, Campylobacter
Oxygen Requirements
ANAEROBES
 do not require O2 for life and reproduction
 Subclasses depend on sensitivity to O2
 Obligate anaerobe
 unable to grow in O2,
 Eg. Clostridia
 Aerotolerant anaerobe
 does not require O2
 grows better in the absence of oxygen but can
survive in atmosphere containing O2
 Eg. Lactobacilli
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Oxygen Requirements
ANAEROBES
 do not require O2 for life and reproduction
 Subclasses depend on sensitivity to O2
 Facultative anaerobe
 capable of surviving in the presence or absence of O 2
(0% to 20-21% O2)
 Eg. Enterobacteria, streptococci, staphylococci
 Capnophiles– grow better in the presence of
increased concentrations of CO2
Extremophile Organisms - Archaea

 Halophiles are salt loving and thrive in high salty environment
like evaporation ponds or salt lakes; Great Salt Lake, Dead
Sea.
 Osmophiles are microorganisms adapted to environments
with high osmotic pressures, such as high sugar
concentrations.
 Thermophiles are microorganisms found in various
geothermally heated regions of the Earth, such as hot
springs like those in Yellowstone National Park deep sea
hydrothermal vents, decaying plant matter; peat bogs and
compost.
 Barophiles (or Piezophiles) are organisms that live in high
pressure environments such as deep sea sediments,
hydrothermal vents, inside invertebrates, and in rock deep
under the Earth.
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Gram-positive and Gram-negative
Bacteria
 Bacteria are classified as being either Gram-positive or

Gram-negative based on differences in the structure of
their peptidoglycan cell wall.

Bacteria
 Gram-positive bacteria have a very thick cell wall made of a

protein called peptidoglycan and teichoic acids.
 Gram negative cell walls contain a thin peptidoglycan layer
(without teichoic acids) and is sandwiched between an inner
cell membrane and a outer membrane.
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Bacteria
 Gram-positive and Gram-negative Bacteria are separated
by a technique developed in the 19th Century by the Danish
bacteriologist, Hans Christian Gram.
 Gram positive – respond positively to Gram’s stains (purple
dye) due to thick peptidoglycan layer
 Gram-negative bacteria are rather stained by counter stain
(safranin), as they are de-stained of the purple dye due to
alcohol wash.
 Thus, they appear as pink color under a microscope.
 Gram-positive bacteria are more receptive
to antibiotics than Gram-negative, due to the absence of
the outer membrane.
 Gram-negative bacteria have a largely impermeable cell
wall, and thus are more resistant to antibiotics.
Gram Stain Procedure (vlab.amrita.edu, 2011)

Basic steps in Gram Staining:
 Smear bacteria inoculum using a sterile loop to form about
one centimeter in diameter circular motion on a slide.
 Pass the air dried slide through the flame of a Bunsen
burner two to three times with the smear-side up.
 Gently flood the heat-fixed smear with crystal violet and
let stand for 1 minute.
 Tilt the slide slightly and gently rinse with tap water or
distilled water using a wash bottle.
 Gently flood the smear with Gram’s iodine and let stand for
1 minute to form the crystal violet-iodine complex.
distilled water using a wash bottle. The smear will appear
as a purple circle on the slide.
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Gram Stain Procedure
 Decolorise using 95% ethyl alcohol or acetone. Tilt the slide
slightly and apply the alcohol drop by drop for 5 to 10
seconds until the alcohol runs almost clear.
 Immediately rinse with water.
 Gently flood with safranin to counter-stain and let stand for
45 seconds.
distilled water using a wash bottle.
 Blot dry the slide with bibulous paper.
 View the smear using a light-microscope under oil-
immersion.
 Cells with purple stain signify Gram Positive bacteria.
 Cells with pink stain represent Gram Negative bacteria
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Bacteria
 Cells with purple stain signify Gram Positive bacteria, eg.,
Bacillus and Clostridium, Streptococcus and Staphylococcus.
 Cells with pink stain represent Gram Negative bacteria; eg.
Escherichia coli, Salmonella, Pseudomonas, Helicobacter.
Nutrition
Source of Energy
 Phototrophs - light
 Chemotrophs – breaking down of inorganic or
organic compounds
 Lithotrophs - inorganic compounds
 Organotrophs - organic compounds
Source of Carbon
 Autotrophs - CO2
 Heterotrophs - organic compounds
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Nutrition
Energy Source and Carbon Source
 Photoautotrophs
 Light + CO2
 Eg. Plants, algae, cyanobacteria, purple and green
sulfur bacteria
 Photoheterotrophs (Photoorganotrophs)
 Light + organic compounds,
 Eg. Green and purple non-sulfur bacteria
 Chemoautotrophs
 Chemical + CO2
 Eg. Nitrifying, hydrogen, iron and sulfur bacteria
Nutrition
Energy Source and Carbon Source
 Chemolithotrophs
 Chemical + inorganic compound
 Chemoheterotrophs
 Chemical + organic compound
 Eg. All animals, protozoa, fungi, most bacteria
 Photolithotrophs
 Light + inorganic compound
 Eg. Plants and algae - producers of food and O2
chemoheterotrophs
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Redox Reactions in Chemotrophs
 Chemoheterotrophs
 Electron donors - organic compounds
 Electron acceptors - O2, Mn4+, Fe3+, SO42-, CO2
 Chemolithotrophs
 Electron donors – H2, NH3, H2S, S, S2-
 Electron acceptors – O2, Mn4+, Fe3+, SO42-, CO2
 For aerobes - oxygen is used
 For anaerobes - oxidants other than oxygen, eg.,
(Mn4+, Fe3+, SO42-, CO2)
 Facultative anaerobes - O2 and other oxidants eg.,
NO3-, Mn4+, Fe3+, SO42-
Metabolism
 Metabolism comprises all the physical and chemical
processes that occur within a living cell or organism in order
to maintain the living state of the cells and the organism.
 In metabolism some substances are broken down to yield
energy while other substances, necessary for life, are
synthesized.
 Metabolism can be conveniently categorised into two:
 Catabolism
 Anabolism
 Catabolism (destructive metabolism) is the breakdown of
complex molecules in living organisms to form simpler ones,
together with the release of energy for vital processes and
other components, eg., lactic acid, acetic acid, carbon
dioxide, ammonia and urea.
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Metabolism
 Two classes of energy producing catabolic pathways are
fermentations and respirations:
 Respirations are catabolic pathways by which organic
compounds are completely oxidised to CO2 due to the
presence of exogenous terminal electron acceptor.
 Fermentation is the process which occurs when there are
no terminal electron acceptors, and so carbon
compounds are rearranged thereby releasing energy.
 Anabolism is the process by which the body utilises the
energy released by catabolism to synthesize complex
molecules from small and simple precursors that act as
building blocks.
 These complex molecules synthesized are then utilized
in forming cellular structures.
Microbial (Bacterial) Growth

 Increase in the number of cells in a population rather
than in the size of individual cells.
 Growth of an individual cell continues until the cell
divides into two new cells, by binary fission, at an
average rate of twenty minutes.
 The growth rate is the change in cell number or cell mass
per unit time.
 The interval for the formation of two cells from one is
called a generation.
 The time required for the cell population to double is
called the generation time or doubling time.
 The generation time is highly variable and may be as
little as 10 minutes to several hours or days.
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Bacterial Growth Curve
 When bacteria are grown in a batch culture, a
predictable pattern of growth in the population occurs.
Lag Phase
 In the lag phase, bacteria are metabolically active and grow
in size but do not divide or increase in number of cells.
 Bacteria take some time to adjust to the new environment
and prepares for the synthesis of enzymes and co-enzymes
for physiological activities.
 Duration of lag phase depends on conditions and species of
bacteria.
 Inoculum from an older culture will take a longer
duration than that from a newer culture.
 Inoculum transferred into a similar culture medium will
take a relatively shorter period to adjust.
 At the end of lag phase, bacteria become fully prepared for
cell division.
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Log Phase or Exponential Phase
 This is the phase in which all bacteria are in their rapid
stage of cell division.
 Bacteria divides continuously at constant rate and the
number of cells increase exponentially.
 Generation time is shortest during log phase, and thus
bacteria have the smallest sizes.
 Log phase is essential for:
 identification of bacteria using biochemical and
physiological characteristics.
 determination of generation time.
 Duration of the log phase depends on the type of
organism, conditions of growth and density of organism.
Stationary or Plateau Phase

 This is the phase in which there is no net increase in
bacterial population due to a balance between cell
division and cell death.
 Stationary phase is generally triggered by:
 increased bacterial cell density.
 depletion of nutrient in media.
 accumulation of toxic secondary metabolic wastes.
 Stationary phase is characterised by:
 production of antibiotics such as penicillin,
streptomycin and enzymes by certain bacteria.
 Sporulation by endospore-forming bacteria.
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Death Phase or Decline Phase
 Death phase appears to be the inverse of log phase.
 The number of viable bacteria decrease continuously
and exponentially.
 Though total count of bacteria may remain constant,
the viable count decreases.
 Death phase is caused by conditions such as depletion
of nutrition and accumulation of toxic wastes.
 The rate at which bacteria die depends on the
resistance of species to harsh growth conditions; for
example, spore-forming bacteria have relatively higher
resistance.
Growth Phase and Metabolites

 Metabolism leads to the production of primary and
secondary metabolites.
 Primary metabolites
 Produced during exponential growth phase where there
is synthesis of microbial cells.
 Products include amino acids, nucleotides, fermentation
end products, and exoenzymes.
 Secondary metabolites
 Produced during stationary phase, often by spore-
forming microbes during sporulation.
 Usually accumulate in the period of nutrient limitation
or waste product accumulation that follows active
growth.
 Products include antibiotics and mycotoxins.
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Primary and Secondary Metabolites
Buying and Culturing Bacteria
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Spores in Freeze-Dried State
Culturing in Liquid/Broth Medium
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Agitation Unit for Shake Culturing
Liquid sample
Sample holder
Orbital shaker
Stationary Culturing on Moist Agar
Petri
dish
Bacterial
colonies
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Bacterial Enumeration
 Bacterial enumeration is normally achieved by serial
dilution, and the two main ways are:
 spreading on a moist medium in a plate.
 dipping in a liquid medium in a tube.
 The dilution is done by reducing the concentration of
microbial cells by 10-fold in the next inoculation.
 After the incubation period, counting can be done by the
following, taking into consideration the dilution factor.
 Microscopic counts – used for Total cell counts (both
dead and living cells) and Viable cell count (only living
cells that can grow into colonies).
 Plate count – relies on colonies visible to the naked eye.

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Bacteria Enumeration
The countable dilution

is the dilution that
produces an average
of 30 to 300 colonies
per plate.
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Colony Counts on Spread Plate
Final dilution Number of colonies on triplicate Average

on plates spread plates number of
colonies
10-2 TNTC TNTC TNTC TNTC
10-3 TNTC TNTC TNTC TNTC
10-4 493 516 309 439.3
10-5 73 65 65 67.7
10-6 14 10 11 11.7
The countable dilution is the dilution that produces an

average of 30 to 300 colonies per plate
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Ass
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Growth in Batch Cultures

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(201
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379
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Growth in Batch vs. Continuous
Biofilms and Microbial Interactions

 Bacterial can live in two states:
planktonic and sessile.
 Planktonic cells are free
flowing bacteria in suspension.
 Sessile state defines microbial
habitation in biofilms.
 Biofilms are extracellular
polymeric substances
(EPSs) within which microbial
cells adhere to one another or
to a surface.
 Biofilms are responsible for
several chronic diseases that
are difficult to treat.
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Biofilm Development
 In nature, bacteria often grow in biofilms as populations
attached to surfaces.
Impact of Biofilms
 Biofilms generally form on surfaces immersed in fluid and are
generally resistant to antibiotics, disinfectants and cleaning
agents.
 Biofilms can form on the teeth of most animals as dental
plaque and cause tooth decay and gum disease.
 Biofilms can lead to:
 biocorrosion of metal pipes, tanks and implants;
 slippery rock in streams;
 infections that are difficult to treat;
 urinary tract infections by forming on catheters and other
implants;
 good performance in biooxidation reactions.
 Excessive agitation in CSTRs thus disturbs biofilm formation
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Biogeochemical
Cycles
77
Biogeochemical Cycles
 Biogeochemical cycles or substance turnover is a pathway
by which a chemical substance moves through living
organisms and the nonliving environment.
 The cycle thus involves biotic (biosphere) and abiotic
(lithosphere, atmosphere, and hydrosphere) compartments
of the Earth.
 The cycles have a state of equilibrium occurs when there
is a balance in the cycling of the elements between
compartments.
 Chemicals absorbed or ingested by organisms are passed
through the food chain and returned to the soil, air, and
water by such mechanisms as respiration, excretion, and
decomposition.
 Examples are water, oxygen, carbon, nitrogen, sulphur,
phosphorus, nutrient. 78
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Water Cycle
 The water cycle involves evaporation, condensation,
precipitation and collection, and describes how water:
 evaporates from the surface of the earth and rises into
the atmosphere,
 cools and condenses into rain or snow in clouds,
 falls again to the surface as precipitation, and
 collects in the oceans, rivers, lakes, streams, with most
infiltrating the ground as underground water.
Water Cycle
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Oxygen Cycle
 The oxygen cycle is the biogeochemical cycle of oxygen
within its four main reservoirs:
 the atmosphere (air),
 the total content of biological matter within the
biosphere (the global sum of all ecosystems),
 the hydrosphere (the combined mass of water found
on, under, and over the surface of planet Earth),
 and the lithosphere/Earth's crust
Oxygen Cycle
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Oxygen Cycle
Carbon Cycle
 Carbon is the most essential constituent of all the organic
compounds like carbohydrates, proteins, fats and nucleic
acids present in the living organisms, and thus plays a crucial
role in the health of all living things.
 Carbon cycle is the cyclic process in which carbon element
is circulated continuously through the living and non-living
components of the biosphere.
 The cycle works to ensure the stability of variables such as
the Earth’s atmosphere, the acidity of the ocean, and the
availability of carbon for use by living things.
 Carbon dioxide present in the atmosphere is the main
reservoir of carbon.
 The cycle ensures continuous exchange of carbon dioxide
between atmosphere, water bodies and living beings.
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Carbon Cycle
 The main steps of the carbon cycle in nature are:
 Photosynthesis by green plants to convert carbon dioxide
to carbohydrate.
 Through the food chain, plant carbohydrate is converted
into animal carbohydrate.
 Respiration (catabolism) by plants and animals to oxidise
carbohydrates in their cells to produce energy, and release
carbon dioxide.
 Decay of dead plants and animals by decomposers,
releasing carbon dioxide into the atmosphere.
 Some of the dead plants and animals get buried deep
under the earth, and get converted into fossil fuels like
coal and petroleum.
Carbon Cycle
 Burning of Fossil Fuels to relaease carbon dioxide into the
atmosphere
 Conversion of dissolved carbon dioxide in water bodies
into calcium carbonate in carbonate rocks, like limestone.
 Weathering of carbonate rocks or acid treatment of
carbonate minerals to release carbon dioxide.
 Action of acid rain on carbonate rocks
 Volcanic eruptions leading to the release of carbon
dioxide into the atmosphere.
 If more carbon enters a pool than leaves it, that pool is
considered a net carbon sink
 If more carbon leaves a pool than enters it, that pool is
considered net carbon source
43
Sulphur Cycle
 The sulfur cycle is made up of atmospheric and terrestrial
processes.
 Terrestrial cycle consists of the following:
 Weathering of rocks to release the stored sulphur.
 Oxidation of sulphur to sulphate.
 Conversion of sulphate by plants and microorganisms into
organic forms.
 Consumption of organic sulphur by animals through the
food chain.
 Release of sulphur as sulphate by the decomposition of
dead organisms.
 Entry of sulphate into the tissues of microorganisms.
44
Sulphur Cycle
 A variety of natural sources such as volcanic eruptions also
emit sulphur directly into the atmosphere.
 The atmospheric sulphur eventually settles back into the
earth or comes down through rainfall.
 A continuous loss of sulphur from the atmosphere and
terrestrial ecosystem runoff occurs through drainage into
lakes and streams, and finally the oceans.
 Sulphur in the ocean cycles through marine communities,
moving through the food chain.
 Part of the sulphur is emitted back into the atmosphere
while part is lost to the ocean depths.
Sulphur Cycle
45
Sulphur Cycle
 Microbial-mediated activities:
 Sulphur and sulphides to sulphate
 Thiobacillus thiooxidans, Thiomicrospira,
Sulfolobus
 Sulphate to hydrogen sulphide
 Proteus vulgaris
 Organic sulphur to hydrogen sulphide
 Proteus vulgaris
 Sulphate to sulphur
 Desulphovibrio desulfuricans, Desulfotomaculum
91
Notes on Sulphur cycle

 Decomposers like Aspergillus (aerobic fungi), Neurospora (aerobic fungi)
and Escherichia (anaerobic bacteria) act on the dead and decaying
organic matter of plants and animals, releasing hydrogen sulphide (H2S)
to the environment.
 Heterotrophic bacteria like Desulfavibrio and Acetobacter reduce
sulphates to elemental sulphur or sulphides under anaerobic conditions.
Though sulphides are harmful to most organisms, sulphur bacteria
oxidise them to sulphate and bring back the element to cycle.
 Oxides of sulphur (SO2 and H2S) in the atmosphere, gets dissolved in rain
water and return to soil as sulphates and sulphuric acid.
 From the reservoir pool in deep sediments in the sea, sulphur reaches
the land through food chains, sea sprays and geological upheavals.
 Hydrogen sulphide is released into atmosphere from lakes, marshes and
water logged soils, which is oxidised to sulphur dioxide in the
atmosphere.
 Elemental sulphur or gaseous compounds of sulphur (H2S and SO2) in the
atmosphere, is oxidised to SO3, which combines with water to form
sulphuric acid and comes down on land as acid rain.
46
Nitrogen Cycle
 Nitrogen is an essential component of many body cells that are
the building blocks of life.
 All organisms require nitrogen to live and grow.
 Although the majority of the air we breathe is N2, most of the
nitrogen in the atmosphere is unavailable for use by organisms.
 This is because the strong triple bond between the N atoms in
N2 molecules makes it relatively inert.
 In order for plants and animals to be able to use nitrogen, N2
gas must first be converted to a more chemically available
form such as ammonium (NH4+), nitrate (NO3-), or organic
nitrogen (e.g. urea - (NH3)2CO).
 The inert nature of N2 means that biologically available
nitrogen is often in short supply in natural ecosystems, limiting
plant growth and biomass accumulation.
93
Nitrogen Cycle Steps

 Nitrogen Fixation
 In nitrogen fixation, conversion of atmospheric NO₂ by
lightning or bacteria enzyme called “nitrogenase” into
ammonia.
 Ammonia is a nitrogen compound that can dissolve in water,
and is easier for other organisms’ enzymes to interact with.
 Nitrification
 In nitrification, a host of chemotrophic soil bacteria
metabolise nitrogen with oxygen thereby converting ammonia
into nitrate in two steps:
 conversion of ammonia into nitrogen dioxide by
Nitrosomonas or Nitrococcus.
 Conversion of nitrite to nitrate by Nitrobacter.
 Nitrification converts nitrogen to a form that can be used by
plants and animals.
47
Nitrogen Cycle Steps
 Assimilation
 In assimilation, plants consume the nitrates made by soil
bacteria through their roots and use them to make amino
acids, nucleotides, and other vital chemicals for life.
 Animals that eat the plants are then able to use these amino
acids and nucleic acids in their own cells.
 Ammonification
 Soil bacteria in decomposing dead plants and animals, break
down amino acids and nucleic acids into nitrates and
ammonia, and release them back into the soil.
 Denitrification
 In the final step of the nitrogen cycle, anaerobic bacteria turn
nitrates back into nitrogen gas.
 This process occurs deep in the soil, or in wet environments which
are devoid of oxygen.
Nitrogen Cycle
48
Phosphorus Cycle
 Phosphorus is essential for the growth of plants and animals,
including microorganisms as it is required for the formation of
nucleotides, which comprise DNA and RNA molecules.
 The phosphorus cycle is the process by which phosphorus
moves through the lithosphere, hydrosphere, and biosphere.
 The main source of phosphorus is found in rocks
 Weathering of phosphorus from rocks and erosion into the soil
and water bodies.
 Absorption of phosphorus from the soil or water by plants,
fungi, and microorganisms. Animals can also obtain phosphorus
from drinking water and eating plants.
 Decomposition of dead plants and animals lead to the return
of phosphorus back to the environment.
 Human activities such as addition of fertilizers containing
phosphorus increase phosphorus levels in the soil.
 Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 97
Phosphorus Cycle
49
Bioremediation
Bioremediation; Why the Need?

 Technological advancement and industrialization of the
world have created many sites that are contaminated.
 Contamination may be by heavy metals, organic
compounds, radioactive waste and a variety of
industrial, agricultural and domestic waste materials.
 Many of these materials are toxic to organisms of any
kind, particularly human beings.
 At low concentrations, metals may be useful to living
organisms but in high concentrations they are very toxic
 It is therefore important to remove these contaminants
or at least minimize their concentrations and hence
their danger to health.
50
Conventional Metal Removal Methods
The conventional metal removal methods include:
 Chemical precipitation and filtration
 Chemical oxidation and reduction
 Electrochemical treatment
 Reverse osmosis
 Ion exchange
 Adsorption and
 Evaporation
 The conventional methods become inefficient and

expensive at low metal ions concentration (<200 ppm).
 At that level biological systems work best
Bioremediation
 Bioremediation is a branch of biotechnology which makes
use of microoganisms (microbial) or plants (phyto) to
remove or neutralise pollutants from
contaminated/polluted environment.
 Examples of contaminants in the environment include oil
spills, toxins, pesticides, petroleum hydrocarbons, metals
and chlorinated solvents.
 Microbial bioremediation uses microorganisms to break
down contaminants/pollutants by using them as a food
source.
 Phytoremediation uses plants to bind, extract, and clean up
contaminants/pollutants.
 Remediation can be accomplished using the biomass of
organisms or their by-products like enzymes.
51
Bioremediation
Bioremediation
 Bioremediation can rely on Natural Attenuation, but this
process is slow and may not complete within a realistic time
frame.
 The current method thus relies on stimulating the growth of
specific microorganisms by the addition of nutrients and the
contaminant as electron donor to serve as source of energy.
 In some cases, bioaugmentation (inoculation with microbes)
is necessary when metabolic capabilities are not naturally
present.
 The microbes consume the contaminants, converting them
into water and harmless gases like carbon dioxide.
 To create optimum conditions for microbes to flourish and
complete the bioremediation process, a conducive
environment comprising the right temperature, nutrients,
pH, oxygen are required.
52
Advantages of bioremediation
 Bioremediation:
 only uses natural processes, and thus accepted as a
relatively green method that causes less damage to
ecosystems.
 does not cause much disruption to nearby communities as
it can take place in situ.
 is useful for the complete destruction of a wide variety of
contaminants.
 creates fewer harmful byproducts, since contaminants and
pollutants are converted into water and gases like carbon
dioxide.
 can be less expensive as it does not require a great deal
of equipment or labor.
105/16
Disadvantages of Bioremediation
 Bioremediation is limited to only biodegradable compounds;
compounds that are susceptible to rapid and complete
degradation.
 Microbial processes are often highly specific; thus suitable
environmental growth conditions and appropriate levels of
nutrients and contaminants are required for growth.
 There are concerns about long term stability of products of
biodegradation.
 It is difficult to extrapolate from bench- and pilot-scale
studies to full-scale field operations.
 Bioremediation often takes longer period to complete than
other treatment options; a few months to several years,
depending on size of contaminated area, environmental
conditions and microbes employed.
53
Microorganisms for Bioremediation
 Microorganisms can be isolated from almost every
environment, with the main requirements being energy and
carbon source.
 The sources include from subzero temperatures to extreme
heat; in water, soil, air, and desert conditions; aerobic and
anaerobic conditions; and in the presence of different types of
waste and hazardous materials.
 To isolate a microbe, identify an area with a particular
contaminant to degrade.
 Feed nutrients and allow improved biological activity.
 Take samples and try to isolate related microbes that grow
directly on the item under consideration.
 Feed the isolates to known volumes/weights of the material.
 Monitor changes in volume/weight/chemistry of the material.
In Situ Bioremediation
 Bioremediation can either be done at the site of the
contamination (in situ), or at a location away from the
original place (ex situ).
 In situ biodegradation involves the infiltration of water-
containing nutrients and oxygen or other electron
acceptors through contaminated soils to stimulate
naturally occurring microbes to degrade contaminants.
 In situ process is generally more desirable due to lower
cost and less disturbance; no excavation and transport of
contaminants.
 It is however limited by the depth of the soil that can be
effectively treated; with the best being up to 30 cm.
 In situ bioremediation includes Bioventing, Biosparging and
Bioaugmentation.
54
 Bioventing
 It is an in situ treatment in which air at low flow rates and
nutrients are supplied through wells to contaminated soil to
stimulate the growth of indigenous bacteria.
 Biodegradation thus takes place with minimum
volatilisation and release of contaminants to the
atmosphere.
 Bioventing can be used for the treatment of simple
hydrocarbons and can be used where the contamination is
deep under the surface.
 Biosparging
 This process involves the injection of pressurised air
below the water table to increase groundwater oxygen
concentrations and enhance the rate of degradation of
contaminants by naturally occurring microbes.
 Biosparging increases the mixing in the saturated zone
and thereby increases the contact between soil and
ground water.
 The ease and low cost of installing small-diameter air
injection points allows considerable flexibility in the
design and construction of the system.
55
 Bioaugmentation
 This process involves the addition of microorganisms
(indigenous or exogenous) to the contaminated sites to
enhance the rate of biodegradation.
 This process should be engineered well to ensure that:
 non-indigenous cultures either outcompete or grow
synergistically with indigenous population to effectively
degrade.
 nhe augmented indigenous microorganisms grow
effectively for degradation.
Ex Situ Bioremediation
 Ex situ bioremediation normally require digging up soil and
cleaning it above ground, and this comes with extra cost.
 Ex situ is more convenient when the nature of the site does
not make microbial growth conducive.
 The processes allow for an efficient biostimulation through
optimisation of incubation parameters such as nutrients,
moistening, pH, aeration, agitation, solvents or surfactants.
 It also allows for proper control of microorganisms addition
(bioaugmentation).
 Ex situ bioremediation can be grouped into Land farming,
Composting, Biopiles and Bioreactors.
56
 Landfarming
 This technique involves excavation and spreading of
contaminated soil over a prepared bed. The soil is
periodically tilled until pollutants are degraded.
 The goal is to stimulate indigenous biodegradative
microorganisms and facilitate their aerobic degradation
of contaminants.
 In general, the practice is limited to the treatment of
superficial 10–35 cm of soil.
 Since landfarming has the potential to reduce monitoring
and maintenance costs, as well as clean-up liabilities, it
has received much attention as a disposal alternative.
 Composting
 Composting is a technique that involves combining
contaminated soil with nonhazardous organic amendants
such as manure or agricultural wastes.
 The presence of these organic materials supports the
development of a rich microbial population and elevated
temperature, which is good for composting.

57
 Biopiles
 Biopiles involve the construction of engineered cells of
aerated composted piles.
 They are a refined version of landfarming that tend to
control physical losses of the contaminants by leaching
and volatilisation.
 Typically used for treatment of surface contamination
with petroleum hydrocarbons
 Biopiles provide a favorable environment for indigenous
aerobic and anaerobic microorganisms.

 Bioreactors
 Bioremediation in reactors is an ex situ technique which
involves the processing of contaminated solid material (soil,
sediment, sludge) or water through an engineered
containment system.
 A slurry bioreactor may be defined as a containment vessel
and apparatus used to create a three-phase (solid, liquid
and gas) mixing condition.
 The mixing condition serves to promote contact between
solid, water, gas and microorganisms, capable of degrading
the target contaminants.
 In general, the rate and extent of biodegradation are
greater in a bioreactor systems as the contained
environment can be better controlled.
58
 Bioreactors
Bioreactor treatment comes with extra cost of excavation,
transportation, soil washing, extraction prior to
bioremediation.
Microbial Interaction with

Contaminants
59
Mechanisms of Microbial
Interaction with Substrates
Mechanism of microbial interactions include:
 Biosorption is a physiochemical process by which biomass
passively concentrate and bind contaminants onto its
cellular structure.
 Biotransformation is the modification of a chemical
compound by an organism.
 Biomineraliation is a transformation process which
produces mineral compounds like CO2, NH4+, or H2O.
 Bioaccumulation is the absorption of substances in an
organism at a rate faster than can be metabolised.
 Biodegradation is the breakdown of material by
microorganisms.
 Bioleaching is the process of extracting metals from ores or
waste by using microorganisms.
PHYTOREMEDIATION
 Phytoremediation mimics the action of plants in natural
ecosystems, where they act as filters and metabolize
substances generated by nature.
 Phytoremediation is a bioremediation process that uses
various types of plants to remove, transfer, stabilize,
and/or destroy contaminants in the soil and groundwater
 The root exudates of the plants play an important role in
phytoremediation by activating the surrounding
microorganism.
 Rhisosphere biodegradation or Rhizodegradation is a
symbiotic relationship that has evolved between plants and
microbes. Plants provide nutrients necessary for the
microbes to thrive, while microbes provide a healthier soil
environment.
60
PHYTOREMEDIATION
 Phytoremediation techniques can be classified into several
groups based on the mechanism employed:
 Phytoextraction or phytoaccumulation is the process
whereby plants (roots, shoots or leaves) are used to remove
contaminants. The contaminants-loaded portion can be
transported for disposal or recycling.
 Phytostabilization is a technique in which plants use
chemical compounds to bound leachable and mobile
constituents by adsorbing them into their structure.
 Phytotransformation is the use of plants to remove
contaminants, and subsequently, change them into a more
stable, less toxic, or less mobile form.
 For example, reduction of hexavalent chromium to trivalent
chromium, which is less mobile and noncarcinogenic
PHYTOREMEDIATION
61
PHYTOREMEDIATION
 Phytovolatilization is a process, whereby plants take up
water containing organic contaminants and release the
contaminants into the air through their leaves.
Phytodegradation is the breakdown of contaminants by the
presence of proteins and enzymes produced by the plants
or by soil organisms such as bacteria, yeast, and fungi.
 Rhizofiltration is a water remediation technique that
involves the uptake of contaminants by plant roots, and this
reduces contamination in natural wetlands and estuary
areas.

PHYTOREMEDIATION
Phytoremediation is well suited for use:
 At very large field sites where other methods of
remediation are not cost effective or practicable;
 At sites with a low concentration of contaminants where
only polish treatment is required over long periods of time;
 In conjunction with other technologies where vegetation is
used as a final cap and closure of the site.
Limitations of phytoremediation include:
 long duration of time for remediation,
 potential contamination of the vegetation and food chain,
 difficulty establishing and maintaining vegetation at some
sites with high toxic levels.

62
Acid Mine Drainage
Acid Mine/Rock Drainage

 Acid Mine Drainage (AMD) or Acid Rock Drainage (ARD)
occurs when exposed sulphide minerals undergo weathering
and release acidic waters.
 In areas where the drainage is caused by the presence of a
mine, it is referred to as Acid Mine Drainage.
 AMD/ARD is bioleaching in the natural environment rather
than in a metal recovery plant
 The major ingredients required for acid generation are
reactive sulphide minerals such as pyrite and arsenopyrite,
oxygen, water and sulphide-oxidising microbes.
 The sulphides may be exposed in a tailings environment,
mined-out pits or rocks in their natural states, road
construction, refill sites, etc.
63
Oxidation of Sulphides
Pyrite
2FeS2 + 7O2 + 2H2O  2Fe2+ + 2SO42- + 2H+
4Fe2+ + 7O2 + 4H+  Fe3+ + 2H2O
Fe3+ + 3H2O  Fe(OH)3 + 3H+
FeS2 + 14Fe3+ + 2H2O  12Fe2+ + 2SO42- + 16H+
 AMD/ARD can pose several problems including:

Chemical, Physical, Biological and Ecological
Problems posed by AMD/ARD

 Chemical
 pH reduction in the soil and surface/ground waters
 Destruction of carbonate buffers
 Increase in soluble metal concentration
 Increase in particulate metals
 Fe(OH)3 ppt gives the characteristic yellow colour
(ochre)
 Physical
 Substrate modification
 Sedimentation of coarse precipitates
 Adsorption of dissolved metals on sediments
 Turbidity resulting from suspended solids
 Decrease in light penetration
64
Problems posed by AMD/ARD
 Biological
 Respiratory challenges for aquatic lives
 Osmoregulation due to increase salt levels
 Acute and chronic toxicity
 Death of sensitive species
 Migration or avoidance
 Acid-base balance failure
 Ecological
 Habitat/Niche modification
 Loss of food source
 Food chain modification
 Bioaccumulation
Prediction of AMD/ARD
 Controlling AMD requires the use of predictive tools before
major excavations.
 A study of the mineralogy and the analysis of the relative
percentages of both acidic and basic minerals is used as a
predictive tool.
 Acid-base accounting is used to define the geochemical
character of different rocks, which is then utilised to
ascertain and predict the likely discharge of acidic waters
in ores and mine wastes.
 The amount of acid-producing rocks is compared with the
amount of acid-neutralizing rocks.
 This should be done prior to mining and other large-scale
excavations.
 Preventive measures can be put in place once the acid
production and drainage potential exist
65
Microbe Used to Mitigate AMD
 Desulfovibrio desulfuricans
 The bacterium is an anaerobic chemoheterotroph, and
an example of sulfate-reducing bacteria (SRB).
 Requires environment starved of molecular oxygen, eg.
Use of cow dung
 Requires electron donors, eg., ethanol, spent
mushroom, compost, acetic acid.
 Sulphate acts as electron acceptors.
 SO42- + CH3COOH + 2 H+ → HS- + 2 HCO3- + 3 H+

 HS- + Me2+ → MeS + H+
66
Application of
Microbial Technology
in the Extraction of
Mineral/Metals
Pretreatment Processes
 Pyrometallurgical methods
 Roasting
 Hydrometallurgical methods
 Chlorine oxidation
 Pressure oxidation (pyro-hydro)
 Bacterial oxidation (bio-hydro)
67
Abiotic Pretreatment Processes
Roasting T=450-700oC
4FeS2  11O2  2Fe2O3  8SO 2
4FeAsS 10O2  2Fe2O3  4SO 2  2As2O3
C  O2  CO2
Gaseous products are of environmental concern
Pressure oxidation T = 180-225oC; P =1500-3200 kPa
 2FeSO 4  2SO 24-  4H

2FeS2  7O2  2H2O 
No gaseous product BUT operational safety concerns due to
high temperature and pressure
Chlorine Oxidation
2FeS2  15HOCl  7H2O  2Fe2 (OH)3  4H2SO 4  15HCl
Corrosion problems, and possible gold dissolution
Why Hydro-process ?
 Pyrometallurgical processes:
 emit significant levels of SO2 gas that is a primary
cause of acid rain
 are not effective with low grade ores and
concentrates containing impurities like arsenic
 Hydro processes allow recovery of gold, silver, platinum
group metals (PGMs) and base metals such as nickel,
cobalt and zinc from low grade ores.
 Hydro processes allows for microorganisms to be
employed.
68
Biological Beneficiation
 Bio-processing is the acceleration of a natural
environmental process using naturally occurring organisms.
 Bioleaching is the extraction of metals from their ores
through the use of living organisms.
 Bioleaching is the biological conversion of an insoluble
metal (eg, CuFeS2) compound into a water-soluble form
(CuSO4).
 Biological beneficiation:
 Provides independence for on-site metal production in
small scale.
 Produces no noxious gases
 Is a simple technology for both small and large projects
Biological Beneficiation of Metal Sulphides
 Use of microorganisms to catalyze the oxidation

of iron sulphides to create ferric sulphate and
sulphuric acid
 Bioleaching can be used in the treatment of:
 Secondary sulphide ores in heaps
 Low-grade sulphide ores in dumps
 Sulphide concentrates in reactors at high
temperatures
69
History of Bioleaching for
Commercial Beneficiation
 Natural bioleaching dates back to about 200 BC.

 The occurrence of copper-bearing waters were described at
Rio Tinto, in Spain, in the 17th-century, as a result of acid
mine drainage.
 When these occurrences were accredited to bacteria in
1947, it generated the interest to commercialize the
process.
 Commercial application of bacterial leaching began in the
late 1950s at the Kennecott Utah Copper Company's
Bingham Canyon Mine near Salt Lake City, Utah.
 It was a management response to the detection of blue
solution of copper sulphate from waste copper sulphide
dump.
History of Bioleaching for

Commercial Beneficiation
 Investigations revealed oxidation of iron sulphides
producing ferric sulphate and sulphuric acid which acted as
oxidizer and leachant for copper sulfides respectively.
 The powerful oxidizing agents were resident bacteria,
which innovated a natural metallurgical processing plant.
 The bacteria were given the names ferrooxidans and
thiooxidans for their ability to respectively oxidize iron
sulphides and oxidize
sulphur to yield
sulphuric acid.
70
Bacterial Oxidation/Leaching (BIOX)
 The processes make use of chemolithotrophic or
‘rock eating’ microbes.
 The original biomining microbes were
Acidithiobacillus thiooxidans, Acidithiobacillus
ferrooxidans and Leptospirillum ferrooxidans.
 These catalyse the oxidation of Fe2+ and S0 in
sulphide minerals to Fe3+ and sulphuric acid.
 In biooxidation, the process ends after iron and
sulphur are oxidized.
 In bioleaching, the metal of interest is solubilised
by the oxidation products of Fe2+ and S0.
Biooxidation Pretreatment of
Sulphide Ores
 Employs iron and sulfur oxidizing bacteria
 Acidithiobacillus thiooxidans; oxidize elemental
sulphur
 A. ferrooxidans; oxidize iron(II) and elemental
sulphur
 Leptospirillum ferrooxidans; oxidize iron (II)
 2Fe2  4SO 24-  4H

2FeS2  7O2  2H2O 
4Fe2  O2  4H  4Fe3  2H2O
2S0  3O2  2H2O  4H  2SO 24
FeS2  2Fe3  3Fe2  2S0
71
Generally …
 Bacteria produces ferric iron and sulphuric acid for the
leaching.
 Bacteria action can be improved by appropriate nutrient or
discouraged by poison.
 Sulphide minerals get oxidised by ferric to produce water-
soluble compound of sulphate.
 Thus metal is leached by sulphuric acid in bioleaching.
 For non-sulphide ores, pyrite is added to be oxidised by
bacterial action.
 Bacterial action on pyrite supplements sulphuric acid
consumption.
MS 2Fe3  H 2O  3 2 O2  2Fe2  MSO4  2H 
M is a metal (eg, U, Cu, Zn, Ni)
Biooxidation/Bioleaching Chart
72
Biobeneficiation
of Copper
Beneficiation of Copper Sulphides

 Pyrometallurgical beneficiation of copper:
 Conventional process
 Accounts for about 75% copper production
 Requires high grade sulphide ores
 Crushed, ground, concentrated, smelted and refined
 Produces large quantities of sulphur dioxide
 Low grade ores cannot be processed directly.
 Low grade sulphides requires flotation followed by:
 Sulphating roasting of concentrate and leaching.
 Pressure oxidation/leaching
 Bio-oxidation/leaching can be used to recover copper
directly from low grade sulphides.
 Leaching (agitation, heap, dump, in situ).
73
Grade/size/process graph
 Leaching (agitation, heap, dump, in situ)
Pyro- and Hydrometallurgy
Biohydrometallurgy of copper accounts for over 25 % of

annual copper production worldwide
74
Biobeneficiation of Gold
Biooxidation of Gold Ores
Gold and its Ore (Host Material)

 Gold exist as very tiny particles (< 75 µm) in the ore.
 Fold is present in minute concentration (<0.001%).
 Gold is highly disseminated in the gangue (unwanted)
materials (99.999%).
 Recovery depends on particle size of gold and degree
of association with unwanted materials.
 Gold ores can be non-refractory (mainly free milling)
or refractory depending of ease of gold extraction.
 Refractory ores are very difficult to treat, and
recovery can be < 50%.
75
Refractory Gold Ores
 Refractoriness is due to the presence of:
 Sulphides
 Carbonaceous matter
 Cyanicides
 Tellurides
Table 1 Classification of Refractory Gold Ores
Classification Gold recovery

Free milling More than 95%
Mildly refractory 80 - 95%
Moderately refractory 50 - 80%
Highly refractory Less than 50%
Simplified gold beneficiation flow-sheet

Gold Ore
Difficult to treat Easier to treat
Refractory Free milling

Metal sulfides and CM (DRGO) Metal oxides and silicates
Grinding to < 75 µm Grinding to < 75 µm
Finer gold particles locked in sulfide matrix

CM adsorbs gold Gold is
liberated
Pretreatment process
Cyanidation
Gold recovery
76
Pretreatment Processes
 Pyrometallurgical methods
 Roasting
 Hydrometallurgical methods
 Chlorine oxidation
 Pressure oxidation (pyro-hydro)
 Bacterial oxidation (bio-hydro)
Environmental Issues of Roasting

 Major components targeted and reactions
 Pyrite
4FeS 2  11O2  2Fe2O3  8SO2
 Arsenopyrite
2FeAsS  5O2  Fe2O3  As2O3  2SO2
 Carbonaceous matter
C  O2  CO2
 Acidic gases
 Carcinogenic substances
 Herbicides
77
Pluses for Bio-oxidation
 Shorter plant construction time
 Robust process
 Simple operation, requiring less skilled labour
 Lower capital and operating costs
 Greater gold recovery
 Less laborious environmental requirements
 Higher arsenic stability
 Safer and healthier plants
 The consortium is non-pathogenic and harmless to
organic life
Is It Worth Comparing?
Environment before BIOX
Environment after BIOX
A method of choice due to its environmental friendliness

78
Gold Processing Plant with
Bio-oxidation Circuit
Ore  Comminution Circuit

Flotation  Tailings

Concentrate (sulfides)

Bio-oxidation

Washing  waste liqour  neutralization  sludge  dam

Oxidized ore

Leaching/Adsorption circuit  cyanidation tailings  dam

loaded carbon

Elution and electrowinning circuit

Smelting  Gold bullion
157
Simplified Process Flow-sheet Employing

Biooxidation for Refractrory Gold Ores
Gold Ore
Comminution
Waste Flotation
Flot Conc
Biooxidation
Waste Washing
BIOX Conc
Waste Cyanidation
Various Waste Treatment

Gold recovery
79
The BIOX Plant
 BIOX process was commercialized in 1986
 A BIOX plant generally consists of:
 BIOX modules/trains
 CCD thickeners
 Neutralisation section
 Leaching/CIL circuit
 Ancilliaries
 Cooling towers
 Blowers/HP compressors
 Nutrient mixing/dosing
Complete BIOX Circuit (Gold Fields

BIOX®, Australia)
80
‘Rock Eating’ Bacteria
Acidithiobacillus sp Leptospirillum
ferrooxidans
Biooxidation pretreatment
of refractory ores
 Employs iron and sulfur oxidizing bacteria
 Acidithiobacillus thiooxidans; oxidize elemental sulfur
 Acidithiobacillus ferrooxidans; oxidize iron(II) and
elemental sulfur
 Leptospirillum ferrooxidans; oxidize iron (II)
 Electron donors- Fe(II), S

 Electron acceptor – O; Fe (III)
 Carbon source – CO2
81
Defining Microbes in the BIOX Process
Bacteria Meaning Implications on biooxidation
Chemotrophic Obtain energy from Mediation of chemical
chemical reaction reactions
Lithotrophic Gain energy by oxidizing Able to oxidize iron and
inorganic compounds sulfur; generates heat
Autotrophic Synthesize needed cell Synthesis consumes some of
carbon from carbon dioxide the excess heat.
Organic carbon in the ore is
not used
Mesophilic Operate at temperatures High temperatures and
between 25-45oC pressures are not required
Acidophilic Thrives at extremely low pH Can survive in acid generated
(pH 1–2) by pyrite oxidation
Airophilic Require oxygen as electron Adequate supply of
acceptor air/oxygen
Nitrophilic Fixes organic nitrogen from Air may be supplied instead
the atmospheric nitrogen of oxygen
Direct Bacteria Mediated Reactions

 Pyrite
4FeS 2  15O2  2H 2O   2Fe2 (SO4 )3  2H 2 SO4

A. f , L. f , A.t
 Arsenopyrite
2FeAsS  7O2  2H 2O  H 2 SO4   Fe2 (SO4 )3  2H 3 AsO4

A. f , L. f , A.t
 **Lixiviant (Fe(III) and SO4) is generated in situ and

recycled.
82
Mechanism of Biooxidation of Pyrite
4FeS 2  15O2  2H 2O   2Fe2 (SO4 )3  2H 2 SO4

A. f , L. f , A.t
FeS 2  Fe2 (SO4 )3 chemical

  3FeSO4  2S
2S  3O2  2H 2O 
A.t
2H 2 SO4
4FeSO4  O2  2H 2 SO4   2Fe2 (SO4 )3  2H 2O

A. f , L. f
Operating Conditions
 pH range: 1.2 - 1.8

 Temperature range: 35oC - 45o
 [Dissolved oxygen]: > 2 ppm
 Pulp density: 15-20%
 Particle size: 95%<45 µm
 Residence time: 3 - 5 days
83
Food for the ‘Bugs’
 Energy: Sulfides
 Carbon: Carbon dioxide
 Supplements: Nitrogen and Phosphorus

3.177 kg N and 0.477 kg P
 per tonne of pyrite
 Nitrogen and phosphorus are added as fertilizer grade

ammonium sulphate and potassium phosphate
Flotation Concentrate -Feed to BIOX
84
The Biox® Reactors (Trains)
A Biox® Reactor (Internal)
85
Proposed Mechanisms of Bacterial
Oxidation of Sulphides
 There are three mechanisms based on whether there is
participation of ferric ions in leaching the mineral.
Indirect mechanism
Indirect contact mechanism
Direct contact mechanism
Indirect Mechanism
 bacteria oxidize ferrous ions in the bulk

solution to ferric ions and ferric ions
leach the mineral
86
Indirect Contact Mechanism
 attached bacteria oxidize ferrous ions to

ferric ions within layer of bacteria and
exopolymeric material, and the ferric ions
within this layer leach the mineral
Direct Contact Mechanism
 bacteria directly oxidise the mineral by

biological means, without any
requirement for ferric or ferrous ions.
87
Biooxidation Reactions (Direct)
4FeS 2  15O2  2H 2O  2Fe2 (SO4 )3  2H 2 SO4
2FeAsS  7O2  H 2 SO4  Fe2 (SO4 )3  2H3 AsO4
4FeS  9O2  H 2 SO4  2Fe2 (SO4 )3  2H 2O
2S  3O2  2H 2O  2H 2 SO4
2H 3 AsO3  O2  2H 3 AsO4
Biooxidation Reactions (Indirect)
FeS 2  7 Fe2 (SO4 )3  H 2O  15FeSO4  8H 2 SO4
FeS 2  Fe2 (SO4 )3  3FeSO4  8S
4FeAsS  4Fe2 (SO4 )3  3O2  6H 2O  12FeSO4  4H3 AsO3  4S
88
Monitoring of Bacterial Activity:
Fe(II) to Fe(III) ratio
2+ 3+
Fe /Fe Concentration as a function of time
12
Fe2+/Fe3+ Concentration
3+
10
Fe
2+
0
Fe
0 1 2 3
Time, days

Fe(II) to Fe(III) Ratio and Redox Potential
89
% Sulphide Sulphur Oxidation
12
10
8
% Sulfur
% Sulphide sulphur
0
0 1 2 3 4 5
Time, days
Items Toxic to Bacteria

 Bactericides, fungicides, and descalants normally
used for the treatment of water
 Nitrates and chlorides
 Heavy metals
 Cyanides and cyanates
 Oil, grease and degreasing compounds
 Detergents.
90
Treatment of
BIOX Products
Counter Current Decantation

(CCD) Circuit
 BIOX product is washed with water in CCD thickeners.
 Soluble species (sulphuric acid, arsenic acids, ferric and
ferrous compounds) are washed off, and neutralised.
 Solid products are thickened.
Solid to liquid
ratio depends on
levels of arsenic
and sulphuric
acid.
91
Neutralization Circuit
 Liquor contains dissolved arsenic which has to be neutralised
before disposal for environmental reasons.
 First stage: pH is raised from 2.5 to 4.5 to form stable ferric
arsenates
2 H 3 AsO4  Fe2 ( SO4 )3  3CaCO3  7 H 2O 
2 FeAsO4  2 H 2O  3CaSO4  2 H 2O  3CO2
 Second stage: neutralisation of excess acids to pH 6.5-7.0

H 2 SO4  CaCO3  H 2O  CaSO4  2H 2O  CO2
The formation of As(V) in the

BIOX liquor is key to suitable
neutralisation
Treatment of Thickened Slurry

from CCD
 Next process is cyanidation
 The thickened slurry has pH of about 2.5
 pH is adjusted to about 11 using lime to avoid
hydrolysis of cyanide
 Cyanide is added to dissolve gold in the
presence of oxygen
 Gold is recovered from solution by various
methods
92
Major Shortcoming of Biooxidation
 Carbonaceous matter is not attacked by bacteria and thus preg-
robs gold during cyanidation
BIOX concentrate
Cyanide Cyanidation Solid residue to tailings dam

Gold pregnant solution
Activated carbon Gold adsorption
Loaded carbon
Elution Barren carbon to CIL
Eluate
Electrowinning
Loaded cathode
Calcination and Smelting
Gold bullion
Fungal-Biotransformation
of
Refractory Gold Ores
93
Fungi Usage in Hydrometallurgy
 Biosorption of Cu
 Aspergillus niger, Pleurotus pulmonarius and
Schizophyllum commune
 Biosorption of Cr
 Rhizopus cohnii, Rhizopus arrhizus
 Biosorption of Cd, Cu, Hg, Ni, Pb, Cr and Zn
 Phanerochaete chrysosporium, Trametes versicolor
 Leaching of metals from electronic scrap (Cu, Sn, Al,
Ni, Pb, and Zn)
 Aspergillus niger, Penicillium simplicissimum
 Leaching of Al, Cd, Fe, Pb, Mn and Zn from municipal
solid wastes incineration fly ash
 Aspergillus niger

 Leaching of nepheline Ni, Fe, Al, V, Mo, and Sb
from fluid catalytic cracking catalyst
 Aspergillus niger
 Leaching of U from low grade uranium ore
 Cladosporium oxysporum, Aspergillus flavus
and Curvularia clavata
 Leaching of phosphate from aluminium phosphate
 Candida krissii, Penicillium expansum and
Mucor ramosissimus
 Leaching of low-grade chromite overburden
 Aspergillus niger and Aspergillus fumigatus
 Leaching of Zn, Fe, Co, Mg, Mn and Ni from silicates
 Aspergillus and Penicillium species
94
 Oxidation of sulphur and sulphides
 Aureobasidium pullulans, Rhizopus oryzae ,
Paecilomyces sp, Phanerochaete
chrysosporium
 Reduction in preg-robbing
 Phanerochaete chrysosporium, Trametes
versicolor, Aspergillus bruneio, Penicillium
citrinum
Proposed Mechanisms Utilized in

Fungi-Hydrometallurgy
 Sorption of metals
 Bioaccumulation
 Metal leaching
 Secretion of organic acids
 Oxidation of sulphur and sulphides
 Secretion of oxidative enzymes
 Reduction in pregrobbing
 Surface oxidation by oxidative enzymes
 Blocking of pores and blanking by organic
substance
95
Mycohydrometallurgy
 Mycology –the study of the properties of fungi, their

beneficial and adverse effects on humans and the
environment
 Hydrometallurgy – branch of extractive metallurgy
which focuses on the use of aqueous chemistry to
recover minerals from ores and metallurgical
products
 ‘Mycohydrometallurgy’ – adopted to define the link
between mycology and hydrometallurgy, thus the
application of fungi in mineral recovery
Current Research Background

 Double refractory gold ores contain sulfides and
carbonaceous matter (CM):
 Sulphides encapsulate gold
 CM confines gold and also adsorbs dissolved gold
during cyanidation
 Sulfides have to be oxidized to release gold
 Carbonaceous matter has to be oxidized or passivated
before gold leaching to prevent preg-robbing
 Methods used include roasting, blanking with organic
solvents, microbial techniques, etc
96
Biooxidation Pretreatment of
Refractory Ores
 Employs iron and sulphur oxidizing bacteria (eg, A.
thiooxidans, A. ferrooxidans and L. ferrooxidans) to
catalyze oxidation of sulphides
 2Fe2  4SO 24-  4H

2FeS2  7O2  2H2O  [1]
2  3
4Fe  O2  4H  4Fe  2H2O [2]
2S0  3O2  2H2O  4H  2SO 24 [3]
FeS2  2Fe3  3Fe2  2S0 [4]
 Does not deactivate carbonaceous matter (CM)

 CM picks up gold from solution during the subsequent
cyanidation
 Loss of gold-loaded carbon leads to reduced overall gold
extraction
Major Shortcoming of Biooxidation

BIOX concentrate
Cyanide Cyanidation Solid residue to tailings dam

Gold pregnant solution
Activated carbon Gold adsorption
Loaded carbon
Elution Barren carbon to CIL
Eluate
Electrowinning
Loaded cathode
Calcination and Smelting
Gold bullion
Carbonaceous matter preg-robs gold during cyanidation

97
Carbonaceous Matter (CM) in Gold Ore
90
85
 CM is naturally part of
the ore
80
 Behaves like activated
Gold recovery (%)
75
carbon
70
65
 Has ability to adsorb gold
after gold dissolution
60
 The loaded gold cannot
55
be recovered
50
0 5 10 15 20 25 30 35 40  Reloading leads to
Cyanidation time (hr) reduced gold recovery
After Osseo-Asare et al., 1984  This phenomena is
termed preg-robbing
Carbonaceous Matter (CM) in Gold Ore

100  Research on preg-robbing
dates back into several
80
decades
Gold adsorption (%)
60  CM is characterized using coal

 Maturity ranges from lignite-
40 to anthracite-grade coal
 Adsorption capacity increases
20
with increasing maturity
0  Anthracite-grade CM
constitutes about 50% of CM in
refractory gold ores
CM maturity  Anthracite-grade CM adsorbs
10 times more than other
After Ofori-Sarpong et al., 2010 coals
98
Coal as Surrogates for
Carbonaceous Matter in Gold Ores
O
OH
O
OH
O OH
O OH
O O Me
OH
O
OH
O OH N O
O S
OH N
Me O
OH O
O
HO O O
O
O O
O Me
OH
Lignite O
Anthracite
Adsorption of Gold Cyanide on

Carbonaceous Matter
 Several theories have been proposed
 Three most important facts about adsorption of
aurocyanide on carbon:
 Porousity/Surface area of the carbon
 Graphitic structure of carbon
 Surface oxygen groups on carbon
 Current concensus indicate graphitic structure is the
most important parameter for gold adsorption
99
Interaction of Gold and Graphitic
Structure
After Jones et al. 1989; Klauber, 1991; Ibrado and Fuerstenau; 1992; 1995; Poinen and Thurgate, 2003
 Au(CN)2- complex adsorbs intact on the surface of

graphitic planes via donation of delocalized pi electrons
from the graphitic planes to the gold ion
 Destruction in graphitic planes will lead to reduction in
gold adsorption
Importance of Graphitic Structure

of Carbon in Gold Adsorption
 Anthracite adsorbs
many times more thatn
lignite and bituminous
 Gold adsorption is
favored by high degree
of graphitization and
low oxygen groups on
carbon
Ofori-Sarpong et al., 2010
100
Reduction in Preg-robbing
 Objective is to disturb the parameters that promote gold
adsorption
 Destruction of graphitic layer of carbon
 Reduction in the surface area and porousity of
carbon
 Increase in surface oxidation of carbon
 Methods used include blanking with organic agents,
roasting to oxidize/destroy carbon, microbial
degradation
 Current research is geared towards the use of microbial-
aided systems
 Findings on biotreatment of CM show promising results
Microbial transformation
of carbonaceous matter
leading to reduction in
preg-robbing of gold
101
Microorganisms Exploited
 A number of bacteria and fungi have been tested for the
deactivation of different grades of CM, and there are
reports of increase in overall gold extraction
 Bacteria used include:
 Streptomyces setonii, Pseudomonas spp., Achromobacter
spp., Arthrobacter spp. and Rhodococcus spp.
 Fungi used include:
 Trametes versicolor, Aspergillus bruneio-uniseriatus and
Penicillium citrinum, Phanerochaete chrysosporium
 Most of the microoganisms used have been more
successful with CM of lignite-grade and bituminous-grade
than anthracite-grade maturity
Some Case Studies
102
Microbial Coal Degradation
 Alkaline metabolites
 polypeptides
 polyamines
 Enzymatic
 peroxidase (lignin and manganese)
 laccase
 hydrolase
Streptomyces setonii
 Chemoheterotroph
 Neutrophile
 Soil microbe
 Degrades softwood, hard wood and coal
 Generates alkaline metabolites
103
Phanerochaete chrysosporium
 Grows rapidly over wide temperature; 25-
40oC and pH; 3-7
 Secretes enzymes lignin peroxidase,
manganese peroxidase, and H2O2
 Capable of degrading lignin and a host of
aromatic substrates, e.g. coal
 Capable of solubilizing sulfides
Reaction of Fungal-Biotransformation
of Carbonaceous Matter (R)
O
OH
OH
Andrawis et al, 1988; Banci et al., 1992; Edwards et al., 1992; Gold and Alic, 1993
 Hypothesis
 Oxidative enzymes can destroy graphitic planes necessary
for adsorption
 Destruction of planes leads to decrease in surface area
and increase in pore diameter
 This will enhance entrance of hyphea/spores leading to
bockage of spores and reduced pore volume
104
General Approach
Materials
Anthracite Gold concentrate Pyrite/Arsenopyrite

CM surrogate CM and Sulfides Sulfide surrogate
Fungal Treatment (Incubation, washing and drying)
Analysis of incubation solution and residual solids
Anthracite Gold Concentrate Pyrite/Arsenopyrite
Gold cyanidation As, Fe, S in solution,

Gold adsorption
sulfide sulfur in solids
Incubation of Materials with

P. chrysosporium
A B C D E F
A: Millet medium; B: Millet and wheat bran (MWB) medium;

C: 1 wk culture in millet; D: 1 wk culture in MWB;
E: 1 wk treatment in millet; F: 1 wk treatment in MWB
Incubation at 60% solids for up to 21 days at 37 oC and pH 4
105
Assessment of Fungal Action on
Anthracite
 Residual solid material is analyzed by various methods:
 Deactivation of carbon – Gold adsorption
 IC  FC  g 
 mol of gold   mL  1 
PEC  in   25 mL x   WC g 
 g of carbon   197 g  
 mol 
 Surface oxidation of carbon – XANES, infrared
 Surface area and pore volume – Porosity analysis
Effect of Processing Time on

Degradation (14 Days, 5% Solids)
1200
Mass degraded, mg
800
lignite
bituminous
anthracite
400
0
0 14 28 42
Time, days
(Amankwah and Yen, 2006)
106
Preg-robbing by Gold Ores
100
as-received sample
56 days, 23 deg
75
% gold sorbed
50
25
0
A B
Sample
Amankwah and Yen, 2007
Two-stage Microbial Pretreatment

Of Gold Ores
Flotation concentrate containing gold, sulfides
and carbonaceous matter
Biooxidation of sulfides using

chemolithotrophic bacteria
Washing of product Wastewater
Biodegradation of carbonaceous matter using

Streptomyces setonii
Washing of product Wastewater
Cyanidation and gold recovery
General process proposal by Amankwah and Yen, 2007

107
Reduction in Gold Adsorption by
Anthracite following Fungal-treatment
100
80
 Fungal-treatment led
Gold adsorption (%)
to over 95% reduction

60
in gold uptake by
anthracite after 5
40
days of treatment
20
0
As-received 3 days 5 days 7 days 14 days
Fungal treatment time (days)
(Ofori-Sarpong et al., 2010)
Surface Area and Pore

Characteristics of Anthracite
120
As-received Treated
 Biotreatment
Percentage value of parameter (%)
100
decreases pore
80
volume
60  Reduction in surface
40 area limits
20
accessibility to sites
of adsorption
0
BET surface Micropore Micropore Average pore  Reduction in
area area volume diameter
micropore volume
Parameter reduces available
(Ofori-Sarpong et al., 2011) sites for adsorption
108
XANES of As-Received and
Biotreated Anthracite
Biotreated
anthracite
0.6
 Peak of 288.2-290
units
As-received
anthracite eV represents
carboxylic acids
Absorbance
xµ (E)
0.4
Carbon nano-
powders
 Braun et al., 2005
0.2
280 285 290 295 300 305

E (ev)
Energy, eV
Summary of Findings on Fungal-

Transformation of Anthracite
 P. chrysosporium is capable of reducing the gold ‘up-
take’ ability of anthracite by over 90% in MWB
medium (5-7 days)
 Reduction in gold adsorption is due to the reduction
in surface area, pore volume and increase in oxygen
content and thus amorphous nature
 These were confirmed by techniques including
XANES, surface area and porosity analysis
109
Assessment of Fungal Action on
Sulfides
 Dissolved constituents; As, Fe, S – ICP-AES
V (mL) x C  μg 
 mL 
Fungal dissolution of constituents, FDC (wt%) 
Pm % x W g 
 Sulfide sulfur in residual solids - LECO volumetric

combustion technique
(SI  SF) (g)
Conversion of sulfur, CoS (wt%) 
SI g 
 Phase changes in residual solids – XRD
Fungal Biotransformation of
Sulphides
 Some results
Sample Concentration of Overall sulfide

constituents in sulfur
incubation solution degraded, wt%
(wt%)
As Fe S
 Pyrite 1.2 6.0 15
Arsenopyrite 7.2 1.5 10.3 35
Gold 6.1 1.8 10.7 57
concentrate
110
Fungal-oxidation of Sulfide in
Gold Concentrate
15 60
 Fungal-transformation
Unconverted sulfide sulfur in gold concentrate (g)
Sulfide sulfur converted (wt % of initial)

50 of sulfide destroyed
12.5
40
sulfides in the gold
Residual sulfide sulfur concentrate
10 Converted sulfide sulfur 30
20  This led to liberation

7.5 of gold
10
5 0
0 5 10 15 20 25
Fungal -treatment time (days)
XRD of As-Received and Fungal-

Treated Flotation Concentrate
As-received FC
300 1
Treated FC
1 – quartz
2 – pyrite
 Fe2O3 and FeAsO4
Intensity (counts)
200 3 – arsenopyrite
4 – hematite
were detected in
5 – ferric arsenate
6 – muscovite
this work
1 2
100 5 4
6 2
1
2
2 3
6 1 6 4 1
5 1 3
1
0
10 20 30 40 50 60 70
2 Theta (deg)
111
Summary of Findings on Fungal-
Transformation of Sulfides
 P. chrysosporium can oxidize sulfides by 10-57% (3-21
days)
 Oxidation products include sulfates, arsenates,
oxides and elemental sulfur
 This was confirmed by XRD and volumetric
combustion analysis
Assessment of Fungal Action on Gold

Liberation and Cyanide Amenability
 Gold liberation – Cyanidation
 Total gold determination in solids– Fire assaying

( g ) xV (mL) xC ( g )
1 1
mL
Gold recovery, GR( wt %)  W
HG( g )
t

Cyanidation was conducted at 30 % pulp density for 24 h at
pH 11 and cyanide strength of 10 g/L
112
Correlation of Sulfur Oxidation
and Gold Extraction
80 70
Sulfide sulfur converted (wt % of initial)

60  Extension of fungal-
70 treatment time
Gold recovery (%)
50
 More sulfide oxidation
40
60  More gold released
30
 Increased contact of
50
20 gold and cyanide
Gold recovery (%) 10  More gold extracted
Sulfide converted (%)
40 0
0 5 10 15 20 25
Fungal-treatment time (days)
(Ofori-Sarpong et al., 2013a)
Motivation for Future Research

 Major drawback of in vivo fungi treatment
– separation of treated material
from biomass
113
Objective is to Address Major
Drawback On In-vivo Fungal
Process
 Assess in-vitro pretreatment of refractory gold

ores using cell-free culture of P. chrysosporium
Decomposition of Sulphide
Sulphur in Flotation Concentrate
30
Sulfide sulfur converted (wt % of
25
20  (Experiments were
conducted at 15%
initial)
15 solids, 37oC and

10 up to 72 hr)
0
4 hr 24 hr 72 hr
Treatment time
(Ofori-Sarpong et al., 2013b)
114
Cyanidation of As-Received Flotation
Concentrate and the Bio-products
70
Gold recovery (% of initial weight)
60  Conclusion
50
 In vitro processing
is a promising
40 alternative to in
30
vivo or whole cell
processing of
20 refractory gold
As-received H2O2 Fungal extracts ores using P.
chrysosporium.
Treatment method
(Ofori-Sarpong et al., 2013b)
On-going and Future Work

 Characterise the activity of enzymes in the in-vivo
process
 Use cell-free extracts, and ultimately, enzymes to
eliminate biomass
 Need for a better understanding of the kinetics
 Systematic quantification and purification of the
extracts to obtain the pure enzyme
 These may then be used in estimating the actual
amount of enzyme required to decompose a given
amount of sulphides and carbonaceous matter in gold
ores.
 Use continuous process to improve the kinetics
115
Genetic Engineering
 A GMO (genetically modified organism) is the result of a
laboratory process where
 Genetic engineering’modification is the process is the
process of transferring genes from the DNA of one species
into the genes of an unrelated plant or animal.
 The foreign genes may come from bacteria, viruses,
insects, animals or even humans.
 Genetic engineering is accomplished in three basic steps.
 Isolation of DNA fragments from a donor organism.
 Insertion of an isolated donor DNA fragment into a
vector genome.
 Growth of a recombinant DNA in an appropriate host.
References
 Anon.(2018a),https://www.copper.org/publications/newsletters/innovations/2004/05/producing_co
pper_natures_way_bioleaching.html. Assessed 26th October, 2018
 Anon. (2018b), http://www.onlinebiologynotes.com/bacterial-growth-curve, Assessed 12th
September, 2018
 Anon. (2018c), Tortora, G.J., Funke, B.R., Case, C.L. and Johnson, T.R., 2004. Microbiology: an
introduction (Vol. 9). San Francisco, CA: Benjamin Cummings, Assessed 15th Sept., 2018.
 Anon. (2018d), https://en.wikipedia.org/wiki/Biofilm, Assessed 15th Sept., 2018.
 Anon. (2018e), https://www.tes.com/teaching-resource/edexcel-a-level-carbon-cycle-lesson-2-
11660732. Assessed 15th September, 2018.
 Anon. (2018f), https://biologydictionary.net/peptidoglycan/#ftoc-function-of-peptidoglycan,
Assessed 17th September, 2018.
 Anon. (2018g), https://www.jove.com/science-education/10100/bacterial-growth-curve-analysis-
and-its-environmental-applications. JoVE, Cambridge, MA, (2018), Assessed 17th September, 2018.
 Anon. (2018h), http://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/21-molecules-
to-metabolism/anabolism-and-catabolism.html, Assessed 17th September, 2018.
 Anon. (2018i), http://www.shareyouressays.com/knowledge/5-main-steps-that-are-involved-in-
carbon-cycle-explained/112952, Assessed 17th September, 2018.
 Falkowski, P. (2000). The Global Carbon Cycle: A Test of Our Knowledge of Earth as a System.
Science, 290(5490), 291-296. doi:10.1126/science.290.5490.291, Assessed 21st Sept., 2018.
 vlab.amrita.edu,. (2011). Gram Stain Technique. Retrieved 15 September 2018.
 https://responsibletechnology.org/gmo-education/the-ge-process, Assessed 27th Oct., 2018.
116

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Faculty of Mineral Resources Technology

Department of Minerals Engineering

UNIVERSITY OF MINES AND TECHNOLOGY, TARKWA UMaT

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 2

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 4

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 5

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 7

 Study of microorganisms, and their beneficial and

 Can be grouped broadly into:

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout

 a) Microbial diversity and versatility

 Basic concepts of microorganisms

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout

 Sulphur, carbon, nitrogen cycles

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 11

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 12

 a) Acid mine drainage and mitigation

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 13

Assignment One - Microorganisms

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 16

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 17

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 18

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 19

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 20

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 21

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 22

Properties of useful microbes

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 24

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout

Genera and Species

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 28

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 29

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 30

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 31

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 32

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 33

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 34

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 35

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 37

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 38

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 39

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 40

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 41

Extremophile Organisms - Archaea

 Bacteria are classified as being either Gram-positive or

Gram-positive and Gram-negative

 Gram-positive bacteria have a very thick cell wall made of a

Gram Stain Procedure (vlab.amrita.edu, 2011)

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 48

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 49

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 51

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 52

Microbial (Bacterial) Growth

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 57

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 58

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 59

Stationary or Plateau Phase

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 60

Assoc Prof Grace Ofori-Sarpong (2018), MR 379 Microbial Technology Handout 61