PowerPoint PresentationOleh
Laboratory, Biosciences & Biotechnology Research Center (BBRC)
Department of Metallurgical Engineering, Faculty of Mining &
Petroleum Engineering
Institut Teknologi Bandung
[email protected];
[email protected]
Cell phone: 087878590709 (WhatsApp)
macromolecules • Polysaccharides • Lipids • Nucleic Acids (DNA,
RNA) • Proteins
Structure of a fungal cell wall
Chitin (C8H13O5N)n (/katn/ 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,
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)
Oil droplet C,H,O,N,S
Ti2+ Cr3+
Cu2+ Zn2+
0.01~10 mg/l Ti, Cr, Ni, Cu, Zn 0.01~ 0.5 mg/l Co 0.01~25 mg/l Ca
0.01~100 mg/l Si (stimulate bacterial growth)
Aerobic condition pH 5-8
Bacteria were identified as: Pseudomonas aeruginosa, Bacillus
cereus or Bacillus thuringiensis, Paracoccus seriniphilus or
Paracoccus marcusii
transmembrane transport
Sea wate
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 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
Asexual spores called conidia
• 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).
– 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 Aerobic condition
Precipitates Microbes
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.
Chelation describes a particular way that ions and molecules bind
metal ions
a ligand /lnd/ is an ion or molecule (functional group) that binds
to a central metal atom to form a
coordination complex
• 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 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
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.
Methylation: the addition of a methyl-group (-CH3) to a
compound
Dealkylation: the removal of a methyl-group from a compound
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.
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
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The main organic components comprising algal and fungal walls
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the general metabolic relationship between organic acids (gluconic,
citric and oxalic acids) produced by A. niger
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Fungal Leaching Mechanisms
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