Metal Toxicity In Leaching Bacteria

Transcription

1 Lecture 8 Metal Toxicity In Leaching Bacteria Keywords: Metal Toxicity, At.ferrooxidans, Adaptation Bioleaching in the presence of At.ferrooxidans generates toxic metal ions which could act as poisons for bacterial growth. The role of metal toxicity in leaching, possible mechanisms as well as development of metal-tolerant At.ferrooxidans are discussed in lectures 8 9 [36 41]. Why metal tolerant strains? Bioleaching of Sulfide mineral concentrates such as chalcopyrite, Arsenopyrite, sphalerite etc. Gold-containing pyrite-arsenopyrite concentrates. Accumulation of toxic metal ions, such as Cu ++ Fe ++, Fe +++ As +++, As accumulate in the bioleached leach liquor, introducing heavy metal ion toxicity. Inhibitory concentrations of some metal ions towards At.ferrooxidans: Fe M Cu ++ 5 x 10-2 M Zn ++ 5 x 10-2 M Ag M As M 1

2 At.ferrooxidans is more tolerant to heavy metals than most of the other heterotrophic bacteria.[36-37] It has been suggested that plasmids are also involved in their metal resistance Some reported inhibitory levels of metals during bacterial ferrous oxidation are: Cobalt, Nickel and Zinc > 10g/L Copper > 1g/L Uranium < 0.7 g/l Silver < 0.05g/L Molybdenum < 0.005g/L The most toxic metals are uranium, silver and molybdenum. Adaptation of bacterial cells to various metal ions will enhance their tolerance (Table 8.1) Table 8.1: Heavy metal tolerance of At.ferrooxidans Natural limits (mol/l) Highest tolerance achieved through adaptation (g/l) Cu Zn Ni U (without adaptation) (after adaptation) Mo Metal tolerance depends on relative metal ion toxicity. Uranium and molybdenum exert highest toxicity. 2

3 Necessity for adaptation? To enhance bacterial efficiency in the leaching of sulfide concentrates such as chalcopyrite, arsenopyrite, sphalerite, millerite, pentlandite. etc., Dissolved metal ions Two types of adaptation Solid substrate (ores and concentrates) a) Repeated subculturing in presence of increased levels of added toxic metal ions in 9K medium. - Increase dosage of toxic metal in instalments - In one instalment of higher dosage and wait till lag period is overcome. b) Subculturing at increasing pulp densities of sulphide mineral concentrates. Some important factors concerning metal toxicity and metal tolerance. [38-41] Metal ion / substrate tolerance reversible. Need to preserve adapted strains under induced stress Cells in a population develop metal tolerance after some structural rearrangement of the cell material associated with cytoplasmic membrane. Protecting mechanism is metal-specific Cell surface membrance changes to detoxify Metal-specific proteins? Chelation? Cell surface changes as well as proteinaceous compounds secreted in the metabolite? Adaptation to mineral sulphide switching from Fe ++ oxidation machinery to S-oxidation- Prolonged lag periods for ferrous ion oxidation exhibited by cells grown on S and mineral sulfides. Surface changes make cells more hydrophobic Better/more efficient mineral adhesion by adapted bacteria 3

4 Copper adapted cells more hydrophobic Difference FTIR-spectra indicated significant presence of NH 3, NH 2, NH CONH 3, COCH 3, CH 2, CH and COOH groups (proteinaceous). Presence of more surface proteins increased cell surface Hydrophobicity. Increased adhesion of Cu-adapted cells on pyrite and chalcopyrite. Cu-adapted cells exhibit IEP shift to higher ph values (2 to 4.7) on treatment with proteinase K, reverts to initial value. Cu-adapted cells adsorb more Cu ++ on surface. Copper toxicity [38 39] Ferrous ion oxidation in 9K medium by an unadapted strain of At.ferrooxidans in the presence and absence of cupric ions is illustrated in Fig In the absence of toxic cupric ions, it took about 42 hours to completely, biooxidize all ferrous ions. It took about 80 hours and 280 hours respectively to oxidize all the ferrous ions in the added presence of 10 and 20 g/l of cupric ions, indicating the toxic and inhibitory role of copper (toxicity increasing with higher copper concentrations). A prolonged lag phase for ferrous oxidation corresponds to the period during which some sort of rearrangement of cell material associated with its membrane takes place. Once this lag phase is crossed, ferrous ion oxidation proceeds at the same rate as in control. Completion of one such cycle of iron oxidation is an indication that the entire cell populations have achieved the ability to overcome metal toxicity. 4

5 Fe ++, g/l Lecture 8: Metal Toxicity In Leaching Bacteria 8 Control 1st subculture (10 g/l Cu ++ ) 2nd subculture 1st subculture (20 g/l Cu ++ ) 2nd subculture t (h) Fig. 8.1: Ferrous ion oxidation in 9K media by unadapted Acidithiobacillus ferrooxidans in absence and presence of 10 and 20 g/l Cu ++. Repeated sub culturing enables the cells to gain tolerance and get adapted to the toxic metal ion content and adaptation is considered achieved if the iron oxidation rate in the presence of toxic metal ions reaches the same level as that of the control (in absence of toxic ions). Repeated subculturing in progressively increasing concentrations of the toxic metal ion will lead to bacterial adaptation. However, adaptation may be possible in a single subculturing, if the cells are allowed to oxidize all the iron, however prolonged the lag phase may be. Exposing cells to higher toxic metal ion levels till they are capable of oxidizing all the iron could be yet another single step procedure for adaptation of At.ferrooxidans. Cell surfaces preadapted to cupric ions were also found to be more hydrophobic than those which were unadapted. Bacterial growth in the presence of toxic ions such as Cu ++ results in the generation of stress-induced proteins. Even growth of At.ferrooxidans in the presence of chalcopyrite and pyrite minerals induced such protein secretion which renders the cell surfaces more hydrophobic. Such enhancement of cell surface hydrophobicity will enable higher 5

6 Electrophoretic mobility Lecture 8: Metal Toxicity In Leaching Bacteria adsorption (attachment) of At.ferrooxidans onto sulfides such as pyrite and chalcopyrite. Copper and mineral-adapted bacterial cells are more efficient in the leaching of chalcopyrite. Electrophoretic mobilities of unadapted and copper-adapted cells of At.ferrooxidans as a function of ph are shown in Fig Compared to unadapted cells, those preadapted to copper extribited significant shifts in isoelctric point (IEP) towards higher ph values. Increased positive surface charges on adapted cells are attributed to enhanced secretion of amino-group containing proteins. FTIR spectra taken on cell surfaces which were grown in the presence of copper-containing medium revealed the prominent presence of NH 2 -groups. Surface-chemical changes due to the presence of newly secreted proteins are brought about due to preadaptation to toxic metal ions such as copper C B -2.4 A ph Fig. 8.2: Electrophoretic mobility as a function of ph for cells of At.ferrooxidans grown in the (A) absence and presence of (B) 10g/L and (C) 20 g/l of copper sulfate. Another important aspect regarding development of metal-tolerant strains of At.ferrooxidans is that the acquired toxic metal tolerance is found to be a temporary trait brought about by membrane-related changes due to exposure to toxic stress. For example, when copper-tolerant strains were back-cultured repeatedly in a 9K medium in the absence of cupric ions, the initially 6

7 acquired metal tolerance was found to be lost. In order to preserve such metal-tolerant strains, it becomes necessary to store and resubculture them in the presence of the same concentration levels of the toxic metal ions (such as copper). Same observation was found to be true in case of bacterial strains preadapted to sulfide mineral concentrates as well. Arsenic toxicity [40] Biooxidation using At.ferrooxidans is used in the liberation of encapsulated gold particles from arsenopyrite. Interaction of At.ferrooxidans with arsenic and iron sulfides and the role of arsenic toxicity are therefore practically significant. Arsenic is toxic to At.ferrooxidans, resulting in significant decrease in the growth rate and eventual death. The levels at which arsenic becomes toxic to At.ferrooxidans are not clearly known. Bacterial cells can be adapted to tolerate much higher concentrations of arsenic species than it naturally would allow. Tolerance levels for arsenic ranging anywhere from 1mg/L upto 40g/L have been reported. Arsenic present in the trivalent form is found to be three to eight times more toxic to At.ferrooxidans than As (V). Toxicity of As (III) and As (V) on cells is dependent on the availability of an energy source. It has been documented that At.ferrooxidans does not oxidize arsenite to arsenate, but they can oxidize arsenic-containing minerals such as arsenic sulfide, arsenopyrite and enargite. Arsenate formed in the cultures was either a result of oxidation from ferric iron or autoxidation in conjunction with the metabolite, but not by the bacteria itself. Exposure of cells of Acidithiobacillus ferrooxidans to arsenic (as As +++ and As ) resulted in increased cell surface hydrophobicity and decreased electrophoretic mobility. Strains grown in presence of arsenic exhibited stronger surface adsorption on arsenopyrite indicating that hydrophobic interactions alone are not principally responsible for the initial adsorption of adapted strains of At ferrooxidans. 7

8 Bacterial resistance to arsenic was found to be mainly due to active efflux system and not because of decreased membrane permeability. Neither the bacterial cells nor the ferric ions were capable of oxidizing or precipitating arsenite ions directly. Presence of ferric ions in the EPS was necessary for binding or entrapment of arsenic ions in the EPS during the growth of At.ferrooxidans. Bacterial EPS of ferrous-grown unadapted cells were able to uptake arsenate ions due to the strong affinity of ferric ions towards arsenate ions. Both arsenate and arsenite ions were co-precipitated in the presence of ferric ions, formed during bacterial growth, resulting in the formation of crystalline jarosite, scorodite and amorphous ferric arsenate or jarosite, tooeliete and amorphous ferric arsenite phases respectively. Co-precipitation of arsenic with the ferric hydroxide precipitate formed during growth of At. ferrooxidans can be made use of for remediation of arsenic in arsenic contaminated soils and waters. 8