Microbial mining

TIBS - April 1979

77

Acknowledgements

28 Stephens, R. E. (1976) Physiol. Rev. 56, No. 4,

Part of this paper was given at an EMBO workshop on Principles of Biological Assembly, held at Hirschhorn, Germany, in September 1977. This study was aided by U.S.P.H.S. Grant No. AM17346, N.S.F. Grant No. PCM76-10558, and a grant from the Muscular Dystrophy Associations of America.

709-777

29 Ishikawa, H., Bischoff, R. and Holtzer, H. (1969) J. Cell Biol. 43, 312-328 30 Lazarides. E. and Weber, K. (1974) Proc. Nat. Acad.

Sci. U.S.A.

71, 2268-2272

31 Tilney, L. G. (1975) in InouC, S. and Stephens, R. E. (eds), op. cit., pp. 339-398 32 Schroeder, T. E. (1975) in Inoue, S. and Stephens, R. E. (eds), op. cit., pp. 305-334 33 DeRosier, D., Tilney, L. G. and Flicker, P. (in preparation)

34 DeRosier, D., Mandelkow, E., Silliman, A., Tilney, L. G. and Kane, R. (1977) J. Mol.

References 1 Gurdon,

J. B. (1974) The Control

Expression

in Animal Development,

of Gene

Clarendon

Press, Oxford 2 Waddmgton, C. H. (1969) Behind Appearance, Edinburgh University Press 3 Child, C. M. (1941) Pattern and Problems in University of Chicago Press Development, 4 Spemann, H. (1938) Embryonic Development Yale University Press, New and Induction, Haven 5 Oppenheimer, J. M. (1967) in Essays in the M.T.T. History of Embryology and Biology, Press, Cambridge, Mass. 6 Boveri, T. (1910) in Festschrift z. 60. GeburtsVol. 3, pp. 133-214, tug R. Hertwigs, Gustav Fischer, Jena Harrison, R. G. (1918) J. Exp. Zool. 25,

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37 Adeistein, R. S. (1978) TZBS 3, No. 2, 27-30 38 Craig, R. and Megerman, J. (1977) J. Cell Biol. 75, 990-996

39 Weber, K. (1976) in Cell Motility (Goldman, R., Pollard, T. and Rosenbaum, J. (eds), Cold Spring Harbor Labs., New York, pp. 403417

40 Inoue, S. and Ritter,

Richard

A. and Meinhardt, H. (1972) Kyberas Biological Cybernetics) 12,

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H. (1974) Lect 10 Gierer, A. and Meinhardt, Math Life Sci. 7, 163-182 11 Gierer, A. (1977a) Current Topics in Devel. Biol. 1 I, 17-59 10, 12 Gierer, A. (1977b) Q. Rev. Biophys. no. 4, 529-593 Srructurelle et 13 Thorn, R. (1972) Stabilitk Morphogenese, Benjamin, New York (French 1972; English trans. by D. A. Fowler, 1975) Theory, 14 Zeeman, E. C. (1977) Catastrophe Addison-Wesley, Reading, Mass. 15 Jacob, F. (1970) in La Logique du Vivant, Paris (The Logic of Life, tr. 9. Spillander’ New York, 1973) 16 Harrison, R. G. (1936) Proc. Nut. Acud. Sci. U.S.A. 22, 238-247 17 Harrison, R. G. (1907) Anat. Rev. 1, 116-I 18; also Proc. Sot. Exp. Biol. Med. (1907), 4,

140-143 18 Harrison, R. G. (1921) J. Exp. Zool. 32, 1-136 19 Harrison, R. G. (1945) Tr. Corm., Acad. Arts and Sci. 36, 277-330 (reprinted in Organization and Development of the Embryo (1969)

20 21 22 23 24

by R. G. Harrison (ed. S. Willens), Yale University Press, New Haven) Harrison, R. G., Astbury, W. T. and Rudall, K. M. (1940) Z. Exp. Zool. 85, 339-366 Cohen, C. (1977) TIBS 2, No. 3, 51-55 Wegner, A. (1976) J. Mol. Biol. 108, 139-150 Hanson, J. and Huxley, H. E. (1953) Nature (London) 172, 530-532 Huxley, A. F. and Niedergerke, R. (1954)

Nature (London) 173, 971-973 25 Huxley, H. E. (1969) Science 164, 1356-1366 26 Cohen, C. (1975) Sci. Am. 233, No. 5, 36-45 27 Gibbons, I. (1975) in Inoue, S. and Stephens, R. E. (eds), Molecules and Cell Movement,

Raven Press, New York, pp. 207-232

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43 Burton,

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47 Osborn, M., Franke, W. W. and Weber, K. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 24902494 48 Hynes, R. 0. and Destree,

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50 Yamada, K., Yamada, S. and Pastan, I. (1976) Proc. Nat. Acad. Sci. U.S.A.73, 1217-1222 51 Ali, I. U., Mautner, V., Lazza, R. and Hynes, R. 0. (1977) Cell 11, 115-126 52 Hynes, R. O., Ah, 1. U., Mautner, V. and Destree, A. (1978) in The Molecular Basis of (Lerner, R. A. and Cell-Cell Interuction

Bergsma, D., eds), Alan Liss, Inc., New York, pp. 139-153 53 Cohen, C. (1979) TIBS 4, No. 5, (in press)

Microbial mining

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36 Wehland, J., Osborn, M. and Weber, K. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 5613-

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and Stephens, R. E. (eds), op. cit., pp. 3-30 41 Borisey, G. G. and Olmsted, J. B. (1972) Science 177, 196-197 42 Mandelkow, E.-M. and Mandelkow, E.

Manchee

The use of micro-organisms in the recovery of commercially valuable minerals from low grade ores is rapidly expanding. Uranium mines in Canada have already adopted microbial methods on a large scale, and if present trends continue, conventional chemical extraction techniques may become obsolete in the foreseeable future. Man has exploited the activities of microorganisms for many centuries, in particular in such well-known activities as baking and brewing, but it is only during the last 150 years that measures have been taken to realize their full potential: modern methods of genetic engineering are likely to extend their range of activities still further. One group of bacteria has evolved a unique mode of existence and although their activities have also assisted man during the last 2000 years it is only during the last two decades that scientists have appreciated the revolutionary changes these bacteria might have on the mining of metallic minerals. I refer to the acidophilic thiobacilli which thrive in an environment which would normally be considered as extremely hostile to life. Their natural growth environment consists of a solution of metals, particularly iron, in 0.05 M sulphuric acid; they will tolerate a range of temperature between 0 and 60°C. Richard Manchee is Research Establishment, SP4 OJG, U.K.

at the Microbiological Porton Down, Snlisbury,

The most important member of this group as far as the mining engineer is concerned is Thiobacillus ferrooxidans [ 11, which obtains its energy by oxidizing sulphur, sulphides and ferrous iron [2]. Carbon is obtained from atmospheric CO, and nitrogen from dissolved ammonia or nitrates : if dissolved sources of nitrogen are in short supply the organism is capable of fixing atmospheric nitrogen [3]. In spite of the fact that the primitive metabolism of this organism places it among the earliest life forms present on the earth, it was not until 1947 [4] that it was isolated from the flood waters issuing from an abandoned coal mine in West Virginia. Although it is ubiquitous, its activity is normally limited by the small quantities of substrate exposed to the atmosphere, because it requires an abundant oxygen supply. The activities of miners, however, have ensured that the organism is frequently confronted by an environment which is abundant both in suitable mineral substrates, oxygen and moisture and the unchecked growth which ensues is responsible for the chronic @ Elsevicr/North-Holland

Biomedical Press 1979

78

TIBS - April 1979

TABLE I Some of the minerals leached by microbial action AsFeS As& B&S, Cu,(As,Sb)S, CuFeS, Cu,FeS, cus C&S Cu,,Sb,S, CuSeO,.ZH,O

Arsenopyrite Orpiment Bismuthite Enargite Chalcopyrite Bornite Covellite Chalcocite Tetrahedrite Chalcomenite

pollution of rivers and lakes with acid and basic ferric sulphate which occurs in many parts of the world. One of the most important growth substrates is irofi pyrites which often occurs in association with other, more valuable minerals like copper and uranium. Pyrite is virtually insoluble under sterile conditions [5] but the presence of T. ,ferrooxiduns may increase the dissolution rate by as much as 1 million-fold. The way in which it attacks the mineral is unknown but viable T. ferrooxidans organisms adsorb tenaciously to pyrite and it is possible that the interface between mineral and organism contains high local concentrations of ferric iron which would cause rapid oxidation. The reactions involved can be expressed as a series of simple equations : 2FeS, + 70, + 2H,O = 2FeS0, 2H,SO, 4FeS0, + 0, + 2H,SO, 2H,O FeS, + Fe,(SO&

+

= 2Fe,(SO,),

= 3FeS0,

2S + 30, + 2H,O = 2H,SO,

(1) + (2)

+ 2S

(3) (4)

FeS Fe& MoS, (NiFe).&

Marcasite Pyrite Molybdenite Pentlandite

NiS

Miller&e

PbS Sb,S, uo, V,V,0,,.8H,O? ZnS

Galena Stibnite Uraninite Vanoxite Sphalerite

still used today and similar methods are used in copper-mining areas throughout the world. It should be stressed that, as far as copper is concerned, deposits of highgrade ore are probably extracted most efficiently by smelting, but even in rich deposits there are areas which do not contain sufficient mineral to make smelting an economic proposition. It is to this lowgrade material that bacterial leaching methods have been applied, with highly successful results. The method used to treat this ore is called heap leaching. Roughly fist-sized lumps are piled in heaps on an impervious surface making sure,that the top of the heap is permeable to water which is applied by a sprinkler system. It is unnecessary to inoculate the heap with T. ferrooxidans organisms ; they will already be present. Leaching will occur by two methods. First, in areas where oxygen is plentiful, iron pyrites will be attacked directly by the bacteria, producing ferric sulphate and sulphuric acid. In additiov the sulphide moiety of other mineral sulphides will also be attacked directly, releasing sulphuric

acid and simultaneously dissolving the metallic mineral. Copper sulphides, for instance, will end up as a solution of copper sulphate. As well as this direct mineral attack, which occurs only in the presence of there is a second important oxygen, mechanism which occurs in anaerobic situations such as the centre of a heap of ore. This purely chemical, indirect leaching of minerals results from the reaction of the highly oxidizing acidic ferric sulphate solution, produced by direct bacterial attack on iron pyrites, with mineral sulphides and oxides. The effluent from a heap leach will contain, therefore, a solution of the commercially valuable mineral together with a mixture of ferric and ferrous sulphates in sulphuric acid. The heaps are carefully constructed so that only a certain proportion of the ferric iron is reduced to ferrous during its passage through the ore. Complete reduction to ferrous would indicate that mineral oxidation, particularly in the lower levels of the heap, was incomplete. The treatment of the effluents varies according to the metal, but the usual process for copper recovery is cementation. Briefly, the copper-rich waters are passed slowly over quantities of scrap iron and the copper deposits as a layer which can be scraped off at intervals. The combination of heap leaching and cementation is used at Rio Tinto and also in the U.S.A. where it accounts for about 10% of the total production of copper. After the copper has been recovered, the barren liquor, rich in ferrous iron, is usually collected in a lagoon where it becomes reoxidized to ferric by bacterial action, thus creating an ideal

T. ferrooxidans obtains energy from reactions (2) and (4); reaction (3) is entirely chemical. Reaction (1) occurs extremely slowly in the absence of bacteria but it may be important in providing the solution of ferrous iron necessary to initiate bacterial growth. The products of these reactions are, therefore, ferric sulphate and sulphuric acid, a mixture capable of oxidising and dissolving many, otherwise insoluble minerals (Table I). It is the application of this phenomenon which has given rise to

microbial mining. Practical applications The ancient Romans are believed to have been the first to recover copper from areas where natural sulphide leaching was occurring but it was not until 1670 that details of a copper recovery process, at Rio Tinto in Spain, were documented. The are methods used there three centuries

Copper mine waste dump under leach. Bacterial activity generates the ferric ion in solution which solubilizes the copper contained in the waste rock.

TIBS - April 1979

79 pillars to support the roof of the workings. To extract the uranium the pillars and walls are hosed down with water, thus producing ideal conditions for the growth of T. ferrooxidans. The acid ferric sulphate produced is capable of penetrating the pores and grain boundaries within the rock, and in due course over 90% of the residual uranium dissolves and can be recovered from solution. In certain localities bacterial leaching has superseded conventional chemical techniques, the operation at Stanrock Uranium Mines being an outstanding example. The mine began operating in 1958 and by 1960 the underground water had become so acid due to bacterial oxidation of pyrite that severe corrosion problems had arisen. Naturally the acidic ferric mine water also leached out uranium and in 1962 13,000 kg of uranium oxide were recovered from this source. In 1963, high-pressure hosing of the stopes was commenced, and this operation was so successful that conventional mining was terminated and thereafter all the stopes were hosed down at regulat intervals. By 1966 the change in methods had cut the cost of production by 25 “/,. In situ and microbial plant processes

leaching solution heaps of ore.

for application

to fresh

Uranium leaching The double-pronged attack of direct and indirect leaching does not occur with most uranium ores because they are often in the form of insoluble oxide. The oxide cannot be directly attacked by the bacteria but it is inevitably closely associated with iron pyrites and susceptible to the indirect attack of bacterially produced ferric sulphate as follows: UO, + Fe,(SO,),

= UO,SO,

+ 2FeS0,

The uranyl sulphate can be recovered from solution by ion-exchange or solvent extraction.

At present, bacterial leaching of uranium is still considered by many as a secondary recovery process. The richer ores are extracted by conventional chemical techniques (usually hot-acid slurry with oxidizing agents) whereas the lower grade material which would otherwise be discarded is heap leached in the same way as copper ore, although the final recovery process is different. Uranium mining, however, is exhibiting signs of a wholesale conversion to bacterial leaching methods. This contention is supported by examining the mining techniques presently employed in the Elliot Lake area of Canada. When uranium ore is brought to the surface, vast underground caverns are produced and about 30 % of the ore body is left behind as

One of the most recent and exciting developments is in situ leaching. The most obvious advantage of this method is that the ore is never brought to the surface but remains below the ground. This produces an enormous saving in the cost of transporting, crushing and, grinding the ore. Minimal damage to the surface environment is caused, and miners’ lives are not at risk. If the ore body is porous, bacterially produced ferric sulphate solution is allowed to percolate from the surface until it reaches impermeable rock where it collects and can later be pumped to the surface through boreholes for recovery of the dissolved mineral. If the ore body is impervious the rock can be shattered by underground explosions so that the leaching liquor can filter through. The processes mentioned so far are currently used for mineral recovery (a) from low-grade ores which cannot be economically treated by conventional methods or (b) as a secondary process to recover the residual metals from ore which has already been treated or which has been rejected by a purification process such as oil flotation. The important question is whether microbial techniques can be developed to a state which would render them suitable for use in a primary recovery process. It is unlikely that microbial

TIBS - April 1979

80 References

I-. Fig. I. Simplified diagram of microbially assisted uranium leaching plant. Column 1 contains ore which has been leaching for &IO days and column 5, ore leached for O-2 days. Every 2 days a fresh column of ore is added on the right and column 1 is removed. Thus ore and leaching fluid flow counterfluid. currently, the oldest ore receiving the freshest The ore-leaching temperature is 50°C and the other stages are at 30°C.

methods will prove more economical than the conventional methods, such as smelting, which are used for high-grade ores of metals like copper and aluminium. In the case of uranium, however, I have already indicated that microbial processes are beginning to supplant traditional methods, and a plant process has now been developed for the primary recovery of uranium from Elliot Lake ore (Fig. 1) [6, 71. This process has a number of advantages over conventional methods: it is cheaper to construct the plant, grinding costs are eliminated, less acid and oxidizing agents are required, and heating costs are less. Its main disadvantage is that the ore treatment time is over twice as long as that required in a conventional process. Uranium recovery in both processes is similar at 94-95 %. As yet this novel microbial process has not been adopted by mining companies, but the technology exists and it is hoped that in the near future someone will have the courage to try it on the commercial scale. ‘The future What then is the future for this ancient, yet so recently understood technology? Will it forever remain in the shadows of the vast smelting and chemical extraction plants of today? I think not; already new mineral extraction processes utilizing other species of micro-organisms are being developed [8-lo]. The ore extraction plant of the future could have the appearance of a present-day water-treatment plant: situated in an environment of comparative quiet and tranquillity and free from the dirt and spoil heaps normally associated with mining operations, while far below ground millions of microbes are carrying out the tasks which today are characterized by the roar of machinery and the ring of pick and shovel on rock.

1 Temple, K. L. and Calmer, A. R. (1951) J. Bact. 62, 605-611 2 Tuovinen, 0. H. and Kelly, D. P. (1974) 2. Allg. Mikrobiol. 12, 3l,l-346 3 Mackintosh, M. E. (1978) J. Gen. Microbial. 105, 215-218 4 Calmer, A. R. and Hinkle, M. E. (1947) Science 106, 253-256 5 Winchell, A. N. (1907) Econ. Geol. 2, 29&294 6 Derry, R., Garrett, K. H., Le Roux, N. W. and Smith, S. E. (1977) in Geology, Mining and Extractive Processing of Uranium (Jones, M. J. ed.), pp. 56-62, Institution of Mining and Metallurgy, London

I Manchee, R. J. (1977) Trans. Inst. Min. Metall. 86, Cl26133 8 Agate, A. D. and Deshpande, H. A. (1977) in Bacterial Leaching, 1977, Conference (Schwartz, W. ed.), pp. 243-250, G. B. F. Monograph Series, no. 4. Verlag Chemie, Weinheim 9 Berthelin, J., Belgy, G. and Magne, R. (1977) in Conference Bacterial Leaching, 1977, (Schwartz, W. ed.), pp. 251-260, G. B. F. Monograph Series, no. 4, Verlag Chemie, Weinheim 10 Kiel, H. (1977) in Conference Bacterial Leaching, 1977, (Schwartz, W. ed.), pp. 261-270, G.B.H. Monograph Series, no. 4, Verlag Chemie, Weinheim

Conformation and moleculati biology of polypeptide hormones II. Glucagon* Tom Blundell Monomeric glucagon has an extended flexible structure. Self-association to trimers and higher oligomers is accompanied by the formation of helix. It is probable that a helical conformer is also stabilized when glucagon binds its membrane receptor.

The first step in the activation of adenylate cyclase by glucagon is the recognition of a membrane receptor [ 1,2,3]. As glucagon has little secondary structure at high dilutions [4,5] and probably exists as many different flexible conformers in equilibrium with each other, the receptor, or its environment, must stabilize, or even induce, the conformer found in the receptor complex. In this review I discuss the evidence for the conformation in solution, in the presence of lipid micelles and bilayers, and in the crystals where a detailed description has been obtained by X;ray analysis in our laboratory. I then consider the relevance of conformational studies to glucagon in storage granules and at the receptor, and contrast this hormone with insulin, which was the subject of a previous article. The conformation of glucagon Glucagon is a 29 amino acid polypeptide, shown in Fig. la, which has no disulphide cross-links to stabilize its structure [6]. Crystals (see Fig. 2b) of the hormone were obtained at an early stage and preliminary studies showed that they were rhombic dodecahedra containing 12 mole*This is part II of a two-part review. Part I, subtitled Insulin, insulin-like growth factors and relaxin, was published in TIBS March 1979,5 l-54. Tom Blundell is’at the Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WClE 7HX. U.K.

cules packed with cubic symmetry [7]. A medium resolution X-ray study [8] indicated an a-helical conformation (Fig. lb) between residues 6 and 27 resulting in the formation of one mainly hydrophobic region by Phe 6, Tyr 10, Tyr 13 and Leu 14, and another by Ala 19, Phe 22, Val23, Trp 25, Leu 26 and Met 27. Parts of these hydrophobic regions interact to form two kinds of trimers in the crystals. One trimer (Fig. lc) involves close contacts between equivalent residues (Phe 22, Val23, Leu 26, Met 27) of three identical molecules leaving the N-terminal halves free. The second type of trimer (Fig. Id) involves contacts between Trp 25 and Phe 22 of one molecule and Phe 6, Tyr 10 and Tyr 13 of another, as suggested earlier by Blanchard and King [9]. The centre of the trimer is mainly hydrophilic, involving charged residues such as Asp 15, Arg 17, Arg 18 and Asp 21. The two kinds of intermolecular contacts found in the trimers are not mutually exclusive and in the crystals both coexist so that the trimers become part of an extended oligomer of cubic symmetry. The formation of crystals containing such an arrangement at pHs from 3 to 9.5 indicates that self-association of helical conformers to trimers and oligomers occurs over a wide range of pH. At neutral pH, glucagon has a very low solubility due to extensive oligomerization which, unless the approach to supersaturation is carefully controlled, gives an amorphous precipitate. 0 Elsevier/NortJ+Holland Biomedical Prear 1979