LOVELY PROFESSIONAL UNIVERSITY

PHAGWARA (PUNJAB)

MSc.MICROBIOLOGY

TERM PAPER

MICROBIAL PHYSIOLOGY AND METABOLISM

TOPIC- “INDUSTRIAL IMPORTANCE OF

THIOBACILLUS FERROXIDANS”

SUBMITTED BY:-

SHASHI SHARMA

Roll no. RP8003B15

CONTENTS:

1. Introduction about Thiobacillus ferroxidans.

2. Why this bacteria is having industrial importance?

3. Industrial importance:

Treatment of leather industry wastewater by aerobic biological and Fenton oxidation process

Modeling and analysis of biooxidation of gold bearing pyrite-arsenopyrite concentrates by Thiobacillus ferrooxidans

Suppression of pyrite oxidation by iron 8- hydroxyquinoline

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite:

Role of Ferrous Ions in Synthetic Cobaltous Sulfide Leaching of Thiobacillus ferrooxidans

Thiobacillus ferrooxidans involved in the biohydrometallurgical extraction processes

The role of Thiobacillus ferrooxidans in the bacterial leaching of zinc sulphid

The role of Thiobacillus ferrooxidans in the bacterial leaching of zinc sulphide

Bacterial leaching OXIDATION OF SULPHIDIC MINERALS:

Novel mineral processing by flotation using Thiobacillus ferrooxidans

4. Scope of Thiobacillus ferroxidans for further research.

5. Refrences.

Introduction about Thiobacillus ferroxidans :

The genus Thiobacillus is also known under the name of Acidithiobacillus.

Thiobacillus ferrooxidans are airborne bacteria.

This genus is thermophilic, preferring temperatures of 45-50 degrees Celsius.

In addition, this is an acidophilic genus, preferring a pH of 1.5 to 2.5.

A few species only grow in a neutral pH.

Thiobacillus are strictly aerobic bacteria. All species are respiratory organisms.

Thiobacillus are obligate autotrophic organisms, meaning they require inorganic molecules as an electron donor and inorganic carbon (such as carbon dioxide) as a source.

They obtain nutrients by oxidizing iron and sulfur with O2.

Thiobacillus do not form spores; they are Gram-negative Proteobacteria.

Their life cycle is typical of bacteria, with reproduction by cell fission.

Thiobacillus are colorless, rod-shaped, Gram-negative bacteria with polar flagella.

They possess an iron oxidase, which allows them to metabolize metal ions such as ferrous iron.

Fe2+ + 1/2 O2 + 2H+ --> Fe3+ + H2O

ECOLOGY:

Thiobacillus ferrooxidans is the most common type of bacteria in mine waste piles.

This organism is acidophilic (acid loving), and increases the rate of pyrite oxidation in mine tailings piles and coal deposits.

It oxidizes iron and inorganic sulfur compounds.

The oxidation process can be harmful, as it produces sulfuric acid, which is a major pollutant.

However, it can also be beneficial in recovering materials such as copper and uranium.

It has been suggested that T. ferrooxidans forms a symbiotic relationship with members of the genus Acidiphilium, a bacterial capable of iron reduction.

Other species of Thiobacillus grow in water and sediment; there is both freshwater and marine strain.

A colony of Thiobacillus ferrooxidans. The reddish color is the result of iron production.

Why this bacteria is having industrial importance?

Sulfur-binding protein of flagella of Thiobacillus ferrooxidans.

The sulfur-binding protein of Thiobacillus ferrooxidans ATCC 23270 was investigated

. The protein composition of the bacterium's cell surface changed according to the culture substrate.

Sulfur-grown cells showed greater adhesion to sulfur than iron-grown cells

The sulfur-grown cells synthesized a 40-kDa surface protein which was not synthesized by iron-grown cells.

The 40-kDa protein had thiol groups and strongly adhered to elemental sulfur powder.

However, adhesion was disturbed by 2-mercaptoethanol, which broke the disulfide bond.

The thiol groups of the 40-kDa protein formed a disulfide bond with elemental sulfur and mediated the strong adhesion between T. ferrooxidans cells and elemental sulfur.

The 40-kDa protein was located on the flagella. The location of the protein would make it possible for cells to be in closer contact with the surface of elemental sulfur powder .

Thiobacillus ferroxidans.

INDUSTRIAL IMPORTANCE:

1. Treatment of leather industry wastewater by aerobic biological and Fenton oxidation process.

Degradation of leather industry wastewater by sole aerobic treatment

incorporating Thiobacillus ferrooxidans, Fenton's reagents, and

combined treatment was investigated in this study.

The sole treatment by Fenton's oxidation involving the introduction

of 6g FeSO(4) and 266 g H(2)O(2) in a liter of wastewater at pH of

3.5 and 30 degrees C for 30 min at batch conditions and T.

ferrooxidans alone showed maximum reduction to an extent of 77, 80,

85, 52, 89, in 21 d treatment at pH 2.5, FeSO(4) 16 g/L and

temperature of 30 degrees C.

The combined treatment at batch conditions involving 30 min

chemical treatment by Fenton's oxidation followed by 72 h

biochemical treatment by T. ferrooxidans at batch conditions gave rise

up to 93%, 98%, 72%, 62% and 100% removal efficiencies of COD,

BOD, sulfide, chromium and color at pH of 2.5 and 30 degrees C.

Decrease in photo absorption of the Fenton's reagent treated samples,

as compared to the banks, at 280, 350 and 470 nm wave lengths was

observed. This may be the key factor for stimulating the

biodegradation by T. ferrooxidans.

2. Modeling and analysis of biooxidation of gold bearing pyrite-arsenopyrite concentrates by Thiobacillus ferrooxidans.

The results of modeling the biooxidation of a mixed sulfidic

concentrate by Thiobacillus ferrooxidans is reported here.

A kinetic model, which accounts for the dissolution of sulfide matrix

due to both bacterial attachment onto the mineral surface and indirect

leaching, has been proposed..

The bacterial balance accounts for its growth, both on solid substrate

and in solution, and for the attachment to and detachment from the

surface.

This model was tested in both laboratory scale batch and continuous

biooxidation processes.

A further analysis of the model was carried out to predict the

conditions for efficient biooxidation.

Studies on the effect of residence time and pulp density on steady-

state behavior showed that there is a critical residence time and pulp

density below which washout conditions occur.

Operation at pulp densities lower than 5% and residence times lower

than 72 h was found unfavorable for efficient leaching.

3. Suppression of pyrite oxidation by iron 8- hydroxyquinoline.

One of the important approaches to prevent pyrite (FeS(2)) oxidation and subsequent formation of acid mine drainage (AMD) is to create a surface coating on pyrite.

A coating of iron 8-hydroxyquinoline was formed by leaching pyrite with a 0.10 M H(2)O(2)/0.0034 M 8-hydroxyquinoline solution;

The results showed that iron 8-hydroxyquinoline coating could significantly suppress further pyrite oxidation by both chemical (H(2)O(2)) and biological ( e.g., Thiobacillus ferrooxidans) processes

At pH from 3.0 to 5.0 and temperature from 10-40 degrees C, the amount of SO(4)(2-) leached out by 0.10 M H(2)O(2) from the coated pyrite samples was 54.8-70.1% less than that from the uncoated controls.

Thus, the coating decreased the leachability of pyrite by 97% in the inoculated systems. In comparison to the more widely studied iron phosphate coating, the advantage of iron 8-hydroxyquinoline coating was that it inhibited both chemical and biological pyrite oxidation, whereas iron phosphate coating could only inhibit chemical pyrite oxidation.

4. Selective Adhesion of Thiobacillus ferrooxidans to Pyrite: Bacterial adhesion to mineral surfaces plays an important role not

only in bacterial survival in natural ecosystems, but also in mining industry applications

Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena.

Escherichia coli was used as a control bacterium.

Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria.

The contact angle of E. coli in a 0.5% sodium chloride solution was 31 degrees , and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23 degrees . E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite.

Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion.

When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions.

5. Role of Ferrous Ions in Synthetic Cobaltous Sulfide Leaching of Thiobacillus ferrooxidans

Microbiological leaching of synthetic cobaltous sulfide (CoS) was

investigated with a pure strain of Thiobacillus ferroxidans

The strain could not grow on CoS-salts medium in the absence of

ferrous ions (Fe2+). However, in CoS-salts medium supplemented with

18 mM Fe2+, the strain utilized both Fe2+ and the sulfur moiety in CoS

for growth, resulting in an enhanced solubilization of Co2+.

Cell growth on sulfur-salts medium was strongly inhibited by Co2+,

and this inhibition was completely protected by Fe2+.

Cobalt-resistant cells, obtained by subculturing the strain in medium

supplemented with both Fe2+ and Co2+, brought a marked decrease in

the amount of Fe2+ absolutely required for cell growth on CoS-salts

medium.

Since a similar protective effect by Fe2+ was also observed for cell

inhibition by stannous, nickel, zinc, silver, and mercuric ions, a new

role of Fe2+ in bacterial leaching in T. ferrooxidans is proposed

.

T.ferrooxidans metabolizes metal ions, such as those found in this iron sculpture.

6 Thiobacillus ferrooxidans involved in the biohydrometallurgical extraction processes.

The microbiological leaching techniques are currently practiced at industrial-scale, especially for recovery of copper and uranium from low-grade materials.

Application of microorganisms in leaching metal sulfides requires a more knowledge about the interactions of the physical and chemical factors with the growth of T. ferrooxidans in pure and mixed cultures including heterotrophic and thermophilic cohabitants.

Altogether, the future industrial exploitation of these microbiological leaching techniques are very attractive in many countries of the world.

7. The role of Thiobacillus ferrooxidans in the bacterial leaching of zinc sulphide

The role of Thiobacillus ferrooxidans in the bacterial leaching of mineral sulphides ..

The role of the bacteria would be more easily discernible if the concentrations of ferric and ferrous ions are maintained at a set value throughout the experimental period

This apparatus is designed to control the redox potential in the leaching compartment of an electrolytic cell by the reduction or oxidation of dissolved iron.

By controlling the redox potential and the pH the concentrations of ferrous and ferric ions are maintained at their initial values.

Leaching experiments have been conducted in the presence of T. ferrooxidans and in sterile conditions.

At high concentrations of ferric ions, the conversions of zinc sulphide in the absence and presence of the bacteria are the same.

However, at high concentrations of ferrous ions, the conversion of sphalerite in the presence of bacteria is higher than in their absence.

The results show that at higher concentrations of ferrous ions, diffusion of ferrous ions through the sulphur product layer becomes important in leaching under sterile conditions.

However, bacteria oxidise the sulphur layer, which removes this diffusional resistance, and thereby increase the rate of leaching.

8. Bacterial leaching :

Thiobacillus ferrooxidans is a bacterium typically used in what's called

bioleaching, a bioprocess in which microbes are used to leach out metals

from mineral deposits. It is the most commonly used bacteria in biomining

It is a commercially successful process used for pretreatment of

refractory gold-bearing sulfides.

The role of Thiobacillus ferrooxidans in bacterial leaching of mineral sulfides is controversial

. Much of the controversy is due to the fact that the solution conditions, especially the concentrations of ferric and ferrous ions, change during experiments.

The role of the bacteria would be more easily discernible if the concentrations of ferric and ferrous ions were maintained at set values throughout the experimental period.

The constant redox potential apparatus is designed to control the redox potential in the leaching compartment of an electrolytic cell by reduction or oxidation of dissolved iron.

By controlling the redox potential the apparatus maintains the concentrations of ferrous and ferric ions at their initial values.

EXPERIMENTS CONDUCTED:

Experiments were conducted in the presence of T. ferrooxidans and under sterile conditions. Analysis of the conversion of zinc sulfide in the absence of the bacteria and analysis of the conversion of zinc sulfate in the presence of the bacteria produced the same results. This indicates that the only role of the bacteria under the conditions used is regeneration of ferric ions in solution. In this work we found no evidence that there is a direct mechanism for bacterial leaching.. The first commercial operation, at Fairview Gold Mine (South Africa), began in 1986 with a capacity of 10 tons per day. This plant now treats 35 tons of a pyrite-arsenopyrite concentrate per day. The largest bacterial leaching plant, at Ashanti in Ghana, presently treats 1,100 tons of gold-bearing pyritic concentrate per day. There are also operations at Sao Bento in Brazil and at Harbour Lights in Australia. More recently, a bacterial leaching project has been set up in Uganda to extract cobalt from a colbaltiferous pyrite ore.

Thiobacillus ferrooxidans, the microorganism that is associated with these leaching processes, is able to oxidize ferrous ions and reduced sulfur compounds.

The product of oxidation of ferrous ions, ferric ions, is a strong oxidant that is capable of oxidizing sulfide minerals.

One of the two dominant views on the mechanism of bacterial leaching is that the overall leaching process occurs by bacterial oxidation of ferrous ions and chemical leaching of the mineral.

This process is described by the following set of reactions for bacterial leaching of zinc sulfide:

2Fe2+ +2H+ +0.5O2 = 2Fe3+ + H2O

ZnS +2Fe3+ = Zn2++ S + 2Fe2+

This mechanism is often referred to as the indirect mechanism, and it is clear that the iron couple plays a central and critical role in this mechanism. The second proposed mechanism involves bacterial catalysis of the dissolution of sulfide minerals. It has been proposed that the bacteria are able to directly interact with the mineral and enhance the rate of dissolution of the mineral above the rate achieved during chemical leaching by ferric ions under the same conditions. This mechanism is often referred to as the direct mechanism. In the case of zinc sulfide, the direct mechanism may be represented as follows:

Experimental apparatus.

The working compartment is the compartment in which leaching experiments are conducted. The flow of current is regulated by adjusting the variable resistor so that the redox potential remains at the setpoint value

The objective of this study was to investigate the mechanism of dissolution of sulfide ores in the presence of T. ferrooxidans under conditions under which the concentrations of ferric and ferrous ions were controlled for the duration of the experiment. In this paper we report the results of a study aimed at determining the mechanism of bacterial leaching of zinc sulfide (sphalerite).

Bacterial leaching of ores and other materials

The principal bacteria which play the most important role in solubilizing sulfidic metal minerals at moderate temperatures are species of the genus Thiobacillus.

They are gramnegative rods, either polarly or nonflagellated. Most species are acidotolerant, some even extremely acidotolerant and acidophilic.

Some grow best at pH 2 and may grow at pH 1 or even at pH 0.5. Most species are tolerant against heavy metal toxicity. 

Thiobacilli are chemolithoautotrophs, that means CO2 may be the only source of carbon and they derive their energy from a chemical transformation of inorganic matter.

All Thiobacilli oxidize sulfur or sulfur compounds to sulfate or sulfuric acid.

Microbial leaching of ores depends primarily on bacterial processes which are the essential causes of natural weathering of sulfidic minerals.

If sulfidic heavy metal minerals come into contact with air and water they begin to decay with the formation of sulfate, sometimes sulfuric acid, and water soluble heavy metal cations. 

Weathering of an ore body results in a typical picture

a)An upper oxidation zone, being in contact with atmospheric oxygen and rain water, which contains secondary minerals formed by oxidation of the primary ore minerals and in most cases a remarkable enrichment of ferric iron,minerals,limonite.  (b) An underlying cementation zone just below the groundwater level, in which minerals, formed by the reaction of primary ore minerals with the constituents of the leaching solution descending from the oxidation zone, are accumulated.   (c) A zone in which the primary ore minerals are unchanged. 

But there are some other bacteria which may also be involved. For example the thermophilic Sulfolobus plays a role in leaching at elevated temperatures.

Thiobacillus thiooxidans, which oxidizes merely sulfur and sulfur compounds but not iron, and Leptospirillum ferrooxidans, which contrarily oxidizes only ferrous iron, may play a role if they work together or with other bacteria. 

Oxidation of hydrogen sulphide by Thiobacilli

Oxidation of elemental Sulfur by Thiobacilli

If they oxidize hydrogen sulfide, thiosulfate, polythionates or elemental sulfur they produce hydrogen ions and so they lower the pH of the medium, often below pH 2, in some cases below pH 1. 

HS- + 2O2 --> S04-- + H+

S° + H20 + 1½O2 à S04-- + 2 H+

In addition to the oxidation of sulfur and sulfur compounds Thiobacillus ferrooxidans is able to oxidize ferrous to ferric iron and so derive its energy from this exergonic reaction.

In this reaction hydrogen ions are consumed and so the pH of the medium should rise.

But at pH values higher than 2 the ferric iron precipitates as ferric hydroxide, jarosites or similar compounds and this results in the formation of hydrogen ions, so that the pH of the medium is lowered as is the case with oxidation of sulfur compounds: 

2Fe++ + 2H+ + ½O2  ---->    2Fe+++ + H20

2Fe+++ + 6H20       ---->    2Fe(OH)3 + 6H+

2Fe++ + 5H20 + ½O2   ---->    2Fe(OH)3 + 4H

Oxidation of ferrous ions by Thiobacillus ferrooxidans. 

  

9. OXIDATION OF SULPHIDIC MINERALS:

Some Thiobacilli, especially T. ferrooxidans, are able to oxidize sulfide and some heavy metals -mainly iron but also copper, zinc, molybdenum and presumable some other metals - in the form of sulfidic heavy metal minerals which are of very low solubility in water, practically insoluble. These oxidations result in a solubilization of the minerals. This is often seen in the case of pyrite or marcasite, both FeS2, minerals which are oxidized very easily by Thiobacilli: 

FeS2 + H20 + 3½O2 = Fe++ + 2 SO4-- + 2 H+

But also in the case of other minerals. Oxidation of the sulfide of a divalent metal: 

MeIIS + 2O2 = Me++ + SO4—

Thiobacillus thiooxidans, an extremely acidophilic but not ferrous iron oxidizing species of the Thiobacilli, is not able to solubilize sulfidic heavy metal minerals in pure culture. Nevertheless T. thiooxidans plays a role in metal leaching. The solubilization of sulfidic minerals by Thiobacillus ferrooxidans is increased by cooperation with T. thiooxidans as compared with the effect of T. ferrooxidans alone. We can assume that the cause of this enhancement is the oxidation of elemental sulfur and hydrogen sulfide which is formed as a result of the oxidation by ferric iron . 

CARBON LEACHING.

10. Novel mineral processing by flotation using Thiobacillus ferrooxidans :

Oxidative leaching of metals by Thiobacillus ferrooxidans has proven

useful in mineral processing.

It involves a new use for T. ferrooxidans whereby bacterial adhesion

is used to remove pyrite from mixtures of sulfide minerals during

flotation.

Under control conditions, the floatabilities of 5 sulfide minerals tested

(pyrite, chalcocite, molybdenite, millerite and galena) ranged from 88

to 99%.

Upon addition of T. ferrooxidans, the floatability of pyrite was

significantly suppressed to less than 20%.

In contrast, addition of the bacterium had little or no effect on the

floatabilities of the other minerals.

T. ferrooxidans thus appears to selectively suppress pyrite

floatability.

As a consequence, 84 to 95% of pyrite was removed from mineral

mixtures, while 73 to 100% of non-pyrite sulfide minerals were

recovered.

The suppression of pyrite floatability was caused by bacterial

adhesion to pyrite surfaces.

The number of cells adhering to pyrite was significantly larger than

the number adhering other minerals.

These results suggest that flotation with T. ferrooxidans may provide

a novel approach to mineral processing in which the biological

functions involved in cell adhesion play a key role in the separation of

minerals.

SCOPE FOR FUTURE RESEARCH:

Thiobacillus ferrooxidans can be used for further research because of

its strain diversity and its significance in biohydrometallurgy.

Structural changes in chromosomal DNA of Thiobacillus ferrooxidans

strains that occur under the influence of varied growth conditions

were studied by pulsed-field gel electrophoresis. Strain diversity of T.

ferrooxidans was manifested in different growth rates and oxidation

rates of inorganic substrates under extreme conditions, in different

resistance to metal ions and low pH values, and also in polymorphism

of the chromosomal DNA fragments generated by the

macrorestriction endonucleases. Adaptation of some strains to growth

on media containing new substrates was accompanied by changes in

the number and size of restriction fragments.The data obtained on the

natural and experimental genomic variability of T. ferrooxidans

strains provide biotechnologists with practical recommendations for

selection aimed at the intensification of bioleaching processes and

testify about possibilities of strain monitoring in natural and

technological conditions. Strains with the labile genome have an

advantage in biohydrometallurgy.

T. ferrooxidans' successful growth rate, the bacterium can be used in

the technology of desulphurization of fuels and industrial gases. The

efficiency of this process is competitive with the traditional methods

of desulphurization, and the side products are sulphur, sulphuric acid

or gypsum.

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