ADVANCES IN BIOCHEMICAL ENGINEERING Volume 6 Editors" .T K. Ghose, A. Fiechter, N. Blakebrough Managing Editor" A. Fiechter With 82 Figures Springer-Verlag Berlin Heidelberg New York 7791 ISBN 3-540-08363-4 Springer-Verlag Berlin Heidelberg New York ISBN 0-387-08363-4 Springer-Verlag New York Heidelberg Berlin This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. @ by Springer-Verlag Berlin • Heidelberg 1977 Library of Congress Catalog Card Number 72-152360 Printed in Germany The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting, printing, and bookbinding: Briihlsche Universitg.tsdruckerei GieBen. 2152/3140-543210 Editors Prof. Dr. T. K. Ghose Head, Biochemical Engineering Research Centre, Indian Institute of Technology Hauz Khas, New Delhi 110029/India Prof. Dr. A.Fiechter Eidgen. Techn. Hochschule, Mikrobiologisches Institut, Weinbergstrage ,83 CH-8006 Ziirich Prof. Dr. N. Blakebrough University of Birmingham, Dept. Chemical Engineering, P.O.B. 363, Birmingham B15 2TT/England Managing Editor Professor Dr. A.Fiechter Eidgen. Techn. Hochschule, Mikrobiologisches Institut, WeinbergstraBe ,83 CH-8006 Ziirich Editorial Board Prof. Dr. .S Aiba Prof. Dr. R. M. Lafferty Biochemical Engineering Laboratory, Institute of Applied Techn. Hochschule Graz, Institut fiir Biochem. Technol., Microbiology, The University of Tokyo, Bunkyo-Ku, Tokyo, Schl6gelgasse 9, A-8010 Graz Japan Prof. Dr. M. Moo-Young Prof. Dr. B. Atkinson University of Waterloo, Faculty of Engineering, Dept. Chem. University of Manchester, Dept. Chemical Engineering, Eng., Waterloo, Ontario N21 3 GL/Canada Manchester / England Dr. I. N~iesch Dr, J.B6ing Ciba-Geigy, K 4211 B 125, CH-4000 Basel R6hm GmbH, Chem. Fabrik, Postf. 4166, D-6100 Darmstadt Dr. L. K. Nyiri Prof. Dr. J.R.Bourne Dept. of Chem. Engineering, Lehigh University, Whitaker Eidgen. Techn. Hochschule, Techn. Chem. Lab., Lab., Bethlehem, PA 18015/USA Universit~itsstraBe ,6 CH-8006 Ziirich Prof. Dr. H.J.Rehm Dr. E. Bylinkina Westf. Wilhelms Universitiit, Institut fiir Mikrobiologie, Head of Technology Dept., National Institute of Antibiotika, TibusstraBe 7--15, D-4400 M~nster 3a Nagatinska Str., Moscow M-105 / USSR Prof. Dr. P. L. Rogers Prof. Dr. H.Dellweg School of Biological Technology, The University of Techn. Universit~it Berlin, Lehrstuhl fiir Biotechnologie, New South Wales, PO Box ,1 Kensington, New South Seestral3e ,31 D-1000 Berlin 65 Wales, Australia 2033 Dr. A.L.Demain Prof. Dr. W. Schmidt-Lorenz Massachusetts Institute of Technology, Dept. of Nutrition Eidgen. Techn. Hochschule, Institut ftir Lebensmittelwissen- & Food Sc., Room 56-125, Cambridge, Mass. 02139/USA schaft, Tannenstral3e ,1 CH-8006 Ztirich Prof. Dr. R.Finn Prof. Dr. H. Suomalainen School of Chemical Engineering, Director, The Finnish State Alcohol Monopoly, Alko, Olin Hall, Ithaca, NY 14853/USA P.O.B. 350, 00101 Helsinki 10/Finland Dr. K. Kieslich Prof. Dr. F. Wagner Schering AG, Werk Charlottenburg, Max-Dohrn-Strage, Ges. .f Molekularbiolog. Forschung, Mascheroder Weg ,1 D-1000 Berlin 10 D-3301 St6ckheim Contents The Role of Thiobadllus ferrooxidans ni HydrometaUurgicul Processes .A E, Torma, Socorro/New Mexico (USA) Cellulase Biosynthesis and Hydrolysis of Cellulosic 93 Substances T. K. Ghose, New Delhi (India) Metabolism of Methanol by Yeasts 77 H. Sahm, Braunschweig (Germany) Control of Antibiotic Synthesis by Phosphate 501 J. F. Martin, Salamanca (Spain) The Role of ThiobaciUus ferrooxidans in Hydrometailurgical Processes Arpad E. Torma Department of Metallurgical and Materials Engineering, New Mexico Institute of and Mining Technology, Socorro, New Mexico 87801, USA Contents 1. Introduction ............................................. 2 2. Microbiological Background ........................ ~ ........... 3 a) Morphology of T. ferrooxidans ................................ 3 b) Physiology of T. ferrooxidans ................................. 3 c) Chemical Composition and Structure of T. ferrooxidans .................. 5 d) Mechanisms of Bacterial Action ................................ 5 e) Kinetics of Microbial Growth ................................. 7 3. Factors Influencing Bacterial Activity .............................. 10 a) Effect of Environment ..................................... 10 b) Effect of pH ........................................... 11 c) Effect of Temperature ..................................... 11 d) Effect of Nutrients ....................................... 13 e) Effect of Ferric Ion ....................................... 15 f) Effect of Particle Size and Surface Area ........................... 15 g) Effect of Regrinding of the Leach Residue .......................... 17 h) Effect of Surface Active Agents and Organic Solvents ................... 18 i) Effect of Adaptation of Bacteria to the Specific Substrate ................. 20 4. Microbiological Leach Techniques ................................. 20 5. Microbiological Leaching of Metal Sulfides ............................ 21 a) Copper Sulfides Leaching .................................... 21 b) Cobalt and Nickel Sulfides Leaching ............................. 22 c) Zinc Sulfide Leaching ...................................... 23 d) Lead Sulfide Leaching ..................................... 24 e) Uranium Extraction ....................................... 24 f) Leaching of Other Metal Sulfides ............................... 25 6. Biodegradation of Non-Sulfide Materials ............................. 27 7. Different Aspects of Bacterial Leaching ............................. 27 8. Conclusion .............................................. 28 9. Nomenclature ............................................ 28 10. References .............................................. 29 The present article illustrates the increased interest which is manifested in the microorganisms, Thiobacitlus ferrooxidans, involved in the biohydrometatlurgical extraction processes. The wide varieties of problems currently studied are very important in order to gain a better understanding about the factors which are governing the growth of microorganisms, and as a consequence, the metal dissolution phenomena. In several mining sites, the microbiological leaching techniques are currently practiced at industrial-scale, especially for recovery of copper and uranium from low-grade materials. However, an accurate assessment of further potential possibilities for the application of 2 .E daprA Torma smsinagroorcim ni gnihcael fundamental more a requires sulfides metal egdelwonk about the inter- actions of physical the dna factors chemical with growth the of .T snadixoorref ni pure dna mixed cultures gnidulcni heterotrophic dna the future Altogether, cohabitants. thermophilic lairtsudni exploitation of these lacigoloiborcim gnihcael techniques era attractive very ni countries many of the world. 1. Introduction In spite of the fact that bacterial oxidation of sulfide minerals has been occurring for centuries, microbiological leaching is only a recent development. The microorganisms, sullicaboihT ferrooxidans, responsible for this oxidation were first isolated in 1947 from the acid mine drainage of bituminous coal mines ]. 1 [ The presence of copper in mine drainage waters was observed by the Phoenicians, Romans, Arabs and Spaniards. The earliest leaching of copper from copper sulfide-bearing materials was recorded in 1970 at Rio Tinto in Spain [2]. However, the presence of bacteria in leach waters of the Rio Tinto mines was not confirmed until 1963 [3]. Dump leaching techniques were practised in the United States of America, Peru, Canada, Africa and in many other locations without knowing about the contribution of the microorganisms in these processes. The earliest report on microbiological leaching of metal sulfides was published in 1922, using some non identified autotrophic bacteria [4, 5] and suggesting that the biological treatment might be an economical way for the extraction of metals from low-grade sulfide-bearing ores. This idea was neglected for the next twenty five years until the discovery and characterization of the chemolithotrophic .T ferrooxidans [ 1,9-I ]. 1 These bacteria can tolerate exceptionally high metal and hydrogen ion concentrations, for example, 120 1/g of zinc [ 12], 72 1/g of nickel [ 13], 30 1/g of cobalt 13 [ ], 1/12 g U3Oa [ 14], 55 1/g of copper [15 ], 160 g/t of iron (II) [ 16] and an acid medium of pH 1.0 to 5.0 [ 17]. These facts are of considerable economic significance from metallurgical point of view and because, unlike many other fermentations, the bacterial leaching does not require an expensive sterilisation of the medium prior to inoculation. .T ferrooxidans are virtually ubiquitous. They can be found everywhere in nature, wherever an acidic environment is maintained in the presence of sulfide minerals [6-8]. The chemolithotrophic microorganisms 18] [ have the ability to utilize energy released from the metabolic oxidation of inorganic substrates 19, [ 20] such as reduced-valence inorganic sulfur compounds [21-23] and ferrous ion [24]. The chemical energy is converted by oxidative phosphorylation to ATP [25]. This is universally recognized to be the form of metabolic energy which can be utilized by the cell [26] for transportation work (substrate and nutrients into the cell and product out of the cell), mechanical work (muscle work for vibration and locomotion) and biosynthesis work (synthesis of cellular material). In this process the ATP is hydrolyzed to ADP and inorganic phosphate. These latter two species will be recombined into high energy carrier ATP in the follow- ed-up respiration. The carbon metabolism by the chemolithotrophic bacteria may be either autotrophic, or facultative which represents a nutritional mode between the autotrophic and heterotrophic metabolism. The autotrophic capabilities of bacteria ehT Rote of sullicaboihT snadixoorref in lacigrullatemordyH sessecorP 3 were established in 1887 [27] and can be defined as the ability to grow on strictly inorganic substrates providing energy for growth and carbon dioxide as the main source of carbon for the biosynthesis of cell materials [28, 30, 13 .] The discovery of .T ferrooxidans, opened up an area of research which has had and will continue to have considerable economic significance. It represents a potential solution to the problem faced in many countries where continuing depletion of high-grade ore deposits has created a need to develop effective methods for recovering metals from low-grade sulfide ores. The microbiologiclaela ching of metal sulfides can be defined as a biochemical oxidation process catalyzed by living organisms. However, only the insoluble sulfides are of com- mercial consequence. This process can be represented by the following simplified equation: SM + 2 02 microorganisms MSO4, (1) where is a M bivalent metal. When the oxidation product is insoluble, as it is the case, i.e., for the lead sulfide leaching, this fact can be used for selective leaching purpose [30] to separate the insoluble from the solubilized metals. The microbiological leaching processes involve complex interactions between the micro- organisms, substrates and the nutrient concentrations, which are not yet completely understood. Altogether, a more economic use of these leaching processes require a better understanding of the various factors influencing bacterial growth and as a con- sequence, the microbiological metal dissolution processes. 2. Microbiological Background a) Morphology of T ferrooxidans .T ferrooxidans possesses the following morphological characteristics: it is a motile, flagellated [32-34], non spore forming, Gram-negative, rod shaped (0.1 by 1.5/.tm) bacterium occurring single or occasionally in pairs [9, 10]. The growing cell goes through lag, log, stationary and death phases. When cell growth reaches about the double of a single cell size, it divides by binary fission. In the death cell, the mechanisms which regulate the permeability of the cell wall and cytoplasmic membrane do not function and the cell is plasmolyzed and broken downu nder the influence of the acid medium [35]. b) Physiology of ferrooxidans .T The microorganisms, .T ferrooxidans, derives the necessary energy for its life processes from oxidation of ferrous ion and of reduced-valence inorganic sulfur compounds and utilizes carbon dioxide forg rowth [33]. It is morphologically and, in some aspects, physiologically similar to .T thiooxidans, which is often present in acid mine drainage [36, 37]. The fundamental difference between the two species is generally recognized 4 daprA E. Torma to be the inability of .T thiooxidans to oxidize ferrous iron and insoluble metal sulfides [9, 10, 36]. Other bacteria have been identified from acid mine waters, oxidizing ferrous iron but not elemental sulfur or thiosulfate. It was considered to be a new genus and assigned the name of Ferrobaciltus ferrooxidans [38, 39]. Similarly, the name Ferrobacitlus sulfo- oxidans was assigned to a microorganism which utilized ferrousi ron and elemental sulfur but not thiosulfate [40]. Subsequent investigations [41-45] indicated that the microorganisms (T. ferrooxidans, F. ferrooxidans and F. sulfooxidans) were identical and should be called .T ferrooxidans. All these organisms were capable of oxidizing elemental sulfur and thiosulfate in addition to ferrous ion [44]. The earlier, apparent fragmentation of the nomenclature and classification for this single species resulted from the use of different techniques in studying it. A new approach in the naming and classifying of bacteria is to reflect the manner in which present organisms are related by virtue of descent [53]. The increasing know- ledge of comparative cytology and biochemistry led to the characterization of bacteria and blue-green algae as being procaryotic cells [54], possessing a simpler and an evolu- tionary more primitive structure than do all other cells (eucaryotic, i.e., possessing true mitotically dividing nuclei). The latest edition (8th) of the Bergey's Manual carries now the GC content of the DNA of each described nomenclatural type of organisms. The studies of DNA base composition appear to be the most beneficial when the cultures analysed are characterized by other biochemical means [55-57]. The DNA base composition of ferrous iron grown .T ferrooxidans, which belongs to the procaryote group of microorganisms, has been found to be in a narrow range of 56.0-57.0% GC [33]. However, recent studies [58, 59] on ferrous iron, chalcopyrite and lead sulfide grown .T ferrooxidans indicated 56.0, 60.1 and 54.4% GC respectively. The relatively important variations obtained for .T ferrooxidans grown on different substrate seemed to be not attributable to the analytical methods (melting temperature, cesium chloride density gradient centrifugation and ultraviolet absorbancy ratios) because of the good reproducibility of the results (5 t.0, 51.5, and 51.8% GC respectively) obtained for E. coli reference DNA. These variations cannot be explained by the adaption of .T ferrooxidans to the specific substrate. This problem is very complex and consi- derable caution must be exercised in extrapolating laboratory observations to micro- organisms in their natural habitat [60]. Under natural conditions pure culture do not occur, growth rates are very slow by laboratory standards. In the development of heavy metal resistance in various organisms, it is difficult to decide whether adaptation, mutation, cohabitation or a combination of these is involved. However, the possibility exists that in these studies a kind of selection of microorganisms took place as suggested by other investigators [61-63] who isolated new species of bacteria from cultures of ."7 ferrooxidans by changing environmental conditions and substrates. Data are available indicating that in the relatively strong sulfuric acid media [64] other (mixotrophic) microorganisms (algae, molds, protozoa and bacteria) than .T ferrooxidans can develop simultaneously. This view is supported by recent studies on microbial mutualism in ore leaching [65, 66], i.e., use of an aerobic, nitrogen fixing Beijerinckia lacticogenes in presence of .T ferrooxidans. Further, it has been pointed out [67], that considerable caution must be exercised when GC-data are compared from different laboratories. ehT Role of sullicaboihT snadixoorref ni lacigrullatemordyH sessecorP 5 c) Chemical Composition and Structure of .T ferrooxidans The first report [46] on cell composition of .T ferrooxidans indicated that it contained approximately 20% protein which consisted of 13 amino acids, and two B-vitamins: riboflavin and thiamine. These results have been refined [47] and the following cell composition has been obtained: 44% protein, 26% lipid, %51 carbohydrate, %01 ash, and at least, the two B-vitamins mentioned before. The cell structure of .T ferrooxidans has been found to be similar to that of other Gram-negative bacteria [47, 48]. The cell envelope, which is semi-permeable to nutrient [49, 50], appears to be composed of three osmophilic and three osmophobic layers, the total thickness measures 125 to 215 A [51, 52]. These layers are composed of lipo- protein, lipopolysaccharide, globular protein and peptidoglycan passing from outer to inner layers [51, 68]. The lipopolysaccharide layer consists of heptose, glucose, galac- tose, mannose and 2-keto-3-deoxyoctutosonate. Iron, mostly in the ferric form is associated with the lipopolysaccharide, suggesting it might serve as the initial binding site for the substrate. The peptidoglycan layer consists of glutaminic acid, a-e-diamino- pimelic acid, alamine, glucosamine and muramic acid [69]. These two layers are of similar composition as those ofE. coli strains [70-72]. Phospholipids and neutral lipids are also related to the cell envelope structure [73]. The cytoplasma of .T ferrooxidans contains ribosomes, nuclear materials and cell inclusions [52, 68, 69]. At the present time, the relation between the structure and the function of cell envelope of .T ferrooxidans remains to be obscure. However, a better understanding of these problems would be very important from the point of view of evolution of chemolitho- trophs and elucidation of oxidation pathways of insoluble inorganic sulfur substrates. d) Mechanisms of Bacterial Action The fact that .T ferrooxidans is capable of oxidizing ferrous ion and the reduced-valence forms of inorganic sulfur compounds, is an indication that it should have an enzymatic system similar to those of the iron and sulfur oxidizing bacteria. With regard to the mechanisms of metabolism of these substrates, there exist still different opinions over the basic concepts of oxidation pathways. There is agreement however, that the solid substrates must be rendered soluble before the bacterial oxidation could take place 17, [ 74] and the same time, nutrients have to be available in the environment of the contacted mineral surface. d. I) Oxidation of Inorganic Sulfur Compounds. The metabolism of inorganic sulfur compounds has been studied extensively [20, 21, 75, 76, 77, 80, 81]. The inorganic sulfide to sulfate oxidation can be represented by a simplified schema as follows: S -2 ~ +6 S + 8 e-. (2) In this reaction 8 electrons are removed from the substrate and will be carried out through a series of intermediate products: Sx SO , 2 -2, 2OS -2, $20~ 2- , $2042, SxO~ ,2- SO~ ,2 and so on. However, many of these products are highly unstable [78, 92] and probably 6 daprA .E Torma could not exist under physiological conditions. It has been proposed [79] that the first intermediate of sulfur oxidation is sulfite: + O S 02 + H20 bacteria 3OS~H (3) which is probably the key reaction in the pathways of the oxidation of sulfides, poly- sulfides or polythionates. On the basis of the available data [22, 82-85] the following scheme can be suggested for sulfides oxidation by .T ferrooxidans: 6 t t 7 S -2 ~ 0 S ~ 52032 ,i $4062 ~1 ~OS 2" ,~ S042. (4) 1 2 3 4 5 ~8 9 $3062 ]0 Reactions 1-4 are catalyzed by the sulfur-oxidizing enzyme [89, 83, 90, 91] where sulfite is the product of the reaction. Sulfite is oxidized to sulfate [85] by sulfite-oxi- dase (reaction 5) with the formation of ATP [88]. Thiosulfate is formed [84] by oxida- tion of elemental sulfur (reaction 7). It can be cleaved [82] by rhodanese (reaction 8) to sulfite and elemental sulfur. The thiosulfate is oxidized to tetrathionate (reaction 3) by the thiosutfate-oxidizing enzyme [84]. The reactions 8-10 may exist if .T ferro- oxidans species behave like other Thiobacilli [86, 87]. d. 2) Oxidation of Ferrous Ion The oxidation of ferrous ion has been studied by many investigators [34, 93-99] using .T ferrooxidans: Fe 2+ ~ Fe a+ + e-. (S) A mechanism of iron oxidation is proposed [24] in which the complexing of ferrous ion with molecular oxygen precedes the electron transport involving sulfate [ 100-102] and phosphatidylserine [73, 103] in the oxidation. The isolation of cytochrome c and cyto- chrome a from .T ferrooxidans 104] [ allowed the postulation of iron oxidation through the cytochrome system with oxygen as the final electron acceptor [96, 104-106]: ) ) ) (6) Fe 3+ reduced \ oxidized H20 cytochrome c cytochrome a It has been reported that the cytochrome c reductase is associated with DNA [107] and the nucleic acid involved was RNA 108]. [ Further, the iron-cytochrome c reductase was