1. Applied Environmental Geoscience (ESE 3.22)
Dr. Bill Dudeney
Room B339 RSM
Department of Earth Science and Engineering
Spring term 2006
Tuesday – Keynote lectures
Friday – Coursework
One day field work at Silwood Park
Session 3 (3 hours) – Microbe-mineral interactions (1)

2. Objective
Understanding biological (mainly microbial) activities with mineral species through earth’s evolution and at the present time, emphasising:
Geological impacts (genesis and degradation of minerals; soil and sediment formation and transformation) - geomicrobiology
Practical impacts (biosorption, bioprecipitation, bioleaching) - biohydrometallurgy

3. Microbes in geology
Microbes are assumed to have been the first life-forms to emerge during development of the early earth. The extreme conditions thought to exist during those times were suited to so-called extremophiles, presumably ancestors of current strains of archaea, e.g., Methanopyrus which grows at 110º C and Halobacterium which grows in high salt concentrations
Microbes have continued to mutate, diversify and influence their environment ever since, e.g.,
- oxygenic photosynthesis in the cyanobacteria is believed to be responsible for oxygenation of the atmosphere some 3 billion years ago
- microbial adsorption of metal ions and precipitation (or leaching) of minerals has continued through much of geological time. Many mineral deposits can be associated with microbial influence, such as sedimentary sulphide ores, carbonates, diatomite, oil and gas – see later

4. Surface chemical processes
Reactions involving minerals or microbes usually occur at interfaces, particularly the interface between an aqueous medium and a solid phase, e.g., a pyrite surface, a microbial cell wall or an extra-cellular bio-polymer
Solid surfaces usually have a small net electrical charge in aqueous medium as a result of selective adsorption of cations or anions from the bulk solution, e.g., hydrated iron oxide is usually +ve and humate -ve. This gives rise to the so-called ‘electrical double layer’ and can have large effects, such as coagulation and joint sedimentation of small (1-10 m) and colloid-sized (< 1 m) particles - see later
More generally, dissolved species (including reactants) undergo transport by diffusion and/or physical mixing to the mineral or biological surface where initial physical or chemical adsorption occurs
The adsorbed species may be stable as such and form a so-called monolayer coverage, i.e., occupy all available surface sites on the solid. Alternatively, they may form a base for further reaction:
- building up successive layers of adsorbate, thus producing a surface precipitate, e.g., iron oxide encrustation on limestone or ‘iron’ bacteria, or
- dissolving or mobilising substance from the solid, e.g., leaching iron from pyrite or corroding steel pipes
Stable adsorption, progressive precipitation and dissolution can be understood via
the separate diagrams provided. Thermodynamics and kinetics decide which occurs

5. Microbe-mineral interactions
In addition to utilisation of carbon, energy and nutrient sources, microbes take part in a range of interactions with hydrated cations and anions, neutral molecules, minerals and organic matter, in particular by:
- biosorption (particularly ion exchange or complexation between cations or anions with oppositely charged species in the cell structure)
- bioprecipitation (reaction between adsorbed species and precipitation of mineral phases once the solubility product has been ‘exceeded’)
- bioleaching (dissolution or mobilisation of mineral phases under microbial influence)
Microbes may also become immobilised on surfaces or congregate together in aggregates
Microbe-mineral interactions are dependent partly upon the organisation of the biomolecules making up the cell structure. Two major divisions of cell structure are recognised: Gram +ve and Gram –ve (see separate diagrams)
Interactions influenced by microbes may occur in different locations with respect to cell structure
- within the bulk phase
- at the cell surface
- within the periplasmic space
- within the cytoplasm

6. Biosorption
Microbes metabolise, or otherwise associate with, a wide variety of molecules, e.g., glucose and ions, e.g., NH4+, HPO32-, K+, Mg2+, transported from the surrounding aqueous medium to the cell surface or into the cell structure (intracellular uptake)
Biosorption is usually regarded as a rapid ion exchange or complexation with similarly charged species on biomolecules, e.g., peptidoglycan (bacteria) or chitin (fungi), at the cell surface. For instance, K+ might replace for H+ on a carboxylate group (-CO2-), or H2PO3- might replace Cl- on a protonated amine group (-NRH2+)
As a chemical equilibrium, biosorption is usually considered to be independent of whether the cell is alive or dead and may be reversible, e.g., by adding acid. A close analogy sometimes exists with ion exchange resins, although the active groups are much more variable in biomass

7. Adsorption/desorption isotherms
Biosorption is studied quantitatively by means of equilibrium adsorption/desorption isotherms and plots of rates of adsorption/desorption – see separate diagrams
Different masses of biomass (usually g) are typically equilibrated or reacted for set time periods with 50 ml of solutions containing known initial concentrations of target species at set pH and temperature. After reaction the phases are filtered and analysed
As all microbes contain similar biomolecules the same theory applies in principle to all - see next slide

8. Biosorption theory (UO22+ interaction with A. niger )
The rate of adsorption of UO22+ = kU[UO22+]nH+ = 0.5 x rate of desorption of H+ and
rate of adsorption of H+ = kH[H+]nUO22+ = 2 x rate of desorption of UO22+,
where kU and kH are rate constants for adsorption and nH+ and nUO22+ are the numbers of hydrogen and uranyl ions per unit surface area of biomass
At equilibrium: kU[UO22+]eqnH+(eq) = 0.5kH[H+]eqnUO22+(eq) and
K = 0.5kH/kU = [UO22+]eqnH+(eq)/{[H+]eqnUO22+(eq)}, where K is the equilibrium constant
The total number of (univalent) exchange sites nT is given by
nT = nH+ + 2nUO22+ = 2kB x maximum mass of UO MUO22+(max) - adsorbed per unit mass of biomass, where kB is a constant. At any state less than maximum adsorption:
nUO22+ = 2kB x instantaneous mass of UO22+ - MUO22+ - adsorbed per unit of biomass
Substituting these equations into that of K, taking logs and rearranging:
Log{[H+]eq/[UO22+]eq} = Log{(2MUO22+(max) – 2MUO22+)/MUO22+} – LogK
Thus, a simple plot should give a straight line of unit slope - see next slide

9. Biosorption: plot verifying UO22+ ion exchange mechanism

10. Biosorption applications
Waste biomass (particularly from fungal fermentations, e.g., A.niger from citric acid production and S. cerevisiae from brewing) can be considered as a biosorption reagent if converted into a suitable physical form to contact with metal-bearing process solutions or effluents. Metal concentrations up to 1 g per unit biomass can be achieved.
For biosorption, contaminated water can be passed through:
- pelletised biomass packed in columns or canisters (cf resin ion exchange systems)
- biomass (alive or dead) dispersed in tanks, ponds or streams
Biomass should be less expensive than commercial ion exchange resins or activated carbon. However, it may not represent a consistent supply
Metals having sufficient value, e.g., Au, may be recovered from the loaded biomass by incineration or acid elution. Spent biomass, e.g., with mixed Pb, Zn and As adsorbed, having no market value needs discarding, perhaps after incineration, taking account of any toxic content
Much research and pilot scale demonstration have been carried out, but only a few sustainable applications have been developed, e.g., uranium bearing minewater has been purified in canisters with Rhizopus fungi and marine plankton in Canada and Zn/Pb bearing mine effluent has been treated in stream meanders with Cladophora, Spirogyra and other algae at the Buick Mine in the USA – see next slide

11. Purification of mine effluent by algae

12. Bioprecipitation
Under certain circumstances microorganisms cause relatively large quantities of substances to be precipitated:
- within cells, e.g., calcium phosphate, metal polyphosphate or sulphur as storage granules, nickel carbonate as a protection against Ni toxicity, metallic gold as a product of protein reduction of adsorbed gold species and magnetite for cell navigation
- near to cells and on cell walls, e.g., hydrated iron(III) oxide on sheathed ‘iron bacteria’ and metal sulphides around sulphate reducing bacteria
Biosorption is taken to be the initial process in many such bioprecipitation reactions
The variety of biosorption and bioprecipitation products are illustrated on the next two slides
Mineral deposits can result from the combined effects of huge microbial numbers, long deposition times (e.g., on geological timescales) and accompanying biodegradation of organic cell components
Biosorption and bioprecipitation (collectively bioaccumulation) are widespread in environmental systems and can occur rapidly. Numerous schemes have been proposed to intensify them for use in industry. Several applications will be discussed

13. Metal uptake processes
M = metal
R = alkyl group
After Hughes and Poole

14. Mineral formation by microbial cells
Cytoplasm
Periplasm
Cell surface
Bulk phase
After H.L. Ehrlich

15. Iron precipitation and dissolution
Iron forms more than 5% of the earth’s crust and is a major constituent of many rocks and soils
The element exists is two major oxidation states which have variable solubility under near-neutral environmental conditions:
- iron(II) or ferrous iron is often soluble (as Fe2+), although precipitation is likely in the presence of some anions, particularly carbonate and sulphide at pH >6
- iron(III) or ferric iron is often insoluble (e.g., as hematite, goethite or amorphous analogues), but may dissolve when complexed with organic substances, e.g., humates or oxalates, or at pH <3
Iron is readily oxidised or reduced (Eo = 0.77 V) under mild redox conditions suited to microbial growth. A microbial Eh-pH diagram is shown on the next slide
All organisms require iron for one purpose or another (although dissolved iron is sometimes toxic to microbes). For instance, some gain energy for metabolic processes from the Fe(III)/Fe(II) redox couple, particularly when an insoluble iron product forms, e.g., hydrated iron(III) oxide (oxidative) or iron(II) carbonate or sulphide (reductive)
For these and other reasons, iron plays a large role in microbial metabolism and ecology. Conversely, microbes figure significantly in the so-called iron cycle – see two and three slides on.

16. Eh-pH diagram for microbes

17. Qualitative Eh-pH diagram for the Fe-S-C system including chemical and microbial influences

18. The iron cycle as a ‘food web’
After Johnson 1991

19. Kinetics of iron oxidation
Aqueous ferrous iron forms in various reducing environments but oxidises and hydrolyses in air:
Fe O2 + H+ = Fe H2O
Fe3+ + 3H2O = ‘Fe(OH)3’ + 3H+
The classic rate equation corresponding to the first of these equations is
-d[Fe2+]/dt = k[Fe2+][OH-]2p(O2), where k is the rate constant
As Kw = [H+][OH-] = 10-14, the rate of iron oxidation in air therefore depends strongly on pH:
- at pH<6 the rate is very slow (unless catalysed) – see later
- at pH>8 the rate is extremely rapid
- at pH 6-8 the rate is readily measurable (8.0 x 1013 min-1atm-1litre-1 at 20ºC pH 7)
Many microbes gain energy from iron(II)/iron(III) redox couple in this pH range, and the rate of oxidation may be further increased by microbial catalysis (perhaps enzymatically). However, this is difficult to prove. Thus, at pH 6-8 various ‘iron bacteria’ are likely to benefit from iron oxidation but they may or may not themselves have a significant impact on the reaction

20. Examples of bioprecipitation of iron minerals
Hydrated iron(III) oxide is considered to be formed via enzymatic oxidation of iron(II) by G. ferruginea and by non-enzymatic disruption of iron(III) chelates by L. ochracea
Many organisms passively accumulate iron(III) oxides from other sources on their surfaces by adsorption onto extracellular carbohydrate-based polymer in the form of sheaths, capsules or slime
Magnetotactic bacteria take up complexed iron and partially reduce it to magnetite (in the form of single domain crystals). The organisms use lines of so-called magnetosomes rather like compass needles for navigation in the earth’s magnetic field – see next slide
Microbes have played a role in the formation various sedimentary iron deposits
- iron(III): Precambrian banded iron ores through to modern bog and lake iron ores (partially influenced by the ‘iron bacteria’ – see two slides on)
- iron(III)/(II): magnetite deposits (partially attributed to magnetotactic bacteria)
- iron(II): siderite, pyrrhotite and pyrite deposits (partially influenced by sulphate reducing bacteria)

21. Magnetotactic bacterium
Magnetosomes: single domain magnetite
crystals with roughly hexagonal habit and
length some 90 nm
1 μm
Magnetotactic bacterium: two lines of magnetosomes are just visible running almost vertically through the organism. Several thick flagella are attached near the upper bacterial pole and many thinner fimbriae or pili around the perifery of the organism

22. Iron(III) oxide deposition on Leptothrix ochracea from Fe2+ rich mine water

23. Recent studies on iron bacteria
Iron is considered to be the most important environmental metal according to a paper by Edwards, et al., in Geomicrobiology Journal, 21: , 2004; title ‘Neutrophilic iron–oxidising bacteria in the ocean: their habitats, diversity and roles in mineral deposition, rock alteration, and biomass production in the deep sea’
Iron oxidation influenced by microbes on the sea floor is apparently widespread and analogous to that already discussed. Such microbes are also implicated widely in promoting rock and mineral weathering
Such references will be useful in the first coursework assignment

24. Gold precipitation
Gold dissolves at low concentrations (approximately 10-7 m) in sea water, mainly as the chloride complexes AuCl2- and AuCl4-, but can become relatively concentrated in salty hydrothermal systems and in precious metal process solutions
There is geomicrobiological evidence that plants and microbes have a role in formation of some gold deposits
As a result, it is of interest to understand the mechanisms involved and to investigate whether traces of gold can be recovered from gold process effluents

25. Gold bioprecipitation from AuCl4-
Four separate handouts show experimental data needed for a mechanistic evaluation:
- adsorption isotherms for the algae C. vulgaris and S. platensis
- adsorption/desorption characteristics for these organisms
- adsorption profiles at set times for C. vulgaris
- gold precipitation profile with time for C. vulgaris
A 3-step mechanism consistent with these data is shown on the next slide. This mechanism involves:
- rapid reversible biosorption of AuCl4- for Cl- at protonated amine groups
- slow irreversible reduction of Au(III) to Au(I) by an amino acid sulfhydryl group
- very slow irreversible disproportionation of three adjacent Au(I) complexes to form Au(III) and crystalline gold
Images of crystalline gold produced on C. vulgaris are shown in two further slides

26. Mechanistic steps for the accumulation of Au on
C. vulgaris

27. Gold on algae
Left: flat triangular and hexagonal gold crystals and cells of Chlorella vulgaris in transmitted light
Right: similar view of gold crystals in reflected light

28. Gold crystallization within a channel through 1 mm pellets of the alga Spirulina platensis tightly packed in an adsorption column

29. Biodegradation of metal cyanide
At the Homestake Mine in South Dakota gold cyanidation effluents used to kill trout in the receiving river
The effluents contained base metal cyanide complexes, thiocyanate and low levels of Au(CN)2-
Environmental bacteria, acclimatised to the effluent (including strains of Pseudomonas and Nitrosomonas) were taken from the stream and immobilised on rotary biological contactors
When the effluent was passed over these bacteria prior to discharge, cyanide and thiocyanate were oxidised in stages to nitrate, which was much less polluting – see next slide
The receiving stream was able to support trout populations once more
Metals, including 45 g/tonne gold, could be recovered from the biomass by incineration

30. Homestake gold operation
‘M(CN)’ + 2H2O + 0.5O2 + H-biofilm M-biofilm + HCO3- + NH4+
SCN H2O + 2O SO42- + H2CO3 + NH4+
Pseudomonas
NH O NO H+ + H2O
NO O NO3-
Nitrosomonas
Homestake biodegradation plant Pseudomonas and Nitrosomonas on contactors