Bioleaching Mineral Oksida & Silicate

Mekanisme Bioleaching oleh fungi

Oleh

Siti K Chaerun
Geomicrobiology-Biomining & Biocorrosion Laboratory, Microbial Culture Collection
Laboratory, Biosciences & Biotechnology Research Center (BBRC)
Department of Metallurgical Engineering, Faculty of Mining & Petroleum Engineering
Institut Teknologi Bandung
skchaerun@metallurgy.itb.ac.id; skchaerun@gmail.com
Cell phone: 087878590709 (WhatsApp)
monomers macromolecules
• Sugars • Polysaccharides
• Fatty acids • Lipids
• Nucleotides • Nucleic Acids (DNA, RNA)
• Amino acids • Proteins
 Chitin (C8H13O5N)n (/ˈkaɪtɨn/ KY-tin) is a long-
chain polymer of a N-acetylglucosamine, a
derivative of glucose, and is found in many
places throughout the natural world. It is the
main component of the cell walls of fungi,

Structure of a fungal cell wall

The main organic components comprising algal and fungal
walls
The mitochondrion (plural mitochondria) is a
membrane bound organelle found in most
eukaryotic cells

The ribosome is a large and complex

molecular machine, found within all living
cells, that serves as the primary site of
biological protein synthesis (translation).
Ribosomes link amino acids together in the
order specified by messenger RNA (mRNA)
molecules
 Ni2+ Oil droplet
Cl- C,H,O,N,S
ta er Na+
K+
eaw
S Mg2+
0.01~10 mg/l Ti, Cr, Ni, Cu, Zn
0.01~ 0.5 mg/l Co
0.01~25 mg/l Ca
Ca2+ 0.01~100 mg/l Si
(stimulate bacterial growth)
Co2+ transmembrane transport

Br- Extracellular polymeric substances (EPS)

i.e., exopolysaccharides
Sr2+ Cr3+
2+ Cu2+
2+
Ti SiO2 Zn Aerobic condition
pH 5-8

Bacteria were identified as:

Pseudomonas aeruginosa, Bacillus cereus or Bacillus thuringiensis,
Paracoccus seriniphilus or Paracoccus marcusii
 Fungi
• Fungi are heterotrophic eukaryotes which lack
of chlorophyll and require pre-formed organic
carbon and energy source
• It contains a real nucleus and normally forms
mycelia.
• Fungi typically reproduce asexually and/or
sexually by producing spores
• They are aerobic organisms and thus oxygen
supply is necessary.
• Heterotrophic fungi can Asexual spores called conidia

withstand a much wider

pH range compared to
chemolithoautotrophic
bacteria
• Filamentous fungi which
are capable of
bioleaching include the
genus Aspergillus which
have black, brown or
green spores, and the
genus Penicillium which is
well-known in producing
Penicillin as an effective
treatment of infectious
diseases
• Fungi are capable of oxidizing substrate only
partially and then secreting it. This incomplete
oxidation causes the accumulation of organic
acids, which are able to extract metals from
solid materials.
• These organic acids may be categorized into
two groups:
– those derived from sugars by simple oxidation
(gluconic acid = C6H12O7, kojic acid = C6H6O4),
– and those which are related to tricarboxylic acid
intermediates (citric acid, oxalic acid and malic
acid).
the general metabolic relationship between organic acids (gluconic, citric and
oxalic acids) produced by A. niger
• Gluconic acid results from the
conversion of glucose by glucose
oxidase

• the biosynthesis of citric acid in

fungi involves:
– glycolysis
–tricarboxylic acid cycle (TCA
cycle).
 Glycolysis
• Is the conversion of glucose to pyruvate by:
– The Embden-Meyerhof Parnas (EMP) Pathway
– Pentose Phosphate (PPP) Pathway or Enter-
Doudoroff (ED) Pathway.
(Of these, EMP is the most common and important
pathway)
• Pyruvate is then oxidized to carbon dioxide
and water in the TCA cycle and at the same
time, accumulation of citric acid occurs within
the cycle (Figure 2.2b).
 The oxalate biosynthesis in fungi
• Three possible pathways may be involved:
– formation of oxalate by splitting of the
oxaloacetate which does not enter the TCA
cycle
– formation of oxalate by splitting of the
oxaloacetate which arises from the TCA
cycle
– Formation of oxalate from glyoxylate via the
glyoxylate cycle
[Gadd, 1999]
 Fungal Leaching Mechanisms
• Metal leaching by heterotrophic microbes
generally involves an indirect process with
microbial production of organic acids (e.g.
lactic acid, oxalic acid, citric acid, gluconic
acid), amino acids, and other metabolites via:
– Acidolysis
– Complexolysis
– Alkalolysis
– bioaccumulation
 Acidolysis
• Acidolysis is a fast and dominant leaching mechanism in fungi
bioleaching
• It is a process which involves the protonation of oxygen atoms
in the metal compound.
• The protonated oxygen then combine with water, resulting in
the metal oxide being detached from the solid surface and
being solubilised
• The amount of metal oxides solubilized results from the
maximum amount of protons obtained from the organic acids.
• Acidolysis reaction in general:
• The common acids secreted by heterotrophs
are lactic, oxalic, gluconic, acetic, citric,
succinic, pyruvic and formic acids.
• These assist in creating a low pH environment
which enhances the bioleaching of metals
• Organic acids produced which decrease the
availability of anions to the cations in metal
compounds, thus causing the solubilization of
metal ions
 Complexolysis
• While the organic acids formed from the heterotrophs
participate in acidolysis, the relatively slower mechanism
of complexolysis also takes place because the organic
acids, some of which are powerful natural chelating
agents, form a metallic complex with the metals from the
material to be bioleached.
• The solubilization of metal ions is based on the complexing
capacity of a molecule.
• If the bonds between metal ions to ligands are stronger
than the lattice bonds between metal ions with solid
particles, metal will be successfully leached out from solid
particles
Chelation describes a particular way that ions and molecules bind metal ions

a ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex
• Complexolysis adalah mekanime pelarutan logam yang diinduksi oleh ligan.
Dalam mekanisme ini terjadi mikroba membentuk pengompleks atau chelating
agents yang menyebabkan peningkatan mobilitas logam.
• Complexolysis, which often combines with
acidolysis, stabilize the metal ions that are
solubilized into solution by acidolysis.
• The stability of metal complexes also reduces the
toxicity of metal ions to the microbes
• Stable complexes could be formed between the
organic ligands with metal ions, such as oxalic
acid and iron, citric acid and magnesium, phenol
derivatives and some amino acids with metal
ions.
• The production of low molecular weight iron
chelating siderophores by fungi or bacteria also
enables solubilization of iron (III)
 Bacterial Cell Fe+3
Aerobic condition
Neutral pH (Insoluble form in solution)
Precipitates
Chelation describes a particular way that ions and Microbes
molecules bind metal ions

Soluble form in solution

a ligand /lɪɡənd/ is an ion or molecule (functional
group) that binds to a central metal atom to form a
coordination complex

Fe
+3-Siderophores

Transport into cell

Fe+2-Siderophores (enzymatically)

Fe+2 ---assimilated into protein

Desferrisiderophore

Siderophores (Greek: "iron carrier") are small, high-affinity iron chelating compounds secreted by microorganisms
such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known.
• It has been found that amino acids are released
together with organic acids.
• Both groups of compounds provide protons and
complexing capacity for metal solubilization
• In most cases of metal solubilisation by
heterotrophic microorganisms, the organic acids
are the lixiviant (leaching agent) and excreted
amino acids are also able to solubilize metals.
• However, it is found that leaching through amino
acids is not of great importance for fungi in
general as amino acid efflux is rare among
filamentous fungi
• The complexation of metal ions with citrate was
found to result in the formation of highly mobile
species and therefore allows transport and
activity of toxic metals at a distance from their
source.
• Metals interaction with oxalic acid could
ultimately lead to the formation of insoluble
oxalates which could immobilize toxic metal
species.
• Most metal oxalates are immobile and resistant
to further solubilization, with only a few species
of anaerobic bacteria, aerobic actinomycetes,
bacteria and fungi able to degrade them readily
 Alkalolysis
• The enzymatic hydrolysis of urea or
deamination of amino acids by microbes,
when these compounds are used as the
energy source, results in the production of
ammonia which is able to leach metals by the
alkalolysis process.
• This mechanism is very effective in mobilizing
metals in silicates or aluminosilicates
• This mechanism enables bioleaching to take
place at high pH.
 Bioaccumulation
• Bioaccumulation is the only mechanism that does not involve
the excretion of metabolites
• Bioaccumulation occurs when the solubilised metal ions
accumulate within the mycelia of the heterotrophic fungi
through passive adsorption and active metabolic reactions,
thereby enhancing further metal solubilisation into the
solution
solubilised metal ions
• This could be interpreted as the mycelium functioning somewhat as
a sink for metal ions.
• Fungal cell wall contains many different functional groups (e.g.
hydroxyl, amine, carboxyl, phosphate and sulfate groups) which are
able to bind metal ions to a greater or lesser extent.
• Among the filamentous fungi, the genera of Aspergillus and
Penicillium have been reported to have high ability to accumulate
heavy metals and radionuclides from their external environment.
• The fourth advantage is energy source. Fungi leach metals by the
excretion of metabolites, and involve several indirect leaching
mechanisms such as acidolysis and complexolysis of metal ions.
Energy for growth and organic acid excretion is externally supplied
through the organic substrates in the medium.
• The main drawback on the use of heterotrophic fungi is the need for
a significant amount of organic carbon source for growth and for the
production of leaching agents. Thus, bioleaching using heterotrophs
needs a higher operating cost compared with
chemolithoautotrophs.
Bioaccumulation
The accumulation process involves the biological
sequestering of substances that enter the organism through
respiration, food intake, epidermal (skin) contact with the
substance, and/or other means.

The level at which a given substance is bioaccumulated

depends on:
the rate of uptake
the mode of uptake
how quickly the substance is eliminated from the organism
transformation of the substance by metabolic processes
the lipid (fat) content of the organism
the hydrophobicity of the substance
environmental factors
other biological and physical factors
Biosorption

The ability of certain types of inactive, dead, microbial biomass to

bind and accumulate heavy metals from aqueous solutions
through non metabolically mediated or physico-chemical
pathways of uptake.

Materials which exhibit biosorptive behaviour (biosorbents)

include certain algae, fungi and bacteria.

Interactions of metal and fungal cells
Methylation: the addition of a methyl-group (-CH3)
to a compound
Dealkylation: the removal of a methyl-group
from a compound
oligomer is (chemistry) a compound intermediate between a
monomer and a polymer, normally having a specified number
of units between about five and a hundred while polymer is
(organic chemistry) a long or larger molecule consisting of a
chain or network of many repeating units, formed by
chemically bonding together many identical or similar small
molecules called monomers a polymer is formed by
polymerization, the joining of many monomer molecules.
Metallothioneins (MTs) are a family of small, highly
conserved, cysteine-rich metal-binding proteins that
are important for zinc and copper homeostasis,
protection against oxidative stress, and buffering
against toxic heavy metals.
Phytochelatins are oligomers of glutathione, produced by
the enzyme phytochelatin synthase. They are found in
plants, fungi, nematodes and all groups of algae including
cyanobacteria. Phytochelatins act as chelators, and are
important for heavy metal detoxification. They are
abbreviated PC2 through PC11
 A vacuole
• The fungal vacuole is a large, membrane-bounded organelle
that functions as a reservoir for the storage of small molecules
(including polyphosphate, amino acids, several divalent cations
(e.g. calcium), other ions, and other small molecules) as well as
being the primary compartment for degradation.

• It is an acidic compartment, containing an ensemble of acid

hydrolases (An acid hydrolase is an enzyme that works best at acidic pHs. It is commonly
located in lysosomes, which are acidic on the inside).

• A vacuole is a membrane-bound organelle which is present in

plant and fungal cells and some animal and bacterial cells.

• A space or vesicle within the cytoplasm of a cell, enclosed by a

membrane and typically containing fluid.
Uptake nutrisi secara extraselular
(oleh fungi)

Fig 1
Fig 2
• Fungi are in direct contact with their nutrients
in the environment.
• Smaller molecules (such as simple sugars and
amino acids) in solution in the watery film
surrounding the hyphae can be directly
absorbed by the hyphae.
• Larger insoluble polymers such as cellulose,
starch, and proteins must undergo a
preliminary digestion before they can be used.
• Molecules that are too large to be absorbed by
the fungus are attacked by extracellular
enzymes (Figs. 1 and 2).
• Like all digestive enzymes, the digestive enzymes of fungi-
control hydrolysis reactions that cleave the large
molecules into simpler components.
• The digestive enzymes are highly specific and are able to
control hydrolysis of particular molecules only.
• Complete digestion of a large polymer is a stepwise
process involving different enzymes until finally a simple,
soluble molecule is released. It is this simple molecule
that is taken up by the fungus. Once it is absorbed into
the cell, this small molecule is further acted upon by
intracellular enzymes.
• The ability to utilize large molecules ultimately depends
on the ability of the fungus to digest them, which in turn
depends on the enzymes with which the fungus is
equipped.
• Fungi typically have a large number of enzymes but for the
most part, many of them are inactive until the fungus comes
into contact with a substrate on which particular enzymes can
act.
• Growth of the fungus occurs equally well on a medium
containing either complex or simple nutrients.
• The necessary enzymes may be entirely lacking, however, and
the fungus may be unable to grow on a medium that contains
an undigestible substrate.
• All ions and molecules entering the fungal cell must pass
through both the cell wall and the plasmalemma.
• The wall itself is somewhat porous, allowing ions and molecules
to pass through if perhaps through minute pores or channels.
• The plasmalemma is a semipermeable membrane that can
regulate the movement of solutes into the cell